Orchid Biochemistry

Orchid
Biochemistry
Edited by
Jen-Tsung Chen
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International Journal of Molecular Sciences
www.mdpi.com/journal/ijms
Orchid Biochemistry
Orchid Biochemistry
Editor
Jen-Tsung Chen
MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin
Editor
Jen-Tsung Chen
Department of Life Sciences
National University of
Kaohsiung
Kaohsiung
Taiwan
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Contents
About the Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Preface to ”Orchid Biochemistry” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
Jen-Tsung Chen
Orchid Biochemistry
Reprinted from: International Journal of Molecular Sciences 2020, 21, 2338, doi:10.3390/ijms21072338 1
Jean Carlos Cardoso, Cesar Augusto Zanello and Jen-Tsung Chen

An Overview of Orchid Protocorm-Like Bodies: Mass Propagation, Biotechnology, Molecular
Aspects, and Breeding
Jie Gao, Rui Ren, Yonglu Wei, Jianpeng Jin, Sagheer Ahmad, Chuqiao Lu, Jieqiu Wu,
Chuanyuan Zheng, Fengxi Yang and Genfa Zhu
Comparative Metabolomic Analysis Reveals Distinct Flavonoid Biosynthesis Regulation for
Leaf Color Development of Cymbidium sinense ‘Red Sun’
Mummadireddy Ramya, Seonghoe Jang, Hye-Ryun An, Su-Young Lee, Pil-Man Park and
Pue Hee Park
Volatile Organic Compounds from Orchids: From Synthesis and Function to Gene Regulation
Yingdan Yuan, Bo Zhang, Xinggang Tang, Jinchi Zhang and Jie Lin
Comparative Transcriptome Analysis of Different Dendrobium Species Reveals Active
Ingredients-Related Genes and Pathways
Björn Bohman, Alyssa M. Weinstein, Raimondas Mozuraitis, Gavin R. Flematti and

Anna-Karin Borg-Karlson
Identification of (Z)-8-Heptadecene and n-Pentadecane as Electrophysiologically Active
Compounds in Ophrys insectifera and Its Argogorytes Pollinator
Ying Zhang, Yuanyuan Li, Xiaomei Chen, Zhixia Meng and Shunxing Guo
Combined Metabolome and Transcriptome Analyses Reveal the Effects of Mycorrhizal Fungus
Ceratobasidium sp. AR2 on the Flavonoid Accumulation in Anoectochilus roxburghii during
Different Growth Stages
Surendra Sarsaiya, Archana Jain, Qi Jia, Xiaokuan Fan, Fuxing Shu, Zhongwen Chen, Qinian
Zhou, Jingshan Shi and Jishuang Chen
Molecular Identification of Endophytic Fungi and Their Pathogenicity Evaluation Against
Dendrobium nobile and Dendrobium officinale
Yiyi Zhang, Tinghong Zhou, Zhongwu Dai, Xiaoyu Dai, Wei Li, Mengxia Cao, Chengru Li,
Wen-Chieh Tsai, Xiaoqian Wu, Junwen Zhai, Zhongjian Liu and Shasha Wu
Comparative Transcriptomics Provides Insight into Floral Color Polymorphism in a Pleione
limprichtii Orchid Population
v
Lei Zhang, Chunyan Jiao, Yunpeng Cao, Xi Cheng, Jian Wang, Qing Jin and Yongping Cai
Comparative Analysis and Expression Patterns of the PLPd eCGenesinDendrobium officinale
Reprintedf rom : International Journal of Molecular Sciences2019, 21, 54, doi : 10.3390/ijms21010054 161
Fusheng Jiang, Meiya Li, Hongye Wang, Bin Ding, Chunchun Zhang, Zhishan Ding, Xiaobo
Yu and Guiyuan Lv
Coelonin, an Anti-Inflammation Active Component of Bletilla striata and Its Potential
Mechanism
Reprinted from: International Journal of Molecular Sciences 2019, 20, 4422, doi:10.3390/ijms20184422177
Anjin Zhao, Zheng Cui, Tingge Li, Huiqin Pei, Yuhui Sheng, Xueqing Li, Ying Zhao, Yang
Zhou, Wenjun Huang, Xiqiang Song, Ting Peng and Jian Wang
mRNA and miRNA Expression Analysis Reveal the Regulation for Flower Spot Patterning in
Phalaenopsis ‘Panda’
Reprinted from: International Journal of Molecular Sciences 2019, 20, 4250, doi:10.3390/ijms20174250195
vi
About the Editor
Jen-Tsung Chen
Jen-Tsung Chen is currently a professor at the National University of Kaohsiung in Taiwan. He
teaches cell biology, genomics, proteomics, medicinal plant biotechnology, and plant tissue culture
in college. Dr. Chen’s research interests include bioactive compounds, chromatography techniques,
plant tissue culture, phytochemicals, and plant biotechnology. He serves as an editorial board
member of Plant Methods, Biomolecules, International Journal of Molecular Science, and a guest editor
in Frontiers in Plant Science, Frontiers in Pharmacology, Journal of Fungi.
vii
Preface to ”Orchid Biochemistry”
Orchidaceae is the second largest family among the angiosperms and it consists of over
twenty-eight thousand species worldwide across a wide range of habitats. It is well known that
orchids possess attractive flowers that have high economical value in the global horticultural market.
What often receives less attention is the fact that some orchids are edible or scented, and, more than
this, many species have long been used in preparations as traditional medicine. In the past two
decades, extensive studies have been conducted on genetic as well as functional genomics to achieve
certain progress in biotechnology and breeding programs for orchids. However, in-depth research on
bioactive compounds, flower pigments, and floral scents is still limited to several species. This book
gives emphasis to molecular insights into orchid biology, biotechnology, and biochemistry based
on advanced and high-throughput technologies. It offers a critical reference for students, teachers,
researchers, and professionals in the fields of orchid biology. As the editor of this book, I greatly
appreciate the invaluable contribution of all authors and reviewers as well as the efforts of Editors in
the International Journal of Molecular Sciences: Molecular Plant Sciences.
Jen-Tsung Chen
Editor
ix
International Journal of
Molecular Sciences
Editorial
Orchid Biochemistry
Jen-Tsung Chen
Department of Life Sciences, National University of Kaohsiung, Kaohsiung 811, Taiwan; [email protected]

Received: 25 March 2020; Accepted: 26 March 2020; Published: 27 March 2020
Orchids belong to Orchidaceae which is one of the largest families in flowering plants. This family
comprises over twenty thousand members, and many of them are fascinating with attractive flowers
that sell in the markets with increasing demand around the world. What often receives less attention is
the fact that some orchids are edible or scented, and more than this, many species have long been used
in preparations in traditional medicine. The Special Issue “Orchid Biochemistry” collected original
research and review articles that explore molecular aspects and insights into pigment formation, floral
scent and pollination, bioactive compounds, plant-microbial interaction as well as biotechnology in
orchid species.
1. Pigment Formation
Orchid populations have always been good materials for revealing the secrets of plant evolution.
Zhang et al. studied species evolution using comparative transcriptomics in the P. limprichtii population
that has a wide range of floral color varieties [1]. They proposed that the distribution pattern of
different color morphs may be considered as a reproductive strategy that plays an important role in
maintaining the population size. In this study, a molecular mechanism of color variation was proposed
in which a crucial gene PlFLS interacts with a putative MBW protein complex (MYB, bHLH, and WDR),
which may sever as a repressor of anthocyanin accumulation.
Flower spot patterning could affect the ornamental value of some orchids and may play a
significant role in the interaction with pollinators. Zhao et al. used a transcriptome analysis in
the anthocyanin biosynthetic pathways of Phalaenopsis “Panda” to identify differentially expressed
genes (DEGs) [2]. They further confirmed that some candidate structure genes among the DEGs
expressed in significantly higher levels in spot tissues using qPCR analysis. Eventually, differentially
expressed miRNAs (DEMs) were analyzed and 40 DEMs target transcription factor genes were found
to express in significantly different levels in the spot sepal. According to the results, they proposed a
microRNA-suppressing model for explaining the regulation in flower spot formation.
A comparative metabolomic study was made by Gao et al. and aims to reveal the regulation of
flavonoid biosynthesis that contribute to leaf color formation in a foliage orchid, Cymbidium sinense
“Red Sun” [3]. They identified 196 flavonoid-related metabolites using a UPLC-MS/MS-based method
and revealed that the trends of leaf color changing from red to yellow and eventually to green,
were mainly contributed by down-regulated anthocyanin biosynthetic enzymes.
2. Bioactive Compounds
Dendrobium orchids possess a number of bioactive compounds that have been used as traditional
Chinese medicine for thousands of years. In recent years, a number of Dendrobium transcriptomes
have been announced, and it opens a way to predict gene functions via in silico analysis. Zhang et al.
performed a comparative analysis of PLP_deC genes in D. officinale, and the results showed that
they may be involved in the responses of abiotic stress and consequently affect the biosynthesis of
secondary metabolites [4]. Yuan et al. used weighted gene co-expression network analysis to predict
crucial gene modules that may be involved in the regulation and biosynthesis of active compounds in
Dendrobium orchids [5].
Int. J. Mol. Sci. 2020, 21, 2338; doi:10.3390/ijms21072338 1 www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2020, 21, 2338
Bletilla striata (Thunb.) Reichb.f is an important traditional Chinese herb with multi-bioactivities.
A dihydrophenanthrene compound, coelonin, was isolated and identified by Jiang et al. [6].
This compound mainly has anti-inflammatory activity, and the negative regulator phosphatase
and tensin homologue on chromosome ten (PTEN) may play a crucial role in inhibiting macrophage
proliferation and inflammatory factor secretion when treated silicosis.
3. Flower Scent
Bohman et al. studied the mechanism of pollination in European Ophrys orchids, and they
identified two new pollinator attractants, including (Z)-8-Heptadecene and n-pentadecane, and gained
insights into the biosynthesis of semiochemicals [7]. Ramya et al. contributed a review to summarize
the advances of volatile organic compounds in orchids mainly focusing on their gene expression
patterns in different tissues and developmental stages of Cymbidium orchids as well as their key role in
pollination ecology [8]. They proposed a molecular breeding strategy through the manipulation of
floral traits to improve the quality of orchids in the future.
4. Plant-Microbial Interaction
Orchids commonly have a symbiotic relationship with mycorrhizal fungi that benefit seed
germination, seedling growth, and development. Sarsaiya et al. identified five species of
myco-endophytes in Dendrobium orchids, and subsequent in vitro testing showed that they could
affect seedling growth, especially at the stage of protocorms [9]. Zhang et al. profiled metabolome
and transcriptome in the symbiosis between a mycorrhizal fungus, Ceratobasidium sp. AR2, and a
medicinal orchid, Anoectochilus roxburghii [10]. They concluded that C. sp. AR2 could induce
differential expressions, particularly in flavonoid biosynthetic genes and accomplished an increase in
the accumulation of some flavonoids. They proposed that C. sp. AR2 has a high potential to enhance
the quality of A. roxburghii.
5. Biotechnology
Orchids have a unique structure that induces from explants in vitro, namely protocorm-like bodies
(PLBs) that resemble or equate to somatic embryos. Cardoso et al. provided an overview of PLBs in
aspects of biotechnology and molecular biology [11]. Commonly, orchid PLBs are adequate materials
for studying developmental biology and breeding techniques. In this review, they suggested that
techniques using induction, proliferation, and regeneration of PLBs could be applied in the commercial
mass propagation of orchids in the future.
6. Conclusions and Perspectives

Overall, this Special Issue collected recent advances in orchid biochemistry, including original
articles and reviews. It provides in-depth insights into the biology of pigment and flower scent
formation, bioactive compounds, and plant-microbial interaction as well as the biotechnology of PLBs.
With the rapid progress of high-throughput technologies and integrative omics, scientists may have
more opportunities to reveal the secret of orchid biology in the future.
Funding: This research received no external funding.

Conflicts of Interest: The author declares no conflicts of interest.
2
Int. J. Mol. Sci. 2020, 21, 2338
References
1. Zhang, Y.; Zhou, T.; Dai, Z.; Dai, X.; Li, W.; Cao, M.; Li, C.; Tsai, W.-C.; Wu, X.; Zhai, J.; et al. Comparative
transcriptomics provides insight into floral color polymorphism in a Pleione limprichtii orchid population.
Int. J. Mol. Sci. 2020, 21, 247. [CrossRef] [PubMed]
2. Zhao, A.; Cui, Z.; Li, T.; Pei, H.; Sheng, Y.; Li, X.; Zhao, Y.; Zhou, Y.; Huang, W.; Song, X.; et al. mRNA and
miRNA expression analysis reveal the regulation for flower spot patterning in Phalaenopsis ‘Panda’. Int. J.
Mol. Sci. 2019, 20, 4250. [CrossRef] [PubMed]
3. Gao, J.; Ren, R.; Wei, Y.; Jin, J.; Ahmad, S.; Lu, C.; Wu, J.; Zheng, C.; Yang, F.; Zhu, G. Comparative metabolomic
analysis reveals distinct flavonoid biosynthesis regulation for leaf color development of Cymbidium sinense
‘Red Sun’. Int. J. Mol. Sci. 2020, 21, 1869. [CrossRef] [PubMed]
4. Zhang, L.; Jiao, C.; Cao, Y.; Cheng, X.; Wang, J.; Jin, Q.; Cai, Y. Comparative analysis and expression patterns
of the PLP_deC genes in Dendrobium officinale. Int. J. Mol. Sci. 2020, 21, 54. [CrossRef] [PubMed]
5. Yuan, Y.; Zhang, B.; Tang, X.; Zhang, J.; Lin, J. Comparative transcriptome analysis of different Dendrobium
species reveals active ingredients-related genes and pathways. Int. J. Mol. Sci. 2020, 21, 861. [CrossRef]
[PubMed]
6. Jiang, F.; Li, M.; Wang, H.; Ding, B.; Zhang, C.; Ding, Z.; Yu, X.; Lv, G. Coelonin, an anti-Inflammation active
component of Bletilla striata and its potential mechanism. Int. J. Mol. Sci. 2019, 20, 4422. [CrossRef] [PubMed]
7. Bohman, B.; Weinstein, A.M.; Mozuraitis, R.; Flematti, G.R.; Borg-Karlson, A.-K. Identification of
(Z)-8-heptadecene and n-pentadecane as electrophysiologically active compounds in Ophrys insectifera
and Its Argogorytes pollinator. Int. J. Mol. Sci. 2020, 21, 620. [CrossRef] [PubMed]
8. Ramya, M.; Jang, S.; An, H.-R.; Lee, S.-Y.; Park, P.-M.; Park, P.H. Volatile organic compounds from orchids:
From synthesis and function to gene regulation. Int. J. Mol. Sci. 2020, 21, 1160. [CrossRef] [PubMed]
9. Sarsaiya, S.; Jain, A.; Jia, Q.; Fan, X.; Shu, F.; Chen, Z.; Zhou, Q.; Shi, J.; Chen, J. Molecular identification of
endophytic fungi and their pathogenicity evaluation against Dendrobium nobile and Dendrobium officinale.
Int. J. Mol. Sci. 2020, 21, 316. [CrossRef] [PubMed]
10. Zhang, Y.; Li, Y.; Chen, X.; Meng, Z.; Guo, S. Combined metabolome and transcriptome analyses reveal the
effects of mycorrhizal fungus Ceratobasidium sp. AR2 on the flavonoid accumulation in Anoectochilus roxburghii
during different growth stages. Int. J. Mol. Sci. 2020, 21, 564. [CrossRef] [PubMed]
11. Cardoso, J.C.; Zanello, C.A.; Chen, J.-T. An overview of orchid protocorm-like bodies: Mass propagation,
biotechnology, molecular aspects, and breeding. Int. J. Mol. Sci. 2020, 21, 985. [CrossRef] [PubMed]
© 2020 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
3
Molecular Sciences
Review
An Overview of Orchid Protocorm-Like Bodies: Mass
Propagation, Biotechnology, Molecular Aspects,
and Breeding
Jean Carlos Cardoso 1 , Cesar Augusto Zanello 2 and Jen-Tsung Chen 3, *
1 Laboratory of Plant Physiology and Tissue Culture, Department of Biotechnology,
Plant and Animal Production, Centro de Ciências Agrárias, Universidade Federal de São Carlos,
Rodovia Anhanguera, km 174, CEP 13600-970 Araras, SP, Brazil; [email protected]
2 Masterscience degree by Programa de Pós Graduação em Produção Vegetal e Bioprocessos Associados,
Centro de Ciências Agrárias, Universidade Federal de São Carlos, CEP 13600-970 Araras, SP, Brazil;
[email protected]
3 Department of Life Sciences, National University of Kaohsiung, Kaohsiung 811, Taiwan
* Correspondence: [email protected]; Tel.: +886-7-591-9453

Received: 1 January 2020; Accepted: 28 January 2020; Published: 2 February 2020
Abstract: The process through induction, proliferation and regeneration of protocorm-like bodies
(PLBs) is one of the most advantageous methods for mass propagation of orchids which applied
to the world floricultural market. In addition, this method has been used as a tool to identify
genes of interest associated with the production of PLBs, and also in breeding techniques that use
biotechnology to produce new cultivars, such as to obtain transgenic plants. Most of the molecular
studies developed have used model plants as species of Phalaenopsis, and interestingly, despite
similarities to somatic embryogenesis, some molecular differences do not yet allow to characterize
that PLB induction is in fact a type of somatic embryogenesis. Despite the importance of species for
conservation and collection purposes, the flower market is supported by hybrid cultivars, usually
polyploid, which makes more detailed molecular evaluations difficult. Studies on the effect of plant
growth regulators on induction, proliferation, and regeneration of PLBs are the most numerous.
However, studies of other factors and new technologies affecting PLB production such as the use of
temporary immersion bioreactors and the use of lighting-emitting diodes have emerged as new tools
for advancing the technique with increasing PLB production efficiency. In addition, recent studies
on Phalaenopsis equestris genome sequencing have enabled more detailed molecular studies and the
molecular characterization of plantlets obtained from this technique currently allow the technique
to be evaluated in a more comprehensive way regarding its real applications and main limitations
aiming at mass propagation, such as somaclonal variation.
Keywords: biotechnology; breeding; mass propagation; Orchidaceae; protocorm-like bodies;

somaclonal variation; somatic embryogenesis
1. Introduction
Orchids (Family Orchidaceae) represent one of the two largest plant families, including from
736 [1] to 899 genera and 27,800 accepted species names [2] and over 100,000 hybrids produced by
artificial pollination [3]. In addition to their unquestionable botanical and ecological importance,
orchids participate in current cultivation systems using high-tech horticulture, grown in environments
with good climate control, especially temperature, which allows the induction of flowering regardless of
the time of year, especially aiming at the scheduled supply of potted and cut flowers in the competitive
world flower market. Some species of orchids, such as the genera Dendrobium, Gastrodia, and Bletilla,

Int. J. Mol. Sci. 2020, 21, 985
have also been used for medicinal purposes, using the basis of traditional Chinese medicine [4] and
some Vanilla species is also used for food purposes [5].
In this economic context, family Orchidaceae currently represents one of the most important in
the world commercial floriculture, with emphasis on the genus Phalaenopsis as well as its interspecific
hybrids, which is currently the main potted flower marketed in the main world flower markets. To have
an idea of the importance of this genus in the expansion of world floriculture, only in the Dutch market,
the largest in the world, in 2014, 121 million pots of Phalaenopsis were sold generating approximately
US$ 500 million [6]. In addition to Phalaenopsis, other genera of economic importance to floriculture
include the genera Cattleya, Dendrobium, and Oncidium and their hybrids [7–9] as well as Cymbidium
and Vanda used for production of potted or even cut flowers.
Despite the individual importance of these genera, a commercial classification for orchids must
be set separately from the botanical classification. This is because although genera have a greater
genetic and morphological contribution to commercial plants, most commercial flower production of
these genera occurs through the production of hybrids from interspecific crosses, which include the
use of crosses between species of the same genus, but also species of different genera (intergeneric
hybrids) [9]. An example of this case is the very frequent use of Doritis in crossings with Phalaenopsis,
generating the hybrid genus known as Doritaenopsis [10,11]. Nevertheless, commercially these hybrids
are all called Phalaenopsis because considering the morphological similarity and commercialization
value, there is no commercial justification for separation into two classes.
Another justification for the separation of botanical and commercial classification is the recent
changes of genera in many species, including those of commercial importance and resulting from
the advancement of available molecular techniques that allow genetic rather than just morphological
comparisons [1]. An example would be the genera Laelia and Sophronitis, commonly used in crossings
with the genus Cattleya to incorporate hybrids with red, yellow and orange flowers, little present
in Cattleya. Both Laelia and Sophronitis have undergone more than one change in their names in the
last decade, with new changes possibly still remaining due to advances in molecular markers and
phylogenetic aspects related to this complex and diverse plant family [12,13].
Thus, it is important to highlight this botanical difference from the commercial one, due to the
complexity of the family and its high hybridization capacity. Thus, using as an example the commercial
classification encompassing these genera includes not only the genus, but its many hybrids used for the
genetic improvement and development of new cultivars for the world floriculture. When mentioning
Cattleya, this includes genera such as Laelia, Sophronitis, Broughtonia, Epidendrum, Encyclia, Caularthron,
among other correlates and with possible hybridization with Cattleya. The same occurs in Oncidium, in
which plants of different genera such as Brassia, Ionopsis, Odontoglossum, Miltonia, among others [14] are
used for breeding intergeneric hybrids and many commercial hybrids are the result of combinations of
more than two genera.
In few plant families it is possible to obtain so many viable and fertile combinations of progenies
from very different morphologically species and genera. This allows breeders to incorporate numerous
traits of interest into a single plant, which brings the innovative aspect of flower production as well as
the advance in breeding, using these same mostly fertile hybrids for the advancement of generations of
crosses and obtaining new hybrids. This high hybridization capacity may be a result of the specific
process of embryogenic development and later protocorm development that occur in orchids [15]. In
other species, it has been reported that lack of hybridization and hybrid seed abortion is associated
with disruption of proper endosperm development or mismatch between endosperm development
and embryo [16]; and zygotic embryogenesis in family Orchidaceae, embryo development occurs in
the absence of endosperm [15].
After obtaining the hybrid of commercial interest, propagation is the factor that defines the time
for this hybrid to be available in the market for clonal propagation, which ensures the maintenance of
the selected characteristics in propagated plants, quickly, on a large scale and allowing the production
of plantlets throughout the year. These propagation characteristics, in addition to ensuring the quality
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Int. J. Mol. Sci. 2020, 21, 985
of the plantlets produced, also aim to maintain the commercial scale necessary to meet the target market.
The only viable technique that combines all these characteristics has been in vitro micropropagation of
orchids [17].
Among the in vitro cultivation techniques used for the in vitro seedling or plantlets production of
orchids, it can be used the in vitro asymbiotic germination and micropropagation techniques aiming at
the large-scale production of clonal plantlets.
Asymbiotic germination involves the in vitro inoculation and germination of orchid seeds with
the aid of a sucrose-containing culture medium [18,19], under conditions free of microorganisms;
including those symbionts that assist in germination, especially under natural conditions, a technique
known as symbiotic germination, which can be done in vitro [19,20], ex vitro, or in situ and which,
unlike asymbiotic, considers the use of symbiotic microorganisms to assist in the germination and
early development of newly germinated seedlings, and lacking nutritional reserves to support early
seedling development [20,21].
Techniques involving the germination of orchid seeds under in vitro conditions are especially used
in: Conservation and production of seedlings of native species; germination of seedlings from crosses
aiming at genetic improvement and production of new orchid cultivars [8]; aiming at the production
of protocorms in order to study somatic embryogenesis in vitro, also known as protocorm-like bodies
or simply PLBs [17,22]. They can also be used for commercial propagation and seedlings production,
but with high genetic variability inherent in the family Orchidaceae, including commercial groups
used for flower production [8].
In vitro germination of orchids makes it possible to increase the efficiency of conservation and
breeding programs, since in vitro germination rates higher than 70% are commonly reported [23],
while in ex vitro conditions under natural environmental conditions, these rates hardly exceed 5%
germinated seeds [24]. This is especially due to the fact that orchid seeds do not contain nutritional
reserves [25], and the embryo and seedlings at early germination are highly dependent on symbiosis
with microorganisms known as mycorrhizae, which nutritionally supply these plants during a long
time until the complete establishment of the seedling in the natural environment [26]. In Serapias
vomeracea orchid, in symbiosis with Tulasnella calospora there was observed a differential gene expression
related to organic nitrogen transport and metabolism, showing the nutritionally supply of fungus to
orchids in early development of protocorms [27].
A characteristic of the in vitro asymbiotic germination of orchids is the formation of the so-called
protocorms, prior to budding, mainly containing the first leaves and undeveloped stem, followed by
the roots [25] and later on with the development of the leaf and pseudobulb.
The term protocorm-like bodies (PLBs) is used as a reference to this type of protocorm-producing
germination, characteristic of orchids. The main difference between the germination and the sexual
reproduction process, which includes the fertilization process, zygotic embryogenesis, followed by the
germination and formation of protocorms, is that PLBs comes from somatic tissues, therefore being
considered a type of vegetative propagation.
The production of PLBs, therefore, can be compared to a specific type of somatic embryogenesis
that occurs in orchids, and the anatomy, development and characteristics of cells and some cell wall
markers at the beginning of PLB formation are similar to those in the development of protocorms
in orchids [28]. These authors observed that in non-embryogenic callus of Phalaenopsis orchids, the
inability to synthesize some cell wall components such as the JIM11 and JIM20 epitopes resulted
in loss of morphogenic capacity of these calli, and the correct formation of the cell wall is directly
associated with the ability of cell division and elongation in these cell types. In contrast, embryogenic
calli synthesized these components, similar to what occurred in zygotic embryogenesis [28].
Despite these anatomical and cellular similarities between PLB induction and zygotic
embryogenesis, molecularly, zygotic embryogenesis in Phalaenopsis aphrodite is considered different
from PLB formation, and that induction of PLBs follows a different route from the embryogenic
program [29]. One explanation for these differences is a consequence of the degree of speciation for
7
Int. J. Mol. Sci. 2020, 21, 985
the development of the embryogenic program in orchids, which follows a very specific pattern and
different from the conventional embryogenic program occurring in species of other families, such as
the absence of endosperm development and gene expression for establishing symbiotic relationships
during seed germination process [15].
Due to these still-present doubts regarding comparisons of zygotic embryogenesis with induction
of PLBs in orchids, we have adopted the term IPR–PLB (induction, proliferation, and regeneration
of PLBs) as the standard to describe this technique in this paper. IPR–PLBs in orchids have different
applications in the world flower industry. Undoubtedly the one with the largest commercial application
is aimed at the mass propagation of clonal plants to meet the world’s demanding flower production
market, in which orchids play a significant part in both the pot and cut flower market [6,30]. However,
other applications such as for species conservation purposes [31] and obtaining transgenic plants [32]
can be found in the literature.
Despite a significant amount of studies with IPR-PLB in different orchid species and hybrids, such
as Coelogyne cristata and C. flaccida [33,34], Cyrtopodium paludicolum [35], Grammatophyllum speciosum [36]
among others, this review has as its main objective to compile the recent studies and advances found
in the induction, proliferation and regeneration of PLBs from the two most important genera in the
world flower market, especially Phalaenopsis and Oncidium hybrid groups.
2. Genus Phalaenopsis and Related

The limited efficiency of clonal multiplication by the induction of shoots from floral stems
cultivated in vitro has been one of the main difficulties faced in micropropagation of Phalaenopsis,
resulting in an increase in the production cost of micropropagated plantlets [37] and associated
with falling prices in the international market [6] place in vitro plantlets as the current major cost of
producing Phalaenopsis. In this sense, the IPR–PLBs can be an important tool in the micropropagation of
commercial hybrids of this genus aiming to increase the production efficiency, being necessary to know
the main factors involved in each phase of plantlets from PLBs production, e.g., induction, proliferation,
and regeneration, which result in efficient clonal and mass propagation techniques for Phalaenopsis.
The first studies involving clonal micropropagation of Phalaenopsis were conducted by [38–40]
using Phalaenopsis amabilis as a model. Soon after, [41] concluded that leaf segments obtained from
inflorescence buds grown in vitro when grown in New Dogashima Medium (NDM) [41] medium
supplemented with 0.1 mg L−1 NAA (Naphthaleneacetic Acid) and 1.0 mg L−1 BA (6-Benzyladenine)
could generate up to 10,000 PLBs within a year. Ref. [42] also reported PLB regeneration from a callus
induction phase (indirect somatic embryogenesis) using Vacin Went medium [43] supplemented with
20% coconut water and 4% sucrose with the hybrid Phalaenopsis Richard Shaffer ‘Santa Cruz’.
In orchids, PLBs are suggested to be somatic embryos due to the morphological similarity
and developmental pattern observed between them and the zygotic embryos [42,44]. Besides that,
ontogenetic studies based on histological and histochemical methods developed by [28] compared the
early developmental pattern of zygotic embryos and PLBs, which led to the conclusion that cytological
characteristics and cell wall markers were similar in the early developmental stages of both zygotic
embryos and PLBs, which would justify saying that PLBs are somatic embryos. Still, histological
analyses made by [45] also showed that the formation of PLBs occurs directly on the epidermal surface
of the leaf segment with a cluster of meristem cells in constant division and without connection with
the leaf vascular system, which is interesting from a commercial point of view, since it ensures the
health of plants obtained through PLBs [46–49] and enable success of genetic transformation [50,51].
In several plant species, some genes that are involved in somatic embryogenesis, known as SERK
(somatic embryogenesis receptor-like kinase), are described. Ref. [52] characterized and analyzed the
expression of 5 of these genes in Phalaenopsis and which were described by the authors as PhSERK.
According to this study, the expression of these 5 genes was observed in various parts of plants
(root, leaf, apical bud, and flower meristem) as well as during seed germination and PLB induction.
According to the authors, PLBs segmented and grown in secondary PLB-inducing medium showed
8
Int. J. Mol. Sci. 2020, 21, 985
high PhSERK5 expression during the third week, when secondary PLBs became visible, suggesting that
this SERK transcription may be closely associated with the acquisition of embryogenic competence
during formation of PLBs. It is noteworthy that transformed Arabidopsis plants with overexpression
of the AtSERK1 gene showed high capacity for induction of somatic embryos in in vitro culture [53],
showing that this gene is indeed involved in somatic embryogenesis, at least in Arabidopsis.
Although cytological features indicate that a PLB is a somatic embryo and studies have shown
PhSERK gene expression during PLB induction [52], transcriptome studies developed by [29] analyzing
gene expression in Phalaenopsis aphrodite concluded that PLBs are molecularly distinct from zygotic
embryos. According to the authors, PLBs share different transcriptomic signatures from zygotic
embryos, and early processes of PLB development show a distinct regeneration program, not following
the embryogenesis program. In addition, the authors report that the SHOOT MERISTEMLESS gene, a
class I KNOTTED-LIKE HOMEOBOX gene, probably plays an important role in PLB regeneration and
should be further investigated.
The genetic transformation with AtRKD4 gene, which encode proteins with RWP-RK transcription
factor and is associated to early embryogenic pattern in Arabidopsis thaliana [54], also increases the
number of PLBs produced in leaves of this Phalaenopsis ‘Sogo vivien’ [55] and Dendrobium phalaenopsis [56]
transgenic plants.
Recent studies with Phalaenopsis equestris genome sequencing [57], with 2n = 2x = 38 and 29,431
predicted protein-coding genes and Phalaenopsis Brother Spring Dancer ‘KHM190’ [58], 2n = 2x = 38
and 41,153 protein coding genes, make room for further detailed studies on the identification and
expression of genes involved in the production of PLBs from different types of somatic tissue in
orchids, which can be compared with other model species and in which the embryogenic pathway is
already better elucidated, similar to the studies already carried out that brought new discoveries about
flowering and the development of floral organs [58].
Among the several factors that regulate somatic embryogenesis in Phalaenopsis, the absence of
light is described as responsible for the PLB induction step [59]. After maintaining the leaf segments for
60 days in the dark, it is possible to observe at the ends of the segments the formation of embryo-like
structures, still with a yellowish-white color (Figure 1A). After about 15 days under 14 h light
photoperiod, PLBs change color to light green and dark green (Figure 1B) and after 90 days subjected
to light there is the onset of differentiation of PLBs with leaf primordia to their complete differentiation
with leaf and root formation. The PLBs also could be induced from shoots and proliferate in solid
(Figure 1C) or liquid medium under shake agitation (Figure 1D).
From these observations, it is possible to infer that the absence of light plays an important role in
the induction of PLBs, just as light influences the differentiation of PLBs into plantlets. Also, according
to [60], the type of light used can also optimize the regeneration of PLBs, with the use of red and white
LED combined with sucrose as a carbohydrate source, or blue and white LED with trehalose as the
carbohydrate source, which had the best response for the regeneration of PLBs. However, only 17.5%
of papers described a dark-period to induce PLBs, while 67.5% used light period (12-16-h photoperiod)
to induce and regeneration of PLBs in Phalaenopsis (Table 1).
9
Int. J. Mol. Sci. 2020, 21, 985
ȱ
Figure 1. Induction, proliferation and regeneration of protocorm-like bodies in Dendrobium and
Phalaenopsis orchids. Protocorm-like bodies (PLBs)-directly induced from leaf segments of Phalaenopsis
hybrid ‘501’ (A) obtained from young in vitro shoots from inflorescence nodal segments and details
of secondary PLBs (B) obtained in New Dogashima Medium (NDM) culture medium. Proliferation
of PLBs in agar (C) and liquid (D) MS1⁄2 culture medium of Dendrobium ‘Hybrid 3’. Bars = 1 cm.
Unpublished photos of Cesar A. Zanello (A,B) and Jean C. Cardoso (C,D).
Besides the influence of light, another admittedly important factor in the induction of PLBs in
Phalaenopsis and orchids in general is the genotype [61]. This means that under the same cultivation
condition, the induction responses of PLBs may be significantly different [62], which is still considered
a limitation of the technique. Ref. [30] evaluated the induction of PLBs in two commercial hybrids
(Ph908—red-painted yellow flowers and RP3—dark red) of Phalaenopsis and reported significant
differences in both percentage of PLB leaf segments (45% and 10%, respectively) as in the number of
PLBs per leaf segment (25 and 2 PLBs, respectively).
Regarding the type of explant, leaf segments of plants grown in vitro have been the most suitable
for induction of PLBs in Phalaenopsis (45% of papers; Table 1), but there are reports of protocols that
used in vitro roots of P. ‘Join Angle × Sogo Musadian’ cultivated in MS1⁄2 medium supplemented with
NAA, BAP, and IAA (0.5 ppm, 5 ppm, and 0.5 ppm, respectively) and up to 49.33 PLBs/explant [63].
10
Int. J. Mol. Sci. 2020, 21, 985
Table 1. Compliance of studies with induction, proliferation and regeneration of PLBs (IPR-PLBs) with Phalaenopsis and Doritaenopsis.
Species or Hybrids Origin and Age of Explants Culture Media Growth Conditions Main Results Evaluation and Detection of SV Reference
NDM added 10 g L−1 93–100% survival rate of explants,
Shoot tips derived from sucrose, 2 g L−1 Gelrite, 23 ± 1 ◦ C, 14-h photoperiod, 33–40% PLB formation, green color
12 cultivars of Phalaenopsis Non-evaluated [41]
flower stalk buds 0.1 mg L−1 NAA and 33 μmol m−2 s−1 of PLBs showed multiplication,
1–5 mg L−1 BA 27–28% PLBs formed shoots
MS1⁄2 + 100 mg L−1
myo-inositol + 0.5 mg L−1 Both TDZ and BA were able to
niacin and pyridoxine + induce PLBs in calluses, but Not observed any phenotypic
Calluses derived from 1–2 0.1 mg L−1 thiamine + 26 ± 2 ◦ C, 16-h photoperiod, interestingly equal number of PLBs abnormality and no chromosome
Phalaenopsis Nebula [64]
months protocorms 2.0 mg L−1 glycine + PPFD 28–36 μmol m−2 s−1 per callus (74) was obtained when number alterations were
170 mg L−1 NaH2 PO4 + callus was transferred to free-PGR observed in 2–3 months plantlets
20 g L−1 sucrose + 2.2 g L−1 medium
Gelrite, pH 5.2
100 ml medium per 0.5 g inoculum
25 ± 2 ◦ C, 16-h photoperiod, under agitation (9.2 PLBs/PLB
Section transversely cutted Liquid Hyponex modified PPFD 60 μmol m−2 s−1 , section) or air-lift balloon with 10.0 g
from apical meristems medium (Kano, 1965—1 g white fluorescent light, inoculum (12.6 PLBs/PLB section);
Phalaenopsis Hybrid with
(2-mm in size) of PLBs L−1 of 6.5N − 4.5P − 19K + 1 under shaker at 100 rpm or charcoal filter attached to bioreactor Non-evaluated [65]
pink striped flowers
obtained from leaf g L−1 20N − 20P − 20K + 1% temporary our continuous increased to 17 PLBs/PLB section;
segments potato homogenate) immersion bioreactor Hyponex medium increased
system percentage of PLB regeneration,
rooting and fresh weight of plantlets
11
Shoot tips to PLBS, NDM +
44.4% PLB formation from shoot tips The type and frequency of
2 g L−1 Gellan gum, pH 5.4; 23 ± 1 ◦ C, 14-h photoperiod, were obtained with 0.5 μM NAA and morphological variants were
Shoot tips from flower stalk Cell suspension, liquid 4.44 μM BA and 29.2 mM sucrose; large dependent on genotype: in
33 μmol m−2 s−1 , cell
9 genotypes of Phalaenopsis buds and callus from cell NDM + 58.4 mM sucrose; increases in sucrose concentration P. Snow Parade and P. Little [66]
suspension culture were
suspension cultures Induction of PLBs from (58.4 mM increased callus formation); Steve any variants was reported,
obtained in liquid medium
calluses, NDM + 29.2 μM calluses could induced to PLBs with while 47.9% variants were
under agitation 0f 80 rpm
sucrose + 2 g L−1 Gellan 29.2 mM sucrose observed in P. Reichentea
gum
70–90% of explants with PLBs
depending on cultivar; 85% explants
induction of PLBs: MS1⁄2 + with PLBs and 12 PLBs/explant with
10% coconut 88.8 μM BA + 5.4 μM NAA; 45 g L−1
Temp 25 ± 1 ◦ C, 16-h
Phalaenopsis Tinny Sunshine water/Proliferation of PLBs: sucrose showed highest number
Young leaf segments (10 × 5 photoperiod by cool white
‘Annie’; ‘Taisuco Hatarot’; different saline formulation PLBs per explant (6) and low light
mm) derived in vitro shoots fluorescent lamps, PPFD 30 Non-evaluated [67]
Teipei Gold ‘Golden Star’; + 2 g L−1 peptone + 3% intensity (10 μmol m−2 s−1 ) resulted
from flower stalk nodes potato homogenate + 0.05% μmol m−2 s−1 ; liquid media
Tinny Galaxy ‘Annie’ in best PLBs induction (90%) and
activated charcoal + 30 g in shaker at 50 rpm
number of PLBs/explant (12); liquid
L−1 sucrose with cotton raft support Hyponex
medium increased PLBs proliferation
(20.5 PLBs)
Int. J. Mol. Sci. 2020, 21, 985
Table 1. Cont.
Irregular shaped bodies (CLBs)
1 week in dark at 27 ◦ C 9.0 μM TDZ resulted in best PLB increased with increases in
Leaf segments 1 mm thick MS1⁄2 + 20% coconut water + followed by 25 ± 1 ◦ C, 16-h formation (72.3%); Thin leaf concentrations of TDZ (0.57% at
Doritaenopsis ’New Candy’
from three months old 10 mg L−1 adenine sulphate photoperiod by cool white segments—1 mm— resulted in best free-PGR to 11.56% at 22.5 μM)
× (D. ’Mary Anes’ × D. [68]
leaves from in vitro fluorescent lamps, PPFD PLB formation (>50%) than thick leaf and BA (32.14% at 4.4 μM);
’Ever Spring’ + 2.3 g L−1 Gelrite, pH 5.5
plantlets sections—5 mm (10%) and are However, no phenotypic
10 μmol m−2 s−1 correlated with ethylene content (ppm) variations were observed in
vegetative growth in greenhouse
Temp 25 ◦ C, cool white TDZ at 2.3 μM showed best PLB
Doritaenopsis ’New Candy’ Root tips (<0.5 cm) from MS + 20% coconut water + formation (47.2% of root tips with
fluorescent lamps, PPFD
× (D. ’Mary Anes’ × D. 3-months old in vitro 10 mg L−1 adenine sulphate 2–6 PLBs each) compared to BA and Non-evaluated [69]
’Ever Spring’ plantlets 30 μmol m−2 s−1 , 16-h Zea; most of PLBs originated from
+ 2.3 g L−1 Gelrite, pH 5.5
photoperiod cortex tissues of root
The response were
23 ± 1 ◦ C, 14-h photoperiod, genotype-dependent: Glucose at 58.4
Phalaenopsis Snow Parade mM and sucrose at 29.2 mM showed
and Wedding Promenade, Cell suspension from 33 μmol m−2 s−1 , cell several increases in number (>2000)
NDM + 2 g L−1 gellan gum,
suspension culture were Non-evaluated [70]
Doritaenopsis New calluses pH 5.4 and fresh weight of PLBs for P. Snow
Toyohashi obtained in liquid medium Parade, while glucose at 14.6–29.2
under agitation 0f 80 rpm mM showed highest number of PLBs
in P. Wedding Promenade
MS1⁄2 added 4.54 μM TDZ,
100 mg L−1 myo-inositol +
12
Leaf explants (1cm2) 0.5 mg L−1 niacin + 0.5 mg
derived from flower stalk L−1 pyridoxine + 0.1 mg L−1 Dark for 2 months followed 40% explants with PLBs; not reported
Phalaenopsis ’Little Steve’ Non-evaluated [71]
buds eighteen-month-old thiamine + 2.0 mg L−1 by 16-h photoperiod the number of PLBs per explant
in vitro plants glycine + 1000 mg L−1
peptone + 2.2 g L−1 Gelrite
+ 20 g L−1 sucrose, pH 5.2
M1⁄2 S added 3 mg L −1 TDZ,
100 mg L−1 myo-inositol +
0.5 mg L−1 niacin + 0.5 mg 93.8% explants with PLBs and 19.4
Leaf tip segments obtained
PLBs per explant for leaf tip
Phalaenopsis amabilis var. from in vitro germinated L−1 pyridoxine + 0.1 mg L−1 Temp 26 ± 1 ◦ C; 16-h
segments; 5.4 proliferation rate and Non-evaluated [72]
formosa seedlings and leaf-derived thiamine + 2.0 mg L−1 photoperiod
13.8 PLBs per explant for leaf-derived
nodular masses glycine + 1000 mg L−1 embryogenic masses
Trimmed protocorms increased PLBs proliferation (56.8%) and number of
Temp 25 ± 2 ◦ C, under
Trimmer base protocorms 1 XER medium (Ernst, 1994) + continuous illumination PLBs/protocorm (4.24) using 15% coconut water and 2.5 g L−1 activated
Phalaenopsis gigantea mm from 150-d in vitro 20 g L−1 fructose + 1% agar, from cool fluorescent lamps, charcoal, compared to untrimmed (4.56% and 0.56 PLB/protocorm) and [73]
germinated protocorms pH 5.7 shoot regeneration from PLBs were increased using only coconut water at
PPFD 20–50 μmol m−2 s−1
10% (33.56% shoot formation)
Int. J. Mol. Sci. 2020, 21, 985
Table 1. Cont.
NDM culture medium
added 1.0 mg L−1 BA and
0.1 mg L−1 NAA, 100 mg
L−1 myo-inositol + 1.0 mg
Alba flower hybrid’ of L−1 (niacin, pyridoxine, Not reported growth 8.5 PLBs per explant; not reported
Nodular masses Non-evaluated [74]
Phalaenopsis thiamine, cysteine, calcium conditions percentage of explants with PLBs
pantothenate) + 0.1 0 mg
L−1 biotin + 20 g L−1
sucrose + 2.0 g L−1
Phytagel, pH 5.8
MS1⁄2 added 2,0 mg L−1 BA,
Phalaenopsis amabilis cv. Inflorescence axis thin 0,5 mg L−1 NAA, 2%
sucrose, 10% coconut water, 20 PLBs/explant after 12 weeks Non-evaluated [75]
’Cool Breeze’ sections
2 g L−1 peptone and 1 g L−1
activated charcoal
NDM culture medium
Phalaenopsis var. Hawaiian added 1 mg L−1 TDZ, 10 g
Clumps of callus (8 mm
Clouds × Phalaenopsis Temp 25 ± 2 ◦ C, in the dark 52.5% callus with PLBs Non-evaluated [76]
diameter) L−1 maltose, 2.8 g L−1
Carmela’s Dream
Gelrite
MS1⁄2 added 3 mg L−1 TDZ,
−1
100 mg L myo-inositol +
Cut end of leaf explants (1.0
13
0.5 mg L−1 niacin + 0.5 mg 50% explants with PLBS and 8.2
cm length); clonal plantlets Temp 26 ± 1 ◦ C; dark for
Phal. amabilis; Phal. L−1 pyridoxine + 0.1 mg L−1 PLBs/explant for P. amabilis; 80%
of P. amabilis and in vitro 60-d (induction) 45-d for Non-evaluated [59,77,78]
’Nebula’ thiamine + 2.0 mg L−1 explants with PLBs and 3.5 PLBs for
germinated seedlings for P. subculture period;
glycine + 1000 mg L−1 P. ’Nebula’
’Nebula’
3.5 g L−1 HyponexTM #1 +
1 g L−1 tryptone + 0.1 g L−1 High endopolyploidy were
Basal portion of sectioned observed in Phalaenopsis
citric acid + 1 g L−1
horizontally protocorms Temp 25 ± 2 ◦ C, 16-h 22% of sectioned protocorms induced protocorms; from 22 diploid
activated charcoal + 20 g
10 genotypes of Phalaenopsis (3–5 mm) were placed photoperiod with PPFD 10 PLBs and 17.5 PLBs per responsive protocorms used as explant, [79]
upward in contact with the L−1 sucrose + 20 g L−1 protocorms were obtained 34.1% of derived-PLBs were
μmol m−2 s−1
culture medium homogenized potato + 25 g polyploidy at first cycle and 51.7%
L−1 homogenized banana + at second cycle of proliferation
7.5 g L−1 agar, pH 5.5
70% of leaf segments formed PLBs
Temp 25 ◦ C, 16-h with 0.8 μM BAP, while TDZ were
Leaf segments (1 × 1 cm) photoperiod, PPFD 40
12
able to induce PLBs only in 40% of
Phalaenopsis violacea from in vitro shoots derived MS ⁄ + 5% banana extract Non-evaluated [80]
μmol m−2 s−1 by white explants and BAP (0.6 μM) was more
from flower stalks
fluorescent tubes effective to PLBs proliferation than
TDZ and Zea
Int. J. Mol. Sci. 2020, 21, 985
Table 1. Cont.
MS1/2 + 2% sucrose + 10% 2.0 mg L−1 BA and 0.5 mg L−1 NAA

coconut water + 2 g L-1 Temp 24 ± 1 ◦ C, cool white resulted in 75.5% explants formed
Phal. amabilis cv. Lovely Young emerging leaves fluorescent light, PPFD 30 PLBs and 10 PLBs/explant; MS1⁄2 +
peptone + 1 g L-1 activated Non-evaluated [81]
(purple flowers) from in vivo plants μmol m−2 s−1 , 16-h 10% coconut water + 150 mg L−1
charcoal + 2.2 g L-1 Gelrite, photoperiod glutamine showed best proliferation
pH 5.6;
rate of PLBs (200.5 PLBs/explant)
MS1⁄2 + 100 mg L−1
myo-inositol + 0.5 mg L−1
niacin and pyridoxine + 0.1 PLBs from leaf (S0), PLBs Minimal dissimilarity in P. bellina
Efficiency of induction and
mg L−1 thiamine + 2.0 mg proliferation after 3 months by RAPD markers; S0 presented
Phalaenopsis bellina In vivo leaf regeneration of PLBs not presented [82]
L−1 glycine + 3.0 mg L−1 (S1) and after six months 96% similarity, S1 87% and S2 80%
by authors
TDZ + 2% sucrose + 3.0 g (S2) similarity to the mother plant
L−1 Gelrite + 10% fresh
banana extract, pH 5.6
MS1⁄2 + 2% sucrose + 100
mg L−1 myo-inositol + 0.5
mg L−1 niacin and
pyridoxine + 0.1 mg L−1 71.9–78.1% explants with PLBs;
Temp 25 ± 2 ◦ C, 14-h
Young leaves (1.5 cm2 ) of a 14.3–14.8 PLBs per flask; MS1/2 was
Phalaenopsis bellina thiamine + 2.0 mg L−1 photoperiod for 12–16 Non-evaluated [83]
nursery plant the best for PLB proliferation
glycine + 10% fresh ripen weeks
compared to VW
banana extract + 3.0 mg L−1
14
TDZ + 3.0 g L−1 Gelrite, pH
5.6
NDM culture medium, The authors only report that NAA
Leaf tip segments (1.0 cm sucrose 20 g L−1 + 1.0 mg Temp 25 ± 2 ◦ C, 12-h and TDZ treatment was the best for
Phalaenopsis gigantea length) from in vitro Non-evaluated [84]
L−1 NAA and 0.1 mg L−1 photoperiod for 6 weeks callus induction and PLBs after 6
germinated seedlings
TDZ weeks of culture.
BA at 2.0 mg L−1 combined with
MS1⁄2 + 2% sucrose + 10% NAA 0.5 mg L−1 resulted in 80.5%
coconut water + 2 g L−1 Temp 24 ± 1 ◦ C, cool white
fluorescent light, PPFD 30 explants with PLBs and 15
Phalaenopsis amabilis cv. Young emerging leaves
peptone + 1 g L−1 activated PLBs/explant; MS1⁄2 + 10% coconut Non-evaluated [85]
’Golden Horizon’ from in vivo plants μmol m−2 s−1 , 16-h
charcoal + 2.2 g L−1 Gelrite, photoperiod water + 150 mg L−1 glutamine
pH 5.6 showed best proliferation rate of
PLBs (250.5 PLBs/explant)
Although higher Fresh weight of
PLBs was obtained with 0.1
Temp 25 ◦ C, 16-h Tesla–South (237.4 g), best number of
Miracle Pack®culture photoperiod, PPFD 45
Longitudinally bisected PLBs was obtained in control
Phalaenopsis Gallant Beau system with liquid VW +
PLBs (2–3 mm in diameter) μmol m−2 s−1 , plant growth without magnetic fields; 0.15 Tesla Non-evaluated [86]
’George Vasquez’ 20% coconut water without
and 2-months old fluorescent lamps, under for 7 weeks (South) also increased
sucrose, pH 5.3
magnetic fields PLB fresh weight, control treatment
not differed from the best results
using magnetic fields
Int. J. Mol. Sci. 2020, 21, 985
Table 1. Cont.
Leaf segments from in vitro MS1⁄2 added 0.1 mg L−1
Temp 25 ± 1 ◦ C; 16-h 100% explants with PLBs; 35 PLBs
Phalaenopsis cornu-cervi germinated seedlings with NAA, 0.1 mg L−1 TDZ and Non-evaluated [87]
photoperiod for 45 days per explant
2-months 15% coconut water
Temp 25 ± 2 ◦ C, under 16-h
PLBs obtained from leaf tip photoperiod using VW medium with 10 mg L−1 chitosan ISSR, non-detected somaclonal
Liquid medium with 20% fluorescent lighting 30
Phalaenopsis gigantea segments (1.5 cm length) resulted in higher number of PLBs variations in P. gigantea related to [88]
coconut water, pH 5.4.
from young leaves μmol m−2 s−1 , 60 rpm (177) and fresh weight of PLBs (8.4 g) mother plants
rotary shaker
MS1⁄2 + 15% coconut water
Stem segments were interesting
+ 0.01% activated charcoal +
Leaves, root tips and stem explant for PLB induction; sucrose at
0.03% polyvinylpyrrolidone Temp. 25 ◦ C, 16-h
Phalaenopsis ’R11 × R10’ explants from eight months 3% (71.2 PLBs) was more effective Non-evaluated [89]
(PVP) + 88.8 μM BA + 5.37 photoperiod
(plantlets or seedlings?) than maltose (39 PLBs) in PLBs
μM NAA + 0.025%
proliferation
Phytagel, pH 5.6–5.8
PLB induction: 14
macroelements and
full-strength microelements,
glycine and vitamins of MS Basal part of sectioned of bi or
+ 30 g L−1 sucrose + 0.5 mg trisectioned PLBs resulted in highest
Young etiolated shoots L−1 TDZ + 7 g L−1 agar / ◦
Temp 25 ± 2 C, under 12-h explants with PLB formation
leaves segments (5 × 10 photoperiod by cool white
PLB Proliferation: 3 g L−1 (46.8–96.3%) and number of
Phalaenopsis Tropican Lady mm) from flower stalk fluorescent lamps, PPFD Non-evaluated [90]
Hyponex (7-6-19) + 1 g L−1 PLBs/explant (15.4–22.9); wounding
15
nodes for induction and
tryptone + 50 g L−1 potato 23.2 μmol m−2 s−1 , stimulate ethylene production and
PLBs for proliferation
homogenate + 50 g L−1 gene expression for stimulation of
banana homogenate + 30 g cell division
L−1 sucrose + 2 g L−1
activated charcoal +
7.5 g L−1 agar, pH 5.6
Temp 25 ± 1 ◦ C, under 16-h Highest percentage of explants with
Whole leaves and photoperiod, cool white PLBs (30%) and number of PLBs per
MS1⁄2 + 3% sucrose + 15% fluorescent lamps, PPFD 20
leaf-segments (proximal, leaf segment (5.3) were obtained with
Phalaenopsis cornu-cervi coconut water + 0.23% Non-evaluated [91]
middle and distal regions) μmol m−2 s−1 or pre-treated 9 μM of TDZ under without dark
Gelrite, pH 5.6
from 120-d old seedlings with 1 week in the dark period. Dark period reduced number
before photoperiod of PLBs/explant
NDM medium added Temp 25 ± 2 ◦ C, 16-h
Leaf tip segments from
0.1 mg L−1 TDZ, 10 mg L−1 353 PLBs per explant and 4.8 g PLBs ISSR, SV detected after the
Phalaenopsis gigantea young leaves of in vitro photoperiod, 33 μmol m−2 [92]
chitosan, 0.2% Gelrite and fresh weight subculture four (5 to 20%)
seedlings s−1
pH 5.7
No PLBs formed in intact
protocorms; Middle and Basal part of
Intact and transversally Temp 25 ± 2 ◦ C, 16-h sectioned protocorms showed 40 and
divided protocorms (two or MS + 15% coconut water + 44% PLB formation and 11.7 and 13.3
Phalaenopsis hybrids photoperiod, 25 μmol m−2 Non-evaluated [93]
four divisions) 1.0–1.5 mm 7.0 g L−1 agar PLBs per explant in Free-PGR culture
width s−1 medium, respectively; Four division
of protocorms increased PLBs
formation and number of PLBs
Int. J. Mol. Sci. 2020, 21, 985
Table 1. Cont.
Using 2-step method:
Liquid MS1⁄2 for 2 months
and then transferred to
in vitro germinated solid MS (half strength) Temp 25 ± 2 ◦ C; followed
P. aphrodite subsp. formosana 44 PLBs per seedling Non-evaluated [94]
seedlings with 2-months with 1 cm of medium by 16-h photoperiod
Liquid MS (half strength)
for a further 2 months. All
media with 1 mg L−1 TDZ.
Leaf segments from in vitro 5 mg L−1 BA + 2 mg L−1 NAA non reported by authors that
Temp 25 ± 1 ◦ C; 16-h
Phalaenopsis amabilis cv. shoots obtained from produced 8.7 number of PLBs and acclimatized and cultivated
- photoperiod, subcultured [95]
’Surabaya’ inflorescence stalk TDZ at 3.0 mg L−1 showed 22.45 regenerated plantlets until
each 14-d
segments PLBs flowering stage
MS with 412.5 mg L−1 Hyaluronic acid 9 and 12, at 0.1 mg
NH4 NO3 and 950 mg L−1 of L−1 , increased percentage of explants
with PLBs (100%), PLB number (18.2 no malformation was observed in
Phalaenopsis ’Fmk02010’ Single PLBs KNO3 + 20;0 g L−1 sucrose - [96]
to 23.3) and fresh weight of PLBs regenerated plantlets
+ 2.0 g L−1 Phytagel, pH (0.291 to 0.596 g) compared to control
5.5–5.8 (86.7%, 12.9 and 0.198 g)
Temp 26 ± 1 ◦ C; dark for 1
MS1⁄2 added NAA (0.5
P. ‘Join Angle × Sogo month (induction) followed 49.33 PLBs per explant; not reported
In vitro roots ppm), BA (5 ppm) and IAA Non-evaluated [63]
Musadian’ by 16-h photoperiod percentage of explants with PLBs
(0.5 ppm)
(4 weeks)
2
16
12
MS ⁄ added NAA The percentage of explants in
leaf segments (1.0 cm ) with Temp 25 ± 2 ◦ C, dark for
Phalaenopsis Classic Spoted
90-d obtained from in vitro (0,537μM) and TDZ 90-d (induction) followed regeneration and the number of Non-evaluated [45]
Pink
shoots (13,621μM) by 16-h photoperiod PLBs/explant were not described
Leaf segments (1 cm × 0.5
Phalaenopsis amabilis var. MS added 15 mg L−1 BA Temp 25 ± 1 ◦ C; 16-h
cm) obtained from in vitro 50.65 PLBs per explant after 6 weeks Non-evaluated [97]
’Manila’ and 3 mg L−1 NAA photoperiod
flower stalk nodes
Protocorm: 100% explants with PLBs
and 23.3 PLBs/explant; Leaf: 100%
Protocorms (4 weeks-old), NP (New Phalaenopsis) 25 ± 1 ºC with 1000 lux explants with PLBs and 7.75
roots, leaves and stems
Phalaenopsis amabilis medium added 3 mg L−1 intensity of continuous PLBs/explant; Root: 80% explants Non-evaluated [98]
(6-month-old) cut
TDZ light; 8 weeks with PLBs and 8.25 PLBs/explant;
transversely
Stem: 100% explants with PLBs and
28.25 PLBs/explant
Highest number of PLBs (54.13) were
MS with 412.5 mg L−1 obtained with Red-White LEDs and
Temp 25 ± 2 ◦ C, 16-h
NH4 NO3 and 950 mg L−1 of photoperiod, PPFD 54 with sucrose at 20 g L−1 and highest
Phalaenopsis ’Fmk02010’ Single PLBs Non-evaluated [60]
KNO3 + 2.2 g L−1 Phytagel, fresh weight of PLBs (0.167 g) was
μmol m−2 s−1 obtained with Red-Blue-White LEDs
pH 5.5–5.8
and trehalose (20 g L−1 )
NDM culture medium
Leaf segments (0.4–0.5 cm2 ) added 0.25 mg L−1 TDZ Temp 25 ± 2 ◦ C, dark for 45% (908) and 10% (RP3) explants
Phalaenopsis ’RP3’ and ’908’ obtained from in vitro (908) or 1.0 mg L−1 NAA, 60-d (induction) followed with PLBs; 25 and 2 PLBs/explant Non-evaluated [30]
shoots 20.0 mg L−1 BA and 0.125 by 14-h photoperiod respectively
mg L−1 TDZ (RP3)
NDM: New Dogashima Medium [41]; MS: Murashige and Skoog Medium [99]; Hyponex medium: [100]; XER medium: [101]; VW: Vacin Went medium [43]; NP: New Phalaenopsis
medium [102]. 2,4-D, 2-4-Dichlorofenoxiacetic acid; BA, 6-Benzyladenine; IAA, 3-Indoleacetic acid; IBA, Indole-3-butyric acid; NAA, Naphtaleneacetic acid; PPFD: Photosynthetically
Photon Flux Density; Temp, Temperature; TDZ, Thidiazuron.
Int. J. Mol. Sci. 2020, 21, 985
Segmentation made in leaf segments of Phalaenopsis to induce PLBs results in a process called
phenolic oxidation, which is the release of polyphenol oxidase (PPO) [103] and other compounds toxic
to plant tissue, which may cause its death [74], consequently reducing the induction of PLBs. The
immersion of leaf segments in solution of cystine and ascorbic acid during the leaf segmentation stage
is reported as a way to reduce the release of these compounds capable of impairing the formation of
PLBs [74].
One of the influential factors in the induction of PLBs that has been widely evaluated is the
concentrations and possible combinations of plant growth regulators (PGRs). Based on the current
literature, successful induction of PLBs seems to be mainly influenced by cytokinin BA (6-benzyladenine)
and cytokinin-like compound TDZ (thidiazuron), and in some cases the combination of these cytokinins
with an auxin [30,45] also proved beneficial. Protocols citing the use of cytokinin BA recommend
concentrations between 0.5 mg L−1 [78] and 20 mg L−1 [67]. For the induction of PLBs with the use
of TDZ, the recommended concentrations range from 0.25 mg L−1 [30] to 3.0 mg L−1 [72]. With the
combined use of cytokinins and auxins, the most commonly used auxin is NAA, which varies in
concentration from 0.1 mg L−1 [45,74] to 1.0 mg. L−1 [30,82].
Ref. [104] reviewed the influence of auxins in orchids, including in PLBs and concluded that
auxins is important for callus induction and PLB formation and proliferation, while is inhibitory for
PLB regeneration into shoots.
As already described, the addition of PGRs is critical to the success of the PLB induction and
regeneration technique in Phalaenopsis. Cytokinin-like compound such as TDZ (47.5%) and BA (35%)
was the most PGRs used to IPR-PLB technique (Table 1). Nevertheless, the use of these regulators may
also result in somaclonal variation. This variation can be assessed by morphological, physiological,
biochemical traits or molecular markers [105]. Using Random Amplified Polymorphic DNA (RAPD)
markers, [82] reported 17% dissimilarity between PLBs and the parent plant in P. bellina. Ref. [89]
observed 20% dissimilarity after 20 weeks of cultivation in P. gigantea using ISSR (Inter Simple Sequence
Repeats) markers, leading to the conclusion that PLB proliferation should be done for up to 16 weeks
to reduce somaclonal variations and morphological changes. It should be noted that changes in alleles
will not always result in phenotypic changes [106], so the variations observed by the markers will not
always cause some kind of morphological change in plants.
According to [107], the combination of red light and far red contributes to decrease
endoreduplication rates during PLB induction and regeneration, and consequently may reduce
somaclonal variations during mass propagation processes.
Bioreactors could be used to improve the proliferation of PLBs in Phalaenopsis. The authors of [108]
obtained 18,000 PLBs from 1000 PLBs sections using 0.5 or 2.0 L volume of air per volume of medium min−1 .
3. Oncidium Hybrids Group

