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International Journal of
Molecular Sciences
Molecular Advances
in Wheat and Barley
Edited by
Manuel Martinez
Printed Edition of the Special Issue Published in
International Journal of Molecular Sciences
www.mdpi.com/journal/ijms
Barley
Barley
Special Issue Editor

Manuel Martinez
MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade

Special Issue Editor
Manuel Martinez
Universidad Politecnica de Madrid
Spain
Editorial Office
MDPI
St. Alban-Anlage 66
4052 Basel, Switzerland
This is a reprint of articles from the Special Issue published online in the open access journal
International Journal of Molecular Sciences (ISSN 1422-0067) from 2018 to 2019 (available at: https:
//www.mdpi.com/journal/ijms/special issues/Wheat Barley)
For citation purposes, cite each article independently as indicated on the article page online and as
indicated below:
LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. Journal Name Year, Article Number,
Page Range.
ISBN 978-3-03921-371-9 (Pbk)

ISBN 978-3-03921-372-6 (PDF)

c 2019 by the authors. Articles in this book are Open Access and distributed under the Creative
Commons Attribution (CC BY) license, which allows users to download, copy and build upon
published articles, as long as the author and publisher are properly credited, which ensures maximum
dissemination and a wider impact of our publications.
The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons
license CC BY-NC-ND.
Contents
About the Special Issue Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Manuel Martinez
Editorial for Special Issue “Molecular Advances in Wheat and Barley”
Reprinted from: Int. J. Mol. Sci. 2019, 20, 3501, doi:10.3390/ijms20143501 . . . . . . . . . . . . . . 1
Claus Krogh Madsen and Henrik Brinch-Pedersen

Molecular Advances on Phytases in Barley and Wheat
Mercedes Diaz-Mendoza, Isabel Diaz and Manuel Martinez

Insights on the Proteases Involved in Barley and Wheat Grain Germination
Iris Koeppel, Christian Hertig, Robert Hoffie and Jochen Kumlehn

Cas Endonuclease Technology—A Quantum Leap in the Advancement of Barley and Wheat
Genetic Engineering
Muhammad Amjad Ali, Mahpara Shahzadi, Adil Zahoor, Abdelfattah A. Dababat, Halil
Toktay, Allah Bakhsh, Muhammad Azher Nawaz and Hongjie Li
Resistance to Cereal Cyst Nematodes in Wheat and Barley: An Emphasis on Classical and
Modern Approaches
Reprinted from: Int. J. Mol. Sci. 2019, 20, 432, doi:10.3390/ijms20020432 . . . . . . . . . . . . . . . 51
Tuo Qi, Jia Guo, Huan Peng, Peng Liu, Zhensheng Kang and Jun Guo
Host-Induced Gene Silencing: A Powerful Strategy to Control Diseases of Wheat and Barley
Alexey V. Pigolev, Dmitry N. Miroshnichenko, Alexander S. Pushin, Vasily V. Terentyev,

Alexander M. Boutanayev, Sergey V. Dolgov and Tatyana V. Savchenko
Overexpression of Arabidopsis OPR3 in Hexaploid Wheat (Triticum aestivum L.) Alters Plant
Development and Freezing Tolerance
Malika Ayadi, Faiçal Brini and Khaled Masmoudi

Overexpression of a Wheat Aquaporin Gene, TdPIP2;1, Enhances Salt and Drought Tolerance
in Transgenic Durum Wheat cv. Maali
Madhav Bhatta, P. Stephen Baenziger, Brian M. Waters, Rachana Poudel, Vikas Belamkar,
Jesse Poland and Alexey Morgounov
Genome-Wide Association Study Reveals Novel Genomic Regions Associated with 10 Grain
Minerals in Synthetic Hexaploid Wheat
Ryo Nishijima, Kentaro Yoshida, Kohei Sakaguchi, Shin-ichi Yoshimura, Kazuhiro Sato and
Shigeo Takumi
RNA Sequencing-Based Bulked Segregant Analysis Facilitates Efficient D-genome Marker
Development for a Specific Chromosomal Region of Synthetic Hexaploid Wheat
v
Jinghuang Hu, Jingting Li, Peipei Wu, Yahui Li, Dan Qiu, Yunfeng Qu, Jingzhong Xie,
Hongjun Zhang, Li Yang, Tiantian Fu, Yawei Yu, Mengjuan Li, Hongwei Liu, Tongquan Zhu,
Yang Zhou, Zhiyong Liu and Hongjie Li
Development of SNP, KASP, and SSR Markers by BSR-Seq Technology for Saturation of Genetic
Linkage Map and Efficient Detection of Wheat Powdery Mildew Resistance Gene Pm61
Harsimardeep S. Gill, Chunxin Li, Jagdeep S. Sidhu, Wenxuan Liu, Duane Wilson, Guihua
Bai, Bikram S. Gill and Sunish K. Sehgal
Fine Mapping of the Wheat Leaf Rust Resistance Gene Lr42
Kateřina Perničková, Veronika Koláčková, Adam J. Lukaszewski, Chaolan Fan, Jan Vrána,
Martin Duchoslav, Glyn Jenkins, Dylan Phillips, Olga Šamajová, Michaela Sedlářová, Jozef
Šamaj, Jaroslav Doležel and David Kopecký
Instability of Alien Chromosome Introgressions in Wheat Associated with Improper Positioning
in the Nucleus
Haimei Du, Zongxiang Tang, Qiong Duan, Shuyao Tang and Shulan Fu
Using the 6RLKu Minichromosome of Rye (Secale cereale L.) to Create Wheat-Rye 6D/6RLKu
Small Segment Translocation Lines with Powdery Mildew Resistance
Yinping Liang, Ye Xia, Xiaoli Chang, Guoshu Gong, Jizhi Yang, Yuting Hu, Madison Cahill,
Liya Luo, Tao Li, Lu He and Min Zhang
Comparative Proteomic Analysis of Wheat Carrying Pm40 Response to Blumeria graminis f. sp.
tritici Using Two-Dimensional Electrophoresis
Cyrine Robbana, Zakaria Kehel, M’barek Ben Naceur, Carolina Sansaloni, Filippo Bassi and
Ahmed Amri
Genome-Wide Genetic Diversity and Population Structure of Tunisian Durum Wheat Landraces
Based on DArTseq Technology
Jianxin Bian, Pingchuan Deng, Haoshuang Zhan, Xiaotong Wu, Mutthanthirige D. L. C.

Nishantha, Zhaogui Yan, Xianghong Du, Xiaojun Nie and Weining Song
Transcriptional Dynamics of Grain Development in Barley (Hordeum vulgare L.)
Veronika Kapustová, Zuzana Tulpová, Helena Toegelová, Petr Novák, Jiřı́ Macas, Miroslava
Karafiátová, Eva Hřibová, Jaroslav Doležel and Hana Šimková
The Dark Matter of Large Cereal Genomes: Long Tandem Repeats
Wei Xi, Zongxiang Tang, Shuyao Tang, Zujun Yang, Jie Luo and Shulan Fu
New ND-FISH-Positive Oligo Probes for Identifying Thinopyrum Chromosomes in
Wheat Backgrounds
vi
About the Special Issue Editor
Manuel Martinez is a senior researcher in the Plant Genomics and Biotechnology Center (CBGP). He
received a PhD from the Universidad Complutense de Madrid working in cereal cytogenetics (1999).
Then, he moved to the Biotechnology Department at the Universidad Politécnica de Madrid, with a
Post-Doc fellowship from 1999 to 2002 to work in plant molecular biology. At the same Institution, he
received a Ramón y Cajal research contract in 2003, a contract as Assistant Professor from December
2005, and a permanent contract as Associate Professor in 2012. In September 2008, he moved to the
Plant Genomics and Biotechnology Center to carry out his research activity. At present, he belongs
to the UPM research group “plant-pest molecular interactions”. His research activity is focused
on the molecular characterization of defense genes against pathogens and pests, the relationships
between proteases and their inhibitors with senescence and germination mechanisms in cereals, and
the bioinformatic analysis of the evolution and composition of gene families. He has coauthorized
more than 70 research articles in international journals and participates as Guarantor Researcher in the
Severo Ochoa grant awarded by the CBGP. Currently, he carries out his teaching activity in different
courses related to plant biotechnology and bioinformatics.
vii
Molecular Sciences
Editorial
Editorial for Special Issue “Molecular Advances in
Wheat and Barley”
Manuel Martinez 1,2
1 Centro de Biotecnologia y Genomica de Plantas (CBGP, UPM-INIA), Universidad Politecnica de,
Madrid (UPM)- Instituto Nacional de Investigacion y Tecnología Agraria y Alimentaria (INIA), Campus,
Montegancedo, Pozuelo de Alarcon, 28223 Madrid, Spain; [email protected]; Tel.: +34-910679149
2 Departamento de Biotecnologia-Biologia Vegetal, Escuela Tecnica Superior de Ingenieria Agronomica,
Alimentaria y de Biosistemas, UPM, 28040 Madrid, Spain
Received: 10 July 2019; Accepted: 15 July 2019; Published: 16 July 2019
Along with maize and rice, allohexaploid bread wheat and diploid barley are the most cultivated
crops in the world (FAOSTAT database, http://www.fao.org/faostat, accessed on 22 June 2019). Their
economic importance and close relationship supports a parallel study of both cereals. Nowadays,
analyses based on high-throughput sequencing have become a key approach in genome-wide biology
for crop improvement. Advances in genomics have resulted in the development of new technologies
and strategies that give support to experimental research. In this context, the release of the genomic
sequences of wheat and barley has permitted the application of genome-scale approaches, such as those
related to metabolomics, proteomics, transcriptomics, and phenomics analyses. Additionally, new tools
for gene identification, such are Genome-Editing and Genome-Wide Association Studies, are being
developed. Modern research based in this new technological scenario is focused on understanding
regulatory systems in order to improve crop productivity. The final goal should be the functional
genomic analysis of genes and regulatory networks that control important agronomic traits and
biological processes, such as yield, grain quality, disease and pest resistance, nutrient-use efficiency,
and abiotic stress resistance. This Special Issue aimed to report novel molecular research and reviews
related to wheat and barley biology using these new technologies. The Special Issue presents a total of
18 articles (Table 1).
Five articles are reviews covering different aspects of both wheat and barley crops. Two of these
reviews are focused on understanding the role of phytases and proteases in the grain using novel
technologies with an agronomical goal. In the case of phytases, single-stomached animals and humans
depend on phytase supplied through the diet to hydrolyze phytate and make associated nutrients, such
as phosphorous, iron, and zinc, bioavailable. This review highlights advances in the understanding
of the molecular basis of the phytase activity and how understanding the function and regulation
of the PAPhy_a gene may support the development of improved wheat and barley with even higher
phytase activity [1]. Proteases are crucial for the continuous release of nutrients from the endosperm
to the embryo to achieve the correct development of the new plant and to avoid agronomical losses
due to the absence of seed germination. Many advances have been made in understanding the role of
proteases in the grain due to their potential value for the brewing industry and their relationship with
celiac disease. Novel technologies have permitted the application of genome-scale approaches, such as
those used in functional genomics and proteomics, to increase the repertoire and knowledge on the
barley and wheat proteases involved in germination [2].
Int. J. Mol. Sci. 2019, 20, 3501; doi:10.3390/ijms20143501 1 www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2019, 20, 3501
Table 1. Contributors to the Special Issue “Molecular Advances in Wheat and Barley”.
Publication Species Topic/Molecular Advance Reference