According to the World Checklist of Selected Plant Families of the Kew Botanical Garden, in
December 2019, there are 374 accepted names of Oncidium species with more than 90% of accepted
names allocated in Southern America and the last in Northern America. In addition to the species,
thousands more interspecific and intergeneric hybrids have been registered with the Royal Horticultural
Society and are used in the commercial production of cut and pot flowers worldwide [9,109]. Different
chemical and physical factors alter the response to PLB induction in Oncidium. Using Oncidium ’Gower
Rampsey’ shoot tips, [109] observed a higher percentage of shoot tips induced to produce PLBs (96.7%)
in monochromatic red-light emitting diodes (RR), compared to blue LED (83.3%) and fluorescent white
light (76.7%) used as control. However, the use of RR, as well as green LEDs, increased in inhibition
of differentiation of PLBs into green buds, while blue LEDs enhanced differentiation. Associated
with this response, the authors also observed that in blue light, PLBs contained higher contents of
carotenoids, chlorophyll, soluble proteins, lower amounts of soluble sugars and carbohydrates. The
authors further argue that in red LEDs, where a higher PLB induction response was obtained, there
was a greater accumulation of soluble sugars, starch and carbohydrates, while in blue light, where
17
Int. J. Mol. Sci. 2020, 21, 985
there was a greater differentiation of PLBs, there was a greater accumulation of proteins and pigments
such as chlorophylls and carotenoids.
PGRs are one of the most tested factors in IPR–PLBs in Oncidium (Table 2). Benzyladenine
(BA) at 2.0 mg L−1 + 0.2 mg L−1 Naphthaleneacetic Acid (NAA) has been shown to be the most
efficient treatment for inducing PLBs in Oncidium ’Sweet Sugar’ apical and axillary buds [110] and
the combination of 0.1 mg L−1 BA + 0.2 mg L−1 ANA resulted in better response for Oncidium Aloha
’Iwanaga’ [111]. In this context, BA can be used efficiently to obtain PLBs in Oncidium in 31.8% of the
papers, and auxin NAA is the one most used along with BAP (Table 2).
Interestingly, [112] reported the individual and combined effects of BA and NAA PGRs at different
stages of in vitro induction, proliferation and regeneration of PLBs on Oncidium sp. These authors
identified that previous callus production in culture medium containing 2,4-D at 1.0 mg L−1 , prior
to induction, was beneficial for the production of PLBs from in vitro shoots, and from callus it was
possible to observe up to 98 PLBs/callus cluster using 0.75 mg L−1 NAA, while only 28.2 PLBs/shoot
cluster were directly obtained using the combination of 0.5 + 0.5 mg L−1 NAA and BA, respectively. The
use of 1.0 mg L−1 NAA alone allowed PLB proliferation (up to 79.2 PLBs/sample), while the addition
of 1.0 mg L−1 BA resulted in shoot bud formation (up to 12.4 shoots/PLB). Similarly, [113] observed
that the concentration of 2.0 mg L−1 BA resulted in the highest number of shoot buds obtained from
PLBs (4.3/PLB) in Oncidium ‘Sweet Sugar’.
Thidiazuron (TDZ) also appears to have a pronounced effect on direct induction of PLBs in
Oncidium leaf segments and were reported in 54.5% of the papers (Table 2), being higher for the
percentage of explants directly forming PLBs (60–75%) and number of PLBs per explant (10.3–10.7)
compared to other cytokinins such as kinetin, zeatin, 2-isopentenyladenine and BA itself [114]. Ref. [115]
reported direct regeneration of PLBs from mainly the epidermis and cut regions of young leaf segments
of Oncidium ‘Gower Ramsey’ using TDZ alone (0.3–3.0 mg L−1 ), rather than BA in the culture medium,
while the combination 2,4-D and TDZ was not beneficial for induction of PLBs. The production of
PLBs from tissue damaged regions of inflorescence segments (65%) of Oncidium ‘Gower Ramsey’ using
3 mg L−1 TDZ [116] has also been reported. A similar experiment using the same cultivar observed
that calli from root apexes and stem segments produced PLBs in medium containing 0.3–3.0 mg L−1
TDZ, being beneficial the addition of NAA for the formation of embryos n root and leaf calli [117],
being a tissue-specific response.
Other PGRs as GA3 is reported as an inhibitor of PLB induction in Oncidium, while the use of
antigibberellins, as ancymidol and Paclobutrazol, increased the percentage of leaf explants with PLBs
and the number of PLBs obtained [118].
The use of liquid medium, rather than semi-solidified with Agar, is also an alternative for
in vitro PLB proliferation (Figure 2). Ref. [113] used 5 L balloon-type air-lift bioreactor to provide
mass propagation of Oncidium ‘Sweet Sugar’, and show that this system provides 326.3 g PLBs and
growth ratio of 10.2, and is more efficient than semi-solid (2.7 g PLBs and Growth ratio of 3.4) and
liquid-agitated flask culture (3.5 g PLBs and growth ratio of 4.4). In bioreactor, the lag phase was
observed in the first 10-d culture, accompanied by a sharp drop in pH (5.7 to 4.7) and EC (3.2 to
1.5 mS cm−1 ) in the first 20-d of cultivation, followed by an intense mass growth from 10 to 40 days of
cultivation, when the pH increased again to 5.9. An interesting fact was the dynamics of sugars in the
culture medium, and a fast and drastic reduction of sucrose in the medium was observed, from 27
(day zero) to 5.5 (day five), 1.2 (day 10) and zero (day 20), associated with a substantial increase in
glucose and fructose in the first 10 days of cultivation, with the exhaustion of these sugars at 40 days of
cultivation, when the PLBs entered the stationary phase, demonstrating that during a certain period
the PLBs release invertases in the culture medium to reduce sugars, and these are metabolized during
the exponential phase of production of PLBs [113].
18
Int. J. Mol. Sci. 2020, 21, 985
Table 2. Compliance of studies with induction, proliferation and regeneration of PLBs (IPR-PLBs) technique used with Oncidium species and hybrids.
Modified VW (replace
Fe2 (C4 H4 O6 )3 by 27.8 mg L−1 Only one callus formed PLB and
25 ± 1 ◦ C, Gro-lux bulbs
Root tips 1.5 mm long from Fe-EDTA + 15% coconut water proliferation of PLBs occurred only
Oncidium varicosum with 16-h photoperiod and Non-evaluated [119]
seedlings (PLBs proliferation from PLB) + in liquid medium with 15%
500 lux
1.25 mg L−1 NAA (callus and coconut water
PLB induction), pH 5.5
Donor leaves with 5–7 cm long
MS1⁄2 + 100 mg L−1 inositol + showed higher percentage formed
niacin and pyridoxine (0.5 mg PLBs (25–35%) and number of
Leaf segments 5 mm in L-1 ) + thiamine (0.1 mg L-1 ) + Temp 26 ± 2 ◦ C, PPFD PLBs/leaf segment (17–24.4) than
length from in vitro 28–36 μmol m−2 s−1 , 2–4 cm donor leaves (15–25% and
Oncidium ‘Gower Ramsey’ glycine (2.0 mg L-1 ), peptone Non-evaluated [115]
plantlets leaves of 2–4 cm daylight fluorescent tubes, 5.3–13.0) using 1–3 mg L−1 TDZ;
and 5–7 cm (1000 mg L-1 ), NaH2 PO4 (170 mg proliferation of PLBs was highest
16-h photoperiod
L-1 ), sucrose (20,000 mg L-1 ) + with 0.3 mg L−1 TDZ, and
Gelrite (2,500 mg L-1 ), pH 5.2 regeneration of PLBs showed best
response in absence of PGRs
MS1⁄2 + 100 mg L−1 inositol +
niacin and pyridoxine (0,5 mg
L-1 ) + thiamine (0.1 mg L-1 )+ 10% and 25% callusing from stem
glycine (2.0 mg L-1 ), peptone Temp 26 ± 1 ◦ C, PPFD Different callus lines showed
and root tips, 3.38 and 3.86 callus
Leaves 2–4 and 5–7 cm, (1000 mg L-1 ), NaH2 PO4 (170 mg large differential response to
28–36 μmol m−2 s−1 white proliferation rate from stem and
Oncidium ‘Gower Ramsey’ stem internodes 5mm and PLBs induction (0% to 93.8%) [117]
19
L-1 ), sucrose (20,000 mg L-1 ) + cool fluorescente, 16-h root tips, until 93.8 callus forming
root tips 1 cm and number of PLBs/explant (0
Gelrite (2200 mg L-1 ), pH 5.2: photoperiod embryos and 29.1 embryos/callus
to 29.1)
callus phase, 3.0 mg L−1 2,4-D + from roots
3.0 mg L−1 TDZ; PLBs, 0.1 NAA
+ 3.0 mg L−1 TDZ
MS1⁄2 + 100 mg L−1 inositol + TDZ 1–3 mg L−1 increased
niacin and pyridoxine (0,5 mg explants produced PLBs directly in
L-1 ) + thiamine (0.1 mg L-1 )+ Temp 26 ± 1 ◦ C, PPFD O. Sweet Sugar, but not in O.
Internodes 5 mm length
Oncidium ‘Gower Ramsey’ 28–36 μmol m−2 s−1 , Gower Ramsey. Callus from
from 15–20 cm glycine (2.0 mg L-1 ), peptone Non-evaluated [116]
and O. ‘Sweet Sugar’ daylight fluorescent tubes, explants on NAA + TDZ both at
inflorescence length (1000 mg L-1 ), NaH2 PO4 (170 mg
16-h photoperiod 1.0 mg L−1 showed 19 PLBs/callus.
L-1 ), sucrose (20,000 mg L-1 ) + PLBs regeneration into shoots
Gelrite (2200 mg L-1 ), pH 5.2 occurred in free-PGR MS1⁄2
MS1⁄2 + 100 mg L−1 inositol + Auxins IAA, NAA, IBA and 2,4-D
niacin and pyridoxine (0,5 mg inhibited direct PLB induction,
L-1 ) + thiamine (0.1 mg L-1 )+ Temp 26 ± 1 ◦ C, PPFD while cytokinins promoted; TDZ
Leaf explants 1 cm in length
Oncidium ‘Gower Ramsey’ from two-month old donor glycine (2.0 mg L-1 ), peptone 28–36 μmol m−2 s−1 , 0.3–3.0 mg L−1 increased Non-evaluated [114]
in vitro plantlets daylight fluorescent tubes, percentage of explants formed
(1000 mg L-1 ), NaH2 PO4 (170 mg
16-h photoperiod PLBs (60–75% in leaf tips and
L-1 ), sucrose (20,000 mg L-1 ) + 25–40% in adaxial surfaces, with
Gelrite (2200 mg L-1 ), pH 5.2 9.5–10.7 PLBs/explant
Int. J. Mol. Sci. 2020, 21, 985
Table 2. Cont.
MS1⁄2 + 2% sucrose + 2 g L−1
Leaf segments 4 × 4 mm 25.5% of leaf segments formed
Oncidium bifolium Phytagel + 1.0 mg L−1 TDZ, 27 ± 2 ◦ C, 14-h photoperiod Non-evaluated [120]
from germinated seedlings PLBs and 12 PLBs/explant
pH 5.5
Leaf tips and leaves with adaxial
surface in contact with culture
Temp 26 ± 1 ◦ C, PPFD medium was the best region for
Leaf explants 1-cm length PLB induction, sucrose at 10–20 g
Oncidium ‘Gower Ramsey’ from two month old in vitro 28–36 μmol m−2 s−1 , Non-evaluated [121]
MS1⁄2 + 1.0 mg L−1 TDZ, pH 5.2
daylight fluorescent tubes, L−1 , NaH2 PO4 170 mg L−1 ,
donor plantlets
16-h photoperiod peptone 1.0 g L−1 (65–80% explants
with PLBs and 10.7 to 11.2
PLBs/explant);
MS1⁄2 + 100 mg L−1 inositol + GA3 inhibited PLB formation,
niacin and pyridoxine (0,5 mg while anti-gibberellins Ancymidol
L-1 ) + thiamine (0.1 mg L-1 )+ Temp 26 ± 1 ◦ C, PPFD
Leaf tips 1-cm length from (2.5 mg L−1 ) and paclobutrazol
Oncidium ‘Gower Ramsey’ two month old in vitro glycine (2.0 mg L-1 ), peptone 28–36 μmol m−2 s−1 , Non-evaluated [118]
daylight fluorescent tubes, (10 mg L−1 ) increased explants
donor plantlets (1000 mg L-1 ), NaH2 PO4 (170 mg formed PLBs (80–87.5% leaf tips
16-h photoperiod
L-1 ), sucrose (20,000 mg L-1 ) + formed PLBs and 154.8–193.2
Gelrite (2200 mg L-1 ), pH 5.2 PLBs/petri dish)
MS + 3% sucrose + 0.7% agar,
pH 5.7–5.8. PLBs induction at 26 ± 2 ◦ C, 12-h photoperiod,
Axillary buds 0.5–1.0 cm 90% explants with PLBs and 9.4
Oncidium taka 1.0 mg L−1 BA + 0.5 mg L−1 3000 lux cool white Non-evaluated [122]
lenght shoots per culture
20
NAA; PLBs regeneration at 2.0 fluorescent light
mg L−1 BA + 1.0 mg L−1 BA
MS1⁄2 + thiamine (1.0 mg L-1 ) +
nicotinic acid and pyridoxine
(0.5 mg L-1 ) + glycine (2.0 mg callus induction and 680–732 g callus FW (1.0 mg L−1
L-1 ) + inositol (100 mg L-1 ) + 2% proliferation in dark for
Shoot tips 2–3 mm length 2,4-D and 0.5–1.0 mg L−1 TDZ);
60-d (induction),
for callus induction and sucrose + 7.5 g L−1 Agar, pH 5.7: 1478 PLBs/0.25 g callus (Sucrose
Oncidium ‘Gower Ramsey’ subcultured every 2-weeks; Non-evaluated [123]
9-months age callus line for callus proliferation, 1.0 mg L−1 PLBs induction, Temp 26 ± 10–20 g L−1 ); 24 to 52.9 efficiency of
PLBs induction 2,4-D + 0.5–1.0 mg L−1 TDZ / plantlet conversion from PLBs
2 ◦ C, PPFD 57 μmol m−2
PLBs induction, 0.1 mg L−1 s−1 , 16-h photoperiod (trehalose at 20 g L−1 )
NAA and 0.4 mg L−1 BA with
sucrose, maltose or trehalose
Leaf tips and Adaxial region of
leaves showed most response to
◦ PLB formation; 95% explants with
Temp 26 ± 1 C, PPFD
Oncidium ‘Gower Ramsey’ Leave tips 1-cm long from 28–36 μmol m−2 s−1 PLBs with 20 g L−1 fructose in two
MS1⁄2 + 1.0 mg L−1 TDZ non evaluated [124]
and O. ‘Sweet Sugar’ in vitro plantlets daylight fluorescent tubes, cultivars; 31.1 (O. Sweet Sugar) to
16-h photoperiod 33.7 (O. Gower Ramsey)
PLBs/explant with 20 and 30 g L−1
sucrose, respectively
Int. J. Mol. Sci. 2020, 21, 985
Table 2. Cont.
MS + 25 g L−1 sucrose + 10%
25 ± 2 ◦ C, 10–12-h
Cut flower varieties of coconut water + 7.5 g L−1 agar +
New lateral buds photoperiod, 2000–2500 lux proliferation of 2.96 Non-evaluated [125]
Oncidium 3.0 mg L−1 BA + 0.3 mg L−1 cool white fluorescent light
NAA, pH 5.6
Callus at 26 ± 2 ◦ C in the
darkness; PLBs from callus
Method described by ref. [123] 2986 PLBs under fluorescent lamps
in 50 μmol m−2 s−1 for 16-h
Oncidium ‘Gower Ramsey’ PLBs from callus statistically equal to red + blue + Non-evaluated [126]
using 10 g L−1 maltose photoperiod, under blue
far red LEDs (3114 PLBs)
(455 nm), red (660 nm) and
Far-red (730 nm)
MS1⁄2 + 30 g L−1 sucrose + Darkness for 90-d before
myo-inositol 100 mg L−1 + 5 g photoperiod increased explants
−1 Temp 25 ± 2 ◦ C, PPFD regenerating PLBs from 5 (Light)
Leaf apices 0.5 cm in length L agar + nicotinic acid and
to 80% (Dark) and 10.8 PLBs per
Oncidium flexuosum 40 μmol m−2 s−1 , 16-h Non-evaluated [127]
from 4-m seedlings Pyridoxine (0.5 mg L-1 ) +
photoperiod explant using 1.5 mg L−1 TDZ.
thiamine (0.1 mg L-1 ) + glycine Until 29.3 PLBs/explant 60-d after
(2.0 mg L-1 ), pH 5.8 transfer PLBs to free-PGR MS
Callus: MS1⁄2 +2.0 mg L−1 BA +
0.3 mg L−1 NAA + 30 g L−1
sucrose + 7.0 g L−1 agar, pH 5.7; PLBs proliferation: 25 ◦ C,
PLBs proliferation in MS + 30 g 16-h photoperiod, white 3335.5 g fresh weight PLBs per
Shoot tips 0.5 mm length fluorescent light at 30 μmol vessel and 16.8 growth ratio; until
21
Oncidium ‘Sugar Sweet’ for callus induction and L−1 sucrose + 1.0 mg L−1 BA + Non-evaluated [113]
m−2 s−1 at 5 l balloon type 4.3 shoots/PLB and 1.17 g fresh
PLBs obtained from callus 0.2 mg L−1 NAA, pH 5.8; PLBs
air lift bioreactor, 20 g fresh weight per explant
regeneration, MS + 2.0 mg L−1 weight PLBs per bioreactor
BA + 0.1 mg L−1 NAA + 30 g L−1
sucrose + 7.0 g L−1 agar
PLBs induction: MS1⁄2 + 30 g L−1 Red LEDs (660 nm) resulted in best
sucrose + 6.0 g L−1 agar + 1.0 induction rate (83.3% explants),
Shoot tips 5 mm for PLB ◦ Fresh weight (≡ 20 g) and
mg L−1 BA / PLBs proliferation: Temp 25 ± 2 C, 16-h
Oncidium ‘Gower Ramsey’ induction and PLBs propagation rate (>6) of PLBs, Non-evaluated [109]
MS + 30 g L−1 sucrose + 6.0 g photoperiod
sections 3–4 mm diameter while Blue LEDs showed 90% of
L−1 agar + 1.0 mg L−1 BA + 0.5 differentiation rate of PLBs into
mg L−1 NAA shoots
Age of callus from 0.5 to 2 years Different callus lines showed
resulted in best percentage large differential response to
Callus induction: MS1⁄2, pH 5.2 /
Root tips segments 1 cm in (80–100%) of callus produced PLBs PLBs induction. However,
PLB induction: MS1⁄2 +
Oncidium ‘Gower Ramsey’ length from 6-months old Temp 25 ± 1 ◦ C, darkness and number of PLBs/callus 3-years old plantlets greenhouse [128]
0.1 mg L−1 NAA + 3.0 mg L−1
in vitro plantlets (6.2–6.6); the increase in age of cultivated showed same color,
TDZ
callus reduced it embryogenesis size and morphology of O.
capacity Gower Ramsey
Int. J. Mol. Sci. 2020, 21, 985
Table 2. Cont.
Lateral thin cell layers in culture
Temp 25 ± 1 ◦ C/19 ± 1 ◦ C medium with BA at 2.0 μM
Transverse and lateral Thin
(day/night), 16-h increased PLB induction in 64 to
Oncidium forbesii cell layers 1mm thickness WPM + 3% sucrose + 0.6% agar,
photoperiod, white 82% explants and both from lateral Non-evaluated [129]
(Brasilidium forbesii) from in vitro germinated pH 5.8
fluorescent tubes and transversal TCL at 1.0 μM
protocorms
40 μmol m−2 s−1 promoted the number of PLBs
obtained/explant (17.1–24.6)
MS1⁄2 (full strength MS vitamins) PLBs regeneration from PLBs
+ 1 g L−1 tryptone + 20 g L−1 section increased with addition of
PLBs sections obtained sucrose + 1 g L−1 activated chloro or methyl or nitro
Temp 22 ± 2 ◦ C, 16-h
Oncidium ‘Gower Ramsey’ from nodal explants from charcoal + 65 g L−1 potato tuber derivatives (compounds 5a–5c) Non-evaluated [130]
photoperiod
inflorescences + 8 g L−1 agar + 5 μM TDZ using 2–5 μM, from 41 (control)
(TDZ, vitamins and glycine were until 95 plantlets per culture bottle
filter sterilized) using 5 μM of 5c compound
NAA 0.75 mg L−1 produced
Temp 26 ± 2 ◦ C, PPFD highest number of PLBs/callus (98)
Oncidium sp. (Vu Nu MS1⁄2 + 20 g L−1 sucrose + 10%
In vitro shoots 22.2 μmol m−2 s−1 , 12-h and 1 mg L−1 BA promoted PLBs Non-evaluated [112]
Orchids) coconut water + agar, pH 5.8
photoperiod regeneration into shoots
(12.42/PLB)
Leaves from 1–2 cm plantlet height
showed highest explants induced
MS1⁄2 (with Fe-NaEDTA, PLBs using 2.0 mg L−1 BA (16.7%),
22
vitamins and glycine at Temp 25 ± 2 ◦ C, 8-weeks in Plants were transferred to plastic
but highest number of embryos
Leaf segments from full-strength MS) + 100 mg L−1 dark and transferred to dim pots and flowered after one-year
Tolumnia Snow Fairy different in vitro plantlets light, PPFD 5 μmol m−2 s−1 , was obtained with 4.0 mg L−1 BA without reports of somaclonal [31]
myo-inositol + NaH2 PO4 and from plantlets with 2–3 cm (41
height and leaf positions cool white fluorescent variations in vegetative and
(170 mg L−1 ), 30 g L−1 sucrose + tubes, 12-h photoperiod PLBs/explant), upper wounding reproductive phase
8.0 g L−1 agar, pH 5.2 region of bigger PLBs improved
PLBs proliferation and number of
PLBs per explant
MS: Murashige and Skoog Medium [99]; VW: Vacin Went medium [43]; WPM: Wood Plant Medium [131]. 2,4-D, 2-4-Dichlorofenoxiacetic acid; BA, 6-Benzyladenine; IAA, 3-Indoleacetic
acid; IBA, Indole-3-butyric acid; NAA, Naphtaleneacetic acid; PPFD: Photosynthetically Photon Flux Density; Temp, Temperature; TDZ, Thidiazuron.
Int. J. Mol. Sci. 2020, 21, 985
Another study conducted in a gelled medium by [124] observed that the use of 2% fructose
resulted in 95% explants containing PLBs in Oncidium Gower Ramsey or 2% glucose resulted in 85%
explants containing PLBs in Oncidium Sweet Sugar [124]. However, for the number of PLBs per explant,
the best results were obtained with 2–3% sucrose (31.1–33.7 PLBs/explants), demonstrating that sucrose
is the most suitable sugar for IPR–PLB. The use of other types of sugars, cellobiose, maltose and
trehalose do not result in benefits for number of PLBs from callus in Oncidium Gower Ramsey [122] or
for direct production of PLBs from young leaves [124].
There are no doubt about the application of PLBs in mass clonal production of Oncidium [132], but
recent studies also showed and confirmed the presence of somaclonal variation in Oncidium obtained
from IPR–PLBs [133], similar to observed with Phalaenopsis genus.
4. Some News with Cymbidium, Dendrobium, and Others

The most of results obtained with Phalaenopsis and Oncidium were similar to reported with other
species of orchids of importance in floriculture, as Cymbidium and Dendrobium genera, such as the main
PGRs used for IPR–PLBs. As example, the combination of cytokinin BA (5.0 mg L−1 ) and auxin NAA
(2.5 mg L−1 ) were used to induce PLBs (20.55 PLB per primary protocorm) in Cymbidium mastersii
protocorms [134]. Thin cell layers (TCL) from different types of tissues was a technique used to improve
the production of PLBs in Cymbidium [135], Dendrobium [136,137], Oncidium [129], and Phalaenopsis [93].
In Dendrobium, a wide and complete study about molecular research was exhaustively carried
out by [138], and considered especially the identification, classification and breeding of Dendrobium.
Similarly, other study with micropropagation of Dendrobium was realized by [17] and concluded that
PLBs were used as explants in 21.8% of studies, and together with nodal or nodal segments explants is
one of the major method used for Dendrobium micropropagation.
Thidiazuron was also an important PGR for induction of PLBs in Dendrobium orchids, but the
response to different cytokinins depends on genotype. In Dendrobium aqueum, only the cytokinin 2iP
[N-6-(2-isopentyl) adenine] at 1.5 mg L−1 proved it efficiency in production of PLBs (42.7 PLBs per
explants) from callus, compared to other cytokinins BA, Kin and Zea, and cytokinin-like compound
TDZ. These authors also observed that arginine at 25 mg L−1 increased direct somatic embryogenesis,
instead of callus derived PLBs [137]. Meta-Topolins, a natural aromatic type of cytokinin, were also
reported used in induction and regeneration of PLBs in D. nobile, which combined with 0.5 mg L−1
NAA resulted in best PLBs formation (92%) and shoots/explants (9.2) [139]. These same authors
observed that addition of polyamines, such as spermidine and putrescine increased regeneration of
shoots from PLBs and secondary PLB formation.
In our laboratory, PLBs of Dendrobium Hybrid ‘H3’, could be induced and proliferated in one-step,
and obtained from in vitro shoots, using liquid MS1⁄2 medium with 1.0 mg L−1 BA, and under agitation
of 80 rpm (Figure 1D).
5. Applications of IPR–PLB Technique on Orchid Propagation and Breeding and Main

Limitations of the Technique
Induction, proliferation, and regeneration of PLBs in orchids have many advantages to
conventional micropropagation by shoot proliferation or use of shoots from inflorescence stalk
segments as in Phalaenopsis [140], as increased rate of proliferation/multiplication [141] and single-cell
derived PLBs [123], which could be used for propagation, but also for breeding purposes and to obtain
disease free plantlets.
In breeding programs using in vitro techniques, PLBs could be used to obtain autotetraploid
plants with use of anti-mytotic agents as oryzalin [142] and colchicine [143], and to obtain mutants by
the use of chemical mutagens as sodium azide [144] or physical mutagens as gamma-irradiation [145].
PLBs can be also used for transformation protocols and successful protocols were developed
and obtained stable transgenics with target characteristics for floriculture [146,147]. In genetic
transformation of orchids, the use of PLBs derived directly from individual epidermal cells resulted
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Int. J. Mol. Sci. 2020, 21, 985
in solid transgenic plants with clonal identity of Oncidium Sharry Baby ‘OM8’ [32], an exceptional
advantage over PLBs from callus and with multicellular origin [126], which may result in the
emergence of somaclonal variants [42] and chimeric tissues when used for genetic transformation,
which are difficult to characterize and separate [32]. Using this technique, these authors reported
33–43% PLBs expressing the β-glucuronidase gene (GUS) and obtained six lineages that amplified the
transgenes pepper ferredoxin-like protein (pflp) and hygromycin phosphotransferase (hpt) using the
particle bombardment technique. Agrobacterium tumefasciens-mediated transformation has also been
successfully used in the production of transgenic plants of Oncidium ‘Sharry Baby OM8’ and Oncidium
Gower Ramsey using the induction of secondary PLBs from in vitro-maintained PLBs [148,149].
From a phytosanitary point of view, it is known that the use of seeds for in vitro asymbiotic
sowing of orchids is a real way to obtain virus-free seedlings in orchids from contaminated mother
plants, as observed for Cymbidium species [150,151]. Ref. [152] confirmed on a large scale (1000 plants)
that in vitro plants from seeds are free of Cymbidium Mosaic Virus (CyMV) and Ondontoglossum Ringspot
Virus (ORSV).
The technique of culturing apical meristems may also be effective in eliminating viral diseases in
orchids, but it requires great manual skill for excision of tiny meristems leading to contamination-free
tissue [153]. These requirements and the individual characteristics of viral diseases may lead to
breakthroughs in the technique, which may result in in vitro plantlets containing viral diseases, as
reported in Brassolaeliocattleya, Cattleya, Dendrobium, Epicattleya, Oncidium, and Mokara grown in vitro,
for which CyMV virus was reported to be present in 27.6% of 880 plantlets evaluated, while ORSV was
not detected in these samples [152].
Furthermore, in genera such as Phalaenopsis, the most commercially important in the world,
only stem apex culture may not be effective in completely eliminating important viral diseases in the
crop [140], and may still result in the need to kill the mother plant to obtain the apical meristem, since
these plants are monopodial and have poorly developed stem [150]. In this sense, in vitro IPR–PLBs
is an alternative to the production of virus-free clonal plants in orchids. In Phalaenopsis hybrid ‘V3’,
Ref. [140] obtained PLBs from stem apexes of donor plants contaminated with Ondontoglossum ringspot
virus and Cymbidium mosaic virus, and observed that the first PLBs produced directly from the stem
apex had 31.25% PLBs with viruses, identified by the enzyme-linked immunosorbent assay (ELISA)
and RT-PCR and were only eliminated in the process after some subcultures. The PLBs identified
as virus-free were subcultured in PLB proliferation medium, and in the second subculture 18.18%
positive PLBs were identified for both viruses. Only in the third subculture of PLB proliferation, it was
possible to obtain 100% virus-free PLBs, which remained until the end of the experiment.
PLBs can also be used for orchid propagation using the synthetic seed technique and for
cryopreservation. In Dendrobium ‘Sonia’, the use of PLBs stored at 4 ◦ C for 15 days in the pro-meristematic
and leaf primordium stages and encapsulated with 3–4% sodium alginate + 75–100 mM CaCl2 *2H2 O
resulted in 100% germinated PLBs, with the appearance of the first leaf at 22–27 days and the first root at
30–35.8 days, and the technique can be replicated with similar results for Oncidium ‘Gower Ramsay’
and Cattleya leopoldii [154].
In Dendrobium candidum and Dendrobium nobile, PLBs have also been used to increase the
production of bioactive compounds. In D. nobile, an increase was observed in the production of
secondary metabolites such as phenols, flavonoids and alkaloids extracted from PLB-micropropagated
plants, when compared to the mother plant [139]. In D. candidum, the increase in methyl-jasmonate
elicitor concentrations, although resulting in a proportional reduction in PLBs mass gain, increased
the concentrations of alkaloids, polysaccharides, phenols and flavonoids when used between 75 and
100 μM [155].
Although the IPR–PLB technique is widely used for large scale plantlet production, breeding
and conservation, some difficulties still limit the wider use of the technique on a commercial scale.
Among the main limitations are the high genotype-dependence of PLB induction and proliferation
responses in vitro, and the occurrence of undesirable somaclonal variations, which greatly hinder the
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Int. J. Mol. Sci. 2020, 21, 985
proliferation of clonal propagation of PLBs for a wide range of commercial cultivars available and
required by the market.
Ref. [30] used NDM culture medium plus TDZ (0.25 mg L−1 ) and NAA (1.0 mg L−1 ) and observed
distinct responses between ’908’ genotype (45% explants with PLBs and up to 25 PLBs/leaf segment)
and ’RP3’ genotype (10% explants with PLBs and only 2 PLBs/leaf segment), the latter being highly
recalcitrant to the induction and proliferation of PLBs from leaf segments of plants grown in vitro.
A study by [59] also noted important differences between the PLBs induction responses between
P. amabilis (up to 50% explants with PLBs and 15.6 PLBs/explant) and the commercial cultivar P. nebula
(80% explants with PLBs and up to 5.3 PLBs/explant). The same occurred in another study with the
same cultivars, in which the cytokinin types and concentrations that resulted in the highest percentage
of explants with PLBs were 13.32 μM BAP in P. amabilis (80%) and 13.62 μM TDZ in P. nebula (65%). The
largest number of PLBs per explant was obtained with 13.62 μM TDZ in P. amabilis (7.8 PLBs/explant)
and 4.65 μM Kin in P. nebula (16 PLBs/explant) [77].
Ref. [156] point out that one of the biggest difficulties in Phalaenopsis micropropagation by PLBs
is that not all genotypes respond to a single protocol and the same cultivation conditions, and often
result in plants with undesirable characteristics. Ref. [41] compared eight cultivars of Phalaenopsis
and Doritaenopsis to obtain PLBs from shoot tips of inflorescence stalk buds with best percentage
of PLB formation in four genotypes using 1.0 mg L−1 BAP (26.9–71.4% depending on genotype),
while two respond better with 2.0 mg L−1 (60–75% explants with PLBs) and one produced 50% PLBs
independently of the concentration of BAP (1, 2, or 5.0 mg L−1 ). Testing other four genotypes authors
reported ranges from 7.1% to 40% of PLBs formation only in NDM culture medium, while in 1⁄2MS
only two cultivars produced PLBs [41].
Ref. [156] have been associated undesirable characteristics observed in some plantlets with the
identification of somaclonal variants from PLBs, which can be morphologically identified even at the
shoot bud regeneration and in vitro plantlet production stage. According to [157], the occurrence of SV
in the IPR–PLBs technique is higher than that observed from adventitious bud propagation, and that
most commercial laboratories use a maximum of three generations of PLBs subcultures to avoid high
frequencies of somaclonal variations in this type of propagation.
In our laboratory conditions, using leaf segments from in vitro plantlets to obtain PLBs (Figure 1A,B)
somaclonal variations are observed in rooting phase of PLB-derived plantlets of Phalaenopsis ‘Ph908’,
while were not observed in plantlets derived from shoot-proliferation using inflorescence stem nodal
segments (Figure 2A). The main symptoms were the limited development of plantlets that remains
in acclimatized plantlets, with morphological abnormalities in leaves (Figure 2B), also observed and
called as ‘creased leaves’ by [66] and flowers deformities as absence of lip in some flowers of the
inflorescence (Figure 2C,D) possibly associated with mutations rather than epigenetic variations.
Ref. [139] used induction of PLBs from pseudostems from in vitro germinated Dendrobium nobile
plants in MS + 1.5 mg L−1 TDZ and 0.25% activated charcoal medium and verified 94% explants
producing PLBs and up to 11.6 PLBs/explant. These authors observed a somaclonal variation rate close
to 6% in the obtained plants, being the main cause of the somaclonal variations detected by molecular
markers Random amplified polymorphic DNA (RAPD) and Start codon targeted (SCoT), attributed by
the authors to the use and exposure time to TDZ.
Although the cytokinin-like compound TDZ is appointed as one of the major causes of SV in
orchid PLB induction, there were some contradictory reports.
As example, the cytokinin Kinetin at 1.5 mg L−1 resulted in increases of somaclonal variations
frequency of PLBs in Dendrobium Sabin Blue, detected by ISSR and DAMD molecular markers, when
compared with use of TDZ at 4.0 mg L−1 added activated charcoal [158].
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Int. J. Mol. Sci. 2020, 21, 985
ȱ
Figure 2. Somaclonal variations observed in Phalaenopsis induction, proliferation and regeneration
of protocorm-like Bodies in Phalaenopsis Hybrid “908”. Normal vegetative developed plant (A) and
somaclonal variation observed in vegetative development with “creased leaves” (red arrow) (B);
(C,D), Normal vegetative developed plants with somaclonal variations in flower development, with
first and last flower without of labellum (red arrow, wl) in the same inflorescence with normal flowers
(nf). All figures are unpublished photos from J.C.C.
In addition, [159] observed somaclonal variants in Phalaenopsis True Lady ‘B79-19’, obtained from
the induction of PLBs and from young leaves obtained from in vitro plants in VW culture medium
containing only BA and NAA as phytoregulators, i.e., without using TDZ. These authors also reported
that variant plants were discarded during in vitro subcultures (not quantified), and out of the plants
obtained and without morphological variations in the leaves, only 20 out of a total of 1360 obtained
(1.5%) were somaclonal variants, indicated by the different flowers of the original clone.
Also the use of topolins meta-Topolins (mT) and meta-Topolins Riboside (mTR), a natural aromatic
cytokinin reported as reducing phytotoxic effects in micropropagation, it use not solved the problem of
somaclonal variation obtained in vitro [160] and, although was reported increasing efficiency of PLB
induction it use not resulted in absence of somaclonal variation in orchids [139].
26
Int. J. Mol. Sci. 2020, 21, 985
These observations with other cytokinins PGRs diminish the importance of TDZ as the unique
or main factor for VS inducing in orchid IPR–PLBs, and include other causes, such as the differential
susceptibility of genotype and the number of subcultures under proliferation stage of PLB production.
Genotype susceptibility is appointed one of the main factors lead to VS in Phalaenopsis and
Doritaenopsis orchids micropropagation, ranging from zero to 100% SV depending on genotype and
is not exclusive of the PLB technique [72,161]. Similarly, [70] also observed that some genotypes of
Phalaenopsis not presented any variants, while others showed until 47.9% of variants. Among them,
most of SV in this genus were reported in flowering stage [161], by modification of inflorescence and
flower characteristics, such as the perloric and semi-perloric mutants observed in Phalaenopsis Zuma
Pixie ‘#1’, P. Little Mary and Doritaenopsis Minho Diamond ‘F607’ [162]. Lose of part of flowers were
also reported, such as pollinia [162] and absence of labellum (Figure 2C,D).
Ref. [161] evaluated until the flowering stage (1.0–1.5 years after acclimatization) plants of
10 genotypes of Phalaenopsis and Doritaenopsis hybrids micropropagated by the PLB technique, and
subcultured in vitro for 5 to 10x and identified the presence of seven types of VS, possible to be
identified only at the flowering stage. The plants had deficiencies or divergences in the petals and
sepals or in the development of the inflorescence, but with similar vegetative development in relation
to the mother plant. These authors observed that the produced VS were not polyploid mutants,
maintaining the same amount of genetic material as the mother plants.
Although most of SV was reported in flowering stage, transcript analysis by Real-Time PCR
demonstrated that mutants has also many other alterations in factors of transcription and transcripts
were detailed reported in Phalaenopsis and Doritaenopsis by [162]. In Oncidium ‘Milliongolds’ were also
observed chlorophyll SV (whole yellow or with streaked leaves) in vegetative development of in vitro
plantlets [133].
Another factor related to the origin of VS in PLBs in orchids is the phase in which VS occurs.
It has been reported that in the proliferation phase, undesirable VS induction from PLBs occurs at a
higher intensity and frequency, and it is necessary to establish a number of subcultures to keep the VS
frequencies low in clonal propagation. Ref. [92] reported increases in SV after the third subcultures
of PLBs in proliferation medium (NDM + 0.1 mg L−1 TDZ and 10 mg L−1 chitosan) with same ISSR
profile until third subculture, 95% at fourth and 80% at fifth subculture of PLBs.
The use of RAPD molecular markers (total of 1116 bands) did not allow the identification of
these somaclonal variants in these plants, but isozyme pattern analysis demonstrates the difficulty
of observing mutations in materials obtained from PLBs using RAPD molecular markers and the
occurrence of conclusion errors or even underestimated data of somaclonal variants in the confirmation
of clonal origin in other studies conducted with these markers [159].
Ref. [82] also used RAPD markers to analyze the clonal origin of PLBs and induced seedlings in
in vitro leaf segments of Phalaenopsis bellina in 1⁄2MS medium with 3.0 mg L−1 TDZ. They observed that
most somaclonal variants are obtained at the proliferation/multiplication phase, with no VS observed
in the origin phase of the PLBs of the mother plant.
Analyses of SCoT and Target Region Amplification Polymorphism (TRAP) markers also showed
the presence of somaclonal variants in Dendrobium Bobby Messina PLBs cryopreserved or not [163].
These differences in the frequencies of VS observed in different orchid species and genotypes
are probably associated with higher sensitivity of different genotypes to the occurrence of mutations.
Ref. [164] observed that the frequency of VS at the vegetative and reproductive stages in Phalaenopsis
PLBs was dependent on the genotype used. These authors observed that there was a reduction in DNA
methyltransferase (Dnmt)-related gene expression in Phalaenopsis ‘Little Mary’ VS.
Current advances in molecular marker techniques allow increasing the number of tools and
the accuracy of these analyses and the greater possibility of identifying possible VS. There is little
information about wide molecular genome characterization in Oncidium, and [133] used specific-locus
amplified fragment sequencing (SLAF-seq) to analyze possible variations in single-nucleotide
polymorphisms (SNPs) in Oncidium ‘Milliongolds’ obtained by PLBs grown for 10 years and observed
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Int. J. Mol. Sci. 2020, 21, 985
high rates of variation and that adjacent SNPs adenine and thymine were more frequent than those
related to guanine and cytosine, with prominence of mononucleotideInDels.
Ref. [157] isolated two most expressed transposable elements and identified a new Instability
Factor (PIF)-like, one of which, called PePIF1 was identified by similarity to the Phalaenopsis equestris
genome sequence, and which was transposed in the somaclonal variants of cultivars of Phalaenopsis
from micropropagation, which resulted in the insertion of new genes identified and sequenced by
the authors.
6. Conclusions
Induction, proliferation, and regeneration of PLBs (IPR–PLBs) in orchids is one of the
most promising techniques to replace current conventional micropropagation techniques, in
particular because it has wide application in clonal conservation, propagation, breeding, and
phytossanitary-cleaning of elite plants used in the flower market. Although many authors used
somatic embryogenesis to describe IPR–PLBs technique or their origin, recent molecular studies about
the origin route of PLBs, at least in Phalaenopsis orchids, showed that IPR–PLBs routes are not the
same of somatic embryonic origin. Some limitations of IPR–PLBs in orchids such as low repeatability
of responses due to high genotype dependence and the presence of somaclonal variations (SV) still
limit their large-scale use in the production of clone plantlets. Although the main causes of SV
described in papers were the genotype-sensibility, the use of cytokinin thidiazuron and subsequent
PLBs proliferation, only genotype sensibility looks conclusive, because SV was also observed in
protocols using other cytokinins, such as BA and Kin. Nevertheless, the new findings associated with
the identified instability factors, associated with the recent sequencing of the Phalaenopsis equestris
genome, and the use of new molecular tools that increase the accuracy of quantitative identification
analyses and the causes of somaclonal variation, are in agreement with the evolution of this technique,
which represents the tool of greatest potential today to replace other less efficient micropropagation
techniques in the production of plantlets in orchids.
Author Contributions: J.C.C. and C.A.Z. designed and wrote the manuscript. J.C.C. and J.-T.C. comprehensively
revised and improved the quality of manuscript. All authors have read and agreed to the published version of
the manuscript.
Funding: This study was financed in part (English editing service and scholarship to C.A.Z.) by the Coordenação
de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES) Finance Code 001.
Acknowledgments: J.C.C. thanks to São Paulo Research Foundation for the project number 2018/20673-3 and to
Conselho Nacional de Desenvolvimento Científico e Tecnológico for the project number 311083/2018-8.
Conflicts of Interest: The authors declare no conflict of interest, either financially or otherwise.
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© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
36
Molecular Sciences
Article
Comparative Metabolomic Analysis Reveals Distinct
Flavonoid Biosynthesis Regulation for Leaf Color
Development of Cymbidium sinense ‘Red Sun’
Jie Gao † , Rui Ren † , Yonglu Wei, Jianpeng Jin, Sagheer Ahmad, Chuqiao Lu, Jieqiu Wu,
Chuanyuan Zheng, Fengxi Yang * and Genfa Zhu *
Guangdong Key Laboratory of Ornamental Plant Germplasm Innovation and Utilization, Environmental
Horticulture Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China;
[email protected] (J.G.); [email protected] (R.R.); [email protected] (Y.W.); [email protected] (J.J.);
[email protected] (S.A.); [email protected] (C.L.); [email protected] (J.W.);
[email protected] (C.Z.)
* Correspondence: [email protected] (F.Y.); [email protected] (G.Z.); Tel.: +86-020-8516-1014 (F.Y.)
† These authors contributed equally to this work.

Received: 5 February 2020; Accepted: 6 March 2020; Published: 9 March 2020
Abstract: The colorful leaf is an important ornamental character of Cymbidium sinense (C. sinense),
especially the red leaf, which has always been attracted by breeders and consumers. However, little
is documented on the formation mechanism of the red leaf of C. sinense. In this study, the changing
patterns of flavonoid-related metabolites, corresponding enzyme activities and genes expression in
the leaves of C. sinense ‘Red Sun’ from red to yellow and finally to green was investigated. A total
of 196 flavonoid-related metabolites including 11 anthocyanins metabolites were identified using
UPLC-MS/MS-based approach. In the process of leaf color change, 42 metabolites were identified as
having significantly different contents and the content of 28 differential metabolites turned to zero. In
anthocyanin biosynthetic pathway, content of all 15 identified metabolites showed downregulation
trend in the process of leaf color change. Among the 15 metabolites, the contents of Naringenin
chalcone, Pelargonidin O-acetylhexoside and Anthocyanin 3-O-beta-d-glucoside decreased to zero
in the green leaf stage. The changing pattern of enzyme activity of 10 enzymes involved in the
anthocyanin biosynthetic pathway showed different trends from red leaves that have turned yellow
and finally green, while the expression of genes encoding these enzymes was all down-regulated
in the process of leaf color change. The results of this study revealed the types of flavonoid-related
metabolites and the comprehensive analysis of metabolites content, enzyme activities and genes
expression providing a new reference for breeders to improve the leaf color of C. sinense ‘Red Sun’.
Keywords: metabolomic analysis; differential metabolites; enzyme activity; gene expression; leaf
color; Cymbidium sinense
1. Introduction
As the largest family of monocotyledons, Orchidaceae has a long history of cultivation and
contains abundant varieties. Among these varieties, C. sinense is a unique variety produced in China,
which has been loved by consumers all around the world. The most important phenotypic feature of C.
sinense is that it has a very rich variation in leaf color, which improves its ornamental and economic
values. C. sinense contains a number of varieties based on the location and color of leaf variegation,
including chimera art, claw art, crown art, crane art, middle penetration art, spot art and treasure art
and so on [1]. At present, the leaf variegation varieties of C. sinense are basically yellow-green leaves,
but there are few reports about red leaves. C. sinense ‘Qihei’ is the most common variety of green leaves.

Int. J. Mol. Sci. 2020, 21, 1869
In the process of culture, the color of ‘Qihei’ leaf buds changes to red, and then the leaf color changes
from red to yellow, finally to green, leaving only a little yellow at the tip of the leaf during the growth
and development of leaf buds. The leaf variegation variety is named C. sinense ‘Red Sun’. So far, the
metabolic changes associated with the formation of red leaves in C. sinense ‘Red Sun’ are not known.
Compared with flower color, the influencing factors of leaf color are more complex. Generally, the
greening of leaves is mainly due to the absolute proportion of chlorophyll, and the formation of yellow
leaves is mostly due to the degradation of chlorophyll, leaving the color of carotenoids to dominate in
leaves [1,2]. Anthocyanins are responsible for the red color of leaves, which is helpful for plants against
various biotic and abiotic stresses [3,4]. In Cymbidium orchids, the activation of anthocyanin synthesis
can be restored by introducing MYB and bHLH anthocyanin regulators simultaneously [5]. The
synthesis pathway of anthocyanins begins with the precursor phenylalanine, resulting in the formation
of dihydrokaempferol (DHK) by the catalysis of six enzymes. After the catalysis of four enzymes, DHK
produces three kinds of steady-state anthocyanins, including pelargonidin, cyanidin and delphinidin,
respectively [6–8]. At present, more than 600 anthocyanins found in nature are derived from these
three substances and the accumulation of anthocyanins in plants has two main functions: one is to
produce rich and colorful visual signals to promote pollination or seed transmission, and the other is
to resist a series of biotic or abiotic stresses [9,10].
Plant metabolomics carries out a qualitative and quantitative analysis of small molecular
metabolites in plants, so as to help researchers understand the synthesis and accumulation patterns
of metabolites [11]. At present, the study of plant metabolites mainly involves the identification of
metabolites, variety differentiation and auxiliary breeding and so on [12–15]. Flavonoids-targeted
metabolomics refers to the analysis of small molecular metabolites of flavonoids (anthocyanins), which
is often used to analyze the formation mechanism of plant color. Analysis of Flavonoids-targeted
metabolomics in the white and purple flowers of Phalaenopsis identified 142 different flavonoid-related
metabolites, of which the most important anthocyanin was the derivative of cyaniding [16]. A study on
green and purple asparagus suggested that the difference in color was mainly caused by the contents of
peonidin and cyanidin and their glycoside derivatives [17]. Metabolomic analysis the color difference
of fig peel showed that the content of anthocyanin derivatives in purple peel was significantly higher
than that in green fruit [18]. These results indicate that metabolomics is an important and effective
method to analyze the formation mechanism of plant color.
In order to understand the key metabolites involved in the process of color change from red to
green in the leaves of C. sinense ‘Red Sun’, flavonoids-targeted metabolomic analysis was used to
analyze the change pattern of flavonoid-metabolites in the process of leaf color change. Physiology,
enzyme kinetics and molecular biology experiments were carried out to explore the mechanism of leaf
color difference. To the best of our knowledge, this study is the first effort to analyze the mechanism of
the formation of C. sinense leaf variegation from the perspective of metabolites, and the results provide
a new train of thought and basis for the study of C. sinense leaf variegation.
2. Results
2.1. Pigments Content Analysis

The leaves of C. sinense ‘Red Sun’ show red at the leaf bud stage. With the development of the
leaves, the leaves gradually change from red to yellow, and finally the mature leaves develop into green
with a little yellow color at the tip, as seen in Figure 1. The color of leaves was affected by a variety of
pigments, including the contents of chlorophyll, carotenoids, total flavonoids and total anthocyanins
in C. sinense ‘Red Sun’. The results showed that the maximum contents of the four pigments were
found at the red leaf stage, while the minimum contents were shown by leaves at yellow stage. The
contents of all four pigments in green leaves were higher than those in yellow leaves, but lower than
those in red leaves (Figure 2).
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Int. J. Mol. Sci. 2020, 21, 1869
Figure 1. Phenotypes of Cymbidium sinense ‘Red Sun’ red leaves (RL) (a), yellow leaves (YL) (b) and
green leaves (GL) (c).
Figure 2. Chlorophyll (a), carotenoids content (b), total flavonoid (c) and total anthocyanin (d) content
of different color leaves of Cymbidium sinense ‘Red Sun’. Bars represent the mean of three biological
replicates ±SE. Lowercase letters indicate significant differences at p < 0.05.
2.2. Qualitative and Quantitative Analyses of Metabolites and Quality Control (QC) Analysis of Sample
Anthocyanin is the cause of red leaf color. From the pigment contents, it can be seen that
the contents of total flavonoids and total anthocyanins change significantly in the process of leaf
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Int. J. Mol. Sci. 2020, 21, 1869
color change. To compare the differences of flavonoid-metabolites, the UPLC (Shim-pack UFLC
SHIMADZU CBM30A)−MS/MS (Applied Biosystems 4500 QTRAP) technique was used to detect
the flavonoid-related metabolites in RL, YL and GL. The total ion flow map of the mixed sample
quality control (QC) sample (Total ions current, TIC, is the map obtained by adding the intensities
of all ions in each time point mass spectrum) is shown in Figure S1a. The multi-peak map of MRM
metabolite detection of multi-substance extraction is illustrated in Figure S1b. Based on the local
metabolic database, the metabolites of the samples were qualitatively and quantitatively analyzed by
mass spectrometry. The multi-peak map of MRM metabolite detection in the multi-reaction monitoring
mode shows the substances that can be detected in the sample, and each mass peak of different
color represents a detected metabolite. A total of 196 flavonoid-related metabolites were detected
(Table S1), including 11 anthocyanins, 3 Chalcone, 9 Dihydroflavonoid, 5 Dihydroflavonol, 12 flavanols,
11 Flavone, 59 Flavonoid, 20 Flavonoidcarbonoside, 3 Flavonoids, 59 Flavonols and 4 Isoflavones. In
the process of instrumental analysis, one QC sample is inserted into every 10 test and analysis samples
to monitor the repeatability of the analysis process. Through the overlap display analysis of the TIC
map of different quality control QC samples (Figure S1c), the results showed that the curve overlap of
metabolite detection of total ion current was high, that is, the retention time and peak intensity were
the same, indicating that the signal stability was good when the same sample was detected by mass
spectrometry at different time points. All the detected metabolite content data were normalized by
range method, and the accumulation patterns of metabolites among different samples were analyzed
by cluster analysis (Hierarchical cluster analysis, HCA) by R software (www.r-project.org/) (Figure 3).
2.3. Formatting of Mathematical Components Principal Component Analysis (PCA) and Orthogonal
Projections to Latent Structures-Discrimination Analysis (OPLS-DA)
Principal Component Analysis (PCA) is a multidimensional data statistical analysis method of
unsupervised pattern recognition. Through principal component analysis of samples (including QC
samples), we can preliminarily understand the overall metabolic differences among samples and the
degree of variability between samples within groups. From the analysis results, it can be observed that
there are significant differences among RL, YL and GL groups, but there is no significant difference
within groups (Figure S2).
Although PCA can effectively extract the main information, it is not sensitive to variables with
small correlation, and Partial Least Squares-Discriminant Analysis (PLS-DA) can solve this problem.
Compared with PCA, PLS-DA can maximize the distinction between groups and facilitate the search
for differential metabolites. Through the analysis of PLS-DA, the orthogonal variables, which are
not related to the classification variables of metabolites are first eliminated, and then the differences
of correlation between groups and within groups are analyzed. According to the OPLS-DA model,
we analyzed the metabolic group data, draw the score chart of each group, and further showed the
differences between each group. The prediction parameters of the evaluation model are R2X, R2Y and
Q2, in which R2X and R2Y represent the interpretation rate of the model to X and Y matrix respectively,
and Q2 indicates the prediction ability of the model. The closer these three indexes are to 1, the
more stable and reliable the model is. Q2 > 0.5 can be regarded as an effective model, and Q2 > 0.9
is an excellent model. From the results, there are significant differences among the three groups of
data, but there is no significant difference between groups (Figure S3a). The alignment verification of
OPLS-DA was carried out (n = 200, that is, 200 permutation experiments were carried out). In the
model verification, the horizontal lines correspond to R2 and Q2 of the original model, and the red
dots and blue dots represent R2’ and Q2’ of the model after Y replacement, respectively. The results
showed that R2’ and Q2’ of each group were smaller than R2 and Q2 of the original model, which
indicated that the model was meaningful and the differential metabolites could be screened according
to VIP value analysis (Figure S3b).
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Int. J. Mol. Sci. 2020, 21, 1869
Figure 3. Hierarchical clustering analysis of all metabolites detected in this study. The abscissa indicates
three biological replicates of red leaves (RL1, RL2, and RL3), yellow leaves (YL1, YL2, and YL3) and
green leaves (GL1, GL2, and GL3), and the ordinate indicates the metabolites detected in this study.
The red segments indicate a relatively high content of metabolites, while the blue segments indicate a
relatively low content of metabolites. The relative metabolite contents represented by color segments at
the corresponding locations are listed in Table S1.
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Int. J. Mol. Sci. 2020, 21, 1869
2.4. Screening Differential Metabolites in the Process of Leaf Color Change

The differential metabolites were screened by combining the VIP values of fold change and
OPLS-DA model. Following was the screening criteria: (1) If the difference of metabolites content
between the control group and the experimental group is more than 2 times or less than 0.5, the
difference is considered to be significant; (2) On the basis of the above, the metabolites with VIP ≥ 1 are
selected. Volcano Plot was used to show the difference of expression level between the two groups
of samples, and the difference was statistically significant (Figure 4). Compared with red leaves, 85
metabolites in green leaves were significantly different (10 up-regulated and 75 down-regulated),
and 61 metabolites in green leaves were significantly different from yellow leaves (16 up-regulated
and 45 down-regulated) (Tables S2 and S3). Compared with red leaves, 86 metabolites showed
significant change in yellow leaves (3 up-regulated and 83 down-regulated) (Table S4). Through
the analysis of Vennn diagram, it was found that there were 42 metabolites with different contents
in the three periods of leaf color change. Except for the content of Liquiritinapioside, the other
41 substances decreased significantly with the change of leaf color (Figures S4 and S5). After a
comprehensive analysis of the contents of all the differential metabolites, we found that the contents of
28 metabolites turned to zero in the process of leaf color change, as shown in Figure 5. Among these
metabolites with drastic changes, the contents of Naringenin chalcone, an important intermediate in
anthocyanin synthesis, and two important anthocyanin metabolites, Pelargonidin O-acetylhexoside
and Anthocyanin 3-O-beta-D-glucoside, all decreased to zero in the green leaf stage.
Figure 4. (a) Volcano plot of differential metabolites for RL vs. GL, RL vs. YL and YL vs. GL. The colors
of the scatter points in Figure 4a indicate the final screening results: red indicates metabolites that
were significantly up-regulated; green indicates metabolites that were significantly down-regulated;
grey indicates metabolites with no significant difference. (b) Venn diagram analysis of differential
metabolites. RL, red leaves; YL, yellow leaves; GL, green leaves.
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Int. J. Mol. Sci. 2020, 21, 1869
Figure 5. Differences in the content of 28 metabolites in the process of leaf color change. Y-scale
represent the integral value of chromatographic peak area.
2.5. Intermediates Content, Enzymes Activities and Genes Expression Associated with the Anthocyanin
Biosynthetic Pathway
In order to further analyze the regulation mechanism of anthocyanin in the process of C. sinense
‘Red Sun’ leaf color change, the detected intermediates of anthocyanin synthesis pathway and all
the enzyme activities involved in anthocyanin synthesis were analyzed [19] (Figure 6). It can be
observed that all 15 detected intermediates show a downward trend in the process of leaf color
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Int. J. Mol. Sci. 2020, 21, 1869
change. In total, 11 kinds of anthocyanins metabolites were detected, and the contents of eight of
them (Cyanidin 3-rutinoside, Cyanidin 3-O-galactoside, Peonidin 3-O-glucoside chloride, Cyanidin
3-O-malonylhexoside, Cyanidin O-acetylhexoside, Anthocyanin 3-O-beta-d-glucoside, Pelargonidin
3-O-malonylhexoside and Pelargonidin O-acetylhexoside) changed significantly during the change
of leaf color, especially the content of Pelargonidin O-acetylhexoside (molecular weight: 474.096
Da), a derivative of Pelargonidin, and Anthocyanin 3-O-beta-d-glucoside (molecular weight: 449.089
Da), a derivative of Cyanidin finally decreased to 0 in green leaves. After measuring the activity of
all the enzymes involved in anthocyanin synthesis, it was found that the activity of henylalanine
ammonia-lyase (PAL), trans-cinnamate 4-monooxygenase (C4H), 4-coumarate-CoA ligase (4CL) and
flavonoid 3’,5’-hydroxylase (F3 5 H) was up-regulated, the activity of chalcone synthase (CHS),
chalcone isomerase (CHI), naringenin 3-dioxygenase (F3H) and flavonoid 3’-monooxygenase (F3 H)
was down-regulated. The activity of DFR was down-regulated at first and then up-regulated, while
the activity of ANS was up-regulated and then decreased. QRT-PCR analysis of the genes encoding
these enzymes showed that all genes were significantly down-regulated (Figure 7). Comprehensive
analysis of enzyme activity and the corresponding gene expression pattern showed that the changing
trend of enzyme activity of CHS, CHI, F3H, F3’H was consistent with that of gene expression pattern,
while that of the other six enzymes was different from that of gene expression pattern.
Figure 6. The changing patterns of enzymes activity and intermediate products contents related to
anthocyanin synthesis in the process of Cymbidium sinense ‘Red Sun’ leaf color change. Red and
blue shading in the lower right corner indicates the relatively high-or low content, respectively.
PAL, Phenylalanine ammonia lyase, C4H, cinnamate 4-hydroxylase, 4CL, 4-coumarate CoA
ligase, CHS, chalcone synthase, CHI, chalcone isomerase, F3H, flavone 3-hydroxylase, F3 H,
flavonoid 3 -hydroxylase, F3 5 H, flavonoid 3 ,5 -hydroxylase, DFR, dihydroflavonol reductase, ANS,
anthocyanidin synthase. Units on y-scale of enzymes activity is U/g.
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Int. J. Mol. Sci. 2020, 21, 1869
Figure 7. Expression pattern of genes coding enzymes related to anthocyanin synthesis in the process
of Cymbidium sinense ‘Red Sun’ leaf color change.
3. Discussion
The regulation of plant leaf color is a complex process. Most of the previous studies analyzed the
mechanism of leaf color formation by methods of physiology, cytology and molecular biology [1], but
the mechanism of leaf color regulation from the perspective of small molecular metabolites needs to be
studied further. In this study, based on UPLC-MS/MS, the changes of metabolites in the process of leaf
color change of C. sinense ‘Red Sun’ were qualitatively and quantitatively analyzed, and the unique
pattern of flavonoid-related metabolites in the process of leaf color change was constructed for the
first time. At the same time, the regulation mechanism of leaf color was further analyzed by enzyme
kinetics and gene expression analysis.
As far as we know, this is the first time to analyze the types of flavonoid-related metabolites
in the leaves of C. sinense ‘Red Sun’ by UPLC-MS/MS (flavonoids-targeted) method. A total of 196
flavonoid-related metabolites were detected. These substances belong to anthocyanin, chalcone,
dihydroflavonoid, dihydroflavonol, flavanols, flavone, flavonoid, flavonoid carbonoside, flavonoids,
flavonols and isoflavones. Based on wide target metabolomics analysis, only 6 and 15 differential
flavonoid-related metabolites were detected in tea leaves and Ginkgo biloba leaves, respectively [20,21].
Based on phenolic-targeted secondary metabolites analysis in purple fig peel, only 15 differential
flavonoid-related metabolites (including four anthocyanins metabolites) were detected [18]. In this
study, 119 kinds of differential flavonoid-related metabolites were found (including 10 kinds of
anthocyanins). The above results show that Flavonoids-targeted metabolites method can identify more
kinds of flavonoid-related metabolites, and has more advantages in mining the types and contents of
flavonoid-related metabolites, especially anthocyanins metabolites.
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Int. J. Mol. Sci. 2020, 21, 1869
Phalaenopsis and the materials of this study belong to Orchidaceae. In the metabolomic analysis
between petals of white and purple Phalaenopsis, 142 differential flavonoid-related metabolites, including
17 anthocyanins metabolites, were detected by flavonoids-targeted metabolomic analysis [16]. In
accordance with the results of this research, among the 119-differential flavonoid-related metabolites
detected, there were 8 differential anthocyanins. The differential metabolites of eight anthocyanins
identified in the leaves of C. sinense ‘Red Sun’ are Cyanidin 3-rutinoside (Keracyanin chloride), Cyanidin
3-O-galactoside, Peonidin 3-O-glucoside chloride, Cyanidin 3-O-malonylhexoside, Pelargonidin
3-O-malonylhexoside, Cyanidin O-acetylhexoside, Pelargonidin O-acetylhexoside and Anthocyanin
3-O-beta-d-glucoside. Compared with the 18 anthocyanin differential metabolites detected in the petals
of Phalaenopsis [16], only Cyanidin 3-O-malonylhexoside and Cyanidin O-acetylhexoside are the same.
Among the three main categories of pigmented glycosides, pelargonidin mainly shows orange/red,
cyaniding mainly shows pink/magenta and delphinidin mainly shows purple/blue [22,23]. The
metabolites with the highest proportion were cyanidin derivatives found in the petals of Phalaenopsis
and leaves of C. sinense ‘Red Sun’. Interestingly, during the process of C. sinense ‘Red Sun’ leaf color
change, the two anthocyanins from existence to absence are Pelargonidin 3-O-malonylhexoside and
Anthocyanin 3-O-beta-D-glucoside; the content of derivatives of six kinds of cyanidin and a kind of
delphinidin was high in purple petals but zero in white petals of Phalaenopsis [16]. The above results
are consistent with the phenotype of the corresponding materials.
As the upstream reaction of anthocyanin and other flavonoids, Phenylalanine was first converted
to P-coumaroyl-CoA under the catalysis of PAL, C4H and 4CL [24]. The activities of PAL, C4H and
4CL were all up-regulated during the change of leaf color, while the coding genes expression of these
three enzymes decreased significantly, which indicated that the activities of these three enzymes
were also subject to post-transcriptional modification or post-translational modification [25]. Then
P-coumaroyl-CoA was transformed into Naringenin under the action of CHS and CHI [24]. The
activities of CHS and CHI were down-regulated in the process of leaf color change, which was
consistent with the change pattern of gene expression. CHS is the first key enzyme in anthocyanin
synthesis, its activity determines the formation of anthocyanin metabolic pathway, and the loss of
its activity will lead to the loss of anthocyanin and other flavonoids [26,27]. From the results of
qRT-PCR, the expression of CHS-3 in the red leaf stage was 813 times higher than that in the green leaf
stage, indicating that CHS may play an important role in the process of leaf color change. Overall,
similar correlations between gene expression and anthocyanin levels were also observed during the
differential pigment deposition in crabapple cultivars with dark red, pink and white petal colors [28].
Next, Naringenin forms DHK under the catalysis of F3H. DHK then forms Dihydroquercetin (DHQ)
and Dihydromyricetin (DHM) under the catalysis of F3 H and F3 5 H, respectively [29]. The enzyme
activity of F3H and F3 H was down-regulated, while that of F3 5 H was up-regulated, and the coding
gene expression of these three enzymes was significantly down-regulated. Both natural mutants and
transgenic studies have proved that the competitions of three enzymes lead to different branching
pathways at this critical point [30], and our results support this argument. DHK, DHQ and DHM
formed unstable anthocyanins under the catalysis of DFR and ANS. The enzyme activity of DFR
decreased at first and then increased, while the activity of ANS increased at first and then decreased,
but the coding genes expression level of the two enzymes was significantly down-regulated. DFR from
different plants has specific substrates biases for DHK, DHQ and DHM [6,31], and the downstream
DFRs and ANSs is necessary for large sum of anthocyanin accumulation in Phalaenopsis [32,33]. These
unstable anthocyanins eventually went through the action of UFGT to form stable anthocyanins [34].
During the period of color change, UFGT activity firstly decreased, and then increased, while the UFGT
gene expression level was significantly down-regulated (Figure S5). Previous studies have found
that overexpression of UFGT causes plants to show darker colors, such as crimson or purple, while
overexpression of DFR or ANS only deepens the color to pink or lavender [34–36]. In this study, the
results of qRT-PCR showed that the expression of ANS in red leaf stage was 833 times higher than
that in green leaf stage, while the expression of UFGT in red leaf stage was 14 times higher than that
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Int. J. Mol. Sci. 2020, 21, 1869
in green leaf stage. The question of whether ANS or UFGT had a greater effect on the leaf color of
Cymbidium remains to be further verified.
4. Materials and Methods
4.1. Plant Materials

The three-year-old C. sinense ‘Red Sun’ planted in the glass greenhouse located in Environmental
Horticulture Research Institute of Guangdong Academy of Agricultural Sciences was used as the
research material. According to the change pattern of leaf color, red leaf samples of three independent
plants (RL1, RL2 and RL3) were taken on 15 January 2019, yellow leaf samples of three independent
plants (YL1, YL2 and YL3) were taken on 25 February 2019 and green leaf samples of three independent
plants (GL1, GL2 and GL3) were taken on 21 March 2019. The samples were quickly fixed with liquid
nitrogen and stored at −80 ◦ C.
4.2. Pigments Content Measurement

The contents of total chlorophyll and carotenoids were determined by spectrophotometric analyses.
About 0.1 g leaves (accurately record the weight) were cut into pieces and rinsed with distilled water.
Add 1 mL extracting solution (95% ethanol) and 50 mg calcium carbonate powder in mortar, the
mixtures were grinded fully in low light conditions and transferred to 10 mL glass test tube. The mortar
was rinsed with the extracting solution, all the washing solution was transferred into the glass tube
and replenished to 10 mL with the extracting solution. Keep the glass tube in the dark until the tissue
is completely whitened. Add 200 μL leach liquor in a 96-well plate, the automatic microplate reader
(Sunrise, TECAN, Switzerland) was set to zero by extracting solution, then the absorbance values of
663 nm, 645 nm and 470 nm were determined and recorded as A663, A645 and A470, respectively.
The calculation formula is as follows: total chlorophyll content (mg/g) = 0.02 × (20.21 × A6458.02 ×
A663) × N/M, total carotenoid concentration (mg/g) = 0.02 × [(1000A470–3.27Ca–104Cb)/229] × N/M
(N represents dilution multiple, M represents sample fresh quantity).
The content of total anthocyanins was determined by colorimetry method. The sample was dried
in the oven (37 ◦ C) to a constant weight and crushed. After passing through a 40-mesh sieve, about 0.1
g sample was weighed (accurately recording the weight). Add 1 mL extracting solution (95% ethanol:
1.5 mol/L HCL = 85:15) in samples and the mixtures were extracted at 4 ◦ C for 24 h. Then the mixtures
were centrifuged at 8000× g for 10 min at room temperature, and the supernatant was taken to be tested.
The automatic microplate reader (Sunrise, Switzerland) was preheated more than 30min, and reagent
A (PH 1.0 buffer, 0.2mol/L KCL : 0.2mol HCL = 25:67) and reagent B (PH 4.5 buffer, 1 mol NaAC : 1 mol
HCL: H2 O = 100:60:90) were preheated more than 10 min. Add 180 μL reagent A to 20 μL supernatant
and let stand for 15 min, then the absorbance values at 530 nm and 700 nm were determined and were
recorded as A1 and A2, respectively. Add 180 μL reagent B to 20 μL supernatant and let stand for 15
min, then the absorbance values at 530 nm and 700 nm were determined and were recorded as A3 and
A4, respectively. The calculation formula is as follows: total anthocyanin concentration (μg/g) = 33.4 ×
[(A1 − A2) − (A3 − A4)] × N/M (N represents dilution multiple; M represents sample dry weight, g).
The content of total flavonoids was determined by colorimetry method. The sample was dried
in the oven (37 ◦ C) to a constant weight and crushed. After passing through a 40-mesh sieve, about
0.1 g sample was weighed (accurately recording the weight). Add 1 mL extracting solution (60%
ethanol), the mixtures A were extracted by ultrasonic method (ultrasonic power: 300 W, crushing time:
5 s, interval time: 8 s) at 60 ◦ C for 30 min. Then, the mixtures were 12,000 rpm for 10 min at room
temperature, and the supernatant A was replenished to 1 mL by extracting solution and taken to be
test. The automatic microplate reader (Sunrise, Switzerland) was preheated more than 30 min, set to
470 nm and zero by distilled water. Reagent A (5% nitrous acid), reagent B (10% aluminum nitrate),
reagent C (4% sodium hydroxide solution) and standard (10 mg/mL tannic acid standard solution)
were prepared. The supernatant was treated according to the instruction manual of the kit for the
47
Int. J. Mol. Sci. 2020, 21, 1869
determination of total flavonoids in plants (Figure S6). The mixtures B was mixed well and placed
in a water bath at 37 ◦ C for 45 min. Then the mixtures B was centrifuged at 10,000× g for 10 min at
room temperature, and 200 uL supernatant B was used to determine absorbance values at A470 in
a 96-well plate. The calculation formula is as follows: the flavonoids concentration (mg/g) = C × N
× V/M (C represents the content of flavonoids observed in the standard curve, mg/mL; N represents
dilution multiple; V represents the total volume of extracting solution, ml; M represents the dry weight
of sample, g).
Flavonoids-targeted metabolomics analysis was performed by liquid chromatography-mass
spectrometry (LC-MS) at Metware Biotechnology Co.,Ltd (Wuhan, China) as described by [37], with
small modifications. Metabolomics analysis includes two parts: metabolomics experiment and
data analysis, based on the metabolite data obtained from experimental design, sample collection
and processing, metabolite extraction and metabolite detection and analysis. It can carry out the
identification of metabolites and the quality control analysis of sample data, and screen out some
differential metabolites, so as to predict and analyze the related functions of the metabolites of
the samples.
4.3. Analysis of Flavonoids-Targeted Metabolomics
4.3.1. Standards and Reagents

The standard (analytical purity) was purchased in BioBioPha (Kunming, China) or Sigma-Aldrich
(St Louis, MO, USA), and dissolved in dimethyl sulfoxide (DMSO) or methanol as solvent and
stored at −20 ◦ C. Before mass spectrometry analysis, 70% methanol was diluted to different gradient
concentrations. Methanol, acetonitrile and ethanol were all analytically pure and purchased in Merck
(Darmstadt, Germany).
4.3.2. Sample Extraction Process

The leaves of C. sinense ‘Red Sun’ were vacuum freeze-dried and ground with a grinder (30 Hz for
1.5 min) to powder. The 0.1 g powder was dissolved in 70% methanol aqueous solution. The dissolved
sample was swirled three times at 4 ◦ C overnight to improve the extraction rate. After centrifugation
(rotating speed 10,000× g, 10min), the supernatant was filtered with a microporous membrane (0.22 μ
m pore size). The sample was stored in a sample injection bottle for UPLC-MS/MS analysis.
4.3.3. Acquisition Conditions of LC-MS

The data acquisition instrument system mainly includes Ultra Performance Liquid
Chromatography (UPLC) (Shim-pack UFLC SHIMADZU CBM30A, http://www.shimadzu.com.cn/) and
Tandem mass spectrometry (MS/MS) (Applied Biosystems 4500 QTRAP, http://www.appliedbiosystems.
com.cn/).
UPLC was run under following conditions: (1) Waters ACQUITY UPLC HSS T3 C18 1.8 μm,
2.1 mm × 100 mm; (2) The aqueous phase was ultra-pure water (0.04% acetic acid is added) and the
organic phase was acetonitrile (adding 0.04% acetic acid); (3) Elution gradients: 95:5 v/v at 0 min, 5:95
v/v at 11.0 min, 12 min, 12.1 min and 15 min; (4) The flow rate was 0.4 mL/min, the column temperature
was 40 ◦ C, and the injection volume was 5 μL. MS/MS worked with these conditions: (1) Curtain gas
(CUR) was set to 25 psi; (2) Electrospray ionization (ESI) was set to 550 ◦ C; (3) the MS voltage was
set to 5500V; (4) Dclusteringotential (DP) was optimized; (5) Collision energy (CE) was optimized; (6)
Collision-activated dissociation (CAD) was set to high.
4.3.4. Data Evaluation

Based on the self-built database MWDB (metware database) and the public database of metabolite
information, the primary and secondary spectral data of mass spectrometry were qualitatively analyzed
by software Analyst 1.6.3. For the qualitative analysis of some substances, the interference from isotope
48
Int. J. Mol. Sci. 2020, 21, 1869
signals are removed, including duplicate signals of K+ , Na+ and NH4 + , as well as duplicate signals of
fragment ions which were derived from other large molecules. The structure analysis of metabolites
refers to the existing mass spectrometry public databases such as MassBank (http://www.massbank.jp/),
KNAPSAcK (http://kanaya.naist.jp/KNApSAcK/), HMDB (http://www.hmdb.ca/) (Wishartetal.2013),
MoToDB (http://www.ab.wur.nl/moto/) and METLIN (http://metlin.scripps.edu/index.php) [38].
The quantification of metabolites was carried out by using multiple reaction monitoring (MRM)
mode. In the MRM model, the quadrupole first selected the precursor ions (parent ions) of the
target substance. While screening the corresponding ions of other molecular weight substances
to initially eliminate the interference, the precursor ions were ionized by the collision chamber to
form a lot of fragment ions. Then, the fragment ions were filtered through the triple quadrupole
to select a characteristic fragment ion, which eliminated the interference of non-target ions, making
the quantitative inference more accurate and the better repeatability. After the metabolic substance
spectrum analysis data of different samples were obtained, the mass spectrometry peaks of all
substances were integrated, and the mass spectrometry peaks of the same metabolite in different
samples were integrated and corrected [39].
4.3.5. Data Analysis

The original data obtained were preprocessed at first (noise filtering, peak matching and peak
extraction) and the data were corrected [40]. Then, the data of quality control entered the stage of
statistical analysis.
Statistical analysis used multivariate analysis. Data were log-transformed and mean-centred
using SIMCA software (V14.1, MKS Data Analytics Solutions, Umea, Sweden) for PCA and OPLS-DA
analysis. PCA analysis was followed by automated modelling analysis [41,42]. The first principal
component (PC1) was subjected to OPLS-DA modelling, and the model quality was tested by 7-fold
cross validation. After that, the resulting R2Y (the interpretability of the model on the categorical
variable Y) and Q2 (the predictability of the model) were used to evaluate the validity of the model.
The permutation test was performed multiple times to generate different random Q2 values, which
were used to further test model validity [43].
R software (version 3.0.3) was used for Hierarchical clustering analysis (HCA) analysis. The
data were log 2 transformed and similarity assessment for clustering was based on the Euclidean
distance coefficient.
Based on OPLS-DA analysis, the differential metabolites were screened by the following criteria:
(1) If the difference of metabolites content between the control group and the experimental group is
more than 2 times or less than 0.5, the difference is considered to be significant; (2) On the basis of the
above, the metabolites with VIP ≥ 1 are selected.
Finally, the Kyoto Encyclopaedia of Genes and Genomes (KEGG) Pathway database (http:
//www.kegg.jp/kegg/pathway.html) [44] and MW-database (Metware Biotechnology Co., Ltd, Wuhan,
China) were centred on metabolic reactions and concatenates possible metabolic pathways.
4.4. Enzyme Activity Determination

The enzyme activities of 10 enzymes were determined by enzyme-linked immunosorbent assay
(ELISA). Leaves were ground to powder in liquid nitrogen and accurately weighed 0.1 g sample in the
centrifuge tube. Then, added the same volume of 0.1 mol/L precooled PBS solution, centrifuged about
3000 rpm/min at low-temperature and low-speed for 10 min. 1 mL of the supernatant was collectedn
to test. The ELISA detection kit manual of the corresponding enzyme (ProNets Biotechnology Co.,
Ltd., Wuhan, China) was used to determine the enzyme.
4.5. Total RNA Extraction and qRT-PCR Analysis

The total RNA of C. sinense ‘Red Sun’ was extracted by RNA extraction kit (TIANGEN, Biotech,
Beijing, China). The content of RNA in three samples was determined by nanodrop2000 (Thermo
49
Int. J. Mol. Sci. 2020, 21, 1869
Fisher, Waltham, MA, USA). 500 ng RNA was taken from each sample for reverse transcription by
HiScript Q RT SuperMix for qPCR kit (Vazyme Biotech Co., Nanjing, China) to obtain cDNA. cDNA
dilution of 10-folds was used for qRT-PCRanalysis.
QRT-PCR used CFX96TM Real-Time System (Bio-Rad, Hercules, CA, USA) following the
instructions based on ChamQ SYBR qPCR Master Mix kit (Vazyme Biotech Co., Nanjing, China).
CsACTIN was used as normalization standard for gene expression. The gene expression was calculated
by 2−ΔΔCT . The primers for qRT-PCR are listed in Table S5.
5. Conclusions
In this study, a LC-MS-based metabolomics approach was used to evaluate the difference in
metabolites during the change of leaf color of C. sinense ‘Red Sun’. This is the first metabolomics study
on C. sinense. A total of 196 flavonoid-related metabolites were detected. 42 metabolites were identified
as differential metabolites during the process of leaf color change. In anthocyanin biosynthetic pathway,
15 metabolites were identified and the contents of them all showed decrease. Especially the contents
of Naringenin chalcone, an important intermediate in anthocyanin synthesis, and two important
anthocyanins metabolites, Pelargonidin O-acetylhexoside and Anthocyanin 3-O-beta-D-glucoside,
decreased to zero in the green leaf stage. The enzyme activity of 10 enzymes related to anthocyanin
synthesis showed different change patterns, while the expression of corresponding encoding genes
was all down-regulated in the process of leaf color change. Overall, this study substantially contributes
to the knowledge flavonoid-related metabolites composition in C. sinense and provides important
reference values for breeders to improve the leaf color of C. sinense.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/5/1869/

s1.
Author Contributions: Conceptualization, F.Y. and G.Z.; methodology, J.G. and R.R.; software, Y.W.; validation, J.J.
and J.W.; formal analysis, J.G.; resources, C.L. and C.Z.; writing—original draft preparation, J.G.; writing—review
and editing, S.A. and R.R. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by grants from National Key Technologies R & D Program 2018YFD1000401,
the National Natural Science Foundation of China (31902065), the Natural Science Foundation of Guangdong
province (2017A030312004), the Orchid Industry Technology Innovation Alliance (2019KJ121), R & D plan in
key areas of Guangdong Province (2018B020202001), Foundation of President of the GuangDong Academy of
Agricultural Sciences (2019B030316033), Guangzhou Key projects of scientific research plan (201904020026) and
team construction project of Guangdong Academy of Agricultural Sciences (201612TD), Discipline team building
projects of Guangdong Academy of Agricultural Sciences in the 13th Five-Year Period (201612TD) and Special
fund for scientific innovation strategy-construction of high level Academy of Agriculture Science(R2016PY-QF015
and R2018QD-103).
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
Abbreviations
PAL Phenylalanine ammonialyase
C4H Cinnamate 4-hydroxylase
4CL 4-coumarateCoA ligase
CHS Chalcone synthase
CHI Chalcone isomerase
F3H Flavonoid 3-hydroxylase
F3 H Flavonoid 3’-hydroxylase
F3 5 H Flavonoid 3’,5’-hydroxylase
DFR Dihydroflavonolreductase
ANS Anthocyanidin synthase
UFGT anthocyanidin 3-O-glucosyltransferase
DHK Dihydrokaempferol
DHQ Dihydroquercetin
DHM Dihydromyricetin
qRT-PCR Quantitative real-time PCR
50
Int. J. Mol. Sci. 2020, 21, 1869
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Molecular Sciences
Review
Volatile Organic Compounds from Orchids: From
Synthesis and Function to Gene Regulation
Mummadireddy Ramya 1 , Seonghoe Jang 2 , Hye-Ryun An 1 , Su-Young Lee 1 , Pil-Man Park 1
and Pue Hee Park 1,3, *
1 Floriculture Research Division, National Institute of Horticultural and Herbal Science, RDA, Wanju-gun,
Jellabuk-do 55365, Korea; [email protected] (M.R.); [email protected] (H.-R.A.);
[email protected] (S.-Y.L.); [email protected] (P.-M.P.)
2 World Vegetable Center Korea Office (WKO), Wanju-gun, Jellabuk-do 55365, Korea;
[email protected]
3 Department of Horticultural Science and Biotechnology, Seoul National University (SNU),
Seoul 08826, Korea
* Correspondence: [email protected] or [email protected]; Tel.: +82-10-4507-8321 or +82-63-238-6842;
Fax: +82-63-238-6805

Received: 31 December 2019; Accepted: 7 February 2020; Published: 10 February 2020
Abstract: Orchids are one of the most significant plants that have ecologically adapted to every
habitat on earth. Orchids show a high level of variation in their floral morphologies, which makes
them popular as ornamental plants in the global market. Floral scent and color are key traits for
many floricultural crops. Volatile organic compounds (VOCs) play vital roles in pollinator attraction,
defense, and interaction with the environment. Recent progress in omics technology has led to
the isolation of genes encoding candidate enzymes responsible for the biosynthesis and regulatory
circuits of plant VOCs. Uncovering the biosynthetic pathways and regulatory mechanisms underlying
the production of floral scents is necessary not only for a better understanding of the function of
relevant genes but also for the generation of new cultivars with desirable traits through molecular
breeding approaches. However, little is known about the pathways responsible for floral scents
in orchids because of their long life cycle as well as the complex and large genome; only partial
terpenoid pathways have been reported in orchids. Here, we review the biosynthesis and regulation
of floral volatile compounds in orchids. In particular, we focused on the genes responsible for volatile
compounds in various tissues and developmental stages in Cymbidium orchids. We also described
the emission of orchid floral volatiles and their function in pollination ecology. Taken together, this
review will provide a broad scope for the study of orchid floral scents.
Keywords: Cymbidium; floral scents; Orchidaceae; pollination; volatile organic compounds
1. Introduction
The Orchidaceae family is one of the largest and widely diverse families of flowering plants,
with more than 28,000 accepted species spanning 763 genera [1]. These plants are absent only in
polar and desert regions, but are particularly abundant in the wet tropics worldwide [2]. However,
a majority of orchids are distributed locally and generally rare [3]. Associated with the massive
number of species in Orchidaceae, orchids display extraordinary floral diversification and represent
a highly advanced and terminal line of floral evolution in the monocotyledons. As fascinating and
highly popular plants, orchids are valued because of their exquisite flowers and long floral lifespan.
These plants consist of great diversity in floral form, size, color, fragrance, and texture. A specific
interaction between a pollinator and orchid flower may be one of the factors that promote orchid
species richness [4]. The Orchidaceae family can be categorized into four subfamilies (Cypripedioideae,

Int. J. Mol. Sci. 2020, 21, 1160
Epidendroideae, Orchidoideae, and Vanilloideae) [5] and comprises a considerable diversity in life
forms, with approximately 30% of species being terrestrial and mainly growing as epiphytes and
lithophytes [6]. Furthermore, commercial production of orchids has greatly expanded and become a
very profitable industry. Dominant species, such as those of Cymbidium, Paphiopedilum, and Phalaenopsis,
are cultivated based on consumer flower preferences [7].
Orchids have complex life histories and diversified adaptation strategies; consequently, researchers
have focused on orchid flower development and orchid pollination interactions. Flower color and
scent are main traits for many floricultural crops. Floral scents emit various types of volatile organic
compounds (VOCs). Orchids currently account for a prominent share of the world’s flower trade, with
annual sales of more than $4 billion (USD). It is widely used in perfumes, cosmetics, florivores, and
medicinal applications. Some are also used as food and traditional medicines in many countries [8].
For example, dried vanilla seed pods (especially Vanilla planifolia) are commercially important as a
flavoring used in baking, as well as for perfume manufacturing and aromatherapy [9]. Gastrodia elata
is one of three orchids listed in the earliest known Chinese Materia Medica, and is used for treating
headaches, dizziness, tetanus, and epilepsy [10]. However, because of its economic value in floral and
pharmaceutical industries, G. elata has suffered great losses in habitat, resulting in a rare species [11,12].
Flower color and volatile compounds are key characteristics for many floricultural crops. Synthesis
of VOCs occur in all plant organs, including roots, stems, leaves, seeds, fruits, as well as flowers, which
are reported to emit the highest amounts and diversity of VOCs [13,14]. To date, more than 1700 floral
VOCs have been identified in around 1000 seed plants [14]. In general, VOCs formed in other organs,
apart from flowers, are involved in defense mechanisms. Although floral volatiles play a crucial role
in reproductive process by attracting pollinators, they also have other adaptive roles [15,16], such
as repellents [17–19] and physiological protectors against abiotic stresses [14,20]. In addition, floral
volatiles are widely used as components of perfumes, cosmetics, flavorings, and even for therapeutic
applications. Together with floral color, volatiles emitted by flowers represent key floral signals used by
insects to detect and select rewarding flower species [21,22]. Floral scents emit different types of VOCs.
VOCs are generally lipophilic and have low molecular weights and high melting points. Based on
their origin, function, and biosynthesis, floral scents are grouped into three major clusters: terpenoids,
phenylpropanoids, and fatty acid derivatives. Floral volatiles with terpene synthases (TPSs) have been
identified in orchids [23,24].
Various species with large genomes are observed in monocots, such as species in Alliaceae,
Asparagaceae, Liliaceae, Melanthiaceae, and Orchidaceae [25]. Among these, Orchidaceae, with
genome sizes in a 168-fold range (1C = 0.33–55.4 pg), are perhaps the most diverse angiosperm
families [25]. Epidendroideae, in Orchidaceae, contain variable genome sizes, with genome sizes in
a range of over 60-fold (1C = 0.3–19.8 pg). Orchidoideae, with the largest descending/offspringing
from species in subtribe Orchidinae, are pictured by a more restricted range of genomes (1C = 2.9–16.4
pg). Cypripedioideae show genome sizes in only a 10-fold range (1C = 4.1–43.1 pg). Cypripedioideae
contain the largest mean genome size (1C = 25.8 pg) among all the subfamilies. Some species in
Vanilloideae have been estimated, ranging from 1C = 7.3 to 55.4 pg. Pogonia ophioglossoides presents
the largest genome size (1C = 55.4 pg) in this family [25]. Apostasioideae, the primitive subfamilies,
contain calculated 1C-values ranging from 0.38 to 5.96 pg in a close to 16-fold range [26].
Orchids are one of the most diversified angiosperms and have mesmerized botanists for centuries.
For orchids, floral color, shape, and fragrance are primary key determinants of consumer preferences.
Many floricultural crops have lost their scents, following traditional breeding. However, only a few
genomic resources are available for these non-model plants. Despite its economic as well as biological
importance, metabolic engineering approaches on floral scents are still at the stage of infancy in orchids.
In this review, we give an overview of orchid floral volatiles with a focus on Cymbidium orchids; we
review their importance in pollination ecology, genes encoding enzymes and transcription factors
(TFs) responsible for the biosynthesis, and the regulation of orchid floral volatiles. We hope that our
information will provide guidance for future studies on orchid floral scents.
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2. Orchid Volatile Compounds and Biosynthetic Pathways

Plant volatile compounds are a complex mixture of low molecular weight lipophilic molecules
that have low melting points [27]. Biosynthesis of VOCs depends on the availability of carbon,
nitrogen, and sulfur together with energy provided from the primary metabolism. Flower color
and volatile compounds are key characteristics in many floricultural crops. Depending on their
origin and functions, floral volatile compounds are categorized into one of three groups: terpenoids,
phenylpropanoids/benzenoids, and fatty acid derivatives [20] (Figure 1).
Figure 1. Floral volatile compound responsible pathways in orchid flowers. Major orchid
floral volatile compounds are highlighted in colors (sesquiterpenes [28], monoterpenes [23,24],
phenylpropanoids/benzenoids [29,30] and fatty acid derivatives/methyl jasmonate [19,31]).
Abbreviations: MVA: mevalonic acid; MEP: methyl erythritol phosphate; LOX: lipoxygenase; PEP:
phosphoenolpyruvate; G3P: glyceraldehyde-3-phosphate; E4P: erythrose 4-phosphate; DMAPP:
dimethylallyl pyrophosphate; FPPS: farnesyl pyrophosphate synthase; FPP: farnesyl pyrophosphate;
GGPP, geranylgeranyl pyrophosphate; GPP, geranyl pyrophosphate; IPP: isopentenyl pyrophosphate;
DAHP: 3-deoxy-D-arabinoheptulosonate-7phosphate; Phe: phenylalanine.
2.1. Terpenoids
Terpenoids, or terpenes, represent the largest group of plant floral volatiles [27]. They play key
roles in attracting pollinators for successful reproduction [32,33] and in defense against pathogens
and florivores [34,35]. Moreover, from their natural roles, terpenoids are widely used in the cosmetic
and perfume industries and as food additives because of their distinctive aromas and flavors [36,37].
Studies on floral scents have mainly focused on isolation and characterization of terpene synthase (TPS)
genes encoding the key enzymes responsible for the synthesis of terpenes. All terpenoids are produced
from isopentenyl diphosphate (IPP) and dimethyl allyl diphosphate (DMAPP), which are five-carbon
(C5) precursors [38]. IPP and DMAPP are derived from two alternative biosynthetic pathways localized
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in different cellular compartments. The classical mevalonic-acid (MVA) pathway, which is localized
in the cytosol, gives rise to IPP from three molecules of acetyl-CoA. In contrast, the methylerythritol
phosphate (MEP) pathway takes place in plastids and produces IPP from pyruvate and glyceraldehyde
3-phosphate. In plants, monoterpenes, diterpenes, carotenoids, ubiquinones, and phytols are produced
in the plastid via the MEP pathway, while all other plant terpenoids (sesquiterpenes, triterpenes, and
polyterpenes) are produced using the MVA pathway. Floral volatiles with TPSs have been identified in
such orchids as P. bellina [24] and C. goeringii [28].
Terpenoids are dominant in floral volatiles including those emitted by orchids. Geraniol, linalool,
and their derivatives are major compounds of scented P. bellina flowers. Monoterpenes (Table 1) play
a key role in the volatile profile [23,24]; in C. goeringii, floral volatile organic compounds include
farnesol, methyl epi-jasmonate, (E)-β-farnesene, and nerolidol. Sesquiterpenes play a key role in the
scent profile [28]. In the Cymbidium hybrid “Sunny Bell,” linalool is the major compound found in the
petal [39]. The volatile floral scents inside species and cultivars of Cymbidium have been reported [28,39].
Among the volatiles α-pinene, eucalyptol, trans-β-ocimene, α-copaene, and β-caryophyllene terpenoid
were leading components in the volatile mixture [40,41]. The Vanda Mimi Palmer flower mainly
contains cimene, linalool oxide, and linalool, which are classified as monoterpenes [42]. In addition,
nerolidol is a sesquiterpene [40,41]. Compared with model plants, there are few reports on floral scent
terpenoids in orchids.
Table 1. Major volatile organic components in orchid flowers.
Compound Structure Species Reference

Terpenoids
P. bellina [23]
Linalool
C. cv. Sunny Bell [39]
Geraniol P. bellina [24]
Ocimene Vanda Mimi Palmer [42]
Farnesol C. goeringii [28]
β-Caryophyllene M. tenufolia [43]
Phenylproponoids/Benzenoids
Eugenol Gymnadenia Species [30]
2-methyl butanal C. cv. Sael Bit [29]
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Table 1. Cont.
Compound Structure Species Reference
Benzyl acetate Vanda Mimi Palmer [42]
Fatty Acid Derivatives
C. ensifolium [31]
Methyl jasmonate
C. faberi [44]
2.2. Phenylproponoids and Benzenoids

Phenylpropanoids and benzenoids are the second most dominant group of volatile compounds [14].
Phenylpropanoids/benzenoids are produced from the aromatic amino acid phenylalanine, which is
produced in plastids via the shikimate pathway and arogenate pathway (Figure 1) through seven
and three enzymatic steps [20]. Phenylalanine ammonia lyase (PAL) catalyzes the conversion of
phenylalanine to trans-cinnamic acid, as a step in the phenylpropanoid pathway of plants [45]. The
conversion of cinnamic acid to phenylpropanoids/benzenoids is followed by a shortening of the propyl
chain via either the β-oxidative pathway or the non-β-oxidative pathway [45]. Recently, it was reported
that the β-oxidative pathway for the formation of benzoic acid (BA) and benzenoids contributes to the
production of volatile benzenoids in petunia flowers [46–49]. Moreover, in the fatty acid metabolism
starting from cinnamic acid activation to its CoA thioester, hydration, oxidation, and thiolysis occurs
in the peroxisome.
The formation of benzenoids (C6-C1) from cinnamic acid requires a shortening of the propyl side
chain by two carbons and has been shown to proceed via a β-oxidative pathway, a non-β-oxidative
pathway, or a combination of these pathways. Benzaldehyde formation, through the non-β-oxidative
pathway, is oxidized by NAD+-dependent benzaldehyde dehydrogenase to benzoic acid, which is
isolated from snapdragon flowers [50]. However, the formation of benzaldehyde in the enzymatic
steps of the non-β-oxidative pathway remains unknown. Furthermore, floral phenylpropanoid and
benzenoid compounds play a crucial role in scent production via two super families, SABATH methyl
transferases and BAHD acyltransferases, in several plants [20]. In contrast, the formation of floral
volatile phenyl propenes such as eugenol and isoeugenol starts with lignin and takes two enzymatic
steps; the oxygen functionality at the C9 position is removed, and coniferyl alcohol is produced [20].
Finally, eugenol and isoeugenol are formed through the conversion of coniferyl acetate. From the
Cymbidium cultivar Sael Bit, benzenes were reported in the full blooming stage [29]. Floral volatiles
such as phenylpropanoids and benzenoids are also emitted from scentless flowers of P. equestris. In
addition, Gymnadenia species quantitatively and qualitatively release diverse blends of nearly 50
volatile compounds and attract different suits of pollinators. Eugenol and benzyl acetate are two
predominant compounds among the scents of these species [51].
2.3. Fatty Acid Derivatives

Among the floral volatile compounds, fatty acid derivatives are the smallest group of volatiles.
They mainly consist of floral fatty acids synthesized from C18 polyunsaturated fatty acids and linolenic
and linoleic acids. Methyl jasmonate is an important volatile fatty acid compound found in orchids. The
biosynthetic process of fatty acid or its derivatives begins from the stereo-specific oxygenation catalyzed
by lipoxygenases (LOXs) to produce 9-hydroxy and 13-hydroperoxy intermediates. These intermediates
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can enter two different batches of the LOX pathway to produce volatile compounds. Allene oxide
synthase (AOS) catalyzes the first step in the biosynthesis of jasmonic acid from lipoxygenase-derived
hydroperoxides of free fatty acids. In addition, the AOS pathway generates the C6 and C9 aldehydes
through condensation of hydroperoxide derivatives by hydroperoxide lyase (HPLS). Limited data
are available regarding the synthesis/pathways of fatty acids and/or their derivatives in flowers. In
Antirrhinum majus flowers, 20 fatty acid derivatives have been identified [52]. Furthermore, methyl
jasmonate and jasmonic acid involved in the floral scent pathway in C. ensifolium and C. faberi have been
identified. Various volatile fatty acids were also found synthesized in the orchid genus Ophrys; among
them, alkenes have an important function in attracting pollinators [17]. Two genes encoding stearoyl-acyl
carrier protein desaturase (SAD) isoforms, SAD1 and SAD2, were reported to be flower-specific, and
these genes broadly parallel alkene production during flower development of Ophrys sphegodes and O.
exalanta; in particular, SAD2 showed a tight association with alkene production [19]. Further study is
required to better understand the floral scent pathways in orchids.
3. Transcriptional Factors in Floral Volatile Regulation

Transcription factors (TFs) are sequence-specific DNA-binding proteins that interact with the
regulatory regions of the target genes and modulate the transcription initiation rate by RNA
polymerases [53]. Although several types of transcription factors have been reported to be involved
in the biosynthesis of volatile compounds and secondary metabolites in plants, a limited number
of TFs involved in the formation of molecules responsible for floral scent have been identified.
Recently, a growing number of research results have reported that several types of TFs, including basic
helix-loop-helix (bHLH), basic leucine zipper (bZIP), ethylene response factor (ERF), NAC, MYB, and
WRKY family members, are involved in regulation for terpene biosynthesis [53].
TFs play a key role in controlling the expression level of genes involved in various developmental
processes and physiological pathways, including plant secondary metabolism [54]. ODORANT1
(ODO1) is the first MYB TF identified in flowers. It is a member of the R2R3-MYB TF family, which
regulates genes involved in floral scent production through the shikimate and phenylpropanoid
pathways [43]. Terpene biosynthesis is regulated by genes encoding required enzymes and their
regulators. Phalaenopsis TFs regulate the terpenoid pathway; PbbHLH4 regulates the geranyl diphosphate
synthase (GDPS) gene for the synthesis of monoterpenoids in P. bellina [55]. Moreover, a higher
expression of five genes encoding TFs (PbbHLH4, PbbHLH6, PbbZIP4, PbERF1, and PbNAC1) was
reported in the scented orchid. Especially, 10-fold higher levels of α-terpineol (a monoterpenoid)
were detected in PbbZIP4-overexpressing flowers of P. aphrodite compared to the control [56]. HY5, an
bZIP TF, is known to play a critical role in mastering both the light and circadian signaling pathway.
Identification of HY5-interacting motifs on the upstream regulatory fragments of PbNAC1 implies that
the light and circadian clock signals are likely to manage monoterpene biosynthesis in P. bellina [56].
Transcriptomic analyses of C. goeringii flowers led to identification of 1179 genes that were
clustered into 64 groups encoding putative TFs with the three largest being bHLH (73 members), ERF
(71 members), and C2H2 zinc finger proteins (65 members). CgbHLH1 and CgbZIP3 are homologs of
AabHLH1 and AabZIP1, respectively. CgbZIP7 is a homolog of PbbZIP4, which regulates monoterpene
biosynthesis in P. bellina (Table 2) [57], while CgERF2 is a homolog of CitAP2.10, which is associated
with sesquiterpene (+)-valencene synthesis in sweet orange [34]. CgNAC5, a homolog of AaNAC4,
controls monoterpene synthesis in kiwifruit [35], and CgWRKY1 and CgWRKY2, which are homologs of
GaWRKY1, regulate sesquiterpene (+)-δ-cadinene synthesis in cotton [36]. Cymbidium Sael Bit MYB1
expression is detected at various flower developmental stages and is highest in petals and columns of the
fully open flower. Cymbidium Sael Bit MYB1 is regarded as a regulator of phenylpropanoid/benzenoid
genes in floral scent profiles. The key component of flower scent in C. faberi is MeJA, which is regulated
by the crosstalk of many plant hormones. A total of 379 TFs identified as belonging to 37 TF families
are differentially expressed between blooming and withered flowers of C. faberi [58], and the top 10
groups belong to the MYB, AP2-EREBP, bHLH, NAC, GRAS, C2H2, C2C2-Dof, MADS, WRKY, and
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ABI3VP1 families. Furthermore, an increase in the levels of floral volatiles in tissues resulted from a
large increase of various transcription factors in orchids.
Table 2. Representative genes responsible for floral scents in orchids.
Floral Scent Gene Metabolism Pathway Species Reference

Genes
PbGDS Terpenoid pathway Phalaenopsis bellina [24]
VMPAAT Terpenoid pathway Vanda species [59]
VMDXS Terpenoid pathway Vanda Mimi Palmer [42]
GdEGS Benzenoid pathway Gymnadenia species [30]
OsSAD1 Benzenoid pathway Ophrys sphegodes [19]
Transcription Factors (TFs)
CsMYB1 Phenylprponid/benzenoid Cymbidium cv. Sael Bit [29]
PbbZIP4 Monoterpene pathway Phalaenopsis aphrodite [60]
PbBHLH2 Monoterpene pathway Phalaenopsis bellina [23]
4. Spatial and Temporal Emission of Volatile Organic Compounds

Scent is an important property of flowers and plays a vital role in the ecological, economic, and
aesthetic properties of flowering plants. Each plant possesses a distinct and unique floral scent. Floral
scent is composed of all the VOCs, including terpenoids, phenylpropanoids, benzenoids, fatty-acids,
and their derivatives, which are emitted by floral tissues (Table 2). In Phalaenopsis bellina, expression
analysis of the PbGDS (geranyl diphosphate synthase) gene encoding a homodimeric GDS showed that
its expression is flower-specific and that maximal expression is concomitant with maximal emission
of monoterpenes on Day 5 post-anthesis [23]. In P. abies, expression of PaIDS1 (isoprenyl diphosphate
synthase 1) encoding a bifunctional GDS and GERANYLGERANYL DIPHOSPHATE SYNTHASE
(GGDS) exhibits a peak in wood where oleoresin, comprising monoterpenes and diterpenes, is
accumulated [24].
Plants emit a large variety of VOCs that are actively involved in plant growth and protection. VOCs
are defined as any organic compound with vapor pressures high enough under normal conditions
to be vaporized into the atmosphere [53]. VOC emissions are strongly dependent on environmental
conditions and developmental stages of the plant tissue. In plants, emission of VOCs is spatiotemporally
regulated; a majority of VOCs are emitted from flowers compared to other plant tissues/organs, and
the level of emission increases when the floral bud is close to opening and decreases as it moves to the
senescence stage [61,62].
In Vanda Mimi Palmer orchids, various types of sesquiterpenes and benzenoids were highly
expressed at the full blooming stage with the expression of floral scent genes [42,59,63]. Floral volatile
emission increased bud to flowering stages in Cymbidium goeringii. Different types of Maxillaria orchids
emit strong vanilla or coffee-like scents, which are responsible for pollinator attraction [64]. M. tenuifolia
Lindl is called a “coconut orchid” due to its strong coconut-like scent, and was recognized as the
best scented orchid in the 18th World Orchid Conference [65]. The sepal is a source of floral volatiles
in Maxillaria species, and the highest level of floral volatiles separated through electronic nose and
GC-MS analyses was detected at the initial flowering stage [43,66]. In addition, methyl jasmonate
(mJA) emission predominantly occurs in sepals and floral parts of C. ensifolium [31]. Phalaenopsis
is undoubtedly the most widely grown orchid in the world, and in P. bellina, various types of
monoterpenes are produced in the full flowering stage [23,24]. Furthermore, in a comparison of
fragrant and non-fragrant Phalaenopsis flowers, terpene compounds were found to be much more
abundant with increased levels of relevant gene expression in flowers of fragrant orchids [67].
Developmental regulation of scent emission occurs at several levels, including orchestrated
expression of scent biosynthetic genes [68], enzyme activities, and substrate availability [69]. Based
on an evolutionary study of floral scent genes in three closely related orchid species of the genus
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Gymnadenia, it is likely that the switch from the production of one to two scent compounds evolved
under relaxed purifying selection [51]. Two major volatile compounds, α-copaene and β-caryophyllene,
have been identified in all floral organs of M. tenuifolia, with the highest levels in the petal. α-copaene
and β-caryophyllene were found to be emitted in all flower developmental stages except the floral bud
stage I [43]. In fact, volatile compounds of M. tenuifolia include α-copaene, β-caryophyllene, 1,8-cineole,
limonene, β-myrcene, α-pinene, β-pinene, sabinene, and δ-decalactone, which is responsible for the
typical coconut aroma. The majority of studies on Maxillaria fragrance reported only the chemical
composition of the floral scent; however, little data are available on the spatiotemporal emission of
the floral volatiles. In addition, sulfur- and nitrogen-containing volatile compounds contribute to
the attraction of pollinators to flowers by mimicking food or brood sources such as carrion or dung.
Besides the importance of floral scents in plant ecology, identification and functional validation of
relevant genes responsible for biosynthetic and/or regulatory pathways of floral volatiles are required
for a better understanding of floral scent production and for the development of novel cultivars with
desirable characteristics. Transcriptomic and metabolic analyses together with genetic engineering
approaches will be of great help in driving towards this goal.
5. Gene Evolution for VOCs

The evolution of orchids has resulted in an immense diversity of flower traits such as color and
scent. Orchidaceae consist of extraordinary adaptations that may have guaranteed its evolutionary
success. To date, most of the examined plant gene families originated through gene duplication. Gene
duplication plays a key role in species evolution because it provides raw materials for the evolution of
new genes and new genetic functions. Multiple mechanisms contribute to gene duplication, including
tandem duplication, segmental duplication, transposon-mediated duplication, and retro duplication.
Studies of floral scent gene duplications in orchids have been limited. Orchid TPSs are the key enzymes
that generate the structure diversity of terpenes. Through the analysis of plant genome, researchers
have shown that the plant TPS gene family have their gene numbers ranging from 20 to 150 and
thus belong to a mid-size family [70]. Phalaenopsis equestris genome has 23 TPSs belonging to TPS-a,
-b, -c, e/f, and -g. Twenty-three TPSs found and predicted as having mono-, di- and sesqui-terpene
synthase evolutionary relationships among orchids and experiencing duplication and then sub- or
neo-functionalization, have occurred during evolution [71]. It has been proposed [72] that diterpene
synthases are the origin of mono- and sesqui-terpene synthases during evolution. P. aphrodite, PaCHS3,
PaCHS4, and PaCHS5 formed a tandemly arrayed gene cluster, and the intervals between the three
CHS genes were approximately 13.3 kb (PaCHS3 and PaCHS4) and 7.7 kb (PaCHS4 and PaCHS5). This
arrangement was also observed in a closely related orchid, P. equestris, in which the three CHS genes
were all positioned on Scaffold 000036. Thus, the tandem array of three CHS genes was probably present
in a common ancestor before speciation within Phalaenopsis. Tandem gene duplications represent a
substantial proportion of all plant genes [73].
6. Functions of Orchid Volatile Compounds

Previous reviews provide a good summary of floral emissions and the involvement of biochemical
processes in the interactions of flowers with their flower visitors [20], their action over pollinator
behavior [21], and the ecological processes that drive their evolution [22], wherein they mediate
intra- and interspecific interactions. The role of vegetative VOCs has been extensively reviewed [20].
Here, we present the functions of the floral VOCs, which are especially involved in the attraction of
pollinators [20]. Floral volatiles have a role in many multifaceted functions that contribute to pollinator
attraction, plant defense, plant reproduction, and plant diversity (Figure 2).
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Figure 2. Functions of floral volatiles in orchid flowers.
6.1. Flower Defense

Generally, flowers have effective physical barriers comprising highly lignified cell walls, although
the generation of these cell walls renders flowers highly vulnerable to pathogens and florivores. Plants
constitutively emit VOCs from flowers, leaves, and roots. Emission usually increases when plants are
attacked by antagonists such as insect herbivores or pathogens [51].
Many VOCs were shown to exhibit antimicrobial and antifungal activities in vitro [74] or inferred
to have these antimicrobial activities based on tissue-specific expression patterns [75]. However, only a
few VOCs have been explored for their role in defense against pathogens. (E)-β-caryophyllene, emitted
from stigmas of Arabidopsis flowers, was shown to limit bacterial growth; furthermore, Arabidopsis
plants lacking (E)-β-caryophyllene emission displayed denser bacterial populations on their stigmas
and reduced seed weight than wild-type plants, indicating that (E)-β-caryophyllene acts in the defense
against pathogenic bacteria and is also important for plant fitness [76]. VOCs emitted by petals of
Saponaria officinalis were also shown to inhibit bacterial growth, supporting their roles in controlling
bacterial community diversity in petals [77].
6.2. Pollinator Attraction

In many flowering plant species, the emission of volatile scents from the flower is important for
attracting insect pollinators. Orchid flowers exhibit visual, chemical, and morphological advertisements
to guide their pollinators, and may offer rewards such as nectar, pollen, fragrance, or oil [78]. Over
the past few years, evidence has supported the role of floral volatiles in pollinator attraction. In
fact, a high occurrence of non-rewarding flowers has been noted in orchids compared to other plant
families [79]. Floral volatile profiles are specific to each species depending on the type of pollinator [80].
However, the selection of pollinator has played a key role affecting the pattern of floral VOC profile
across angiosperms.
Approximately one-third of all orchid species reach pollination over food deception, whereby
flowers contain no nectar or other rewards but resemble or mimic floral signals of rewarding plants to
attract pollinators [78]. Subsequently, intraspecific variation in floral traits is estimated to be high in
food-deceptive orchids, since flowers must delay the avoidance learning of pollinators [79]. Flowers of
the fly-pollinated Satyrium pumilum orchids emit a cocktail of six compounds containing sulfurous oligo
sulfides such as dimethyl disulfide (DMDS) and dimethyl trisulfide (DMTS). Secretion of these volatiles
is also tissue-specific, which is anticipated to be the key olfactory cue for attracting flesh-eating fly
pollinators [80] and lepidopteran pollinators [81]. Floral scent can be distinguished even among closely
related taxa when species differ in pollination systems, such as lepidopteran vs. bee fly pollination in
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Narcissus species [80] and bee vs. hummingbird pollination in two Mimulus species [80], suggesting
that differences in the dominating functional group of pollinators drive divergence in floral scent.
6.3. Plant Reproduction

Pollinator attraction is often intermediated by multimodal signaling mechanisms including floral
morphology, color, and scent [81]. In deceptive species, attractiveness is very important for ensuring
reproductive success. For instance, Ophrys and Neotinea species are known to produce complex
bouquets of volatiles typically consisting of more than 100 chemical compounds [82]. The species
belonging to these genera are all deceptive, but Ophrys species use a sexual deception strategy, while
Neotinea is a food-deceptive genus [83]. Various orchid volatiles play a key role in plant reproduction.
In the Dracula orchid, Dracula lafleurii, the labellum acts as both a visual and an olfactory mimic of
mushrooms that often grow alongside these orchids [84]. The labellum emits an unusual floral volatile
blend of mushroom alcohols, especially (R)-1-octen-3-ol [85]. Cypripedium calceolus is pollinated by
bees. Scent profile consists mainly of aliphatics, terpenoids, and aromatics. In this context, orchids are
highly pertinent models for studying plant reproduction, as they present a great variety of floral traits
and trait associations. This is mirrored by the great diversity of reproductive strategies in orchids. One
of the most intriguing strategies is deceptive pollination (i.e., nectarless flowers), which is found in
about one-third of orchid species. Floral scent analyses in Ophrys orchids [81] showed that their flowers
emit attractive blends of VOCs. Moreover, different Ophrys species, which mainly use alkenes with
certain double-bond positions as key signals for plant reproduction (e.g., non-hydrocarbons with low
molecular weight), were found as “long-range” attractants [85].
Though by no means exclusive to orchids, deceptive pollination approaches are particularly
well-developed in the Orchidaceae, with an estimated one-third of the family (around 10,000 species)
using such strategies [85].
6.4. Evolution
Evolution of angiosperms has resulted in an immense diversity of flower traits such as shape,
size, color, and scent. Remarkably, the quality and quantity of emitted volatiles are species-specific and
vary among different populations within a species [20]. While much effort has so far been invested in
describing scent composition in various flowering species, the mechanisms driving the evolution and
diversification of floral scent remain underexplored. Analysis of the genetic basis for differences in
scent profiles between these two species revealed that only two quantitative trait loci are responsible
for the distinct scent phenotypes [86]. One of these locus maps to the MYB TF ODO1, which controls
flux over the shikimate pathway and, therefore, the amount of precursors available for benzenoid
biosynthesis [87], while the genetic identity of the second locus is presently unknown. Ophrys may
rely on species-specific alkene emission profiles that are distinct in enzyme activity and on the gene
expression of a few stearoyl acyl carrier protein desaturases of the Ophrys genus, and only limited
genetic variation among species and populations was observed with microsatellite markers. These
findings suggest that divergent pollinator-mediated selection rather than genetic drift explains the
strong differences in volatile profiles. Taken together, the above examples demonstrate that small
genetic variations can have large effects on floral scent chemistry and interactions with pollinators.
7. Case Studies of Cymbidium Floral Volatiles

Orchids are the largest, most highly diverse flowering plants, and form an extremely peculiar
group of plants. Cymbidium is one of the most important genera of orchids for the cut-flower and
potted plant markets. Cymbidium spp. have great horticultural value as ornamental plants because
of their beautiful and fragrant flowers. The Cymbidium genus consists of nearly 55 species that are
distributed mainly in tropical and subtropical Asia, reaching as far south as Papua New Guinea and
Australia [88]. The Cymbidium genus can be divided into three subgenera (Cymbidium, Cyperorchis, and
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Int. J. Mol. Sci. 2020, 21, 1160
Jensoa) [89,90] and includes C. sinense, C. goeringii, C. forrestii, C. faberi, C. ensifolium, and C. kanran. C.
sinense is a winter blooming epiphytic orchid usually regarded as a “Spring Festival” flower.
Great efforts have been made to better understand the flowering of orchids such as Cymbidium,
Phalaenopsis, Dendrobium and Cattleya through biotechnological approaches including tissue culture and
transgenic technologies [91–94]. Moreover, while Cymbidium orchid species are not all widely cultivated,
hybrids of Cymbidium orchids lend themselves to cultivation. Some commercially important hybrids
have been created for over 100 years. Because of their ornamental and commercial value, Cymbidium
orchids have been the subject of taxonomic studies and, particularly, species identification [95–97]. In
the past few decades, the application of diverse molecular techniques have contributed to widening
our knowledge in the flowering/flower development, species identification, and volatile compounds
of orchids.
7.1. Floral Volatile Research on Cymbidium

Floral VOCs are important compounds derived from flowers. Floral scent is a key trait for many
floricultural crops. The molecular mechanisms underlying the regulation of biosynthesis and emission
of volatiles from orchids remain largely elusive. For adding commercial value, studies on floral scents
in orchids aim not only to understand the molecular and genetic mechanisms of the biosynthesis and
emission of floral scents but also to assist in Cymbidium breeding programs. Currently, many orchid
researchers are focusing on the development of cultivars with desirable floral scents. Recent reports
on the propagation of sterile seedlings [98–100], leaf and flower morphogenesis [101–103], and the
characterization of volatiles as floral scents [104,105] in Cymbidium spp. also reflect this trend. In fact,
the biosynthesis of widespread VOCs in plant tissues is involved in multiple biological functions such
as defense against pathogens, parasites, and herbivores [106–108]. The development of floral scents is
likely to be a vital event in biological evolution, providing olfactory signals that plants can utilize to
attract pollinators. We are presented major floral scent Cymbidiums (Figure 3).
Figure 3. Cymbidium flowers described to the floral scent. (A) C. goeringii, (B) C. faberi, (C) C. ensiforium,
(D) C. “Sael Bit,” (E) C. “Sunny Bell.”.
7.1.1. Cymbidium goeringii

C. goeringii is one of the most popular terrestrial species indigenous to temperate Eastern Asia,
cultivated as an ornamental, and whose flowers are used as an ingredient in soup, alcoholic drinks, and
tea [88]. Floral fragrance is determined by a mixture of volatile compounds. In C. goeringii, floral scent
pathways have been studied in various developmental stages during flowering [28]. Sesquiterpenes are
the major compounds in the C. goeringii floral scent profile. The dominant floral scent compounds were
identified as farnesol, methyl epi-jasmonate, (E)-β-farrnesene, and nerolidol. In particular, examination
of farnesol emission from the day of anthesis (D0) to the fifth day after anthesis (D+5) demonstrated
that emission had a peak at the D+2 stage. Transcriptomic analyses focusing on floral scent pathways
have been performed using three different stages of flowers in C. goeringii. Most terpenoid pathway
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Int. J. Mol. Sci. 2020, 21, 1160
genes, including 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), 1-deoxy-D-xylulose-5-phosphate

synthase (DXS), and farnesyl diphosphate synthase (FDPS), were expressed at the initial flowering stage
compared to bud and/or full flowering stages [28]. Furthermore, 32 and 38 unigenes known to be
associated with MVA and MEP pathways, respectively, were reported in C. goeringii. Several TPSs and
TFs were also identified in the floral transcriptome of C. goeringii including CgTPS7, which encodes a
key enzyme involved in sesquiterpene synthesis. A putative terpenoid pathway responsible for the
volatile profile in C. goeringii has also been reported.
7.1.2. Cymbidium faberi

C. faberi Rolfe is one of the most significant species with elegant flower scents [44], and is one
of the oriental orchids that has been longest cultivated. There are more than 100 compounds in the
flower scent of blooming C. faberi flowers. Among these, MeJA is the most abundant, but is almost
untraceable in the volatile emission of withered flowers [44]. The major pathways include α-linolenic
acid metabolism, pyruvate metabolism, and fatty acid degradation, which contribute to the conversion
of α-linolenic acid to MeJA. One of the differentially expressed genes (DEGs), jasmonic acid carboxyl
methyltransferase (CfJMT), was highly regulated in the blooming flower of C. faberi. Consequently,
the full-length coding sequence and genomic sequence of CfAOS from C. faberi, which is localized to
the chloroplasts, has no introns, and is one of the most important enzymes in the MeJA biosynthetic
pathways in C. faberi [58], was cloned. CfAOS has numerous roles including insect attraction and
mediation of anti-microbial and stress tolerance. AOS and allene oxide cyclase (AOC) are crucial
enzymes in the MeJA biosynthetic pathway in C. faberi.
7.1.3. Cymbidium ensifolium

C. ensifolium is a popular miniature terrestrial orchid that produces fragrant flowers and is often
marketed as a potted specimen. MeJA was one of the key scent compounds found in C. ensifolium [31].
It is known that MeJA is synthesized via the octadecanoid pathway. MeJA is primarily recognized as a
floral scent compound in flowers of Jasminum grandiflorum, and is also universally distributed among
the plant kingdom including many Cymbidium orchids [31]. MeJA emission was at very low levels in
unopened or half-opened C. ensifolium flowers, and scent emission reached maximal levels between
Days 4 and 6 and decreased from Days 7 to 10 post-anthesis. C. ensifolium tissue-specific manner and
high MeJA emission was found in sepals and petals.
7.1.4. Cymbidium Cultivar Sael Bit

The small green and light yellow colored flowers of Cymbidium cultivar Sael Bit are highly
fragrant [109]. The strong emission of volatiles was detected in petals at the blooming stage. The
dominant floral VOCs in Sael Bit were identified to be alkenes, benzenes, and estaric compounds; i.e.,
2-methyl butanal, 2-methyl pentanal and (Z)-2-octenal at the blooming stage.
Full-length cDNA of Cymbidium Sael Bit MYB1 from the petal has been isolated; fragrance genes
such as MYB1, OOMT, AOS, LOX, and SAMS were expressed during the entire floral developmental
stages, and all genes were highly expressed in the full opening flowers [29]. Sael Bit MYB1 regulates
the floral scent phenylproponoid- and benzenoid-responsible genes during scent emission.
7.1.5. Cymbidium Cultivar Sunny Bell

A Cymbidium variety Sunny Bell (C. karan x C. eburneum) was developed at the National Institute
of Horticultural & Herbal Science, Rural Development Administration, Suwon, Korea in 2013 [39].
Monoterpenes, sesquiterpenes, and aliphatics have been recognized as the major volatile compounds
in Cymbidium Sunny Bell [39]. Twenty-four volatile components were identified in the Sunny Bell
flowers; among the total volatiles, petals produced dominant volatile compounds compared to other
floral tissues such as sepal, labellum, and column. Linalool is the major compound responsible for the
floral volatile profile in Sunny Bell.
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Int. J. Mol. Sci. 2020, 21, 1160
Some species in the genus Cymbidium, including C. floribundum, C. pumilum, and C. suavissimum,
release identical volatiles for pollinator attraction. Various types of alkenes, esters, and fatty acid
derivative compounds are released for pollinator attraction. It has been reported that Cymbidium
flowers are rich in volatile compounds including cineole, isoeugenol, and (-) selinene [104]. Floral scent
and color are major traits for floriculture crops in developing new cultivars of Cymbidium. Furthermore,
21–28 floral scent compounds have been identified as major volatile components in the flowers of three
Cymbidium varieties [105]. The volatiles mainly comprise monoterpenes, aliphatics, and sesquiterpenes,
and their content values have exceeded 90% [105]. Their aromatic characteristics can be determined by
the profiles of each VOC that may vary depending on each genotype [105].
8. Final Remarks and Future Directions for Overcome the Challenges

At present, orchid industries worldwide are facing various difficulties. For developing new
cultivars, physiological and genomic maps have been needed to produce markers. RNA-illumina
sequencing technology has been extensively used for identifying gene expression at a genome-wide
scale in many organisms, including non-model plants. The adoption of this technique, especially
mRNA sequencing from floral tissues and de novo transcriptome construction (Figure 4), has been
performed in several orchid species, with a goal of identifying genes involved in the biosynthesis
and/or biosynthetic pathways of floral volatiles [28,110].
Figure 4. Schematic representation of functional studies for orchid breeding to develop floral scent trait.
Moreover, other strategies include molecular evolutionary analysis tools. For example, testing
for gene duplication and selection signatures in hypothesized pathway genes, from a phylogenetic
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Int. J. Mol. Sci. 2020, 21, 1160
perspective, is frequently used. For the identification of significant candidate genes and pathways,
targeted and strategic transcriptome analyses of fragrant and non-fragrant flower organs and
tissues are often the first key step. Regulation of DEGs between fragrant and non-fragrant
tissues and developmental stages can be investigated. The latter provides a key baseline for
identifying DEGs in fragrant tissues. This approach was utilized for the breakthrough discovery
on volatile biosynthesis [58,111] and may hold potential for elucidating other biosynthetic pathways.
RNA-sequencing analysis across species, with the goal of identifying shared gene expression and
metabolic pathways, may also prove informative. Transformation technology has been developed
for orchids; a few successful methods using virus induced gene-silencing (VIGS) approaches have
recently been demonstrated as efficient strategies for functional studies of genes in orchids (Figure 4).
Furthermore, transgenic approaches, such as the overexpression of floral scent genes and/or genome
editing, have also been recently developed for orchids.
9. Conclusions
Over the last few decades, studies on plant volatile compounds and their biosynthetic processes
have markedly increased. In orchids, volatile compounds play a key role in pollination, which ensures
fertilization. To date, the biosynthesis of orchid floral fragrance is not well understood, with only
some terpenoid pathways reported. Plant volatiles are generally produced at very low concentrations
with low quantity, even in floral tissues. Thus, isolation of each component in the volatile compounds
is inefficient and expensive. Despite the presence of studies of floral VOCs, many aspects of their
biosynthesis together with transcriptional regulation and function require further studies. Further
advances in functional studies on key genes for floral scent may rely on a breakthrough in orchid
transformation technology that may lead to more efficient results. The genome sequences of several
orchids have now been determined [112,113]. Overall, it is clear that genetic manipulation of orchid
volatile compounds may be possible, but requires the selection of the appropriate species. In the future,
studies in scent research may focus on orchid floral traits and on increasing phytochemical compounds,
flavor, and aroma through the regulation of genes by transcription factors in floriculture crops. This
review provides an important theoretical reference for aromatic volatile compound studies in orchids.
Author Contributions: M.R., S.J., and P.H.P., drafted the manuscript. M.R., H.-R.A., P.-M.P., and S.J. collected
the background information. S.J. and S.-Y.L. revised the manuscript. All authors have read and agreed to the
published version of the manuscript.
Funding: This review paper was financially supported by the National Institute of Horticulture and Herbal
Science, RDA, Korea, under the project grant PJ01183202. Funding for the World Vegetable Center co-author (S.J.)
was provided in part by the World Veg Korea Office budget (WKO #10000379) and the long-term strategic donors
to the World Vegetable Center: Republic of China (Taiwan). The authors are also grateful for UK aid from the
UK government, the United States Agency for International Development (USAID), the Australian Centre for
International Agricultural Research (ACIAR), Germany, Thailand, Philippines, Korea, and Japan.
Conflicts of Interest: The authors of this paper declare that there is no conflict of interest in relation to the
publication of this review paper.
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73
Molecular Sciences
Article
Comparative Transcriptome Analysis of Different
Dendrobium Species Reveals Active
Ingredients-Related Genes and Pathways
Yingdan Yuan 1,2 , Bo Zhang 1,2,3 , Xinggang Tang 1,2 , Jinchi Zhang 1,2, * and Jie Lin 1,2, *
1 Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University,
Nanjing 210037, China; [email protected] (Y.Y.); [email protected] (B.Z.);
[email protected] (X.T.)
2 Jiangsu Province Key Laboratory of Soil and Water Conservation and Ecological Restoration, Nanjing
Forestry University, Nanjing 210037, China
3 Department of Environmental Science and Policy, University of California, Davis, CA 95616, USA
* Correspondence: [email protected] (J.Z.); [email protected] (J.L.)