Phytases in grains Madsen et al. [1]
Proteases in grain germination Martinez et al. [2]
Review Wheat/barley Cas endonuclease technology Koeppel et al. [3]
Resistance to nematodes Ali et al. [4]
Host-induced gene silencing Qi et al. [5]
Over-expression of genes for abiotic resistance Pigolev et al. [6]
Over-expression of genes for abiotic resistance Ayadi et al. [7]
GWAS for minerals in grains Bhatta et al. [8]
Bulked segregant analyses for markers Nishijima et al. [9]
BSR-seq for biotic resistance markers Hu et al. [10]
Wheat
Fine-mapping of biotic resistance genes Gill et al. [11]
Article
FISH for instability of alien introgressions Perničková et al. [12]
Wheat–rye small translocations for biotic resistance Du et al. [13]
Proteomic analysis in biotic resistant lines Liang et al. [14]
DArTseq technology for population structure Robbana et al. [15]
Barley Transcriptomics in grain development Bian et al. [16]
Wheat/barley Sequencing of Long tandem repeats Kapustová et al. [17]
Brief report Wheat ND-FISH probes for chromosome identification Xi et al. [18]
GWAS: genome-wide association study; BSR-seq: bulked segregant analysis-RNA-Seq; FISH: fluorescence in
situ hybridization; DArTseq: diversity array technology sequencing; ND-FISH: non-denaturing fluorescence in
situ hybridization.
Three reviews are related to the development of novel techniques with a strong potential to be used
as biotechnological tools, and their specific use against cereal cyst nematodes. One of these reviews
explores the possibilities of the newly emerging Cas endonuclease technology, which allows for the
induction of mutations at user-defined positions in the plant genome. Current trends in the development
of this technology and its biotechnological application in wheat and barley are reviewed [3]. Likewise,
recent studies on host-induced gene silencing (HIGS) technology employing RNA silencing mechanisms
in wheat and barley are reviewed [5]. RNA silencing mechanisms provide a transgenic approach
for disease management. This approach has been successfully applied in crop disease prevention by
silencing the targets of invading pathogens, being a valuable tool to protect wheat and barley from
diseases in an environmentally friendly way. Finally, the use of modern tools for the enhancement of
cereal cyst nematode resistance in wheat and barley is examined [4]. Besides genome-wide association
studies, the application of various transgenic strategies has been exploited, including host-induced
gene silencing, nematode effector genes, proteinase inhibitors, or chemodisruptive peptides, with an
emphasis on the future applicability of Cas endonuclease technology.
Research articles cover most of the modern molecular approaches used to further advance wheat
and barley knowledge. Two articles are focused on transgenic engineering. The overexpression in wheat
of the Arabidopsis AtOPR3 gene, one of the key genes in the jasmonic acid (JA) biosynthesis pathway,
affected wheat development and altered tolerance to environmental stresses [6]. Transgenic durum
wheat overexpressing the wheat plasma membrane aquaporin TdPIP2;1 gene exhibited improved
germination rates and biomass production and retained low Na+ and high K+ concentrations in their
shoots under high salt and osmotic stress conditions [7].
Four articles tried to identify single nucleotide polymorphism (SNP) markers in order to perform
molecular marker-assisted selection in wheat breeding. Synthetic hexaploid wheat was used to quantify
10 grain minerals by an inductively coupled mass spectrometer for a genome-wide association study
(GWAS). For this analysis, 92 marker-trait associations (MTAs) were identified, of which 60 were
novel and 40 were within genes, and the genes underlying 20 MTAs had annotations suggesting
a potential role in grain mineral concentration [8]. Likewise, synthetic hexaploid wheat lines were
used to perform RNA sequencing (RNA-seq)-based bulked segregant analysis (BSA). This analysis
permitted the identification of several SNP markers around the Net2 gene, a causative locus to hybrid
necrosis [9]. The bulked segregant analysis-RNA-Seq technique was also used to find new single
2
Int. J. Mol. Sci. 2019, 20, 3501
SNPs, kompetitive allele specific polymorphisms (KASPs), and simple sequence repeat (SSR) markers
to saturate the genetic linkage map for Pm61, a gene that confers powdery mildew resistance. The
newly saturated genetic linkage map will be useful in molecular marker-assisted selection of Pm61
in breeding for disease-resistant cultivars [10]. With a similar goal, the gene Lr42, which confers
effective resistance against leaf rust, was fine-mapped by using recombinant inbred lines (RILs). The
identified region included nine nucleotide-binding domain leucine-rich repeat genes, and two KASP
markers flanking Lr42 were developed to facilitate marker-assisted selection for rust resistance in
wheat breeding programs [11].
In three papers, chromosomal imaging techniques were used. Somatic nuclei of wheat with
rye introgressions were analyzed by tridimensional fluorescence in situ hybridization (3D-FISH).
While introgressed rye chromosomes or chromosome arms occupied discrete positions similar to
chromosomes of the wheat host, their telomeres frequently occupied improper positions. This feature
probably impacts the ability of introgressed chromosomes to migrate into the telomere bouquet at
the onset of meiosis, leading to their gradual elimination over generations [12]. Non-denaturing
fluorescence in situ hybridization (ND-FISH) was used to identify small segment translocations
after irradiation in a wheat-rye 6RLKu minichromosome addition line. A translocated chromosome
6DL/6RLKu included the powdery mildew resistance from rye, supporting the practical utilization
of the resistance gene on 6RLKu [13]. Additionally, ND-FISH technology provided suitable positive
oligo probes for distinguishing alien Thinopyrum chromosomes in wheat backgrounds [18]. These
oligo probes could be a convenient tool for the utilization of Thinopyrum germplasms in wheat
breeding programs.
Finally, four articles are good examples of the suitability of advanced molecular techniques to delve
into different wheat and barley issues. Proteomics techniques led to the identification of proteins that
were up- and downregulated after powdery mildew inoculation of the wheat line L699, which includes
the Pm40 resistance gene. The identified proteins were predicted to be associated with the defense
response as well as with other physiological processes [14]. The transcriptional dynamics of barley
grain development was investigated through RNA sequencing at four developmental time points.
Transcriptome profiling found notable shifts in the abundance of transcripts involved in both primary
and secondary metabolism during grain development and highlighted the existence of numerous
RNA editing events [16]. Population genetics on durum wheat lines were assessed using diversity
array technology sequencing (DArTseq). Cluster analysis and discriminant analysis of principal
components allowed five distinct groups to be distinguished, thus supporting the importance of
genomic characterization for enhancing knowledge on population structure [15]. Genomic sequencing
was also used to identify missing tandemly organized repetitive sequences in wheat and barley
genomes, which are underrepresented in genome assemblies generated from short-read sequence data.
The authors demonstrated that this missing information may be added to the pseudomolecules with
the aid of nanopore sequencing of individual bacterial artificial chromosome (BAC) clones and optical
mapping [17].
Overall, the 18 contributions published in this Special Issue (Table 1) illustrate research advances
in wheat and barley knowledge using modern molecular techniques. These molecular approaches
at genomic, transcriptomic, proteomic, and phenomic levels, together with new tools for gene
identification and the development of new molecular markers, have contributed to developing a
further understanding of regulatory systems in order to improve wheat and barley performance. In the
near future, the development of novel techniques will permit us to increase our knowledge on the
regulation of important agronomic traits, which will facilitate the breeding of improved wheat and
barley varieties.
3
Int. J. Mol. Sci. 2019, 20, 3501
Acknowledgments: The financial support from the Ministerio de Ciencia y Universidades of Spain (project
BIO2017-83472-R) is gratefully acknowledged.
Conflicts of Interest: The author declares no conflict of interest.
References
1. Madsen, C.K.; Brinch-Pedersen, H. Molecular Advances on Phytases in Barley and Wheat. Int. J. Mol. Sci.
2019, 20, 2459. [CrossRef] [PubMed]
2. Diaz-Mendoza, M.; Diaz, I.; Martinez, M. Insights on the Proteases Involved in Barley and Wheat Grain
Germination. Int. J. Mol. Sci. 2019, 20, 2087. [CrossRef] [PubMed]
3. Koeppel, I.; Hertig, C.; Hoffie, R.; Kumlehn, J. Cas Endonuclease Technology-A Quantum Leap in the
Advancement of Barley and Wheat Genetic Engineering. Int. J. Mol. Sci. 2019, 20, 2647. [CrossRef] [PubMed]
4. Ali, M.A.; Shahzadi, M.; Zahoor, A.; Dababat, A.A.; Toktay, H.; Bakhsh, A.; Nawaz, M.A.; Li, H. Resistance to
Cereal Cyst Nematodes in Wheat and Barley: An Emphasis on Classical and Modern Approaches. Int. J.
Mol. Sci. 2019, 20, 432. [CrossRef] [PubMed]
5. Qi, T.; Guo, J.; Peng, H.; Liu, P.; Kang, Z. Host-Induced Gene Silencing: A Powerful Strategy to Control
Diseases of Wheat and Barley. Int. J. Mol. Sci. 2019, 20, 206. [CrossRef] [PubMed]
6. Pigolev, A.V.; Miroshnichenko, D.N.; Pushin, A.S.; Terentyev, V.V.; Boutanayev, A.M.; Dolgov, S.V.;
Savchenko, T.V. Overexpression of Arabidopsis OPR3 in Hexaploid Wheat (Triticum aestivum L.) Alters Plant
Development and Freezing Tolerance. Int. J. Mol. Sci. 2018, 19, 3989. [CrossRef] [PubMed]
7. Ayadi, M.; Brini, F.; Masmoudi, K. Overexpression of a Wheat Aquaporin Gene, TdPIP2;1, Enhances Salt
and Drought Tolerance in Transgenic Durum Wheat cv. Maali. Int. J. Mol. Sci. 2019, 20, 2389. [CrossRef]
[PubMed]
8. Bhatta, M.; Baenziger, P.S.; Waters, B.M.; Poudel, R.; Belamkar, V.; Poland, J.; Morgounov, A. Genome-Wide
Association Study Reveals Novel Genomic Regions Associated with 10 Grain Minerals in Synthetic Hexaploid
Wheat. Int. J. Mol. Sci. 2018, 19, 3237. [CrossRef] [PubMed]
9. Nishijima, R.; Yoshida, K.; Sakaguchi, K.; Yoshimura, S.I.; Sato, K.; Takumi, S. RNA Sequencing-Based Bulked
Segregant Analysis Facilitates Efficient D-genome Marker Development for a Specific Chromosomal Region
of Synthetic Hexaploid Wheat. Int. J. Mol. Sci. 2018, 19, 3749. [CrossRef] [PubMed]
10. Hu, J.; Li, J.; Wu, P.; Li, Y.; Qiu, D.; Qu, Y.; Xie, J.; Zhang, H.; Yang, L.; Fu, T.; et al. Development of SNP, KASP,
and SSR Markers by BSR-Seq Technology for Saturation of Genetic Linkage Map and Efficient Detection of
Wheat Powdery Mildew Resistance Gene. Int. J. Mol. Sci. 2019, 20, 750. [CrossRef] [PubMed]
11. Gill, H.S.; Li, C.; Sidhu, J.S.; Liu, W.; Wilson, D.; Bai, G.; Gill, B.S.; Sehgal, S.K. Fine Mapping of the Wheat
Leaf Rust Resistance Gene Lr42. Int. J. Mol. Sci. 2019, 20, 2445. [CrossRef]
12. Perničková, K.; Koláčková, V.; Lukaszewski, A.J.; Fan, C.; Vrána, J.; Duchoslav, M.; Jenkins, G.; Phillips, D.;
Šamajová, O.; Sedlářová, M.; et al. Instability of Alien Chromosome Introgressions in Wheat Associated with
Improper Positioning in the Nucleus. Int. J. Mol. Sci. 2019, 20, 1448. [CrossRef]
13. Du, H.; Tang, Z.; Duan, Q.; Tang, S.; Fu, S. Using the 6RLKu Minichromosome of Rye (Secale cereale L.) to
Create Wheat-Rye 6D/6RLKu Small Segment Translocation Lines with Powdery Mildew Resistance. Int. J.
Mol. Sci. 2018, 19, 3933. [CrossRef] [PubMed]
14. Liang, Y.; Xia, Y.; Chang, X.; Gong, G.; Yang, J.; Hu, Y.; Cahill, M.; Luo, L.; Li, T.; He, L.; et al.
Comparative Proteomic Analysis of Wheat Carrying Pm40 Response to Blumeria graminis f. sp. tritici
Using Two-Dimensional Electrophoresis. Int. J. Mol. Sci. 2019, 20, 933. [CrossRef] [PubMed]
15. Robbana, C.; Kehel, Z.; Ben Naceur, M.; Sansaloni, C.; Bassi, F.; Amri, A. Genome-Wide Genetic Diversity and
Population Structure of Tunisian Durum Wheat Landraces Based on DArTseq Technology. Int. J. Mol. Sci.
16. Bian, J.; Deng, P.; Zhan, H.; Wu, X.; Nishantha, M.D.L.C.; Yan, Z.; Du, X.; Nie, X.; Song, W. Transcriptional
Dynamics of Grain Development in Barley (Hordeum vulgare L.). Int. J. Mol. Sci. 2019, 20, 962. [CrossRef]
[PubMed]
4
Int. J. Mol. Sci. 2019, 20, 3501
17. Kapustová, V.; Tulpová, Z.; Toegelová, H.; Novák, P.; Macas, J.; Karafiátová, M.; Hřibová, E.; Doležel, J.;
Šimková, H. The Dark Matter of Large Cereal Genomes: Long Tandem Repeats. Int. J. Mol. Sci. 2019, 20,
2483. [CrossRef] [PubMed]
18. Xi, W.; Tang, Z.; Tang, S.; Yang, Z.; Luo, J.; Fu, S. New ND-FISH-Positive Oligo Probes for Identifying
Thinopyrum Chromosomes in Wheat Backgrounds. Int. J. Mol. Sci. 2019, 20, 2031. [CrossRef] [PubMed]
© 2019 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/).
5
Molecular Sciences
Review
Molecular Advances on Phytases in Barley and Wheat
Claus Krogh Madsen and Henrik Brinch-Pedersen *
Department of Molecular Biology and Genetics, Research Center Flakkebjerg, Aarhus University, 4200-Slagelse,
Denmark; [email protected]
* Correspondence: [email protected]
Received: 20 March 2019; Accepted: 15 May 2019; Published: 18 May 2019
Abstract: Phytases are pro-nutritional enzymes that hydrolyze phytate and make associated nutrients,
such as phosphorous, iron, and zinc, bioavailable. Single-stomached animals and humans depend on
phytase supplied through the diet or the action of phytase on the food before ingestion. As a result,
phytases—or lack thereof—have a profound impact on agricultural ecosystems, resource management,
animal health, and public health. Wheat, barley and their Triticeae relatives make exceptionally good
natural sources of phytase. This review highlights advances in the understanding of the molecular
basis of the phytase activity in wheat and barley, which has taken place over the past decade. It is
shown how the phytase activity in the mature grains of wheat and barley can be ascribed to the
PAPhy_a gene, which exists as a single gene in barley and in two or three homeologous copies
in tetra- and hexaploid wheat, respectively. It is discussed how understanding the function and
regulation of PAPhy_a may support the development of improved wheat and barley with even higher
phytase activity.
Keywords: phytase; wheat; barley; purple acid phosphatase phytase; PAPhy; mature grain phytase
activity (MGPA)
1. Introduction
Phytases (myo-inositol hexakisphosphate 3-,6- and 5-phosphohydrolase, EC 3.1.3.8, EC 3.1.3.26
and EC 3.1.3.72) are phosphatases that can initiate the stepwise hydrolysis of phytate (IP6,
myoinositol-(1,2,3,4,5,6)-hexakisphosphate) and thereby provide phosphate (P), inositol phosphates,
and inositol for a range of cellular activities [1]. In addition to purely scientific inquiries, phytase research
has for many years been driven by the urgent need for improving utilization of phytate-phosphorus in
diets for single-stomached animals, such as pigs and poultry, and to reduce the anti-nutritional effect
of non-digested IP6 chelating micronutrients in the digestive tracts of humans and animals. As such,
phytases can be regarded as tools for managing global phosphate resources and for alleviating human
micro-nutrient deficiencies mainly in the developing world.
IP6 is the main storage form of phosphate in plants, typically amounting 2/3 of the total P
content in the seed (Table 1). IP6 is a strong chelator and exists in the plant seeds as an insoluble
mixed salt with cations called phytin. In cereals and many other plant seeds, phytin forms spherical
crystalloid inclusions called globoids inside protein storage vacuoles. The globoids are the principal
site of phosphorous (P), potassium (K) and magnesium (Mg) in the mature cereal grain but they also
contain calcium (Ca), iron (Fe), zinc (Zn), copper (Cu), manganese (Mn), sodium (Na), sulfur (S),
and protein [2,3].

Int. J. Mol. Sci. 2019, 20, 2459
Table 1. Total P, IP6 bound P, proportion of IP6 bound P, and phytase activity in wheat, barley and
other cereals seeds. The number of samples is n and ± denotes the standard deviation. The range is
given if n ≥ 2. Data from [4] 1 , [5] 2 , [6] 3 .
Total P Total IP6 P Percent IP6 out Phytase Activity

Cereal n
(% of Dry Matter) (% of Dry Matter) of Total P (FTU/kg) *
Wheat 1 13 0.33 ± 0.02 0.22 ± 0.02 67 ± 4.8 1193 ± 223
Wheat 2 18 0.40 ± 0.04 0.29 ± 0.04 73 ± 8.1 2886 ± 645
Wheat 3 30 0.29 ± 0.03 0.23 ± 0.03 79 ± 0.07 1637 ± 275
Barley 1 9 0.37 ± 0.02 0.22 ± 0.01 60 ± 2.4 582 ± 178
Barley 2 15 0.42 ± 0.4 0.26 ± 0.03 63 ± 3.5 2323 ± 648
Barley 3 21 0.31 ± 0.03 0.19 ± 0.02 61 ± 0.04 1016 ± 330
Rye 1 2 0.36 (0.35-0.36) 0.22 (0.20-0.23) 61 ± (56 -66) 5130 (4132-6127)
Rye 2 13 0.36 ± 0.02 0.24 ± 0.2 67 ± 5.0 6016 ± 1578
Rye 3 6 0.34 ± 0.03 0.20 ± 0.01 59 ± 0.02 5147 ± 649
Triticale 1 6 0.37 ± 0.02 0.25 ± 0.02 67 ± 3.7 1688 ± 227
Triticale 2 12 0.40 ± 0.03 0.28 ± 0.03 70 ± 5.4 2799 ± 501
Oats 1 6 0.36 ± 0.03 0.21 ± 0.04 59 ± 11 42 ± 50
Oats 2 6 0.37 ± 0.01 0.25 ± 0.02 67 ± 5.4 496 ± 35
Oats 3 9 0.29 ± 0.02 0.17 ± 0.03 59 ± 0.07 84 ± 39
Maize 1 11 0.28 ± 0.03 0.19 ± 0.03 68 ± 5.9 15 ± 18
Maize 3 7 0.32 ± 0.01 0.18 ± 0.01 78 ± 0.01 70 ± 7
Rice 4 1 72
* One FTU is the amount of enzyme that liberates 1 μmol of inorganic phosphorus per minute from sodium phytate
at pH 5.5 and 37 ◦ C.
Micronutrients are also chelated by phytate in food and feed, and hydrolysis is most wanted for
improving micronutrient bioavailability. The anti-nutritional effect of phytate is in particular regarded
as critical for Fe and Zn, where phytate is considered the single most important anti-nutritional
compound for the bioavailability of these two micronutrients [7]. Iron deficiency is the primary cause
of anemia and ranks among the most widespread nutrient deficiencies, estimated to affect 1.6 billion
people worldwide [8]. Iron deficiency anemia has been linked to maternal and prenatal mortality,
and to impairment of cognitive skills and physical activity [9]. For zinc, around 800,000 child deaths
worldwide per year are attributable to Zn deficiency [10] because it significantly increases the risk of
diarrhea, pneumonia, and malaria. Moreover, Zn deficiency has been linked to the morbidity and
mortality of children younger than five [11].
Humans and single-stomached animals have insufficient phytase activity in their digestive tract
unless it is provided by the diet. Unfortunately, major food and feed components like rice, maize and
soybeans contribute with phytate but negligible phytase [4]. Because of the missing phytase activity,
the phytate passes largely un-digested through the single-stomached animals’ digestive tract and enters
the environment when their manure is spread on agricultural fields. Moreover, to ensure that farm
animals get the phosphate needed, bio-available mined P is added to the feed. However, this strategy
has become critical in many regions of the world where intense livestock production and spreading of
manure with high levels of undigested phytate P on oversupplied agricultural soil leads to run-off of
phosphorus to aquatic ecosystems. The resulting eutrophication is a severe environmental risk [12].
However, also from a resource perspective, inefficient utilization of plant phytate P is inappropriate.
P is a non-renewable resource, essential for efficient agricultural production, and complete depletion
of mined P will have unmanageable consequences for global food production [13].
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Int. J. Mol. Sci. 2019, 20, 2459
2. Plant and Microbial Phytases

IP6 is resistant to most phosphatases whereas the lower inositol phosphates can be degraded by
a wider range of phosphatases. Phosphatases that can initiate the dephosphorylation of IP6 are classified
as phytases. So far, four classes of phytases have been identified: (1) Histidine acid phosphatase (HAP),
(2) purple acid phosphatase (PAP), (3) cysteine phosphatase (CP) and (4) β-propeller phytase (BPP).
Each phytase type has unique structural features due to their distinct catalytic apparatus that allows
them to utilize phytate as a substrate in various environments [14].
For decades, applied phytase research was focusing mainly on microbial phytases and to our
knowledge, all commercial phytases currently used for feed supplementation are microbial enzymes
belonging to the HAP class [14]. Similarly, until recently, microbial HAP phytases were used exclusively
for increasing plant seeds phytase activity through transgenesis. However, scientific achievements in
recent years have led to a substantially increased knowledge based on the complements of phytases,
in particular barley and wheat, and have demonstrated significant potentials of their phytases as highly
stable and potent enzymes with potentials both in feed and food (see later).
3. Mature Grain Phytase Activity

When hydrated, the mature seed tissues activate a battery of preformed hydrolytic enzymes
that degrades the large internal pool of IP6 but also storage compounds like lipids, carbohydrates,
and proteins. When ungerminated seeds are used as feedstuffs, this battery of enzymes constitutes
all plant-derived hydrolytic activities. We refer to this as the first wave of activity and for phytase,
it constitutes what is called the mature grain phytase activity (MGPA). In parallel with imbibition,
the embryo synthesizes and secretes the plant hormone gibberellic acid. The aleurone and the scutellum
layer of the embryo are thereby turned into secretory tissues where a wide range of hydrolytic enzymes
are synthesized and secreted into the endosperm for degradation of cell walls, starch grains and storage
proteins—the second wave of hydrolysis [2].
Cereals generally express phytases to assist in their IP6 metabolism but the MGPA varies several
orders of magnitude. This is in strong contrast to the modest variation in total and proportional content
of seed IP6 (Table1).
4. Classes of Phytases in Barley and Wheat