Received: 24 December 2019; Accepted: 27 January 2020; Published: 29 January 2020
Abstract: Dendrobium is widely used in traditional Chinese medicine, which contains many kinds
of active ingredients. In recent years, many Dendrobium transcriptomes have been sequenced.
Hence, weighted gene co-expression network analysis (WGCNA) was used with the gene expression
profiles of active ingredients to identify the modules and genes that may associate with particular
species and tissues. Three kinds of Dendrobium species and three tissues were sampled for RNA-seq
to generate a high-quality, full-length transcriptome database. Based on significant changes in gene
expression, we constructed co-expression networks and revealed 19 gene modules. Among them, four
modules with properties correlating to active ingredients regulation and biosynthesis, and several
hub genes were selected for further functional investigation. This is the first time the WGCNA
method has been used to analyze Dendrobium transcriptome data. Further excavation of the gene
module information will help us to further study the role and significance of key genes, key signaling
pathways, and regulatory mechanisms between genes on the occurrence and development of medicinal
components of Dendrobium.
Keywords: Dendrobium; comparative transcriptome; active ingredients; different tissues
1. Introduction
Dendrobium is a perennial epiphytic herb of the genus Orchidaceae, with more than 1500 species
in the world which are mostly growing in tropical and subtropical Asia and eastern Australia [1,2].
In China, there are more than 80 species of Dendrobium that had been reported in studies, mainly
distributed in south of the Qinling Mountains [3,4]. As a kind of ornamental and medicinal plants,
more and more researchers have paid attention to it in recent years [5]. Also, Dendrobium is a traditional
Chinese herbal medicine, that has been used for benefiting stomach and clearing heat, nourishing
yin, and promoting fluid [6]. The medicinal ingredients of Dendrobium are very complex, not only
including polysaccharides and alkaloids, but also flavonoids, various amino acids, and trace mineral
elements. However, the most two important components are polysaccharides and alkaloids [7–10].
With the development of high-throughput technology, a variety of data sources have been derived,
including gene expression microarray, RNA-seq, metabolomic and CHIP-seq, these have become
powerful tools for studying plant growth, development, and physiology at the transcriptional and
metabolic levels [11–13]. In order to explore the key genes and regulatory pathways for the synthesis
of its medicinal ingredients, more researchers have done different kinds of Dendrobium transcriptome

Int. J. Mol. Sci. 2020, 21, 861
research [14–16]. However, it has been difficult to systematically explain the relationship between
gene expression or metabolite changes and trait differences [17]. The analysis of correlation networks
can bridge the gap between single gene interpretation and systematic biology research by mining
the link between genes and gene products [18–20], such as integrating a single gene into a co-expression
network based on pairwise gene expression correlation [21]. Gene co-expression analysis has been
used to discover new candidate genes [22,23], identify key modulators of immune responses [24],
and reconstruct regulatory pathways [25]. Genes belonging to the same co-expression sub-network
(or module) are likely to be functionally related [26–28], participate in similar biological processes, or
be part of the same pathway [25].
Weighted gene co-expression network analysis (WGCNA) is one of the most useful methods based
on gene co-expression networks. It focuses on the set of genes other than on a single gene in the observed
gene expression data, and it alleviates the multiple detection problems inherent in chip data analysis
and can be used in unweighted correlation networks [29]. Compared with many other analysis
methods, WGCNA has the advantages of summarizing and standardizing the methods and functions
of integrated R packages, including methods of weighted and unweighted correlation networks [30].
WGCNA is used in combination with gene chip data, transcriptome data, and metabolome data for
metabolic regulation network simulation, mining inter-genetic interactions, screening functional genes,
etc. It has been extensively studied in plant growth, tissue and organ development, pigmentation and
fragrance synthesis [31–34].
To reveal the underlying molecular mechanism of the active ingredients of Dendrobium,
we downloaded three datasets from the NCBI Sequence Reading Archive (SRA) to identify highly
connected hub genes and important modules. This study used three different Dendrobium species and
different tissues as materials to perform transcriptome sequencing data, combined with polysaccharide
and alkaloid content data, and used WGCNA analysis to construct a co-expression gene network.
Correlation analysis was performed between the gene module and the polysaccharide and alkaloid
data, and hub genes related to the main medicinal ingredients were discovered, in order to provide
new clues for further research on the molecular mechanism of medicinal ingredients of Dendrobium.
For the first time, a co-expression network analysis of transcriptome genes in Dendrobium was
constructed, and modules with high correlation in secondary metabolism were analyzed, laying
a foundation for the discovery of functional genes of medicinal ingredients.
2. Results
2.1. Determination of Total Alkaloid and Polysaccharide Contents in Different Species and Different Tissues
Polysaccharides and alkaloids are the main medicinal components of Dendrobium. Therefore,
we determined the polysaccharide and total alkaloid in three different tissues of three Dendrobium
species (Figure 1). The biennial Dendrobium officinale has the highest content in the comparison of
different tissues and species. For polysaccharide content, the stem is the main enrichment tissue,
while for total alkaloid, the leaf is the main enrichment tissue. In the stem, the polysaccharide content
of Dendrobium varied from 23.34 to 37.41%. In the leaf, the total alkaloid content of Dendrobium varied
from 0.0291 to 0.0421%.
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Int. J. Mol. Sci. 2020, 21, 861
Figure 1. Polysaccharide and total alkaloid contents variation in different tissues of different species.
(a) Polysaccharide content. (b) Total alkaloid content. There are three replicates of each sample. 2-Dh:
two-year-old Dendrobium huoshanese, 2-Do: two-year-old Dendrobium officinale, 2-Dm: two-year-old
Dendrobium moniliforme.
2.2. Differential Expression Analysis

In this study, we calculated the FPKM of each sample to regulate the expression and investigate
the gene expression differences among different tissues of different Dendrobium species. We compared
the three data sets from different comparison groups using a Venn diagram (Figure 2a–c). In addition,
we compared the number of DEGs between different species in the same tissue, and many genes were
differentially expressed only in one or two comparisons. Then the edgeR was used to test the differential
expression of the repeated count data. We used “FDR < 0.05 & | log2 FC | ≥1” as the criterion for
significant differences in gene expression. When log2 FC > 1, DEG is considered to be up-regulated.
In contrast, for log2 FC < −1, it is considered a downward adjustment. We did up and down-regulated
analysis of DEGs for three different tissues and three different Dendrobium species, and the results are
shown in Figure 3a–i.
According to the transcriptome data, all DEGs were used for hierarchical cluster analysis of
transcription abundance in three different tissues. The heatmap of DEGs between different tissues and
different Dendrobium species show similar transcriptome profiles for Dh_R, Dh_L, Dh_S, Do_R, Do_L,
Do_S, Dm_R, Dm_L, and Dm_S (Figure 4a). A total of 35,159 DEGs were identified and analyzed
using criteria of log10 (FPKM+1) and p < 0.05. The trend of the specific expression level is shown
in the number under the color bar at the top left. On the left is the gene cluster tree. The closer
the degree of separation between the two genes, the closer their expression is. In order to reflect the main
trends and tissue-specific expression of different Dendrobium species, all DEGs were clustered into ten
expression profiles (Figure 4b) using the K-means method and hierarchical clustering with similar
regulation model and log2 (foldchange). DEGs belonging to cluster 3 were more highly expressed
in leaves than in other tissues. Except for subcluster 1 and subcluster 3, all the other subclusters have
just one gene trend.
Figure 2. Venn diagram of differentially expressed genes (DEGs) in different comparisons. All DEGs
are grouped into three comparison groups represented by three circles. The overlapping portions of
the different circles represent the number of DEGs common to these comparison groups. (a) Venn
diagram of three kinds of Dendrobium leaves. (b) Venn diagram of three kinds of Dendrobium roots.
(c) Venn diagram of three kinds of Dendrobium stems.
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Int. J. Mol. Sci. 2020, 21, 861
Figure 3. Volcano plots of the DEGs in different comparisons. Red dots indicate significant up-regulation
of genes, and blue dots indicate significant down-regulation of genes. Black dots represent non-DEGs.
(a) Dm_L vs. Dh_L volcano; (b) Dm_L vs. Do_L volcano; (c) Do_L vs. Dh_L volcano; (d) Dm_R vs.
Dh_R volcano; (e) Dm_R vs. Do_R volcano; (f) Do_R vs. Dh_R volcano; (g) Dm_S vs. Dh_S volcano;
(h) Dm_S vs. Do_S volcano; (i) Do_S vs. Dh_S volcano.
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Int. J. Mol. Sci. 2020, 21, 861
Figure 4. Expression profiles of all DEGs. (a) The heatmap of DEGs and the FPKM distribution of
all unigenes obtained by hierarchical cluster analysis. Each column in the figure represents a sample,
and each row represents a gene. The colors in the graph indicate the magnitude of gene expression (log10
(FPKM + 1)) in the sample. Red indicates that the gene is highly expressed in the sample, and the blue
indicates that the gene expression is low. (b) K-means clustering analysis of gene expression profiles.
The blue line represents the expression model. The gray lines are the expression profiles of each DEGs.
The x-axis represents different tissues of different Dendrobium plants. The y-axis represents log2 (ratio).
2.3. GO and KEGG of DEGs Annotation and Enrichment Analysis

GO is a database established by the Gene Ontology Consortium. Its purpose is to standardize
biological terms about genes and gene products in different databases, to define and describe gene and
protein functions. Using the GO database, genes can be classified according to biological process, cellular
component, and molecular function. The GO annotation statistics were performed on the differentially
expressed genes in pairs, and one of the samples was used as a control. The obtained results can be
used to plot the GO annotation bar graph of up and down DEGs (Figure 5). Figure 5 only shows
the comparison of the stems of different Dendrobium species, and the other two tissues are shown
in the Supplementary Figure S1.
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Int. J. Mol. Sci. 2020, 21, 861
Figure 5. Gene Ontology (GO) annotations of up and down regulated DEGs. The bottom x-axis
indicates the number of DEGs annotated to a GO term, the upper x-axis indicates the proportion of
DEGs annotated to a GO terms to the total number of all GO annotated DEGs; and the y-axis represents
each detailed classification of GO. (a) Dm_S vs Dh_S GO annotation; (b) Dm_S vs. Do_S GO annotation;
(c) Do_S vs. Dh_S GO annotation.
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Int. J. Mol. Sci. 2020, 21, 861
KEGG focuses on biochemical pathways, especially genes involved in protein, carbohydrate,

and energy metabolism. In the KEGG enrichment analysis, we mapped all these genes to a reference
pathway in the KEGG database to determine the biological pathways in which these genes may be
involved (Figure 6). In terms of the KEGG pathways, “Dm_S vs. Dh_S” comparisons are involved
in 235 pathways (Figure 6a). In “Dm_S vs. Do_S” comparisons, 223 pathways were involved
(Figure 6b). Finally, in “Do_S vs. Dh_S” comparisons, 237 pathways were involved (Figure 6c).
Among these pathways, “flavonoid biosynthesis” was enriched in the Dm_S vs. Dh_S comparison.
“Lipopolysaccharide biosynthesis” related to polysaccharide was the most enriched pathways
in the Do_S vs. Dh_S comparison. Notably, we observed terpenoid biosynthesis in the secondary
metabolites also enriched in the Do_S vs. Dh_S comparison. The KEGG pathways of different
Dendrobium species of the other two tissues are shown in the Supplementary Figure.
Figure 6. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of DEGs. The x-axis
represents the pathway name, and the y-axis represents the enrichment ratio (sample number/background
number). (a) Dm_S vs. Dh_S; (b) Dm_S vs. Do_S; (c) Do_S vs. Dh_S. All pathways in the figure with
asterisks indicate significant KEGG enrichment, with three asterisks indicating p-value < 0.001, two
asterisks indicating p-value < 0.01, one asterisks indicating p-value < 0.05.
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2.4. WGCNA Analysis
2.4.1. Construction of Gene Co-Expression Network of Dendrobium

Consequently, we used the WGCNA package tool to construct a co-expression module that
expressed the expression of 8056 genes in 27 Dendrobium samples. The heat map of the cluster
dendrogram and tissues and species traits are shown in Figure 7a. One of the most critical
parameters is the power value, which mainly affects the independence and average connectivity of
the co-expression modules. First, we selected the appropriate power value. When the power value
was 18, the independence reached 0.6, and the average connectivity was higher. Therefore, the power
values and results of constructing co-expression modules indicate that 19 different co-expression
modules were identified in Dendrobium. These co-expression modules were constructed and are shown
in different colors (Figure 7). The size of these modules depends on the number of genes they contain.
The number of genes and module names are shown in Table 1.
Figure 7. Clustering dendrogram. (a) Clustering dendrogram of 27 samples and heatmaps of species
and tissues traits. The expression is from low to high, and the color transitions from white to red.
(b) Clustering dendrogram of DEGs, with dissimilarity based on the topological overlap, together with
assigned module colors. The clustered branches represent different modules, and each line represents
one gene.
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Table 1. The number of genes in 19 constructed modules.
Module Colors Node Number

Blue 562
Brown 892
Cyan 1132
darkgreen 1016
darkolivegreen 362
darkorange 665
floralwhite 184
greenyellow 440
lightcyan 180
lightgreen 178
lightpink4 40
lightsteelblue1 193
paleturquoise 470
plum2 57
royalblue 275
saddlebrown 179
salmon4 1015
sienna3 109
yellowgreen 107
2.4.2. Interaction Analysis of Co-Expression Module

Subsequently, we analyzed the interactions between the 19 co-expression modules (Figure 8).
The heatmap shows the topological overlap matrix (TOM) of all genes in the analysis. Light color
indicates low overlap, and dark red indicates high overlap. Except for some high-brightness areas,
the overall difference between the modules is not significant, which indicates that the gene expression
between the modules is relatively independent and has a high scale independence. The correlations
between module eigengene and traits: tissues and species, were analyzed and data are shown
in Figure 8b. Two modules are significantly associated with species: Lightgreen (p-value = 1 × 10−5 ,
cor = 0.73) and Lightsteelblue1 (p-value = 2 × 10−6 , cor = −0.76). The modules that highly correlated to
tissues were Yellowgreen (p-value = 1 × 10−6 , cor = 0.79), Salmon4 (p-value = 9 × 10−7 , cor = 0.79),
Blue (p-value = 3 × 10−6 , cor = 0.77), and Sienna3 (p-value =2 × 10−10 , cor = 0.9).
We analyzed the connectivity of eigengenes to find the interactions between these constructed
co-expression modules. First of all, cluster analysis of these eigengenes was performed (Figure 9a).
These 19 clusters were divided into two clusters, including 5 modules (modules 3, 8, 9, 11, and 14)
and the remaining 14 modules. According to that, the connectivity effect between different modules is
obviously different. (Figure 9b).
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Figure 8. Co-expression network analysis across different tissues and different species. (a) Visualizing
the gene network using a heatmap plot. The heatmap depicts the topological overlap matrix
(TOM) among all genes in the analysis. (b) Module-trait associations. Each row corresponds to
a module characteristic gene (eigengene), and each column corresponds to a trait. Each cell contains
a corresponding correlation and p-value. According to the color legend, the table is color-coded
by correlation.
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Figure 9. Analysis of connectivity of eigengenes in different module. (a) Cluster analysis of eigengenes.
(b) The heatmap of connectivity of eigengenes.
2.4.3. Functional Enrichment Analysis of Genes in Interested Modules

We performed pathway enrichment analysis on the genes of each module separately, explored
their related functions, and obtained many meaningful KEGG pathway and GO terms (Figure 10).
It is important to identify the most significant modules related to medicinal components. Sienna3
module showed a significantly high correlation with polysaccharide. In the KEGG pathway, it is
significantly enriched to fructose and mannose metabolism (ko00051), starch and sucrose metabolism
(ko00500), and galactose metabolism (ko00052). The top three GO terms, shown in expanded size, are
glucosyltransferase activity (GO:0046527) and starch synthase activity (GO:0009011). These KEGG
pathways and GO terms are all related to polysaccharide biosynthesis, and polysaccharides are the main
medicinal components of Dendrobium.
In addition, the KEGG pathways regulating Dendrobium polysaccharides and secondary
metabolism biosynthesis were also identified in three other modules, which are the blue
module, the lightsteelblue1 module, and the salmon4 module (Figure 11). In blue module,
genes were significantly enriched in these pathways: anthocyanin biosynthesis (ko00942), drug
metabolism—cytochrome P450 (ko00982) and steroid biosynthesis (ko00100). In lightsteelblue1,
the significantly enriched pathways are as follows: terpenoid backbone biosynthesis (ko00900),
diterpenoid biosynthesis (ko00904), galactose metabolism (ko00052), and glycolysis/gluconeogenesis
(ko00010). In salmon4, genes were significantly enriched in these pathways: the pentose phosphate
pathway (ko00030), terpenoid backbone biosynthesis (ko00900), and diterpenoid biosynthesis
(ko00904). The enriched pathways in these three modules are significantly related to the synthesis of
polysaccharides and secondary metabolites of Dendrobium. Therefore, sienna3, blue, lightsteelblue1
and salmon4 modules were defined as important module and extracted for further analysis.
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Figure 10. GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of module
sienna3. (a) Top 30 of KEGG pathway enrichment. (b) Thumbnails view of directed acyclic graph
(DAG) on molecular function of module sienna3.
Figure 11. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of interested
module. (a) Module blue; (b) module lightsteelblue1; (c) module salmon4.
2.4.4. Module Visualize and Hub Genes

We performed genes trend expression analysis of four modules of interest (Figure 12). Interestingly,
by analyzing the trend expression of genes in sienna3 module, we found that they are specifically
expressed only in the stem, which suggests that they play an important role in the stem. Through further
analysis of this module, several hub genes were discovered, such as beta-glucosidase and granule-bound
starch synthase (GBSSI). This indicates that the specific expression of these polysaccharide-related
genes in stems may be related to the content of polysaccharides in stems significantly higher than
in other tissues.
In the blue module, Do_S shows specific expression in this module, several hub genes were detected
in each important module, with each gene interacting with many other genes. These genes include
bHLH, beta-glucosidase, alpha-L-fucosidase, and other genes related to Dendrobium polysaccharide and
secondary metabolism. In salmon4 module, Do_S shows specific expression in this module, hub genes
include CCR4 (cinnamoyl -CoA reductase), bZIP, and MYB transcription factors were identified.
In lightsteelblue1, a large number of genes were annotated to ketohexokinase (KHK). KHK, also known as
fructokinase, which is a key enzyme in fructose metabolism. Fructose is a kind of plant polysaccharide,
which suggests that this module is related to polysaccharide biosynthesis.
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Figure 12. Expression profile and transcriptional regulatory network associated with the tissue-specific
modules. (a), (c), (e), and (g) heatmaps showing genes in module that were significantly over-represented
in Dm_L, Dm_R, Dm_S, Do_L, Do_R, Do_S, Dh_L, Dh_R, Dh_S and predicted transcriptional regulatory
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network associated with the gene sets showing expression patterns at Dm_L, Dm_R, Dm_S, Do_L,
Do_R, Do_S, Dh_L, Dh_R, and Dh_S. Heatmaps show the expression profile of all the co-expressed
genes (number given on the top) in the modules (labelled on top). Candidate hub genes are shown
in rectangular shapes. Purple in figure (b, d and h) represents genes related to polysaccharides. Red
in figure (d and f) represents genes related to secondary metabolites. The shapes, from big to small,
indicate the weights from big to small. Green in figure d represents bHLH transcription factors. Green
in figure (f) represents bZIP transcription factors and yellow represents MYB transcription factors.
3. Discussion
Traditional biological research focuses on elucidating the effects of individual functional elements
(such as DNA, mRNA, and protein) on life activities at the molecular level. Although those methods
are of great significance for revealing the genetic mechanisms of specific traits, it can only partially
explain the cause of a certain life activities. With the rapid development of sequencing technology,
traditional biological research cannot fully and effectively explore the biological significance contained
in massive data. As a research method of systems biology, the network is widely used in the exploration
of life sciences with the help of data of genome, transcriptome, and metabolome. Compared with other
regulatory networks, WGCNA can screen for genes related to specific traits and perform modular
classification from large samples to obtain highly biologically significant co-expression modules, which
has proven to be an efficient data mining method [35].
WGCNA has been widely used in plants in recent years. In order to obtain key expression modules
and key hub genes related to drought resistance in Brassica napus L., WGCNA was used to analyze
Brassica napus transcriptome data in multiple samples (48 transcriptome data), the well-watered and
droughted networks contained 17 and 20 modules, respectively, suggesting that there are additional
expression patterns in the droughted network because of rearrangement of the transcriptome in response
to the drought treatment. [36]. In the study of Fragaria L. flowers [37], researchers generated different
tissue- and stage-transcriptomic profiling of woodland strawberry (Fragaria vesca) flower development,
they discovered a developing receptacle-specific module exhibiting similar molecular features to those
of young floral meristems and hub genes of the strawberry homologs of a number of meristem regulators,
including LOST MERISTEM and WUSCHEL in the developing receptacle network. [37]. Analysis of
the pollen transcriptome of three male sterile lines using weighted gene co-expression network analysis
revealed that two modules were significantly associated with male sterility and many hub genes
that were differentially expressed in the sterile lines [38]. Farcuh et al. used WGCNA to investigate
sugar metabolism during leaf and fruit development of two Japanese plum varieties, and identified
11 key sugar metabolism-related genes, the results showed that sugar metabolism was reprogrammed
in a non-climacteric bud mutant of a climacteric plum fruit and showed an increase in sorbitol
synthesis [39]. In Ginkgo biloba, a total of 12 gene modules were revealed to be involved in flavonoid
metabolism structural genes and transcription factors by constructing co-expression networks, they
reveal that some hub genes operate during the biosynthesis by identifying transcription factors (TFs)
and structure genes and seven key hub genes were also identified by analyzing the correlation between
gene expression level and flavonoids content [40]. Through these studies, it was found that many
of the hub genes obtained by WGCNA analysis were indeed very important genes. In order to
obtain the hub gene related to the synthesis and regulation of Dendrobium polysaccharides and
alkaloids, we also performed WGCNA analysis.
Therefore, we constructed a Dendrobium gene co-expression network using a WGCNA approach
and identified co-expression modules using transcriptome data from three kinds of Dendrobium
species and three different tissues. Correlation analysis between co-expression modules and two
traits (species and tissues) was carried out, and four highly significant active ingredients-related
modules (p-value < 0.05) were identified. These modules consist of highly connected functional
genes, and different modules appear to be involved in individual functions [41]. Meanwhile, KEGG
pathway enrichment analysis of modules associated with polysaccharide and secondary metabolism
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indicated that these pathways in different Dendrobium species and different tissues are related to
each other at the transcriptomic level. In sienna3 module, we found that several hub genes related
to polysaccharide biosynthesis, such as KHK (ketohexokinase) which is a key enzyme in fructose
metabolism. In the expression trend of this module, the stem has a clear advantage, indicating that
the polysaccharide content is indeed concentrated on the Dendrobium stem, which is consistent with
our previous determination of the polysaccharide content. Meanwhile, in blue module, we found
that not only the hub genes related to polysaccharide, but also related to secondary metabolism.
In the expression trend, we found that the stem of Dendrobium officinale to be the highest. This result
is consistent with the trend in the determination of polysaccharides and alkaloids. The results
also indicated that one component can be regulated by multiple modules, and one module can
simultaneously be associated with multiple components.
Module hub genes are generally considered representative of a given module in a biological
network. Previous studies reported that MYB-bHLH-WDR (MBW) ternary complexes comprise
the essential regulatory machinery for catechin and anthocyanin biosynthesis [42]. In the present
study, transcription factors MYB and bHLH were identified as hub genes in modules related
to secondary metabolism. In addition, several genes involved in polysaccharide and secondary
metabolism biosynthesis (CCR4: cinnamoyl -CoA reductase and KHK: ketohexokinase) were identified
in modules. MYB transcription factors play an important role in the regulation of phenylpropane
biosynthesis. Phenylpropane synthesis is upstream of the regulation of flavonoid biosynthesis,
indicating that MYB is also an important transcription factor for the synthesis of flavonoids. In gentian,
both GtMYBP3 and GtMYBP4 can activate the gene expression of flavonol synthesis, and then
significantly increase the flavonol content in seedlings [43]. Ginkgo GbMYBF2 inhibits the expression
of CHS (chalcone synthase), F3H (flavanone 3-hydroxylase), FLS (flavonol synthase, Flavonol synthase and
ANS genes) on the phenylpropane synthesis pathway, thereby reducing the content of flavonoids and
anthocyanins [44]. Salvia miltiorrhiza SmMYB39 affects the synthesis of rosmarinic acid by regulating
the expression of key enzyme genes of the phenylpropane metabolic pathway [45]. Current studies
indicate that plant bHLH transcription factors are involved in regulating various signal transduction
and anabolic pathways, such as light signal transduction, hormone synthesis, glandular and root hair
development, and stress [46–49].
In this study, the WGCNA method was used for the first time in Dendrobium, and the modules
related to specific tissues and genes related to specific traits were identified. Hub genes were further
analyzed to find related genes and predict gene functions [30]. Combining the WGCNA method
and RNA-Seq data can be used to better mine the genes and transcription factors related to traits.
In strawberry, modules related to tissue specificity such as torus were found in strawberry, and 7 hub
genes were identified in torus tissue [37]; in tomato, genes related to vitamin C biosynthesis were
found [50]; co-expression modules related to acidity and genes related to anthocyanin synthesis were
found in apple [33,51]. In addition, specific modules in other tissues such as roots, leaves, flowers,
and fruits at other periods can be excavated to find relevant metabolic processes and important genes
and potential transcription factors.
4.1. Plant Material

Dendrobium plants were cultivated artificially in a greenhouse located in Anhui Tongjisheng
Biotechnology Company, Lu’an, China. Seed germination, the growth of protocorm-like bodies
protocols and the condition of pots used for planting were described by our previous study [15].
Two-year-old D. huoshanense, D. officinale, and D. moniliforme were selected to provide three replicates
of each sample. Stems, leaves, and roots were collected from three different kinds of Dendrobium
plants for RNA extraction. Then, after drying, they were used to determine polysaccharide and total
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alkaloid contents in March, 2017. The same samples were used for transcriptome sequencing and
polysaccharide and total alkaloid contents determination.
4.2. Polysaccharide and Total Alkaloid Contents Determination

The method for determining the polysaccharide content is consistent with the Chinese
Pharmacopoeia (version 2010), and the details are described in our previous study [15]. The method of
total alkaloids has been modified based on the Bush et al. method and has been described in detail
in our previous studies [52,53]. A part of data of this part has been published [14,15].
4.3. Identification of Orthologous Genes

Datasets for three kinds of Dendrobium species were obtained from the NCBI Sequence Read
Archive (SRA) (https://www.ncbi.nlm.nih.gov/sra) with accessing number SRP122499, SRP139000,
and SRP150489. There was a total of 27 samples of Dendrobium that were taken from three different
species (Dendrobium huoshanense, Dendrobium officinale, and Dendrobium moniliforme) and three different
tissues (stems, leaves, and roots). The data for these samples were submitted by our own laboratory.
The OrthoMCL pipeline [54] was used with standard settings to identify potential orthologous genes
in Dendrobium, and subsequently aligned using the MUSCLE algorithm [55].
4.4. Differential Gene Expression Analysis

The gene expression levels of all samples were estimated by RSEM version 1.2.15 [56],
and the bowtie2 parameter setting mismatch was 0. The R Bioconductor package edgeR [57] was used
to identify differentially expressed genes (DEGs) in two samples. DEGs were used for KEGG and GO
enrichment analyses, which were performed using the KOBAS version 2.0.12 with default settings and
Goatools version 0.5.9 (https://github.com/tanghaibao/Goatools) [58,59].
4.5. WGCNA Analysis
4.5.1. Construction of Weighted Gene Co-Expression Networks and Identification of Modules

We use thresholds for different expression data to calculate the number of genes. The WGCNA
algorithm was applied to the evaluation of gene expression. The flashClust toolkit (R language)
was used to perform cluster analysis on samples and set appropriate thresholds. After background
correction and quantile normalization, the top 50% of genes (8056 genes) with the most variant
in the analysis of variance were selected for WGCNA analysis. The WGCNA package in R was used to
construct the co-expression network of the 8056 genes after verification [30].
The gradient independence method was used to test the scale independence and average
connectivity of different power modules (the power value ranging from 1 to 20). According to
the scale-independent conditions, the appropriate power value was 0.6. After the power value
determined, the module was constructed by the WGCNA algorithm, and the genetic information
corresponding to each module was extracted. For high reliability of the results, the minimum number
of genes per module was set to 50.
4.5.2. Interactions Analysis of Co-Expression Modules

The interaction relationship between different co-expression modules was calculated by WGCNA,
and the topological overlap matrix (TOM) was constructed using the correlation expression values.
The resulting topological overlap is a biologically meaningful measure of gene similarity based
on the co-expression relationship between two genes. Using each TOM as input, the flashClust function
was used for hierarchical cluster analysis. Then the dynamicTreeCut algorithm (minModuleSize = 30)
was used to detect the module as a branch of the dendrogram [30]. Random colors were assigned
to the modules, and the first principal component was used to calculate the module characteristic
genes of each module. The module characteristic genes can be regarded as the representative of
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the gene expression pattern in this module, and the module combining highly relevant characteristic
genes (merge-CutHeight = 0.07). By calculating the Pearson correlation between the module
characteristic genes and the interest characteristics, and using the module characteristic genes to
estimate the module-trait relationship, the samples are classified according to the corresponding
traits, types, and tissues, and the module with a correlation coefficient ≥ | 0.75 | and p-value ≤ 0.01
was selected for further analysis. Through the heat map of the gene network topological overlap,
the structure of co-expression module was visualized. Then through the hierarchical clustering tree
diagram of the characteristic genes and the heat map of the corresponding characteristic gene network,
the relationship between the modules was summarized.
4.5.3. Functional Enrichment Analysis of the Key Module

We performed GO and KEGG enrichment analysis for key modules with most genes by using
the Database for Annotation, Visualization and Integrated Discovery (DAVID, https://david.ncifcrf.gov/
summary.jsp) [60] and KEGG Orthology Based Annotation System (KOBAS, https://bio.tools/kobas).
Analysis results were extracted out under the condition of p < 0.05 after correction [61,62].
4.5.4. Identification of Hub Genes

The hub gene is usually used as an abbreviation for a highly connected gene, which has a high
degree of connectivity in co-expression modules. Module membership (MM) is defined as the correlation
between the expression of each gene and its module eigengenes (MEs). Gene significance (GS) is
defined as the correlation of each gene to the trait of interest. In the module of interest, the genes with
the highest MM and GS were set as candidate genes for further analysis [63]. We chose the intramodular
hub genes by external traits based GS > 0.2 and MM > 0.8 with a threshold of p-value < 0.05 [64].
In our study, based on the size of the module, we classified the top 10% of the most connected
genes in the module as hub genes. We construct and visualize gene–gene interaction networks using
Cytoscape tool [65].
5. Conclusions
Weighted gene co-expression network associated with traits was constructed, and 19 tissue and
species-specific modules were obtained. The biological significance of the tissue and species-specific
module was revealed, the regulatory genes related to the medicinal components of Dendrobium were
identified, and a local biologically significant network was constructed. The results of this study
can provide important experimental data and theoretical basis for the further analysis of the genetic
mechanism of the medicinal ingredients of Dendrobium.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/3/861/s1.

Author Contributions: Conception and design of the research: Y.Y. and X.T.; acquisition of data: B.Z.; statistical
analysis: X.T.; drafting the manuscript: Y.Y. and J.Z.; revision of manuscript for important intellectual content: J.L.
All authors read and approved the final manuscript.
Funding: This project was funded by the Priority Academic Program Development of Jiangsu Higher Education
Institutions (PAPD), National Foundation of Forestry Science and Technology Popularization (Grant No. [2015]
17), Major Fund for Natural Science of Jiangsu Higher Education Institutions (Grant No. 15KJA220004).
Conflicts of Interest: The authors declare no conflict of interest.
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Molecular Sciences
Communication
Identification of (Z)-8-Heptadecene and
n-Pentadecane as Electrophysiologically Active
Compounds in Ophrys insectifera and Its
Argogorytes Pollinator
Björn Bohman 1,2, *, Alyssa M. Weinstein 3 , Raimondas Mozuraitis 4 , Gavin R. Flematti 1 and
Anna-Karin Borg-Karlson 5
1 School of Molecular Sciences, the University of Western Australia, Crawley, WA 6009, Australia
2 Department of Plant Protection Biology, Swedish University of Agricultural Sciences, 23053 Alnarp, Sweden
3 Research School of Biology, the Australian National University, Acton, ACT 2601, Australia
4 Department of Zoology, Stockholm University, 106 91, Stockholm, Sweden
5 Department of Chemical Engineering, Mid Sweden University, 85170 Sundsvall, Sweden
* Correspondence: [email protected]

Received: 25 December 2019; Accepted: 14 January 2020; Published: 17 January 2020
Abstract: Sexually deceptive orchids typically depend on specific insect species for pollination,
which are lured by sex pheromone mimicry. European Ophrys orchids often exploit specific species
of wasps or bees with carboxylic acid derivatives. Here, we identify the specific semiochemicals
present in O. insectifera, and in females of one of its pollinator species, Argogorytes fargeii. Headspace
volatile samples and solvent extracts were analysed by GC-MS and semiochemicals were structurally
elucidated by microderivatisation experiments and synthesis. (Z)-8-Heptadecene and n-pentadecane
were confirmed as present in both O. insectifera and A. fargeii female extracts, with both compounds
being found to be electrophysiologically active to pollinators. The identified semiochemicals were
compared with previously identified Ophrys pollinator attractants, such as (Z)-9 and (Z)-12-C27 -C29
alkenes in O. sphegodes and (Z)-9-octadecenal, octadecanal, ethyl linoleate and ethyl oleate in O.
speculum, to provide further insights into the biosynthesis of semiochemicals in this genus. We
propose that all these currently identified Ophrys semiochemicals can be formed biosynthetically from
the same activated carboxylic acid precursors, after a sequence of elongation and decarbonylation
reactions in O. sphegodes and O. speculum, while in O. insectifera, possibly by decarbonylation without
preceding elongation.
Keywords: Ophrys; sexual deception; semiochemicals; fly orchid; pollination
1. Introduction
Pseudocopulation as a means of pollination was first reported over 100 years ago, in two parallel
systems [1,2]. Correvon and Pouyanne made observations of European Ophrys orchids [1], while in
Australia, Cryptostylis orchids were reported to use the same sexually deceptive strategy, in which insect
pollinators attempt copulatory or courtship behaviour with the flower, thereby transferring pollinia [2,3].
Insect sexual attraction is induced through chemical and physical mimicry of female insects. Pollination
by sexual deception is now known to be a phenomenon that has evolved independently multiple times
on different continents. There are several hundred confirmed cases in the Orchidaceae, with many
more likely to be discovered with future studies [4–6]. There are also single reports of sexual deception
in the Asteraceae [7] and Iridaceae [8], indicating that this pollination strategy may be more common
than is currently known.

Int. J. Mol. Sci. 2020, 21, 620
Following the initial observations of pollination via sexual deception in Ophrys and Cryptostylis
orchids, an intensive Swedish research program was launched in 1948 to investigate the chemical cues
underlying this bizarre pollination strategy. Ophrys insectifera and some southern European Ophrys
and their solitary bee pollinator species were the main study species [9]. In these early studies, field
experiments demonstrated that floral volatiles were the key to pollinator attraction [9,10]. With the use
of electroantennography (EAG), it was later shown that two species of male sphecid wasp pollinator,
Argogorytes mystaceus and A. fargeii, unlike their conspecific females, responded to tentatively identified
alkanes, alkenes, and terpenes in sorption headspace extracts of O. insectifera flowers [11]. A few years
later, the first evidence of chemical mimicry of several species of Andrena bee pollinators by O. fusca
and O. lutea, was found: aliphatic alcohols, monoterpene- and sesquiterpene alcohols, and aldehydes
attracted the patrolling males to varying degrees [12,13].
The first identification of pollinator sexual attractants in the genus Ophrys did not occur until
the late 1990s, with the successful structural elucidation of attractants from O. sphegodes [14,15].
A key to the detection and identification of the semiochemicals from this species was the use of
gas chromatography coupled with electroantennogram detection (GC-EAD), which revealed a set
of 14 electrophysiologically active compounds to be shared among the orchid and the female of its
bee pollinator, Andrena nigroaenea. Before being confirmed as attractants in field bioassays, these
compounds were identified by GC-MS, including microderivatisation experiments, as a series of
long-chained alkanes and alkenes. Furthermore, three (Z)-7 alkenes were discovered to be responsible
for the attraction of male Colletes cunicularius bees to O. exaltata [16]. The chemical stimuli for the sexual
attraction of various Ophrys pollinators also include other types of structures, as shown when a mixture
of hydroxy- and keto acids, together with aldehydes and esters, were identified as the attractants in O.
speculum, which is pollinated by male Campsoscolia ciliata scoliid wasps [17].
In Australian sexually deceptive orchids, 1,3-cyclohexanediones (chiloglottones) have been
identified as pollinator attractants in Chiloglottis [18], as have hydroxymethylpyrazines and
a β-hydroxylactone (drakolide) in Drakaea [19–22], (methylthio)phenols, acetophenones and
monoterpenes in Caladenia [23–25], and tetrahydrofuran acid derivatives in Cryptostylis [26].
Besides the discovery of a broad range of compounds pivotal for pollination in sexually deceptive
orchids, there has also been interest in the biosynthesis of these compounds, with the aim to link
biosynthesis to the evolution and speciation of orchids. Schlüter and Schiestl [27] predicted that, in
Ophrys, the biosynthesis of alkenes would follow the biosynthetic pathway for alkanes [28], but with the
addition of an extra desaturation step, potentially achieved by stearoyl-acyl carrier protein desaturases
(SAD). Later, three putative SAD genes (SAD1-SAD3) were isolated [29]. Transgenic expression and
in vitro enzyme assays revealed SAD2 to be a functional desaturase capable of introducing 18:1 Δ9 and
16:1 Δ4 fatty acid intermediates, from which it was hypothesized that (Z)-9 alkenes and (Z)-12 alkenes
are built. Three additional putative SAD genes (SAD4-SAD6) were also identified from an O. sphegodes
transcriptome [30].
In O. sphegodes and O. exaltata, SAD1 and SAD2 expression levels were shown to be significantly
correlated with (Z)-9 and (Z)-12-alkene production, while high SAD5 expression was correlated with
the (Z)-7-alkene production unique to O. exaltata [31]. In vitro enzyme activity studies further showed
that a putative housekeeping desaturase, SAD3, catalyses the general reactions of stearate to oleate
(18:0-ACP to 18:1 Δ9 -ACP), and palmitate to palmitoleate (16:0-ACP to 16:1 Δ9 -ACP), whereas SAD5 is a
specialized 16:0 Δ9 -ACP enzyme [32]. Subsequent elongation of a 16:1 Δ9 -ACP to a 26:1 Δ19 -coenzyme
A precursor, followed by decarbonylation, would yield the (Z)-7 alkene (25:1 Δ7 ) that characterizes
O. exaltata.
In O. speculum, the pollinator attractants were also identified as carboxylic acid derivatives [17].
The most attractive compounds from both floral extracts and females of the scoliid wasp pollinator
Campsoscolia ciliata were (ω-1)-hydroxy- and -oxo acids. However, it is noteworthy that the
pseudo-copulation rates in field bioassay experiments more than doubled when aldehydes such
96
Int. J. Mol. Sci. 2020, 21, 620
as (Z)-9-octadecenal and octadecanal, together with the esters ethyl linoleate and ethyl oleate, were
added to the dummy female [17].
The phylogenetic relationships within Ophrys are currently under debate [33–37], with some
phylogenetic analyses indicating that the Argogorytes-pollinated O. insectifera group represents a basal
taxon, while the latest studies place the O. fusca complex, including O. iricolor, as ancestral [36,37]. All
studies agree that wasp pollination is ancestral to bee pollination in Ophrys.
To obtain a broader understanding of the chemical details of semiochemicals in the wasp-pollinated
O. insectifera, and sex pheromone candidates in its pollinator A. fargeii, we used GC-EAD, GC-MS,
microderivatisation reactions, and organic synthesis to identify EAD-active compounds. These
semiochemicals were compared with previously identified pollinator attractants from the bee-pollinated
O. sphegodes and wasp-pollinated O. speculum, and biosynthetic relationships within Ophrys
were proposed.
2. Results and Discussion

To identify semiochemicals in O. insectifera, and sex pheromone candidates in Argogorytes fargeii
pollinators, solvent extractions of flowers and insects, and floral headspace sampling, were conducted.
Samples of O. insectifera labella were extracted in solvents of increasing polarity, from n-hexane,
to dichloromethane, to methanol. Headspace volatile sampling was performed using solid phase
extraction (SPME). Furthermore, whole females of A. fargeii were extracted in dichloromethane. Due
to the very limited number of pollinators available, we were restricted to evaluating biological
activity using gas chromatography coupled with electroantennography (GC-EAD). Since we were
unable to locate males of A. fargeii, GC-EAD was used to detect which components of the various
extracts were detected by A. mystaceus, a closely related species that is the second main pollinator
of O. insectifera [9]. Two compounds from the floral extracts were repeatedly EAD-active (elicited
responses in six out of 10, and two out of 10 EAD experiments). These two compounds were tentatively
identified by mass spectrometry (GC-MS) as a C17 alkene and n-pentadecane. In previous studies on
O. insectifera, n-pentadecane (2, Figure 1a) was indeed found to be active in EAG experiments, while
no alkenes were isolated or identified [11]. Here, we found that n-pentadecane and the C17 alkene
were present in the female A. fargeii (six extracts of individual insects) and were also present in only
minor amounts in floral solvent extracts (three extracts of 10 flowers). We investigated the double
bond location by dimethyldisulfide (DMDS) microderivatisation of a semi-preparative GC purified
compound that was extracted from the wasp. The observation of identical retention times and mass
spectra between the semiochemical isolated from the wasp and the synthesized (Z)-8-heptadecene
(1), before and after treatment with DMDS, meant that the double bond position and configuration of
the natural product could be confirmed. Furthermore, a floral extract was treated analogously, and
was confirmed to contain identical mass fragments at the same relative intensity and retention time,
confirming that the compound detected by A. mystaceus was shared between O. insectifera and female
A. fargeii. In addition to the semiochemicals identified from flowers, another two C15-alkenes and
one C17-diene were identified from females of A. fargeii. These compounds were also isolated by
semi-preparative GC and treated with DMDS. Candidate compounds were synthesized and co-injected
with natural extracts (on two GC columns) and tested with GC-EAD. The monoenes were subsequently
confirmed as (Z)-6-pentadecene (3) and (Z)-7-pentadecene (4), while the diene was identified as
(Z,Z)-6,9-heptadecadiene (5) (Figure 1).
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Int. J. Mol. Sci. 2020, 21, 620
Figure 1. (a) Semiochemicals from Ophrys insectifera (1–2; 1 = (Z)-8-heptadecene, 2 = n-pentadecane)

and female Argogorytes mystaceus (1–5; 3 = (Z)-6-pentadecene, 4 = (Z)-7-pentadecene, 5 =
(Z,Z)-6,9-heptadecadiene). (b) GC-MS total ion chromatograms of female A. fargeii (upper trace)
and O. insectifera (lower trace). (c) GC-EAD of SPME extracts of O. insectifera to antenna of A. mystaceus
males. Two replicated analyses are shown. (d) GC-EAD of synthetic standards 1–5 to antenna of A.
mystaceus. Two replicated analyses are shown.
The GC-EAD and GC-MS analyses of the floral extracts showed that n-pentadecane (2) was of low
abundance and was electrophysiologically active in only two experiments, while (Z)-8-heptadecene (1)
was active in six experiments. When tested as synthetics at higher concentrations (100 ng to 1 μg), both
compounds were strongly EAD-active in replicated experiments. However, the additional alkenes 3–5
from A. fargeii, when tested as synthetic samples at the higher concentration, elicited consistently less
frequent and/or weaker EAD responses compared to the orchid-produced 1 and 2 (Figure 1, Table 1).
Table 1. Occurrence of semiochemicals in Ophrys insectifera (SPME extracts) and Argogorytes fargeii
females (solvent extracts), with electroantennographic responses in A. mystaceus males.
Abundance in Abundance in
Compound EAD-Activity
O. insectifera A. fargeii (Female)
1
2
3 – ()
Argogorytes mystaceus visiting 4 – ()
Ophrys insectifera 5 – ()
= very abundant compound (>20% of base peak area); repeated (6 extracts, >6 synthetic samples) strong
EAD-responses. = abundant compound (>10% of base peak area); repeated EAD-responses (2 extracts, >6
synthetic samples). () = occasional weaker EAD-response (generally less than 50% of response of orchid
semiochemicals, >3 synthetic samples). Photo A.M. Weinstein.
By analysing the GC-MS traces of floral extracts, it was observed that larger amounts of compounds
1 and 2 were present in headspace samples of flowers compared with solvent extracts. Although
headspace extractions and solvent extractions are not directly comparable, our findings indicate that
the flowers likely continuously produce compounds (indicated by increasing quantity with an increase
in SPME sampling time), rather than depend on stored compounds (indicated by very low amounts
in solvent extracts) in the floral tissue. This observation is in agreement with earlier studies of O.
insectifera and O. sphegodes, favouring headspace sorption extraction over solvent extraction [14,38].
98
Int. J. Mol. Sci. 2020, 21, 620
In addition to comparing observations between various Ophrys systems, it is of further interest

to extend this comparison to other sexually deceptive orchids with known semiochemistry. Such
cases are predominantly Australian, where the pollinator attractants in hammer orchids and spider
orchids, unlike in Ophrys, have been found to be stored in relatively large amounts within the floral
tissue [21,23,25].
The discovery of (Z)-8-heptadecene (1) in O. insectifera, detected by males of A. mystaceus, provides
important insights about the chemistry of Ophrys orchids. In earlier studies of the biosynthetic
pathways for the longer chained C25 and C27 alkenes from O. exaltata and O. sphegodes, C16 - and C18
activated carboxylic acids have been proposed as intermediates [32] (Figure 2). In fact, it has been
proposed that in the plastid of the lip epidermis cell of the labellum of O. exaltata and O. sphegodes,
16:0-ACP and 18:0-ACP are transformed to 16:1 Δ4 -ACP and 18: 1 Δ9 -ACP by SAD2, before being
elongated in the cuticle [29]. If instead, 16:0-ACP and 18:1 Δ9 -ACP are decarbonylated, the exact
compounds found to be EAD-active in O. insectifera, n-pentadecane (2) and (Z)-8-heptadecane (1),
would be formed (Figure 2).
ȱ
Figure 2. Proposed biosynthesis of bioactive alkenes in Ophrys sphegodes (from [32]) and O. insectifera.
In a similar manner, our results can be compared to the pollinator attractants previously identified
in O. speculum. Out of the blend of eight electrophysiologically active compounds that showed the
highest pollinator attraction in field bioassays, three compounds: hexadecanal, (Z)-9-octadecenal,
and ethyl oleate, show strong structural similarity with the hydrocarbons that we identified in O.
insectifera. In fact, decarbonylation of these semiochemicals, in a similar way as proposed in the case of
O. sphegodes (Figure 2), would yield pentadecane (2) from hexadecanal and (Z)-8-heptadecene (1) from
(Z)-9-octadecenal and ethyl oleate.
Compared to the recent studies of Australian Drakaea and Caladenia orchids, where multiple,
structurally diverse pollinator attractants have been identified in multiple species [21–23,25], the
structural similarities between the semiochemicals of O. insectifera, O. sphegodes, and O. speculum are
evident, all being clearly biosynthetically closely related carboxylic acid derived compounds. It is
also interesting to note the difference in volatility compared to the widely studied Australian systems,
where “traditional” volatiles are used as long-range attractants, while the European systems utilise
less volatile cuticular hydrocarbons, such as the C27 –C29 alkenes in O. sphegodes, which have been
proven sufficiently volatile to lure pollinators from a distance as attractants [15]. Furthermore, it is
relevant to note that in the case of O. insectifera and A. fargeii, the orchid and pollinator share the exact
same semiochemicals, which is in agreement with other investigated Ophrys systems, including O.
sphegodes [39] and O. speculum [17], as well as with most Australian systems [4] (but see [40]).
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Int. J. Mol. Sci. 2020, 21, 620
In conclusion, we have identified (Z)-8-heptadecene (1) and pentadecane (2) as shared

semiochemicals from O. insectifera and A. fargeii. Access to denser populations of A. fargeii or A.
mystaceus would be required to undertake bioassays testing the field activity of these compounds as
pollinator attractants. Nevertheless, this study provides an important first step in the identification of
key compounds that, once pollinator populations have been located, are available to be tested in field
behavioural bioassays. Furthermore, the identification of these semiochemicals and comparison with
related species within the genus shows strong commonalities in structures and suggests a conserved
biosynthetic pathway for semiochemical production within Ophrys.
3.1. General Experimental Procedures

NMR spectra (Supplementary Materials) were acquired on a Bruker Avance (Bruker, Billerica,
MA, USA) 500 or 600 MHz spectrometer with CDCl3 as solvent. Chemical shifts were calibrated to
resonances attributed to the residual solvent signal.
EIMS (70 eV) were recorded on an Agilent 5973 mass detector connected to an Agilent 6890 GC
equipped with a DB5-MS column (Agilent, Santa Clara, CA, USA, 50 m × 0.2 mm × 0.33 μm) using
helium as the carrier gas.
Semi-preparative gas chromatography was performed on an HP 5890 GC (Agilent, USA), equipped
with a three-way glass splitter separating the gas flow post column into the FID and the collector. An
RTX-5 column, 30 m × 0.53 mm id × 5 μm film (Restek, Bellefonte, PA, USA) was used. Samples of 3
μL were injected in splitless mode (1 min) and helium was used as the carrier gas. A custom-made
manual fraction collector was used, with samples collected in glass capillaries (100 × 1.55 mm id,
Hirschmann Laborgeräte, Eberstadt, Germany) positioned in an aluminium holder submerged in a dry
ice/acetone bath. All fractions were eluted with dichloromethane and stored at −20 ◦ C until used for
microderivatisation experiments [26].
GC-EAD data were recorded using an Agilent 6890 GC equipped with an identical column as
the GC-MS and a flame ionization detector (FID) using helium as carrier gas. A GC effluent splitter
(split ratio 1:1) was used to split the flow to the FID and EAD. The split for EAD was passed through a
Syntech effluent conditioner (Syntech, Buchenbach, Germany) containing a heated transfer line, with
the outlet placed in a purified and humidified airstream, where the electrodes holding the antenna
were presented. For each EAD run, an excised antenna with the tip cut off, was mounted on a holder
consisting of two electrodes using electrode gel. The electrode was connected to a PC via a serial
Syntech intelligent data acquisition controller (IDAC) interface for simultaneous recording of the FID
and EAD signals in the Syntech software package.
Solvents for extractions and purifications were of HPLC grade.
3.2. Plant Material and Insects

All plants and insects were collected in June over four years (2016–2019) at various field locations
in Sweden. Ophrys insectifera were sourced from several populations across the central parts of Öland.
Flowers were kept in cooler boxes (ca. 4 ◦ C) while transported to the laboratory, where they were
either sampled with solid-phase microextraction (SPME), extracted with hexane, dichloromethane
or methanol, or kept as baiting flowers to collect male insects. Male Argogorytes mystaceus were
collected from O. insectifera flowers on stems (20 flowers) near Torslunda, Öland or near Södertälje,
Södermanland. Female Argogorytes fargeii were collected from food plants, Pastinaca sativa, near
Långöre, Öland.
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Int. J. Mol. Sci. 2020, 21, 620
3.3. Extraction and Isolation

Flowers for SPME were enclosed in oven bags (Multix 25 cm × 38 cm, McPherson’s Limited,
Kingsgrove, NSW, Australia) and sampled for 24 h (DVB/CAR/PDMS, Supelco, Bellefonte, PA, USA) at
room temperature. For solvent extractions, labella were removed and batches of 20 were extracted in
each solvent (ca. 1 mL) for 48 h. The extracts were concentrated under a gentle stream of nitrogen at
room temperature to a final volume of ca. 0.1 mL, stored at −20 ◦ C and subsequently used for GC-MS
and GC-EAD analyses. Female A. fargeii were extracted in dichloromethane (ca. 0.5 mL) for 48 h. The
extracts were concentrated and treated as for the floral extracts.
3.4. Structure Elucidations of Alkenes

From a concentrated dichloromethane extract of a female A. fargeii, the two fractions containing
C15-alkenes and C17-alkenes were isolated by semi-preparative GC. Each fraction, in hexane (30 μL),
was treated with DMDS (50 μL) and iodine in diethyl ether (5 μL, 60 mg/mL). The reaction mixtures
were left at 40 ◦ C in vials over night before being washed with sodium bisulphite (5%) and concentrated
to ca. 20 μL under nitrogen before being analysed by GC-MS [41]. The fraction containing C15-alkenes
contained two compounds, with characteristic ions for 6-pentadecene (M = 304, fragments m/z = 131,
173) and 7-pentadecene (M = 304, fragments m/z = 145, 159). The fraction containing C17-alkenes
contained one monoene and one diene, with characteristic ions for 8-heptadecene (M = 332, fragments
m/z = 159, 173, also present in O. insectifera) and 6,9-heptadecene (M = 362, fragments m/z = 131, 155,
159, 183, 203 and 231) [42].
3.5. Preparation of Alkenes

All alkenes apart from 7-pentadecene were prepared from the corresponding C16- and
C18-carboxylic acids (oleic acid, linoleic acid, palmitoleic acid) via a modified Barton reductive
decarboxylation [43]. 7-Pentadecene was synthesised via 7-pentadecyne, prepared from
1-bromoheptyne and octylmagnesium chloride [44]. The alkyne was partially reduced to the cis-alkene
in a low yield by the method of Obora et al. [45], although in amounts sufficient to our needs.
Supplementary Materials: Supplementary materials (NMR spectra for all semiochemicals) can be found at
http://www.mdpi.com/1422-0067/21/2/620/s1.
Author Contributions: Conceptualization, B.B. and A.-K.B.-K; methodology, B.B.; R.M. and G.R.F.; investigation,
B.B., A.M.W., A.-K.B.-K.; resources, B.B., R.M. and G.R.F.; writing—original draft preparation, B.B.; writing—review
and editing, all authors.; funding acquisition, B.B. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by the Australian Research Council, DE160101313.
Acknowledgments: B.B. acknowledges the Australian Research Council for funding (DE160101313). D. Bainbridge
is gratefully acknowledged for designing and fabricating the preparative GC collector used in this study. The
authors acknowledge the facilities, and the scientific and technical assistance of the Australian Microscopy &
Microanalysis Research Facility at the Centre for Microscopy, Characterization & Analysis, The University of
Western Australia, a facility funded by the University, State and Commonwealth Governments. The authors thank
Kerstin Persson for allowing the use of her land for this study, Bo G. Svenson for assistance with the identification
of insects, and Monica Ruibal and Rod Peakall for DNA sequencing of several of our wasp samples, which
provided confirmation of our species identification.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
101
Int. J. Mol. Sci. 2020, 21, 620
Abbreviations
EAG Electroantennography
GC Gas chromatography
EAD Electroantennographic detection
SAD Stearoyl-acyl carrier protein desaturase
ACP Acyl-carrier protein
EIMS Electron impact mass spectrometry
GCMS Gas chromatography mass spectrometry
NMR Nuclear magnetic resonance
FID Flame ionization detector
HPLC High-pressure liquid chromatography
SPME Solid-phase microextraction
DVB Divinyl benzene
CAR Carboxen
PDMS Polydimethylsiloxane
DMDS Dimethyl disulphide
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Molecular Sciences
Article
Combined Metabolome and Transcriptome Analyses
Reveal the Effects of Mycorrhizal Fungus
Ceratobasidium sp. AR2 on the Flavonoid
Accumulation in Anoectochilus roxburghii during
Different Growth Stages
Ying Zhang, Yuanyuan Li , Xiaomei Chen, Zhixia Meng * and Shunxing Guo *
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical
College, Beijing 100193, China; [email protected] (Y.Z.); [email protected] (Y.L.);
[email protected] (X.C.)
* Correspondence: [email protected] (Z.M.); [email protected] (S.G.)

Received: 18 November 2019; Accepted: 9 January 2020; Published: 15 January 2020
Abstract: Anoectochilus roxburghii is a traditional Chinese herb with high medicinal value, with
main bioactive constituents which are flavonoids. It commonly associates with mycorrhizal
fungi for its growth and development. Moreover, mycorrhizal fungi can induce changes in
the internal metabolism of host plants. However, its role in the flavonoid accumulation in
A. roxburghii at different growth stages is not well studied. In this study, combined metabolome
and transcriptome analyses were performed to investigate the metabolic and transcriptional
profiling in mycorrhizal A. roxburghii (M) and non-mycorrhizal A. roxburghii (NM) growth for six
months. An association analysis revealed that flavonoid biosynthetic pathway presented significant
differences between the M and NM. Additionally, the structural genes related to flavonoid synthesis
and different flavonoid metabolites in both groups over a period of six months were validated
using quantitative real-time polymerase chain reaction (qRT-PCR) and high-performance liquid
chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). The results showed that
Ceratobasidium sp. AR2 could increase the accumulation of five flavonol-glycosides (i.e., narcissin,
rutin, isorhamnetin-3-O-beta-d-glucoside, quercetin-7-O-glucoside, and kaempferol-3-O-glucoside),
two flavonols (i.e., quercetin and isorhamnetin), and two flavones (i.e., nobiletin and tangeretin) to
some degrees. The qRT-PCR showed that the flavonoid biosynthetic genes (PAL, 4CL, CHS, GT, and
RT) were significantly differentially expressed between the M and NM. Overall, our findings indicate
that AR2 induces flavonoid metabolism in A. roxburghii during different growth stages, especially in
the third month. This shows great potential of Ceratobasidium sp. AR2 for the quality improvement of
A. roxburghii.
Keywords: Anoectochilus roxburghii; Ceratobasidium sp.; metabolome and transcriptome analyses;

flavonoid; HPLC-MS/MS; qRT-PCR
1. Introduction
Anoectochilus roxburghii (Wall.) Lindl., also called “Jin Xianlian” and “Jin Xianlan” is a perennial
herb of the genus Anoectochilus of the Orchidaceae family. It is widespread in southern China and
considered a famous drug in the provinces of Jiangxi, Taiwan, Guangdong, Guizhou, Zhejiang, and
Fujian which is its main markets [1]. The herb is a valuable Chinese medicinal material that is
known as the “King Medicine”, the “Golden Grass”, and the “Bird Ginseng” by countryfolk [2]. The

Int. J. Mol. Sci. 2020, 21, 564
whole plant is used as a medicine and has the efficacy to clear the heat, cool the blood, eliminate
dampness, and detoxification. Many pharmacological studies have demonstrated its wide range
of pharmacological effects including its antioxidant [3,4], hepatoprotective [3], anti-diabetic [5,6],
anti-rheumatoid arthritis [7], anti-hyperglycemic [4,8], anti-inflammatory [9] and many other properties.
Due to the fact of its high medical values, A. roxburghii is not only widely used in medicine and health
care but also in beauty and drinking products with an increasing market demand. The average annual
demand in South Korea and Japan is more than 1000 t, 70% of which depend on imports [10]. The
vigorous market demand results in excessive harvesting and exploitation of the plant, leading to
resource depletion. Thus, tissue culture has become the main source of commercial A. roxburghii.
Most orchids form mycorrhizae with mycorrhizal fungi [11]. The fungal hyphae form coiled
structures termed “peloton” inside the cells of the plant roots which play a key role in the nutrients’
exchange and absorption between the orchid and its mycorrhizal fungi [12]. The process is unique
and complex involving various processes related to growth and development, such as colonization,
increasing the survival rate and morphological growth. Moreover, the mycorrhizal fungal elicitor can
rapidly induce the expression of relative genes that are related to secondary metabolic pathways which
result in a significant accumulation of active ingredients in the host plant [13]; this was demonstrated
in orchid by a few studies. The symbiosis between Dendrobium nobile and Mycena sp. MF23 caused the
accumulation of dendrobine and polysaccharide [14,15]. Mycena sp. MF23 could also stimulate the
accumulation of flavonoid and kinsenoside in A. formosanus [16]. For A. roxburghii, the major bioactive
components include polysaccharides, kinsenosides, steroids, triterpenes, amino acids, alkaloids,
and flavonoids that have been regarded as the quality standard of A. roxburghii [10]. However, A.
roxburghii contains a very limited number of flavonoids, which limits the development and utilization
of A. roxburghii-based medicines. In order to improve the content of flavonoid in A. roxburghii and to
avoid excessive exploitation, many methods have recently been put forward by researchers [17–19];
the role of mycorrhizal fungi in the accumulation of flavonoid in A. roxburghii has been gradually
recognized. Wang et al. reported that flavonoid accumulated significantly in A. roxburghii growth for
8 weeks treated with different fungi such as Rhizoctonia sp. cw-6 and cw-13, Exophila pisciphila (cw-8),
Nemania sp. (cw-10), and Umbelopsis sp. (cw-1) [20]. However, few studies investigated the effects of
mycorrhizal fungi on flavonoid accumulation in A. roxburghii during different growth stages.
The flavonoid biosynthetic pathway has been well characterized in some medicinal plants such as
Gnetum parvifolium [21], Chrysanthemum morifolium [22], Lotus japonicus [23]. Its biosynthesis can be
divided into two stages: phenylpropanoid and flavonoid pathways. Phenylalanine ammonia-lyase
(PAL) is the first enzyme of the phenylpropanoid pathway which can convert phenylalanine into
cinnamic acid [24]. Cinnamic acid is then converted into p-coumaric acid by trans-cinnamate
4-hydroxylase (C4H). Next, 4-coumarate CoA ligase (4CL) converts coumaric acid into its CoA ester.
4CL is one of the key branch point enzymes in the phenylpropanoid pathway and its products
are subsequently used by various oxygenases, reductases, and transferases for the biosynthesis of
lignin, flavonoids, anthocyanins, aurones, stilbenes, coumarins, suberin, cutin, and sporopollenin [25].
Chalcone synthase (CHS) and chalcone isomerase (CHI) are involved in two step condensation reaction,
producing naringenin chalcone and naringenin, respectively. Then, flavanone is catalyzed by flavonoid
3 -hydroxylase (F3 H) and other enzymes. Subsequently, flavanone produces the branches of flavone
and dihydroflavonol under the catalysis of flavone synthase (FNS) and flavanone 3-hydroxylase
(F3H), respectively. Next, flavonol synthase (FLS) catalyzes C-3 hydroxylation in the structure
of dihydroflavonols to form various flavonols, and flavonol-glycosides are formed by flavonoid
3-O-glucosyltransferase (GT) and rhamnosyltransferase (RT) or GT.
With the rapid development of high-throughput sequencing technology and systems biology,
multi-omics technology has become an indispensable research method in the field of life science [26,27].
It can provide the dynamic changes of the plant’s growth and development from the cell to the
individual level. The metabolome is a powerful approach to qualitatively and quantitatively analysis
all the small-molecule metabolites (mass ≤ 1000 Da) in the cells or tissues of an organism at any
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Int. J. Mol. Sci. 2020, 21, 564
physiological period using different analysis technologies including nuclear magnetic resonance (NMR)
spectroscopy, liquid chromatograph-mass spectrometer (LC-MS), and gas chromatography-mass
spectrometer (GC-MS) [28]. In other words, this method could provide the global metabolic changes.
Similarly, the transcriptome means the detection of all RNA transcripts in a sample and reflects gene
expression differences between different treatments [29]. Integrated transcriptome and metabolome
analyses have been successfully applied to study the metabolic pathways of some substances [30,31],
the color formation of vegetables, fruits, and flowers [32,33], the stress resistance mechanisms [34,35],
and the growth and development mechanisms of the plants [36,37]. The combination can not only
elucidate changes in the content of a series of metabolites, but it can also analyze the corresponding
differentially expressed genes.
To investigate the changes of metabolites in A. roxburghii that is infected with mycorrhizal fungus,
we performed metabolome and transcriptome analyses on six-month growth data of the mycorrhizal
A. roxburghii (M) and non-mycorrhizal ones (NM). The results indicated that AR2 significantly promoted
flavonoid biosynthesis in the plant. During the growth stage, the flavonoid content (two flavones:
nobiletin and tangeretin; two flavonols: quercetin and isorhamnetin; five flavonol-glycosides: narcissin,
rutin, isorhamnetin-3-O-beta-d-glucoside, quercetin 7-o-glucoside, and kaempferol 3-O-glucoside) in
different metabolites and the expression of the genes that were related to the biosynthesis of flavonoid
were further tested over a period of six months using high-performance liquid chromatography
coupled with tandem mass spectrometry (HPLC-MS/MS) then quantitative real-time polymerase chain
reaction (qRT-PCR). These will provide valuable information to reveal the effects of Ceratobasidium sp.
AR2 on the flavonoid accumulation in A. roxburghii.
2. Results
2.1. Detecting Mycorrhizal Fungus Colonization in A. roxburghii

Pelotons (hyphae coils) formation is the important characteristic of orchid mycorrhizal
association [38]. In the study, Ceratobasidium sp. AR2 would soon grow over the whole substrate
for about two weeks. At this time, both morphological observation and toluidine blue staining,
which showed no hyphae in the control group (Figure 1A) and that the intracellular pelotons existed
in the treatment (Figure 1B), indicated that a symbiotic relationship had been established between
A. roxburghii and mycorrhizal fungus Ceratobasidium sp. AR2.
Figure 1. The semi-thin sections of root of A. roxburghii after two weeks of symbiotic cultivation. (A) the
control, scale bar = 50 mm; (B) the treatment; arrows represent pelotons, scale bar = 50 mm.
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Int. J. Mol. Sci. 2020, 21, 564
2.2. Identification of Metabolites

The M and NM growth for six months were used for metabolome analysis. Overlapping analysis of
the total ion chromatography (TIC) in different quality control (QC) samples showed that the retention
time and peak intensities were consistent (Figure S1), which indicated that the instrument had a good
stability and could thus be used for subsequent analysis. Principal component analysis (PCA) was
performed on the NM, M, and mixed samples. Principal component 1 (PC1) and principal component
2 (PC2) were 54.7% and 26.3%, respectively (Figure 2A). The metabolite profiles of A. roxburghii were
then subjected to orthogonal partial least squares discriminant analysis (OPLS-DA). The result showed
that the R2X, R2Y, and Q2 were 0.862, 1.000, and 0.995, respectively (Figure 2B), which indicated the
model of OPLS-DA was stable and reliable. The score plots of PCA and OPLS-DA exhibited an obvious
separation between the M and NM, and each formed a cluster. These suggested that mycorrhizal
fungus AR2 affected the metabolism in A. roxburghii.
Figure 2. PCA and OPLS-DA scores plots derived from ultra-performance liquid
chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) profiling
of non-mycorrhizal A. roxburghii (NM) and mycorrhizal A. roxburghii (M) growth for six months.
(A) PCA scores plot of the two samples (NM and M growth for six months) and the quality control
sample (mix, the same volume of sample extract from the NM and M growth for six months was
prepared by mixing); the x-axis represents the PC1 and the y-axis represents PC2. (B) OPLS-DA
scores plot of the putatively annotated metabolites from NM and M growth for six months. The x-axis
represents the score value of main components in the orthogonal signal correction process and the
differences between the groups can be seen from the direction of the x-axis; the y-axis represents the
scores of orthogonal components in the orthogonal signal correction process and the differences within
the groups can be seen from the direction of the y-axis.
A total of 709 metabolites with known structures were identified in M and NM under quality
validation, each of which was analyzed using three biological replicates. Detailed information about
the identified metabolites, including the compounds, classes, molecular weights, ionization models,
Kyoto encyclopedia of genes and genomes (KEGG) pathways, and quantities for each of the three
periods is shown in Table S1. Flavonoid (20.9%), organic acids, and derivatives (15.4%), amino acids
and derivatives (12.8%), lipid (9.6%), and phenylpropanoid (8.7%) accounted for a large proportion of
these 709 metabolites (Figure 3A).
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Figure 3. Component analysis of the putatively annotated metabolites and pathway enrichment analysis
of the DAMs. (A) Component analysis of the putatively annotated metabolites from non- mycorrhizal
A. roxburghii (NM) and mycorrhizal A. roxburghii (M) growth for six months. The percentage of the top
six metabolites are shown in the graph. The percentage after each compound represents the percentage
of the number of DAMs of a certain class of compounds in the total DAMs. (B) Pathway (top 20)
enrichment analysis of the DAMs between the NM and M growth for six months. The x-axis represents
the corresponding rich factor of each pathway. The y-axis represents the name of pathway. The color
of the dot is p-value, and the closer it is to 0, the more significant the enrichment is. The size of the
point represents the number of DAMs enriched in the corresponding pathway. The rich factor is the
ratio of the number of metabolites in the corresponding pathway to the total number of metabolites
detected and annotated in the pathway. The higher the value of rich factor is, the higher the enrichment
degree is.
2.3. Identification of Differentially Accumulated Metabolites and Differentially Accumulated Flavonoids

Differentially accumulated metabolites (DAMs) were defined as those exhibiting a fold change ≥ 2
or ≤0.5 and a variable importance of projection (VIP) ≥ 1. In total, 135 DAMs were identified, among
which 63 and 72 metabolites were upregulated and downregulated, respectively. To further understand
the DAMs function and the related biological processes they participated in, pathway enrichment
analysis of the DAMs was conducted using KEGG. The results showed that the terms “Biosynthesis
of phenylpropanoids”, “Flavonoid biosynthesis”, “Polycyclic aromatic hydrocarbon degradation”,
“Oxidative phosphorylation”, and “Naphthalene degradation” were significantly enriched (Figure 3B).
Among them, the flavonoid biosynthesis began with the biosynthesis of phenylpropanoids. To some
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extent, these data further indicated that the flavonoid metabolism in A. roxburghii was significantly
affected by mycorrhizal fungus AR2.
Furthermore, 148 metabolites involved in the flavonoid metabolism were identified, among
which nine metabolites that belonged to two flavones (i.e., nobiletin and tangeretin), two
flavonols (i.e., quercetin and isorhamnetin) and five flavonol-glycosides (i.e., narcissin, rutin,
isorhamnetin-3-O-beta-d-glucoside, quercetin-7-O-glucoside, and kaempferol-3-O-glucoside) were
obviously different between M and NM.
2.4. Transcriptome Profiles of Mycorrhizal and Non-Mycorrhizal A. roxburghii

The expression profiles of M and NM grown for six months were also analyzed using
RNA-sequencing (RNA-seq). The Q20 and Q30 base percentages were greater than or equal to 97.79%
and 93.61%, respectively (Table 1). The GC contents of the M and NM were in the range of 48.10–49.06%
and 47.93–48.43%, respectively (Table 1). Compared with the control group, 4341 genes were shown to
be differentially expressed, including 2915 upregulated genes and 1426 downregulated genes.
Table 1. Summary of the analysis of transcriptome sequences from the NM and M growth for six months.
Raw Clean Clean Error Q20 Q30 GC

Sample
Reads Reads Base (G) Rate (%) (%) (%) Content (%)
NM1 61,226,728 61,071,914 9.09 0.017 97.79 93.61 48.19
NM2 60,542,772 60,425,910 8.99 0.016 98.11 94.42 47.93
NM3 67,559,786 67,410,292 10.05 0.016 98.07 94.28 48.43
M1 55,632,192 55,492,010 8.28 0.016 98.02 94.20 49.06
M2 65,007,376 64,859,884 9.68 0.016 98.19 94.58 48.10
M3 67,125,158 66,965,132 9.99 0.016 98.09 94.34 48.23
NM: represents non-mycorrhizal A. roxburghii; M: represents mycorrhizal A. roxburghii.
Genes with |log2 (fold change)| > 1 and q < 0.001 were defined as differentially expressed genes
(DEGs). To further understand the functions of DEGs and the related biological processes they have a
role in, gene ontology (GO) and KEGG analyses were conducted. The GO analysis classified DEGs
into three categories: “molecular function”, “cellular component”, and “biological process” with a
total of 45 GO terms (Figure 4A). The enriched GO terms were “binding” and “catalytic activity”
within molecular function, “cell part”, “cell”, and “organelle” within cellular component and “cellular
process”, “metabolic process”, and “response to stimulus” within biological process. The pathway
enrichment analysis of the DEGs using KEGG identified significantly enriched “metabolic pathways”
and “biosynthesis of secondary metabolites” (Figure 4B). As a result, the transcription analysis also
showed that mycorrhizal fungus AR2 significantly affected the metabolic pathways in A. roxburghii.
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Figure 4. The classification column of GO and KEGG from the DEGs between the NM and M growth
for six months. (A) GO classification of DEGs. The x-axis represents the secondary GO item. The y-axis
represents the proportion of the DEGs in the total number of DEGs. The labels above the columns is
the number and proportion of DEGs of this GO item. (B) KEGG classification of DEGs. The x-axis
represents the name of KEGG pathway. The y-axis represents the proportion of genes annotated to the
pathway in the total of annotated genes. The labels above the columns represent the classification of
KEGG pathway.
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2.5. Association Analysis of the DAMs and DEGs

Correlation analysis was carried out on the DAMs and DEGs. The variations in the metabolites
and their corresponding genes with the Pearson correlation coefficient over 0.8 were selected to draw
nine quadrant diagrams and the correlation coefficient cluster heat map. As shown in Figure 5, the
higher number of DAMs and DEGs was in the seventh and ninth quadrants; and they were positively
correlated in the seventh quadrant and negatively correlated in the ninth quadrant.
Figure 5. Quadrant diagrams representing the association of the DAMs and DEGs between the
non-mycorrhizal and mycorrhizal A. roxburghii growth for six months. The x-axis represents that the
log2 ratio of gene and the y-axis represents the log2 ratio of metabolite; black dotted lines represent the
different threshold; each point represents a gene or metabolite; black dots represent the unchanged genes
or metabolites; green dots represent differentially accumulated metabolites with unchanged genes;
red dots represent differentially expressed genes with unchanged metabolites; blue dots represent
both differentially expressed genes and differentially accumulated metabolites; it is divided into
–
1 9 quadrants from left to right and from top to bottom with black dotted lines; the , 1 2 and 4
quadrants indicate that the expression abundance of metabolites is higher than that of genes; the 3
and 7 quadrants indicate that the expression patterns of genes are consistent with the metabolites; the
5 quadrant indicates that the genes and metabolites are not differentially expressed; the , 6 8 and
9 quadrants indicate that the expression abundance of metabolites is lower than that of genes.
2.6. Dynamic Variations in Flavonoid DAMs

In order to measure the dynamic variations of nine flavonoid DAMs in M and NM at different
growth stages, the extracts were detected using HPLC-MS/MS. The optimized parameters for each
analyte are shown in Table 2. Figure 6 showed the TIC diagrams of nine standards in the positive
and negative ion mode. The method validation including linearity, limit of detection (LOD), limit of
quantification (LOQ), stability, precision, and repeatability is shown in Table 3 with an R2 higher than
0.9901, LOD ranging from 0.488 ng/mL to 15.63 ng/mL, LOQ ranging from 0.977 ng/mL to 31.25 ng/mL,
the relative standard deviations (RSDs) of the stability ranging from 1.94% to 4.22%, the RSDs of the
precision ranging from 1.43% to 4.49%, and the RSDs of the repeatability in the range of 1.74% to 4.93%.
Additionally, the recovery rate was determined by adding 50%, 100% and 150% of the standard content
of the sample. The average recovery rate of nine standards was from 86.03% to 99.54% with RSD
between 1.09% and 4.55% (Table S2). These data indicated the reliability of the method.
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Table 2. The optimized parameters for nine flavonoid standards by HPLC-MS/MS.
Precursor Product Ions (m/z) DP CE EP CXP IS Ionization Retention

No. Name
Ion (m/z) q i (V) (V) (V) (V) (V) Mode Time (min)
PI PI
1 nobiletin 402.9 373.1 388.1 20 33 10 13 5500 ESI+ 4.56
2 narcissin 624.9 316.7 479.6 50 25 10 13 5500 ESI+ 3.51
3 isorhamnetin-3-O-beta-d-glucoside 479.3 317 253 150 34 10 13 5500 ESI+ 3.53
4 tangeretin 372.8 343.1 358.1 10 32 10 13 5500 ESI+ 4.67
5 rutin 609 299.9 279.5 −50 −43 −10 −15 −4500 ESI− 3.44
6 quercetin 301 150.8 178.9 −100 −35 −10 −15 −4500 ESI− 3.69
7 isorhamnetin 314.8 300 150.8 −150 −30 −10 −15 −4500 ESI− 3.84
8 quercetin-7-O-glucoside 462.9 300.9 342.9 −50 −28 −10 −15 −4500 ESI− 3.43
9 kaempferol-3-O-glucoside 592.9 284.9 255 −50 −40 −10 −15 −4500 ESI− 3.50
DP de-clustering potential; EP entrance potential; CXP collision cell exit potential; CE collision energy; PI product ions; q for quantification; i for identification; CUR curtain gas; IS ion spray
voltage; TEM temperature; GS1 ion source gas 1; GS2 ion source gas2; CUR = 35 psi; TEM = 500 ◦ C; both GS1 and GS2 = 55 psi.
Table 3. Method validation results including linearity, LOD, LOQ, stability, precision and repeatability.
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Linearity LOD LOQ Stability Precision Repeatability
No. Name
(ng/mL) (ng/mL) (RSD, %) (RSD, %) (RSD, %)
Regression Equations R2 Ranges (ng/mL)
1 nobiletin y = 62,293,800x − 1,576,600 0.9968 7.81–1000 0.488 0.977 3.05 1.43 4.44
2 narcissin y = 3,029,650x − 314,384 0.9942 31.25–4000 7.81 15.63 3.26 3.22 2.26
3 isorhamnetin-3-O-beta-d-glucoside y = 279,387x − 11,500 0.9973 62.5–4000 15.63 31.25 4.22 4.37 4.93
4 tangeretin y = 4,034,570x − 18,086 0.9902 3.91–250 0.488 0.977 1.94 1.76 4.87
5 rutin y = 3,089,740x − 61,316 0.9978 15.63–1000 7.81 31.25 2.99 3.71 1.74
6 quercetin y = 17,556,400x − 169,184 0.9921 15.63–500 7.81 15.63 3.82 4.03 4.9
7 isorhamnetin y = 116,434,300x − 149,754 0.9903 7.81–250 3.91 7.81 2.53 2.94 2.82
8 quercetin-7-O-glucoside y = 5,857,970x − 144,514 0.9989 31.25–1000 7.81 15.63 3.26 3.31 2.39
9 kaempferol-3-O-glucoside y = 4,499,260x − 117,172 0.9901 31.25–1000 7.81 15.63 2.06 4.49 4.9
Int. J. Mol. Sci. 2020, 21, 564
Figure 6. TIC diagrams of nine flavonoid standards in the positive and negative ion mode. (A) the
positive ion mode; A1 represents the chromatograms of narcissin (retention time = 3.51 min); A2 is
the ones of isorhamnetin-3-O-beta-d-glucoside (retention time = 3.53 min); A3 is the ones of nobiletin
(retention time = 4.56 min); A4 is the ones of tangeretin (retention time = 4.67 min); (B) the negative
ion mode; B1 represents the chromatograms of quercetin-7-O-glucoside (retention time = 3.43 min);
B2 is the ones of rutin (retention time = 3.44 min); B3 is the ones of kaempferol-3-O-glucoside
(retention time = 3.50 min); B4 is the ones of quercetin (retention time = 3.69 min); B5 is the ones of
isorhamnetin (retention time = 3.84 min).
For M and NM growth for six months, the tangeretin content was inhibited, while the content of
the other 8 flavonoid DAMs was significantly upregulated which was consistent with the results of
the metabolome and showed that the metabolome data were reliable. The levels of four metabolites
(i.e., isorhamnetin-3-o-beta-d-glucoside, rutin, isorhamnetin, and kaempferol-3-O-glucoside) gradually
increased across the growth time. For narcissin, quercetin, and quercetin-7-O-glucoside, the content
tended to increase at first, then decrease and then increase again. While the tangeretin content reached
the peak of accumulation at day 0 and the fourth month. Flavonoid accumulation in the plantlets
showed significant difference between the fungal and no-fungal inoculations. The AR2 had a positive
effect on narcissin, rutin, quercetin, and quercetin-7-O-glucoside content in A. roxburghii growth for
one month, while it inhibited the accumulation of isorhamnetin-3-O-beta-d-glucoside, isorhamnetin,
and kaempferol-3-O-glucoside and had no significant effect on nobiletin and tangeretin. Compared
with the control group, significantly higher amount of narcissin, rutin and quercetin-7-O-glucoside
accumulated in mycorrhizal plantlets from the first month to the sixth month. Up until the sixth month,
AR2 could significantly promote the accumulation of nobiletin, but there was no significant difference
at other times. The detailed results are shown in Figure 7. In conclusion, AR2 could significantly affect
the accumulation of different flavonoids in A. roxburghii.
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Figure 7. Dynamic variations of 9 flavonoids in the mycorrhizal and non-mycorrhizal A. roxburghii

growth 0 month to 6 months. NM and M represents non-mycorrhizal A. roxburghii and mycorrhizal
A. roxburghii, respectively. Each value is the mean of three replicates, and error bars indicate standard
deviations. Statistical analysis of the data was performed by independent samples t-test using the SPSS
22.0 software (IBM, Chicago, IL, USA). * and ** above the columns are significantly different at p ≤ 0.05
and p ≤ 0.01, respectively.
2.7. Dynamic Variations of Expression Levels of Flavonoid Biosynthetic Genes

The metabolic pathways of three common flavonoids in A. roxburghii are shown in Figure 8.
qRT-PCR was used to assess the relative expression levels of nine key enzyme genes in the conserved
flavonoid biosynthesis pathway in every treatment group. The results showed that the expression of
the 4CL gene in the treatment group was significantly upregulated during the whole growth process,
compared with the control group (Figure 9). The expression of the PAL and CHS genes in the M was
significantly upregulated at the same growth stage (2 months, 3 months, and 4 months), while it was
almost the same at other growth stages. The AR2 showed an inhibitory effect on the expression of the
C4H and F3 H genes in the 1 month old and 2 month old A. roxburghii, while it showed an inhibitory
effect on the expression of the CHI gene in the 1 month old, 5 month old and 6 month old A. roxburghii.
The F3H/FLS gene had an upregulated expression in the M grown for 1 month and 2 months and
the GT gene also had an upregulated expression in the M grown for 1 month, 2 months, 3 months,
5 months, and 6 months. Finally, the expression of the RT gene was upregulated in the M grown for
1 month and 6 months. In conclusion, AR2 could induce the expression of flavonoid synthesis related
genes in varying degrees and at different growth stages.
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Figure 8. The flavonoid biosynthetic pathway in A. roxburghii. Bold words indicate the key enzymes
in flavonoid biosynthesis. Compounds in the box show flavones, flavonols and flavonol-glycosides
studied in this study.
Figure 9. Expression levels of flavonoid biosynthetic genes in the M and NM growth 0 month to
6 months. M and NM represent the mycorrhizal A. roxburghii and non-mycorrhizal A. roxburghii,
respectively. DNxx_Cx_g1 represents the ID of gene. The x-axis indicates the relative expression level
of the genes. Each value is the mean of three replicates, and error bars indicate standard deviations.
Statistical analysis of the data was performed by independent samples t-test using the SPSS 22.0
software (IBM, Chicago, IL, USA). * and ** above the columns are significantly different at p ≤ 0.05 and
p ≤ 0.01, respectively.
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3. Discussion
Symbiotic association of mycorrhizal fungi with plants has been shown to affect flavonoid
content. The application of co-cultivation of plants with orchid mycorrhizal (OM) fungi and arbuscular
mycorrhizal (AM) fungi are progressing gradually. For AM fungi, they can penetrate and colonize
the root of the host to form intracellular haustoria-like structures known as arbuscules, which are
the principal sites of metabolic exchange between the two organisms [39,40]. Flavonoid content in
Medicago truncatula was increased by AM inoculation [41]. Xie et al. [42] reported that AM colonization
in the soybeans attributed to the increase of certain flavonoids in the root exudates. For OM fungi,
they can form pleloton inside the root cells. And the association of OM with the orchid is the focus of
our laboratory. Some studies indicated that flavonoid accumulated significantly in the mycorrhizal
orchidaceae [16,43]. However, the dynamic changes of flavonoid content in mycorrhizal host at
different developmental stages have rarely been studied. In our study, AR2 was belonging to a member
of the orchid mycorrhizal fungi, and the co-cultivation between A. roxburghii and AR2 was performed.
The dynamic changes of several flavonoids showed that the flavonoid had its special accumulation
content at a defined growth time and that AR2 had different effects on different flavonoids at different
growth stages. Also, AR2 induced the narcissin, rutin and quercetin-7-o-glucoside accumulations in
mycorrhizal plantlets across the growth stage. For narcissin and isorhamnetin-3-o-beta-d-glucoside,
their content in mycorrhizal A. roxburghii growth at three months reached the highest and was more
than 420 ng/g and 120 ng/g, respectively. This was the first report regarding the changes of flavonoid
content induced by AR2 in A. roxburghii at different growth stages.
To investigate the effects of mycorrhizal fungi on metabolites in its host, transcriptome and
metabolome analyses were performed. Zhao et al. [29] reported that the secondary biosynthesis
and hormone balance in the Cymbidium hybridum were induced by mycorrhizal fungus through
transcriptome analysis. Schliemann et al. [44] reported that the biosynthesis of some constitutive
isoflavonoids and plastidial metabolism could be activated by mycorrhizal fungus Glomus intraradices
through metabolome analyses. In our study, metabolome analysis revealed that all 709 metabolites and
135 DAMs were putatively annotated among the NM and M. Among them, 148 flavonoid metabolites
and 9 flavonoid DAMs were investigated. Furthermore, transcriptome analysis revealed that 4341 DEGs
were identified between the two groups, of which 2915 DEGs were up-regulated and 1426 DEGs were
down-regulated; KEGG pathways of the more DEGs were involved in the biosynthesis of secondary
metabolites including flavonoid. These results implied that AR2 might change internal metabolism in
A. roxburghii, especially for flavonoids, which would provide a basis for further study on the molecular
mechanisms of AR2 promoting the flavonoid accumulation in A. roxburghii.
PAL, a key enzyme in the first step of the phenylpropanoid biosynthetic pathway, could be
activated by fungal elicitors. Our study also revealed that the PAL gene had a significant upregulation,
especially in the 2, 3, and 4 month mycorrhizal herbs, compared with the uninoculated ones. This
result is in agreement with the results of Zhou et al. [45] and Xu et al. [46]. It is worth mentioning,
the expression of the 4CL gene in the plantlets inoculated AR2 during the whole growth process was
significantly upregulated, with the highest expression being 13.3 fold. This is also consistent with
Wang et al.’s report [20]. These data imply that AR2 might activate the downstream pathways of
phenylpropanoids including flavonoids.
In addition, CHS, is the key enzyme in the flavonoids synthesis pathway [47]. Harrison and
Dixon [48] reported that the expression level of the gene CHS in the roots of Medicago truncatula was
enhanced by mycorrhizal fungus Glomus versiforme. Xie et al. [49] reported that mycorrhizal symbiosis
induced the expression of the CHS gene of Glycyrrhiza uralensis, and the liquiritin accumulation and the
expression of CHS gene showed a positive correlation. In our study, the expression level of the CHS
gene was also upregulated in the mycorrhizal herbs growth for 2, 3, and 4 months; Meanwhile, the
corresponding flavonoids (narcissin, rutin, isorhamnetin and quercetin-7-O-glucoside) accumulated
in different degrees. Our data added new evidence to support mycorrhizal symbiosis induced the
expression of the CHS gene and promoted the flavonoids accumulation. Additionally, our study
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showed that the GT gene expression was significantly upregulated in the 1–4 month mycorrhizal herbs,
while the RT gene was induced in the 1–6 month ones. The corresponding flavonol-glycoside (narcissin,
rutin, isorhamnetin-3-O-beta-d-glucoside, quercetin-7-O-glucoside and kaempferol-3-O-glucoside)
showed basically the same induction trend in mycorrhizal A. roxburghii. These data again indicated
that AR2 might activate the metabolic pathway of flavonoids.
In summary, this study provides much information about the changes that occur in the main
active ingredient flavonoids and its related genes during different growth stages in M and NM. AR2
has different induction effects on flavonoid content and gene expression in A. roxburghii at different
growth stages. These will provide a theoretical basis for reasonable harvest time of A. roxburghii and a
new insight into improving the quality of the A. roxburghii.
4.1. Plant and Mycorrhizal Fungus Materials

Tissue culture plantlets of A. roxburghii were about 3 month-old and 2–3 cm in height from Yongan
city, Fujian province, China. The mycorrhizal fungus AR2 (NCBI accession No: MN068847) was
previously isolated in our laboratory and deposited at the microbiological center of the Institute of
Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical
College, Beijing, China. Before symbiotic cultures, the fungal strain was inoculated on the potato
dextrose agar medium (PDA: potato 200 g·L−1 , glucose 20 g·L−1 , agar 12 g·L−1 , pH 5.2) in darkness at a
temperature of 25 ± 1 ◦ C for 5 days. Two pieces (0.5 cm3 ) of the fungal inoculum were inoculated on
solid matrix medium (sawdust: wheat bran: water = 3:1:1.5, v/v/v; 101.33 kPa and 121 ◦ C for 180 min)
in darkness at a temperature of 25 ± 1 ◦ C for 15 days. After that, the fungal solid substrates were
obtained for subsequent symbiotic culture.
4.2. Symbiotic Cultures of A. roxburghii Plantlets

In symbiotic cultures of plantlets, the substrate (humus soil: vermiculite: water = 3:1:1) was used.
Each culture bottle (9 cm in diameter, 12.5 cm in height) containing 70 g substrate was sterilized at
101.33 kPa and a temperature of 121 ◦ C for 180 min. Six plantlets, derived from asexual reproduction,
were transferred into each culture bottle which was inoculated with 0.5 g of fungal solid substrates,
while the culture bottles without fungal inoculum served as the control. The symbiotic cultures
were placed in the growth room under a 12/12 h photoperiod at a temperature of 25 ± 1 ◦ C and an
illumination intensity of 1500 Lx. The samples (the whole plantlet) were collected once a month from
the beginning of co-culture until the sixth month. Samples were immediately frozen in liquid nitrogen
and stored at −80 ◦ C for later analyses using LC-MS/MS, RNA-seq and qRT-PCR. Among them, the
collected samples grown for six months were used for the metabolome and transcriptome analyses.
All data were obtained based on three independent biological replicates.
4.3. Histological Study

Two weeks after inoculation, fresh root segments were fixed in 50 mM phosphate buffer (pH 6.8)
containing 2.5% glutaraldehyde and 1.6% paraformaldehyde for 4 h at room temperature. After
fixation, the samples were rinsed three times with phosphoric acid buffer (pH 6.8, 0.1 M) for about
15 min each time and then dehydrated with a graded ethanol series (15% ethanol for 30 min; 30%
ethanol for 30 min; 50% ethanol for 30 min; 70% ethanol for 1 h; 85% ethanol for 1 h; 95% ethanol for
1 h; absolute ethanol for 1 h). After dehydration, the samples were embedded in LRwhite gradient
mixture (25% LRwhite for 24 h; 50% LRwhite for 24 h; 75% LRwhite for 24 h; 100% LRwhite for 24 h)
and polymerized for 48 h at 60 ◦ C. Next, three-μm-thick sections were cut using glass knives with a
rotary microtome (Autocut 2040; Reichert-Jung; Germany). The sections were collected on slides and
stained with 0.05% (w/v) toluidine blue O in benzoate buffer for general histology examinations. The
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sections were examined, and images were captured digitally using a digital camera attached to the
microscope (Axio Imager A1; Carl Zeiss, Oberkochen, Germany).
4.4. Sample Extraction and Metabolome Analysis
4.4.1. Sample Extraction

The cryopreserved samples were freeze-dried and then ground for 1 min at 30 Hz using a
MixerMill MM400 (Retsch Technology, Haan, Germany). Subsequently, 100 mg powder was weighed
and extracted for 24 h at 4 ◦ C in 1.0 mL of 70% methanol. During this period, the samples were vortexed
(10 s, 40 Hz) once per 10 min for a total of three times. After extraction, the pellets were centrifuged
at 10,000× g for 10 min at 4 ◦ C and the extracts were then filtered through a 0.22 μm microporous
membrane and stored in a sample vial. The QC was prepared by mixing all the samples. In order
to examine the repeatability of the analysis process, a QC sample was injected after every five test
samples during the instrumental analysis.
4.4.2. Liquid Chromatographic Mass Spectrometry Analysis

The sample extracts were analyzed using a UPLC-ESI-MS/MS system, which mainly includes UPLC
(Shim-pack UFLC SHIMADZU CBM30A, SHIMADZU, Kyoto, Japan) and MS/MS (Applied Biosystems
6500 QTRAP, AB SCIEX, Foster City, CA, USA). The UPLC separation was completed on a Waters
Acquity UPLC HSS T3 C18 column (100 × 2.1 mm, 1.8 μm) (Waters Corp., Milford, MA, USA). The
mobile phase A solvent was water containing 0.04% acetic acid, and the mobile phase B solvent
was acetonitrile containing 0.04% acetic acid. The elution gradient was shown as follows: 0–11 min
95–5% A, 11–12 min 5–5% A, 12–12.1 min 5–95% A and 12–15 min 95–95% A. The flow was 0.4 mL/min
and the injection volume was 2 μL. The column temperature was set to 40 ◦ C.
The effluents were alternatively connected to an electrospray ionization-triple quadrupole-linear
ion trap MS/MS (ESI-QTRAP-MS/MS). Linear ion trap (LIT) and triple quadrupole (QQQ) scans were
carried out using QTRAP. The mass spectrometry conditions were as follows: the ESI temperature was
set to 500 ◦ C, the mass spectrometry voltage was 5500 V, the GS 1 and GS 2 were set to 55 psi and 60 psi,
respectively, and the CUR was set to 25 psi. QQQ scans were obtained as MRM experiments with
collision gas (nitrogen) set to 5 psi. The DP and CE for individual MRM transitions were optimized in
the QQQ. The data were analyzed by the mass spectrometry software (Version 1.6.1 Applied Biosystems
Company, Framingham, MA, USA).
4.4.3. Metabolite Identification

Based on the public database of metabolite information and the MVDB V2.0 database of Wuhan
Metware Biotechnology Co., Ltd. (Wuhan, China), qualitative analysis of the primary and secondary
mass spectrometry data was performed by referencing the existing mass spectrometry databases such
as MassBank, KNAPSAcK, HMDB and METLIN; thus, the structural analysis of metabolites was
determined. Regarding the quantitative analysis of the metabolites, MRM was used, and PCA and
OPLS-DA were then carried out to identify differential metabolites. A VIP ≥ 1 and a fold change ≥ 2 or
≤0.5 were set for metabolites with significant difference.
4.5. Illumina Sequencing
4.5.1. RNA Extraction, cDNA Library Construction and Sequencing

The total RNA was extracted from NM and M using the RNeasy Plant Mini Kit (Qiagen, Hilden,
Germany) following the manufacturer’s instructions, and treated with an RNase-free DNase I digestion
kit (Aidlab, Beijing, China) in order to remove contaminated genomic DNA. RNA degradation
was measured using 1% agarose gel, RNA concentration was measured using NanoDrop 2000
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spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and RNA integrity was assessed on an
Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA, USA).
mRNA was enriched with oligo (dT)-attached magnetic beads and then the fragmentation buffer
was exploited to randomly fragment the mRNA into short fragments. Using these cleaved RNA
fragments as a template, the first cDNA strand was synthesized by random hexamers, then the
second cDNA strand was synthesized by adding buffer, dNTPs, RNaseH and DNA polymerase I and
purification of the double-stranded cDNA was done by NEBNext Ultra RNA Library Prep Kit for
Illumina (NEB, Ipswich, MA, USA). The purified double-stranded cDNA was repaired by end-to-end,
added poly A tail and connected to the sequencing connector. cDNA of about 200 bp in length was
selected by AMPure XP beads, and PCR amplification was performed to enrich the purified cDNA
template. Finally, the libraries were sequenced using an Illumina Hiseq 2000. Sequence data were
deposited in the NCBI SRA database (accession number: PRJNA579778).
4.5.2. De Novo Transcriptome Assembly and Annotation

In order to achieve high-quality and clean data, the raw data was filtered by removing the
reads with adapter sequence and low-quality reads using the fastp software [50], then the clean
reads were assembled into transcriptome by the Trinity v.2.0.6 software (Broad Institute, Cambridge,
MA, USA). Unigene function was annotated based on these databases: NCBI non-redundant protein
sequences (Nr, https://ftp.ncbi.nlm.nih.gov/blast/db/FASTA/), KEGG (https://www.genome.jp/kegg),
Clusters of Orthologous Groups of proteins (COG, https://www.ncbi.nlm.nih.gov/COG/), GO
(https://www.geneontology.org), the Swiss-Prot (http://www.ebi.ac.uk/uniprot/) and the translation of
EMBL (TrEMBL).
4.6. Determination of the Flavonoid Contents During Different Growth Stages
4.6.1. Preparation of Standard Solutions

In order to validate the changes of flavonoids during different growth stages after symbiotic
culture, several monomer flavonoids from DAMs were tested. The standards of nobiletin (478-01-3),
narcissin (604-80-8), isorhamnetin-3-O-beta-d-Glucoside (5041-82-7), tangeretin (481-53-8), rutin
(153-18-4), quercetin (117-39-5), isorhamnetin (480-19-3), quercetin-7-O-glucoside (491-50-9), and
kaempferol-3-O-glucoside (480-10-4) were purchased from the National Institutes for Food and Drug
Control (Beijing, China) and the purity of all of them was over 98%. Each standard sample was
dissolved in methanol to obtain 1 μg/mL of standard working solutions and used within 1 month at
−20 ◦ C.
4.6.2. Preparation of Sample Solutions

The preparation of the sample (approximately 20 mg) followed the same process as the above
mentioned extract. In order to enrich the flavonoids, the extract was processed through C18-SPE solid
phase extraction column (Waters Technology Co., Ltd., USA) and the column was rinsed with 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90% absolute alcohol. The rinse solutions of 70%, 80%, 90%,
and absolute alcohol, that were verified to contain flavonoids, were collected and freeze dried using
a vacuum freeze-drying machine LGJ-18 (Beijing Songyuanhuaxing Technology Develop Co., Ltd,
Beijing, China). The extract was re-dissolved with 2 mL methanol, then the sample solution was passed
through a 0.22 μm syringe nylon filter and stored at −20 ◦ C prior to analysis.
4.6.3. Apparatus and Analytical Conditions

The HPLC separation was performed by an Agilent 1260 Infinity II series HPLC System that was
equipped with Waters Atlantis C18 column (150 × 3.9 mm, 5 μm). The mobile phase consisted of
solvent A (water containing 0.1% formic acid) and solvent B (methanol). The gradient procedure was
120
Int. J. Mol. Sci. 2020, 21, 564
as follows: 0–3 min 60–5% A, 3–5 min 5–5% A, 5–5.02 min 5–60% A, and 5.02–6 min 60–60% A. The
flow rate was 0.5 mL/min and the injection volume was 5 μL.
An Applied Biosystems Sciex QTRAP®4500 MS/MS spectrometer equipped with a version of
1.6 Analyst software (AB SCIEX, Massachusetts, USA) was used for the analysis. The instrument
was equipped with an ESI source, and the targeted analytes were performed in positive and negative
ion modes for all the targeted analytes. Compressed air was used as GS1 and GS2, and high-purity
(99.99%) nitrogen was used as CUR and CAD. The operation conditions were as follows: the EP was
10.0/−10.0 V, the TEM at 500 ◦ C, the IS 5500/–4500 V, GS1 set to 55 psi, GS2 set to 55 psi, CUR set to
35 psi, and the CXP 13.0/−15.0 V for ESI+ /ESI− mode, respectively. The dwell time for each MRM
transition was 10 ms.
4.7. Expression of the Flavonoid Biosynthesis Related Genes During Different Growth Stages
Nine unigenes that were related to flavonoid biosynthesis following Zhang et al. [51] and Park
et al. [52] were selected for validation using qRT-PCR. The RNA was reverse-transcribed to cDNA
using PrimeScriptTM RT reagent Kit (TaKaRa, Dalian, China). The qRT-PCR was performed using
the SYBR®Premix ExTaq TM (TaKaRa, Dalian, China) on the LightCycler®480 II Real-Time PCR
System (Roche, Carlsbad, CA, USA). Three biological replicates and three technical replicates were
performed, and all the primer names and corresponding sequences are listed in Table S3. The qRT-PCR
was performed in a 20 μL reaction volume containing SYBR Premix Ex Taq II (10 μL), forward
prime (10 μM, 0.8 μL), reverse primer (10 μM, 0.8 μL), cDNA template (5 ng/μL, 2 μL), and ddH2 O
(6.4 μL). The PCR conditions were as follows: denaturation at 95 ◦ C for 30 s, followed by 40 cycles of
amplification (95 ◦ C for 5 s, 60 ◦ C for 30 s). The melting curves were measured at 95 ◦ C for 5 s and
60 ◦ C for 1 min. The elongation factor 1 alpha (EF-1α) gene of A. roxburghii was used as the internal
control reference gene. Finally, gene expression was calculated using the 2−ΔΔCt method [53].
4.8. Statistical Analysis

All the transcriptome and metabolome samples were designed for three biological replicates.
Statistical analyses were conducted using the SPSS 22.0 software (IBM, Chicago, IL, USA).
An independent samples t-test was used for statistical evaluations between the control and
treatment groups.

Figure S1: Overlapping analysis of the TIC in QC samples. The x-axis represents the retention time (min) of
metabolite detection, and the y-axis represents the intensity of ion current (cps: count per second). (A) for ESI+
mode; (B) for ESI− mode; Table S1: Total identified metabolites in the NM and M growth for six months; Table S2:
Recovery test for each analyte; Table S3: Primer designed for qRT-PCR.
Author Contributions: Conceptualization, X.C. and Z.M.; Methodology, Y.Z. and Y.L.; Software, Y.Z.; Validation,
Y.Z.; Resources, Z.M. and S.G.; Data curation, Y.Z., Y.L. and X.C.; Writing—Original draft preparation, Y.Z.;
Writing—Review and editing, Z.M. and S.G.; Supervision, X.C.; Funding acquisition, S.G. and X.C. All authors
have read and agreed to the published version of the manuscript.
Funding: This research was funded by the CAMS the Innovation Fund for Medical Sciences (CIFMS)
(2017-I2M-3-013), the National Natural Science Foundation of China (81573526), and the Peking Union Medical
College Discipline Construction Project (201920100901).
Acknowledgments: We thank Ruibin Hu from the Institute of Chemistry, Chinese Academy of Sciences for his
help for data analysis and article logic structure; We thank Yung-I Lee from Biology Department, National Museum
of Natural Science (Taiwan) for providing guidance for semi-thin section observation experiment.
121
Int. J. Mol. Sci. 2020, 21, 564
Abbreviations
CE collision energy
CUR curtain gas
CXP collision cell exit potential
DAMs differentially accumulated metabolites
DEGs differentially expressed genes
DP de-clustering potential
EP entrance potential
ESI-QTRAP-MS/MS electrospray ionization-triple quadrupole-linear ion trap MS/MS
GC-MS gas chromatography-mass spectrometer
GO gene ontology
GS1 ion source gas 1
GS2 ion source gas 2
high-performance liquid chromatography coupled with tandem mass
HPLC-MS/MS
spectrometry
IS ion spray voltage
KEGG Kyoto encyclopedia of genes and genomes
LC-MS liquid chromatograph-mass spectrometer
LIT linear ion trap
LOD limit of detection
LOQ limit of quantification
NMR nuclear magnetic resonance
OPLS-DA orthogonal partial least squares discriminant
PCA principal component analysis
PC1 principal component 1
PC2 principal component 2
PI product ions
QC quality control
QQQ triple quadrupole
qRT-PCR quantitative real-time polymerase chain reaction
RNA-seq RNA-sequencing
RSDs relative standard deviations
TEM temperature
TIC total ion chromatography
ultra-performance liquid chromatography-electrospray ionization-tandem mass
UPLC-ESI-MS/MS
spectrometry
VIP variable importance in project
cps count per second
MRM multiple reaction monitoring
psi pounds per square inch
NM non-mycorrhizal A. roxburghii
M mycorrhizal A. roxburghii
PAL phenylalanine ammonia-lyase
C4H cinnamate 4-hydroxylase
4CL 4-coumarate CoA ligase
CHS chalcone synthase
CHI chalcone isomerase
F3 H flavonoid 3 -hydroxylase
FNS flavone synthase
F3H flavanone 3-hydroxylase
FLS flavonol synthase
GT flavonoid 3-O-glucosyltransferase
RT rhamnosyltransferase
122
Int. J. Mol. Sci. 2020, 21, 564
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Molecular Sciences
Article
Molecular Identification of Endophytic Fungi and
Their Pathogenicity Evaluation Against Dendrobium
nobile and Dendrobium officinale
Surendra Sarsaiya 1,2,† , Archana Jain 1,† , Qi Jia 2 , Xiaokuan Fan 3 , Fuxing Shu 2 ,
Zhongwen Chen 2 , Qinian Zhou 2 , Jingshan Shi 1, * and Jishuang Chen 1,2,3, *
1 Key Laboratory of Basic Pharmacology and Joint International Research Laboratory of Ethnomedicine of
Ministry of Education, Zunyi Medical University, Zunyi 563003, China; [email protected] (S.S.);
[email protected] (A.J.)
2 Bioresource Institute for Healthy Utilization, Zunyi Medical University, Zunyi 563003, China;
[email protected] (Q.J.); [email protected] (F.S.); [email protected] (Z.C.); [email protected] (Q.Z.)
3 College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211800, China;
[email protected]
* Correspondence: [email protected] (J.S.); [email protected] (J.C.)