In wheat and barley, two types of phytases have been described, phytases belonging to the HAP
class and phytases belonging to the PAP class of phosphatases [1]. The HAP phytases belong to the
multiple inositol polyphosphate phosphatase (MINPP) group [15]. MINPP phytases have been reported
to be expressed both during grain development, and thereby, potentially contribute to the MGPA,
and during germination contributing to the second wave of phytate hydrolysis. The PAP phytases
are represented by the TaPAPhy_a/bs from wheat and the HvPAPhy_a/bs from barley, respectively.
Expression analysis showed that PAPhy_a genes are preferentially expressed during grain filling
whereas the PAPhy_b genes are preferentially expressed during germination [16]. The Km value with
phytate as a substrate for recombinant wheat MINPP rTaPhyIIa2 phytase and barley rHvPhyIIb phytase
is around ten-fold higher than for the rTaPAPhy_a/b and rHvPAPhy_a/b PAP phytases, indicating
PAPhys to be more potent phytases than the HAPhys (Table 2).
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Int. J. Mol. Sci. 2019, 20, 2459
Table 2. Kinetic parameters from HAP and PAPhy phytases from wheat, barley, and Aspergillus ficuum.
Data from 1 [15], 2 [16], 3 [17].
Km Vmax Kcat Kcat/Km pH

Class Enzyme
(μM) (μmol/(min × mg)) (s−1 ) (s−1 M−1 ) Optimum
rTaPAPhy_a1 2 35 223 279 796 × 104 5.5
PAP rTaPAPhy_b1 2 45 216 270 600 × 104 5
Phytases rHvPAPhy_a 2 36 208 260 722 × 104
rHvPAPhy_b 2 46 202 253 550 × 104
rTaPhyIIa2 1 246 4.5
HAP
Phytases rHvPhyIIb 1 334 4.5
A. ficuum
27 - 348 129 × 105 5.5
phytase 3
The contribution of HvPAPhy_a and HvPAPhy_b to the total MGPA in barley was recently
evaluated using CRISPR/Cas and TALEN [18]. TALEN- and CRISPR/Cas9 were used for introducing
targeted mutations in the promoter of the barley phytase gene HvPAPhy_a. Barley lines with substantial
deletions in the HvPAPhy_a promoter and 5’CDS retained <5% normal MGPA. This confirms that the
barley PAPhy_a enzyme is the main contributor to the MGPA and can be regarded as the main target
for modulating MGPA.
5. Biochemical Properties and Storage of the PAPhys

Wheat and barley mainly store phytate in the protein storage vacuoles (PSVs) of the aleurone layer.
PAPhy accumulated during grain filling is localized in the same organelles [16]. This suggests that some
mechanism protects the phytate from hydrolysis during grain filling. The PSV’s are rapidly acidified
in response to gibberellic acid as germination commences. A decrease from pH 6.6 to 5.9 was reported
in the PSV’s of barley protoplasts incubated with 5 μM GA3 and the authors speculated that pH might
play a crucial role in regulating vacuolar hydrolases [19]. Recombinant wheat phytases rTaPAPhy_a1
and rTaPAPhy_b1 showed pH optima of 5.5 and 5, respectively [16]. Optima of pH 5 and 6 were
measured for seed purified barley phytases P1 (=HvPAPhy_b) and P2 (=HvPAPhy_a) respectively [20].
This shows that the PAPhy’s are most active when the PSV is in the acidified lytic, state and the higher
pH optimum of preformed PAPhy_a may even be an adaptation, which enhances activity in the earliest
stages of germination. Nevertheless, rTaPAPhy_a retained some activity up to pH 7.5 so pH regulation
of the enzyme alone does not offer a satisfying explanation for the protection of phytate against
premature hydrolysis. The second layer of pH-dependent protection is provided by the substrate’s
organization into globoids, which provides some degree of water exclusion and steric hindrance [3].
A membrane surrounds the globoids and immuno-gold localization suggests that PAPhy is located
outside this membrane [16,21]. The membrane, therefore, seems to provide an additional layer of
protection, by physical separation. The temperature optimum of the recombinant wheat enzymes was
50 and 55 ◦ C, respectively. The temperature curves showed a broad peak with 50% activity already at
30–35 ◦ C but decreasing sharply around 60 ◦ C. Optima of 55 and 45 ◦ C were reported for the seed
purified HvPAPhy_a and HvPAPhy_b respectively [20]. The kinetic parameters of recombinant wheat
and barley phytases at 36 ◦ C and pH 5 are summarized in Table 2. The corresponding values for
Aspergillus ficuum phytase are given for comparison [17]. The values for the PAPhys are very similar.
6. PAPhy Genetics
The PAPhy_a and PAPhy_b genes are paralogs, which originate from gene duplication in a common
ancestor of wheat, barley, and rye (i.e., the Triticeae tribe) [22]. Rice, maize, and sorghum diverged from
the Triticeae earlier and carry only one PAPhy gene whereas Brachypodum distachyon has the duplication
but lack the conserved PAPhy_a promoter of the Triticeae (Figure 1) [22]. Allopolyploidzation has
united the A and B genomes to form tetraploid wheat (e.g., durum wheat). Triticum urartu and
9
Int. J. Mol. Sci. 2019, 20, 2459
Aegilops speltoides are the closest living relatives of the A and B genomes, respectively. Additional
hybridizations have added the Ae. tauschii derived D genome to produce hexaploid wheat (bread
wheat, spelt) and the rye derived R genome to produce triticale (Figure 1) [23]. Allopolyploidization
results in large-scale gene duplication because most genes in one parent will have a homolog in the
other parent species. In allopolyploids, such sets of genes are termed homeologs. Some homeologs
may be lost or translocated as the polyploid species continues to evolve but the Triticeae PAPhy gene
copy number and chromosomal localization are highly conserved. A single locus of PAPhy_a and
PAPhy_b resides on chromosome 5 and 3, respectively in barley and on the homologous chromosomes
on the three subgenomes of wheat [22]. The sequenced diploid members of the Triticum and Aegilops,
i.e., Ae. tauschii, Ae. speltoides, Ae. sharonensis, T. Urartu, and T. monococcum also have one PAPhy_a and
one PAPhy_b [24]. The chromosomal localization in these relatives has not been determined but it is
reasonable to expect a conserved synteny between wheat and its ancestors T. urartu, Ae. Speltoides,
and Ae. tauschii. Secale provides an exception since some members of this tribe have two PAPhy_a loci.
In the case of domesticated rye, one and two PAPhy_a variants were isolated from the cultivars Imperial
and Picasso, respectively [25]. Rye is an outbreeding species, unlike wheat and barley, and tends to
have higher allele heterogeneity. Therefore, it cannot be excluded that the two variants from Picasso are
alleles of the same gene even though phylogeny suggests that one allele may have been introgressed
from S. strictum [25]. Thus, it is not known with certainty if domesticated rye has one or two PAPhy_a
loci or whether it is cultivar-dependent.
Figure 1. Key events in PAPhy evolution. Gene duplication and divergence of the PAPhy_a/b promoters
and further duplications through polyploidization. For simplicity, rye is assumed to have just one
PAPhy_a locus, and phylogenetic distance is not drawn to scale.
The intron/exon structure and the respective promoters of PAPhy_a and PAPhy_b are highly
conserved. PAPhy_a has four introns and PAPhy_b has five. The position and, to a large extent, the size
of the introns is conserved between Triticeae species and between the two paralogs [22]. Both genes have
a core promoter of 3–400 base pairs which is conserved in all studied Triticeae. Both promoters have two
TATA-boxes and upstream of those reside cis-acting regulatory elements consistent with the differential
expression pattern of the two genes (Figure 2). For PAPhy_b, they are ABRE (abscisic-acid-responsive),
TGACG (methyl-jasmonate-responsive) and—conserved in all examined PAPhy_b promoters—GARE
(gibberellic acid responsive) [22]. The PAPhy_a promoter lacks these hormone responsive elements,
10
Int. J. Mol. Sci. 2019, 20, 2459
except TGACG which is found at the very beginning of the conserved sequence. Instead, the most
notable feature is a composite element with the consensus 5’ GAACATGAGTCATGCATG 3’ which is
made of the GAMYB binding motif (bold) [26], the odd base palindrome/GCN4 (underlined) [27,28]
and the RY element (italic) [29]. These are all elements associated with seed development and storage
proteins. Between the composite element and the first TATA box is a G-box motif [22]. Deletion of
the odd base palindrome and the RY-element reduce barley MGPA by approximately 40% whereas
deletion of the whole element and an additional 10 base pairs 3’ reduce MGPA by 75%. Deletions
immediately 3’ of the composite element have even more severe effects [14]. It is not clear if this is
caused by an unknown cis-acting regulatory element at that position or by the change in distance
between the composite element and elements further downstream.
Figure 2. Consensus promoters of PAPhy_a and PAPhy_b, respectively, showing the most conserved
cis-acting regulatory elements.
7. Applied Potentials of the PAPhys

Although the PAPhy phytases appear to be somewhat less active than fungal HAP phytases,
they certainly have technological potential. Transformation of barley with a genomic clone of
HvPAPhy_a, including its native promoter (cisgenesis), more than doubled the MGPA [30]. Moreover,
overexpressing HvPAPhy_a using the 35S promoter resulted in a line with impressive 40.000 FTU [31].
This level of MGPA represents more than a 20 fold increase and provides so much activity that <2% of the
recombinant grains in a feed mixture could theoretically replace conventional phytase supplementation.
Moreover, the enzyme activity was highly stable, mature leaves and straw accumulated PAPhy_a and
high phytase activity remained after three years of storage. The PAPhy_a was easily extracted in water
and could be added to feed or other processes.
The efficacy of a phytase in the digestive tract depends in part on how well intrinsic biochemical
parameters, such as pH optimum, match the environment but also on resistance to proteolysis
and inhibition. Feeding experiments are, therefore, necessary to prove efficacy. Early evidence
for the efficacy of PAPhy_a was provided by comparing phosphorous and calcium utilization in
pigs fed maize and triticale, respectively [32]. In this case, triticale-based feed performed better
on both parameters, indicating an effect of the higher MGPA associated with triticale. In humans,
an intervention study compared wheat bran with or without phytase (native vs. heat treated) in
ileostomy patients. This resulted in recoveries of 40% vs. 95% phytate in the ileostomy content [33].
It is also possible to utilize the MGPA to achieve dephytination during food or feed preparation. Up to
96% of the phytate in whole grain wheat bread could be removed by simply adjusting the pH during
proofing to five [34]. Similarly, fonio (Digitaria exilis) porridge could be dephytinized in one hour at
50 ◦ C and pH 5 when 25% of wheat was included. This was demonstrated to significantly increase iron
absorption in West African women [35]. Taken together, these studies demonstrate a lot of potential for
the utilization of endogenous Triticeae PAPhy_a in food and feed.
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8. Achieving Higher MGPA with the PAPhys

Given the high conservation of the PAPhy_a coding sequence and highly similar enzymatic
properties of all examined PAPhys, including PAPhy_b as well as rice and maize PAPhy, it seems
unlikely that alternative PAPhy_a alleles, encoding decisively better enzymes, exist. On the other hand,
increasing PAPhy_a expression has proven a viable strategy, as discussed. Efforts to increase MGPA by
conventional breeding should, therefore, focus on the discovery of more highly expressed PAPhy_a
alleles and the elimination of defective alleles.
Polyploids (e.g., wheat) are prone to acquire defective alleles because homeologs on the other
subgenomes provide functional redundancy. Nevertheless, defective PAPhy_a alleles may reduce
MGPA because the gene copies appear to function in an additive manner as demonstrated by cisgenic
gene duplication [30]. Further support for this hypothesis is provided by the discovery of a defective
TaPAPhy_a2 gene in the wheat cultivars Skagen and Bob White that correlated with a lower MGPA
compared to Chinese Spring, which has three normal loci [22,25]. A similar reduction of phytase activity
has been observed in wheat lines with induced deleterious mutations in the PAPhy_a genes (author’s
unpublished results). It is not known how commonly and how many different defective alleles exist
in the elite wheat gene pool. A systematic investigation of this combined with the development of
markers for the defective alleles would be helpful in ensuring a minimum MGPA in new cultivars.
More active alleles might be found in crop relatives. Wild emmer (T. dicocoides) can be crossed
directly with other tetraploid wheat (e.g., durum wheat) and with hexaploid wheat trough synthetic
hexaploids. Alleles from Ae. tauscii can also be introduced by this route or through the “octo-amphiploid
bridge” [36]. A higher allele diversity is expected for these species because they have not gone through
the bottlenecks of domestication and hexaploidization [37]. A small sampling of wheat and the closest
relatives support this assumption [25]. Furthermore, it was found that at least one exotic allele had
already been introduced in the elite wheat gene pool in the pursuit of other breeding goals—from T.
timopheevii. In addition to the abovementioned, einkorn (wild and domesticated), T. urartu, rye, and Ae.
speltoides could be used but here the introgression is more complicated.
Another strategy is mutagenesis. A wheat mutant with two to three times increased MGPA
has been patented by the authors and co-workers [38]. This “HighPhy” wheat has an SNP in the
composite element discussed above. Incremental replacement (1/3, 2/3, and 3/3) of conventional wheat
with HighPhy all improved calcium and phosphorous digestibility compared to the control diet in
a comprehensive feeding experiment with broiler chicken. Complete replacement with HighPhy
wheat even significantly outperformed a control to which the standard dose of microbial phytase was
added [39].
With the exception of the Triticeae cereals, none of the major food and feed grains accumulate
nutritionally sufficient amounts of phytase during grain development [4,5]. Rather, phytase is
synthesized de novo during germination in response to gibberellic acid. Introducing a transgene driven
by a seed development specific promoter has, therefore, been considered the only realistic approach to
introduce preformed phytase in major grains, such as maize, rice, and soybean. However, the recent
advances in genome editing may change this because they enable targeted modification of endogenous
genes [40]. In theory, this can introduce preformed phytase activity in one of two ways (a) by modifying
the promoter of endogenous phytase genes to become active during grain development or (b) by
modifying the active site of phosphatases already expressed during grain development so they become
phytases. It seems possible to pursue option “a” by combining the lessons of the natural evolution
of the PAPhy_a promoter from the PAPhy_b counterpart with target species-specific information on
cis-acting regulatory elements involved in seed development-specific expression. The pursuit of option
“b” would have to be guided by structural biology but has the advantage that mutations could be
tested in a heterologous system before the more laborious plant gene editing. It is also likely that this
approach would require smaller changes to the target gene sequence because a single base mutation is
enough to change one critical amino acid.
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Int. J. Mol. Sci. 2019, 20, 2459
Proteinaceous inhibitors are known for many hydrolases including amylases, proteases,
and xylanases. To our knowledge, such an inhibitor has never been reported for a phytase of
microbial or plant origin. However, it was recently discovered that barley grain extracts do inhibit
Aspergillus phytase. The inhibition could be reduced by the addition of pepstatin A. This suggests
that the apparent inhibition is caused by an aspartic acid proteolytic activity [41]. It would represent
a new and important variable, if plants use specialized proteases rather than conventional inhibitors to
counteract phytases, e.g., from pathogens.
In conclusion, preformed wheat and barley phytase has the potential to counter the negative
effects of phytate in the nutrition of single-stomached farm animals and humans alike. Realizing this
potential depends on awareness because minor adjustments to processing may be needed. Generally,
these adjustments should ensure that the phytase is not inactivated before ingestion or, alternatively,
that the phytase has optimal conditions to perform the dephytination before inactivation. The higher
the MGPA, the better the chances of a successful dephytination. Characterization of the PAPhy_a gene
over the past decade should be most helpful for plant breeders who make MGPA a priority.
Funding: This research received no external funding.

Conflicts of Interest: The authors are co-inventors of patent WO 2012/146597Al.
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© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
15
Molecular Sciences
Review
Insights on the Proteases Involved in Barley and
Wheat Grain Germination
Mercedes Diaz-Mendoza 1 , Isabel Diaz 1,2 and Manuel Martinez 1,2, *
1 Centro de Biotecnologia y Genomica de Plantas (CBGP, UPM-INIA), Universidad Politecnica de
Madrid (UPM)- Instituto Nacional de Investigacion y Tecnología Agraria y Alimentaria (INIA), Campus
Montegancedo, 28223 Pozuelo de Alarcon, Madrid, Spain; [email protected] (M.D.-M.);
[email protected] (I.D.)
2 Departamento de Biotecnologia-Biologia Vegetal, Escuela Tecnica Superior de Ingenieria Agronomica,
Alimentaria y de Biosistemas, UPM, 28040-Madrid, Spain
* Correspondence: [email protected]; Tel.: +34-910679149
Received: 26 March 2019; Accepted: 24 April 2019; Published: 28 April 2019
Abstract: Seed storage proteins must be hydrolyzed by proteases to deliver the amino acids essential
for embryo growth and development. Several groups of proteases involved in this process have
been identified in both the monocot and the dicot species. This review focuses on the implication
of proteases during germination in two cereal species, barley and wheat, where proteolytic control
during the germination process has considerable economic importance. Formerly, the participation of
proteases during grain germination was inferred from reports of proteolytic activities, the expression
of individual genes, or the presence of individual proteins and showed a prominent role for papain-like
and legumain-like cysteine proteases and for serine carboxypeptidases. Nowadays, the development
of new technologies and the release of the genomic sequences of wheat and barley have permitted the
application of genome-scale approaches, such as those used in functional genomics and proteomics.
Using these approaches, the repertoire of proteases known to be involved in germination has increased
and includes members of distinct protease families. The development of novel techniques based on
shotgun proteomics, activity-based protein profiling, and comparative and structural genomics will
help to achieve a general view of the proteolytic process during germination.
Keywords: barley; wheat; protease; germination; grain
1. Introduction
Barley is considered a model organism for the investigation of the cereal germination process.
Along with maize and rice, allohexaploid bread wheat and diploid barley are the most cultivated crops
in the world (FAOSTAT database, http://www.fao.org/faostat, access on 22 April 2019). Their economic
importance and close relationship support a parallel study of both cereals. The role of plant proteases
in the mobilization of storage proteins that have accumulated in seeds has been largely established in
both the dicotyledonous and the monocotyledonous species [1–3]. Storage proteins must be degraded
to sustain embryo growth and development until an autotrophic growth is reached. Thus, a controlled
proteolysis is crucial for the accurate delivery of amino acids in the initial stages of seed germination.
Several protease families are involved in the germination process. Cysteine proteases (CysProt) of
the C1A family, which are known as papain-like, and the C13 family, alternatively called legumains
or vacuolar processing enzymes (VPEs), are the main proteases involved in the germination of both
dicot and monocot species [1,2,4]. In dicot species, storage proteins are placed in the mesophyll of the
cotyledons and in the embryonic axis. Members of the papain-like, legumain-like, and subtilisin-like
(S8) families have been demonstrated to participate in the breakdown and mobilization of reserve
proteins from seeds to cotyledons during germination [5–7].

Int. J. Mol. Sci. 2019, 20, 2087
Monocot seeds include proteins with many different functions. Around 80% of these proteins
are storage proteins, packed in the endosperm together with starch and lipids. These proteins are
synthesized during grain development and maturation and consequently are involved in germination.
Among the proteases involved in the germination process, CysProt are responsible for around 90%
of the proteolytic activity [8]. Other than CysProt from the papain family (C1A) and the legumain
family (C13), members of the S10 serine carboxypeptidases (SCP) have also been implicated in the
germination process in cereal grains. Papain-like CysProt participating in different stages of the
germination process include the cathepsin L-like proteases identified in rice (oryzains α and β)
and triticale (EP8), the cathepsin H-like proteases (oryzain y) from rice, and the cathepsin B-like
proteases (BdCathB) from Brachypodium distachyon [9–12]. Among legumains, the OsVPE-1 protease
was described in the degradation of stored proteins in the rice grain [13], and the REP-2 rice legumain
was suggested as an activator of other CysProt during rice germination [14]. In this process, the SCP46
serine carboxypeptidase from rice regulates grain filling and seed germination upon hormonal
induction [15,16]. Besides, serine carboxypeptidases I and III from triticale grains effectively degraded
storage proteins that were proteolytically modified by the cathepsin L-like protease EP8 [17,18].
The participation of proteases in the germination processes of barley and wheat will be widely
described in following sections.
2. Mobilization of Stored Proteins During the Germination of Barley and Wheat

Monocot species like barley and wheat have caryopses or cereal grains as propagation units.
Cereal grains are endospermic seeds, meaning that the storage proteins are accumulated in the
endosperm tissue (Figure 1A). This tissue consists of the starchy endosperm, which is a dead storage
tissue, and the aleurone layer, which is formed by living cells. The main tissues found in the barley
embryo are the coleoptile, the scutellum, and the radicle. During seed development and maturation,
deposition of reserves within the storage tissue takes place. Starch, proteins, and lipids are mainly
accumulated in the endosperm tissue but are also found in axis organs like the radicle and the
embryonic shoot, or in the outer aleurone layer [19]. Cereal grains contain relatively little protein,
with an average stored amount of 10–12% of the dry weight [19]. This storage fraction represents
80–85% of the total protein content. The main seed storage proteins are classified as albumins,
globulins, and prolamins, on the basis of their solubility [20]. Most storage proteins in barley and
wheat are prolamins [19], which are named hordeins in barley and gliadins in wheat. The antagonism
between gibberellins (GA) and abscisic acid (ABA) is an important factor regulating the developmental
transition from seed maturation to seed germination [21]. In terms of physiological and morphological
changes, seed germination typically begins with dry mature seed imbibition and ends with radicle
protrusion (Figure 1B). The embryo is responsible for the synthesis of GA after imbibition in water.
This hormone reaches the aleurone layer via the scutellum, where it induces the expression of genes
encoding α-amylases and proteases. The combination of the CysProt stored in the protein bodies and
de novo-formed proteolytic enzymes, which are spread into the endosperm, triggers the hydrolysis
of most proteins in the storage tissue. The resulting amino acids and small peptides are absorbed by
the scutellum, which delivers them to the growing seedling (Figure 1A). Once the radicle breaks and
protrudes from the seed coat, the germination process is accomplished [22].
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Int. J. Mol. Sci. 2019, 20, 2087
Figure 1. Cereal seed germination. (A) Schematic representation of the main events during storage
protein remobilization in cereal seeds. (B) Photographs of the germination process of barley grains.
HAI, hours after imbibition.
3. Investigation of Proteases in the Germination of Barley and Wheat before High-Throughput

Technologies
Historically, the participation of proteases during grain germination was inferred from reports on
proteolytic activities, the expression of individual genes during the process, or the presence of individual
proteins in the germinating grain (Figure 2). Zhang and Jones [8], using bidimensional gel analyses
and class-specific protease inhibitors, found 42 different spots with protease activity in the barley
germinating grain, putatively corresponding to 27 cysteine proteases, 8 serine proteases, 4 aspartic
proteases, and 3 metalloproteases. Similarly, Dominguez and Cejudo [23] used polyacrylamide
gels copolymerized with gelatin to detect proteolytic activities in germinating wheat grains. Again,
putative CysProt activity was preferentially observed, although serine, aspartic, and metalloprotease
activities were also present. In parallel, individual proteases were isolated from barley and wheat
germinating grains and identified by sequencing the corresponding cDNAs. The first barley CysProt
described participating in the proteolytic degradation of the storage proteins in the grains [24–27]
were the cathepsin L-like proteases EP-A and EP-B from the C1A family. Besides, a cathepsin H-like
protease, aleurain, and a cathepsin B-like protein, HvCathB, were detected in the aleurone of the
barley grain [28,29]. In wheat, several cathepsin L-like CysProt, WEB-1, WEP-2, EP-A, WCP-2, and
gliadain, and the cathepsin B-like proteases Al16, Al20, and Al21 were described as participants in
the proteolysis processes of the germinating grain [30–34]. In addition, members of the S10 serine
carboxypeptidase family were implicated in the germination of barley and wheat [35–37], and some
aspartic proteases were identified in barley and wheat seeds [38,39].
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Int. J. Mol. Sci. 2019, 20, 2087
Figure 2. Global scheme of the main techniques used to analyze the implication of proteases during
the germination of barley and wheat grains.
Nowadays, advances in high-throughput sequencing have resulted in the development of new

technologies and strategies that give support to experimental research. The release of the genomic
sequences of wheat and barley has permitted the application of genome-scale approaches, such as
those used in functional genomics and proteomics. These technologies have been employed, mainly in
barley, in the study of proteases during the germination process (Figure 2).
4. Functional Genomic-Based Advances in the Identification of Proteases in the Germination of

Barley and Wheat Grains
The extremely large number of expressed sequence tags (EST) and cDNA sequences in barley
permitted the first approximation of the protease repertoire. Using the Affymetrix Barley1 GeneChip
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Int. J. Mol. Sci. 2019, 20, 2087
22K [40], the expression of about 20 C1A proteases could be checked. This GeneChip has been
used in several studies on gene expression during germination and seedling elongation (reviewed
in [41]). Among these studies, a detailed transcriptome analysis of barley grain germination can be
highlighted [42]. In this analysis, several C1A papain-like CysProt, such as EP-A, EP-B1, and EP-B2,
were expressed in both the aleurone and the embryo during grain germination. In addition, transcripts
of cathepsin B-like (HvPap-19 and -20), cathepsin H-like (aleurain), cathepsin F-like (HvPap-1), and
some cathepsin L-like proteases (HvPap-4 and -6) were abundant during seed germination in both the
aleurone and the embryo, but were also expressed during seed maturation. Besides, transcripts for
several serine carboxypeptidases were highly detected during germination in both the embryo and the
aleurone [42].
A second step in the identification process came from the public release of the barley genome
sequence [43]. Previous analysis from EST collections showed the presence of 32 C1A papain-like
members [44], which increased to 41 members upon genomic mining [45]. A former analysis of
the expression of cathepsin-L like proteases from different phylogenetic groups showed that four of
the five selected cathepsin L-like genes (HvPap-4, HvPap-6, HvPap-10, and HvPap-17) were primarily
transcribed in germinating embryos [46]. The most abundant transcripts were those of HvPap-10,
which had a seed-specific pattern of expression, followed by HvPap-6 and HvPap-4. Whereas a lower
expression of the HvPap-17 gene was detected in germinating embryos, HvPap-16 was exclusively
transcribed in barley leaves. No expression of any of the cathepsin L-like genes studied was detected in
the developing barley endosperms. During germination, GA treatment of the aleurone layer increased
the quantity of transcripts from HvPap-6 and HvPap-10 but had no effect on the expression level of the
HvPap-4 gene. Likewise, de-embryonated barley grains showed that the HvPap-1 gene was expressed
during grain germination and that GA treatment induced a remarkable increase in its expression [47].
The genomic content of C13 legumain-like genes has also been addressed. Formerly, five legumains
were reported from EST collections [44], two additional legumains were later detected [48], and finally,
a novel protein was added after an in-depth search of the barley genome [49]. Regarding the barley
legumains group, it has been suggested that legumains are able to process other CysProt in order to
activate them to take part in the proteolytic degradation of the storage proteins [50]. For example, the
HvLeg-2 legumain of barley, which is highly expressed during germination, could be involved in the
mobilization of storage proteins, either by direct proteolytic degradation or by the processing and
activation of other CysProt [49,50]. In fact, the capacity of HvLeg-2 to degrade storage seed globulins
was demonstrated, confirming its role as a hydrolytic enzyme against storage proteins. Likewise,
HvLeg-2 could participate in the processing of other peptidases, such as the papain-like CysProt
induced by GA, which degrade hordeins [46,47]. This is similar to the action of the legumain REP-2 on
the papain-like peptidase REP-1 in germinating rice seeds [14].
Recently, a new methodology has been developed for isolating fragments of aleurone, starchy
endosperm, embryo, scutellum, pericarp–testa, husk, and crushed cell layers from barley germinated
grain [51]. This method is based on rapid fixation of the intact grain, followed by dissection for
subsequent transcriptomic analyses. Using this technology, the expression profiles of many genes were
precisely defined during the first 24 h of germination [51]. Interestingly, an analysis of the differential
expressed genes (DEG) in the aleurone fragment nearest the embryo after 24 h of germination showed
the induction of many different proteases, including papain-like CysProt, legumain-like CysProt, and
serine carboxypeptidases, and also several aspartic proteases, metalloproteases, and subtilisin-like
serine proteases. Many of these proteases were exclusively upregulated in this proximal part of the
aleurone layer, and none of them were overexpressed in the distal fragment of the aleurone. Following
24 h of germination, the set of upregulated proteases in the embryo and scutellum comprised several
proteases exclusively upregulated in these tissues and some of the proteases overexpressed in the
aleurone. Recently, the upregulation of several subtilisin-like serine proteases during grain germination
has been confirmed by RT-qPCR assays [52].
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Int. J. Mol. Sci. 2019, 20, 2087
In contrast to wide knowledge on the gene expression in the barley germinating grain, studies
on wheat are scarce. To date, transcriptome expression profiles during seed germination have been
performed using the GeneChip® Wheat Genome Array [53,54]. In whole germinating grains, the
expression of several cysteine, aspartic, and serine proteases increased significantly between 24 h and 48
h after imbibition, with a peak at 36 h for the most expressed class, the CysProt [54]. Efforts on optimizing
RNA-seq analyses clashed with the poor quality of the first draft of the wheat genome released by the
International Wheat Genome Sequencing Consortium [55] (IWGSC, 2014). However, a tremendous
effort using novel approaches generated the recently published high-quality linear reference assembly,
IWGSC RefSeq v1.0 [56]. Likewise, many RNA-seq analyses have been performed and implemented
in different portals, such as the Wheat Expression Browser (www.wheat-expression.com, [57]) which
includes 850 wheat RNA-sequencing samples derived from 32 tissues at different growth stages
and/or challenged by different stress treatments. Unfortunately, these analyses did not cover the
germinating grain.
5. Proteomic-Based Advances on Proteases in the Germination of Barley and Wheat Grains

Wide proteomic analyses of barley and wheat grains were formerly based on two-dimensional gel
electrophoresis, which was subsequently coupled with mass spectrometry. In the absence of a genome
sequence, barley gene and EST sequences, combined with information from other cereals, facilitated
the identification of barley proteins. From this wide range of proteomic analyses, many barley seed
proteins were identified (reviewed by [58]). Besides, to avoid the masking of low-abundant proteins by
large amounts of starchy endosperm storage proteins, isolated aleurone layers were used in proteomic
analyses. When treated with GA, several C1A cysteine proteases, S10 serine proteases, and A1 aspartic
proteases were identified in this tissue [59].
In contrast, relatively few proteomic studies have been performed during wheat seed germination.
Several analyses combined two-dimensional electrophoresis (2-DE) with MALDI –TOF/TOF MS to
explore the proteomic changes in the embryo and endosperm that occur throughout the germination
period. From these analyses, distinct differentially expressed proteins were found in the seed embryo
and endosperm, which presumably cooperate in seed germination [3,60,61]. Although the authors
claimed that some of these proteins were related to storage protein metabolism, no individual proteases
were identified. Besides, the proteome of isolated aleurone layers has only been addressed during
seed development [62]. Recently, a gel-free proteomics approach was performed to obtain a dynamic
proteome survey during barley malting. This shotgun proteomic technique entails the in-solution
tryptic digestion of precipitated proteins and an analysis of peptides by nanoLC–MS/MS. A high number
of proteins were identified [63], including several aspartic, cysteine, metallo, and serine peptidases,
which demonstrates the strength of this technique for identifying low-expressed proteins. Although
similar proteomics approaches have been performed in wheat, no one has analyzed grain germination.
On the other hand, protease activity in the barley grain has been analyzed using two different
approaches. A classical approach is based on the capacity of recombinant proteases to degrade storage
compounds. In a first analysis, whereas recombinant HvPap-10 protease was able to completely
degrade all electrophoretic bands corresponding to B, C, and D hordeins from grain extracts, HvPap-6
only partially reduced the presence of those bands [46]. In addition, the capacity of HvPap-10 to
hydrolyze the recombinant hordeins (B1, B3, and γ1) expressed in Escherichia coli was tested. After 2 h
of incubation with HvPap-10, an almost complete degradation of γ1 and a partial digestion of hordein
B1 and B3 were observed [64]. Besides, recombinant HvPap-1 was able to degrade different barley
proteins (hordeins, albumins, and globulins) stored in the barley endosperm [47]. Further insight on the
role of proteases in grain germination in transgenic plants was provided by silencing or overexpressing
a specific protease. Only the silencing or overexpressing of the cathepsin F-like HvPap-1 protease by
barley plants has been described. These plants showed differential accumulation of storage molecules
such as starch, proteins, and free amino acids in the grain, as well as disturbed electrophoretic patterns
of hordeins, globulins, and albumins during the germinating process. Silencing lines showed a drastic
21
Int. J. Mol. Sci. 2019, 20, 2087
delay in the grain germination process. Remarkably, this phenotypic feature could not be directly
related to cathepsin F-like deficiencies, as alterations in the cathepsin L/F-like proteolytic activities
were also accompanied by changes in cathepsin B-like and trypsin-like proteolytic activities [65].
In wheat, the C1A protease gliadain, purified from E. coli, was able to hydrolyze the storage α, β,
and γ gliadins, but not the glutenins from grain extracts [34]. Gluten is a heterogeneous mixture of
insoluble storage proteins, called gliadins, which contain proline-rich and glutamine-rich repetitive
sequences. The fact that several of the peptides derived from gluten are toxic for humans with celiac
disease led to the identification of proteases with the ability to degrade it [66,67]. Proteases from
protein extracts of germinated barley and wheat grains showed the ability to degrade gliadin-derived
toxic peptides [68]. In particular, the C1A protease Triticain-α, formerly thought to participate in seed
germination by digesting storage proteins [69], was shown to possess glutenase activity in vitro [70].
Triticain-α cleavage sites were found in the majority of the previously identified gluten-derived toxic
peptides, including the major 33-mer α-gliadin-derived peptide. These findings support the potential
of Triticain-α as a basic compound for the development of drugs against celiac disease [70].
The second approach is based on the development of activity-based proteomics, also known as
activity-based protein profiling (ABPP). This method uses molecular probes which bind irreversibly
to the reactive site of members of specific groups of enzymes. The results provided information on
enzyme activity, not just protein abundance [71], allowing differentiation between the inactive plant
proteases synthesized as zymogens and the active proteases, after proteolytic processing. As the
specificity of many commercial protease inhibitors is inaccurate, specific fluorescent probes were
developed for ABPP. When applied on Arabidopsis germinating seeds, the fluorescent activity-based
probes specifically targeted three distinct cysteine protease subfamilies, revealing the dynamic activities
of aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes during the
remobilization of stored proteins [72]. This technology has recently been applied to monitor the activity
of different enzymes in the germination process of the barley grain. Using specific probes and ABPP to
detect the active enzymes extracted from the aleurone layers of a commercial malting barley variety,
several active proteases were found to be induced by GA, such as putative aleurains, cathepsin- B-like
proteases, and serine hydrolases [73].
6. Conclusions and Future Perspectives