Received: 4 November 2019; Accepted: 2 January 2020; Published: 2 January 2020
Abstract: Dendrobium are tropical orchid plants that host diverse endophytic fungi. The role
of these fungi is not currently well understood in Dendrobium plants. We morphologically and
molecularly identified these fungal endophytes, and created an efficient system for evaluating the
pathogenicity and symptoms of endophytic fungi on Dendrobium nobile and Dendrobium officinale
though in vitro co-culturing. ReThe colony morphological traits of Dendrobium myco-endophytes
(DMEs) were recorded for their identification. Molecular identification revealed the presence
of Colletotrichum tropicicola, Fusarium keratoplasticum, Fusarium oxysporum, Fusarium solani, and
Trichoderma longibrachiatum. The pathogenicity results revealed that T. longibrachiatum produced the
least pathogenic effects against D. nobile protocorms. In seedlings, T. longibrachiatum showed the
least pathogenic effects against D. officinale seedlings after seven days. C. tropicicola produced highly
pathogenic effects against both Dendrobium seedlings. The results of histological examination of
infected tissues revealed that F. keratoplasticum and T. longibrachiatum fulfill Koch’s postulates for the
existence of endophytes inside the living tissues. The DMEs are cross-transmitted inside the host
plant cells, playing an important role in plant host development, resistance, and alkaloids stimulation.
Keywords: Dendrobium; molecular identification; endophytic fungi; pathogenicity; protocorm;

seedling
1. Introduction
The Orchidaceae is the chief family of plants, with over 25,000 plant species worldwide. They are
also some of the most vulnerable flowering plants, as numerous genera are endangered and nearly
all genera are at risk of habitat harm and over-assortment [1]. China is a rich source of orchid plants,
with over 1447 species, mostly located in the subtropical and tropical provinces in the southwest and
south [1,2]. Many Dendrobium orchids are horticultural plants and have been used for profitable trade
due to their flowering profusion, extensive variety of flower colors, shapes, sizes, and year-round
producibility, along with long lifespan [3]. Most species are in danger of extinction. Orchids extend
their lives as herbaceous plants using two evolutionary methods: sympodial growth and monopodial
development, which are influenced by the many endophytic fungal species as main pathogens of

Int. J. Mol. Sci. 2020, 21, 316
orchids [4,5]. Dendrobium officinale has been extensively used in traditional medicines for over 2000 years
to decrease fever, inhibit tumors, increase antioxidant activity, treat hypoglycemia, recover from loss of
eyesight, and control the immune system, according to the best of China Pharmacopeia [6,7].
Dendrobium plants parts, for example, roots, stems, buds, and leaves of tropical orchid, harbor
diverse fungal taxa, including mutualistic mycorrhiza, and endophytic fungi and considerably diverse
nonmycorrhizal fungal associates. The role of the root-allied fungi is not well understood. They typically
originate in the velamen, without causing any disease symptoms [8]. They may encourage the growth
of Dendrobium by activating soil chemicals in the rhizosphere. The impact of quantities or variations of
secondary metabolite have been investigated. At large, they act as a supply for bioactive molecules
that defend the host from rhizospheric pathogens [8]. Endophytic fungal groups commonly establish
a sole host specificity at the species level, which can be additionally encouraged by microclimatic
conditions and microhabitat [9]. The relationship is chiefly stimulated by the endophyte fungi, yielding
an overabundance of natural compounds as soon as endophytic fungi are cultured in the external
environment of their ordinary hosts or environmental niches under in vitro test conditions [10].
Protocorm and seedling expansion is an important step in commercial orchid production, and
its conservation is crucial tool for maintaining the genetic diversity of the orchid plant [11]. Orchid
(Dendrobium nobile and D. officinale) protocorms initiating from the orchid seeds are typically very
small, similar to dust, and deficient an endosperm. Subsequently, orchid seed incubation and seedling
expansion require well-matched endophytic fungi to deliver the carbon, nutrients, and water to the
seeds under usual plant conditions [3]. Consequently, an in vitro proliferation method could be a
useful for the mass measure proliferation of these orchids for their marketization. The protocorms
and seedlings rarely survive after relocation into nature from well mature sterile culture. The orchid
protocorm may be reliant on appropriate endophytic fungi for seedling existence [12].
Most examinations of Dendrobium myco-endophytes (DMEs) focused on symbiotic in vitro
practices using fungal endophyte strains obtained from fully-grown dendrobium roots, buds, stems,
and leaves [3]. Understanding whether these endophytic fungi are pathogenic, conditional pathogenic,
or non-pathogenic for the host plant is important. DMEs can be transmitted horizontal or vertically.
Vertical transmission occurs when the seeds are contacted through the fungal endophytes and are
transmitted to the host plant. Horizontal transmission involves the formation of exterior spores and
their airborne dispersal infects many other hosts [13].
A steady state between the fungal pathogen and its host plant is achieved when the pathogen
resides in equilibrium with the surrounding host tissues and causes little damage, which is also
called least pathogenic. The virulence expression of the pathogen is dependent on the particular
host environment. When the pathogen is isolated from an asymptomatic host and introduced into
a new host, strong pathogenic reactions may be observed [14]. Commonly, fungal endophytes have
functions ranging from latent pathogens to mutualistic symbionts. Reliant on the host genome
type, some endophytic fungi may be pathogenic in stressed hosts, whereas they can be helpful in
other conditions due to conditional pathogenic properties [15]. In the environment, orchids are
chiefly dependent on these fungal endophytes for their nourishment and propagation along with
the succeeding seedling (protocorm) phases. However, only imperfect quantitative approaches for
assessing the Dendrobium–fungus connections at the protocorm and seedling phase are available at
present, which places major constraints on understanding the host–endophyte relationships [16].
In this study, we focused on species-specific endophytic fungi pre-isolated from D. nobile plant
parts, which were inoculated onto well-developed protocorms and seedlings of D. nobile and D. officinale
for the evaluation of pathogenicity using in vitro inoculation and histopathological examination of
infected tissues. The test endophytic fungus was re-isolated from the leaves tissues for examining
Koch’s postulates.
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2. Results
2.1. Isolation and Molecular Identification of Endophytic Fungi

Five DME isolates were recovered (ZF01, ZF02, ZF03, ZF04, and ZF05) from leaf segments of
the wild D. nobile. The colony morphological traits of the DME isolates, such as hyphal structures
and spore arrangements, were used for their identification. The isolated endophytic fungal colony
characteristics and microscopic characteristics findings were found as follows: (1) ZF01: fast grower
(35 ± 0.2 mm), flat and buff growth surface, whitish color, reverse whitish to grey/orange, margin
irregular mycelium with conidiogenous cells hyaline to pale brown, conidia hyaline with round
ends, smooth-walled; (2) ZF02: moderate grower (16 ± 0.2 mm), colony flat velvety, white color with
no diffusing pigment coloration, margin irregular, white rims, mycelium with microconidia oval
and elongated and clavate, sickle-shaped conidia containing numerous septations, septate hyaline
hyphae; (3) ZF03: fast grower (25 ± 0.2 mm), colony cottony white to light violet color, aerial white
margins septate and hyaline, macroconidia slightly curved and thick with an attenuated apical cell,
three to five septa, short aerial conidiophore, unbranched, microconidia non-septate, aerial conidia,
ellipsoidal to cylindrical, slightly curved; (4) ZF04: fast grower (28 ± 0.2 mm), colony cottony white
to light white with creamy to white-greyish and reverse light yellow color, irregular white margins
with cream yellow edges with long and narrow macroconidia, cylindrical, dorsal and ventral surface
parallel, three to five septa, phyalide long and thin; and (5) ZF05: fast grower (34 ± 0.2 mm), colony
cottony off white to greenish, later green tufts sporulation, irregular white margins with very hyaline
hyphae, long conidiophores, flask-shaped single phialides, small-walled ellipsoidal conidia, formed
chlamydospores (smooth walled) in terminal or intercalary (Table S1). Molecular characterization was
used to confirm isolate identification in which the rDNA (recombinant deoxyribose nucleic acid)-ITS
(internal transcribed spacer) region was amplified. The sequences data were aligned using BLAST
(basic local alignment search tool). The endophytic fungi were found to be the closest homologs of
Colletotrichum tropicicola (ZF01), Fusarium keratoplasticum (ZF02), Fusarium oxysporum (ZF03), Fusarium
solani (ZF04), and Trichoderma longibrachiatum (ZF05) (Figure 1). Results from BLAST were categorized
for the isolated endophytes under ascomycota, which coincided with the morphological identification.
These were represented by class Sordariomycetes, including isolates belonging to Colletotrichum and
Trichoderma, and Hypocreales included isolates belonging to Fusarium. These were the most frequent
and dominant endophytic fungi isolated from D. nobile.
2.2. Evaluation of Protocorm and Seedling Pathogenicity

The DME pathogenicity results with D. nobile and D. officinale protocorm revealed that
T. longibrachiatum (ZF05) showed the least pathogenicity (11%) against the D. nobile protocorm
after 7, 14, and 21 days, whereas it showed moderate pathogenic (33%) effects at 21 days of incubation
against D. officinale. We found that most of the plant pathogens are latent pathogens or conditional
pathogens, causing symptoms some weeks later when the plant defense mechanism has weakened.
Many environmental factors function together to trigger the virulence factors or genes of the endophytic
fungi. T. longibrachiatum (ZF05) started to change the color of the D. officinale protocorm from green
to light yellowish at 14 days, which changed to brown to blackish at 21 days; whereas no color
change symptoms were observed in the D. nobile protocorm. The second least pathogenic strain was
F. keratoplasticum (ZF02), which was found to be non-pathogenic against D. nobile after 7 and 14 days of
incubation, and least pathogenic (11%) against D. officinale after seven days of incubation, but showed
moderate pathogenic effect after 21 days incubation on both D. nobile and D. officinale. The symptoms on
the protocorms of D. nobile and D. officinale by F. keratoplasticum (ZF02) included the color of D. officinale
protocorm starting to change from green to light yellowish at 14 days, then turning yellow at 21 days,
whereas no color change symptoms were observed in the D. nobile protocorm due to F. keratoplasticum
(ZF02). F. oxysporum (ZF03) and F. solani (ZF04) were moderate pathogens at seven days of incubation
and strong pathogens (100%) at 21 days of incubation. C. tropicicola (ZF01) showed high pathogenicity
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(77–100%) effects against both D. nobile and D. officinale seedlings. F. oxysporum (ZF03) produced no
symptoms on the protocorms of D. nobile and D. officinale at 7 and 14 days, indicating these are latent
pathogens because they demonstrated high pathogenicity at 21 days and their symptoms included
color change of the green protocorm to brownish for both D. nobile and D. officinale. F. solani (ZF04)
produced moderate symptoms on the protocorms of both D. nobile and D. officinale by changing their
green color to light reddish to brown in the initial days and then turned dark reddish-brown at 21 days.
C. tropicicola (ZF01) significantly affected the protocorms of D. nobile and D. officinale by changing their
green color to dark brown at 7, 14, and 21 days (Figures 2 and 3).
Figure 1. Phylogenetic identification of the endophytic fungi based on ITS4 and ITS5 regions of DNA
sequences. The evolutionary detachments were calculated using the Kimura two-parameter method.
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Figure 2. Co-culturing pathogenic effects on protocorm of (A) D. nobile and (B) D. officinale with
endophytic fungi. Means values are significantly different at p = 0.05 according to Duncan’s multiple
range test.
The DME pathogenicity results for D. nobile and D. officinale seedling revealed that T. longibrachiatum
(ZF05) produced the least pathogenic effects on D. officinale protocorm after seven days and increased
gradually with increasing incubation period (at 14 days; 33% disease index), which was stable at 21 days
(33% disease index). For D. nobile, the fungus showed highly pathogenic (77% disease index) effects from
the start to the end of the incubation period. The second least pathogenic strains were F. keratoplasticum
(ZF02) and F. solani (ZF04), which showed least pathogenic effects against D. officinale, whereas
F. keratoplasticum (ZF02) and F. oxysporum (ZF03) showed stable moderate pathogenic (33%) effects
against D. nobile and D. officinale, respectively, throughout the incubation period. C. tropicicola (ZF01)
showed high pathogenicity (77–100%) against both D. nobile and D. officinale seedlings. The seedlings
symptoms included a color change of D. nobile seedlings from green to black spots and brownish
to blackish spots on the D. officinale seedlings with T. longibrachiatum (ZF05) at 7, 14, and 21 days.
F. keratoplasticum (ZF02) produced symptoms on the D. nobile and D. officinale seedlings including
color change of the D. officinale seedlings from green to yellowish at 7 days, then to light brown at
14 days, then to black at 21 days; similar symptoms were observed in the D. nobile seedlings with
F. keratoplasticum (ZF02). F. oxysporum (ZF03) produced light brown color symptoms on the seedlings
of D. nobile and D. officinale at 7 and 14 days, which later changed to brownish to yellowish. F. solani
(ZF04) produced moderate symptoms on the seedlings by changing its green color to light yellowish to
brown at 7 days, changing to dark brown at 14 days, then to blackish brown at 21 days on the seedlings
of D. nobile and D. officinale. C. tropicicola (ZF01) produced significant symptoms on the seedlings of
D. nobile and D. officinale by changing the seedling color from green to brown at 7, 14, and 21 days
(Figures 4 and 5).
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Figure 3. Least pathogenicity by Trichoderma longibrachiatum (ZF05) against the D. nobile protocorm
after 21 days. (A) Control (D. nobile protocorm with water agar media); (B) pure culture of Trichoderma
longibrachiatum (ZF05); (C) D. nobile protocorm with Trichoderma longibrachiatum (ZF05) after 21 days;
(D) Single D. nobile protocorm with Trichoderma longibrachiatum (ZF05) growth indicated by the arrow.
Figure 4. Co-culturing pathogenic effects of endophytic fungi on seedlings of D. nobile and D. officinale.
Means values are significantly different at p = 0.05 according to Duncan’s multiple range test.
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ȱ
Figure 5. Moderate pathogenicity by Trichoderma longibrachiatum (ZF05) against the D. officinale
seedling after 21 days. (A) Control (D. officinale seedling with water agar media); (B) pure culture of
T. longibrachiatum (ZF05); (C) D. officinale seedling with T. longibrachiatum (ZF05) after 21 days; (D)
Single D. officinale seedling with T. longibrachiatum (ZF05) growth indicated by the arrow.
2.3. Re-Isolation of Endophytic Fungi from Co-Culturing Seedling Tissues

After the coculturing incubation periods (21 days), the endophytic fungi were re-isolated from
the plant tissues to confirm the presence of fungi inside the plant tissues by re-inoculating the
samples into the growth media (PDA: potato dextrose agar media). The results showed that the same
endophytic fungi were re-isolated from the uninoculated plant tissues, which were identified based
on characteristics: C. tropicicola (ZF01), F. keratoplasticum (ZF02), F. oxysporum (ZF03), F. solani (ZF04),
and T. longibrachiatum (ZF05). All isolated endophytic fungi were confirmed as the same fungi via
microscopic examination as described in Table S1 and Figure 6.
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Figure 6. Re-isolation of test endophytic fungi from the uninoculated tissues of D. nobile and D. officinale.
2.4. Histological Examination of the Least Pathogenic Fungal Endophyte Seedling Samples
The results showed that the thin sections stained with lactophenol cotton blue showed the presence
of endophytic fungi in the intercellular spaces of D. nobile and D. officinale seedling tissues (Figure 7).
The transverse section of D. nobile seedling stems at lower magnification showed dense blue Trichoderma
longibrachiatum mycelia in the epidermis and mesophyll region (Figure 7A) and, at high magnification,
we observed sprinkled colonies proximal to vascular bundles (Figure 7B). The transverse section of the
D. officinale seedling stem at lower magnification showed T. longibrachiatum mycelia in the intercellular
mycelia and a lower amount in epidermal region (Figure 7C); at higher magnification, we observed a
large amount of T. longibrachiatum mycelium in the phloem region with slightly less in the epidermis
(Figure 7D).
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Figure 7. Histopathological examination of D. nobile and D. officinale stem infected by the least
pathogenic test endophytic fungi, T. longibrachiatum (ZF05). (A,B) Transverse section of D. nobile stem
showing the presence of T. longibrachiatum (ZF05). (A) T. longibrachiatum in epidermal region (10×).
(B) Dense colonies of T. longibrachiatum (ZF05) in phloem with spores (40×). (C,D) Transverse section
of D. officinale stem showing the presence of T. longibrachiatum (ZF05). (C) T. longibrachiatum (ZF05)
in intercellular mycelia with less in epidermal region (10×). (D) Dense colonies of T. longibrachiatum
(ZF05) in phloem with spores (40×).
3. Discussion
Dendrobium is largest genera of Orchidaceae, with more than 1000 species globally [3]. D. nobile
and D. officinale are a wild epiphytic orchid found in the tropical rain forests in China, especially in
Guizhou province, China. The solitary and attractive inflorescence of these orchids is slightly exclusive
and valued amongst orchid cultivators. Due to their high commercial demand, they are being removed
from their usual habitat, placing them at high risk of becoming extinct. To preserve and reinstate
threatened and rare orchid plants, these plants must be reintroduced with fungal co-culturing. Hence,
some pure endophytic fungal strains that stimulate or decrease Dendrobium protocorm and seedling
growth must be determined in vitro [17]. For this purpose, co-culturing will be essential for D. nobile
and D. officinale success. Biosynthesis of many active compounds will be necessary, predominantly
where specific fungal strains are vital for actively providing molecules to the protocorm with or without
producing symptoms.
This study provides valuable information about the orchid bionetwork with endophytic fungi
associated with D. nobile and D. officinale under laboratory environments. The in vitro pathogenicity
evaluation process was used to effectively assess well-suited and species-specific endophytic fungi
in terms of their symptoms and pathogenicity index for D. nobile and D. officinale. For the first
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time, we found that T. longibrachiatum (ZF05) produced the least pathogenic effects on D. nobile
and D. officinale, which provided the plants with nutrition and helped them build an active defense
mechanism to survive without the presence of any nutrients in the media. This phenomenon was
observed as asymptomatic colonization due to the balanced antagonism between the host and the
endophyte [13]. Endophytes also possess various virulence factors that are contradicted by host plant
defense mechanisms. If endophyte virulence and host Dendrobium defense are well balanced, the
relationship is avirulent and asymptomatic. This stage is only a transitory time where environmental
influences play a key role in destabilizing the delicate equilibrium of antagonisms. [18]. Endophytic
fungi can deliver appropriate carbon sources, amino acids, vitamins, and hormones that are important
for seedling and protocorm development [19]. Khamchatra et al. [3] also stated that Beauvaria and
Fusarium species are endophytic fungi, which we also recovered, that may play a role in the growth and
survival of the plants like D. friedericksianum. Though grown in orchid stems and roots, the fungi may be
non-casual and non-mycorrhizal endophytes. Many of these endophytes were reported in many white
rot fungi and are incapable of phytopathogenicity [3]. In another study, Meng et al. [20] found that
several fungal species recovered from some species of Dendrobium protocorms and seedlings have the
ability to cause disease symptoms. Zi et al. [21] described Epulohiza, an anamorphic phase of Tulasnella,
in the Dendrobium protocorms. Athipunyakom et al. [22] reported that Trichosporiella multisporum is
present in Paphiopedilum roots. In an ecosystem, orchids depend on orchid fungal endophytes to provide
needed nutrients for growth, a process termed an asymptomatic relationship. The symbiotic plant
relationship, specifically in vitro approaches, is adopted because it enables higher growth rates and/or
symbiotic seedlings and protocorms progress faster than without this relationship [11]. Although
extensive information about the co-culturing expansion is not available, the process was endorsed as
an effective process for improving the growth of many orchids [19].
From this study, we found that C. tropicicola (ZF01) is a highly pathogenic strain for the protocorms
and seedlings of both D. nobile and D. officinale. Dendrobium endophyte contact might, in addition
to balancing between defense and virulence, might more precisely control this complex contact [10].
Plant–pathogen association may be responsible for the growth conditions for plant disease. Because
several fungal endophytes may be latent plant pathogens, certain inherent or environmental influences
may prompt pathogenic effects [18]. Many fungal endophytes are silent/conditional pathogens, only
resulting in disease as the plant ages or under stress conditions. Fungal endophytes accomplish
asymptomatic colonization through a stable neutral antagonism between the fungal virulence and its
response to the plant defense mechanism [23]. We hypothesized from our research that T. longibrachiatum
(ZF05) mycelium may migrate from inoculated to uninoculated tissues of host plants. The histological
image of D. nobile and D. officinale seedlings clarified that the fungus infects tissues. This paper
is the first report on the cross-transmission of T. longibrachiatum (ZF05) from inoculated tissues
to uninoculated tissues confirmed by re-isolated from host plant segments that fulfilled the Koch
postulates. Colletotrichum, Fusarium, and Trichoderma include morphologically similar taxa that are
commonly found as endophytes, saprobes, and plant pathogens [13–15].
As endophytes exist in within plant tissues and endlessly network with their host tissues, fungal
endophytes may be linked intracellularly, which is responsible for the cross-transmission of the fungus
into the new cells. Along this line, we strongly suggest that endophytic fungus is re-isolated from
the uninoculated infected segments of the D. nobile and D. officinale seedlings. This means that these
endophytic fungi are cross-transmitted intracellularly from one cell to another healthy cell, creating
comprehensive endophytic molecular connections, cross-species appearance, and on/off switching of
the compulsory gene cascades that constantly yield a chosen molecule [10].
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4.1. Isolation of Endophytic Fungal Strains

Endophytic fungi were isolated from leaves parts. Five healthy wild D. nobile plants were collected
(from agricultural farm single location) from Jinshishi, Chishui, Guizhou, China and processed in
the Bioresource Institute for Health Utilization, Zunyi Medical University, Zunyi, China, and then
inoculated aseptically into PDA media. The samples were processed according to a method modified
from Novotná et al. [8]. All the leaves segments of wild D. nobile were used for surface sterilization
processing. All the leaves samples were washed with tap water to remove the dirt spots and connective
tissue of stems. Thereafter, the leaves were washed with tap water to further clean the surface. After
cleaning, all the plant samples were placed into laminar air flow and exposed to UV rays for 20 min.
Under sterilized conditions in the laminar air flow, the samples (1.5 cm pieces) were inoculated onto
PDA media plates. The sample plates were placed in a fungal incubator at 25 ◦ C for 5 days. After
5 day of incubation, the fungal hyphal tips emerging from the samples were recovered and purified by
sub-culturing from the grown hyphal tips.
4.2. Morphological and Molecular Identification of Plant Endophytic Fungi

The endophytic fungal genus was identified using lactophenol cotton blue staining according to the
protocol followed by Barnett and Hunter [24] and Ellis [25]. The DNA was extracted from approximately
100 mg of fungal mycelia using the DNAsecure Plant Kit according to the manufacturer’s instructions
(Tianjin Biotech Co. Ltd., Beijing, China). PCR was performed using a standard protocol [26].
The reactions were prepared in 25 μL volumes constituting 2.0 μL primer, 12.5 μL 2× TsingKe (Blue)
Master Mix (TSINGKE Biological Technology Co., Ltd, Beijing, China), 9.5 μL RNase-free distilled
water, and template DNA (2 μL). A non-template tube was used as a negative control during PCR
amplification to monitor purity. Amplifications were completed using a PCR thermal cycler equipment
(Bio-Rad C1000 Thermal Cycler, Minnesota, USA). PCR cycling was performed using the conditions
and primer sequences [27] presented in Table S2.
4.3. Electrophoresis of PCR Products

The PCR samples were analyzed using electrophoresis on a 1% (w/v) agarose gel (Agarose
G-10, Gene Company Ltd., Hong Kong, China) [28]. A DNA marker (100 to 1000 bp molecular
weights) (Beijing TransGen Biotech Co. Ltd., Beijing, China) was added to the first well of the gel.
Electrophoresis was conducted in a horizontal electrophoresis system (Bio-Rad PowerPacTM Basic,
Henderson, Singapore) for 30 min at 120 V and 250 mA using 1× TAE (Tris-acetate-EDTA) buffer.
The gel was marked with ethidium bromide (0.1 μg/mL). A Molecular Imager Gel DocTM XR+ with
Image Lab Software system (Bio-Rad Gel DocTM XR+ Imaging System, Hercules, CA, United States)
was used to capture images using Image Lab software (version 4.1).
4.4. Sequence Analysis of PCR Products

The amplified ITS regions of PCR samples were sequenced by Tsingke, Beijing, China. Sequence
data were subjected to BLAST analysis on the NCBI (national center for biotechnology information)
web tool to validate the characteristics of the amplified sequences along with isolates. All the sequences
have been deposited in the GenBank under accession numbers i.e., Colletotrichum tropicicola ZF01:
MN826680; Fusarium keratoplasticum ZF02: MN832906; Fusarium oxysporum ZF03: MN826681; Fusarium
solani ZF04: MN826682; and Trichoderma longibrachiatum ZF05: MN826683. MEGA 7.0 software was
used for phylogeny tree study.
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4.5. Collected Samples (Protocorm and Seedling) and Sample Processing

Water agar (0.65% w/v) was used as media and relocated into the glass bottles for the coculturing
experiments. No additional nutriments were added into the media; only twofold distilled water
and agar were used. D. nobile and D. officinale (both protocorms aged 4 months) and seedlings
(aged 8 months) were obtained from the Bioresource Institute for Healthy Utilization (BIHU), Zunyi
Medical University (ZMU; Zunyi, Guizhou, China) for experiments and authenticated per previous
work [29–32]. After the sterilization of the media bottles, the protocorms of the D. nobile and D. officinale
were inoculated into separate bottles under aseptic conditions. For seedlings, we cut marks into the
top leaf of the seedlings where it joined then stem using a sterilized scalpel blade over sterilized paper,
as shown in Figure 8. Three seedlings were transferred into each bottle under aseptic conditions for
pathogenicity and symptoms evaluation.
ȱ
Figure 8. Different phases of coculturing experiment for pathogenicity evaluation. (a) Healthy seedlings;
(b) cut marks at the top leaf and inoculation of mycelial plug of endophytic fungi; (c) symptoms
developed for pathogenicity evaluation; and (d) histopathological examination of infected tissues.
4.6. In Vitro Inoculation for Seedling Pathogenicity Assay

For pathogenicity experiments, the identified endophytic fungal mycelium was used for the
pathogenicity evaluation. For protocorms, 0.5-cm fungal discs were transferred into the protocorm
bottles of D. nobile and D. officinale. The control was set in triplicates under the same conditions
without added any endophytic fungal cultures. After the inoculation, the test co-culturing bottles were
transferred into the plant growth chamber instrument and maintained at 25 ◦ C along with a 14/10 h
light/dark cycle with 2000 lux light intensity. All the test bottles were examined in 7-day intervals up
to 21 days. For D. nobile and D. officinale seedlings, the contents of endophytic fungal flask cultures
were filtered to separate the mycelium suspension. The mycelium was concentrated and injected onto
cut marks at terminal leaves at the link to the stem. The sterilized cotton plug served as a control
for seedlings co-culturing pathogenicity and symptoms evaluation experiments. The disease index
was measured using the disease scale and disease index formula. The scale of the disease index is
described as follows: 0 indicates no disease (non-pathogens), 1 indicates small spots < 1% (pin brown
spots: fewest pathogens), 3 indicates spots (1–11%: least pathogens), 5 indicates spots with 12–25%
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coverage (moderate pathogens), 7 indicates circular spots (26–55% coverage: moderate pathogens),
and 9 indicates circular to irregular spots (>75% coverage: highly pathogenic) [33].
Sum o f disease rating

Disease index percentage = × 100%
Total number o f rating × Maximum disease grade
4.7. Re-Isolation and Identification of Endophytic Fungi from Seedlings

All the samples (seedlings) were washed with sterilized distilled water to eliminate surface fungal
growth. Thereafter, the seedlings were placed into laminar air flow and exposed UV rays for 20 min
to eliminate surface fungi. After UV exposure, 75% alcohol was added into the sample bottles for
30 s and mixed using gentle shaking, which was then washed twice with sterilized distilled water
under laminar airflow. Thereafter, HgCl2 was immersed into the glass bottle for 3 min with continuous
shaking. The HgCl2 water was removed after 3 min and then the samples were washed 3 times using
sterilized distilled water. Brown paper sterilized in a hot air oven at 160 ◦ C for 1.5 h was used for
cutting the samples into smaller pieces. The samples (seedling) were moved onto the potato dextrose
agar media plates. Two pieces of samples were transferred onto each Petri plate with the surface
touching the media. The plates were incubated for five days at 25 ◦ C in the incubator. After the fifth
day of incubation, fungal hyphal tips emerging from the samples were recovered and purified using
sub-culturing from growing hyphal tips. The growth, colony properties, and microscopic features
of DMEs were recorded from the strains grown on the PDA media [34,35]. Microscopic details were
obtained via a slide culture practice. Fungal hyphae stained with lactophenol cotton blue were assessed
microscopically using light microscopy (Nikon Eclipse E200MV R with DS-1600 Panasonic CMOS
Sensor, Nikon Corporation, Tokyo, Japan).
4.8. Histological Examination of Least Pathogenic Fungal Endophyte Samples

Infected D. nobile and D. officinale samples were used for visual histopathological assessment
of endophytic fungi by staining the infected tissues using a method modified from Ding et al. [36].
Infected seedling segments were stained using lactophenol cotton blue to identify the tissue infected
with endophytic fungi. The distribution of fungal endophytes and their localization was studied using
a microscope (Nikon Eclipse E200, Model Eclipse E200MV R, Nikon Corporation, Tokyo, Japan) with
DS-1600 Panasonic CMOS sensor. The endophytic fungal colonies were observed as blue after staining
with lactophenol cotton blue in vascular bundles and cortex region of D. nobile and D. officinale leaves
and stems. Photographs were captured under different magnifications (10× and 40×).

Statistical calculations were completed using SPSS11 software (USA) and the Fisher’s least
significant difference procedure. Correlations of disease index caused by C. tropicicola (ZF01),
F. keratoplasticum (ZF02), F. oxysporum (ZF03), F. solani (ZF04), and T. longibrachiatum (ZF05) were
calculated using SAS CORR procedure.
5. Conclusions
In this study, we evaluated fungal pathogenicity and colonization inside plant tissues under
in vitro conditions. Firstly, T. longibrachiatum (ZF05) was found to be the least pathogenic or a
conditional pathogen that supports the development of the D. nobile protocorms and D. officinale
seedlings without the presence of any nutrients in the media. C. tropicicola (ZF01) is a highly pathogenic
strain, responsible for the host death. We concluded that endophytic fungi were cross-transmitted from
host plant inoculated to uninoculated cells, which was confirmed by histopathological examination
and re-isolation of the same endophytic fungi from uninoculated plant tissues. Future investigations
should determine what role, if any, the plant host specificity plays in the interior plant passage and
differential tissues establishment by test fungal endophytes. How fungi are able to precisely move
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Int. J. Mol. Sci. 2020, 21, 316
within tissue (host plant) should also be examined. The symbiotic seedling and protocorm growth are
advantageous and expedient method to improve orchid growth under the experimental conditions
and could help with the reintroduction of Dendrobium orchids to the natural environment.
Supplementary Materials: The following are available online at http://www.mdpi.com/1422-0067/21/1/316/s1.

Author Contributions: S.S., A.J., X.F. performed experiments. J.C. and J.S. designed and supervised the project.
Q.J., F.S., Z.C. and Q.Z. arranged the materials for experiments. S.S. and J.C. wrote and edited the manuscript.
All authors have read and agreed to the published version of the manuscript.
Acknowledgments: The authors are grateful for the financial support under Distinguished High-Level Talents
Research Grant from a Guizhou Science and Technology Corporation Platform Talents Fund (Grant No.:
[2017]5733-001 & CK-1130-002), National Natural Science Foundation of China (31560079), the Jiangsu Synergetic
Innovation Center for Advanced Bio-Manufacture (XTD1825), Cultivation of Academic New Seedlings and
Exploration of Innovative Specialties(CK-1130-012), and Scientific and Technological Projects in Honghuagang
District of Zunyi City (2018):10. We are also thanks to our all laboratory colleagues especially Xiwu Pan for their
experimental help.
Conflicts of Interest: The authors declare no conflicts of interest.
Abbreviations
DME Dendrobium myco-endophytes
PDA Potato dextrose agar
DNA Deoxyribose nucleic acid
PCR Polymerase chain reaction
ITS Internal transcribed spacer
NCBI National centre for biological information
BLAST Basic local alignment search tool
UV Ultra Violet
HgCl2 Mercury chloride
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Molecular Sciences
Article
Comparative Transcriptomics Provides Insight into
Floral Color Polymorphism in a Pleione limprichtii
Orchid Population
Yiyi Zhang 1 , Tinghong Zhou 2 , Zhongwu Dai 1 , Xiaoyu Dai 1 , Wei Li 1 , Mengxia Cao 1 ,
Chengru Li 1 , Wen-Chieh Tsai 1,3 , Xiaoqian Wu 1 , Junwen Zhai 1 , Zhongjian Liu 1, * and
Shasha Wu 1, *
1 Key Laboratory of National Forestry and Grassland Administration for Orchid Conservation and Utilization,
College of Landscape Architecture, Fujian Agriculture and Forestry University, Fuzhou 350002, China;
[email protected] (Y.Z.); [email protected] (Z.D.); [email protected] (X.D.);
[email protected] (W.L.); [email protected] (M.C.); [email protected] (C.L.);
[email protected] (W.-C.T.); [email protected] (X.W.); [email protected] (J.Z.)
2 Huanglong National Scenic Reserve, Songpan 623300, China; [email protected]
3 Institute of Tropical Plant Sciences and Microbiology, National Cheng Kung University,
Tainan City 701, China
* Correspondence: [email protected] (Z.L.); [email protected] (S.W.); Tel.: +86-13622392666 (Z.L.);
+86-15280430239 (S.W.)

Received: 4 December 2019; Accepted: 26 December 2019; Published: 30 December 2019
Abstract: Floral color polymorphism can provide great insight into species evolution from a genetic
and ecological standpoint. Color variations between species are often mediated by pollinators and
are fixed characteristics, indicating their relevance to adaptive evolution, especially between plants
within a single population or between similar species. The orchid genus Pleione has a wide variety of
flower colors, from violet, rose-purple, pink, to white, but their color formation and its evolutionary
mechanism are unclear. Here, we selected the P. limprichtii population in Huanglong, Sichuan
Province, China, which displayed three color variations: Rose-purple, pink, and white, providing
ideal material for exploring color variations with regard to species evolution. We investigated the
distribution pattern of the different color morphs. The ratio of rose-purple:pink:white-flowered
individuals was close to 6:3:1. We inferred that the distribution pattern may serve as a reproductive
strategy to maintain the population size. Metabolome analysis was used to reveal that cyanindin
derivatives and delphidin are the main color pigments involved. RNA sequencing was used to
characterize anthocyanin biosynthetic pathway-related genes and reveal different color formation
pathways and transcription factors in order to identify differentially-expressed genes and explore their
relationship with color formation. In addition, qRT-PCR was used to validate the expression patterns
of some of the genes. The results show that PlFLS serves as a crucial gene that contributes to white
color formation and that PlANS and PlUFGT are related to the accumulation of anthocyanin which
is responsible for color intensity, especially in pigmented flowers. Phylogenetic and co-expression
analyses also identified a R2R3-MYB gene PlMYB10, which is predicted to combine with PlbHLH20 or
PlbHLH26 along with PlWD40-1 to form an MBW protein complex (MYB, bHLH, and WDR) that
regulates PlFLS expression and may serve as a repressor of anthocyanin accumulation-controlled color
variations. Our results not only explain the molecular mechanism of color variation in P. limprichtii,
but also contribute to the exploration of a flower color evolutionary model in Pleione, as well as other
flowering plants.
Keywords: Pleione limprichtii; flower color polymorphism; variation within populations; metabolome
analysis; anthocyanin biosynthetic pathway; RNA sequencing; transcription factor

Int. J. Mol. Sci. 2020, 21, 247
1. Introduction
Flower color is one of the most attractive characteristics of plants in nature. With such massive
variation, flower color is regarded as an evolutionarily labile trait and has been shown to contribute to
plant evolution [1–3]. In particular, flower color adaptive mutations mediated through pollinators
are directly relevant to phenotypic evolution [4]. Color variation is considered a fixed difference
between species and promotes the formation of population polymorphism [5,6]. Among angiosperms,
sister species always display differences in flower hue and intensity, This pattern of flower color
polymorphism is used as a model trait in the study of ecology, evolution, and gene regulation [7].
Color changes are related to flower pigment content. To date, the molecular mechanism of flower color
transition has been investigated in several species, owing to the main floral pigments having been
well characterized in many plants [8–13] providing sufficient information for studying floral color
formation in non-model species and the opportunity to explore the relationship between phenotypic
evolution and color variations.
Although flower color is influenced by many factors, flavonoids, especially anthocyanins which
are produced by the anthocyanin biosynthesis pathway (ABP), are the primary components that
contribute to floral pigments and they are produced by highly conservative structural and regulatory
components [14]. The ABP involves multi-metabolic processes which mainly consist of seven core
structural genes: CHS, CHI, F3H, F3 H, F3 5 H, DFR, and ANS, and several branch-enzyme genes [15].
Due to the instability of anthocyanidins, they exist mainly as anthocyanins, which are formed by
anthocyanidins and various glycosides [16]. They play an irreplaceable role in the color development
of plants and are primarily derived from three main anthocyanidins: Pelargonidin (brick red to scarlet),
cyaniding (red to magenta), and delphinidin (purple to violet) [16]. Studies have shown that blocking
the ABP can lead directly to variations in pigment production and affect flower color [17]. In addition
to the structural genes in the ABP, transcription factors also contribute to flower color transition by
regulating the spatial and temporal expression of the structural genes [18,19]. The ABP is regulated
by three complex, interacting transcription factors: R2R3-MYB, basic helix–loop–helix (bHLH), and
WD40-repeat (WDR) [20]. These transcription factors activate or suppress the transcription and
expression of target genes, thereby regulating anthocyanin synthesis [21]. Generally, the structural
and regulatory genes involved in the ABP have provided a number of targets to reveal the diversity
of mutations that could block the ABP [22]. For flower polymorphism within populations, locating
the blockage could elucidate the cause of flower color transition at the biochemical and molecular
scales [8,10,11]. In addition, understanding their specific ABP is of benefit for predicting evolutionary
influences from a genetic perspective.
The genus Pleione (Orchidaceae) comprises nearly 30 species of terrestrial, lithophytic, and
epiphytic plants with high ornamental value [23]. There are 27 species in China, while Yunnan is
the world biodiversity distribution center of this genus [24,25]. Pleione possesses different flower
colors ranging between white, pink, lavender, magenta, light purple, and yellow [26]. In particular,
populations of Huanglong, Sichuan Province, there remain a color polymorphism population, consisting
of pink flowers of different intensities along with white mutant individuals, which can be considered
an ideal situation to study the polymorphism formation mechanisms of Pleione, as well as benefit to
explore potential correlation between color pattern and the species evolution.
The focus of our study was to understand the molecular mechanism of color polymorphism,
including how the white individuals formed and the main reason caused pink flowers intensities, as well
as summarize the general rules of the color distribution pattern. We aimed to investigate distribution
of color monomorphic in the Huanglong P. limprichtii population and examine the transcriptome and
biochemistry of their color polymorphic petals. RNA sequencing (RNA-seq) and ultra-performance
liquid chromatography (UPLC) were used to identify the variation of related genes and the differences
in flavonoid intermediates in the ABP that cause color transition, respectively.
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Int. J. Mol. Sci. 2020, 21, 247
2. Results
2.1. Color Differences and Quantity Distribution Pattern

After applying quantitative statistics to the individuals with the three distinct flowers at the
full bloom stage randomly distributed in three rock populations, we concluded that the rose-purple
individuals account for nearly 60%, the pink ones occupied about 30%, and the white ones not more than
10% (Table 1). The ratio of the number of the three differently flowered plants (rose-purple:pink:white)
is roughly 6:3:1. Thus rose-purple is the main flower color in the Huanglong population, while white
is the rarest color.
Table 1. Number of P. limprichtii individuals of each flower color polymorph in three selected
populations at the opening stage.
Population Rose-Purple Pink White

population1 409 (58%) 227 (32%) 70 (10%)
population2 31 (65%) 15 (31%) 2 (4%)
population3 92 (60%) 54 (35%) 8 (5%)
The percentage indicates the proportion of individuals with a given color account for the total number of individuals
in the population.
According to principle component analysis, there were significant differences in the a*(redness
and greenness) values of the three colors (Figure 1. A comparison of L* (lightness) and C* (chroma)
values among the three color groups indicated that C* values can also be used as an indicator to
distinguish these three colors, since the rose-purple group has the highest C* value, the white group
the lowest, and the pink group an intermediate value (Table 2).
Figure 1. Principal component analysis of petal color of P. limprichtii. Distribution based on bivariate
values of a* (redness and greenness) and b* (yellowness and blueness). a* means difference in red and
green, b* means difference in yellow and blue.
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Table 2. Color parameters of three distinct colors of P. limprichtii petal.
Flower Color RHS L* C* b*/a*

67.92 ± 10.27 49.64 ± 9.41 −1.68 ± 0.61
76.66 ± 4.51 52.88 ± 4.18 −2.35 ± 0.23
purple red Purple-group N78B 67.35 ± 10.34 57.13 ± 6.24 −1.68 ± 0.61
81.31 ± 8.72 51.89 ± 7.42 −1.91 ± 0.44
79.68 ± 15.55 46.28 ± 11.76 −1.85 ± 0.79
61.08 ± 5.29 26.71 ± 1.82 −1.20 ± 0.36
66.05 ± 6.63 35.45 ± 9.60 −1.77 ± 0.71
Pink Purple-group 75A 66.35 ± 2.73 31.72 ± 3.14 −1.57 ± 0.29
58.80 ± 5.20 29.66 ± 6.05 −2.20 ± 1.07
62.80 ± 5.78 25.43 ± 4.99 −2.56 ± 0.89
93.08 ± 5.57 13.31 ± 9.67 −4.04 ± 2.06
88.12 ± 8.13 18.53 ± 7.31 −13.60 ± 5.70
White Purple-group 76D 95.85 ± 4.48 22.41 ± 6.77 −7.97 ± 3.88
87.52 ± 11.45 25.27 ± 10.21 −6.11 ± 2.61
87.07 ± 7.97 21.36 ± 6.12 −9.28 ± 4.73
RHS, Royal horticulture society color chart evaluation index; L*, lightness; a*, b*, chromatic components; C*, chroma
(brightness).
2.2. Major Anthocyanin Compounds in P. limprichtii

UPLC analysis revealed that four anthocyanins and derivatives were detected in both
the pigmented and white flower petals: Cyanidin 3-O-glucosyl-malonylglucoside, cyanidin
3-O-malonylhexoside, cyanidin, and delphinidin (Table 3). The cyanidin accumulation and delphinidin
accumulation branches of the ABP in P. limprichtii indicate that anthocyanins and their derivatives are
the main flower color pigments.
Table 3. Anthocyanin content of petal in P. limprichtii.
Index Ion Mode Molecular Weight Substance

pme3609 positive 287.24 Cyanidin
pme0442 positive 303.24 Delphinidin
pmb0541 positive 697.1 Cyanidin 3-O-glucosyl-malonylglucoside
pmb0542 positive 535.1 Cyanidin 3-O-malonylhexoside
2.3. RNA-Seq and Annotation of Unigenes

To understand the molecular basis of flower polymorphism in P. limprichtii, three distinctly colored
flower petals and lips were used for RNA-seq. A total of 80,525 unigenes with a mean length of 856
bp were obtained by de novo assembly. We assessed the quality of the unigenes in the transcriptome
library. The length of N50 (sequence length of the shortest contig at 50%) was 1470 bp, the Q20 and Q30
percentages were 98% and 95%, the GC content was 40%, and the unigenes were generally distributed
between 200 bp and 3000 bp in length. A total of 69,004,304 base pairs were aligned. These data show
that the throughputs and sequencing quality were high enough to ensure further analysis.
According to the BLASTx results, a total of 33,724 unigenes were annotated, accounting for 41.88%
of all the unigenes. Among these, 33,459 could be annotated using the Nr database (Non-redundant
protein database, 41.55%), 21,177 using the Swiss-prot database (Swiss-Protein protein database,
26.30%), 11,067 using the KEGG database (Kyoto encyclopedia of genes and genomes, 24.68%), and
19,871 using the KOG database (Eukaryotic orthologous groups, 13.74%). In addition, there were 8396,
151, 42, and 23 unigenes annotated only using the Nr, Swiss-protein, KEGG, and KOG databases,
respectively (Figure 2).
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Figure 2. Venn diagram of the number of unigenes annotated by BLASTx with four protein databases.
Statistical analysis of the E-value (The probability due to chance) characteristics of the
Nr annotations revealed that 32.05% of the mapped sequences showed strong homology
(E-value < 1 × 10-3 ), while 29.48% in the Swiss-protein database, 44.77% in the KEGG database,
and 30.07% in the KOG database showed strong homology (Figure 3).
ȱ
Figure 3. E-value distribution of top BLASTx hits against four protein databases for each unigene.
(a) distribution E-values in non-redundant protein database; (b) distribution E-values in Swiss-protein
database; (c) distribution E-values in the Kyoto Encyclopedia of Genes and Genomes database;
(d) distribution E-values in the Eukaryotic Orthologous Group database.
Based on the Nr annotations, 6495 unigenes were classified into 53 functional categories, belonging
to three functional terms: molecular function, cellular component, and biological process. The largest
percentages of unigenes identified within each of the three functional terms were metabolic process
(3602 unigenes), cell and cell part (2237 and 2233 unigenes), and binding and catalytic activity (2481
and 3409 unigenes), corresponding to biological process, cellular component, and molecular function,
respectively (Figure 4).
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Figure 4. Function classification of the gene ontology of all unigenes based on Nr annotation.
To exhaustively explore the potential functions of the annotated unigenes, 11,067 unigenes
were mapped onto 131 KEGG pathways, including metabolic pathways, biosynthesis of secondary
metabolites, biosynthesis of antibiotics, and many other important metabolic pathways. Metabolic
pathways and biosynthesis of secondary metabolites were enriched in the most unigenes, with 2450
and 1294 unigenes annotating to these two pathways, respectively, while the anthocyanin pathway
was enriched in only one unigene. Among these 131 pathways, there are two color formation-related
pathways, the flavonoid biosynthesis pathway (ko00941) to which 36 unigenes were mapped, and the
ABP (ko00942) to which one unigene was mapped (the pathways are listed in Supplementary Table S3).
2.4. The Expression Patterns of ABP Genes

Genes involved in the flavonoid biosynthesis and ABPs that are related to color formation were
isolated, including structural and regulatory genes, in order to identify those involved in the regulation
of flower polymorphism in P. limprichtii. There were 21 ABP-related structural unigenes, which encoded
10 enzymes, expressed both in pigmented flowers and white flowers, with a total of 14 R2R3-MYB, 32
bHLH, and 23 WD40 regulatory unigenes. With regard to structural unigenes, six of the 10 belong
to multigene families, with the exception of F3H, F3 5 H, ANR, and FLS, which were single copy
genes (Table 4). All of these unigenes were used to analyze the expression pattern of the flower color
polymorphism of P. limprichtii.
Table 4. Tentative anthocyanin biosynthesis pathway-related genes in P. limprichtii.
Pathway Gene Encoding Enzyme Number

CHS Chalcone synthase 5
CHI Chalcone isomerase 2
F3H Flavanone 3-dioxygenase 1
F3 H Flavonoid 3’-hydroxylase 3
Flavonoid biosynthesis F3 5 H Flavonoid 3’,5’-hydroxylase 1
DFR Dihydroflavonol 4-reductase 3
ANS Anthocyanidin synthase 2
ANR Anthocyanidin reductase 1
FLS Flavonol synthase 1
Anthocyanin biosynthesis UFGT Anthocyanidin 3-O-glucosyltransferase 2
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Since petal anthocyanins are detectable in different color petals, we inferred that floral color
differences were caused by different expression patterns of ABP-related genes. The results show that
the genes encoding flavonol synthese (FLS, PlFLS), anthocyanin synthese (ANS, PlANS1, PlANS2),
and UDP-glucose anthocyanidin 3-O-glucosyltransferase (UFGT, PlUFGT1, PlUFGT2) were expressed
at a higher level in pigmented flowers (rose-purple and pink) than in white flowers, and were more
up-regulated in the rose-purple flowers than in the pink flowers (Figures 5a and 6). These genes
were correlated with flower color intensity and color phenotypes. Besides these directly affected
genes, other genes that have different expression patterns between pigmented flowers and white
flowers also influence color formation, such as the flavanone 3 -hydroxylase gene (F3 H; Pl F3 H3) and
dihydroflavonol 4- reductase (DFR, PlDFR3). These were both up-regulated in rose-purple flowers
and contribute to red color formation. According to the ABP-related gene expression patterns and
metabolites detected in the three distinct flower groups, we drew a putative ABP of P. limprichtii.
Figure 5. Expression pattern analysis base on RNA sequencing of anthocyanin biosynthesis

pathway-related genes and transcription factors in P. limprichtii. (a) Anthocyanin biosynthesis
pathway-related unigenes; (b) bHLH unigenes; (c) R2R3-MYB unigenes; (d) WD40 unigenes. W-pe
(petal of white flower), P-petal (petal of pink flower), R-petal (petal of rose -purple flower).
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Figure 6. Real time quantitative reverse transcription-PCR of several genes in P. limprichtii. Each value
is shown as average ± standard deviation from three biological replicate sampling.
The expression patterns of anthocyanin regulatory genes, including R2R3-MYB, bHLH, and
WD40 were also investigated (Figure 5b–d). Phylogenetic analysis (Figure 7) shown that PlMYB13
(unigene0062421) and PlMYB4 (unigene0039181) were clustered with AtMYB75, AtMYB90, and
AtMYB113, which belong to subgroup 6 of A. thaliana [27], and have been demonstrated to activate
anthocyanin accumulation, while PlMYB10 (Unigene0058559) was homologous to AtMYB11, AtMYB12,
and AtMYB111, which belong to subgroup 7 in A. thaliana, and have been suggested to control flavonol
biosynthesis [28]. The expression pattern of PlMYB13 was consistent with anthocyanin accumulation,
while PlMYB10 exhibited an inverse relationship between its expression and flower color intensity.
Figure 7. Phylogenetic analysis of R2R3-MYB DNA binding domains for P. limprichtii and Arabidopsis
thaliana. (a) Circular phylogenetic tree; (b) amplification of S4, S6, and S7 branches. The R2R3
domains of the 14 MYBs identified in P. limprichtii petal transcriptome were aligned and analyzed using
neighbor-joining phylogenetic methods.
2.5. The Relationship between Structure Genes and TFs

Through the analysis of the expression patterns of ABP-related genes and the phylogenetic tree
of R2R3-MYB, we obtained several candidate genes that correlate with floral color intensity. We
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constructed a co-expression network (Figure 8) to identify the interactions between ABP-related genes
and MBW complex proteins (MYB, bHLH, and WDR). The results show that ten structure unigenes
and five MBW complex transcriptional unigenes composed of two R2R3-MYB, two bHLH and one
WD40 unigene exhibited interaction relationships. The expression pattern of PlMYB10 coincided with
those of PlbHLH20 (Unigene0062784) and PlbHLH26 (Unigene00660), while PlbHLH20 and PlbHLH26
also correlated with PlWD40-1 (Unigene0002153). PlMYB10 also showed a linear relationship with
PlFLS. We therefore inferred that PlMYB10 may interact with PlbHLH20 or PlbHLH26, and PlWD40-1
to form a MBW transcriptional complex, and regulate the expression pattern of PlFLS, finally affecting
flower polymorphism in P. limprichtii.
Figure 8. A co-expression network of anthocyanin biosynthesis pathway-related genes and transcription

factors involved in pigmentation. Rose-purple circle represents structural genes; dark-gray circle
represents R2R3-MYB; light-gray circle represents bHLH; pink circle represents WD40.
3. Discussion
We investigated the distribution of color polymorphic individuals in three rock populations of P.
limprichtii within Huanglong District and combined chemical detection and transcriptomic analysis to
isolate the main pigment compounds and candidate genes that determine flower color intensity. We
present a putative biosynthesis pathway and discuss the regulatory mechanisms of color formation.
To explore color variation formation factors, we used the CIELAB evaluation system to distinguish
rose-purple, pink, and white flower color, and then counted the number of individuals of each of
these phenotypes in each rock population. In all three populations the color distribution pattern was
nearly consistent, with a color ratio of rose-purple 6:pink 3:white 1. This distribution pattern is very
rare in natural color polymorphic populations. Studies have shown that intraspecific flower color
variation is often attributed to genetic drift, pollination-mediated selection, environmental conditions,
or herbivory [13,29–31]. In our experiments, the populations grow across a small range, with the whole
population occurring on similar rocks and exposed to the same climatic conditions. Environmental
elements such as temperature, drought stress, and ultraviolet radiation were therefore not considered
to be crucial promoters of color variation. Pollinator-mediated selection plays an important role
in color variation, especially for deceptive pollination species in which competition for pollinators
in sympatry promotes flower color divergence [32], and shifts in pollinators also contribute to the
macro-evolution of flowers color [22]. Thus, we inferred that the flower color polymorphism within
these populations might have been induced by pollinators. Color polymorphism may be a consequence
of pollination competition or specific adaptations to pollinators, and pollinator behavior exerts strong
selection stress on color variations. Some research also showed adaptive selection for pigmented
flowers because colored flowers are less likely to be disrupted by herbivories than colorless ones [3].
According to our field observations, we found that white-flowered individuals were more susceptible
to damage than individuals with pigmented flowers, and that white flower petals and cores were
severely foraged when blooming. We thus inferred that the dominant pigmented color was beneficial to
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avoid herbivory, and reduce damage by herbivores to the population. When individuals were damaged
they also suffered reduced attractiveness to pollinators, which is not conductive to the stability and
development of the population. This explains the distribution pattern of the number of rose-purple
flowers in the population. There is another view that flower color may not be the primary goal of
natural selection, nor the initial choice of pollinators. Indeed, the biosynthetic precursors of pigments
not only display color variations, but also serve other physiological functions [14]. Studies have
shown that secondary metabolites associated with plant defense functions share the same biosynthetic
pathway, the flavonoid synthesis pathway, correlating pigment with defense ability [33]. Therefore,
colorful-flowered individuals were more resistant to some adversities. In summary, the phenomenon
that rose-purple flowers were frequent and white ones rare within the population may be mediated by
the pollinators and herbivores, and also related to survival adaptability of the P. limprichtii. The 6:3:1
distribution pattern of color polymorphism might be a reproductive strategy for the population to
maintain the maximum population density, but further evidences should be investigated.
In theory, the transition from pigmented to white flowers could involve any mutations that
block one or more steps in the anthocyanin pathway. This includes loss-of-function mutations in any
pathway enzyme-coding genes, as well as the cis-regulatory genes that influence any of the pathway
enzymes [34,35]. In our study, expression analysis identified several obvious differentially expressed
genes in the petal which were down-regulated in white samples compared to pigmented samples, but
metabolite detection found that Cy- and Del-derivatives existed in both white petals and pigmented
petals, indicating that the color variations, especially the white petals, do not lacking any steps of the
anthocyanin pathway. The cis-regulation of transcription factors is a crucial element to promote color
divergence. This result is similar with the white color formation in Primula vulgaris which is caused
by different genes expression pattern rather than loss- of-function mutations leading to the lack of
anthocyanin [36].
For ABP-related genes, we isolated 21 transcripts which encode ten enzymes. Seven of the ten
were flavonoid synthase genes, including PlCHS, PlCHI, PlF3H, PlF3 H, PlF3 5 H, PlDFR, and PlANS;
one was a proanthocyanidin synthase gene, PlANR; one was a flavonol synthase gene, PlFLS; and
one was an anthocyanin synthase gene, PlUFGT. Most of them are multi-gene families, only PlF3H,
PlF3 5 H, PlFLS, and PlANR are single copy. To clearly illustrate the catalyzation steps of the ABP, we
regarded Cy-related and Del-related biosynthesis processes as independent branches [8]. Thus we
did not have to measure the content of each anthocyanin compound. Each branch of anthocyanin
synthase was considered to make an equivalent contribution to pigmentation. Considering that
both the Cy-related and Del-related branches may share the majority of enzymes, here, we selected
ABP-related genes on the Cy-related branches to analyze their expression patterns.
The expression analysis of early step structural genes revealed a high level of PlCHS and PlCHI
expression in white petals and a low level in pigmented petals, suggesting that white petals can
produce a large amount of naringenin but cannot eventually flux this into anthocyanin synthase.
Meanwhile, the expression levels of PlF3 H, PlF3 5 H, PlDFR, PlANS, and PlUFGT in pigmented flower
petals was high, compared with white ones, and we inferred that downstream structural genes make a
large contribution to coloration. Analysis also showed that PlFLS was significantly more upregulated
in white petals than in pigmented petals. FLS encoding enzymes lead substrate into the flavones and
flavonols pathway [37]. It has been suggested that the competition between the anthocyanin synthesis
pathway and the flavone and flavonols pathways mainly results in substrate competition between
FLS with DFR, while the FLS enzyme strengthens the metabolic flux toward the flavonols and limits
anthocyanin accumulation [38]. This situation has also been reported in other species, such as in
Paeonia ostii, a higher expression of PoFLS4 in the nearly white flowers promotes dihydroflavonols
transition into flavonols [11]. In onions, enhanced AcFLS could maximize flavonol production in the
sheath [39]. Finally, in Muscari armeniacun, the conversion of substrate between FLS and DFR facilitates
the elimination of blue pigmentation [8]. Thus, we confirmed PlFLS as one of the candidate genes for
white color formation in P. limprichtii. The up-regulation of PlDFR in pigmented flowers is closely
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accompanied by a decrease of PlFLS; hence, more dihydroflavonols flow into anthocyanin production
in pigmented flowers. From our analysis, the expression patterns of PlANS and PlUFGT are correlated
with color intensity, and they showed their highest expression levels in the rose-purple flowers and
their lowest in the white flowers. The ANS (Anthocyanidin synthase) encoding enzyme catalyzes
the conversion of colorless leucocyanidin into colored anthocyanin [40], and anthocyanin is further
glycosylated by different UFGT (UDP flavonoid glucosyl transferase) encoding enzymes that convert
the anthocyanidins to different anthocyanin derivatives, exhibiting the final color [41]. We therefore
speculate that PlANS and PlUFGT are two crucial genes that determine color intensity in P. limprichtii.
It has been revealed that the MBW protein complex, a combination of R2R3-MYB and bHLH
transcription factors, along with WD40 proteins, play an important role in regulating the transcription
of structural genes [41–45]. The activities of R2R3-MYB factors have distinct roles in determining the
action of the complexes, either to promote or inhibit the transcription of anthocyanin biosynthesis
genes [46,47]. By combining R2R3-MYB phylogenetic and co-expression network analyses, we isolated
PlMYB10, which was homologous with the AtMYB11, AtMYB12, and AtMYB111 belonging to S7 in
Arabidopsis and that have been demonstrated to contribute to the regulation of genes that account for
anthocyanin accumulation in all tissues [27,28]. The expression pattern was high expression in nearly
white flower petals, gradually reducing in flowers with increasing color intensity. It also corresponded
with the color polymorphism phenotypes. In other species, overexpression of AmMYB330, a negative
regulator of the flavonoid biosynthesis, has been proven to inhibit phenylpropanoid metabolism in
transgenic tobacco (Nicotiana tabacum) plants [48]. The co-expression network showed that PlbHLH20
and PlbHLH26, along with PlWD40-1 have a strong relationship to PlMYB10. It is likely that this
potential MBW complex PlMYB10/PlbHLH20/PlWD40-1 or PlMYB10/PlbHLH26/PlWD40-1 may serve
as a repressor responsible for the variation in color intensity in P. limprichtii. Co-expression network
revealed PlFLS is the most likely target gene interacting with PlMYB10. Previous studies have verified
that R2R3-MYB can regulate the expression pattern of FLS through the overexpression of PsMYB114L
(from Paeonia suffruticosa) in Arabidopsis [49], which is consistent with our results. Thus, we tentatively
speculate about the ABP of P. limprichtii (Figure 9). Further studies examining sequencing differences
in these candidate genes and PlMYB10 are necessary to assess our speculations. Metabolic substance
quantification is also necessary to confirm the leading pigments in P. limprichtii.
Figure 9. Tentative pathways for P. limprichtii color variations. The colored bar is the value of
log2 (RPKM + 1), represented using the depth of color, with purple representing the up-regulated
expression genes and pink representing the down-regulated expression genes. RPKM means the
reads per kb per million reads mapped. CHS, chalcone synthase; CHI, chalcone-flavanone isomerase;
F3H, flavanone-3-hydroxylase; F3’H, flavonoid 3’-hydroxylase; F3’5’H, flavonoid 3’5’-hydroxyla;
DFR, dihydroflavonols 4-reductase; ANS, anthocyanidin synthase; UFGT, UDP flavonoid glucosyl
transferase; FLS, flavonol synthase; The three gene complex consist of a MYB, bHLH and WD40 in
most angiosperm.
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Pleione limprichtii, belongs to the Orchidaceae family, and is distributed in the Huanglong Nature
Reverse in Sichuan Province, China, with three distinct color variations including rose-purple, pink,
and white (Figure 10a–c). The study site was located at 32.68◦ N, 104.04◦ E, and 1851.61 m above
sea level (Figure 10d). The plants are generally found living on rocks along streams. The sexual
reproduction of Pleione is commonly dependent on deceptive pollination and each individual usually
grows only one flower. The majority of individuals in the population have rose-purple flowers, while
white flowers are relatively less common.
Figure 10. Flower polymorphism of Pleione limprichtii in Huanglong population. (a) rose-purple
flower; (b) pink flower; (c) white flower; (d) one of the polymorphic populations on a rock.
4.2. Quantitative Statistics and Flower Colorimeter Analysis

Three rocks with P. limprichtii populations which contained individuals with all three color
variations were selected, the number of each color individuals were counted. In addition, in order to
evaluate the flower color objectively, a hand spectrophotometer (CS-280, Hangzhou Color Spectrum
Technology Co., Ltd., Hangzhou, China) was used to measure the CIE L*a*b* color components with
five technical repetitions using five different
parts of the selected petals, in order to evaluate the flower
color objectively. The formula C* = a ∗2 + b∗2 ), the L* (lightness) and C* (chroma) through the
petals were measured using the lightness coefficient ‘a*’, which indicates greenness to redness as the
value increases from negative to positive, and ‘b*’, which represents blueness to yellowness. Principle
component analysis was performed using the a* and b* values as the principle components [32].
4.3. Sampling and RNA Extraction and cDNA Synthesis

Petals from rose-purple, pink, and white flowers at the full bloom stage were sampled, immediately
frozen in liquid nitrogen, and preserved at −80 ◦ C before pigment analysis and RNA extraction. RNA
was isolated using a Quick RNA isolation Kit (Huayueyang Co., Ltd., Beijing, China), according to the
manufacturer’s instructions. The concentration and purity of the RNA was measured with a NanoDrop
2000 (Thermo Fisher Scientific Co., Ltd., Waltham, MA, USA) and Agilent 2100 (Agilent Technologies
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Co., Ltd., Palo Alto, CA, USA) to verify RNA integrity. A total of nine samples, including three
biological replicates, with high concentrations of RNA for each of the three color morphs were selected,
then the strand cDNA synthesis was performed using a Revert Aid First Strand cDNA Synthesis Kit
(Thermo Fisher, Foster City, CA, USA), according to the manufacturer’s instructions, and was stored at
−80 ◦ C for RT-qPCR assays.
4.4. Measurement of Flower Anthocyanin

Sample preparation and extraction methods were as follow, the freeze dried tissues were crushed
using a mixer mill (MM 400, Verder Shanghai Instruments and Equipment Co., Ltd., Shanghai, China)
with a zirconia bead for 1.5 min at 30 Hz. A 100 mg sample of the powder was weighed and extracted
overnight at 4 ◦ C with 1.0 mL 70% aqueous methanol. Following centrifugation at 10, 000× g for
10 min, the extracts were absorbed and filtered. Then the sample extracts were analyzed using a
UPLC system, the analytical conditions were as follow, HPLC: column, Waters ACQUITY UPLC HSS
T3 C18 (1.8 μm, 2.1 mm × 100 mm); solvent system, water (0.04% acetic acid): acetonitrile (0.04%
acetic acid); gradient program, 95:5 v/v at 0 min, 5:95 v/v at 11.0 min, 5:95 v/v at 12.0 min, 95:5 v/v
at 12.1 min, 95:5 v/v at 15.0 min; flow rate,. 0.40 mL/min; temperature, 40 ◦ C; injection volume:
2 μL [32,50]. The mass spectrometry data was analyzed using the software Analyst 1.6.1 (AB Sciex Pte.
Ltd., Concord, Ontario, Canada) and based on the Metware Database (MWDB, Metware Biotechnology
Co., Ltd., Wuhan, China), a local self-built database, and a public metabolite information database, to
identify anthocyanin compounds. The experiments were repeated three times. 1.8 μm
4.5. Library Preparation and Sequencing

The construction of the libraries and RNA-seq were performed by the Omicshare Biotechnology
Corporation (Guangzhou, China). The mRNA was enriched with Oligo (dT) beads, then broken into
short fragments and reverse transcribed into cDNA as templates. First and second-strand cDNA were
then synthesized. The cDNA fragments were purified with a QiaQuick PCR extraction kit (Qiagen,
Valencia, CA, USA) and end repaired. Poly (A) was added and ligated to Illumina sequencing adapters.
They were then sequenced using the Illumina HiSeqTM 4000 (Illumina Inc., Centre Drive, San Diego,
CA, USA).
4.6. De Novo Transcriptome Assembly Annotation

Transcriptome de novo assembly was performed with clean data, filtered from the raw data by
removing adaptors and unknown nucleotides (>10%), and those with low quality reads. The data were
assembled using the Trinity platform [51] with the parameters ‘K-mer = 31 and ‘K-mer cover = 6 .
First, short reads of a certain length were combined with overlap to form longer contigs. Then, based
on their paired-end information, clean reads were mapped back to the corresponding contigs. Thus
the sequences of the transcripts were finished, and defined as unigenes. All assembled unigenes
were then annotated using BLASTx (E-value ≤ 1 × 10-5 ) against protein databases, including the
National Center for Biotechnology Information non-redundant (Nr, ftp://ftp.ncbi.nih.gov/blast/db/),
Swiss-Protein (https://www.uniprot.org/), Kyoto Encyclopedia of Genes and Genomes (KEGG, https:
//www.genome.jp/kegg/), and Gene Ontology (GO, http://geneontology.org/) databases. while Nr
and Swiss-Protein annotate gene function, KEGG is used to understand biological systems and GO
divides genes into different categories. BLASTx was used to search for the unigenes against the
public databases with the following order of priority: Nr, Swiss-protein, KEGG, and COG. When a
unigene could not be aligned to any of these protein databases, the protein code sequence and sequence
direction was confirmed using the ESTscan program.
4.7. Expression Profile and RT-qPCR

To compare color-related unigenes expression divergence, they were first normalized to RPKM
(reads per kb per million reads). After that, different sample ratios of RPKM values were calculated.
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The FDR (false discovery rate) value was used to identify the threshold of the p-value in multiple tests
in order to compute the significance of the differences among unigenes. Here, only FDR significance
scores < 0.05, and log2 ratios > 1 were regarded as differentially expressed genes and used in subsequent
analysis. In order to validate the expression pattern of the RNA-Seq results, ten important different
expression genes (the primers used are listed in Table S1) were selected and measured using RT-qPCR on
a Quant Studio 5 Real-Time PCR System (Thermo Fisher, Foster City, CA, USA) using the PowerUpTM
SYBRTM Green Master Mix (Thermo Fisher, Foster City, CA, USA), according to the manufacturer’s
instructions. PlUBC34 and PlUBC35 (ubiquitin-conjugating enzyme; primers are also listed in Table S1)
actin genes from Pleione were used as the internal control for the normalization of gene expression.
Each sample (including three biological repetitions) was quantified in triplicate.
4.8. Genes Related to the ABP and Phylogenetic Analyses

The ABP-related structure genes and transcription factors including CHS, CHI, F3H, F3 5 H, DFR,
FLS, ANS, FNS, UFGT, MYB, bHLH, and WD40 were used as queries to retrieve the corresponding
unigenes from the P. limprichtii libraries. Meanwhile, every reference gene from model plants obtained
from KEGG were aligned with the libraries using BLASTx to search for more related unigenes.
For the MYB genes, to determine which of the genes belonged to which R2R3-MYB subfamily,
the R2R3-MYB genes from Arabidopsis thaliana were used to conduct phylogenetic analyses (Amino
acid sequence obtained from Gene Bank, accession number is listed in Supplementary Table S2), and
MEGA7.0 software (Institute of Molecular Evolutionary Genetics, PA, USA) [52] was used to perform
sequence aligning and construct a circular phylogenetic tree according to neighbor joining method
with 1000 interactions.
4.9. Structure Genes and Tfs Co-Expression Network

The String online database (https://string-db.org/) was used to search for the interaction
relationships between structure genes and transcription factors. Then we used Cytoscape software
(National Institute of General Medical Sciences, Bethesda, MD, USA) to construct a co-expression
network to identify their main impact factors [53].
5. Conclusions
The ratio of 6:3:1 distribution patterns of P. limprichtii within the population in Huanglong
District seems to reveal that pollinators, herbivores, and survival adaptability could promote the
development of such a reproductive strategy in the population to maintain the maximum population
density. Function and expression patterns indicated that PlFLS probably is a crucial gene in the
formation of white and pigmented flowers. PlANS and PlUFGT were found to determine the color
intensity in pigmented flowers. In addition, a putative MBW complex, PlMYB10/PlbHLH20/PlWD40-1
or PlMYB10/PlbHLH26/PlWD40-1 may serve as a repressor of regulated PlFLS expression that is
responsible for variation in the color intensity of P. limprichtii. Our results provide valuable molecular
information on floral color variations in Pleione, and also provide inspiration to further explore the
relationship between color polymorphism and species evolution and to study its contribution to
color evolution.

Author Contributions: This study was conceived by S.W. and Z.L. Y.Z. performed the transcriptome and gene
expression analysis and wrote the manuscript. T.Z. and Z.D. helped with the sampling and field work. X.D., W.L.,
and M.C. helped to perform the experiment. C.L. and X.W. revised the manuscript. W.-C.T. and J.Z. provided
writing guidance. All authors contributed to revising the manuscript. All authors have read and agreed to the
published version of the manuscript.
Funding: This work was supported by the Fujian Natural Science Foundation Project (grant no. 2019J01410), the
Fujian Agriculture and Forestry University 2015 Outstanding Youth Fund Project (grant no. xjq201620), and the
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Fujian Agriculture and Forestry University Science and Technology Innovation Special Fund Project (grant no.
KFA17331A).
Acknowledgments: The authors thank Bai-Jun Li, Jie-Yu Wang, Yan-Qiong Chen, Guo-Qiang Zhang, Si-jin Zeng,
Zhi-yao Ren, and Xueyan Yuan who helped with this research. We also thank Qi-Xuan Peng and other staff of
Huanglong National Scenic Reserve for assisting sampling.
Conflicts of Interest: The authors declare no conflict of interest. Huanglong National Scenic Reserve help to
sample, and OmicShare company help to analysis raw data.
Abbreviations
UPLC Ultra performance liquid chromatography
ABP Anthocyanin biosynthesis pathway
CHS Chalcone synthase
CHI Chalcone isomerase
F3H Flavanone 3-hydroxylase
F3 H Flavonoid 3’-hydroxylase
F3 5 H Flavonoid 3’5 -hydroxyla
FLS Flavonol synthase
DFR Dihydroflavonol 4-reduct
ANS Anthocyanidin synthase
ANR Anthocyanidin reductase
UFGT UDP flavonoid glucosyl transferase
RHS Royal horticulture society
RHSCC The royal horticulture society color chart
NR Non-redundant protein database
Swiss-Prot Swiss-Protein protein database
KEGG Kyoto encyclopedia of genes and genomes
KOG Eukaryotic orthologous groups
GO Gene ontology
RPKM reads per kb per million reads
FDR false discovery rate
qRT-PCR Real-time reverse transcription-PCR
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Molecular Sciences
Article
Comparative Analysis and Expression Patterns of the
PLP_deC Genes in Dendrobium officinale
Lei Zhang 1,2 , Chunyan Jiao 1,2 , Yunpeng Cao 3,4 , Xi Cheng 1,2 , Jian Wang 1,2 , Qing Jin 1,2, *
and Yongping Cai 1,2, *
1 School of Life Sciences, Anhui Agricultural University, Hefei 230036, China; [email protected] (L.Z.);
[email protected] (C.J.); [email protected] (X.C.); [email protected] (J.W.)
2 Anhui Provincial Engineering Technology Reserach Center for Development & Utilization of Regional
Characteristic Plants, Anhui Agricultural University, No. 130, Changjiang West Road, Hefei 230036, China
3 Key Laboratory of Cultivation and Protection for Non-Wood Forest Trees, Ministry of Education,
Central South University of Forestry and Technology, Changsha 410004, China; [email protected]
4 Key Lab of Non-wood Forest Products of State Forestry Administration, College of Forestry,
Central South University of Forestry and Technology, Changsha 410004, China
* Correspondence: [email protected] (Q.J.); [email protected] (Y.C.);
Tel.: +86-551-65786907 (Q.J.); +86-551-65786137 (Y.C.)

Received: 15 November 2019; Accepted: 17 December 2019; Published: 20 December 2019
Abstract: Studies have shown that the type II pyridoxal phosphate-dependent decarboxylase
(PLP_deC) genes produce secondary metabolites and flavor volatiles in plants, and TDC (tryptophan
decarboxylase), a member of the PLP_deC family, plays an important role in the biosynthesis of
terpenoid indole alkaloids (TIAs). In this study, we identified eight PLP_deC genes in Dendrobium
officinale (D. officinale) and six in Phalaenopsis equestris (P. equestris), and their structures, physicochemical
properties, response elements, evolutionary relationships, and expression patterns were preliminarily
predicted and analyzed. The results showed that PLP_deC genes play important roles in D. officinale
and respond to different exogenous hormone treatments; additionally, the results support the selection
of appropriate candidates for further functional characterization of PLP_deC genes in D. officinale.
Keywords: Dendrobium officinale; PLP_deC; bioinformatics; expression pattern analysis; evolution
1. Introduction
Dendrobium officinale Kimura et Migo (also known as D. catenatum) is a perennial herb that is
commonly used as a valuable Chinese herbal medicine and has a long evolutionary history among
orchids. D. officinale is rich in alkaloids [1,2], and its genome, transcriptome, and metabolome indicate
that D. officinale may also contain terpenoid indole alkaloids (TIAs) [3–5]. The common precursor
of TIAs is strictosidine, which is formed by the combination of tryptamine and secologanin [6,7].
Tryptophan decarboxylase (TDC), which catalyses the formation of tryptamine, belongs to the type II
pyridoxal phosphate-dependent decarboxylase (PLP_deC) family [8]. To date, the actual roles of many
PLP_deCs in plants are still unknown due to a lack of relevant protein sequences and information
about the biochemical properties. In particular, the role of PLP_deC in the alkaloid synthesis pathway
of D. officinale has not been reported.
Pyridoxal 5 -phosphate (PLP) is the active form of vitamin B6 and is used by a variety of enzymes
in all organisms [8]. Previously, we classified all PLP-dependent enzymes into at least five structural
groups based on their protein structures [8,9]. Among them, the type I group is the most common and
contains aminotransferases, decarboxylases, and an enzyme that catalyzes α-, β-, or γ-eliminations.
Type II encodes the enzymes involved in β-elimination reactions. Type III is primarily alanine-racemase

Int. J. Mol. Sci. 2020, 21, 54
specific, while type IV enzymes typically include D-alanine aminotransferases. Type V enzymes are the
most diverse, including glycogen and starch phosphorylases. One important group of PLP-dependent
enzymes belongs to the PLP_deC family, which includes aromatic-L-amino acid decarboxylase (AAD),
glutamic acid decarboxylase (GAD), and histidine decarboxylase (HDC) [10]. The biological functions
of plant and animal AADs are closely related to their corresponding substrate selectivity and catalytic
reactions; thus, some AADs, such as tyrosine decarboxylase (TYDC) and tryptophan decarboxylase
(TDC), are further annotated based on their principal substrates [8,11]. These enzymes catalyze the
decarboxylation of aromatic L-amino acids and are primarily involved in the synthesis of secondary
metabolites in plants [12,13].
Numerous data indicate that PLP_deC exhibits tissue-specific and inducible transcript accumulation
during plant development. In addition, several roles of PLP_deC in plant development have been
identified. For example, TDC is a key enzyme that links primary and secondary metabolism with high
substrate specificity [14,15]. In addition, the transcript levels of PLP_deC genes are affected by abscisic
acid (ABA), methyl jasmonate (MeJA), salicylic acid (SA), and abiotic stress [16,17].
In 2015, Chinese scientists announced that they had completed the genome sequence of the
orchid Phalaenopsis equestris [18]. D. officinale and P. equestris (Schauer) Rchb.f. are epiphytes in the
family Orchidaceae. The draft of the D. officinale genome sequence was reported recently [4,19].
To further understand the PLP_deC gene family in orchids, we identified 8 and 6 PLP_deC genes from
the genomes of D. officinale and P. equestris, respectively, and analyzed their phylogenetic relationships,
gene structures, cis-regulatory elements, tissue expression patterns, and expression profiles under
MeJA, ABA, and SA treatments. Our results may not only improve the current understanding of the
evolutionary expansion, sequence conservation, and functional differentiation of PLP_deC genes but
also provide in-depth basic biological information for further studies of the evolution of these genes
in Orchidaceae.
2. Results
2.1. Sequence Analysis of PLP_deC Family Members

HMMER software was used to identify candidate genes in the genomes of D. officinale and P.
equestris. All candidate genes were submitted to Pfam and SMART for verifying the presence of the
PLP_deC domain. The sequences without the conserved domain and the redundant sequences were
removed. Finally, a total of 14 PLP_deC-family sequences were obtained in D. officinale and P. equestris.
The basic information of each PLP_deC was listed in Table 1. We found that in D. officinale, the molecular
weights ranged from 34.48 kDa (DoGAD4) to 79.29 kDa (DoAAD2), with an average molecular weight
of 57.64 kDa, and the theoretical isoelectric points ranged from 5.27 (DoHDC1) to 7.52 (DoAAD1),
with an average value of 6.12. In P. equestris, the molecular weights ranged from 53.44 kDa (PeHDC1) to
57.26 kDa (PeGAD3), with an average molecular weight of 55.53 kDa, while the theoretical isoelectric
points ranged from 5.53 (PeGAD1) to 8.55 (PeGAD3), with an average of 6.52. These results showed
that most genes have an acidic pI. We analyzed the subcellular localization of 8 and 6 PLP_deC-family
protein sequences in D. officinale and P. equestris by Target P 1.1. and the results showed the presence of
a signal peptide in DoAAD1 and DoAAD3, suggesting their cellular localization in extracellular with a
probability of 0.735 and 0.561, The proteins of DoHDC1, PeAAD1, PeAAD2 and PeHDC1 are possibly
located in other cellular compartments, e.g. chloroplast transit peptide and mitochondrial, whereas the
location of the remaining eight proteins is unknown (Table 2).
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Int. J. Mol. Sci. 2020, 21, 54
Table 1. Sequence analysis of pyridoxal phosphate-dependent decarboxylase (PLP_deC) family members.
Gene Name Gene ID Location pI MW (kDa) Pyridoxal_deC Domain

√
PeGAD1 PAXXG012610 scaffold3 5.53 55.66 √
PeAAD1 PAXXG064070 scaffold28 6.43 54.31 √
PeAAD2 PAXXG110580 scaffold67 6.68 55.69 √
PeHDC1 PAXXG233810 scaffold291 5.96 53.44 √
DoAAD2 Dendrobium_GLEAN_10136748 scaffold166 6.32 79.29 √
DoGAD3 Dendrobium_GLEAN_10126118 scaffold506 5.88 68.10 √
DoHDC1 Dendrobium_GLEAN_10045094 scaffold7529 5.67 73.82 √
DoGAD1 Dendrobium_GLEAN_10044649 scaffold7738 5.27 47.84
√
Note: shows conserved domain of Pyridoxal_deC.
Table 2. Subcellular localization of PLP_deC proteins.
Chloroplast Transit Mitochondrial

Gene Name Signal Peptide Other Location Reliability Class
Peptide Targeting Peptide
DoAAD1 0.040 0.026 0.735 0.346 S 4
DoAAD2 0.093 0.239 0.029 0.806 * 3
DoAAD3 0.039 0.109 0.561 0.525 S 5
DoGAD1 0.218 0.279 0.203 0.148 * 5
DoGAD2 0.135 0.068 0.180 0.727 * 3
DoGAD3 0.570 0.190 0.106 0.161 * 4
DoGAD4 0.015 0.261 0.229 0.061 * 5
DoHDC1 0.096 0.194 0.134 0.846 _ 2
PeAAD1 0.038 0.201 0.091 0.893 _ 2
PeAAD2 0.128 0.098 0.088 0.900 _ 2
PeGAD1 0.324 0.135 0.110 0.383 * 5
PeGAD2 0.460 0.188 0.134 0.243 * 4
PeGAD3 0.283 0.595 0.056 0.072 * 4
PeHDC1 0.064 0.116 0.219 0.845 _ 2
Note: Confidence levels range from 1 to 5, and 1 shows the highest predicted reliability with difference > 0.8; 2:
0.8 > difference > 0.6; 3: 0.6 > difference > 0.4; 4: 0.4 > difference > 0.2; 5: 0.2 > difference. Location: C, M, and S,
represent chloroplast, mitochondrial and extracellular, respectively. _ other location, * unknown location.
2.2. Analysis of the Gene Structures, Conserved Motifs, and Phylogenetic Relationships of PLP_deC Genes
To clarify the evolutionary relationships among the PLP_deC genes, we compared the PLP_deC
proteins from Arabidopsis thaliana (A. thaliana), Oryza sativa (O. sativa), D. officinale and P. equestris.
We used the maximum likelihood (ML) method to construct a phylogenetic tree using IQ-TREE
software. As shown in Figure 1, the 42 PLP_deC genes could be divided into three subfamilies: GAD,
HDC, and aromatic-L-AAD. The PLP_deC genes in D. officinale and P. equestris were named according
to their relative homology with A. thaliana and O. sativa genes. Among them, the GAD subfamily was
the largest, with 18 PLP_deC genes, and the HDC subfamily was the smallest, with 6 members.
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Figure 1. Phylogenetic analysis of PLP_deC genes from Dendrobium officinale, Phalaenopsis equestris,
Oryza sativa, and Arabidopsis thaliana. The maximum likelihood (ML) tree was created using IQ-TREE
with 8 D. officinale (Do), 6 P. equestris (Pe), 15 O. sativa (Os) and 12 A. thaliana (At) PLP_deC protein
sequences. The red, green, and blue colors indicate GADs, HDCs, and AADs, respectively. Bootstrap
supports are indicated at each branch.
To further analyze the gene structures and conserved motifs of the PLP_deC family members in
D. officinale and P. equestris, a total of 10 motifs were identified from the amino acid sequences of the
PLP_deC family members using the Multiple EM for Motif Elicitation (MEME) software (Figure 2).
We checked these motifs to verify they are known domains by pfam and the motif logos were generated
using online MEME program (Figure S1). Group I, which includes GAD sequences, harbors all
the motifs; group II, which includes AAD sequences, harbors motifs 5, 6, 9, and 10; and group III,
which includes HDC sequences, harbors motifs 4, 5, and 10. These results show that most PLP_deC
genes in the same subfamily have highly similar motifs, which supports their close evolutionary
relationships and the reliability of the constructed phylogenetic tree. Remarkably, motif 10 and motif 5
are present in all subfamilies, but they are not part of the PLP_deC domain. Thus, we speculate that
they may perform other specific functions. In addition, we used the online Gene Structure Display
Server to analyze the gene structures. The results showed that the number of exons in PLP_deC genes
ranged from 2 to 14. For example, there are 3–8 exons in subfamily GAD, 2–14 exons in subfamily AAD,
and 5 or 7 exons in subfamily HDC. These results indicate that the number of exons in the PLP_deC
gene family has increased or decreased during evolution, providing a basis for functional differences
among the homologous PLP_deC genes (Figure S2).
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Int. J. Mol. Sci. 2020, 21, 54
Figure 2. Distribution of 10 putative conserved motifs in PLP_deC proteins.
2.3. Analysis of Evolutionary Patterns of PLP_deC Genes

We analyzed the relationships of D. officinale and P. equestris homologous gene pairs to further
analyze the evolutionary patterns of PLP_deC genes. We obtained six homologous gene pairs
(PeGAD1–DoGAD1, DoGAD2–DoGAD4, PeAAD1–DoAAD3, DoAAD2–PeAAD2, PeHDC1–DoHDC1,
and PeGAD2–PeGAD3) and calculated their Ka, Ks, and Ka/Ks values. The estimation of the
synonymous (Ks) and nonsynonymous (Ka) nucleotide substitution rates is one of the important
parameters for molecular evolutionary analyses, which are, respectively, defined as the number of
synonymous substitutions per synonymous site and the number of nonsynonymous substitutions per
nonsynonymous site per year or per generation. It is generally recognized that Ka/Ks > 1, Ka/Ks = 1,
and Ka/Ks < 1 indicate positive, neutral, and purifying selection, respectively [20]. The experimental
results showed that the Ka/Ks values of two homologous pairs (DoAAD2–PeAAD2, PeHDC1–DoHDC1)
were lower than 0.3, the Ka/Ks values of the three homologous pairs (PeAAD1–DoAAD3,
DoGAD2–DoGAD4, and PeGAD1–DoGAD1) were between 0.3 and 1, and the Ka/Ks value of one of the
gene pairs (PeGAD2–PeGAD3) was greater than 1 (Table 3). These data indicated that most homologous
PLP_deC gene pairs are subjected to purifying selection. To further understand the influence of selection
on the homologous gene pairs, we performed a sliding window analysis. The grey regions shown in
Figure 3 represent the conserved domains. The Ka/Ks values for the conserved domains were typically
less than 1, indicating that these pairs have purifying selection. These findings suggest that purifying
selection may have played a key role in the evolution of this gene family.
Table 3. The Ka, Ks, and Ka/Ks values of gene pairs.
Gene Pair Ka Ks Ka/Ks

PeGAD1 DoGAD1 0.6314 0.8256 0.7645
DoGAD2 DoGAD4 0.194 0.37 0.5243
PeAAD1 DoAAD3 0.3143 0.5474 0.5742
DoAAD2 PeAAD2 0.1117 0.76 0.147
PeHDC1 DoHDC1 0.1642 0.5726 0.2868
PeGAD2 PeGAD3 0.2015 0.1384 1.4559
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Int. J. Mol. Sci. 2020, 21, 54
Figure 3. Sliding window analysis of Ka/Ks for each gene pair. The window size is 150 bp, and the step
size is 9 bp. The grey region represents the conserved domain.
2.4. D. officinale PLP_deC Gene Expression in Different Tissues

To understand the gene expression pattern of the PLP_deC genes in D. officinale, we performed an
overall in silico analysis of gene expression profiles in eight tissues (root, root tip, stem, leaf, sepal,
column, lip, and flower bud). These PLP_deC genes exhibited distinct organ-specific expression and
could be divided into three groups. As shown in Figure 4, in group A, two genes (DoAAD1 and
DoAAD2) were highly expressed in the flower bud and lip columns, indicating that these PLP_deC genes
may be involved in the development of these tissues. In addition, DoAAD2 had a high expression level
in the sepal, indicating that DoAAD2 may be involved in sepal development. The four PLP_deC genes in
group B were generally expressed in low amounts in all eight tissues. Remarkably, a homologous gene
pair (DoGAD2–DoGAD4) exhibited similar patterns of expression and had relatively low transcript
abundance in different tissues. In group C, DoAAD3 had low expression in all tissues. DoGAD3 had
higher expression levels in the column and sepal. Overall, the PLP_deC genes with high expression
levels may be involved in tissue growth and differentiation in D. officinale.
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Int. J. Mol. Sci. 2020, 21, 54
Figure 4. Expression profiles of PLP_deC genes in different tissues. (a) Heatmap of the in silico expression
analysis in different tissues and organs. Blue and red indicate lower and higher transcript abundance,
respectively. (b) PLP_deC genes expressed in different tissues and organs. Green, yellow, and red indicate
low (0.01–0.71 FPKM), medium (1–1.71 FPKM), and high (3.42–28.4 FPKM) expression, respectively.
2.5. Identification of Cis-Acting Elements of PLP_deC Genes

Plant growth and development are regulated by different cis-elements in genes. Therefore,
we used the PlantCARE database to identify and analyze cis-elements in the PLP_deC genes and
identified three categories of cis-elements: plant growth and development, phytohormone response,
and biotic and abiotic stress response. The cis-acting elements in the growth and development category
included the CAT-box for meristem expression, O2 -site for zein metabolic regulation, MRE and Box 4
for light response, and others. We identified 35 Box-4 motifs, which compose the largest portion of
the growth and development category. All the PLP_deC genes contained Box-4 elements (Figure 5a),
indicating that the expression of all these genes is closely related to light. In the phytohormone response
category, TCA elements for SA response and P-boxes and TATC-boxes for gibberellic acid response
were identified. Notably, TGACG motifs for MeJA response accounted for 47% of the phytohormone
response category (Figure 5c), while ABRE elements for ABA response accounted for 33%. The last
category was biotic and abiotic-stress-response elements, including AREs and GC motifs for anerobic
response, TC-rich repeats for defence and stress response, and MBSs and LTRs for low temperature
response. Our data suggested that the PLP_deC genes may respond to abiotic stresses in D. officinale.
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Int. J. Mol. Sci. 2020, 21, 54
ȱ
Figure 5. Numbers of cis-acting elements in all the PLP_deC genes of D. officinale and P. equestris. (a) The
different colors and numbers in the grid indicate the numbers of different promoter elements in each
PLP_deC gene. (b) The different colored histogram represents the numbers of cis-acting elements in the
different categories. The red, blue, and green indicate plant growth and devolepment, phytohormone
response, and biotic and abiotic stress, respectively. (c) The pie charts indicate the percentages of
different promoter elements in the different categories.
2.6. Analysis of the Expression Patterns of PLP_deC Genes in D. officinale

In plants, many stress responses are modulated or mediated by various signaling pathways that
are inseparable from gene expression and regulation [21]. To investigate the responses of the PLP_deC
genes to different hormone treatments, we used qRT-PCR to analyze their expression under MeJA,
ABA, and SA treatments.
In the ABA treatment, we found that DoAAD1, DoAAD2, DoAAD3, DoGAD1, DoGAD3,
and DoGAD4 reached their highest expression levels after 72 hours, and DoGAD1 and DoGAD3
were strongly upregulated (by more than 110-fold and 50-fold, respectively). The expression of
two PLP_deC genes (DoGAD2 and DoHDC1) peaked at 96 h, and DoGAD2 was strongly upregulated
(more than 250-fold) (Figure 6). In the MeJA treatment, three PLP_deC genes (DoAAD3, DoGAD3,
and DoHDC1) showed strong upregulation at 2 h (Figure 7), while five PLP_deC genes (DoAAD1,
DoAAD2, DoGAD1, DoGAD2, and DoGAD4) were strongly upregulated after 4 h of treatment. In the
SA treatment, the expression levels of DoAAD1, DoAAD3, DoGAD1, DoGAD2, and DoHDC1 were
strongly upregulated at 2 h (Figure 8). However, DoAAD2 and DoGAD3 were strongly upregulated
after treated with SA for 72 h of treatment (over 645- and 508-fold, respectively).
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Figure 6. The expression levels of PLP_deC genes in D. officinale under abscisic acid (ABA) treatment.
The x-axis represents the treatment time, and the y-axis represents the gene expression level. Error bars
indicate the mean and standard deviation (SD). The asterisks indicate significant difference relative to
the time 0. ** significant difference (p < 0.01), * significant difference (p < 0.05).
Figure 7. The expression level of PLP_deC genes in D. officinale under methyl jasmonate (MeJA)
treatment stress. The x-axis represents the treatment time, and the y-axis represents the gene expression
level. Error bars indicate the mean and standard deviation (SD). The asterisks indicate significant
difference relative to the time 0. ** significant difference (p < 0.01), * significant difference (p < 0.05).
Figure 8. The expression levels of PLP_deC genes in D. officinale under salicylic acid (SA) treatment.
The x-axis represents the treatment time, and the y-axis represents the gene expression level. Error bars
indicate the mean and standard deviation (SD). The asterisks indicate significant difference relative to
the time 0. ** significant difference (p < 0.01), * significant difference (p < 0.05).
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3. Discussion
The type II PLP_deC enzymes are an important group of carboxylases among the PLP-dependent
enzymes. Many data indicate that PLP_deCs show developmental, tissue-specific, and inducible
transcript accumulation during plant development [19,22]. In this paper, we identified 8 and 6 PLP_deC
genes from the D. officinale and P. equestris genomes, respectively. According to the phylogenetic analysis,
all the PLP_deC genes from A. thaliana, O. sativa, D. officinale, and P. equestris were clustered into GAD,
AAD, and HDC subclasses based on their high sequence similarity, which is consistent with the ML
tree of PLP_deC genes from the genomes of 18 species and previously published articles [17]. However,
some of these genes might have evolved with different functions. For example, in tomato, SlHDC19 and
SlHDC6 do not act on histidine but prefer tyrosine as their substrate [17]. Furthermore, many HDCs are
biased toward serine rather than histidine based on biochemical analysis [18]. Therefore, although their
sequences have high similarity, PLP_deC genes have individual substrate specificities; we should
perform an in-depth biochemical characterization to understand their precise functions [23].
Gene duplication is a common phenomenon in species and contributes to the generation of
biodiversity during evolution [24]. To date, the chromosome assemblies of the D. officinale and
P. equestris genomes have not yet been finished [25], and thus, the homologous genes of D. officinale and
P. equestris cannot yet be clearly shown on the chromosomes. Therefore, we are unable to determine
the type of replication events that have occurred between these species. To further understand the
evolutionary patterns of the PLP_deC genes, we calculated the Ka and Ks values of homologous gene
pairs. We predicted that two gene pairs (PeGAD1–DoGAD1 and PeAAD2–AAD2) are evolved from
the genome-wide duplication events shared by D. officinale and P. equestris, because their values of
Ks are 0.7 to 1.1 [26]. The Ka/Ks values in this experiment were less than 1 for all the homologous
gene pairs except for PeGAD2–PeGAD3, implying that these gene pairs have undergone purifying
selection during evolution. In addition, we noticed four homologous gene pairs (PeGAD1–DoGAD1,
PeAAD2–AAD2, PeAAD1–DoAAD3, and PeHDC1–DoHDC1) had the comparatively high Ka/Ks
values (>0.5), showing that these gene pairs have undergone rapid evolutionary diversification after
duplication events in the course of evolution [24].
Analysis of D. officinale PLP_deC gene expression in different tissues can help us better understand
the tissue specificity of the PLP_deC genes. Therefore, expression profiles for all the PLP_deC genes
were established using published RNA-sequence data. Among them, DoAAD1, DoAAD2, and DoGAD3
were highly expressed in different tissues, indicating that these PLP_deC genes play important roles
during D. officinale growth and development. For example, GADs are involved in many cellular
processes, including pollen-tube development in Arabidopsis and Picea wilsonii [25,27]. In this study,
some cis-acting elements associated with particular tissues were identified in the PLP_deC gene
promoter regions, such as the O2 -site required for seed expression and the CAT-box required for
meristem organization. The corresponding PLP_deC genes (such as DoAAD1 and DoAAD2) might play
important role in the formation of reproductive organs.
Many studies have suggested that the expression levels of PLP_deC genes are also influenced
by abiotic and biotic stresses [28–30]. Furthermore, plant hormones such as ABA, SA, and ethylene
also modulate the expression of these genes [17,29,30]. In this study, we identified a number of
cis-acting elements in the promoter regions of PLP_deC genes in both D. officinale and P. equestris, such as
MBS, MRB, Box 4, and ABRE. We found that these PLP_deC genes contain at least one abiotic stress
cis-element, which showed that they may contribute to biotic and abiotic stress responses. To further
investigate the responses of the PLP_deC genes to different hormones, we analyzed their expression
with the treatments of MeJA, ABA, and SA by qRT-PCR. We observed that the PLP_deC genes had
significantly differential expression patterns under different treatments. Some of the PLP_deC genes
showed strong upregulation under the treatments, indicating that these genes play key roles in the
abiotic stress responses of D. officinale. For example, DoAAD2 was strongly upregulated (645-fold) after
72 h of SA treatment. Overall, we found that the PLP_deC genes of D. officinale responded to abiotic
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stress, such as MeJA, ABA, and SA stresses. These results provide strong evidence that the PLP_deC
genes in plants are involved in abiotic stress responses.
4.1. Materials and Treatments

Seedlings of D. officinale were planted on Murashige and Skoog (MS) medium and placed in a
tissue culture chamber at a constant temperature of 25 ◦ C (16 h light/8 h dark) for 1 month. The tissue
culture seedlings were then transferred to MS medium containing 1.0 mg/L 6-BA (biosharp, Shanghai,
China), 0.1 mg/L NAA (Aladdin, Shanghai, China), and 30 g/L sucrose (Aladdin, Shanghai, China).
The induced protocorms (PLBs) were supplemented with 1/2 MS liquid medium containing 0.1 mg/L
α-naphthylacetic acid (NAA), 0.1 g/L whey protein hydrolysate, and 30 g/L sucrose (pH 5.8), and then
cultured in darkness at 25 ◦ C for 2 months. The PLBs were cut into 0.5 cm × 0.5 cm pellets, and 7 g of the
pellet was inoculated into an Erlenmeyer flask containing 40 mL of MS medium. MeJA (100 μM methyl
jasmonate; Aladdin, Shanghai, China), SA (100 μM salicylic acid; Aladdin, Shanghai, China), and ABA
(100 μM abscisic acid; Aladdin, Shanghai, China) were filtered through 0.22 μm filter membrane and
added to the MS medium, based on previously published articles [31]. After induction with SA and
MeJA, samples were harvested at 0, 2, 4, 8, 24, 48, and 72 h. The original bulbs under ABA induction
were harvested at 0, 24, 48, 72, 96, and 168 h. All samples were immediately stored at − 80◦ C after
harvesting for RNA extraction. All results were based on three biological repeats and each biological
repeat had three technical replicates. The extraction of total RNA from PLBs was carried out with Plant
Total RNA Isolation Kit (Sangon Biotech, Shanghai, China) using 300 mg tissue homogenized in liquid
nitrogen according to the manufacturer’s protocol, which was subsequently reverse transcribed into
the first DNA strand using a One Step RT-qPCR Kit (BBI Life Science, Shanghai, China).
4.2. Screening and Identification of the PLP_deC Genes

We obtained the HMM (Hidden Markov Model) configuration file for the PLP_deC domain
(Pfam00282) from Pfam (http://pfam.xfam.org/). All PLP_deC genes were then identified in the
D. officinale and P. equestris genomes using HMMER software (E-value = 0.001) [32]. All candidate genes
were submitted to Pfam and SMART for verifying the presence of the PLP_deC domain. The sequences
that did not contain the conserved domain and the redundant sequence of the repeat were removed,
and finally, the PLP_deC genes were obtained. In Table S1 are listed the sequences of the PLP_deC
genes of O. sativa and A. thaliana described in previous studies (add citations) and used in the present
work [17].
4.3. Sequence Attribute Analysis and Phylogenetic Tree Construction of PLP_deC Genes
To analyze the sequence attributes and characteristics of the amino acids of the PLP_deC family
members, the isoelectric points (pIs) of the obtained PLP_deC amino acid sequences were determined
using online analysis with the ProtParam tool (https://web.expasy.org/protparam/) [33] and subcellular
localization of 8 and 6 PLP_deC-family protein sequences in D. officinale and P. equestris by Target
P 1.1 (http://www.cbs.dtu.dk/ services / Target P). Properties such as molecular weight (MW) were
predicted. The obtained PLP_deC protein sequences were aligned using Clustal X [34], implemented
in MEGA 5.0 [35], and a maximum likelihood (ML) phylogenetic tree was generated using IQ-TREE
software [36] with 1000 bootstrap replicates. The PLP_deC genes were classified according to the
phylogenetic relationships. If two different species of genes are located in the phylogenetic tree at the
same node and the sequence similarity is more than 80%, we consider two of these are homologous
genes [37]. The conserved motifs on the orchid sequences of PLP_deC were defined by MEME
(http://meme-suite.org/) using the following parameters: maximum number of motifs = 10, number of
repetitions—any, and only motifs with an E-value < 0.01 were retained for further analysis. The motif
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Int. J. Mol. Sci. 2020, 21, 54
logos of the PLP_deC domains were generated using online MEME program (Figure S2) [38], and GSDS
was used to determine the exon–intron structure (http://gsds.cbi.pku.edu.cn /) [39].
4.4. Calculation of Ks and Ka Values of the PLP_deC Genes

The protein sequences of the gene pairs were first aligned using Clustal X 2.0, and then the
multiple sequence alignments of proteins and the corresponding cDNA sequences were converted
to codon alignments using PAL2NAL (http://www.bork.embl.de/pal2nal/) [40]. Finally, the resulting
codon alignment was used to calculate Ks and Ka using DnaSP 5.0 (http://www.ub.edu/dnasp/) [41].
4.5. Analysis of Cis-Acting Elements of the PLP_deC Genes

A 2000 bp sequence upstream of the translation start site (ATG) of each PLP_deC gene was
obtained, and an analysis of the upstream regulatory promoter elements was performed using the
online tool PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [42].
4.6. Expression of the D. officinale PLP_deC Genes in Different Tissues

To obtain expression data for D. officinale, we searched the NCBI SRA database (PRJNA348403)
for RNA-sequence data from different tissues [27] (Additional file: Table S2). The raw data were
stripped of adapters and low-quality reads (and bases) and then rRNA and virus reads were filtered
out by using Trimmomatic software with the default parameters. The clean reads were aligned to
the D. officinale genome using Hisat2 [43] with the options -dta and -no-unal. The aligned outputs
were converted from SAM to BAM format by using SAMtools [44]. Then, the Stringtie software was
used to estimate the transcript abundances with FPKM method. The heat map of the PLP_deC genes
expression profiles was obtained by TBtools software (https://github.com/CJ-Chen/TBtools/releases).
4.7. Quantitative Fluorescence Analysis of the PLP_deC Genes

We used the CFX96 TouchTM Real-Time PCR Detection System (Singapore) to perform a
quantitative fluorescence analysis of the PLP_deC genes in the cDNA samples from D. officinale
protocorms with three replicates. β-actin [45–47] was used as an internal reference, the relative
expression levels of genes were calculated using the 2−ΔΔCT method [48], and the primers were
designed using Primer Premier 5.0 software (Table S3). Each reaction contained the following: 8 μL of
SYBR Premix Ex Taq II (2x), 2 μL of template cDNA, 1 μL of forward and reverse primers, and addition
of ddH2 O to a final volume of 20 μL. The reaction conditions were as follows: 95 ◦ C for 3 min followed
by 40 cycles of 95 ◦ C for 10 s, 52 ◦ C for 15 s, and 72 ◦ C for 30 s.
To determine whether the differential expression was significant, the difference in the relative
expression of each target gene in the different treatment groups was analyzed by Student’s t-test in
SPSS 25.0 software [49].
5. Conclusions
Overall, we conducted a comprehensive analysis of PLP_deC genes in both D. officinale and
P. equestris. Comparative analysis has shown that eight PLP_deC genes from D. officinale could be
divided into three subfamilies: GAD, HDC, and AAD. Most genes have an acidic pI. Purifying selection
may have played a key role in the evolution of this PLP_deC genes in D. officinale based on the Ka/Ks
value. Among them, both DoAAD1 and DoAAD2 were highly expressed in the column, flower bud,
and lip. Under three hormone treatments, MeJA, ABA, and SA, the PLP_deC genes responded to
abiotic stresses. These results provide preliminary biological information for further studies of the
evolution of PLP_deC genes in Orchidaceae.
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Author Contributions: Y.C. (Yongping Cai) and Q.J. conceived and designed the experiments; L.Z., C.J., Y.C.
(Yunpeng Cao), X.C., and J.W. performed the experiments; L.Z. and C.J. analyzed the data; L.Z. wrote the paper.
All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Acknowledgments: This work was supported by the Universities Natural Science Research Project of
Anhui, China (Project number KJ2016A224), the University Synergy Innovation Program of Anhui Province
(Project number GXXT-2019-043), and the Major Science and Technology Project in Anhui Province (Project number
17030701031).
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175
Molecular Sciences
Article
Coelonin, an Anti-Inflammation Active Component of
Bletilla striata and Its Potential Mechanism
Fusheng Jiang 1,† , Meiya Li 2,† , Hongye Wang 3 , Bin Ding 1 , Chunchun Zhang 4 , Zhishan Ding 5 ,
Xiaobo Yu 3, * and Guiyuan Lv 4, *
1 College of Life Science, Zhejiang Chinese Medical University, Hangzhou 310053, China
2 Academy of Chinese Medical Sciences, Zhejiang Chinese Medical University, Hangzhou 310053, China
3 State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein
Sciences (PHOENIX Center, Beijing), Beijing Institute of Lifeomics, Beijing 102206, China
4 College of Pharmaceutical Science, Zhejiang Chinese Medical University, Hangzhou 310053, China
5 College of Medical Technology, Zhejiang Chinese Medical University, Hangzhou 310053, China
* Correspondence: [email protected] (X.Y.); [email protected] (G.L.)

Received: 7 August 2019; Accepted: 6 September 2019; Published: 8 September 2019
Abstract: Ethanol extract of Bletilla striata has remarkable anti-inflammatory and anti-pulmonary
fibrosis activities in the rat silicosis model. However, its active substances and molecular mechanism
are still unclear. To uncover the active ingredients and potential molecular mechanism of the
Bletilla striata extract, the lipopolysaccharide (LPS)-induced macrophage inflammation model and
phospho antibody array were used. Coelonin, a dihydrophenanthrene compound was isolated
and identified. It significantly inhibited LPS-induced interleukin-1β (IL-1β), interleukin-6 (IL-6)
and tumor necrosis factor-α (TNF-α) expression at 2.5 μg/mL. The microarray data indicate that
the phosphorylation levels of 32 proteins in the coelonin pre-treated group were significantly
down-regulated. In particular, the phosphorylation levels of the key inflammatory regulators factor
nuclear factor-kappa B (NF-κB) were significantly reduced, and the negative regulator phosphatase
and tensin homologue on chromosome ten (PTEN) was reduced. Moreover, the phosphorylation
level of cyclin dependent kinase inhibitor 1B (p27Kip1 ), another downstream molecule regulated by
PTEN was also reduced significantly. Western blot and confocal microscopy results confirmed that
coelonin inhibited LPS-induced PTEN phosphorylation in a dose-dependent manner, then inhibited
NF-κB activation and p27Kip1 degradation by regulating the phosphatidylinositol-3-kinases/ v-akt
murine thymoma viral oncogene homolog (PI3K/AKT) pathway negatively. However, PTEN inhibitor
co-treatment analysis indicated that the inhibition of IL-1β, IL-6 and TNF-α expression by coelonin
was independent of PTEN, whereas the inhibition of p27Kip1 degradation resulted in cell-cycle arrest
in the G1 phase, which was dependent on PTEN. The anti-inflammatory activity of coelonin in vivo,
which is one of the main active ingredients of Bletilla striata, deserves further study.
Keywords: coelonin; Bletilla striata; anti-inflammation; signal pathway; cell-cycle arrest; PTEN
1. Introduction
Bletilla striata (Thunb.) Reichb.f is a famous traditional Chinese herb that is widely used in the
treatment of lung and stomach diseases such as pneumogastric hemorrhage, silicosis, tuberculosis,
and gastric ulcer; it can also be used for the treatment of skin cracks, burns and freckles when
combined with other traditional Chinese medicines. Numerous compounds have been identified
from Bletilla striata, such as benzyls, phenanthrenes, dihydrophenanthrenes, anthracene, phenolic
acid and polysaccharides [1,2]. Among these, polysaccharides are the most extensively and deeply

Int. J. Mol. Sci. 2019, 20, 4422
studied, and their anti-ulcer [3], wound healing [4], homeostasis [5] and immune regulation [6] effects
have represented most of the efficacy of Bletilla striata. However, in recent years, the pharmacological
activities of the small molecular components in Bletilla striata have also attracted much attention.
Liu [7] reported that the 80% ethanol elunt fraction of D101 macroporous resin significantly reduced
bleeding time and increased the maximum platelet aggregation rate. Our previous research showed
that the ethanol extract of Bletilla striata dose dependently inhibited alcohol induced gastric ulcer and
silica induced silicosis in rats [8,9]. Furthermore, the ethanol extract of Bletilla striata significantly
down regulated the serum level of IL-1β, TNF-α, transforming growth factor-β (TGF-β) and other
inflammatory factors in rats with silicosis [9], thereby reducing the degree of pulmonary fibrosis,
and this effect is far more effective than the polysaccharide of Bletilla striata [10]. However, its active
components and underlying molecular mechanisms are unclear.
Silicosis is a type of systemic disease, characterized by chronic persistent inflammation
and progressive fibrosis in lung tissue. The innate immune response mediated by alveolar
macrophage plays a very important role in inflammatory reaction during the process of silicosis.
The activated macrophages release proinflammatory mediators such as IL-6, IL-1β, TNF-α,
TGF-β and platelet-derived growth factor (PDGF), etc. [11]. These inflammatory factors are
recognized as key factors in pulmonary fibrosis, and the interruption of these factor pathways
can alleviate or prevent fibrosis [12–14]. The classic LPS-induced RAW264.7 macrophage
model can mimic the process of macrophage activation in vitro. One active compound
2,7-dihydroxy-4-methoxy-9,10-dihydrophenanthrene (coelonin) from Bletilla striata was separated and
identified under the guidance of this cell model and combined with column chromatography.
Although few studies have described the anti-inflammatory effect of coelonin, but we found that
this compound significantly down regulated IL-1β and IL-6 expression at 2.5 μg/mL on LPS-induced
RAW264.7 cell. Hence, coelonin may be one of the main active components contributing to the
anti-silicosis effect of Bletilla striata. In this study, we used a Phospho Explorer Antibody Array PEX100
to discover the potential target of the anti-inflammation effect of coelonin. The microarray results imply
that coelonin may play an anti-inflammatory and cell-cycle regulation role through the PTEN/AKT
pathway. Examining the down-stream signaling profile and cytokines secretion in RAW264.7 cells
induced by LPS with or without coelonin or the PTEN inhibitor SF1670 suggests that coelonin blocked
RAW264.7 cells in the G1 phase cell cycle in a PTEN- dependent manner, and PTEN may partially
participated in coelonin inhibition on the secretion of inflammatory factors. Therefore, the potential
molecular mechanism of the anti-inflammatory effect of coelonin remains to be addressed. Furthermore,
as one of the main active ingredients of Bletilla striata, the anti-inflammatory activity of coelonin in vivo
deserves further study.
2. Results
2.1. Separation, Purification and Identification of Active Components from Bletilla striata
The ethanol extraction of Bletilla striata tuber was separated into five fractions using the polyamide
adsorption method, then they were characterized by the high performance liquid chromatography
(HPLC) method (see Figure 1A). The results indicated that there were few common peaks in each fraction,
which shows the effective enrichment effect of the polyamide column. The anti-inflammation activity of
the five fractions was screened on the LPS-induced RAW264.7 cell model, and the real-time polymerase
chain reaction (RT-PCR) results indicate that except F0 and F80, the fractions dose-dependently
inhibited IL-1β expression, whereas F80 showed inhibition activity at low dosage, but the messenger
RNA (mRNA) expression level of IL-1β was dose-dependently increased to even higher than the
LPS-treated group at 30 μg/mL (see Supplementary Figure S1). F40 showed remarkable inhibition
activity and 83.07% of IL-1β mRNA expression was inhibited at a concentration of 10 μg/mL (see
Figure 1B).
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Figure 1. (A) HPLC characterization of the five fractions. A total of 10 μL each sample (1 mg/mL)
was injected and analyzed using a Dionex UltiMateTM 3000 HPLC system with photodiode array
detection (PAD) at 259 nm. A Symmetrix ODS-RC18 (25 × 4.6 mm, 5 mm) HPLC column protected
with a Phenomenex security guard column (C18, 4 × 3.0 mm) operated at 30 ◦ C was used, and the flow
rate was maintained at 1 mL/min. The elution solvents were acetonitrile (a) and 0.1% acetic acid (b).
Samples were eluted according to the following gradient: 0–35 min 30% a isocratic, 35–45 min 30%
to 40% a, 45–55 min 40% a isocratic, and finally washing and recondition of the column. (B) Relative
expression of IL-1β mRNA after treatment with F40. RAW264.7 cells were pretreated with different
concentration of F40 for 1 h and then treated with 200 ng/mL LPS for 6 h. Total RNA was extracted and
genes expression level were analyzed by RT-PCR in triplicate. The expression level of each gene was
normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Data are expressed as
mean ± SD (n = 6). ** p < 0.01 vs. LPS treatment group.
The F40 fraction was further separated by silica gel chromatography. Dry silica gel was packed
into a glass column (diameter via height ratio 1:10), then dry sample (sample silica gel ratio 1:3)
was loaded and eluted with chloroform-methanol (50:1, V/V) at 2 mL/min. Fractions of 10 mL were
collected and monitored by thin-layer chromatography (TLC), visualized by iodine vapor and those
possessing similar Rf values were combined and six sub-fractions of F40 were obtained (see Figure 2A).
Anti-inflammation assay manifested that all sub-fractions inhibited the expression of IL-1β in a
dose-dependent manner (see Supplementary Figure S2), and the sub-fractions of F40-3 and F40-4
showed significant inhibition ratio at 20 μg/mL (see Figure 2B). HPLC analysis revealed that F40-3 and
F40-4 were mainly composed of two identical compounds (see Figure 3A), and the two compounds
were purified by Dionex UltiMateTM 3000 semi-prepared HPLC system. A Welch Ultimate® XB-C18
(10 × 250 mm, 10 μm) HPLC column operated at 30 ◦ C was used, and the flow rate was maintained at
5 mL/min. Samples were isocratic eluted with acetonitrile (30%) and 0.1% acetic acid (70%).
The results of mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectra are as
follows: Compound I (HPLC > 98%) electrosprary ionization-mass spectrometry (ESI-MS) m/z: 245.1108
(M + H)+ . 1 H-NMR (CD3 OD) δ: 2.79 (4H, m, CH2 ), 3.71 (3H, s, OCH3 ), 6.19 (1H, dd, J = 1.8, 2.4 Hz,
H-4), 6.25 (2H, dd, J = 1.8, 1.8 Hz, H-2, 6), 6.63 (3H, m, H-2’, 4’, 6’), 7.08 (1H, dd, J = 7.8, 8.4 Hz,
H-5’); 13 C-NMR (CD3 OD) δ: 160.83 (C-5), 157.98 (C-3), 156.93 (C-3’), 144.06 (C-1), 143.29 (C-1’), 128.84
(C-5’), 119.43 (C-6’), 114.93 (C-2’), 112.36 (C-4’), 107.63 (C-2), 105.12 (C-6), 98.54 (C-4), 54.10 (OCH3 ),
37.80 (CH2-a’), 37.47 (CH2 -a). Compared with the data shown in literature [15], the compound was
identified as batatasin III. Compound II (HPLC > 98%) ESI-MS m/z: 243.1006 (M + H)+ . 1 H-NMR
(CD3 OD) δ: 2.64 (4H, s, H-9, 10), 3.83 (3H, s, 4-OCH3 ), 6.32 (1H, d, J = 2.4 Hz, H-1), 6.41 (1H, d,
J = 2.4 Hz, H-3), 6.64 (1H, d, J = 1.8 Hz, H-6), 6.63 (1H, dd, J = 6, 2.4 Hz, H-8), 8.02 (H, d, J = 9 Hz, H-5).
13 C-NMR (CD OD) δ: 157.68 (C-2), 154.63 (C-7), 156.04 (C-4), 140.42 (C-10a), 139.08 (C-8a), 128.63 (C-5),
3
113.61 (C-4a), 124.77 (C-5a), 112.17 (C-6), 115.37 (C-8), 106.90 (C-1), 97.83 (C-3), 54.45 (-OCH3 ), 29.80
(C-9), 30.38 (C-10). Comparison with the data shown in literature [16], led to the compound being
identified as coelonin.
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Figure 2. (A) Sub-fractions separated from F40 by silica gel chromatography. TLC was performed on
precoated silica gel 60 F254 plates (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), developed
with chloroform-methanol (4:0.1, V/V) and then exposed to the iodine vapor in a dark enclosed chamber
for 10 min. (B) The relative expression of IL-1β mRNA after treatment with sub-fractions of F40-3 and
F40-4. RAW264.7 cells were pretreated with different concentration of different sub-fractions of F40 for
1 h, then treated with 200 ng/mL LPS for 6 h. Total RNA was extracted and genes expression levels
were analyzed by RT-PCR in triplicate. The expression level of each gene was normalized to GAPDH
mRNA. Data are expressed as mean ±SD (n = 6). ** p < 0.01 vs. LPS treatment group.
The anti-inflammation activity was verified (see Figure 3B) and both coelonin and batatasin III
showed dose-dependent inhibition activity. A total of 93.1% of IL-1β mRNA expression was inhibited
by coelonin at 2.5 μg/mL, which was significantly better than that of batatasin III at 10 μg/mL (62.3%).
This result implies that coelonin is probably the main anti-inflammatory component of Bletilla striata.
2.2. Inhibitory Effect of Coelonin on LPS-Induced IL-1β, IL-6, TNF-α Gene Expression and Protein Secretion in
RAW264.7 Macrophages
LPS can induce the expression and secretion of a variety of inflammatory factors, such as IL-1β,
IL-6, TNF-α, monocyte chemo-attractant protein-1 (MCP-1), inducible nitric oxide synthase (iNOS)
and cyclooxygenase 2 (COX2) et al. Using IL-1β as an index, a highly active ingredient, coelonin, was
isolated from Bletilla striata by the RAW264.7 cell inflammation model. Here, two other important
inflammatory factors, IL-6 and TNF-α were examined as well. We confirmed that coelonin was without
a significant cytotoxic at a concentration of up to 5 μg/mL [17] (see Supplementary Figures S3 and S4).
LPS markedly elevated IL-1β, IL-6 and TNF-α mRNA expression and protein secretion, but coelonin
dose-dependently lowered these levels in macrophages (see Figure 3C,D). These results confirmed the
anti-inflammation effect of coelonin. However, there was almost no anti-inflammation report of this
compound; therefore, the underlying mechanism is worth further research.
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Figure 3. (A) HPLC characterization of the sub-fractions F40-3, F40-4 and two purified compounds.
10 μL each sample (0.1 mg/mL) was injected and analyzed using a Dionex UltiMateTM 3000 HPLC
system with PAD at 193 nm. A Symmetrix ODS-RC18 (25 × 4.6 mm, 5 mm) HPLC column protected
with a Phenomenex security guard column (C18, 4 × 3.0 mm) operated at 30 ◦ C was used, and the
flow rate was maintained at 1 mL/min. The elution solvents were acetonitrile (a) and 0.1% acetic
acid (b). Samples were eluted according to the following gradient: 0–5 min 10% to 35% a, 5–12 min
35% a isocratic, 12–16 min 35% to 45% a, 16–22 min 45% a isocratic, 22–35 min 45% to 80% a, and
finally washing and recondition of the column. (B) The relative expression of IL-1β mRNA treated
by active compounds. (C) Inhibitory effect of coelonin on LPS-induced gene expression in RAW264.7
macrophages. RAW264.7 cells were pretreated with different concentration of compound I or II for
1 h, then stimulated with 200 ng/mL of LPS for 6 h. Total RNA was extracted and genes expression
level were analyzed by RT-PCR in triplicate. (D) Inhibitory effect of coelonin on LPS-induced cytokine
secretion in RAW264.7 macrophages. RAW264.7 cells were pretreated with coelonin for 1 h and then
treated with 200 ng/mL LPS. 12 h after LPS stimulation, culture supernatants was collected for IL-6
and TNF-α detection by cytometric bead array (CBA) method; for IL-1β detection, the remaining cells
were following treated by 1 mM adenosine triphosphate (ATP) for an additional 15 min at 37 ◦ C [18],
then supernatants were collected and analyzed by the CBA method in triplicate. Data are expressed as
mean ± SD (n = 6). ** p < 0.01 vs. LPS treatment group.
2.3. Identifying Differentially Expressed Protein Phosphorylation Sites Induced by Coelonin Treatment
To identify differentially expressed signaling-associated phosphorylated proteins between
coelonin-treated and untreated RAW264.7 cells induced by LPS, the expression levels of
phospho-antibody specific proteins were compared. Of the 1318 antibodies analyzed in microarray
experiments, a total of 32 different phosphorylation proteins showed downregulated expression using
a fold ratio ≥2 as the cutoff criterion (Figure 4, Table 1). In addition, we performed a protein network
analysis using REACTOME (http://reactome.ncpsb.org/) to identify major interactions, three major
cellular processes were significantly enriched (p value < 0.05) as follows: (1) Immune system, (2) signal
transduction, and (3) cell cycle. LPS is the component of the outer membrane of Gram-negative bacteria,
and is one of the most well characterized pathogen-associated molecular patterns (PAMPs), which can
be recognized by Toll-like receptor 4 (TLR4), and trigger innate responses [19], such as enhancing the
secretion of cytokines and chemokines, and promoting macrophages migration and proliferation [20].
Therefore, it is evident that the protein network analysis results imply coelonin may block LPS induced
RAW264.7 cell signal transduction, immune response, and proliferation.
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Figure 4. Protein and phosphorylation altered after coelonin treatment. From a total of 1318 differentially
phosphorylated proteins, 32 were selected as significantly downregulated by coelonin using a volcano
plot analysis.
Table 1. The phosphorylation of 32 proteins were significantly downregulated by coelonin treatment

(p < 0.05).
Phosphorylation Phosphorylation
Name Gene ID Name Gene ID
Site or Antibody Site or Antibody
HSP90B Ser254 3326 Trk A Tyr791 4914
NFκB-p65 Ser536 5970 PKC α/β II Ab-638 5578
Ezrin Thr566 7430 MAP3K8/COT Thr290 1326
FLT3 Ab-599 2322 NFκB-p105/p50 Ab-932 4790
p53 Ser33 7157 p27Kip1 Thr187 1027
CK2-b Ab-209 1460 Shc Tyr349 6464
ATF1 Ab-63 466 Smad1 Ab-187 4086
AXL Tyr691 558 ERK3 Ab-189 5597
Rac1/cdc42 Ser71 5879 Caveolin-1 Tyr14 857
PTEN Ser380 5728 MARCKS Ser163 4082
CDK2 Ab-160 1017 GRK2 Ser685 156
DNA-PK Ab-2056 5591 Ephrin B1 Ab-317 1947
EGFR Ab-1069 1956 Estrogen Ser104 2099
Receptor-α
Keratin 8 Ser73 3875 BRCA1 Ser1524 672
ATPase Ab-16 476 PTEN Ser380/Thr382/Thr383 5728
CDC25A Ab-75 993 eIF4B Ser422 1975
Moreover, we entered the ENTREZ Gene IDs of the 32 genes into the DAVID Bioinformatics
Resources 6.8 database. As depicted in Figure 5, the numbers of changed genes in the PI3K/AKT
signaling pathway, the mitogen-activated protein kinase (MAPK) signaling pathway, the neurotrophin
signaling pathway, the Sphingolipid signaling pathway and Ras signaling pathway were ranked in the
top 15. In addition, we found that within these genes, the maximum number of genes belonged to the
PI3K/AKT signal pathway. It is noteworthy that the phosphorylation of both subunits p65 and p105/50
of transcription factor NF-κB in this signaling pathway was reduced, which was closely related to the
regulation of inflammatory gene expression [21].
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Figure 5. Number of genes in signaling pathways changed by coelonin pretreatment.
2.4. Validation of Proteomic Findings–Coelonin Treatment Inhibits Inflammatory Cytokines Secretion by

Blocking NF-κB Activation
NF-κB is the most important signaling molecule induced by LPS through TLR4. The
phosphorylated NF-κB can translocate to the nucleus, interacts with the κB elements and cause
numerous cytokines secretion such as IL-1β, MCP-1 and TNF-α et al. [21]. As observed in Table 1,
both NF-κB p65 and p105/50 phosphorylation levels were significantly reduced by 2.5 μg/mL coelonin
treatment. Therefore, we performed western blot and immunofluorescence assays by nucleus
translocation of p65 for validation. As shown in Figure 6A, the western blot results indicated that LPS
stimulation significantly increased p65 accumulation in the nucleus, but the coelonin dose dependently
reduced the effect of LPS. Moreover, confocal microscopic analysis reconfirmed that LPS stimulation
significantly induced p65 translocation from the cytoplasm to the nucleus, which was remarkably
inhibited by 2 μM of ammonium pyrrolidine dithiocarbamate (APDC), an inhibitor of NF-κB, and
5.0 μg/mL of coelonin pre-treatment (Figure 6B). In addition to cytokines, numerous studies have
shown that NF-κB also regulates the expression of iNOS and COX2 genes [22,23]. Furthermore, over
expression of iNOS and COX2 can lead to inflammation, tissue damage and even tumorigenesis [24,25].
As observed in Figure 6A, 200 ng/mL LPS treatment for 24 h caused a remarkable increased expression
of iNOS and COX2. Pre-treatment with coelonin dose dependently reversed LPS-induced iNOS
and COX2 expression. These results were consistent with the microarray results and confirmed that
coelonin exerts its anti-inflammatory effect by inhibiting NF-κB activity.
2.5. Colonin May Partially Inhibit the Activation of NF-κB through PTEN/AKT Pathway
It is well know that LPS can activate NF-κB through the Toll-like receptor 4/myeloid differentiation
factor 88/IL-1 receptor associated kinase/TNF receptor associated factor 6/TGF beta-Activated
Kinase 1/inhibitor of nuclear factor-κB kinase (TLR4/MyD88/IRAK/TRAF6/TAK1/IKKs) pathway [26]
and now, several lines of evidence suggest that LPS can also activate NF-κB through the
TLR4/MyD88/PI3K/AKT/IKKs pathway [27,28]. However, the PI3K/AKT pathway is negatively
regulated by the PTEN [29,30]. Previous research has shown that the down regulation of PTEN
can activate NF-κB activity by increasing p65 nucleus translocation in mouse mesangial cells and
bovine alveolar macrophages, contrary, activate PTEN would reverse the effect [29,31]. The microarray
results indicated that coelonin treatment significantly down-regulated the phosphorylation of PTEN at
Ser380/Thr382/Thr383 (see Table 1), which implies that coelonin may inactivate NF-κB by restoring the
activity of PTEN, as phosphorylation of PTEN will make it inactivated [30]. Thus, western blotting
was carried out to verify this presumption. As show in Figure 6A, LPS significantly increased the
phosphorylation of PTEN, AKT and inhibitor of NF-κB (IκBa), which was dose-dependently reduced
by coelonin pre-treatment (Figure 6A). In order to further confirm the possible inhibition activity of
coelonin against LPS-induced NF-κB activation mediated by PTEN/AKT pathway, RAW264.7 cells
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were pre-treated with PTEN inhibitor SF1670. As show in Figure 7A, pre-treatment with SF1670
significantly increased LPS-induced AKT phosphorylation, which could not be downregulated by
additional coelonin. Meanwhile, SF1670 also dramatically promoted LPS-induced secretion of IL-1β,
IL-6 and TNF-α, and significantly reduced but could not completely block the inhibitory activity
of coelonin. This result further indicates that PTEN did participate in PI3K/AKT/NF-κB activation
pathway. However, it is note worthy that most levels of IL-1β and IL-6 were still significantly inhibited
by coelonin co-treated with SF1670, suggesting that more critical pathways need to be identified besides
the PTEN/AKT pathway.
ȱ
Figure 6. (A) Coelonin inhibits LPS induced NF-κB activation in macrophage. RAW264.7 cells were
pretreated with coelonin for 1 h and then treated with 200 ng/mL LPS for 30 min. Then, the nuclear
protein was extracted, the content of p65 in the nucleus was determined, the total cellular protein was
extracted, and the levels of phosphorylated PTEN, AKT and IκBα were determined. For iNOS and
COX2 detection, RAW264.7 cells were pretreated with coelonin for 1 h, then treated with 200 ng/mL
LPS for 24 h, then total cellular protein was extracted and analyzed. (B) Confocal microscopy analysis
of p65 nucleus translocation. RAW264.7 cells were incubated with solvent (a–c) or 200 ng/mL LPS for
1 h in the absence (d–f) or presence 2 μM of the NF-κB inhibitor APDC (g–i) or 5 μg/mL of coelonin (j–l).
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Figure 7. (A) Western blotting result of pAKT. RAW264.7 cells were incubated with solvent or 200 ng/mL
LPS for 30 min in the absence or presence 5 μg/mL of coelonin or 2 μM of the PTEN inhibitor SF1670
or 2 μM of SF1670 combined with 5 μg/mL of coelonin. Then total cellular protein was extracted and
analyzed by a simple western immunoblotting technique on a Peggy Sue system. (B–D) Effect of PTEN
inhibitor SF1670 on anti-inflammatory activity of coelonin. RAW264.7 cells were incubated with solvent
or 200 ng/mL LPS for 12 h in the absence or presence 2 μM of the PTEN inhibitor SF1670 or 5 μg/mL of
coelonin or 2 μM of SF1670 combined with 5 μg/mL of coelonin. Culture supernatants was collected for
IL-1β, IL-6 and TNF-α detection by CBA method. Data are expressed as mean ± SD (n = 6). ** p < 0.01.
2.6. Coelonin Treatment Leads to G1 Cell Cycle Arrest through PTEN