Germination is a key process in the life cycle of plants. During this period, a new plant starts
its development from the embryo with the help of the compounds stored in the seed endosperm.
The continuous release of nutrients from the endosperm to the embryo is crucial to achieve the correct
development of the new plant and to avoid agronomical losses due to the absence of seed germination
in the field. Therefore, knowledge on the regulatory mechanisms that take part during this process
must be improved to establish the best conditions for a correct germination. In the case of barley and
wheat, many advances have been made in understanding the role of proteases in the grain due to their
potential value for the brewing industry and their relationship with the celiac disease. Using different
technologies, many proteases have been identified and closely associated to the grain germination.
The development of novel techniques based on shotgun proteomics, ABPP, and comparative and
structural genomics will lead to a more comprehensive understanding of this process and the specific
roles of the proteases involved. For example, the identification by ABPP of active proteases during the
barley grain germination process will increase the selection of efficient prolamin-degrading proteases.
To reduce costs, the brewing industry is replacing barley malt with unmalted grains [74]. Thus,
these proteases may be included in the commercial brewing enzyme cocktails used to improve the
wort obtained from unmalted barley grains. Likewise, technical advances will allow the selection
of high gliadin-degrading efficient proteases, which could be combined with the low-gliadin wheat
plants obtained by genome edition [75], as therapeutic alternatives in the treatment of celiac disease.
Furthermore, silencing or overexpressing a specific protease will contribute to the knowledge on the
role of the protease and also in understanding the intricate network of proteolytic reactions combining
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Int. J. Mol. Sci. 2019, 20, 2087
different classes of proteases and protease inhibitors involved in the remobilization of storage proteins.
The required technology has recently been developed, and the challenge is to combine research efforts
to address key questions concerning the control of proteolysis during seed germination.
Author Contributions: M.M. conceived the review. M.D-M., I.D., and M.M. wrote the manuscript.
Acknowledgments: The financial support from the Ministerio de Economía, Industria y Competitividad (project
BIO2014-53508-R) is highly acknowledged.
Conflicts of Interest: The authors declare no conflict of interest.
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© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
26
Molecular Sciences
Review
Cas Endonuclease Technology—A Quantum Leap in
the Advancement of Barley and Wheat
Genetic Engineering
Iris Koeppel † , Christian Hertig † , Robert Hoffie † and Jochen Kumlehn *
Plant Reproductive Biology, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben,
06466 Seeland, Germany; [email protected] (I.K.); [email protected] (C.H.);
hoffi[email protected] (R.H.)
* Correspondence: [email protected]
† These authors contributed equally to this work.
Received: 8 May 2019; Accepted: 24 May 2019; Published: 29 May 2019
Abstract: Domestication and breeding have created productive crops that are adapted to the climatic
conditions of their growing regions. Initially, this process solely relied on the frequent occurrence
of spontaneous mutations and the recombination of resultant gene variants. Later, treatments
with ionizing radiation or mutagenic chemicals facilitated dramatically increased mutation rates,
which remarkably extended the genetic diversity of crop plants. However, a major drawback of
conventionally induced mutagenesis is that genetic alterations occur simultaneously across the
whole genome and at very high numbers per individual plant. By contrast, the newly emerging Cas
endonuclease technology allows for the induction of mutations at user-defined positions in the plant
genome. In fundamental and breeding-oriented research, this opens up unprecedented opportunities
for the elucidation of gene functions and the targeted improvement of plant performance. This
review covers historical aspects of the development of customizable endonucleases, information on
the mechanisms of targeted genome modification, as well as hitherto reported applications of Cas
endonuclease technology in barley and wheat that are the agronomically most important members of
the temperate cereals. Finally, current trends in the further development of this technology and some
ensuing future opportunities for research and biotechnological application are presented.
Keywords: cereals; CRISPR; crops; genetic engineering; genome editing; plant; Triticeae
1. Introduction
1.1. Historical View of Genetic Modification in Crop Plants

For more than 10,000 years, plants have been cultivated to feed humans and their livestock,
as a source of raw materials and to generate energy. Over time, the driving forces of evolution,
domestication, and breeding of cultivated plants have changed according to environmental conditions
and societal demands. To cope with the growth of the world’s population and the consequent
increase in demand for food, the total production needs to be substantially increased during the
next decades [1,2]. Not only is the growing world population a challenge, but also the expected
climatic changes [3,4]. Thus, the plants of the future also have to be better adapted to harsh weather
conditions [5,6]. In addition, the demands of modern society are increasing; food should be of better
and consistent quality, healthier, and more diverse. At the same time, society calls for a substantially
reduced application of fertilizers and pesticides to render agriculture more sustainable [2].
In nature, mutations occur spontaneously and alterations that provide an advantage under the
given conditions are likely to prevail over time. For modern plant breeding, however, the rate of

Int. J. Mol. Sci. 2019, 20, 2647
spontaneous mutations is too low to keep up with the demands for crop improvements. Moreover, it
is in the nature of spontaneous mutations that they are unpredictable in terms of both position and
resultant nucleobase sequence. In the 1930s, mutation breeding emerged, in which mutations are
deliberately induced with chemicals such as ethylmethanesulfonate (EMS) or with ionizing radiation.
Induced mutagenesis breeding has so far produced over 3000 approved varieties [7] and for many
further cultivars it has not been documented which induced mutations they have inherited from
germplasms they derive from. Using this technology, however, thousands of mutations occur at
different sites in a single plant’s genome at a time. Therefore, a vast number of undesirable mutations
have to be eliminated via cumbersome and time-consuming back-crossing procedures [5]. The method
of Targeted Induced Local Lesions in Genomes (TILLING) represented a significant progress in the
detection of mutations. With TILLING or Eco-TILLING, respectively, it is possible to readily identify
induced and spontaneous mutations in known genes of interest [8,9].
The method of gene transfer using Agrobacterium tumefaciens was introduced in the 1980s [10]. Ever
since, genes derived from unrelated organisms or genetically modified gene variants can be introduced
into plant genomes. This technique makes it possible to modify or introduce performance-determining
genes into cultivated plants more quickly and in a more targeted manner. However, there are some
limitations, such as the fact that the transferred gene is integrated at a random position in the genome
and that changes to the plant’s own genes are only possible to a very limited extent. The insertion site
in the genome then also determines whether and how effectively the introduced gene is expressed, and
thus to what extent the respective trait will be modified. In addition, not all plants can be transformed
equally well by the use of Agrobacterium, with cereal species being a particular challenge in this
respect [11].
With the new methods for targeted genome modification that are based upon customizable
endonucleases, it is possible to modify DNA sequences at a previously defined site of choice in the host
genome. The standard application of this technology is site-directed mutagenesis, in which the location
of the mutation can be precisely determined, whereas the resulting nucleobase sequence is random [12].
However, the precision can be increased by a variety of approaches. The most sophisticated one
involves the use of an artificial DNA repair template implemented via homology-directed DNA repair.
By this still very challenging principle, any sequence of choice can be created at any predefined
genomic locus.
Wheat (Triticum aestivum) and barley (Hordeum vulgare) are among the most important cereals
in the world. Among all crops, wheat occupies the largest cultivated area in the world, and barley
is one of the oldest domesticated crops, currently being the fourth most frequently cultivated cereal
(http://faostat/fao.org). With a size of about 17 Gbp, wheat has a very large and complex genome. Only
recently has the wheat genome been almost completely read out and this data has been made publicly
available [13]. Hexaploid bread wheat evolved via hybridizations from several ancestors. This process
involved allopolyploidization, resulting in a total of three diploid subgenomes [14]. In contrast to
wheat, barley has a genome with a size of 5.1 Gbp. Already in 2012, a detailed draft of the barley
genome was published [15], which was recently complemented by much improved data, including
genomic sequences of a large number of representative gene bank accessions [16,17].
1.2. Platforms of Customizable Endonucleases

In plant research and biotechnology, four platforms of customizable endonucleases have
been used so far; meganucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector
nucleases (TALENs), and the RNA-guided, clustered, regularly interspaced, short palindromic repeats
(CRISPR)-associated (Cas) endonucleases.
Meganucleases are naturally occurring endonucleases that recognize and cut comparatively
long (>12 bp) DNA sequences. The most commonly used meganuclease is the I-SceI from
Saccharomyces cerevisiae [18,19]. The ability of this endonuclease to induce double-strand breaks
in plants was first demonstrated by Puchta et al. [20] using Nicotiana protoplasts. Meganucleases
28
Int. J. Mol. Sci. 2019, 20, 2647
are very specific and efficient. It is, however, very difficult to reengineer these enzymes for target
sequences other than their native ones [21]. Hence, their use is fairly restricted as compared to the
alternative endonuclease platforms [22,23]. As a consequence, meganucleases have been used in plants
almost exclusively for basic research, in particular for DNA repair mechanisms [24].
Zinc-finger nucleases are hybrid proteins with a DNA recognition domain consisting of at least
three zinc-fingers combined with a FokI restriction endonuclease domain [25]. Each zinc-finger
specifically interacts with three base pairs (bp) of the genomic target sequence and several zinc-fingers
can be consecutively assembled to recognize and bind a total of 9 to 12 bp of DNA [26]. Zinc-finger
nucleases must always be used in pairs, since their FokI endonuclease domain is only catalytically
active if it is present as a dimer [25]. The target motifs on the DNA are selected in such a way that the
two zinc-finger nuclease monomers bind to the target DNA in antiparallel orientation and at a suitable
distance to each other. A DNA double-strand break (DSB) is then catalyzed in the interspace between
the two binding sites [27,28]. In addition to the particularly complex production process of zinc-finger
nucleases, there are limitations in the selection of possible binding sites, as well as unpredictable
neighbor effects between adjacent zinc-fingers on the DNA binding specificity [22].
In 1989, it was shown for the first time how transcription activator-like effector (TALE) proteins
are transferred from a pathogenic bacterium of the genus Xanthomonas into a host plant [29].
The TALE-based nucleases (TALENs) act, similarly to zinc-finger nucleases, as dimers [30]. The
DNA-binding domains consist of up to 30 copies of highly conserved repeats of typically 34 amino
acids. Only the amino acid positions 12 and 13 (called repeat-variable di-residues) of each repeat are
not uniform, because they specify the different DNA base pairs to bind [31]. A FokI endonuclease
bound to such a TALE binding domain induces the double-strand break in the target motif [30]. Since
each repeat recognizes just one nucleobase of the DNA target, the design and assembly of the binding
domain is more straightforward and versatile than with zinc-finger nucleases [31].
The platform of RNA-guided Cas endonucleases is derived from the CRISPR/Cas adaptive
immune system of microbes. The most commonly used Cas endonuclease is from Streptococcus pyogenes
(Sp). The two-component system consists of the Cas restriction enzyme and an artificial guide RNA
(gRNA) that navigates the Cas protein to a cognate DNA sequence motif [12]. The gRNA consists of a
structurally functional and a variable part. The 3 -tail forms a spatial structure required for binding
with Cas to form a ribonucleoprotein complex. The variable part of the gRNA located at the 5 -end
usually comprises 20 nucleotides and defines the DNA-binding specificity of the gRNA according
to the principle of complementary base pairing. The genomic target motif of such ribonucleoprotein
complex includes the same nucleotide sequence as the variable part of the gRNA, which enables the
gRNA to specifically bind the opposite DNA strand. This part of the target DNA is often referred to as
the protospacer, a term adopted from the microbial immune system these molecules originate from.
In addition, this major part of the target motif is complemented by the so-called protospacer-adjacent
motif (PAM), which is recognized and bound by the Cas protein. In the case of SpCas9, the nucleobase
triplet NGG, in which N stands for any of the four nucleobases, represents the PAM site [12]. The high
versatility of the gRNA 5 -end allows a wide variety of target sequences of choice to be addressed. The
comparatively simple application and the efficiency and reliability achievable in higher organisms
have rendered this platform the most popular and frequently used tool for site-directed genome
modification today [32,33].
2. Methodological Aspects of Cas Endonuclease Technology
2.1. System Components

When employing Cas endonuclease technology in plants, there are a number of opportunities and
some specific requirements in terms of the construction of transformation vectors. Cas9 endonucleases
have been modified in various ways for use in plants. Importantly, the coding sequences were
complemented by one or two nuclear localization signals (NLSs) and the codon usage of the protein
29
Int. J. Mol. Sci. 2019, 20, 2647
biosynthesis was optimized for use in plants in general, for monocots and dicots, or even more
specifically for individual species, such as wheat and barley [34–36].
Depending on the host organism, also various promoters have been used to drive endonuclease
expression. In crop plants, the doubled enhanced cauliflower mosaic virus (2x35S) promoter has been
mainly used for test systems [34,37,38] and UBIQUITIN promoters for the generation of heritable
modifications. Accordingly, the maize POLYUBIQUITIN1 promoter (ZmUBI1) has been commonly
used for cas9 expression to generate heritable changes in barley and wheat [37,39,40]. Alternatively,
Cas endonuclease-encoding genes can also be expressed by self-replicating virus particles in the plant
cell [41]. This expression system ensures a comparatively high gene dosage, which may be particularly
useful for homology-based approaches that are described in more detail below. Gil-Humanes et al. [42]
have shown that the Wheat Dwarf Virus, a replicon-based geminivirus, can be modified to express
gRNA and cas9 in wheat, albeit this approach has not yet been shown to be applicable for the generation
of plants with heritable modifications.
The expression cassette for a gRNA typically consists of plant-derived RNA polymerase III
(Pol III)-processed promoters and terminators derived from small nuclear RNA (snRNA)-encoding
genes. Somehow surprisingly, it also proved to be sufficient in plants to use the transcriptional
termination signal of bacterial CRISPR RNAs, which solely consists of a stretch of five to seven
thymidines [34,43]. A comparative test in maize protoplasts had shown that wheat and rice U3
promoters were more efficient than the Arabidopsis U6 promoter that has been preferentially used in
dicots [44]. The wheat U6 promoter has hitherto been mostly used for barley and wheat [37,39,40].
More recently, a study of Kumar et al. [45] indicated that the barley U3 promoter might be more
efficient in site-directed mutagenesis of barley than the frequently used rice U3 promoter.
The preference of the U3 and U6 promoters for A and G as the first transcribed nucleotides limits
the selection of target motifs to GN20GG and AN20GG, respectively [43]. However, there are thus
far no examples where gRNAs were directly driven by Polymerase II-compatible promoters. The
mRNAs otherwise expressed by Pol II promoters are processed at both ends and these modifications
significantly alter the structure of the gRNA, and thus compromise the ability to bind both Cas9 and
the target motif that is to be processed with sufficient efficiency [43].
Several systems have been developed for the expression of multiple gRNAs. Mostly, each gRNA
is expressed by a separate Pol III promoter [44,46,47]. Alternatively, multiple gRNAs can be located
on only one transcript and post-transcriptionally separated, as has already been demonstrated with
wheat [48]. However, such expression systems have not yet led to improved efficiency of the technology
with regard to individual target motifs in plant genomes [43,49,50].
2.2. Criteria for Target Motif Selection and in Silico gRNA Design
Thanks to the opportunity to equip the gRNA with any target-specific 5 -sequence, the plant
genomic target site of the gRNA/Cas complex can, in principle, be chosen at will. However, the Cas9
endonuclease requires the aforementioned two guanines that are part of the protospacer-adjacent
motif (PAM) at the 3 -end of the target sequence. In addition, there are further preferences of the
target-specific part of the gRNA, some of which can have a considerable impact on the functionality of
the gRNA/Cas9 complex. For instance, it was determined for the so-called seed region, that is, the
six nucleotides residing immediately upstream of the PAM site, that a GC content above 50 percent
increases the probability of sufficient gRNA functionality [51]. With regards to the entire target motif,
an enrichment of guanines and low adenine content was shown to cause increased binding stability
and activity of the gRNA/Cas9 complex [52].
The target sequence-specific part of the gRNA usually has a length of 20 nucleotides [12,53]. As
previously stated, the transcriptional start defined by the U3 and U6 promoters, which are preferred
to drive gRNAs in plants, are A and G, respectively, which requires the selection of genomic targets
having the same nucleobases at their 5 -end [38]. In human cell lines and zebrafish, it has been shown
that the length of the target-specific part of the gRNA can be shortened to 18 nucleotides without
30
Int. J. Mol. Sci. 2019, 20, 2647
a noticeable effect on mutation efficiency. Surprisingly, such shorter gRNAs even had increased
specificity for the on-target sequence as compared with off-targets [52,54,55]. Guide-RNAs with less
than 20 target-specific nucleobases also showed high performance in Arabidopsis and barley [56,57].
On the other hand, an extension of the gRNA 5 -part beyond 20 nucleotides was reported to cause
reduced cleavage efficiency [58].
Quite a number of online platforms have been developed for the selection of target motifs and
corresponding gRNAs. For instance, CRISPR-Plant was especially developed for plants [59,60]. While
a whole array of plant reference genomes can be directly screened in this platform, the barley and
wheat genomes are not implemented. However, for the selection of gRNAs in cereals, DeskGen is
a highly instrumental alternative, as these cereal genomes are available for target validation [61,62].
This platform offers a comparatively large scope of options, including various Cas endonucleases
and a useful range of lengths of the target-specific gRNA 5 -part. Also, the CRISPOR online tool
can be specifically used for barley and wheat. It comprises a comprehensive choice of different Cas
enzymes [63,64]. In addition to the selection of gRNAs predicted to be well-performing at their target
motif, all above online tools are also capable of indicating potential off-targets in the chosen reference
genome. WU-CRISPR is another useful platform, as its algorithm is comparatively strict in selecting
particularly useful target motifs. However, this tool is confined to SpCas9 and does not offer off-target
screens in reference genomes [65]. A general disadvantage of all these platforms is that their algorithms
have been established using data from other organisms than plants, which is thought to be one of the
reasons that the reliability of their results is still fairly limited.
Liang et al. [66] have focused on the preservation of the gRNA secondary structure. They
determined that three of the stem loops frequently occurring in the gRNA 3 -part are essential
to appropriately bind to the Cas9 protein, and hence are also of decisive importance to the overall
functionality of the gRNA/Cas complex. These authors also found out that 98 percent of well-performing
gRNAs have no more than a total of 12 base pairs formed between their target-specific and 3 -parts
and no more than 7 target-specific nucleobases consecutively involved in intra-molecular base pairs.
In addition, no more than 6 base pairs should be formed within the target-specific part of the gRNA.
In order to ensure these gRNA features, it is recommended to thoroughly investigate the secondary
structure of candidate gRNAs. For the prediction of RNA secondary structures, online platforms such
as mfold [67–69] or RNAfold [70,71] are available.
In cases where genetic modifications are intended to be performed in genotypes other than those
with reference genomes, it is recommended to countercheck pre-selected target motifs for their presence
and integrity, because even single nucleotide polymorphisms typically result in a dramatic drop in
gRNA/Cas efficiency.
2.3. Delivery of Cas Endonucleases and Associated Reagents into Plant Cells
A number of methods are available for the transfer of DNA, RNA, and proteins into plant cells,
which in principle can also be used for Cas endonucleases, customized gRNAs, DNA repair templates,
and any further reagents that may be used to support site-directed genome modification. Nowadays,
the most widely used approach is based on the genomic integration of expression units with gRNA
and Cas-encoding DNA sequences. However, the production of stably transgenic plants is a particular
challenge for cereals, because agrobacteria, which are mostly used for plant transformation, have a very
limited compatibility with these non-host plants. Moreover, the formation of adventitious shoots from
leaf or shoot explants, which is readily achieved in the context of DNA transfer methods applicable
to most dicotyledonous species, has only been successful in exceptional, hardly reproducible cases
in cereals [72]. On the basis of special methodological approaches, routine genetic transformation of
barley and wheat is nevertheless possible, at least by using selected accessions that are comparatively
amenable to methods of DNA transfer. Such experimental model genotypes are, for example, the
barley cultivar Golden Promise and the Mexican wheat breeding line Bobwhite SH9826 [11]. For
stable DNA transfer in cereal crops, immature zygotic embryos are most widely used. Importantly,
31
Int. J. Mol. Sci. 2019, 20, 2647
such embryos must be of a developmental stage at which highly totipotent cells are still abundantly
present. In immature embryos of barley and wheat, such cells are preferentially residing in the area
that surrounds the shoot apical meristem and the scutellum, respectively. Stable transgenic wheat and
barley plants were already produced by ballistic transfer of plasmid-coated gold particles to immature
embryos in the early 1990s [73,74]. Using hypervirulent bacterial strains, which had initially been used
for the transformation of rice [75], thereafter it was soon also possible to generate transgenic barley and
wheat plants using this principle [76,77]. Today, it is routinely possible to generate stable transgenic
plants from over 10% of inoculated immature embryos of barley [78], while in wheat, efficiencies of
about 5 percent have been achieved both via Agrobacterium-mediated and ballistic DNA transfer [79,80].
However, a largely improved protocol was more recently demonstrated to allow for much improved
transformation efficiency in wheat ([81,82]. The comparative examination of a variety of genotypes has
shown that although some can indeed be transformed in addition to the model lines, the efficiency,
however, is much reduced [83,84]. Previous studies on Cas9-induced mutagenesis of barley have been
based without exception on Agrobacterium-mediated DNA transfer. In contrast to this, site-directed
mutagenesis in wheat has mostly relied on ballistic gene transfer [85].
Another particularly interesting principle of DNA transfer into cereal cells is the use of immature,
single-celled pollen (microspores), which can undergo cell proliferation and embryogenic development
under suitable culture conditions [86]. Since pollen consists of haploid cells, it is possible that
homozygous transgenic plants can be directly produced via this developmental pathway in association
with DNA transfer and whole genome duplication. Agrobacterium-mediated DNA transfer in
embryogenic pollen cultures of barley has the further advantage that a winter barley variety can
be used, which is comparatively difficult via DNA transfer to immature embryos [87,88]. A similar
transformation method has been reported also for wheat, which is, however, based on ballistic DNA
transfer [89]. In the authors’ laboratory, barley mutants have already been produced via the pollen
embryogenesis pathway using TALENs [90], as well as by Cas endonucleases. Furthermore, the
production of stable transgenic barley and wheat by ballistic DNA transfer into meristematic tissue
from the shoot apex of embryos prepared from mature grains was also successful [91,92]. This method
has potential because of particularly low genotype dependence, since plant regeneration is based
on conventional germination of the embryos, and therefore no formation of adventitious shoots is
necessary. More recently, Hamada et al. [93] have also demonstrated that the production of plants with
site-directed modifications using gRNA/cas9 constructs is possible using this method.
After alteration of the genomic target motif, the presence of transgenes coding for gRNA and
endonuclease is not only unnecessary but also undesirable, as off-targets can still be mutated, even
if they are not identical with the on-target. In this context, an advantage of the application of Cas
endonucleases compared to conventional genetic engineering is that the integration site of the transgenes
is mostly not coupled with the site of the desired genetic modification. Thus, the elimination of these
transgenes by genetic segregation of progeny is comparatively straightforward while maintaining
the desired modification. Endonuclease-triggered genetic alterations in primary transgenic plants
are often heterozygous and restricted to tissue sectors. Therefore, progeny needs to be screened in
order to select homozygously mutated individuals. Consequently, no extra time is required to obtain
transgene-free mutants. Haploid technology is a particularly elegant solution for the separation of
achieved genetic modifications from unnecessary transgene insertions or for the segregation of several
mutations present in chimeric plants of the M1 generation. With this technology, populations of
completely homozygous recombinants can be obtained, in which desired genotypes are present at a
much higher frequency than among offspring derived from conventional selfing [94,95].
In addition to the various possibilities of genomic integration of gRNA and cas endonuclease
genes, methods for transient expression have also been established, which is of considerable value
for the preliminary validation of target-specific gRNAs, Cas endonuclease variants, and any further
components used in this context. The most widely used transient expression method is based on
the transfection of isolated mesophyll protoplasts, whose plasma membrane is rendered porous
32
Int. J. Mol. Sci. 2019, 20, 2647
by application of polyethylene glycol. This enables transgenic plasmid DNA, in vitro transcribed
RNA, bacterially pre-produced Cas protein, or ribonucleoprotein complexes to be taken up by the
protoplasts. This has been exemplified, amongst others, in wheat and barley [39,57,96]. The activity
of the transferred components can then be checked after amplification of the genomic target regions
using the T7E1 assay, by Sanger or deep sequencing, as is described in more detail further down
below. Another method for the functional validation of gRNA/cas constructs by transient expression is
based on the ballistic transformation of leaf epidermis. Epidermal cells are comparatively well suited
to be screened microscopically, for instance in regards to Cas-induced restoration of a reporter gene
construct [97]. Barley leaf tissue has been used to demonstrate precise editing via homology-based
DNA repair using a synthetic DNA repair template [98].
As an alternative to the expression of gRNA- and Cas-encoding DNA, in vitro transcribed RNA
or Cas protein can be transferred to plant cells in order to make specific modifications to the genome.
Guide RNA and Cas endonuclease can also be pre-assembled to form gRNA/Cas9 ribonucleoprotein
complexes prior to their transfer into plant cells. The use of pre-produced RNA and protein molecules
or complexes thereof has the advantage over DNA that their effective amount is independent of the
expression profile and strength of plant promoters. In addition, the activity of these components is
limited in time, as they are not continuously delivered by gene expression but are subject to cellular
degradation. Accordingly, fewer mutations will occur in off-target motifs during the subsequent
development of the plants. A further advantage is that mutated progeny do not have to be examined
for the loss of integrated gRNA- and Cas9-encoding DNA sequences, as all mutated individuals can be
used without restriction due to their transgene-free nature. In wheat, the GW2 gene was used as an
example to show that the transfer of in vitro transcribed RNA coding for both gRNA and Cas9 leads
to mutations in one percent of ballistically transformed cells [99]. About one third of the resulting
plants were mutated in all six copies of the hexaploid genome. Compared to the transfer of gRNA
and cas9 transgenes, however, this method had a mutation rate that was about 60 percent lower.
Liang et al. [96] achieved an improvement in mutation efficiency by assembling gRNA and Cas9 into
ribonucleoprotein complexes before ballistic transfer into immature embryos. After selection-free plant
regeneration, mutants were identified with a frequency of more than four percent, which is on a par
with the efficiency of conventional wheat transformation.
2.4. From Site-Directed Mutagenesis to Precise Genome Editing

Current utilization of Cas endonuclease technology is still largely limited to random alterations
of the DNA sequence at the user-defined genomic sites. This comparatively simple approach of
site-directed mutagenesis relies on the error rate of the DNA repair mechanism, called non-homologous
end-joining (NHEJ), which is predominant in plant cells. In this process, the two DNA ends resulting
from a double-strand break are recognized and relegated irrespective of their nucleobase sequence.
Increased predictability of site-directed modifications was shown to result, for example, from
two simultaneously induced DNA breaks, which can lead to the precise deletion of the interjacent
fragment [22]. In addition, microhomology-based DNA repair produces predictable deletions in a
comparatively simple way, provided that identical sequence repeats are present at both DNA ends that
are to be relegated [100].
A more ambitious approach is the so-called base editing, in which a single nucleotide is specifically
converted into another so that no more than one amino acid of the encoded protein is altered at
a time [101]. For this purpose, a Cas-based nickase, that is, a Cas endonuclease derivative that
cuts only one strand of DNA due to the mutative alteration of one of its two nucleolytic domains,
is bound to a natural or artificial nucleobase deaminase enzyme [102]. Cytidine deaminases can
convert C/G basepairs to T/A in the target region, whereas adenosine deaminases induce A/T to G/C
conversions [102,103]. The functionality of cytidine and adenosine deaminases has already been
demonstrated in several plant species, including wheat [104,105]. The effective base editing area ranges
in dependence of the base editor used, e.g., the cytidine deaminase used by Zong et al. [104] can
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Int. J. Mol. Sci. 2019, 20, 2647
convert positions 1 to 17 within the target site and the adenosine deaminase used by Li et al. [105] is
capable of converting positions 4 to 8.
In addition to the aforementioned error-prone non-homologous end-joining (NHEJ) DNA repair
mechanism that predominates in plant cells, homology-directed repair (HDR) can also be used
to generate precise genome alterations, albeit this process is much less active in somatic cells.
Homology-directed repair naturally involves the sister chromatid of the same chromosome or the
homologous chromosome as the correct sequence template, allowing the original sequence to be
restored, even in cases of comparatively severe DNA damage. By using artificial DNA repair templates
that are partially complementary to the site of the plant genome to be modified, it is possible to make
even fairly large modifications precisely, as specified by the experimenter [106]. However, due to the
methodological challenges, there are very few examples of this precise editing in plants published
thus far. A first experimental approach involved the stable integration of the repair template together
with the gRNA- and Cas-encoding expression units into the plant genome. The integrated repair
template is flanked by the target sequences of the gRNA-mediated endonuclease used, so that it is
cut out by the Cas restriction enzyme. This principle has proven to be sufficient in Arabidopsis [107].
The use of paired Cas9 nickases, which generate two single-strand breaks on the two opposite DNA
strands, has increased the efficiency of homology-directed DNA repair [50,108,109]. With geminivirus
replicons as carriers of the repair template, the dose per cell is increased. In this case, a strain of the
Bean Yellow Dwarf Virus was modified so that only the elements essential for the method remained
and the artificial repair template was amplified with high frequency [110]. In barley and wheat, precise
editing has been achieved either only at the cellular level [98] or has the limiting prerequisite that the
obtained genetic modification leads to an in vitro selectable trait, e.g., an herbicide resistance [111].
2.5. Identification and Characterization of Site-Specifically Modified Plants

The presence of gRNA- and Cas-encoding expression units is usually tested by PCR
amplification [112,113]. In some studies, the expression of gRNA and cas9 was additionally assessed
by RT-PCR, which can be very informative, especially in the process of method establishment [37]. For
the detection of site-directed modifications, PCR products derived from the genomic target region
can be analyzed using various methods. Digestion of the PCR product using conventional restriction
enzymes is the most straightforward approach. However, the prerequisite for this is that the restriction
site of the Cas endonuclease lies within a recognition sequence of the conventional restriction enzyme
that then can be used for the test. In contrast to the wild-type sequence, PCR products of mutated
target motifs cannot be digested by this enzyme [34]. A disadvantage of this method is that the choice
of target motifs is substantially limited. This can be circumvented using the T7E1 assay, in which a
mixture of PCR products of mutant and wild-type alleles is digested by the mismatch-sensitive T7
endonuclease I [39,114]. Another disadvantage these methods have in common is that their sensitivity
is low, i.e., if mutated alleles of a plant are limited to small tissue sectors, detection may be not possible.
This leads to a certain number of mutants not being identified.
To avoid this problem, Liang et al. [115] used target-specific synthetic gRNA and Cas9 protein for
in vitro digestion of PCR products from genomic targets. This method is independent of conventional
restriction sites in the target motif and was reported to be extremely sensitive. However, a large-scale
application of this approach is hampered by the fact that recombinant Cas protein is fairly expensive.
As compared with the aforementioned restriction-based assays, sequencing of PCR product
derived from the genomic target region is more informative. In heterozygous or chimeric mutated
plants, the sequencing chromatogram shows double and multiple peaks at the individual base
positions, which usually starts at the restriction site of the Cas endonuclease and continues in the
same direction as the sequencing was conducted. Various bioinformatic tools, such as Tracking of
Indels by DEcomposition (TIDE) [116] and DSDecode [117], provide the opportunity to read out such
peaks, thereby determining the different allele variants present in the analyzed plant sample. A more
reliable approach involves the analysis of several single clones via Sanger sequencing, as this provides
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Int. J. Mol. Sci. 2019, 20, 2647
a detailed breakdown of the mutations present in the analyzed sample. In addition, more conclusive
information on the proportions of the mutated alleles can be obtained. In this context, it is important to
consider that Cas endonuclease-triggered mutations are usually heterozygous and often limited to
sectors of the plant. Moreover, leaf samples are not necessarily representative for the whole plant. This
phenomenon is reflected by the observation that not all mutations detected in primary mutants are
inherited by subsequent generations. In addition to Sanger sequencing, deep sequencing methods
can be used to analyze mutations in individual plants, which is particularly informative owing to the
obtained amount of data [48,118]. In practice, it is typically necessary to find a reasonable compromise
between workload, conclusiveness of assays, and costs. Therefore, a combination of different methods
is often the most suitable solution [40,42].
Figure 1. General workflow of targeted genome modification in barley and wheat, including target
motif selection, vector construction, vector tests, gRNA and Cas9 delivery, plant regeneration, mutant
detection, and genotypic and phenotypic analyses.
35
Int. J. Mol. Sci. 2019, 20, 2647
The molecular analysis of candidate plants can also address accidental modification of so-called
off-target sites, which can cause undesired side effects and also influence the efficiency of on-target
mutagenesis. In plants, the proportion of off-target mutations is typically below 1% [119]. In contrast
to human cells, this does not pose a serious problem. These mutations are often confined to small
sectors that are usually not passed on to offspring. Furthermore, due to the high specificity of the
gRNA/Cas complex, possible off-target sites are well predictable so that progeny can be readily tested
to eliminate segregants with unwanted mutations.
Since primary mutants are often chimeric or heterozygous and integrated cas9 and gRNA
transgenes can be inherited, the analysis of progeny is essential to identify homozygous mutants that
are null segregants in regards to the transgenes [35,112].
It is not recommended to spend too much effort for phenotypic analyses of primary mutants,
because in addition to their uncertain genetic homogeneity and zygosity, the general fitness of these
individuals is typically strongly affected owing to the manipulation and culture procedure they have
gone through [120]. Importantly, it is advisable to use non-transgenic, non-mutant segregants as
wild-type controls for the analysis of plant traits, because the comparison with other plants bears a
fairly high risk of obtaining misleading results. A survey of the general workflow of targeted genome
modification in barley and wheat is depicted in Figure 1.
3. Applications
While in this section only some representative examples for applications of Cas endonuclease
technology in barley and wheat are presented in more detail, Table A1 provides a comprehensive
overview of studies hitherto published on these crops.
In 2013, Upadhyay et al. [37] were the first to show that Cas endonucleases are in principle
applicable in a Triticeae species and especially in the large and complex genome of wheat. However,
this study was still confined to site-directed mutagenesis in a cell suspension, from which no plants
can be regenerated. Mutations were detected in 18 to 22 percent of the sequenced amplicons derived
from target regions of the INOSITOL OXYGENASE (TaINOX), and PHYTOENE DESATURASE
(TaPDS) genes. The first plants carrying Cas endonuclease-induced mutations were presented by
Wang et al. [39]. While heritable loss-of-function mutations were induced in all three homoeologues of
the MILDEW-RESISTANCE LOCUS O (TaMLO) gene by the use of TALENs, Cas9-triggered mutations
were still confined to the A subgenome. Using two wheat backgrounds, the authors produced a total
of 687 gRNA/cas9 primary transgenic plants, of which about 5 percent proved to carry mutations in
the target motif. However, in contrast to the TALEN-induced ones, the heritability of these mutations
was not shown.
The first published use of Cas endonucleases in barley aimed to induce mutations in HvPM19 [35],
which encodes an ABA-induced plasma membrane protein previously described in wheat as a positive
regulator of dormancy. Four copies of this gene are present in barley. For two of those, knockout
mutants were generated. Amongst 13 primary transgenic plants, three carried mutations in HvPM19-1,
whereas one HvPM19-3 mutant was found amongst ten T0 plants. While this study was the first
to provide evidence for the heritability of Cas9-induced mutations in a Triticeae crop, a resultant
phenotype was not described.
Once the applicability of Cas endonuclease technology to achieve heritable mutations was shown
for barley and wheat, this principle was used for many further approaches. Holme et al. [40] used
Cas endonuclease-induced mutations to investigate the function of the PHYTASE GENE A of barley.
HvPAPhy_a acts as the main regulator of phytase content in the barley grain. Phytases are important
sources of bioavailable phosphorus, and therefore particularly important for germination. To reduce
the activity of HvPAPhy_a independently of HvPAPhy_b, which is very similar in its gene sequence,
Holme et al. specifically mutated the promoter of HvPAPhy_a. While small insertions and deletions in
homozygous mutant progeny had only little effect, larger deletions, which also affected exon 1 of the
target gene, caused a significant reduction of phytase activity.
36
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minnares, en te zweren, dat hij nimmer weder als mededinger van Dr.
Fox zou optreden!
Maar hoe kwam die man hier?
Raffles had hem toch zooeven medegedeeld, dat hij die twee kerels, na
hen zoogenaamd te hebben gearresteerd, bewusteloos had gemaakt,
en hij zou toch wel zoo verstandig geweest zijn, dit niet op den
openbaren weg te doen?
Hoe was het mogelijk, dat die man nog altijd bewusteloos was, en dat
geen der doctoren nog naar hem was komen zien?
Hij zou spoediger antwoord op die vraag krijgen dan hij wel vermoedde,
en wel uit den mond van de hoofdverpleegster zelve, een praatgrage
dame, die volstrekt niet kon denken, dat Beaupré deel uitmaakte van
een dievenbende, en meende, dat hij inderdaad het slachtoffer [7]van
een laaghartige poging tot afzetterij was geweest—hetgeen in zekeren
zin ook de waarheid was!
Zij had het bed van den bewustelooze verlaten en trad nu snel op dat
van Beaupré toe.
Toen zij zag, dat hij klaar wakker en blijkbaar volkomen bij zijn
positieven was, begon zij:
—Nu moet ik u toch iets heel bijzonders mededeelen, mijnheer Dubois!