The PI3K-Akt pathway has been shown be involved in a variety of cellular processes, including
inflammation response [27], cell survival and proliferation [32]. Many reports indicate that the
PI3K/AKT pathway plays a pivotal role in regulating cell cycle progression through phosphorylation
and degradation of cell cycle regulator p27Kip1 [33,34]. P27Kip1 can interact with cyclin-dependent
kinase 2 (CDK2) and cyclinE to prevent cell entry into the S phase, and over-expression p27Kip1 would
promote cell G1 phase cell cycle arrest [35]. In many cancer cells, such as cervical cancer cells, oral
squamous cell carcinoma cells, and prostatic carcinoma cells, the expression protein of p27Kip1 was
significantly reduced, the invasion and migration ability was enhanced, and the expression of p27Kip1
was negatively correlated with survival [33,36]. We noticed that the microarray results showed that
coelonin significantly down regulated the phosphorylation of p27kip1 , and interestingly, a previous
study indicated that the up-regulation of PTEN prevents p27Kip1 phosphorylation and proteolysis [37].
Hence, we speculate that coelonin may inhibit p27Kip1 phosphorylation and degradation mediated by
PTEN. Western blot confirmed that 5 μg/mL coelonin treatment significantly recovered the p27Kip1
level, which was even higher than the un-treatment group, while LPS stimulation significantly reduced
the p27Kip1 level, and PTEN inhibitor SF1670 almost completely abrogated the effect of coelonin (see
Figure 8A). Theoretically, high levels of p27Kip1 can block cells in the G1 phase cell-cycle, so we used
flow cytometry to verify whether coelonin could induce G1 phase cell cycle arrest in RAW264.7 cells.
As observed in Figure 8B, coelonin pre-treatment remarkably induced G1 phase cell cycle arrest and
SF1670 completely inhibited the effect of coelonin. These data suggest that coelonin inhibits p27Kip1
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degradation in a PTEN-dependent manner, thereby exerting its G1 phase cell cycle arrest effect in
RAW264.7 cells.
Figure 8. (A) Western blotting result of p27kip1 . RAW264.7 cells were incubated with solvent or
200 ng/mL LPS for 12 h in the absence or presence 5 μg/mL of coelonin or 2 μM of the PTEN inhibitor
SF1670 or 2 μM of SF1670 combined with 5 μg/mL of coelonin. Then total cellular protein was extracted
and analysed by a simple western immunoblotting technique on a Peggy Sue system. (B) Coelonin
induce G1 cell cycle arrest through PTEN analysed by flow cytometry. RAW264.7 cells were incubated
with solvent or 200 ng/mL LPS for 12 h in the absence or presence 5 μg/mL of coelonin or 2 μM
of the PTEN inhibitor SF1670 or 2 μM of SF1670 combined with 5 μg/mL of coelonin. Cells were
harvested, fixed and stained with PI, and cell cycle distributions were analyzed on a BD Accuri™ C6
flow cytometer in triplicate.
3. Discussion
To date, over 200 phenanthrene compounds have been isolated and identified, most of
which come from the Orchidaceae family [38]. In Bletilla striata, a member of Orchidaceae,
more than 30 phenanthrenes have been isolated from its pseudobulbs and fibrous roots, most
of which showed antitumor and antimicrobial activities [39,40]. In addition, numerous studies have
shown that phenanthrene and dihydrophenanthrene derivatives with remarkable anti-inflammation
activity [38,41,42]. In this study, coelonin, a compound with a strong anti-inflammatory activity,
was isolated and identified from the pseudobulbs of Bletilla striat under the guidance of biological
activity screening. Few studies have reported its anti-inflammatory activity, and our studies show
that coelonin can dose-dependently inhibit LPS-induced expression and secretion of IL-1β, IL-6
and TNF-α in RAW264.7 cells. Furthermore, we found that Bletilla striata has a high content of
coelonin (0.020%–0.301% in different samples) [43]. Therefore, coelonin is probably one of the main
anti-inflammatory active components of Bletilla striata.
In order to better elucidate the potential anti-inflammatory molecular mechanism of coelonin,
PEX100 protein microarrays containing 1318 antibodies were used. The results indicated that 32 different
phosphorylated proteins were significantly downregulated by pre-treatment with coelonin, which
were closely related to the response of LPS-induced signal transduction, immune response and cell
proliferation. Additionally, most of these genes were focused on the PI3K/AKT signal pathway, and we
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performed verification on the phosphorylation levels of PTEN, NF-κB and p27Kip1 . NF-κB is considered
as a central regulator of LPS-induced pro-inflammatory response in macrophage activation [44], which
is usually formed as p65:p50 heterodimer. In addition, PEX100 microarray results indicated that the
phosphorylation levels of both p65 and p50 were significantly downregulated by coelonin pre-treatment
(Table 1), which was further confirmed by western blot and confocal microscopic analysis on p65
nucleus translocation (Figure 6). Studies have shown that numerous dihydrophenanthrene derivatives
exert anti-inflammatory effects by inhibiting NF-κB pathway [38,41,42], which was significantly
correlated with the presence of phenolic hydroxyl groups [45]. Interestingly, coelonin has two phenolic
hydroxyl groups, which is in accordance with its anti-inflammatory effect. However, few reports
have studied the upstream targets that regulate the activity of NF-κB by dihydrophenanthrene
derivatives. According to the results of the PEX100 microarray, we propose that coelonin may play an
anti-inflammatory role by inhibiting the activity of NF-κB through the PTEN/AKT pathway. However,
some studies have indicated that the PI3K/AKT pathway negatively regulates LPS induced NF-κB
activation [46]. In contrast, other studies have shown that the PI3K/AKT pathway does positively
regulate LPS-induced gene expression [27]. We found that LPS stimulation could dramatically induce
AKT phosphorylation, IκBa degradation and NF-κB nucleus translocation (Figure 6), and the PI3K
inhibitor LY294002 significantly inhibited LPS inducted IL-1β, IL-6 and TNF-α expression in RAW264.7
cells (see Supplementary Figure S5), which is consistent with the findings that the PI3K/AKT pathway
is required for LPS induction of gene expression in RAW264.7 cells. Meanwhile, PTEN inhibitor
SF1670 significantly increased LPS-induced AKT phosphorylation and cytokines secretion (Figure 7),
which is consistent with the report of Zhao et al. [47]. These results indirectly supported that both
PI3K/AKT and PTEN/AKT pathways are involved in LPS inducted NF-κB activation. However, the
phosphorylation level of Akt did not decrease after co-treatment with PTEN inhibitor SF1670 and
coelonin, but coelonin still could significantly down-regulate the expression of inflammatory cytokines
induced by LPS. This contradictory result not only indicated that coelonin has other pathways to exert
its anti-inflammation activity, but also indicated that AKT pathway has complex mechanisms in the
regulation of inflammation. In fact, the exact mechanism of activation of NF-κB by AKT pathway
remains controversial. Some reports indicated that activation AKT lead to IKK-dependent IκBα
degradation and nucleus translocation of NF-κB, while others shown that AKT-dependent activation
of NF-κB by stimulating the transactivation potential of the p65 subunit, rather than inducing IκBα
degradation [48]. Obviously, although coelonin could inhibit IκBα phosphorylation and degradation
in a dose-dependent manner, it is not clear whether coelonin plays a role through the AKT pathway or
TLR4/MyD88/TRAF6/TAK1 pathway, the canonical pathway of NF-κB activation (Figure 9). Therefore,
it is difficult to draw a definite conclusion only through the intervention of inhibitors, and more
scientific and reasonable experiments need to be designed to elucidate the alternative mechanism of
coelonin inactivating NF-κB.
In addition, our study also detected the degradation of p27Kip1 after the LPS challenge, which is a
downstream target of PTEN/AKT. This effect was dramatically inhibited by coelonin pre-treatment.
However, the PTEN inhibitor SF1670 completely abrogated the effect of coelonin (Figure 8). We noticed
that LPS treatment also induced RAW264.7 cells G1 phase arrest (Figure 8B), which contradicts a
previous report [20], but is consistent with report [49]. The exact molecular mechanism underlying
these contradictory results need to be further studied, but it is clear that LPS-induced G1 phase arrest
in RAW264.7 cells did not occur through the up-regulation of p27Kip1 . However, LPS treatment
did promote p27Kip1 degradation (Figure 8A), and which may be dependent on PTEN inactivation
(Figure 9). While coelonin could block the LPS-induced degradation of p27Kip1 by restoring PTEN
activity (Figure 9). Studies have shown that alveolar macrophage proliferation may play an important
role in the formation of multinucleated giant cells and granuloma induced by silica or asbestos [50].
These results suggest that, in addition to inhibiting the secretion of inflammatory factors by macrophages,
inhibiting the proliferation of macrophages may also play a role in alleviating silica or asbestos-induced
lung pathological changes to an extent.
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Int. J. Mol. Sci. 2019, 20, 4422
ȱ
Figure 9. Proposed mechanism of coelonin inhibiting LPS-stimulated activation of NF-κB and inducing
G1 phase cell-cycle arrest in RAW264.7 cells. Coelonin inhibits the expression of inflammatory cytokines
IL-1β, IL-6 and TNF-α in RAW264.7 cells treated by LPS may partially through the PTEN/AKT pathway.
However, it induces G1 cell cycle arrest of RAW264.7 cells by inhibiting the degradation of p27Kip1 in a
PTEN-dependent manner. The black arrow section has been verified, while the red arrow section needs
further validation. The question mark indicated that coelonin may inactivate NF-κB by inhibiting
TLR4/MyD88/TRAF6/TAK1 signal pathway, the canonical pathway of NF-κB activation.
4.1. Active Component Separation, Purification and Identification

A modified polyamide adsorption separation method was used [51]. An amount of 100.0 g
tuber powder of Bletilla striata (collected from Meichuan, Wuxue, Hubei province, China) was reflux
extracted by 1 L 80% ethanol, and the process was repeated three times. The filtrate was concentrated
under vacuum to the volume of 600 mL, and an equal volume of distilled water and 30.0 g polyamide
(100–200 mesh, Taizhou Luqiao Sijia Biochemical Plastics Factory, Taizhou, China) was added, then the
suspension was concentrated under vacuum to the volume of 600 mL. Finally, the mix suspension was
packed, and successively eluted with water, 20%, 40%, 60% and 80% ethanol, and each elution was
concentrated and dried under vacuum to obtain the fractions F0, F20, F40, F60 and F80, respectively.
The IL-1β mRNA expression level, detected by a RT-PCR as the index of anti-inflammation activity
on the LPS-induced RAW264.7 cell model was carried out to evaluate the anti-inflammatory effect
of the fractions. Then, the active fraction was further separated using silica gel (200–300 mesh,
Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) column chromatography, the subfractions
were collected after monitoring by TLC (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China),
and the anti-inflammation effect was analyzed. The final active compound was purified by Dionex
UltiMate™ 3000 semi-prepared HPLC system (Dionex, Sunnyvale, CA, USA) following the guidance of
anti-inflammation assay. Furthermore, the molecular weight of the active compounds were determined
on an ACQUITY UPLC system coupled to a SYNAPT-G2-Si high-definition mass spectrometer (Waters,
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Int. J. Mol. Sci. 2019, 20, 4422
Milford, MA USA), and their NMR spectra were measured on a Bruker DRX-600 spectrometer (Bruker,
Rheinstetten, Germany) with CD3 OD as the solvent.
4.2. Cell Culture

RAW264.7 cells (ATCC) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing
10% heat inactivated fetal bovine serum (Gibco, Waltham, MA, USA), 100 units/mL penicillin and
100 μg/mL streptomycin. The cells were grown at 37 ◦ C in a 5% CO2 incubator.
4.3. RNA Isolation, cDNA Synthesis and RT-PCR

RAW264.7 cells were pre-treated with fractions or active components (dissolved in Dimethyl
Sulfoxide, DMSO and diluted with DMEM medium) for 1 h and then stimulated with LPS (200 ng/mL)
for 6 h, 0.05% DMSO was applied as the parallel solvent control. Total RNA was extracted using
TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol, and subsequently
used to obtain cDNA with a reverse transcription polymerase chain reaction following the protocol
of PrimeScript reverse transcription reagent kit with genomic DNA (gDNA) Eraser (TaKaRa, Dalian,
China) in a 20 μL volume. Levels of IL-1β, IL-6 and TNF-α were determined using specific primers (see
Supplementary Table S1) by RT-PCR on a 7500 Real-Time PCR System (Applied Biosystems, Foster City,
CA, USA). The expression of each gene was normalized relative to the GAPDH expression level, and
relative expression levels were determined using the 2−ΔΔCt method.
4.4. Cytokine Assays

RAW264.7 cells were pre-treated with coelonin for 1 h and then stimulated with LPS (200 ng/mL)
for 12 h, 0.05% DMSO was applied as the parallel solvent control. The culture supernatant was
collected for IL-6 and TNF-α detection. Then, the remaining cells were treated with 1 mM ATP
for an additional 15 min at 37 ◦ C [18], and supernatants were collected for IL-1β detection. IL-6,
TNF-α and IL-1β levels were quantified using the cytometric beads array (CBA) method, according to
the manufacturer’s protocols (BD, Franklin Lakes, NJ, USA), performed on a BD Accuri™ C6 flow
cytometer (BD, Ann Arbor, MI, USA).
4.5. Protein Extraction

The cells were washed three times with phosphate-buffered saline (PBS) chilled to 4 ◦ C. Whole-cell
proteins were extracted with M-PER Mammalian Protein Extraction Reagent (78503, Thermo Fisher
Scientific, Waltham, MA, USA), containing protease and phosphatase inhibitor (Roche, Mannheim,
Germany), at 4 ◦ C for 30 min. Then, the samples were centrifuged at 14,000× g for 10 min, and
the supernatant was transferred to a new tube for analysis. Nuclear proteins were extracted in
accordance with the instructions of the Nuclear and Cytoplasmic Protein Extraction Kit (P0027,
Beyotime Biotechnology, Shanghai, China) for detection of the activity of NF-κB.
4.6. Signal Pathway Phosphorylation Antibody Array Screening

Cell lysates obtained from RAW264.7 cells treated with LPS (200 ng/mL) and with or without
coelonin (2.5 μg/mL) were applied to a Phospho Explorer antibody Array PEX100, which was designed
and manufactured by Full Moon Biosystems, Inc. (Sunnyvale, CA, USA). The microarray contains
1318 antibodies [52], each of which has two replicates that are printed on a coated glass microscope
slide, along with multiple positive and negative controls. The antibody microarray experiment was
performed according to the manufacturer’s protocol. The protein phosphorylation level was measured
as a ratio of the phospho and unphospho values.
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Int. J. Mol. Sci. 2019, 20, 4422
4.7. Automated Western Immunoblotting

Before blotting, the protein was quantified using the bicinchoninic acid (BCA) method.
Simple western immunoblotting was performed on a simple wes system (ProteinSimple, San Jose,
CA, USA) using a Size Separation Master Kit with Split Buffer (12–230 kDa) according to the
manufacturer’s standard instruction and using specific antibody. Anti-PTEN (phospho S380) antibody
[EP2138Y] (ab76431), Anti-PTEN antibody [Y184] (ab32199), Anti-AKT1 (phospho S473) antibody
[EP2109Y] (ab81283), Anti-AKT1/2/3 antibody [EPR16798] (ab179463), Anti-IκBα (phospho S32+S36)
antibody (ab12135), Anti-IκBα antibody (ab32518), Anti-NF-κB-p65 antibody (ab32536), Anti-iNOS
antibody (ab178945), Anti-COX2 antibody (ab179800) were purchased from Abcam(Cambridge, USA),
Anti-p27kip1 antibody (2552), Anti-Lamin A/C antibody (2032S,) and anti-β-actin (4970S) antibody were
obtained from CST (Danvers, MA, USA). Compass software (version 4.0.0, ProteinSimple, San Jose,
CA, USA) was used to program the Peggy Sue and for presentation (and quantification) of the western
immunoblots. Output data were displayed from the software calculated average of seven exposures
(5–480 s).
4.8. Confocal Microscopy Analysis

RAW264.7 cells were grown on glass coverslips. After the treatment with LPS (200 ng/mL) with or
without pre-treatment with coelonin or NF-κB-inhibitor APDC, cells were fixed with 4% formalin in
PBS for 20 min at room temperature, permeabilized with 0.1% Triton X-100 for 15 min, and nonspecific
protein binding sites were blocked with 10% FBS at room temperature for 1 h. Then samples were
incubated overnight at 4 ◦ C with a p65 (ab32536, Abcam, Cambridge, MA, USA) specific Antibody
(1:100 dilution in PBS), washed three times with PBS, and then incubated with a Alexa Fluor® 488-labeled
secondary antibody (ab60314, Abcam; 1:600 dilution in PBS) for 2 h at room temperature. The coverslips
were rinsed three times with PBS, then stained with 4 ,6-diamidino-2-phenylindole (DAPI) for 10 min.
Again, the coverslips were rinsed with PBS three times and then mounted on glass slides using
Antifluorescence Quenching Sealing Solution. The coverslips were analyzed by confocal microscopy
(LSM880; ZEISS, Upper Cohen, Germany).
4.9. Cell Cycle Analysis

After treatment, the RAW264.7 cells were washed three times with chilled 1 × PBS (pH 7.4) and
fixed overnight with 70% ethanol at 4 ◦ C followed by centrifugation at 2,000 rpm for 8 min. The cells
were re-suspended in 1 × PBS (pH 7.4) with PI/RNase Staining Buffer (BD, 550825, San Diego, CA,
USA) for 30 min. The cell cycle distributions were analyzed on a BD Accuri™ C6 flow cytometer (BD,
Ann Arbor, MI, USA).

Data are presented as the mean ± SD derived from at least three independent experiments.
ANOVA analysis was used to examine the statistical significance of the differences between the groups,
and the criterion for significance for all the experiments was p < 0.05.
5. Conclusions
Our study indicated that coelonin is one of the active components of Bletilla striata. Furthermore,
we showed that using a PEX100 antibody microarray, a total of 32 different phosphorylation proteins
were downregulated by coelonin pre-treatment on LPS-induced RAW264.7 cells. The maximum
number of proteins belonged to the PI3K/AKT signal pathway, and three of them, PTEN, p65 and p27
Kip1 were confirmed by western blot, and more proteins and signaling pathways need to be verified.
Western blot and confocal microscopy analysis revealed that coelonin inhibits the expression of IL-1β,
IL-6 and TNF-α in a dose-dependent manner by eliminating lipopolysaccharide-induced NF-κB activity.
However, besides inhibiting IκBα degradation, which pathways coelonin mainly inhibits the activation
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Int. J. Mol. Sci. 2019, 20, 4422
of NF-κB still need to be further studied. While, we did confirm that coelonin inhibit LPS-induced p27
Kip1 degradation and block RAW264.7 cells in the G1 phase in a PTEN dependent manner (Figure 9).
Overall, our results suggest that traditional Chinese medicine Bletilla striata has anti-inflammatory
activity, and coelonin is one of the main active components. It may play a potential role in treating
silicosis by inhibiting the proliferation of macrophages and the secretion of inflammatory factors.
Additionally, PTEN may play an important role during this process, our following work will use
modified RAW264.7 cells to address this issue.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/18/

4422/s1.
Author Contributions: Conceptualization: G.Y.L., methodology: X.B.Y. and Z.S.D.; software: F.S.J. and H.Y.W.;
formal analysis: M.Y.L. and B.D.; writing—original draft preparation: F.S.J. and M.Y.L.; writing—review and
editing: F.S.J., M.Y.L. and C.C.Z.; supervision: G.Y.L. and X.B.Y.; funding acquisition: F.S.J., M.Y.L. and Z.S.D. All
authors have read and approved the final version of the manuscript.
Funding: This research was funded by the National Natural Science Foundation of China (81603360, 81503329,
81673672) and the Natural Science Foundation of Zhejiang province (LY16H310005).
Acknowledgments: We thank the public platform of the Medical Research Center, Academy of Chinese Medical
Sciences, Zhejiang Chinese Medical University for its instrumentation and equipment, and Jiangjiang Qin for his
guidance in revising the manuscript.
Conflicts of Interest: The authors declare no conflicts of interest.
Abbreviations
TLR4 Toll-like receptor 4
MyD88 myeloid differentiation factor 88
IRAK IL-1 receptor associated kinase
TRAF6 TNF receptor associated factor 6
TAK1 TGF beta-Activated Kinase 1
IKKs inhibitor of nuclear factor-κB kinase
PTEN phosphatase and tensin homologue on chromosome ten
PI3K phosphatidylinositol-3-kinases
AKT v-akt murine thymoma viral oncogene homolog
p27Kip1 cyclin dependent kinase inhibitor 1B
NF-κB nuclear factor-kappa B
IκBa inhibitor of NF-κB
IL-1β interleukin-1β
iNOS inducible nitric oxide synthase
COX2 cyclooxygenase 2
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Molecular Sciences
Article
mRNA and miRNA Expression Analysis Reveal the
Regulation for Flower Spot Patterning in
Phalaenopsis ‘Panda’
Anjin Zhao 1 , Zheng Cui 1 , Tingge Li 1 , Huiqin Pei 1 , Yuhui Sheng 1 , Xueqing Li 1 , Ying Zhao 1,2 ,
Yang Zhou 1,2 , Wenjun Huang 3 , Xiqiang Song 1,2 , Ting Peng 4 and Jian Wang 1,2, *
1 Key Laboratory of Ministry of Education for Genetics and Germplasm Innovation of Tropical Special Trees
and Ornamental Plants, Hainan Key Laboratory for Biology of Tropical Ornamental Plants Germplasm,
College of Forestry, Hainan University, Haikou 570228, China
2 Research Center for Terrestrial Biodiversity of the South China Sea, College of Forestry, Hainan University,
Haikou 570228, China
3 Department of Development and Design, Hainan University, Haikou 570228, China
4 Key Laboratory of Germplasm Innovation on Protection and Conservation of Mountain Plant Resources,
Ministry of Education/College of Agriculture, Guizhou University, Guiyang 550025, China
* Correspondence: [email protected]; Tel.: +86-6627-7907

Received: 14 July 2019; Accepted: 26 August 2019; Published: 30 August 2019
Abstract: Phalaenopsis cultivar ‘Panda’ is a beautiful and valuable ornamental for its big flower
and unique big spots on the petals and sepals. Although anthocyanins are known as the main
pigments responsible for flower colors in Phalaenopsis, and the anthocyanins biosynthetic pathway
in Phalaenopsis is generally well known, the detailed knowledge of anthocynins regulation within
the spot and non-spot parts in ‘Panda’ flower is limited. In this study, transcriptome and small
RNA libraries analysis from spot and non-spot sepal tissues of ‘Panda’ were performed, and we
found PeMYB7, PeMYB11, and miR156g, miR858 is associated with the purple spot patterning
in its sepals. Transcriptome analyses showed a total 674 differentially expressed genes (DEGs),
with 424 downregulated and 250 upregulated (Non-spot-VS-Spot), and 10 candidate DEGs involved
in anthocyanin biosynthetic pathway. The qPCR analysis confirmed that seven candidate structure
genes (PeANS, PeF3 H, PeC4H, PeF3H, PeF3H1, Pe4CL2, and PeCHI) have significantly higher
expressing levels in spot tissues than non-spot tissues. A total 1552 differentially expressed miRNAs
(DEMs) were detected with 676 downregulated and 876 upregulated. However, microRNA data
showed no DEMs targeting on anthocyanin biosynthesis structure gene, while a total 40 DEMs target
transcription factor (TF) genes, which expressed significantly different level in spot via non-spot
sepal, including 2 key MYB regulator genes. These results indicated that the lack of anthocyanidins in
non-spot sepal may not directly be caused by microRNA suppressing anthocyanidin synthesis genes
rather than the MYB genes. Our findings will help in understanding the role of miRNA molecular
mechanisms in the spot formation pattern of Phalaenopsis, and would be useful to provide a reference
to similar research in other species.
Keywords: Phalaenopsis; transcriptome; microRNA; anthocyanin biosynthesis; molecular mechanism
1. Introduction
Flower spots are heterochromatic dots or streaks with a specific texture and pattern appearing on the
corollas of plants, which can affect the behavior of pollinators and the ornamental value of flowers [1–4].
Previous studies have confirmed the flower spot is caused by the accumulation of anthocyanins
in a specific area of corollas. For example, peonidin-3-O-glucoside, malvidin-3-O-glucoside,

Int. J. Mol. Sci. 2019, 20, 4250
delphinidin-3-O-diglucoside, and cyanidin-3-O-glucoside are the main anthocyanins found in petal

and sepal spots in Oncidium [5], and cyanidin and delphinidin are the main anthocyanidins in the spot
of pansy (Viola × wittrockiana Gams.) petals [6].
Molecular mechanism of anthocyanins accumulation has been clearly studied in some plants,
such as Dianthus hybrida [7], Antirrhinum majus [8], Petunia hibrida [9], Platycodonis Radix [10],
Gerbera jamesonii [11], and Phalaenopsis equestris [12], and the anthocyanin pathway, which is a
branch of the flavonoid pathway, has been elucidated as well [13,14]. Three kinds of transcription
factor genes families—MYB transcription factor, bHLH transcription factor, and WD40 repeat protein
family (MBW)—were also found to regulate the anthocyanin biosynthesis genes [15].
As for the accumulation of anthocyanin in specific regions of colloras, the direct cause attributes
to the specific expression of the biosynthesis genes involved in the anthocyanin pathway. For example,
high level expression of the genes of OgCHI and OgDFR results in anthocyanin accumulation and
pigmented spot formation in yellow lip in Oncidium [5,16]. Upregulation of LhCHSA, LhCHSB,
and LhDFR is detected within the spots located in the center of the petals, in comparison to
the low expression levels in the margin in Lilium ‘Sorbonne’ [17]. In Dendrobium moniliforme,
pigment accumulation in the base of the column has been caused by a consequence of preferential
expression of DmF3 5 H [18]. In Clarkia gracilis, precise spatiotemporal regulation of the expression of
the anthocyanin genes F3 H, F3 5 H, DFR1, and DFR2 produces spotted petals [19]. In pansy, VwDFR,
VwF3 5 H and VwANS have more significantly higher level expression in cyanic flower areas [6].
Moreover, the MYB genes also play an important role for the production of flower spot by regulating the
anthocyanin biosynthesis genes. For example, the large purple spots in Phalaenopsis ‘Everspring Fairy’
was mainly caused by the expression of MYB [12], and the LhMYB6 and LhMYB12 positively regulate
anthocyanin biosynthesis and determine organ- and tissue-specific accumulation of anthocyanin in
Asiatic hybrid lily ‘Montreux’ [20]. Hsu also conducted a detailed study on three MYBs in Phalaenopsis,
and found that the color patterning of flower sepals, petals and lips is regulated by different MYB
genes combinations, and the pigmented veins and spots on the petals are also regulated by these three
MYB genes [21].
RNA interference, regulating gene expression by post-transcriptional mechanisms, has also
been recognized to play an important role in the color special patterning model of some plants [22].
Koseki et al. [23] found that the star-type color pattern of Petunia hybrida Red star’ flowers is induced by
sequence-specific degradation of chalcone synthase RNA. Their further study found that the formation
of bicolor flower types of petunia was due to RNA interference in two CHSA alleles (PhCHS-A1
and PhCHS-A2) [23]. In Arabidopsis thaliana, miRNAs may function as regulators in anthocyanin
biosynthesis by targeting on related transcription factors and lead to the different accumulation of
anthocyanin [24].
Phalaenopsis spp. have become important ornamental plants worldwide for their long-lasting
and various colorful flowers [25]. There are varieties of flower colors and corolla pigmentation
patterning styles of Phalaenopsis spp., so it is very important to find more details about the flower color
patterning for its breeding and production. However, there was little research in this field because of
the complex anthocyanin synthesis pathways and the gene expression networks in Phalaenopsis spp.
RNA sequencing can effectively identify the subtle differences in gene expression and find the targeted
genes of small RNA in transcription level, so it has been used to study flower color patterning recently,
such as in Lilium ‘Tiny Padhye’ [26], monkeyflowers (Mimulus) [27], and tree peony [28]. In this study,
the transcriptome sequencing and small RNA sequencing in spot and non-spot sepal of Phalaenopsis
‘Panda’ were carried out by genomics Illumina sequencing platform, and the expression levels of
candidate genes, as well as microRNAs, were verified by qPCR. Meanwhile, the model of flower spot
formation pattern was predicted by the interactions of the structural genes, regulatory genes together
with small RNA. This study created a joint research of transcriptome sequencing and small RNA
sequencing to explain the spot pigmentation in Phalaenopsis. spp., and the results will be helpful for
the breeding of new colorful cultivars of Phalaenopsis spp.
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2. Results
2.1. Anthocyanin Accumulation Patterns in Phalaenopsis ‘Panda’

The examination by light microscope showed that anthocyanin accumulated in the upper
epidermal cells of spot area, while no anthocyanin accumulated in cells in non-spot area (Figure 1A–C).
This result indicated that the visible spots resulted from accumulation of anthocyanin in cells of
the upper epidermis. Furthermore, the anthocyanin content of the spot in Phalaenopsis ‘panda’
sepals was significantly different from that of the sepal according to the results by the UV-visible
spectrophotometer scanning detection (Figure 1D,E). Anthocyanin content measured by the pH
differential method revealed that high content of anthocyanin (1.0798 mg/g) was accumulated in spot
area, while was barely detectable in the acyanic parts extraction.
Figure 1. Anatomical structures and anthocyanin content of Phalaenopsis ‘Panda’ sepal. (A) Sepal
(bar = 1 cm); (B) Upper epidermal cell in spot area (bar = 10 μm); (C) Upper epidermal cell in non-spot
area (bar = 10 μm); (D) UV-visible spectrophotometer scanning of non-spot area; (E) UV-visible
spectrophotometer scanning of spot area. Red number of 531 nm represents the absorption wavelength
of anthocyanin.
2.2. Construction of cDNA Library and Gene Mapping to the Reference Genomes
Four cDNA libraries were constructed using Illumina Hiseq 2000 platform (Illumina, San Diego, CA,
USA) and 44,632,028, 45,110,536, 45,380,086, 44,434,116 high-quality reads were obtained, respectively
(Table S1 in supplememntary materials). The sequencing raw data has been deposited into the Short
Reads Archive (SRA) database under the accession number SRP166213. These clean reads were mapped
to reference genome of Phalaenopsis equestris and the average gene mapping ratio of each sample was
55.47%. We considered that the low homology between Phalaenopsis ‘Panda’ and Phalaenopsis equestris
result to the low mapping ratio, but it has no effect on RNAseq quantitative analysis owing to the high
clean reads quantity and enough sequencing data (Table S2).
2.3. Functional Annotation and Classification

To annotate the gene with putative functions, the assembled genes were searched against the
public databases of NR. Among them, 17,871 genes were annotated to the NR database. To further
illustrate the main biological functions of the transcripts, GO (15,588 genes) and KEGG pathway
(19,864 genes) analyses were performed (Table S3).
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2.4. Identification of Differentially Expressed Genes and KEGG Enrichment Analysis of DEGs
A total of 674 DEGs were detected in pairwise comparison with 424 downregulated and
250 upregulated genes (Non-spot-VS-Spot) (Figure 2). A total of 543 DEGs were mapped to
all 106 pathways. Notably, the Flavonoid biosynthesis (ko00941, 12 DEGs of 105 gene with
Q-value = 0.0005279107) and Phenylalanine metabolism (ko00360, 8 DEGs of 132 gene with
Q-value = 1.900591 × 10−1 ) were most significantly enriched in top20 pathways (Figure 3, Table S4).
Figure 2. Volcano plot of differentially expressed genes (DEGs). X axis: log2 transformed fold change;
Y axis: −log10 transformed significance; Red points: upregulated DEGs; Blue points: downregulated
DEGs. Gray points: non-DEGs.
7KHPRVWHQULFKPHQWSDWKZD\
Figure 3. The most enrichment pathway of DEGs (TOP20). X axis: enrichment factor; Y axis:
pathway name; The color: the q-value (high: white, low: blue), the lower q-value indicates the more
significant enrichment; Point size: DEG number (The bigger dots refer to larger amount); Rich Factor:
the value of enrichment factor, which is the quotient of foreground value (the number of DEGs)
and background value (total Gene amount), the larger the value, the more significant enrichment.
Red arrows represent the pathways related directly to the anthocyanin biosynthesis.
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2.5. DEGs in Anthocyanin Biosynthesis and MBW Genes

There were 10 DEGs in flavonoid biosynthesis which are directly related to spot pattern,
including PeANS (PEQU_25924), PeF3 H (PEQU_00400), PeC4H (PEQU_12025), PeDFR (PEQU_34933),
PeCHI (PEQU_22606), PeF3H1 (PEQU_38891), PeF3H (PEQU_22432), PePAL (PEQU_01877), Pe4CL2
(PEQU_00756), Pe4CL (PEQU_07458), and most of them presented higher expression level in spot
areas than non-spot areas (Table 1, Figure 4).
Table 1. Putative anthocyanin structural genes identified from differentially expressed genes (DEGs).
FPKM FPKM
Gene ID Annotation Log2FC Padj Up/Downregulation
(non-spot) (spot)
PEQU_25924 PeANS 119.9303758 21,612.25078 7.493508409 4.71 ×10−130 Up
PEQU_00400 PeF3 H 31.02321 6117.42 7.623432 5.88 × 10−88 Up
PEQU_12025 PeC4H 2272.365 8325.431 1.87333 1.79 × 10−8 Up
PEQU_34933 PeDFR 17.40537 5973.723 8.422954 1.13 × 10−117 Up
PEQU_22606 PeCHI 2467.193 707.8831 −1.80129 1.13 × 10−6 Down
PEQU_07458 Pe4CL 11.87304 253.4331 4.415844 3.15 × 10−18 Up
PEQU_38891 PeF3H1 52.59874 5401.439 6.682172 2.59 × 10−29 Up
PEQU_22432 PeF3H 21.49244 3076.48 7.161307 2.17 × 10−65 Up
PEQU_01877 PePAL 14783.45 6368.878 −1.21487 8.83 × 10−8 Down
PEQU_00756 Pe4CL2 383.56 1290.653 1.750578 0.000153 Up
ȱ
Figure 4. Heatmap of differentially expressed structure genes (DEGs) related to anthocyanin biosynthesis
in Phalaenopsis. Bold arrow means up/downregulations of genes. (PAL, phenylalanine ammonia lyase;
C4H, cinnamate-4-hydroxylase; 4CL, 4-coumarate–CoA ligase; CHS, chalcone synthase; CHI, chalcone
isomerase; F3H, flavanone 3-hydroxylase; F3 H, Flavonoid 3’-hydroxylase; DFR, dihydroflavonol
4-reductase; ANS, Anthocyanidin synthase; DHK, dihydrokaempferol; DHQ, dihydroquercetin; DHM,
dihydromyricetin; LC, leucocyanidin; Cy, cyaniding).
As for the MBW transcriptor genes related to the anthocyanin synthesis, 4 unigenes were found
having significant expression difference including 3 MYB uingenes and 1 bHLH unigene (Table 2).
In these transcriptor unigenes, 2 MYB unigenes and 1 bHLH unigene were upregulated and 1 MYB
unigene was downregulated.
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Table 2. Putative MBW genes identified from differentially expressed genes (DEGs).
FPKM FPKM
Gene ID Annotation Log2FC Padj Up/Downregulation
(non-spot) (spot)
PEQU_03393 PeMYB7 29.41538126 113.6604505 0.04965 0.00303 UP
PEQU_10361 PeMYB11 10.58835723 1693.628622 7.32149 8.71 × 10−62 UP
PEQU_10362 PeMYB11 4.49564678 216.34208328 5.58864183 2.35 × 10−18 UP
PEQU_09064 PeMYB16 1022.11354 508.0418102 −1.00853 0.03605 DOWN
PEQU_19747 PebHLH1 149.2074009 801.9464953 2.42618 2.57 × 10−18 UP
2.6. Data Analysis of Small RNA Sequencing

Four sRNA libraries were constructed and 23,352,936, 24,896,769, 26,605,438, 25,926,370 high-quality
tag were obtained, respectively (Table S5). The sequencing raw data has been deposited into the Short
Reads Archive (SRA) database under the accession number SRP161646. These clean tags were mapped
to reference genome of Phalaenopsis equestris and other sRNA databases. The average gene mapping
ratio of each sample was 77.38% (Table S6).
A total of 1552 DEMs were detected in pairwise comparison with 676 downregulated
and 876 upregulated (Non-spot-VS-Spot) (Figure 5). miRNA target gene prediction resulted
in 1396 common unigenes between TargetFinder (http://http://targetfinder.org/) and psRobot
(http://http://omicslab.genetics.ac.cn/psRobot/), 1648 by TargetFinder and 7652 by psRobot, and totally
1307 target unigenes were aligned to KEGG database. However, there were no DEMs were found
targeting on anthocyanin biosynthesis structure gene. As for the regulator genes, we found totally
12 microRNAs targeting on 10 significant different expressed MYB, bHLH or WRKY unigenes (Table 3).
However, only PeMYB7 (PEQU_03393), PeMYB11 (PEQU_10361, PEQU_10362) were significantly
upregulated in these TF families according to the transcriptomes data, and these microRNAs belonged
to miR156 or miR858 families.
Figure 5. Volcano plot of DEMs. X axis represents log2 transformed fold change. Y axis represents
−log10 transformed significance. Red points represent upregulated DEMs. Blue points represent
downregulated DEMs. Gray points represent non-DEMs.
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Table 3. Putative miRNAs targeting on MYB, bHLH or WRKY unigenes.
miRNA miRNA Up/Down Up/Downregulation

Target
miRNA id Expression in Expression Regulation p-Value Target Gene of Target Unigene
Unigene ID
Non-spot in Spot of miRNA in Spot
Novel-m1700-5p 12 0 UP 0.000818887 PeMYB39 PEQU_22029 NA
mtr-miR156g-3p 49 1 UP 2.66 × 10−12 PeMYB7 PEQU_03393 UP
Novel-m0210-3p 82 10 UP 1.69 × 10−13 PeMYB7 PEQU_03393 UP
PeMYB11 PEQU_10361 UP
cme-miR858 52 0 UP 1.57 × 10−14 PeMYB8 PEQU_10866 NA
PeMYB12 PEQU_20333 NA
ath-miR858 336 116 UP 4.67 × 10−34 PeMYB11 PEQU_10362 UP
PeMYB12 PEQU_20333 NA
ata-miR528-3p 12 0 UP 0.000818887 PebHLH086 PEQU_08299 NA
zma-miR528a-3p 49 1 UP 2.66 × 10−12 PebHLH086 PEQU_08299 NA
osa-miR162b 82 10 UP 1.69 × 10−13 PebHLH13 PEQU_33912 NA
Novel-m0112-3p 0 52 DOWN 1.57 × 10−14 PebHLH PEQU_26133 NA
gma-miR169v 54 0 UP 2.83 × 10−13 katanin p80 WD40 PEQU_05516 NA
Novel-m0290-5p 24 0 UP 9.92 × 10−7 katanin p80 WD40 PEQU_05516 NA
2.7. qPCR of Key Structural Genes, Regulate Genes and miRNA

The expression levels of 10 anthocyanin genes and 4 transcriptor genes between the non-spot
and spot areas were verified by qPCR. The results showed the genes Pe4CL2, PeANS, PeF3H, PeF3H1,
PeF3 H and PeMYB7, PeMYB11 presented significantly higher expression levels in spot areas than the
non-spot areas (Figure 6), which was generally consistent with the results of the transcriptome data
(Table 1).

Figure 6. The expression level of structure gene and regulate gene Associated with Anthocyanin
Biosynthesis, * represent there are significant differences between spot and non-spot area; ** represent
there are extremely significant differences; red: spot area, blue: non-spot area.
The expression of microRNA of RNA miR858, miR156g were also analyzed by qPCR between the
non-spot and spot areas (Figure 7). The results showed that they presented higher expression levels in
non-spot areas, which means their targeting genes, PeMYB7 and PeMYB11, having less expression
levels in the non-spot areas.
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Figure 7. The expression level of mtr-miR156g-3p and miR858. ** represent an extremly significant
difference (p < 0.01); * represent an significant difference (p < 0.05).
3. Discussion
3.1. Low Expression of Anthocyanin Genes Causing the Lack of Pigments in Non-Spot Areas
In this study, we found anthocyanin accumulated in the upper epidermal cells in spot area,
while no anthocyanin accumulated in cells in non-spot areas of ‘Panda’ (Figure 1). Transcriptome data
and qPCR indicated 7 anthocyanin genes were low expression in the non-spot areas, especially for
the very different expression levels of 4CL2, F3’H, F3H and F3H1 (Table 2, Figure 6). In Phalaenopsis
‘Everspring Fairy’, which also has purple spot on the white petal and sepal, DFR is the main gene
which differently expressed between the purple and white part of the petals and sepals [12]. However,
in Phalaenopsis ‘Panda’ expression of DFR in spot tissue was not significantly changed in comparison
to non-spot area. These results showed that the same phenotype may be caused by different genes in
the pigmentation in Phalaenopsis.
3.2. PeMYB7 and PeMYB11 Are Important Genes in Spot Formation

MYBs have been found to be very important in the floral pigmentation patterning in Phalaenopsis
spp. In the sepals/petals, silencing of PeMYB2, PeMYB11, and PeMYB12 resulted in the loss of the
full-red pigmentation, red spots, and venation patterns, respectively; PeMYB11 was responsive to the
red spots in the callus of the lip, and PeMYB12 participated in the full pigmentation in the central
lobe of the lip [21]. In P. amabilis and P. schilleriana, anthocyanin-specific Myc and Wd were expressed,
however, Myb specific for anthocyanin biosynthesis were undetectable in P. amabilis; in Phalaenopsis
‘Everspring Fairy‘ petals and sepals, high levels of anthocyanin-specific Myb transcripts were present
in the purple, but not in the white sectors [12]. Hsu, et al. [29] verified PeMYB11 as the major regulatory
R2R3-MYB transcription factor for regulating the production of the black color, and a retrotransposon,
named Harlequin Orchid RetroTransposon 1 (HORT1), resulted in strong expression of PeMYB11 and
thus extremely high accumulation of anthocyanins in the harlequin flowers of the P. Yushan Little
Pearl variety.
In our study, PeMYB7 and PeMYB11 expressed significantly different between the spot and
non-spot areas, while PeMYB2 and PeMYB12 had not different expression levels (Table 2 and Figure 6).
This result indicated that the gene of PeMYB11 may also play an important role in spot pigmentation in
Phalaenopsis ‘Panda’, which was similar to these previous researches [12,21,29,30]. PeMYB7 had different
expression levels between the spot and non-spot areas of sepals in this research, which indicated
this gene may also associate with purple spot patterning in Phalaenopsis ‘Panda’. The function of
PeMYB7 is not very clear presently, however, in the phylogenetic tree inferred from MYB genes of
Phalaenopsis equestris and Oryza sativa (Figure 8A), PeMYB7 was in the same clad of PeMYB2 and
PeMYB8, suggesting PeMYB7 may have similar function as PeMYB2 which can activate anthocyanin
synthesis [30].
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3.3. miR156g, miR858 Silence PeMYB7, and PeMYB11

MiRNAs can influence tissue anthocyanin formation in previous studies. In petunia,
the sequence-specific degradation of CHS RNA is the primary cause of the formation of white
sectors in ‘Red Star’ flowers [23]. In potato (Solanum tuberosum L.), small RNAs of miR828 and TAS4
D4(−) can decrease the expression levels of MYB12 and R2R3-MYB genes in purple skin and flesh,
which caused to anthocyanin accumulation [31]. In our research, miR156 and miR858 were predicted
as the main interference RNAs for PeMYB7 and PeMYB11 (Table 3). In order to confirm PeMYB7 and
PeMYB11 are the direct targets of the small RNAs, we download the complete cDNAs of PeMYB7
(GenBank: KF769472.1) and PeMYB11 (GenBank: MH675649.1), and analyzed the target sites of miR156
and miR858 in these two genes (Figure 8B). This finding confirms that miR156g and miR858 can silence
PeMYB7 and PeMYB11.
Figure 8. Identification of PeMYB7, PeMYB11 and their target sites by miR156g, miR858.
(A) Phylogenetic tree inferred from the MYB sequences of Phalaenopsis equestris and Oryza sativa;
(B) Identification of target sites of miR156 and miR858 in PeMYB7 and PeMYB11.
In Arabidopsis thaliana, miR156 negatively regulates the anthocyanin accumulation by regulating

the expression level of target gene MYB113 indirectly [32]. High miR156 level promotes anthocyanin
biosynthesis through the negative regulation of SPL9 gene because SPL9 can destabilize the
MYB-bHLH-WD40 transcriptional activation complex. However, in this study, SPL9 gene in Phalaenopsis
doesn’t express differently, which indicated that mtr-miR156g-3p may be able to directly silence PeMYB7
and caused the anthocyanin lacking on the white sector.
The biological functions of miR858 has not been fully explored, however, small RNA sequencing in
Arabidopsis thaliana [24] and Vitis vinifera L. [33] verified that its target gene is MYB12, which is a positive
transcript factor for the anthocyanin synthesis. Chitwood et al. transferred the constructed binary
vector into wild-type tomato plants to reduced MIR858 expression level, and found the expression
levels of SLY-Myb12 in transgenic plants were lower than wild type, with upregulations of the related
structure gene PAL, DFR, ANS and CHS involved in anthocyanin biosynthesis, and increasing of
anthocyanin content [34]. Ballester et al. [35] used VIGS technology silenced MYB12 and then found
the tomato fruits turned pink. In our study the targeted genes of miR858 were predicted to be both
MYB12 and MYB11 (Table 3), however, the unigene of PeMYB12 didn’t show significantly different
expression levels between the spot and non-spot areas, suggesting that miR858 explored higher effect
on the unigene of PeMYB11 in ‘Panda’.
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3.4. A Proposed Modelsummarizing of Spot Formation Pattern in Phalaenopsis ‘Panda’

Our research confirmed Pe4CL2, PeF3H, PeF3 H, and PeANS expression were low or repressed in
non-spot areas in ‘Panda’ sepals, and MIR156g and MIR858 may silence the expression of PeMYB11
and PeMYB7 in non-spots parts. In conclusion, we illustrated a proposed molecular mechanism in
spots formation in ‘Panda’ (Figure 9). The diagram revealed the unique spot formation in ‘panda’ may
result from the higher expression of MIR156g and MIR858 clusters in non-spot tissue, which targeted
and suppressed the expression of key regulate genes PeMYB7, PeMYB11 and then caused very low
expression of Pe4CL2, PeF3H, PeF3 H, and PeANS in non-spot tissue. These results finally led to the
lack of synthesis and accumulation of anthocyanin in non-spot area.

Figure 9. A proposed model summarizing of spot formation pattern in Phalaenopsis ‘Panda’ sepal.
The purple segment illustrates the accumulation of anthocyanidin in spot area of ‘Panda’. MIR156g,
MIR858 target on key regulate genes PeMYB7 and PeMYB11, suppressed the transcript level of
Pe4CL2, PeF3H, PeF3 H, PeANS and resulted in reduced anthocyanidin production in the non-spot area,
while relatively lower expression levels of MIR156g and MIR858 and high levels of transcription of
these genes in spot area cause accumulation of anthocyanidin. The black dashed arrows represent
low levels of transcription, while the black solid-line arrows represent high levels of transcription.
The yellow solid-line arrows represent promoting transcription, while yellow dashed arrow represent
lack of promotion function. The blue bold solid-line T-arrow represents interfering transcription,
while blue bold dashed arrow represents lack of interference function. The red bold arrow represents
upregulation, and the green bold arrow represents downregulation. DHK, dihydrokaempferol; DHQ,
dihydroquercetin; DHM, dihydromyricetin; LC, leucocyanidin; LD, leucodelphinidin; Cy, cyanidin;
De, delphinidin.

Phalaenopsis ‘Panda’ (Figure 10) were grown at the horticultural farm of Hainan University
(Latitude: 20.03N, longitude: 110.33E), Haikou, Hainan Province, China. The sepal tissues were
divided into two parts, spots area and non-spots area for total anthocyanin analyses and totally RNA
extraction. Three independent biological replicates were collected for each sample. All samples were
immediately frozen in liquid nitrogen and stored at −80 ◦ C until use.
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Int. J. Mol. Sci. 2019, 20, 4250
ȱ
Figure 10. The flower tissue of Phalaenopsis ‘Panda’. Bars = 1 cm. (A) Full bud; (B) Sepal; I. spot area; II.
non-spot area. (C) Full bloom stage.
4.2. Observations of Sepal Anatomy and Determination of Total Anthocyanin Content

Spot and non-spot sepal tissue of full bud were cut into segments longitudinally
(approx.10 mm × 5 mm), and the upper epidermis and dorsal epidermis were separated with forceps.
The sections were observed and photographed with a light microscope (Primo Vert, Zeiss, Germany).
The total anthocyanin content of Phalaenopsis sepal in spot and non-spot areas were determined
by the pH differential method [36].
4.3. RNA Extraction, cDNA Library Construction, and mRNA Sequencing

RNAs were extracted from spot and non-spot parts of sepals of full bud using the modified
Trizol method [6]. Two biological replicates were used for spot sepal and non-spot sepal. A total of
4 RNA-seq libraries were constructed from these sepals. RNA-Seq libraries were constructed using the
RNA Library Prep Kit for Illumina according to the manufacturer’s instructions (NEB, Ipswich, MA,
USA). Library quality was assessed on the Agilent Bioanalyzer 2100 system. The mRNA libraries were
sequenced on the Illumina HiSeq 2000 platform (Illumina, San Diego, CA, USA) based on sequencing
by synthesis with 150 bp paired-end reads (Biomarker Technologies, Wuhan, China).
4.4. mRNA Transcriptome Data Analysis

The reads with adaptors were removed firstly. The reads in which unknown bases comprised
more than 5% of the total and low quality reads (the percentage of the low quality bases of quality
value ≤ 15 is more than 20% in a read) were also removed. The clean reads were aligned to
Phalaenopsis equestris genome accessed from (https://www.ncbi.nlm.nih.gov/bioproject/192198) by
HISAT2 (http://www.ccb.jhu.edu/software/hisat/index.shtml) [37].
Gene alignment rate was counted with Bowtie2 v2.2.5 (http://bowtie-bio.sourceforge.net/bowtie2/
index.shtml), gene and transcript expression levels were calculated with RSEM v1.2.12 (http://deweylab.
biostat.wisc.edu/RSEM). The differentially expressed genes (DEGs) between spot and non-spot tissues
were identified and filtered with the R package DESeq2 based on |log2 (foldchange)| ≥ 1 and Padj
< 0.05 [38]. The heatmap displays of the Trimmed Mean of M-values (TMM) normalized against
the Fragment Per Kilobase of transcripts per Million (FPKM) mapped reads were performed using
the R package pheatmap (https://cran.r-project.org/src/contrib/Archive/pheatmap/). The DGEs were
imported into Blast2GO software v2.5 [39] and in-house perl scripts for gene ontology (GO) term
analysis, while the KEGG pathways were assigned to the assembled genes using the online KEGG
Automatic Annotation Server (KAAS, http://www.genome.jp/kegg/), and then the phyper function
in R software was used for enrichment analysis with FDR ≤ 0.01. To identify transcription factors,
the assembled transcriptome were searched against the Plant Transcription Factor Database PlnTFDB
(http://plntfdb.bio.uni-potsdam.de/v3.0/downloads.php) using hmmseach v3.0 (http://hmmer.org).
205
Int. J. Mol. Sci. 2019, 20, 4250
4.5. qRT-PCR Analyses of mRNA

10 DEGs in anthocyanin biosynthesis and 4 DEGs of transcription factor were chosen for qRT-PCR
analyses. qRT-PCR analyses were performed using SYBR Premix Ex TaqTM II (Tli RNaseH Plus)
(Takara, Dalian, China) according to the manufacturer’s instructions with the following reaction
conditions: denaturation at 95 ◦ C for 30 s and 40 cycles of amplification (95 ◦ C for 5 s, 60 ◦ C for 30 s).
The β-Actin gene was used as an internal control for normalization. Relative expression levels of target
genes were calculated using the 2−ΔΔCt [40] against the internal control. The gene-specific primers are
shown in Table S7. Experiments were performed with three independent biological replicates and
three technical replicates.
4.6. Small RNA Library Construction and Sequencing

RNA was extracted from spot and non-spot sepal using the modified Trizol method [6], and 4 small
RNA libraries from sepal spot and non-spot samples with 2 biological replicates were constructed
and sequenced by SBQ500 sequencing method (BGI, Wuhan, China). Then the low-quality, 5 primer
contaminants, without 3 primers and insert tags, and sequences fewer than 18 nucleotides (nt) were
filtered out to obtain clean reads from raw data reads. The final clean reads of the 4 libraries were
mapped to Phalaenopsis genome and other smallRNA databases using Bowtie2 [41]. All the remaining
clean sequences were annotated into different classes to remove rRNA, scRNA, snoRNA, snRNA,
and tRNA using miRBase, siRNA, piRNA, snoRNA database with Bowtie2 [42] and Rfam database
with cmsearch [43]. The novel miRNAs were predicted by Mireap software (https://sourceforge.net/
projects/mireap/).
4.7. Data Analysis of Small RNA Sequencing

The expression level of miRNAs was compared between spot and non-spot tissues to identify
differentially expressed miRNAs (DEMs). The differentially expressed miRNA between spot and
non-spot tissues were identified and filtered with the R package DESeq2 and Poisson Distribution [38,44].
The DEMs were determined based on FDR ≤ 0.001 and the absolute value of |Log2FC| ≥ 1. The heatmap
displays of the Trimmed Mean of M-values (TMM) normalized against the Fragment Per Kilobase of
transcripts per Million mapped reads (FPKM) were performed created using the R package pheatmap
(https://cran.r-project.org/src/contrib/Archive/pheatmap/).
The potential target genes by differentially expressed miRNAs were predicted and analyzed with
2 different software, including psRobot [45] and Targetfinder [46]. In order to increase the level of
confidence and get more reliable results, we selected only those binding sites that were predicted by
both of two softwares.
All protein-coding target genes regulated by DEMs were annotated against the KEGG databases.
The KEGG enrichment analysis for the target genes of the DEMs was performed by Fisher’s exact test
(P-values) in Blast2GO pipeline, and P-values were used to conduct multiple test correction by FDR.
KEGG terms with FDR< 0.05 were considered to be significantly enriched.
4.8. Verification of miRNA Expression Levels by qPCR

Two miRNA targets on key regulate gene transcription factor PeMYB11 and PeMYB7 were chosen
for qRT-PCR analyses. The qRT-PCR analyses were performed using SYBR Premix Ex TaqTM II
(Tli RNaseH Plus) (Takara, Dalian, China) according to the manufacturer’s instructions with the
following reaction conditions: Denaturation at 95 ◦ C for 30 s and 40 cycles of amplification (95 ◦ C
for 5 s, 60 ◦ C for 30 s). The U6 gene was used as an internal control for normalization. Relative
expression levels of target genes were calculated using the 2−ΔΔCt [40] against the internal control.
The specific stem-ring primers were designed according to the miRNA qPCR primer design method [47],
showed in Table S8. Experiments were performed with three independent biological replicates and
three technical replicates.
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Int. J. Mol. Sci. 2019, 20, 4250
Supplementary Materials: Supplementary Materials can be found at http://www.mdpi.com/1422-0067/20/17/

4250/s1.
Author Contributions: Conceptualization, J.W. and X.S.; methodology, A.Z., Z.C. and T.L.; software, H.P.;
materies culture, Y.S. and X.L.; validation, A.Z., Y.Z. (Ying Zhao) and Y.Z. (Yang Zhou); writing—original draft
preparation, A.Z.; writing—review and editing, J.W., W.H. and T.P.
Funding: This study was supported by the Hainan major science program (zdkj201815) and the National Natural
Science Foundation of China (31760590; 31260488). This research was conducted at Research Center for Terrestrial
Biodiversity of the South China Sea, Hainan University, Hainan, China.
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About the Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

Preface to ”Orchid Biochemistry” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

Jean Carlos Cardoso, Cesar Augusto Zanello and Jen-Tsung Chen

Björn Bohman, Alyssa M. Weinstein, Raimondas Mozuraitis, Gavin R. Flematti and

Int. J. Mol. Sci. 2020, 21, 2338; doi:10.3390/ijms21072338 1 www.mdpi.com/journal/ijms

6. Conclusions and Perspectives

Funding: This research received no external funding.

Keywords: biotechnology; breeding; mass propagation; Orchidaceae; protocorm-like bodies;

Int. J. Mol. Sci. 2020, 21, 985; doi:10.3390/ijms21030985 5 www.mdpi.com/journal/ijms

2. Genus Phalaenopsis and Related

MS1/2 + 2% sucrose + 10% 2.0 mg L−1 BA and 0.5 mg L−1 NAA

3. Oncidium Hybrids Group

4. Some News with Cymbidium, Dendrobium, and Others

5. Applications of IPR–PLB Technique on Orchid Propagation and Breeding and Main

Int. J. Mol. Sci. 2020, 21, 1869; doi:10.3390/ijms21051869 37 www.mdpi.com/journal/ijms

2.1. Pigments Content Analysis

2.4. Screening Diﬀerential Metabolites in the Process of Leaf Color Change

4. Materials and Methods

4.1. Plant Materials

4.2. Pigments Content Measurement

4.3. Analysis of Flavonoids-Targeted Metabolomics

4.3.1. Standards and Reagents

4.3.2. Sample Extraction Process

4.3.3. Acquisition Conditions of LC-MS

4.3.4. Data Evaluation

4.3.5. Data Analysis

4.4. Enzyme Activity Determination

4.5. Total RNA Extraction and qRT-PCR Analysis

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/5/1869/

Keywords: Cymbidium; ﬂoral scents; Orchidaceae; pollination; volatile organic compounds

Int. J. Mol. Sci. 2020, 21, 1160; doi:10.3390/ijms21031160 55 www.mdpi.com/journal/ijms

2. Orchid Volatile Compounds and Biosynthetic Pathways

Table 1. Major volatile organic components in orchid ﬂowers.

Compound Structure Species Reference

Geraniol P. bellina [24]

Ocimene Vanda Mimi Palmer [42]

Farnesol C. goeringii [28]

β-Caryophyllene M. tenufolia [43]

Eugenol Gymnadenia Species [30]

2-methyl butanal C. cv. Sael Bit [29]

Compound Structure Species Reference

Benzyl acetate Vanda Mimi Palmer [42]

Fatty Acid Derivatives

2.2. Phenylproponoids and Benzenoids

2.3. Fatty Acid Derivatives

3. Transcriptional Factors in Floral Volatile Regulation

Table 2. Representative genes responsible for ﬂoral scents in orchids.

Floral Scent Gene Metabolism Pathway Species Reference

4. Spatial and Temporal Emission of Volatile Organic Compounds

5. Gene Evolution for VOCs

6. Functions of Orchid Volatile Compounds

Figure 2. Functions of ﬂoral volatiles in orchid ﬂowers.

6.1. Flower Defense

6.2. Pollinator Attraction

6.3. Plant Reproduction

7. Case Studies of Cymbidium Floral Volatiles

7.1. Floral Volatile Research on Cymbidium