Gij weet, dat ik zooeven aan de telefoon werd geroepen. Wie denkt gij
wel, dat er aan het andere einde van de lijn was?
Beaupré meende wel ongeveer te kunnen zeggen, wie dat was, maar hij
wachtte er zich wel voor, zijn vermoedens te uiten.
Hij haalde de schouders op en zeide glimlachend, wat hem moeilijk

genoeg viel, daar hij begon te begrijpen, dat hij zich in groot gevaar zou
bevinden, zoodra de man daarginds uit zijn bewusteloosheid zou
ontwaken, en zou inzien dat zijn loopbaan der misdaad beëindigd was,
zoodat er geen reden meer was, den Franschen bendechef, die reeds
lang door de Parijsche politie gezocht werd, nog langer te ontzien en
hem dus stellig zou verraden:
—Hoe kan ik dat weten, miss?
—De politie!
Beaupré had dit antwoord verwacht, maar toch liep er een zenuwachtige
rilling over zijn bleek gelaat, welke hij niet geheel en al had kunnen
bedwingen.
—De politie? vroeg hij. Zeker in verband met het bezoek dier beide
kerels, die mij geld wilden afdreigen?
—Juist! Gij zult er van opzien, wat er met hen geschied is! Ik moet u
zeggen, dat ik er niets van begrijp—maar de detective, dien wij hier
verwachten zal wel licht in de duisternis brengen! Hij kan over een
kwartier hier zijn!
Dat was een alles behalve aangename mededeeling voor den

Franschman!
Wel hoopte hij, dat zijn gelaat, zooals het nu was, niet meer zou
overeenkomen met het signalement, dat ongeveer drie jaar geleden
door de Parijsche politie was gezonden aan alle groote politiebureaux
over de geheele wereld, vooral omdat hij zijn fraaien, blonden baard uit
dien tijd had afgeschoren, maar hij was daar verre van zeker van.
Hij wist zich echter te beheerschen en vroeg vrij kalm, alsof de zaak
hem eigenlijk niet al te veel belang inboezemde:
—Wat is er dan eigenlijk gebeurd, miss?
—Dat zal ik u zeggen! Het staat nu wel vast, dat de twee rechercheurs,
die hier gisteren de twee schavuiten in de vestibule van dit gebouw
hebben gearresteerd, in het geheel niet tot de politie behoorden, en ook
geen particuliere detectives waren!
—Dat is niet te gelooven! riep Beaupré met goed gespeelde

verwondering.
—En toch is het de waarheid, riep de hoofdverpleegster zegevierend uit,

alsof zij zelve een rol in dit geheimzinnige drama had vervuld. Die
zoogenaamde detectives waren bedriegers, en zij hebben de twee
mannen, die u hier zijn komen lastig vallen, ontvoerd!
—Maar met welk doel? riep Beaupré uit, die eens wilde zien wat de
politie wel, en wat zij niet wist.
—Ja, het doel—dat is juist het ongehoorde! antwoordde de

hoofdverpleegster. Dat begrijpt niemand! Maar het staat vast, dat hier
een misdrijf gepleegd is, dat niet geheel kon worden volvoerd. Ik zal u
nu zeggen, hoe de politie dit alles weet!
De praatzieke dame was op haar gemak gaan zitten en vervolgde,

terwijl Beaupré als het ware aan haar lippen hing:
—De vier mannen zijn hier voor de deur in een huurauto gestapt en wij
meenden natuurlijk niets anders of de gewaande detectives zouden
hunne arrestanten rechtstreeksch naar Scotland Yard brengen. Er
geschiedde echter iets geheel anders met de twee mannen die hier zijn
geweest! De beide bedriegers hadden hen op de een of andere wijze—
hoe, dat weten de geneesheeren nog niet—bewusteloos gemaakt, en
toen de auto stil stond, hebben zij hen naar een onbewoond huis aan de
Bishop Street gebracht, en daar opgesloten. Maar nu komt het mooie!
De chauffeur, die hen gereden had, was nieuwsgierig uitgevallen! Hij
had een buitengewoon hooge fooi gekregen, om zoo snel mogelijk te
rijden, en dat droeg er niet toe bij, hem te kalmeeren, dat begrijpt gij wel!
—Ik begrijp het volkomen, miss! antwoordde de Fransche Markies, die

brandde van ongeduld om de rest van het verhaal te hooren.
—Hij had duidelijk gezien, dat de twee mannen die hem besteld hadden
de beide anderen onder den arm [8]hadden moeten nemen, en dat die er
al heel gek uitzagen met hun wijd geopende oogen, die echter niets
schenen te zien, en hun automatische bewegingen! Hij kon niet
begrijpen, wat die mannen in dat huis gingen uitvoeren, en omdat hij,
zooals gezegd, heel nieuwsgierig was—zoo reed hij niet weg, maar
plaatste zijn auto om een hoek van een dwarsstraat en stelde zich
verdekt op in een donker portiek!
—En.…..? vroeg Beaupré ademloos.
—Na een half uur kwamen er twee mannen uit het huis—en dat waren
de lieden, die geboeid waren binnengeleid! Maar van boeien was niets
te bekennen en zij liepen ook volkomen recht en natuurlijk! Zij schenen
groote haast te hebben en riepen een auto aan, die juist voorbij reed. En
de chauffeur was zoo overbluft, dat de wagen al uit het gezicht was,
voor hij er aan dacht hen met zijn eigen auto te volgen!
—Merkwaardig! mompelde Beaupré, om iets te zeggen, maar hij was

zeer verschrikt door dezen loop der zaken, waarvan Raffles nog niet het
minste vermoeden scheen te hebben!
—Daar de chauffeur de twee mannen toch niet meer kon achterhalen,

besloot hij de politie te waarschuwen, en die op de hoogte te brengen
van het zonderlinge voorval, waarvan hij zooeven getuige was geweest.
Het duurde eenigen tijd voor er een aantal agenten onder bevel van een
inspecteur ter plaatse waren en de dag begon al aan te breken toen
men eindelijk de buitendeur openbrak, daar er op het herhaaldelijk
schellen niet werd opengedaan en de chauffeur pertinent volhield, dat er
zich nog twee personen in het huis moesten bevinden. En wat denkt gij
wel, dat de agenten vonden?
—De twee rechercheurs? vroeg Beaupré onnoozel.
—Wel neen! antwoordde de hoofdverpleegster ongeduldig. Die waren al

lang weg! Zij vonden daar de twee mannen die hier waren geweest,
geboeid en wel, bewusteloos op een groot bed liggen!
—Dat is kras! bromde de Franschman.
—Niet waar? riep de verhaalster uit. Men deed alles, om de beide

mannen uit hun zonderlinge bewusteloosheid te doen ontwaken, maar
vruchteloos! Toen werd er een dokter bijgehaald—en toen nog een,
maar met hen beiden slaagden zij al evenmin! Zij moesten verklaren
hier voor een raadsel te staan! Zij beproefden alle bekende opwekkende
middelen, maar zij hadden dit evengoed met poppen kunnen doen!
Daarop werd besloten de twee mannen naar een gasthuis te doen
overbrengen! Een hunner ligt daar drie bedden van u af!
—Daar staat mijn verstand bij stil! riep Beaupré uit, ofschoon hij de
geheele zaak volkomen begreep. En waar is de andere?
—In de groote operatiezaal. De geneesheeren zijn op dit oogenblik

bezig allerlei middelen te beproeven om hem tot bewustzijn te brengen.
—En.….. lukt het? vroeg Beaupré snel, terwijl hij de verpleegster met
zijn groote, zwarte oogen vorschend aankeek.
—Neen! Hij ligt daar nog even stil en schijnbaar levenloos, ofschoon het
lichaam warm is, als toen hij hier werd binnen gebracht.
Beiden zwegen en eindelijk vroeg de hoofdverpleegster:
—Kunt gij u in het geheel niet voorstellen wat dit alles te beteekenen
heeft en in welke verhouding die twee zoogenaamde detectives met de
mannen stonden, die u hier gisterenavond zijn komen bezoeken?
—Neen, zeide Beaupré onvervaard, ofschoon hij het zich maar al te

goed kon voorstellen!
—Maar die twee bezoekers van gisteren—die kent gij toch wel?
—Slechts vluchtig, miss!
De hoofdverpleegster wilde nog iets zeggen, toen de deur geopend

werd en er een man met een krachtigen lichaamsbouw, ofschoon niet
zeer groot, met een vierkant, schrander gelaat, waarin twee
donkergrijze, energieke oogen schitterden, de ziekenzaal binnentrad.
Die man was James Sullivan, een der bekwaamste detectives van
Scotland Yard, die reeds eenige malen had deelgenomen aan de jacht
op den Grooten Onbekende, en tot zijn felste vijanden gerekend mocht
worden.
Hij bleef eenige oogenblikken op den drempel staan en trok de dichte,

zwarte wenkbrauwen samen, toen hij de hoofdverpleegster zoo druk in
gesprek zag met den gewonde. [9]
Toen trad hij snel op het bed toe en zeide tamelijk kortaf:
—Vergun mij een oogenblik, zuster.….. Dit is zeker de man, die hier
gisterenavond door messteken zwaar gewond werd binnen gebracht?
—Ja, mijnheer! antwoordde de hoofdverpleegster.
Sullivan trok haar een weinig terzijde en vroeg op zachten toon, zoodat
de zieke hem niet zou kunnen verstaan:
—Hebt gij dien man alles medegedeeld, wat u zooeven per telefoon is
gezegd?
—Ja, mijnheer! antwoordde de zuster aarzelend, en een weinig
schuldbewust, toen zij de ernstige, grijze oogen zoo strak op zich
gevestigd zag.
—Dat doet mij leed! hernam Sullivan en hij klemde de lippen opeen.
—Ik wist niet, dat ik er verkeerd aan deed, stamelde de

hoofdverpleegster. Wie is die man dan?
—Dat weet ik niet, maar hij speelt toch in dit alles een vrij dubbelzinnige
rol en het ware beter geweest, als gij hem onkundig hadt gehouden van
wat wij zoo pas ontdekt hebben! Nu, er is niets meer aan te doen—en ik
zou u nu wel gaarne verzoeken mij den man eenige vragen te laten
stellen. Hij schijnt sterk genoeg te zijn, om een kort verhoor te kunnen
ondergaan!
—Een verhoor! herhaalde de hoofdverpleegster verschrikt. Maar wat

vermoedt gij dan eigenlijk, mijnheer?
—Dat kan ik nu nog niet zeggen, miss! antwoordde Sullivan kortaf. Gij
kunt er trouwens bij tegenwoordig zijn, en goed opletten, of de man zich
zelf wellicht tegenspreekt!
De detective trad nu op het bed toe, zag den gewonde strak aan en
begon:
—Ik ben detective van Scotland Yard en aan mij is de taak opgedragen
onderzoek te doen, naar het geheimzinnig voorval, dat zich deels in dit
ziekenhuis, deels … ergens anders heeft afgespeeld en waarin gij
eveneens een rol hebt gespeeld, misschien ondanks uzelf! Hoe is uw
naam?
Het was een moeilijk oogenblik voor den Franschen Markies.
Hij kende den detective van aangezicht zeer goed en wist wie hij was—
een der beste speurneuzen van de politie der Engelsche hoofdstad.
Maar Sullivan herkende hem niet—dat was duidelijk—en dat was in
ieder geval een goede troef!
De bandiet dwong zich dus tot zoo groot mogelijke kalmte en

antwoordde:
—Mijn naam is Dubois—Pierre Dubois! Ik ben Franschman, als

Engelschman genaturaliseerd.
—Woont gij hier dan al lang?
—Sedert een aantal jaren!
—Men zou naar uw adressen kunnen informeeren?
Dat was een vraag waarop de Franschman niet gerekend had! Want
inderdaad was hij pas drie jaren in Engeland, en daarvan had hij nog
eenige maanden in de Fransche hoofdstad doorgebracht, als chef eener
bende!
Toch antwoordde hij onverschrokken:
—Dat spreekt vanzelf—ofschoon ik mij al die adressen niet al te goed

meer herinner! Ik heb in dozijnen pensions gewoond!
—Ja—dat kan ik mij begrijpen! antwoordde Sullivan droogjes. Men heeft

mij medegedeeld, mijnheer Dubois, dat gij hier zijt binnengebracht,
zwaar gewond door messteken. Zoudt gij mij willen zeggen hoe gij aan
die wonden gekomen zijt?
—Heel eenvoudig—ik ben op straat aangerand!
—Kunt gij het dan verklaren, hoe het komt, dat men u niets ontstolen
heeft? Uw beurs, horloge, uw zilveren sigarettenkoker zijn allen op uw
persoon gevonden!
Een ander zou door die vraag misschien in verwarring zijn gebracht,
maar niet aldus Beaupré!
Hij had zich nu hersteld en was vastbesloten zijn incognito tot het
uiterste te verdedigen.
En zoo antwoordde hij rustig:
—Ik vermoed, dat de straatroovers, die mij hebben overvallen, door de

nadering van agenten op de vlucht zijn gedreven voor zij gevolg hadden
kunnen geven aan hun voornemen mij te berooven.
—Maar gij zijt niet door agenten gevonden!
—Dan hebben zij het zich eenvoudig verbeeld en waren het burgers die
naderden en die mij gevonden hebben! Gij moet mij de opmerking ten
goede houden, mijnheer, maar dit begint veel te gelijken op een verhoor!
Mag ik weten, wat gij eigenlijk denkt of vermoedt? [10]
Sullivan beet zich op de lippen en scheen een oogenblik met zijn

houding verlegen.
Hij wantrouwde dezen man, dat was zeker, maar redenen, deugdelijke
redenen zou hij daarvoor niet kunnen opgeven.
En zoo zeide hij met een lichte buiging:
—Ik denk er niet aan u een verhoor af te nemen, mijnheer Dubois! Ik

wilde slechts eenige inlichtingen uit uw mond vernemen, die mij wellicht
van nut kunnen zijn, bij mijn verdere naspeuringen in deze duistere
zaak! Daarbij wilt gij mij toch zeker wel helpen?
—Ongetwijfeld! Als dit slechts in mijn vermogen is! Vraag vrij uit!
—Als gij mij dit toestaat dan zou ik u willen vragen: wie waren de twee
mannen, die u gisteravond kwamen bezoeken en wat wilden zij van u?
—Het zijn twee schurken, die iets uit mijn verleden weten, waarvan de
openbaarmaking mij groot nadeel zou kunnen berokkenen en daaruit
willen zij munt slaan! antwoordde Beaupré brutaal, ofschoon hij zijn hart
voelde kloppen bij het stellen van deze gevaarlijke vraag. Hun namen
wensch ik om begrijpelijke redenen niet te noemen.
—Maar dan hebben die mannen zich aan een strafbare zaak schuldig
gemaakt! riep Sullivan uit. En als gij een aanklacht in dient, kunnen wij
hen vervolgen wegens poging tot afpersing! Dat kunnen wij slechts dan
doen, als het slachtoffer zelf een klacht bij het parket indient!
—Misschien zal ik dat later ook wel doen, mijnheer! antwoordde

Beaupré. Als ik maar eerst dit ziekenhuis verlaten heb!
Sullivan haalde de schouders op.
—Uw stilzwijgen maakt onze taak niet gemakkelijker! zeide hij. Er heeft
hier een geheimzinnige gebeurtenis plaats gehad, waarin die twee
mannen een gewichtige rol vervullen. En het onderzoek naar de
identiteit van de beide gewaande detectives, die hen zijn komen
arresteeren—met een doel, dat ons volkomen onverklaarbaar is—zou
ons heel wat lichter worden gemaakt, als wij wisten, wie zij zijn!
—Wacht, tot zij uit hun bewusteloosheid ontwaakt zijn, kwam Beaupré
kortaf. Dan zullen zij wel spreken!
Hij had het stoutmoedig gezegd—maar bij zich zelf overwoog hij, dat het
voor hem wel eens zeer onaangename gevolgen zou kunnen hebben,
als de schurken inderdaad begonnen te spreken!
—Dat is ook juist een der meest verrassende zijden van deze gansche
geschiedenis! riep Sullivan uit. Geen der geneesheeren weet te zeggen,
welke eigenaardige verdooving de twee mannen heeft aangegrepen!
Beaupré bromde iets onverstaanbaars in zich zelf, maar hij antwoordde

niet.
Sullivan wierp nogmaals een verstolen, onderzoekenden blik op den
gewonde, en hernam toen:
—Ik wil u thans niet langer lastig vallen, want gij zult wel rust behoeven.
Later echter hoop ik u nogmaals eenige vragen te mogen stellen.
Hij knikte Beaupré toe en stapte vervolgens op het bed toe waar de
bewustelooze ter neder lag.
Eenigen tijd keek hij onafgebroken naar het witte gelaat met de wijd
geopende oogen en toen schudde hij het hoofd en haalde de schouders
op.
—Ik begrijp er niets van! mompelde hij. Het lichaam is blijkbaar warm en
volstrekt niet stijf—het heeft niet weinig van schijndood!
Juist op dit oogenblik ging de deur open en traden twee geneesheeren

binnen. [11]
[Inhoud]
HOOFDSTUK III.
Een raadselachtig geval.
De beide doktoren waren in een druk gesprek met elkander verdiept en

begaven zich dadelijk naar het bed van den bewusteloozen man.
Sullivan maakte dadelijk bescheiden plaats voor hen en bleef een

weinig op een afstand staan.
Wat Beaupré betreft—hij voelde zijn hart in zijn keel kloppen, want als
deze geneesheeren er werkelijk eens in slaagden om den man weder
tot bewustzijn te brengen, dan liep zijn vrijheid groot gevaar!
Want de man daarginds zou zeker wel vastgehouden worden—het zou

spoedig uitkomen wie hij werkelijk was—en dan zou hij den Markies
zeker in het ongeluk willen meeslepen, en diens waren identiteit
verraden.
Een der geneesheeren trad naast het hoofdeinde van het bed en trok
een der oogleden omlaag.
Even bleef het lid in dien zelfden toestand, maar toen schoof het uit zich
zelf langzaam weder naar boven.
De geneesheer lichtte een arm op en liet hem weder vallen, tastte den
pols, opende den mond, niet zonder moeite, en legde een thermometer
onder de tong van den bewustelooze.
Na eenigen tijd trok hij het instrument weder terug en raadpleegde het.
Hij schudde het hoofd, maakte een wanhopig gebaar en zeide op

zachten toon tot zijn collega:
—Normale bloedwarmte—iets meer dan zeven en dertig graden—de

pols een weinig langzaam—maar toch zoo goed als normaal! Geene
reflexbeweging—en een geringe verlamming van de oogspieren—die
blijkbaar slechts van tijdelijken aard is! Volkomen dezelfde
verschijnselen als bij den anderen man. Ik herken het volmondig—ik sta
hier voor een raadsel!
—Hebt gij reeds van alles geprobeerd? vroeg de andere geneesheer.
—Van alles! Onderhuidsche injecties, met een zoutoplossing, morphine

inspuiting, insnijdingen in de voetzool—waarbij ik er op wil wijzen, dat er
geen druppel bloed te voorschijn kwam—electrische bestraling, cocaïne,
cognac, het hielp alles niets! Ik zou zeggen dat wij hier te doen hebben
met atropie van de gevoelszenuwen, een tijdelijke stilstand van het
zintuigelijk waarnemingsvermogen, maar waardoor dat teweeg is
gebracht, en hoe wij het kunnen doen eindigen, dat is mij een raadsel!
—Hoelang zouden lieden zich reeds in dezen toestand bevinden?
—Op dit oogenblik bijna een half etmaal!
Nu trad Sullivan naderbij, stelde zich voor en zeide:
—Ik zou het bijzonder op prijs stellen, als ik u een vraag zou mogen
doen!
—Vraag slechts mijnheer—maar ik vrees, dat ik u wel niet zal kunnen

antwoorden, antwoordde de geneesheer die zooeven den patiënt
onderzocht had.
—Acht gij het mogelijk dat deze man zich uit zich zelf bewogen heeft?
De wenkbrauwen van den geneesheer gingen de hoogte in en als een

antwoord daarop antwoordde hij glimlachend met een kwalijk verborgen
medelijden van den vakman voor den leek:
—Dat is volkomen buiten gesloten!

—Maar hoe verklaart gij het dan, dat deze twee lieden zelf uit een auto
zijn gestapt en een huis zijn binnen gegaan?
—Dat verklaar ik niet, mijnheer—dat ontken ik—antwoordde de

geneesheer kortaf. Dat is onmogelijk! Als er getuigen zijn die verklaren
dat zij dit gedaan hebben, dan kunnen zij niet in de auto bewusteloos
zijn gemaakt, maar dan moet dit in dat huis geschied zijn!
—Ik dank u voor uwe bereidwilligheid om mij te antwoorden, maar ik

ben niet voldaan, antwoordde Sullivan. [12]
—Waarom niet? als ik vragen mag, hernam de geneesheer een weinig

uit de hoogte.
—Omdat ik mij niet kan voorstellen hoe die beide mannen zonder
eenigen tegenstand te bieden, of tenminste hun verbazing te uiten, dat
donkere, onbewoonde huis in de Bishop Street binnen gingen! Zij
moesten immers verwacht hebben naar Scotland Yard of naar een Huis
van Bewaring te worden overgebracht?
De geneesheer gaf niet aanstonds antwoord, maar stond met gefronst

voorhoofd stil, terwijl hij zenuwachtig met de vingers op zijn rug
friemelde.
Toen antwoordde hij een weinig gemelijk:
—Ik kan u op die vraag geen antwoord geven, mijnheer—dat zijn

politiezaken. Wel kan ik u echter zeggen dat er geen sprake van is dat
deze man of zijn metgezel zich zouden hebben kunnen bewegen.
Sullivan haalde vluchtig de schouders op en zeide:
—Als dat uw vaste overtuiging is—dan moet ik mij daar natuurlijk wel bij
neerleggen. Maar het maakt voor mij de zaak des te raadselachtiger.
Beaupré had het geheele gesprek, ofschoon het op tamelijk zachten

toon gevoerd was, gehoord, en hij was door zijn hoop en vrees heen en
weer geschommeld.
Nu waren zijn twee doodsvijanden nog bewusteloos, maar wie weet hoe
lang dat zou duren?
Zij konden weder ieder oogenblik tot het leven terugkeeren—met al de

gevolgen daarvan.
De Franschman zag nu hoe de beide geneesheeren zich verwijderden,

na nogmaals een blik op den bewustelooze geworpen te hebben en het
volgende oogenblik waren zij weg, door Sullivan op den voet gevolgd.
—Ik moet Marthe waarschuwen! mompelde Beaupré zacht. Zij moet

John Raffles op de hoogte brengen. Hij is de eenige die mij kan helpen!
Als die man daarginds spreekt ben ik verloren—en Raffles was de man
die hem bewusteloos heeft gemaakt en hij is dus waarschijnlijk de
eenige die hem weder tot het bewustzijn kan terugroepen. Hij moet hen
hier trachten weg te voeren, voor het te laat is.
En nu begonnen de hersens van den Franschen markies met

koortsachtige haast te werken.
Hij overwoog alle mogelijkheden, en ten slotte meende hij de

eenvoudigste oplossing te hebben gevonden. Hij zou zich houden alsof
zijn toestand plotseling zeer verergerde, en dan zou men zeker niet
weigeren Marthe aanstonds bij hem te laten komen.
Beaupré gaf aanstonds gevolg aan zijn voornemen!
Hij begon te kreunen, wentelde het hoofd van links naar rechts over zijn
kussen en het duurde niet lang of een der verpleegsters kwam
toeloopen, boog zich over hem heen, en vroeg:
—Hebt gij erge pijn?
—Vreeselijk, zuster! antwoordde Beaupré. Ik geloof dat het met mij ten
einde loopt.
—Maar uw toestand was van morgen redelijk, riep de zuster verschrikt
uit. Ik zal aanstonds de hoofdverpleegster roepen.
Deze werd gehaald en kwam haastig op het bed van den gewonde
toeloopen.
Nu was deze inderdaad een weinig uitgeput, door het langdurig en

opwindend gesprek, gevolgd door het verhoor van Sullivan en de vrees
voor zijn vrijheid had het zweet met fijne druppeltjes op zijn slapen doen
parelen.
Hij stak een bevende hand naar de hoofdverpleegster uit en zeide op

heeschen toon:
—Ik geloof dat het met mij mis loopt! Ik smeek u aanstonds mijn vriendin
te laten halen!
—Gij ziet werkelijk heel bleek, zeide de verpleegster verschrikt.
Zij legde den zieke den thermometer aan en bemerkte dat hij hooge
koorts had.
Een oogenblik stond zij in beraad, en toen nam zij een besluit en zeide:
—Geef mij het adres van uw vriendin—ik zal haar laten halen, maar gij
moogt volstrekt niet langer dan vijf minuten met haar spreken.
—Dat beloof ik u, Miss—vijf minuten zijn voldoende om afscheid van

haar te nemen,—kwam de Fransche Markies op dramatischen toon.
Hij noemde een afgelegen straat in een der Noordelijkste wijken van
Londen, een oogenblik later was er een telegram aan het adres van
Marthe Debussy gezonden.
Met koortsachtig ongeduld wachtte Beaupré de komst af van zijn

minnares en zijn vrees en ongerustheid deden hem veel zieker schijnen
dan hij inderdaad was.
Onophoudelijk wendde hij zijn blik naar den bewustelooze, vreezend
hem eensklaps te zien ontwaken. [13]
Het zou niet veel helpen, als hij zich onder de dekens verborg, want „Big
Billy”, zoo was de naam van den bewustelooze, wist zeer goed wie er in
dat bed lag!
Er verliepen twee bange uren—en toen werd de deur opengeworpen en

trad Marthe Debussy doodelijk verschrikt binnen.
Het telegram had haar zeer ontsteld en zij meende niet anders of zij zou
haar minnaar stervende vinden.
Zij snelde op het bed van Beaupré toe, maar deze stelde haar
onmiddellijk met eenige gefluisterde woorden gerust, en hernam daarop
iets luider opdat de verpleegster hem zou kunnen verstaan:
—Ik meende zooeven mijn einde te voelen naderen—en ik wilde je nog

eens zien voor ik stierf! Wij hebben slechts vijf minuten!
De verpleegster verwijderde zich bescheiden, en nu volgde Beaupré

zachtjes:
—„Big Billy” is in deze zelfde zaal gebracht—hij ligt drie bedden van mij
af—kijk aanstonds eens voorzichtig!
Marthe Debussy kon met moeite een kreet van schrik weerhouden, want
ook zij had aanstonds het gevaar begrepen!
Zij kende Big Billy maar al te goed en zij wist dat hij geen medelijden
zou kennen, als hij zelf gearresteerd werd—hij zou trouwens overtuigd
zijn, zijn vriend Dr. Fox een grooten dienst te bewijzen als hij den
Franschen markies, diens mededinger, in het verderf stortte!
Zij keek even schichtig in de aangeduide richting en zeide toen

fluisterend:

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Molecular Advances in Wheat and

Barley Manuel Martinez Editor

Advances in Protein Molecular and Structural Biology

Current Strategies to Improve the Nutritional and

Race and Blood in the Iberian World Max S Hering Torres

Advances in Water Resource Planning and Sustainability

Periodontitis Advances in Experimental Research

Beverage Sensory Modification Manuel Malfeito Ferreira

Advances in Speech and Music Technology Proceedings of

Advances in Cybersecurity Management Kevin Daimi

Special Issue Editor

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade

ISBN 978-3-03921-371-9 (Pbk)

About the Special Issue Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

Claus Krogh Madsen and Henrik Brinch-Pedersen

Mercedes Diaz-Mendoza, Isabel Diaz and Manuel Martinez

Iris Koeppel, Christian Hertig, Robert Hofﬁe and Jochen Kumlehn

Alexey V. Pigolev, Dmitry N. Miroshnichenko, Alexander S. Pushin, Vasily V. Terentyev,

Malika Ayadi, Faiçal Brini and Khaled Masmoudi

Jianxin Bian, Pingchuan Deng, Haoshuang Zhan, Xiaotong Wu, Mutthanthirige D. L. C.

Received: 10 July 2019; Accepted: 15 July 2019; Published: 16 July 2019

Int. J. Mol. Sci. 2019, 20, 3501; doi:10.3390/ijms20143501 1 www.mdpi.com/journal/ijms

Publication Species Topic/Molecular Advance Reference

Received: 20 March 2019; Accepted: 15 May 2019; Published: 18 May 2019

Int. J. Mol. Sci. 2019, 20, 2459; doi:10.3390/ijms20102459 6 www.mdpi.com/journal/ijms

Total P Total IP6 P Percent IP6 out Phytase Activity

2. Plant and Microbial Phytases

3. Mature Grain Phytase Activity

4. Classes of Phytases in Barley and Wheat

Km Vmax Kcat Kcat/Km pH

5. Biochemical Properties and Storage of the PAPhys

7. Applied Potentials of the PAPhys

8. Achieving Higher MGPA with the PAPhys

Funding: This research received no external funding.

Received: 26 March 2019; Accepted: 24 April 2019; Published: 28 April 2019

Keywords: barley; wheat; protease; germination; grain

Int. J. Mol. Sci. 2019, 20, 2087; doi:10.3390/ijms20092087 16 www.mdpi.com/journal/ijms

2. Mobilization of Stored Proteins During the Germination of Barley and Wheat

3. Investigation of Proteases in the Germination of Barley and Wheat before High-Throughput

Nowadays, advances in high-throughput sequencing have resulted in the development of new

4. Functional Genomic-Based Advances in the Identiﬁcation of Proteases in the Germination of

5. Proteomic-Based Advances on Proteases in the Germination of Barley and Wheat Grains

6. Conclusions and Future Perspectives

Received: 8 May 2019; Accepted: 24 May 2019; Published: 29 May 2019

1.1. Historical View of Genetic Modiﬁcation in Crop Plants

Int. J. Mol. Sci. 2019, 20, 2647; doi:10.3390/ijms20112647 27 www.mdpi.com/journal/ijms

1.2. Platforms of Customizable Endonucleases

2. Methodological Aspects of Cas Endonuclease Technology

2.1. System Components

2.4. From Site-Directed Mutagenesis to Precise Genome Editing

2.5. Identification and Characterization of Site-Specifically Modified Plants

Op dit oogenblik kwam er een hulpverpleegster aan, die eenige

Deze wendde zich tot het kleine groepje en zeide:

—Ik word daar juist door hoofdinspecteur Baxter aan de telefoon

Zij knikte de bezoekers vriendelijk toe en volgde de hulpverpleegster

Raffles en Beaupré hadden een eigenaardigen blik met elkander

—Op iets dergelijks zult ge wel niet gerekend hebben.

—Ik vraag verschooning—ik wist natuurlijk integendeel zeer goed wat

Raffles was reeds opgestaan en zeide nu tot zijn metgezellin: