Li et al.

BMC Genomics (2017) 18:896

DOI 10.1186/s12864-017-4290-5

RESEARCH ARTICLE Open Access

Proteomic insight into fruit set of

cucumber (Cucumis sativus L.) suggests the
cues of hormone-independent
parthenocarpy
Ji Li†, Jian Xu†, Qin-Wei Guo, Zhe Wu, Ting Zhang, Kai-Jing Zhang, Chun-yan Cheng, Pin-yu Zhu,
Qun-Feng Lou and Jin-Feng Chen*

Abstract
Background: Parthenocarpy is an excellent agronomic trait that enables crops to set fruit in the absence of
pollination and fertilization, and therefore to produce seedless fruit. Although parthenocarpy is widely recognized
as a hormone-dependent process, hormone-insensitive parthenocarpy can also be observed in cucumber; however,
its mechanism is poorly understood. To improve the global understanding of parthenocarpy and address the
hormone-insensitive parthenocarpy shown in cucumber, we conducted a physiological and proteomic analysis of
differently developed fruits.
Results: Physiological analysis indicated that the natural hormone-insensitive parthenocarpy of ‘EC1’ has broad
hormone-inhibitor resistance, and the endogenous hormones in the natural parthenocarpy (NP) fruits were stable
and relatively lower than those of the non-parthenocarpic cultivar ‘8419 s-1.’ Based on the iTRAQ technique, 683
fruit developmental proteins were identified from NP, cytokinin-induced parthenocarpic (CP), pollinated and
unpollinated fruits. Gene Ontology (GO) analysis showed that proteins detected from both set and aborted fruits
were involved in similar biological processes, such as cell growth, the cell cycle, cell death and communication.
Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that ‘protein synthesis’ was the major
biological process that differed between fruit set and fruit abortion. Clustering analysis revealed that different
protein expression patterns were involved in CP and NP fruits. Forty-one parthenocarpy-specialized DEPs
(differentially expressed proteins) were screened and divided into two distinctive groups: NP-specialized proteins
and CP-specialized proteins. Furthermore, qRT-PCR and western blot analysis indicated that NP-specialized proteins
showed hormone- or hormone-inhibitor insensitive expression patterns in both ovaries and seedlings.
Conclusions: In this study, the global molecular regulation of fruit development in cucumber was revealed at the
protein level. Physiological and proteomic comparisons indicated the presence of hormone-independent
parthenocarpy and suppression of fruit abortion in cucumber. The proteomic analysis suggested that hormone-
independent parthenocarpy is regulated by hormone-insensitive proteins such as the NP-specialized proteins.
Moreover, the regulation of fruit abortion suppression may be closely related to protein synthesis pathways.
Keywords: Cucumber (Cucumis sativus L.), Parthenocarpy, Proteome, iTRAQ, Hormone dependent/independent

* Correspondence: [email protected]
†
Equal contributors
State Key Laboratory of Crop Genetics and Germplasm Enhancement,
Nanjing Agricultural University, Nanjing 210095, China

© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Li et al. BMC Genomics (2017) 18:896 Page 2 of 18

Background Genetic studies have suggested that a majority of

Parthenocarpy is considered the most cost-effective solu- natural parthenocarpic properties in crops are quantita-
tion for improving the fruit set rate when pollination or tive traits regulated by both genetic and environmental
fertilization is suppressed by sub-optimum growth con- factors [6, 22–26]. Photoperiod, temperature, light
ditions, such as low temperature, weak light intensity or intensity and nutritional conditions have considerable
facility environments, and ensuring yields of vegetable influences on parthenocarpy [27–29]. A hypothesis
and fruit crops that are self-sterile or gynoecious. More- proposed in the 1930s suggested that plant developmen-
over, seedless fruits produced by parthenocarpy have a tal responses to environmental stimuli were due to the
better texture, appearance and shelf life [1] and avoid spatiotemporal variations in phytohormone synthesis
yield loss caused by seed development [2–4]. and transport [30, 31]. Studies have suggested that
Parthenocarpy is a widely recognized hormone- short-daylight conditions could enhance parthenocarpy
dependent biological process. Independent evidence indi- by increasing the activity of auxin, while high tempera-
cates that auxins play a vital role in parthenocarpic fruit tures suppressed the parthenocarpy rate by inhibiting
set. The genes linking the auxin signal transduction path- the synthesis of auxin and gibberellin in the ovary of
way to fruit set have been identified in recent decades. cucumber [32, 33]. Kim et al. [34] also found that
The involvement of IAA9, a member of the tomato Aux/ ovaries had twice the auxin content at 15 °C than that at
IAA gene family of transcriptional regulators, was con- 25 °C, resulting in a higher rate of parthenocarpy in
firmed in tomato fruit set. Auxin dose-response assays cucumber. Anyhow, the agricultural application of
showed that the down-regulation of IAA9 led to auxin parthenocarpy was limited by its environmental sensitiv-
hypersensitivity and resulted in parthenocarpy [5]. ity. In practice, the excessive application of exogenous
Another auxin signaling component involved in fruit set is hormones was often used to overwhelm the environ-
ARF8, which was identified as a candidate gene for two mental effects on hormone synthesis, thereby inducing
parthenocarpy QTLs in tomato [6]. Based on the findings environmentally stable parthenocarpy.
of Goetz et al. [7], a model was proposed for the Cucumber (Cucumis sativus L.) is emerging as a model
mechanism of parthenocarpic induction. According to this for plants in the Cucurbitaceae family because of its small
model, ARF8 forms an inhibitory complex together with and fully sequenced genome (2n = 2x = 14, 367 Mb
an AUX/IAA protein, possibly IAA9, to repress the genome) [35]. The mechanism for sex determination,
transcription of the auxin response genes and conse- vascular system development, and typical pepo fruit are
quently induce parthenocarpy. Wittwer et al. [8] showed also well documented in cucumber. The rich partheno-
that a second class of hormone, gibberellins (GAs), could carpic germplasm of cucumber offers an opportunity to
also stimulate parthenocarpic fruit set. The only known investigate the coordination and communication of hor-
gibberellin signaling component shown to be involved in mone signals and genes during parthenocarpic fruit set.
fruit set is DELLA. The reduction in SlDELLA mRNA Genetic studies of parthenocarpy in cucumber started in
levels induces the formation of parthenocarpic tomato 1930s. Early studies suggested that parthenocarpy in
fruit [9]. Null and loss-of-function recessive mutations in cucumber is controlled by single genes [36–40]. While
the DELLA genes of Arabidopsis provoke a constitutive most recent studies confirmed that inheritance of par-
GA-response phenotype, including parthenocarpy [10]. thenocarpy in cucumber is consistent with characteristics
Besides Auxin and GAs, Cytokinin (CK) is also involved of quantitative traits [25, 26, 41–43]. Although cucumber
in parthenocarpy, which accumulates to high levels in is rich in parthenocarpic germplasm resources e.g. main
ovaries during fruit set [11–15]. Recent studies suggested branch specialized/lateral branch specialized partheno-
that CKs may induce parthenocarpy partially through carpy, temperature/photoperiods sensitive parthenocarpy
modulation of IAA and GA metabolisms [16–18]. and parthenocarpy with accelerated ovary expansion
Ethylene and abscisic acid (ABA) also play important roles before anthesis, the agricultural application of partheno-
in the regulation of fruit set and development. Ethylene is carpic cucumber was limited by their environmental
likely involved in the fruit set program by functioning sensitivity [43]. A serial of studies indicated that stable
coordinately with auxin [19–21]. ABA may acts as an parthenocarpy of cucumber can be induced by auxin or
antagonist of GA or auxin to induce and maintain the auxin transport inhibitors [34, 44–48]. Meanwhile artifi-
dormant state of ovaries, likely by repressing their cially increasing of endogenous auxin in the ovary by
transition to fruit [20]. These studies demonstrate the introducing the DefH9-iaaM auxin-synthesizing gene into
complicated and confusing relationships among hormone cucumber might also stimulate parthenocarpy [49].
responses during fruit set. However, the key integrating Besides auxin, application of other hormones such as
molecular players remain largely undiscovered, and a cytokinins, gibberenllins and brassinosteroids (BRs) could
global understanding of the mechanisms underlying also promote parthenocarpy in cucumber [50, 51]. How-
parthenocarpy has yet to be attained. ever, it reported that auxin and GAs had less potential to
Li et al. BMC Genomics (2017) 18:896 Page 3 of 18

induce parthenocarpic fruit growth than CKs in cucumber and non-parthenocarpic cultivar ‘8419 s-1’ during natural/
[34, 51, 52]. Therefore, in practice application of exogen- cytokinin-induced parthenocarpy, pollinated fruit set and
ous cytokinin, particularly CPPU (N-(2-chloro-4-pyridyl)- unpollinated fruit abortion. The detail information of the
N′-phenyl urea, a type of synthetic cytokinin), to induce two cultivars was described in Material and Methods sec-
parthenocarpy is widely used in cucumber production. tion. The longitudinal and radial growth of the natural
In previous studies, an excellent parthenocarpic parthenocarpic fruits of ‘EC1’ and CPPU -induced par-
cucumber cultivar, ‘EC1,’ was found, which showed envir- thenocarpy of ‘8419 s-1’, pollinated and unpollinated fruits
onmentally stable parthenocarpy under different culture of ‘8419 s-1’ were measured (Fig. 1a). Our results showed
conditions [25, 43]. Previous transcriptome studies have that the length and diameter of the parthenocarpic and
demonstrated that the natural parthenocarpy (NP) of pollinated fruits linearly increased from 0 to 6 dpa (days
‘EC1’ has many different aspects compared with post-anthesis), and the natural and CPPU-induced par-
cytokinin-induced parthenocarpy (CP) at the mRNA level thenocarpic fruits showed similar growth curves, wherein
[53]. However, mRNA levels are not always in accordance the fruit size was generally larger than the pollinated
with protein activity. To improve the global understanding fruits. In contrast, the growth of the unpollinated fruits of
of parthenocarpy and address the environmental stability ‘8419 s-1’ was blocked, and the length and diameter of the
of parthenocarpy in cucumber, we conducted a physio- abortive fruits also decreased slightly.
logical analysis and an iTRAQ (isobaric tags for relative Kim et al. [34] suggested that genetic factor for par-
and absolute quantitation)-based proteomic analysis in the thenocarpy in cucumber may be associated with high
natural parthenocarpic fruits of ‘EC1’ and cytokinin- content of IAA in the ovaries at anthesis. In this study,
induced parthenocarpic fruits of ‘8419 s-1’ (a non- endogenous auxins, cytokinins, and gibberellins were
parthenocarpic variety). analyzed in the cucumber fruits noted above (Fig. 1b).
The induction of both naturally occurring and hormone
Results induced parthenocarpy is attributed to the presence of
Physiological comparison of the parthenocarpic and non- sufficient phytohormones in the ovaries [54–58]. How-
parthenocarpic cucumber cultivars ever, parthenocarpic fruits of ‘EC1’ had relatively low
Experiments were conducted to investigate the physio- and stable hormone levels compared with the fruits of
logical differences between parthenocarpic cultivar ‘EC1’ ‘8419 s-1.’ Moreover, the auxin and gibberellin

Fig. 1 Growth curve and endogenous hormone analysis of different cucumber fruits. a The length and diameter of natural parthenocarpic fruits
of ‘EC1,’ CPPU-induced parthenocarpic ‘8419 s-1’ fruits, and the pollinated and unpollinated fruits of ‘8419 s-1’ were measured from 0 dpa (days
post-anthesis) to 6 dpa. Each value represents the mean ± SE (n = 30). b The concentrations of auxins, cytokinins and gibberellins in the natural
parthenocarpic fruits of ‘EC1’ and pollinated/unpollinated fruit of ‘8419 s-1’ at −1 dpa to 3 dpa (analyzed by ELISA). The results are presented as
mean ± SE of three repeated sample pools (n = 10) with three technical replicates
Li et al. BMC Genomics (2017) 18:896 Page 4 of 18

concentrations also decreased unexpectedly during the processes of cell division and cell expansion. In addition,
natural parthenocarpic fruit set of ‘EC1’ (Fig. 1b). fruit growth is tightly related to the availability of carbo-
For a further comparison of the fruit developmental hydrate, because fruit is a very strong metabolic sink.
differences between ‘EC1’ and ‘8419 s-1,’ we conducted Many studies confirmed that CKs was demonstrated to
ovary treatment experiments. The ovaries of ‘EC1’ and regulate carbohydrate allocation in fruit [65, 66]. We
‘8419 s-1’ were treated with hormones, hormone inhibi- thought that maybe another reason why CPPU induced
tors and pollen separately at anthesis. The weight, length parthenocarpic fruit was consistently bigger than the
and diameter measurement of the treated ovaries was fruit induced by other PRGs.
conducted at 4dpa to reveal different phytohormone Martínez et al. [21] have demonstrated that the inhib-
responses between ‘EC1’ and ‘8419 s-1’. In cucumber, ition of ethylene response (STS treatment) is sufficient
etiolation of ovary tips is the principal identifying symbol to induce the set and early development of the fruit in
to identify whether the fruits are set or aborted since 2 absence of pollination in both the parthenocarpic and
dpa. It was showed that the ovaries with etiolated tips the non-parthenocarpic cultivar of zucchini squash.
did not grow or even wilt by comparing with the 0 dpa Coincidentally, it was showed that STS has stimulated
ovaries (Table 1). The non-etiolated tip phenotypes and parthenocarpy in the three non-parthenocarpic cucum-
growth of ovaries suggested that parthenocarpy of ber cultivars; however it had no effect on fruit develop-
‘8419 s-1’ could be induced by all of the exogenous ment of parthenocarpic cultivar ‘EC1’ (Additional file 2:
hormones, including NAA, CPPU, GA3 and EBR; Table S6). Besides, diameter, length and weight of
however, NAA, GA3 and EBR exerted weak effects on ethephon treated fruit of ‘EC1’ showed no significant
fruit growth, causing the treated ovaries to grow slightly differences to the natural parthenocarpic fruits
in length and remain in a dormant-like state (Table 1, (Additional file 2: Table S6). It indicated that neither
Additional file 1: Figure S1). Marcelis et al. [59] ethephon nor STS (ethylene response inhibitor) could
suggested that cell division in fruit of cucumber occurs affect parthenocarpy of EC. Interestingly, the ethephon
about a week after anthesis whereas cell size increases treated ovaries of non-parthenocarpic cucumbers
markedly only after cell division begins to decline. Fruit displayed more severe atrophy of ovaries by comparing
size increases because of increase in both the number with their unpollinated ovaries (Additional file 2: Table S6),
and size of cells. Therefore we speculated that NAA, suggested that the fruit abortion of the non-parthenocarpic
GA3 and EBR might be primarily involved in cell cultivars maybe accelerated by ethephon. Irradiated pollen
division, rather than cell expansion during fruit treatment could promote stenospermocarpy in 8419 s-1,
development of cucumber, thus ovaries treated by these but the stenospermocarpic ovaries were much smaller than
hormones are smaller than pollinated and CCPU-treated the active pollen-treated ovaries, implying that seed set may
fruits. On the other hand, the weak effect of NAA, GA3 be essential for the fruit growth of non-parthenocarpic
and EBR might be due to the low hormone concentration varieties. Interestingly, the seedless fruit of ‘EC1’ formed by
used in this study. Fu et al. [51] showed that 0.2 μM parthenocarpy was much larger than its pollinated fruit.
(20 mg/L) of EBR led to high efficiency of fruit growth in The pollination fruits of ‘8419 s-1’ were blocked by either a
cucumber. But in this study we found 10 mg/L of EBR hormone inhibitor mixture or individual hormone inhibi-
showed weak effect on fruit expansion. However, half con- tors (Table 1). Etiolation was observed in the pollen and
centration of CPPU (50 mg/L) referred to Fu’s method still hormone inhibitor co-treated ovaries. In contrast, the
lead to strong effect on fruit set and growth. natural parthenocarpic fruit set of ‘EC1’ could not be
The strong effect of CPPU on fruit development of blocked by hormone inhibitors, but the growth of the
cucumber was observed and documented in many other ovaries was suppressed (Table 1).
reports [34, 51, 52]. Similar observation was also found
in many other species such as watermelon, apple, iTRAQ-based proteomic study of differently developed
kiwifruit and blueberry [60–63]. Early fruit development cucumber fruits
generally consists of three stages: fruit formation, cell Early fruit development generally consists of three stages:
division and cell expansion [13]. The growth of cucum- fruit formation, cell division and cell expansion [13]. The
ber fruit size is often mirrored by the increase in cell earliest stage, in which the ovary is aborted or allowed to
number and size [64]. Previous cytological observation proceed with fruit development, is referred to as fruit set.
showed that the CPPU-treated cucumber fruits was initi- In this study, we focused on the fruit set stage in cucum-
ated with an increase of cell numbers in the pericarp ber, which is 0 to 2 days post-anthesis [53, 59, 64, 67, 68].
and placenta tissues, and the size of pericarp cells were The natural parthenocarpic fruits of ‘EC1,’ cytokinin-
bigger than natural parthenocarpic fruits, although the induced parthenocarpic fruits of ‘8419 s-1’, the pollinated
number of cell layers was similar [53]. The findings and unpollinated fruits of ‘8419 s-1’ were investigated
suggested that CPPU might be involved in both (described in the M&M section). The proteomes of these
Li et al. BMC Genomics (2017) 18:896 Page 5 of 18

Table 1 The measurement of ovary weight, length and diameter after treated by pollen, exogenous hormones and hormone inhibitors
0 dpa ovary Unpollinationb Pollination Ic Pollination II NAA CPPU GA3 EBR Hormone Mixd
Ovaries of Weight 0.75 ± 0.14 0.66 ± 0.15 D 8.39 ± 1.4 B 5.05 ± 0.94 C 1.01 ± 0.12 D 12.81 ± 1.25 A 1.01 ± 0.17 D 0.86 ± 0.21 D 13.04 ± 3.14 A
8419 s-1 (g) Da
Length 28.4 ± 2.4 E 27.60 ± 3.3 E 69.33 ± 4.25 58.91 ± 3.96 C 34.06 ± 1.91 D 77.76 ± 5.67 A 33.31 ± 2.59 31.31 ± 3.1 81.43 ± 6.77 A
(mm) B D DE
Diameter 6.86 ± 0.33 6.30 ± 0.48 D 13.46 ± 1.38 11.07 ± 2.8 C 6.96 ± 0.7 D 15.87 ± 1.9 A 6.98 ± 0.58 D 6.73 ± 0.33 14.99 ± 2.3 A
(mm) D B D
P/A/Te 0/30/30 – – 31/0/31 30/0/30 36/0/36 34/0/34 35/0/35

TIBA Lovastatin Uniconazole Brz Inhibitor Mixg

Pollinated ovaries Weight 0.67 ± 0.06 0.67 ± 0.05 D 0.69 ± 0.04 D 0.68 ± 0.03 0.62 ± 0.08 D
of 8419 s-1f (g) D D
Length 25.7 ± 26.8 ± 1.11 E 25.9 ± 1.59 E 26.9 ± 1.27 E 26.5 ± 1.40 E
(mm) 0.55 F
Diameter 5.96 ± 5.84 ± 0.05 F 5.70 ± 0.35 F 5.69 ± 0.31 F 6.07 ± 0.19 F
(mm) 0.24 F
P/A/T 0/35/35 0/29/29 0/30/30 0/32/32 0/30/30

0 dpa ovary Unpollination Pollination I TIBA Lovastatin Uniconazole Brz Inhibitor Mix
Ovaries of EC1 Weight 0.83 ± 0.07 8.09 ± 1.51 A 2.64 ± 0.49 1.56 ± 0.23 1.22 ± 0.37 0.93 ± 0.13 E 1.76 ± 0.38 C 1.11 ± 0.2 DE
(g) E B CD CDE
Length 30.6 ± 3.1 F 58.70 ± 2.74 A 45.27 ± 1.51 34.82 ± 1.74 34.52 ± 0.58 33.89 ± 0.55 38.06 ± 1.33 32.3 ± 2.53 EF
(mm) B D D DE C
Diameter 6.87 ± 0.27 10.58 ± 1.11 A 8.39 ± 7.20 ± 0.38 7.17 ± 0.78 8.81 ± 1.33 B 9.16 ± 1.35 B 7.84 ± 0.87D
(mm) E 1.47 BC DE DE
P/A/T 30/0/30 – 30/0/30 30/0/30 30/0/30 30/0/30 37/0/37
The treatments were conducted at the anthesis day (0 dpa), and the measurements were conducted at 4 dpa. Means (±SE) of three independent experiments
were calculated
a
Letters indicate differences between the treated ovaries with statistical significance at P ≤ 0.05 (t-test). The same letter means not significantly different;
different letters means significantly different
b
Underline words means the treatment can induce etiolation of ovary tips at 4dpa and lead to fruit abortion
c
‘Pollination I’ means hand pollination with active pollens; ‘Pollination II’ means hand pollination with irradiated pollens (γ-ray irradiation at a dose of 200 Gy)
d
Mixed solution of NAA (50 mg/L), CPPU (50 mg/L), GA3 (50 mg/L) and EBR (10 mg/L)
e
P/A/T: number of parthenocarpic ovaries/number of abortive ovaries/total treated ovaries; etiolation phenotype of ovary tips is the principal identifying
symbol to identify whether the fruits are set or aborted since 2dpa
f
Ovaries of 8419 s-1 were pollinated at 0 dpa, the hormone inhibitor treatments were conducted 3 h after pollination
g
Mixed solution of TIBA (50 mg/L), Lovastatin (50 mg/L), uniconazole (50 mg/L) and Brz (10 mg/L)

differently developed fruits were analyzed by iTRAQ with genes were actively involved in fruit development at
two technical replicates per sample. The strategy for both mRNA and protein levels.
analysis is shown in Additional file 3: Figure S2.
Protein homolog identification was conducted by Identification and comparison of differentially expressed
BLASTP against the cucumber Refseq database and proteins involved in different fruit developmental
the Arabidopsis thaliana Refseq database (E-value processes
<1E-10). After redundancies were removed, 683 Large numbers of polymorphic SNPs (Single Nucleotide
unique proteins were identified, including 359 fruit Polymorphisms), InDels (Insertion-Deletion Polymor-
set-related proteins (natural/cytokinin-induced par- phisms) and SVs (Structural Variations) were detected
thenocarpic fruit and pollinated fruit) and 377 fruit between ‘EC1’ and ‘8419 s-1’ by genome resequencing
abortion-related proteins. Gene Ontology (GO) ana- study [25]. Besides, 84 differentially expressed proteins
lysis showed that the proteins detected in both set were also identified between 0dpa fruits of ‘EC1’ and
and aborted fruits were involved in similar biological ‘8419 s-1’ (Additional file 4: Figure S3). These findings
processes (Fig. 2a). Proteins related to cell growth demonstrated that the genetic background of the two
(GO:0016049), cell cycle (GO:0007049), cell death varieties was significantly different with each other. In
(GO:0008219) and cell communication (GO:0007154) order to identifiy fruit developmental DEPs (Differentially
were detected that actively expressed during fruit Expressed Proteins), a proteome comparison strategy was
development, that was consistent with our previous employed (Additional file 3: Figure S2). Proteomic com-
transcriptomic study [53], suggesting the cell growth, parisons were not conducted between cultivars, therefore
cell cycle, cell death and cell communication related the differentially expression of identified DEPs were not
Li et al. BMC Genomics (2017) 18:896 Page 6 of 18

Fig. 2 Analysis of iTRAQ-detected proteins involved in the processes of fruit set and fruit abortion. a A total of 359 proteins were detected from
the fruits set by pollination or natural/cytokinin-induced parthenocarpy. Three hundred seventy-seven proteins were detected from abortive fruits.
GO analysis suggeste that the fruit developmental proteins were involved in similar biological processe. b Clustering analysis of differentially
expressed proteins from differently developed cucumber fruits. The cluster distance between the natural parthenocarpic and cytokinin-induced
parthenocarpic fruit was farther than that between cytokinin-induced parthenocarpic and abortive fruit; KEGG analysis of DEPs from different
cucumber fruits. c KEGG pathway analysis was performed using MapMan software (Version 3.5.1R2) according to the biological pathway maps
of Arabidopsis

caused by genetic variations but only correlated with guest/home). Large numbers of DEPs were shown to be
development of fruit. The false discovery rate (FDR) involved in the biological process of protein synthesis
method (FDR ≤ 0.05, |fold ≥ 1.5|, P-value < 0.05) was used and were mainly up-regulated during fruit set and
to determine the significance of the differential protein down-regulated during fruit abortion (Fig. 2c). Tran-
expression. Consequently 138 DEPs were identified and scriptome analysis has shown that amino acid metabol-
four groups of DEPs were screened (Additional file 4: ism, glycolysis and TCA cycle-related genes were
Figure S3). In pollination and CP fruits, most of the DEPs actively expressed during fruit set [53], corroborating
were up-regulated; in contrast, more down-regulated the present proteomic analysis, which also showed that
proteins were detected in the abortive fruit of ‘8419 s-1’ the proteins related to these biological processes were
and the natural parthenocarpic (NP) fruit. Clustering ana- actively expressed (Fig. 2c).
lysis indicated that the DEPs in CP and pollinated fruit The interactions between the DEPs were analyzed
showed similar protein expression patterns, but the pro- based on the reference proteome-wide binary protein-
tein expression profiles of the CP and NP fruits were clus- protein interaction (PPI) map of Arabidopsis [70]. A core
tered into two groups, and the cluster distance between fruit developmental PPI network was revealed, which
CP and NP fruits was greater than that between CP and consisted of 30 DEPs and 19 ‘bridging’ interaction pro-
the abortive fruit (Fig. 2b). teins (Fig. 3, Additional file 2: Table S1). The proteins
Kyoto Encyclopedia of Genes and Genomes (KEGG) within the PPI network were mainly involved in the pro-
pathway analysis of the DEPs was conducted using Map- cesses of protein metabolism (GO:0019538), transport
Man software [69], according to the biological pathway (GO:0006810) and signal transduction (GO:0007165).
maps of Arabidopsis (http://mapman.gabipd.org/web/ Interactions within the PPI network occurred more
Li et al. BMC Genomics (2017) 18:896 Page 7 of 18

Fig. 3 Prediction of protein-protein interactions (PPIs) in cucumber during fruit development. The PPI network was predicted by the reference
proteome-wide binary PPI map of Arabidopsis thaliana. The PPI network consists of 30 DEPs (in black color) and 19 interaction proteins (in blue
color). Within the PPI network, 23 proteins (marked by green circles) were protein metabolism (GO:0019538)-related proteins, nine transport
(GO:0006810)-related proteins (marked by yellow circles) and two signal transduction (GO:0007165)-related proteins (marked by red circles).
Interactions occurring in different fruit developmental processes are presented in Additional file 5: Figure S4. The annotations expressional
information of the proteins are presented in Additional file 2: Table S1

frequently in fruit abortion, while the smallest scale of is involved in cuticle metabolism and the maintenance
interactions was involved in natural parthenocarpy of plant water status [71], and PRL (PROLIFERA,
(Additional file 5: Figure S4). Two interaction proteins Csa7M407650.1), which is specifically expressed in pop-
exhibited specialized expression in natural partheno- ulations of dividing cells in the sporophytic tissues of the
carpy: CER9 (ECERIFERUM 9, Csa7M073540.1), which plant body [72]. TOC159 (Csa1M229500.1, a chloroplast
Li et al. BMC Genomics (2017) 18:896 Page 8 of 18

biogenesis-related protein), IMPA-6 (Csa1M597740.1, a divided into two groups, of which 19 were uniquely
nuclear import protein) and RS6 (Csa1M229500.1, a expressed in NP fruits, while the other 19 existed only in
putative ovule development regulator) showed special- CP fruits (Table 2). These DEPs were mainly involved in
ized up-regulation in CPPU-induced parthenocarpic the biological processes of pollen germination, seed and
fruit set (Additional file 2: Table S1). seedling development, cell proliferation and pro-
The common and specific DEPs in the differently devel- grammed cell death (Table 2). The NP-specialized DEPs
oped cucumber fruits are shown in the Venn diagram in Csa7M073540.1 and Csa7M450640.1, which were related
Fig. 4. No DEPs were commonly expressed in NP, CP and to cell cycle and proliferation, and Csa1M025890.1 and
pollination fruit. Twelve common proteins were identified Csa4M036590.1, the amino acid biosynthesis-related
in the CP and pollinated fruits and showed similar expres- proteins, showed dramatically up-regulated expression
sion patterns (Additional file 2: Table S2). Most of these during fruit set (expression fold >5) (Table 2). Moreover,
CP and pollination-specialized DEPs are closely related to Csa2M139820.1, which has the putative function of
pollen and seed development. Eleven DEPs were com- translational elongation, was the only DEP that was dra-
monly expressed in NP and abortive fruit, most of which matically increased in the CP fruit (Table 2).
were protein metabolism-related proteins and mainly
involved in the biological processes of pollen germination, Expression analysis of NP- and CP-specialized proteins in
gametophyte and endosperm development as well as root response to phytohormones
morphogenesis; however, these proteins showed opposite The transcription profiles of the parthenocarpy-
expression trends during parthenocarpy and fruit abortion specialized DEPs in the hormone- and hormone inhibitor-
(Additional file 2: Table S2). treated fruits were compared (the treated fruits are
Forty-one parthenocarpy-specialized DEPs were identi- described in the 1st part of the results section). Consistent
fied, but only 3 DEPs are commonly found in the NP with the result of iTRAQ, the specialized DEPs from both
and CP fruits (Additional file 2: Table S3). The parthenocarpy groups showed inactive transcription
remaining 39 parthenocarpy-specialized DEPs were during pollinated fruit set (Fig. 5, middle panel). The CP-
specialized DEPs were actively expressed in the cytokinin-
induced parthenocarpic fruits of ‘8419 s-1.’ However, in
the parthenocarpic fruits of ‘EC1,’ including the hormone
inhibitor-treated but not blocked parthenocarpic fruits,
most proteins showed decreased expression (Fig. 5, left
column of the panel). In contrast, the NP-specialized
DEPs showed up-regulated expression in parthenocarpic
fruits of ‘EC1’, whereas many of these DEPs were silenced
in the cytokinin-induced parthenocarpic fruits (Fig. 5,
right column of the panel). Although a few of the NP-
specialized DEPs were differentially transcribed in
cytokinin-induced parthenocarpic fruits, the expression
levels of the genes in these fruits were the same as those
in the parthenocarpic fruits of ‘EC1.’ Moreover, similar
transcription patterns of NP-specialized DEPs between
the natural parthenocarpic and unblocked natural
parthenocarpic fruits were found, indicating that the tran-
Fig. 4 Venn diagram and relative expression of DEPs in natural and scription of these proteins could not be affected by hor-
cytokinin-induced parthenocarpic, pollinated and abortive cucumber
mone inhibitors (Fig. 5, top right of the panel). These
fruits. The differently expressed proteins (DEPs) from the natural par-
thenocarpic fruit of ‘EC1’, cytokinin-induced parthenocarpic fruit of findings, to some extent, indicated that the transcription
‘8419 s-1’, pollinated and unpollinated fruits of ‘8419 s-1’ were com- of NP-specialized DEPs was not sensitive to hormones or
pared. Twelve DEPs were commonly expressed in cytokinin-induced hormone inhibitors.
parthenocarpic and pollinated fruits (the common DEPs are anno- iTRAQ showed that four NP-specialized proteins—C-
tated in Additional file 2: Table S2). Eleven DEPs were commonly
sa7M073540.1, Csa7M450640.1, Csa1M025890.1 and
expressed in natural parthenocarpic and abortive fruits and showed
opposite expression trends (Additional file 2: Table S2). Three DEPs Csa4M036590.1—showed a high abundance in expression
were parthenocarpy-specialized proteins that are commonly during parthenocarpy (expression >5-fold; Table 2,
expressed in both natural and cytokinin-induced parthenocarpic marked by stars). To further investigate the expression
fruits (Additional file 2: Table S3). The natural and cytokinin-induced characteristics of the active parthenocarpy-specialized
parthenocarpy-specialized proteins are individually annotated
proteins, we conducted a western blot analysis. Consistent
in Table 2
with the results of iTRAQ, the NP-specialized proteins
Li et al. BMC Genomics (2017) 18:896 Page 9 of 18

Table 2 DEPs specifically expressed in natural or Cytokinin induced parthenocarpic fruit

Protein ID Top Hita Descriptionb Relative Biological Processesc Expression fold of
DEPs in parthenocarpic
fruitd
Natural parthenocarpy specialized DEPs
Csa1M024830.1 AT1G48630.1 RACK1B, RECEPTOR FOR ACTIVATED Seed Germination and Early Seedling −1.97
C KINASE 1B Development; shoot development
(GO:0048367)
AT5G46180.1 DELTA-OAT, ORNITHINE-DELTA- Pollen germination and tube growth [86]; 85.50
Csa1M025890.1★ AMINOTRANSFERASE cellular amino acid biosynthetic process
(GO:0008652)
Csa1M025980.1 AT3G10920.1 MSD1, MANGANESE SUPEROXIDE Seed Germination [87]; female gametophyte −1.94
DISMUTASE 1 development; programmed cell death
Csa2M223140.1 AT3G04840.1 40S ribosomal protein S3a-like Pollen germination and tube growth [86] 2.58
protein
Csa2M338890.1 AT1G26880.1 60S ribosomal protein L34 Pollen germination and tube growth [86] 2.99
Csa3M002370.1 AT4G35630.1 PHOSPHOSERINE Serine biosynthesis; responses to cytokinin −3.16
AMINOTRANSFERASE 1, PSAT1
Csa3M827370.1 AT3G01280.1 VOLTAGE DEPENDENT ANION Female gametogenesis; pollen germination −2.53
CHANNEL 1, VDAC1 and tube growth [86]
Csa4M001980.1 AT1G02780.1 EMB2386, EMBRYO DEFECTIVE 2386 Pollen germination and tube growth [86]; 3.02
embryonic development (GO:0009790)
Csa4M012460.1 AT1G60710.1 Aldo/keto reductase family protein Seed Germination and Floral Development −2.65
AT4G39660.1 AGT2, ALANINE:GLYOXYLATE Responses to brassinosteroids; cellular amino 8.87
Csa4M036590.1★ AMINOTRANSFERASE 2 acid biosynthetic process (GO:0008652)
Csa4M179090.1 AT4G33680.1 AGD2, ABERRANT GROWTH AND Responses to cytokinin; pollen germination and −2.44
DEATH 2 tube growth (Wang et al. [86]); cell growth
Csa4M290220.1 AT1G09200.1 Histone H3 Cell cycle [88]; cell expansion and proliferation; −3.25
male gametogenesis
Csa4M664520.1 AT1G76550.1 Fructose-6-phosphate 1- seed development [89]; glycolysis (GO:0006096) 2.29
phosphotransferase
Csa6M193590.1 AT1G07660.1 Histone H4 Chromatin organization (GO:0006325) −3.16
Csa6M450410.1 AT2G36460.1 FBA6, FRUCTOSE-BISPHOSPHATE Seed Germination [87]; responses to cytokinin −2.03
ALDOLASE 6
Csa6M451470.1 AT3G52880.1 MDAR1, MONODEHYDROASCORBATE Pollen germination and tube growth [86]; −1.95
REDUCTASE 1 Cell Wall Regeneration
AT4G34100.1 CER9, ECERIFERUM 9 Seed Germination; pollen germination and tube 7.44
Csa7M073540.1★ growth [86]; Cell cycle [88]
Csa7M407650.1 AT4G02060.1 PRL, PROLIFERA Cell cycle and division [72]; GO:0007049) 3.66
AT1G67120.1 MDN1, MIDASIN 1 Seed germination and seedling development; 87.90
Csa7M450640.1★ female gametophyte development;
cell proliferation
Cytokinin induced parthenocarpy specialized DEPs
Csa1M003540.1 AT4G20360.1 RABE1B, RAB GTPASE HOMOLOG E1B Seed Germination and development [87, 89] 2.55
Csa1M031900.1 AT1G48410.1 AGO1, ARGONAUTE 1 Fruit development; cell division −1.67
Csa1M042700.1 AT3G18080.1 BGLU44, B-S GLUCOSIDASE 44 Female gametophyte development; −1.80
Cell wall proteins
Csa1M229500.1 AT5G20250.1 DIN10, DARK INDUCIBLE 10 Seed germination and seedling development; 1.78
pollen germination and tube growth [86]
Csa1M573730.1 AT5G56680.1 EMB2755, EMBRYO DEFECTIVE 2755 Gametogenesis and embryo development 1.95
(GO:0009793)
Csa1M597740.1 AT5G20720.1 CPN21, CHAPERONIN 20 Seed development [89]; pollen germination 1.55
and tube growth
Csa1M604600.1 AT1G50480.1 THFS,10- Seed development [89]; responses to cytokinin 2.88
FORMYLTETRAHYDROFOLATE
SYNTHETASE
AT1G07920.1 EF1α, Elongation factor 1-alpha Pollen development; translational elongation 6.08
Csa2M139820.1★ (GO:0006414)
Li et al. BMC Genomics (2017) 18:896 Page 10 of 18

Table 2 DEPs specifically expressed in natural or Cytokinin induced parthenocarpic fruit (Continued)
Protein ID Top Hita Descriptionb Relative Biological Processesc Expression fold of
DEPs in parthenocarpic
fruitd
Csa2M264020.1 AT4G34880.1 GAtA, Glutamyl-tRNA (Gln) amido Translation (GO:0006412) 2.70
transferase subunit A
Csa2M350200.1 AT1G24510.1 TCP-1/cpn60 chaperonin family Plant cell death [90] 1.95
protein
Csa4M094000.1 AT3G29360.1 UGD2, UDP-GLUCOSE Pollen germination and tube growth [86]; 3.98
DEHYDROGENASE 2 cell wall organization (GO:0007047)
Csa4M496230.1 AT5G63860.1 UVR8, UVB-RESISTANCE 8 Cell cycle (GO:0007049) −3.02
Csa5M623870.1 AT5G07030.1 Aspartic proteinase nepenthesin-1 Re-arrangements of cell wall [91] −2.44
Csa5M644550.1 AT3G02530.1 TCP-1/cpn60 chaperonin family Plant cell death [90] 1.85
protein
Csa6M439410.1 AT5G19440.1 Cinnamoyl CoA reductase-like Seed development [89]; lignin biosynthetic −4.09
protein pathway
Csa7M048110.1 AT3G14940.1 PPC3, PHOSPHOENOLPYRUVATE Development of male gametophyte; Cell cycle 2.75
CARBOXYLASE 3, [88]
Csa7M075590.2 AT5G42650.1 AOS, ALLENE OXIDE SYNTHASE Floral organ development; defense response −2.65
(GO:0006952)
Csa7M390010.1 AT3G02080.1 40S ribosomal protein S19 Translation (GO:0006412) 2.55
Csa7M405310.1 AT4G02290.1 GH9B13, GLYCOSYL HYDROLASE Root development; cell wall organization −2.42
9B13 (GO:0007047)
a
Homologous search was conducted by BLASTP against the Arabidopsis Refseq database (http://www.arabidopsis.org/index.jsp). E-value was set to <1E-
10. The Arabidopsis gene ID with highest score is picked for further analysis
b
The proteins were annotated based on the public databases: Arabidopsis database (http://www.arabidopsis.org/index.jsp) and cucumber Refseq
database (http://cucumber.genomics.org.cn/page/cucumber/index.jsp)
c
The proposed biological processes were refer from the GO terms and related research reports of the top hit Arabidopsis genes
d
The expression fold was calculated as the ratio of the protein expression in 2 dpa fruits vs. protein expression in 0 dpa fruit, P-value <0.05; The
dramatically increased (fold >5) DEPs were marked with stars

Fig. 5 Transcription analyses of parthenocarpy-specialized DEPs in different types of parthenocarpic fruits. Ovaries of ‘EC1’ and ‘8419 s-1’ were
treated by hormones and hormone inhibitors separately as described in the materials and methods section. Thus, different types of partheno-
carpic fruits were induced (Table 1, Additional file 1: Figure S1). Transcription analysis was conducted by quantitative real-time PCR. Transcriptional
profiles of the two groups of parthenocarpy-specialized DEPs in different types of parthenocarpic fruits were clustered. The experiment was re-
peated three times. Each value represents the mean ± SE of three replicates
Li et al. BMC Genomics (2017) 18:896 Page 11 of 18

were up-regulated during natural parthenocarpic fruit set Discussion

but inactively expressed in the cytokinin-induced fruit, Although studies on parthenocarpy have been conducted
while the NP-specialized protein was not detected in the for over 100 years, current understanding of partheno-
NP fruit but was actively expressed in the CP fruit (Fig. 6a). carpy remains at a nascent stage. Cucumber is emerging
Auxins, cytokinins and gibberellins are essential fruit as a model for the Cucurbitaceae family. The rich par-
developmental phytohormones, which can induce par- thenocarpic germplasm of cucumber offers an opportun-
thenocarpy in cucumber. The expression patterns of the ity to investigate the coordination and communication
parthenocarpy-specialized proteins responding to these of environmental factors, hormone signals and genes
hormones were also investigated (Fig. 6b). Our results during parthenocarpy. In this study, we investigated the
showed that the treatment with endogenous hormones proteomes of cucumber fruits to help improve the global
NAA, CPPU and GA3 increased the expression of the CP- understanding of parthenocarpy.
specialized protein Csa2M139820.1 but decreased the
expression of Csa4M036590.1 and Csa7M450640.1 (NP- Post-translational regulation of fruit development in
specialized proteins). Although the NP-specialized cucumber
proteins Csa1M025890.1 and Csa7M073540.1 were up- Proteins are executors with a vast array of functions
regulated by GA3, they were not significantly changed by within organisms. The translational and post-translational
NAA and CPPU (Fig. 6b, Additional file 6: Figure S5A). regulations of proteins such as protein synthesis, proteoly-
The protein expression pattern of Csa2M059750.1 was sis, glycosylation, phosphorylation and folding are
also analyzed, which was up-regulated during natural essential for plant development. Studies on hormone-
parthenocarpy and down-regulated in abortive fruit dependent biological processes have revealed that post-
(Additional file 2: Table S3). Csa2M059750.1 was the translational regulations frequently occur during fruit
unique DEP located in the chromosome region of the development. For instance, in the absence of auxins, the
major parthenocarpic QTL Parth2.1 of ‘EC1’ [25]. function of ARFs (auxin response factors) was inhibited
Western blot analysis showed that Csa2M059750.1 was via heterologous dimerization with Aux/IAA. However, in
degraded during fruit abortion but actively expressed the presence of auxin, the ARFs dissociated from Aux/
during fruit set. However, in contrast to the natural IAA proteins, whose targeting and degradation were
parthenocarpic fruit set, the increasing expression of mediated by the E3 ubiquitin ligase SCFTIR1/AFB, and
Csa2M059750.1 was delayed in the pollinated and CP consequently stimulated fruit set [5, 73–77]. The fruit
fruits until 3 dpa (Fig. 7a). Western blot analysis also developmental responses to ethylene were mediated by
showed that the expression of Csa2M059750.1 was not the SCFEBF1/EBF2-dependent proteolysis of EIN3 (Ethylene
significantly affected by hormones, indicating that insensitive 3) [78]. Furthermore, the function of EIN3 was
Csa2M059750.1 may be a hormone-insensitive protein regulated by the MAPK-dependent phosphorylation
(Fig. 7b, Additional file 6: Figure S5B). within the EPR1 domain of the transcriptional factor [79].

Fig. 6 Western blot analysis of the parthenocarpy-specialized proteins that were actively expressed during NP and CP fruit set. iTRAQ result
showed that Csa1M025890.1, Csa4M036590.1, Csa7M073540.1, and Csa7M450640.1 were dramatically increased in natural parthenocarpic fruits,
while Csa2M139820.1 was highly increased in cytokinin-induced parthenocarpic fruits (Table 2; marked by solid stars, >5-fold). The expression pat-
terns of these proteins were further analyzed by western blotting. a Expression analysis of the parthenocarpy-specialized proteins during NP and
CP fruit set individually. b Expression analysis of the parthenocarpy-specialized proteins in response to hormone treatments in seedlings. The cu-
cumber beta-actin (Csa5M182010.1) was used as reference protein for Western blotting. The experiment was repeated three times. The band in-
tensity analysis of western blots was conducted using ImageJ (Version 1.4), and the data are presented in Additional file 6: Figure S5A. CK:
Seedlings without phytohormone treatment; NAA: treated with 50 μM NAA; CPPU: treated with 10 μM CPPU; GA: treated with 10 μM GA3
Li et al. BMC Genomics (2017) 18:896 Page 12 of 18

Fig. 7 Expression analysis of Csa2M059750.1 during different fruit developmental processes and the response to phytohormone treatments.
Csa2M059750.1 was considered a candidate parthenocarpy regulatory protein by combined analysis of iTRAQ and genetic mapping results (Wu
et al. [25]). The protein expression of Csa2M059750.1 was analyzed by western blotting. a The expression of Csa2M059750.1 during fruit
development; b The expression of Csa2M059750.1 after phytohormone treatment in cucumber seedlings. The cucumber beta-actin
(Csa5M182010.1) was used as a reference protein for western blotting. The experiment was repeated three times. The band intensity analysis of
western blots was recorded using ImageJ (Version 1.4), for which the data are presented in Additional file 6: Figure S5B. CK: Seedlings without
phytohormone treatment; NAA1: treated with 5 μM NAA; NAA2: treated with 10 μM NAA; NAA3: treated with 50 μM NAA; CPPU: treated with
10 μM CPPU; GA: treated with 10 μM GA3

In addition, our previous transcriptome study confirmed Table S1). Protein metabolism related proteins were
that glycosylation reactions were dramatically active commonly expressed in unpollinated and natural
throughout fruit development in cucumber [53]. In the parthenocarpic fruit (Additional file 2: Table S2). The
present proteomic study, the protein folding-related opposite expression patterns of the common proteins
proteins were up-regulated in both pollinated and par- indicated the fate (set or abortion) of the mature ovary
thenocarpic fruits, including Csa1M255160.1, which was in cucumber, which was determined by protein metabol-
annotated as a TCP-1 (T-COMPLEX PROTEIN 1 ALPHA ism pathways.
SUBUNIT), and was actively expressed in natural and
cytokinin-induced parthenocarpic fruits (Additional file 2: The cues of hormone-independent parthenocarpy in
Table S3). Moreover, Csa2M099450.1, also defined as a cucumber
TCP-1-like protein, showed specialized expression in Growth measurement showed that natural and
pollinated and cytokinin-induced parthenocarpic fruits cytokinin-induced parthenocarpic fruits presented simi-
(Additional file 2: Table S1). lar growth curves (Fig. 1a). However, clustering analysis
Many lines of evidence indicate that protein synthesis/ showed that the protein expression profiles of the CP
degradation may function during cell growth [80, 81]. and NP fruits were quite different from each other.
The present proteomic study showed that over 30% of Moreover, the cluster distance between the CP and NP
the differentially expressed proteins during the cucum- fruits was greater than that between the CP fruit and the
ber fruit development were related to the biological abortive fruit (Fig. 2b). The specialized proteins
process of protein metabolism (Fig. 2c). Within the pre- expressed in the parthenocarpic fruits were divided into
dicted IPP network, nearly half of the interaction pro- two individual groups, which were separately involved in
teins were protein metabolism-related proteins, such as the NP and CP fruit set, as shown in the Venn diagram
the three TIF (TRANSLATION INITIATION FACTOR) (Fig. 4; Table 2). These findings suggested that there may
proteins involved in the initiation phase of eukaryotic be individual parthenocarpic pathways in cucumber.
translation, four CSN (COP9 signalosome) multi- Gustafson [54, 55] proposed that plants produce par-
proteins that functioned in the ubiquitin–proteasome thenocarpic fruits because the ovary contains enough
pathway, and three ribosomal proteins (Additional file 2: auxins to promote fruit initiation. Since then, many
Li et al. BMC Genomics (2017) 18:896 Page 13 of 18

studies have confirmed that parthenocarpy is a leads to fruit abortion in a short time (2 days at most).
phytohormone-dependent biological process. In cucum- Although natural parthenocarpic fruits of ‘EC1’ could
ber, polar auxin transport-blocking experiments have not be blocked by hormone inhibitors, these treated
shown that parthenocarpy could be triggered by the fruits stayed in a dormant state for a long time (more
sufficient accumulation of auxin in the ovary [44]. More- than 4 days) (Additional file 1: Figure S1). We speculated
over, the application of exogenous hormones such as that inhibitory regulations of fruit abortion might exist
auxins, cytokinins, gibberellins and brassinosteroids in ‘EC1’, causing the fruits to maintain a dormant state.
could induce parthenocarpy [51]. In this study, hormone Coincidentally, the common proteins that detected in
measurement showed that the endogenous hormone the NP fruits of ‘EC1’ and the abortive fruit of ‘8419 s-1,’
levels increased during fruit set but decreased during showed opposite expression trends. Most of these pro-
fruit abortion in ‘8419 s-1’ (Fig. 1b). However, the teins were down-regulated during fruit abortion but up-
endogenous hormone levels were relatively low and regulated during NP fruit set (Additional file 2: Table
remained stable during natural parthenocarpic fruit set S2). Besides, ethephon treating experiments showed that
in ‘EC1’ compared with ‘8419 s-1.’ Moreover, the NP although fruit abortion of the non-parthenocarpic culti-
fruits showed a broad resistance to hormone inhibitors vars was accelerated by ethephon which had no effect on
(Fig. 1b; Table 1; Additional file 1: Figure S1), indicating fruit development of ‘EC1’, further suggesting inhibitory
the existence of a hormone-independent parthenocarpic regulations of fruit abortion might exist in ‘EC1’.
mechanism in ‘EC1.’ This speculation was supported by Conversely, hormone inhibitor-induced dormant state in
expression analysis in the parthenocarpy-specialized pro- ‘EC1’ indicated that hormone stimuli might be required
teins, whereby the NP-specialized proteins performed for fruit expansion in either parthenocarpic cultivars or
hormone-insensitive transcriptional and translational non-parthenocarpic cultivars.
functions (Figs. 5, 6 and 7; Additional file 6: Figure S5).
Conclusions
Inhibiting the regulation of fruit abortion in cucumber Based on the evidence provided in this study, a working
Dormant fruits, as a result of first-fruit inhibition or nu- hypothesis for the cucumber parthenocarpic fruit set
tritional stress, can always be observed in the field [25, was proposed (Fig. 8), whereby parthenocarpy in cucum-
26]. However, the dormant state of these fruits usually ber may be promoted by a ‘parallel switch,’ namely,

Fig. 8 A proposed model for parthenocarpy in cucumber. Proposed model illustrating the working hypothesis of parthenocarpy, which can be
promoted by either hormone-dependent or -independent pathways. The hormone-unassociated stimulations may be regulated by the NP-
specialized proteins (Table 2) because of their hormone-insensitive expression characteristics. In the presence of sufficient hormone levels
(endogenous or exogenously supplied), the parthenocarpic young fruits can continue to grow. However, in the absence of hormones, hormone-
dependent parthenocarpic fruits will return to the fruit abortion pathway, while the hormone-independent parthenocarpic fruits will stay in a
dormant growth state that may be caused by abortion-inhibiting proteins. Whether the dormant fruits can restart growth or be artificially induced
remains unclear. ‘+’: in the presence of hormones; ‘-’: in the absence of hormones. The plant images were taken by JL in a greenhouse of Jiangpu
experimental station of Nanjing Agricultural University
Li et al. BMC Genomics (2017) 18:896 Page 14 of 18

hormone-dependent and hormone-independent path- The trapped ovaries of ‘EC1’ and ‘8419 s-1’ were also
ways. During hormone-independent parthenocarpy, fruit treated with phytohormones (NAA, 1-naphthaleneacetic
set was promoted by hormone-insensitive regulatory acid, 50 mg/L; CPPU, 100 mg/L; GA3, Gibberellin A3,
proteins, such as the NP-specialized proteins in ‘EC1.’ In 50 mg/L; EBR, Epi-Brassinosteroids, 10 mg/L; Ethephon,
the presence of sufficient hormones, young fruits formed 100 mg/L) and hormone inhibitors (TIBA, 2,3,5-triodo-
through both hormone-dependent and -independent benzoic acid, inhibitor of auxin, 50 mg/L; Lovastatin,
pathways could continuously grow to maturity. In the inhibitor of cytokinin, 50 mg/L; uniconazole, inhibitor of
absence of hormones, the development of hormone- gibberellin 50 mg/L; Brz, Brassinazole, inhibitor of
sensitive fruits proceeds to fruit abortion, whereas the Brassinosteroids, 10 mg/L; STS, silver thiosulphate,
hormone-insensitive fruits remain in a dormant state inhibitor of ethylene, 0.25 mM). The exogenous phyto-
because of the increasing expression of abortion- hormones and hormone inhibitors were separately
inhibiting proteins. However, the expansion of dormant sprayed on the surface of the ovaries at 0dpa. Active
fruits and their further promotion are unknown. Although pollens and irradiated pollens (γ-ray irradiation at a dose
the accurate regulation of parthenocarpy in cucumber of 200Gy) also used to treat the 0dpa ovaries by hand-
remains unclear, our studies provide a theoretical frame- pollination. After spaying and hand-pollination, the
work for understanding the mechanism of parthenocarpy ovaries were trapped again. The weight, length and
for its application in agricultural production. diameter of the ovaries were measured at 4dpa. The
experiments were repeated three times (n = 30). The
Methods treated ovaries were also harvested at 2dpa, of which the
Plant material and growth conditions RNA was isolated for qRT-PCR analysis.
In this study, the cucumber cultivar ‘EC1’ was used as a For western blotting, the seedlings of ‘8419 s-1’ (at the
parthenocarpic sample (Gynoecious inbred line, three true leaf stage) were also treated with exogenous
European Glasshouse type, parthenocarpic rate ≥ 95%) phytohormones (50 μM, 10 μM, or 5 μM NAA; 10 μM
and ‘8419 s-1’ as a non-parthenocarpic sample (Monoe- CPPU and 10 μM GA3) by spraying the solutions on the
cious inbred line, European Glasshouse type, the rare surface of the true leaves. After growing in the growth
occurrence of parthenocarpy is occasionally observed chamber with a 14 h photoperiod and 25 °C for 24 h, in
in the senescence phase of the cultivar). Plants were total 30 true leaves from five individual plants by same
grown in a greenhouse at Nanjing Agricultural Univer- treatment were collected and mixed by grinding in liquid
sity with a 14 h photoperiod, a mean daily air nitrogen, then stored at −80 °C before protein extraction.
temperature of 28/20 °C (day/night).
Protein extraction and quantization
Phytohormone measurement Approximately 1 g of powdered sample was mix with
Phytohormones were separately analyzed through ELISA 3 mL extraction buffer [500 mM Tris-HCl (pH 7.5),
using IAA, ZR and GA3 ELISA Kits (Sangon Biotech 150 mM NaCl, 50 mM ethylene diaminetetraacetic acid
Company) based on Weiler’s method [82]. The results (EDTA), 1% Triton-X-100, 2 mM dithiothreitol (DTT),
are presented as the mean ± SE (n = 10) with three 2 mM phenylmethanesulfonyl fluoride (PMSF)]. Protein
technical replicates. extraction was performed using the methods described
by Omar et al. [83]. The protein precipitation was
Ovary treatments collected and washed with cold methanol containing
The female flowers (at the 12-15th node of the main 10 mM DTT three times, cold acetone containing
stem) of the above cucumber cultivars were previously 10 mM DTT twice and then dried by vacuum freeze.
trapped with bags in order to prevent pollen contamin- The extracted proteins were quantified by using the
ation on the day before anthesis. When anthesis, the Bradford method [84].
trapped ovaries were treated separately: keeping trapping
(unpollination), hand pollination [34] and CPPU treat- Protein digestion and iTRAQ labeling
ment. CPPU (N-(2-chloro-4-pyridyl)-N′-phenyl urea) is One hundred micrograms Proteins from each samples
a kind of synthetic cytokinin which could induce par- were precipitated with five volume of cold acetone at
thenocarpy in cucumber. For CPPU treatment, 20 μL −20 °C for 1, centrifuged by 12,000 rpm for 15 min at
CPPU solution (100 mg/L) was sprayed on the surface of 4 °C, and dried by vacuum freeze dryer (Thermo savant,
the ovaries. All the treated ovaries were harvested at 0, USA). Pellets were dissolved in the dissolution buffer
1, 2 and 3 dpa (days-post-anthesis). Thirty ovaries of with reducing reagent described in iTRAQ Reagent 8-
each treatment were ground into powder with liquid Plex kit (Applied Biosystems, USA), and alkylated by
nitrogen and mix as a sample pool for iTRAQ and cysteine-blocking reagent according to the manufac-
western blot analysis. turer’s instructions [85]. After digestion with 50 μl of
Li et al. BMC Genomics (2017) 18:896 Page 15 of 18

50 ng/μl sequence grade modified trypsin (Promega, specific quantification was adopted to trace the differ-
USA) solution overnight at 37 °C, the peptide samples ences between expressions of various isoforms which
were labeled. The samples were labeled with the iTRAQ was applied to the peptide identification. Protein identi-
tags as described in Additional file 1: Figure S1. fication was performed with emphasis on biological
modifications option. Database search parameters were
SCX chromatography and LC–MS/MS analysis the followings: instrument was TripleTOF 5600, iTRAQ
The vacuum dried iTRAQ labeled samples were re- 8-plex quantification, cysteine modified with iodoaceta-
suspended with 100 μl SCX (Strong cation exchange) mide, biological modifications were selected as the ID
buffer A (10 mM ammonium formate, 20% ACN (aceto- focus, trypsin digestion. An automatic decoy database
nitrile), pH 2.8) and fractionated using a Poly-SEA search strategy was employed to estimate the false dis-
HPLC (High Performance Liquid Chromatography) col- covery rate (FDR) using the Proteomics System Perform-
umn (2.0 × 150 mm, 5 μm particle size, 300 pore size) ance Evaluation Pipeline Software (PSPEP) t was
using at a flow rate of 0.3 ml/min on the Agilent 1200 integrated in the Protein Pilot Software. In this study,
HPLC System (Agilent, USA). The 50 min HPLC gradi- only protein quantification data with the value of global
ent consisted of 100% buffer A (10 mM ammonium for- FDR ≤0.05 were chosen for further analysis, and proteins
mate, 20%ACN, pH 2.8) for 5 min, 0–50% buffer B with a |fold change ≥1.5| were considered to be signifi-
(500 mM ammonium formate, 20%ACN, pH 2.8) for cantly differentially expressed.
25 min, then 50–80% buffer B for 15 min, followed by
80–100% buffer B for 10 min, and lastly 100% buffer B Quantitative real time PCR
for 15 min. Chromatograms were recorded at 215 and Proteins based on their differential expression patterns
280 nm. All the collected fractions were vacuum dried, revealed by iTRAQ were selected for verification by
and re-suspended with Nano- RPLC (Reversed Phase Li- Quantitative real-time PCR (qRT-PCR) with primers
quid Chromatography) buffer A (0.1% FA, folic acid; designed using Primer 5.0 software (Additional file 2:
2%ACN). Samples were desalted with C18 nanoLC trap Table S4). Total RNA of the samples described above
column (100 μm ID × 3 cm, 3 μm particle size, 150 was extracted by Trizol (Invitrogen, USA). After extrac-
pore size) and Nano-RPLC buffer A (0.1%FA, 2%ACN) tion, total RNA was treated with DNase I (Fermentas,
at 2 μl/min for 10 min for LC–MS/MS analysis. UK) according to the manufacturer’s protocol. First-
The mass spectroscopy analysis was performed using a strand cDNA synthesis was carried-out using the Prime-
Triple TOF 5600 System (AB SCIEX, USA), coupled Script™ RT-PCR Kit (TaKaRa, Japan). The real-time
with the Eksigent nanoLC-Ultra™ 2D System (AB SCIEX, qRT-PCR was accomplished in a thermal cycler and
USA).The iTRAQ labeled peptides were separated using analyzed by an IQ5 multicolor Real-time PCR detection
an analytical ChromXP C18 column (75 μm ID × 15 cm, system (Bio-Rad, USA). To determine relative fold differ-
3 μm particle size, 120 Å pore size) (New Objectives, ences for each sample in each experiment, the CT values
USA) with a nanospray emitter (2500 V, 30 PSI (pounds were normalized using Cs-actin as an internal control
per square inch) curtain gas, 5 PSI nebulizer gas, 150 °C and calculated relative to a calibrator using the formula
interface heater temperature) (New Objectives, USA), 2-△△Ct. The experiment was repeated three times.
and analyzed by LC-MS/MS. A rolling collision energy
setting was applied to all precursor ions for collision- Western blot analysis
induced dissociation (CID). For information dependent Proteins extracted from ovaries and leaves as previ-
acquisition (IDA), survey scans were acquired in 250 ms ously described were mixed with protein lysis buffer
and as many as 35 product ion scans were collected if at 4 °C. Total protein lysis was boiled at 98 °C for
they exceeded a threshold of 150 counts per second 10 min and separated with 10% SDS-PAGE (Sodium
(counts/s) with a 2+ to 5+ charge-state. The total cycle dodecyl sulfate-polyacrylamide gel electrophoresis),
time was fixed to 2.5 s. Dynamic exclusion was set for then transferred to nitrocellulose (NC) membranes
one-half of peak width (18 s), and then the precursor (GE Hybond, USA) with semi-dry approach. After 2 h
was refreshed off the exclusion list. The peak areas of of blocking with 5% milk in TBST (Tris Buffered
the iTRAQ reporter ions reflect the abundance of the Saline with Tween), membranes were incubated with
proteins in the samples. polyclonal antibodies against the proteins of cucum-
ber that were raised in rabbit by synthetic peptides
Protein identification (Lufei, P.R.China; Additional file 2: Table S5). Poly-
Mass spectrometric data was processed with Protein clonal antibody against cucumber beta-actin was used
Pilot Software v. 4.0 (AB SCIEX, USA) against Cucum- as internal control. The antibodies were used at 1:300
ber database using the Paragon algorithm, and further dilutions. Membranes were incubated with goat anti-
processed by a Pro Group algorithm where isoform- rabbit IgG (Proteintech, USA) at 1:2000 dilutions in
Li et al. BMC Genomics (2017) 18:896 Page 16 of 18

TBST for 2 h, after that membranes were washed Funding

with TBST for five times. Signals were detected by This work was supported by the National Natural Science Foundation of
China [31430075, 31672168]; Special Fund for Agro-Scientific Research in the
using enhanced electro-chemiluminescence (Beyotime, Public Interest [201403032]; National Key Research and Development
P.R. China). Program of China [2016YFD0101705-5]; Agricultural science and Technology
Innovation Fund of Jiangsu Province [CX(15)1019]. 31430075, 31672168 were
involved in iTRAQ analysis. 201403032, 2016YFD0101705-5 and CX(15)1019
Additional files were involved in sample collection. The funding bodies were not involved in
the design of the study and collection, analysis, and interpretation of data
and in writing the manuscript.
Additional file 1: Figure S1. The typical phenotypes of the treated
ovaries of ‘EC1’ and ‘8419 s-1’ at 4 dpa. (DOCX 196 kb)
Availability of data and materials
Additional file 2: Table S1. The annotation and expressional The datasets generated during the current study are available from the
information of the proteins within the protein-protein interactions (PPIs) corresponding author on reasonable request.
which presented in Fig. 5. Table S2. DEPs commonly expressed in
pollinated and Cytokinin induced parthenocarpic fruit (I), and DEPs Authors’ contributions
commonly existed in unpollinated and natural parthenocarpic fruit (II). JFC and JL conceived the study. JL and JX performed most of the
Table S3. DEPs commonly expressed in natural and cytokinin induced experiments. QWG helped to prepare samples for iTRAQ. ZW, TZ, KJZ and
parthenocarpic fruits. Table S4. The primers of natural and cytokinin CYC carried out the iTRAQ data analysis. PYZ together with JX performed the
induced parthenocarpy specialized protein encoding genes for qRT-PCR. phytohormone measurement. QFL gave useful advices and contributed to
Table S5. The antigenic peptides of parthenocarpy specialized proteins experiments. JL interpreted the results and wrote the paper. All authors read
for producing rabbit polyclonal antibodies. Table S6. The measurement and approved the final version of the manuscript.
of ovary weight, length and diameter after treated by ethephon and
ethylene inhibitors.The treatments were conducted at the anthesis day (0 Ethics approval and consent to participate
dpa), and the measurements were conducted at 4 dpa. Means (±SE) of The plant material used in this work has been generated for research. All the
three independent experiments were calculated.Letters indicate materials were grown in Jiangpu experimental station of Nanjing Agricultural
differences between the treated ovaries with statistical significance at University. The seeds are maintained in State Key Laboratory of Crop
P ≤ 0.05 (t-test).Detail information of EC1 and 8419 s-1 was described in Genetics and Germplasm Enhancement (Nanjing Agricultural University).
M&M section. Both cucumber cultivars CC3 and CCMC were non-
parthenocarpic, monoecious inbred lines, Asia ecotype. (XLSX 35 kb) Consent for publication
Additional file 3: Figure S2. The analytical strategy of the iTRAQ based Not applicable
proteome analysis of cucumber fruits. Total proteins of each sample were
extracted and labeled separately with tags (113 to 118). Proteomic Competing interests
analysis was conducted by iTRAQ. The differentially expressed proteins The authors declare that they have no competing interests.
(DEPs) were identified by comparing proteomes of 0 and 2 dpa ovaries
of each treatment. CK1 and CK2: Control sample 1 and Control sample
2; NP: natural parthenocarpic fruits of EC1; Unp: Unpollination fruits of
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in
8419 s-1 (fruit abortion); P: pollination fruits of 8419 s-1; CP: Cytokinin
published maps and institutional affiliations.
induced parthenocarpic fruits of 8419 s-1. Bar = 20 mm. (DOCX 357 kb)
Additional file 4: Figure S3. Statistics of differentially expressed Received: 5 May 2017 Accepted: 9 November 2017
proteins during different fruit developmental processes of cucumber. NP:
natural parthenocarpic fruits of EC1; Unp: Unpollination fruits of 8419 s-1
(fruit abortion); P: pollination fruits of 8419 s-1; CP: Cytokinin induced References
parthenocarpic fruits of 8419 s-1. (DOCX 247 kb) 1. Pandolfini T. Seedless fruit production by hormonal regulation of fruit set.
Additional file 5: Figure S4. Protein interactions occurred in different Nutrients. 2009;1(2):168–77.
fruit developmental processes based on the predicted PPIs network. The 2. Tiedjens VA. The relation of environment to shape of fruit in Cucumis
interactions marked in red color means the interactions involved in Sativus L. and its bearing on the genetic potentialities of the plants. J Agr
Cytokinin induced parthenocarpy (A), natural parthenocarpy (B), pollination Res. 1928;36:794–809.
fruit set (C) and unpollination fruit abortion (D). (DOCX 349 kb) 3. Denna DW. Effects of genetic parthenocarpy and gynecious flowering habit
on fruit production and growth of cucumber Cucumis Sativus L. J Am Soc
Additional file 6: Figure S5. Band intensity analysis of western blotting
Hortic Sci. 1973;98(6):602–4.
by using the software ImageJ. The relative expression fold of each
4. Falavigna A, Soressi GP. Influence of the pat-sha gene on plant and fruit
parthenocarpy specialized protein was calculated by the formula: (band
traits in tomato (L. esculentum mill.) In: Modern trends in tomato genetics
intensity after hormone treatments/band intensity without hormone
and breeding; 1987. p. 128.
treatment)/(band intensity of beta-actin in hormone treated sample/band
5. Wang H, Jones B, Li Z, et al. The tomato aux/IAA transcription factor IAA9 is
intensity of beta-actin in untreated sample). Each value represents the
involved in fruit development and leaf morphogenesis. Plant Cell. 2005;
mean ± SE of three Western blotting replicates. (DOCX 238 kb)
17(10):2676–92.
6. Gorguet B, Eggink PM, Ocana J, et al. Mapping and characterization of novel
parthenocarpy QTLs in tomato. Theor Appl Genet. 2008;116(6):755–67.
Abbreviations
7. Goetz M, Vivian-Smith A, Johnson SD, et al. AUXIN RESPONSE FACTOR8 is a
Brz: Brassinazole, a kind of inhibitor of Brassinosteroids; CP: Cytokinin induced
negative regulator of fruit initiation in Arabidopsis. Plant Cell. 2006;18(8):
parthenocarpy; CPPU: N-(2-chloro-4-pyridyl)-N0-phenylurea, a diphenylurea-
1873–86.
derived cytokinin; DEP: Differentially expressed protein; dpa: days post
8. Wittwer SH, Bukovac MJ, Sell HM, et al. Some effects of gibberellin on
anthesis; EBR: Epi-Brassinosteroids; FDR: False discovery rate; iTRAQ: isobaric
flowering and fruit setting. Plant Physiol. 1957;32(1):39.
tags for relative and absolute quantitation; NP: NATURAL Parthenocarpy;
9. Martí C, Orzáez D, Ellul P, et al. Silencing of DELLA induces facultative
QTL: Quantitative trait locus; STS: Silver thiosulphate; TIBA: 2,3,5-triodobenzoic
parthenocarpy in tomato fruits. Plant J. 2007;52(5):865–76.
acid, a kind of inhibitor of auxin
10. Carrera E, Ruiz-Rivero O, Peres LEP, et al. Characterization of the procera
tomato mutant shows novel functions of the SlDELLA protein in the
Acknowledgements control of flower morphology, cell division and expansion, and the
We would like to thank Professor Robert N. Trigiano (University of Tennessee, auxin-signaling pathway during fruit-set and development. Plant Physiol.
USA) for critical reading of the manuscript. 2012;160(3):1581–96.
Li et al. BMC Genomics (2017) 18:896 Page 17 of 18

11. Mapelli S. Changes in cytokinin in the fruits of parthenocarpic and normal 40. Meshcherov E, Juldasheva L. Parthenocarpy in cucumber. Trudy Prikl Bot
tomatoes. Plant Sci Lett. 1981;22(3):227–33. Genet Selek. 1974;51:204–13.
12. Bohner J, Bangerth F. Effects of fruit set sequence and defoliation on cell 41. Shawaf EI, Baker L. Inheritance of parthenocarpic yield in gynoecious
number, cell size and hormone levels of tomato fruits (Lycopersicon pickling cucumber for once-over mechanical harvest by diallel analysis of
esculentum mill.) within a truss. Plant Growth Regul. 1988;7(3):141–55. six gynoecious lines. J Am Soc Hortic Sci. 1981;106:359–64.
13. Gillaspy G, Ben-David H, Gruissem W. Fruits: a developmental perspective. 42. Sun ZY, Lower RL, Staub JE. Analysis of generation means and components
Plant Cell. 1993;5(10):1439. of variance for parthenocarpy in cucumber (Cucumis sativus L.). Plant Breed.
14. Srivastava A, Handa AK. Hormonal regulation of tomato fruit development: 2006;125:277–80.
a molecular perspective. J Plant Growth Regul. 2005;24(2):67–82. 43. Yan LY, Lou LN, Li XL, et al. Evaluation of parthenocarpy in cucumber
15. Trueman SJ. Endogenous cytokinin levels during early fruit development of germplasm. Acta Hortic Sin. 2009;36:975–82. (in Chinese with English abstract)
macadamia. Afr J Agric Res. 2010;5(24):3402–7. 44. Robinson RW, Cantliffe DJ, Shannon S. Morphactin-induced parthenocarpy
16. Matsuo S, Kikuchi K, Fukuda M, et al. Roles and regulation of cytokinins in in the cucumber. Science. 1971;171(3977):1251–2.
tomato fruit development. J Exp Bot. 2012;63(15):5569–79. 45. Cantliffe D. Parthenocarpy in the cucumber induced by some plant growth-
17. Ding J, Chen B, Xia X, et al. Cytokinin-induced parthenocarpic fruit regulating chemicals. Can J Plant Sci. 1972;52(5):781–5.
development in tomato is partly dependent on enhanced gibberellin and 46. Quebedeaux B, Beyer E. Chemically-induced parthenocarpy cucumber by a
auxin biosynthesis. PLoS One. 2013;8(7):e70080. new inhibitor of auxin transport. Hortscience. 1972;7:474–6.
18. Li Y, Yu JQ. Photosynthesis and 14C-assimilate distribution as influenced by 47. Elassar GJ, Patevitch D, Kedar N. Induction of Parthenocarpic fruit
CPPU treatment on ovary. Acta Agric Nucleatae Sin. 2001;15:355–9. (in development cucumber by growth regulators. Hortscience. 1974;9(3):238–9.
Chinese with English abstract) 48. Takeno K, Ise H, Minowa H, et al. Fruit growth induced by benzyladenine in
19. Vriezen WH, Feron R, Maretto F, et al. Changes in tomato ovary Cucumis Sativus L.: influence of benzyladenine on cell division, cell enlargement
transcriptome demonstrate complex hormonal regulation of fruit set. New and indole-3-acetic acid content. J Jpn Soc Hortic Sci. 1992;60(4):915–20.
Phytol. 2008;177(1):60–76. 49. Yin Z, Malinowski R, Ziolkowska A, et al. The DefH9-iaaM-containing
20. Pascual L, Blanca JM, Cañizares J, et al. Transcriptomic analysis of tomato construct efficiently induces parthenocarpy in cucumber. Cell Mol Biol Lett.
carpel development reveals alterations in ethylene and gibberellin synthesis 2006;11(2):279–90.
during pat3/pat4 parthenocarpic fruit set. BMC Plant Biol. 2009;9(1):67. 50. Ogawa Y, Inoue N, Aoki S. Promotive effects of exogenous and endogenous
21. Martínez C, Manzano S, Megías Z, et al. Involvement of ethylene gibberellins on the fruit development in Cucumis sativus L. J Jpn Soc Hortic
biosynthesis and signalling in fruit set and early fruit development in Sci. 1989;58(2):327–31.
zucchini squash (Cucurbita pepo L.). BMC Plant Biol. 2013;13(1):139. 51. Fu FQ, Mao WH, Shi K, et al. A role of brassinosteroids in early fruit
22. Fos M, Nuez F, García-martínez JL. The gene pat-2, which induces natural development in cucumber. J Exp Bot. 2008;59(9):2299–308.
parthenocarpy, alters the gibberellin content in unpollinated tomato 52. Hikosaka S, Sugiyama N. Effects of exogenous plant growth regulators on yield,
ovaries. Plant Physiol. 2000;122(2):471–80. fruit growth, and concentration of endogenous hormones in Gynoecious
23. Beraldi D, Picarella ME, Soressi GP, et al. Fine mapping of the parthenocarpic Parthenocarpic cucumber (Cucumis sativus L.). Hortic J. 2015;84(4):342–9.
fruit (pat) mutation in tomato. Theor Appl Genet. 2004;108(2):209–16. 53. Li J, Wu Z, Cui L, et al. Transcriptome comparison of global distinctive
24. Miyatake K, Saito T, Negoro S, et al. Development of selective markers linked features between pollination and parthenocarpic fruit set reveals
to a major QTL for parthenocarpy in eggplant (Solanum melongena L.). transcriptional phytohormone cross-talk in cucumber (Cucumis sativus L.).
Theor Appl Genet. 2012;124(8):1403–13. Plant Cell Physiol. 2014;55(7):1325–42.
25. Wu Z, Zhang T, Li L, et al. Identification of a stable major-effect QTL (Parth 54. Gustafson FG. Further studies on artificial parthenocarpy. Am J Bot. 1938a;
2.1) controlling parthenocarpy in cucumber and associated candidate gene 25:237–44.
analysis via whole genome re-sequencing. BMC Plant Biol. 2016;16(1):182. 55. Gustafson FG. Induced parthenocarpy. Bot Gaz. 1938b;99(4):840–4.
26. Lietzow CD, Zhu H, Pandey S, et al. QTL mapping of parthenocarpic fruit set 56. Schwabe WW. Hormones and parthenocarpic fruit set, a literature survey.
in north American processing cucumber. Theor Appl Genet. 2016;129(12): Hortic Abstr. 1981;51:661–98.
2387–401. 57. Vivian-Smith A, Koltunow AM. Genetic analysis of growth-regulator-induced
27. Smith O, Cochran HL. Effect of temperature on pollen germination and parthenocarpy in Arabidopsis. Plant Physiol. 1999;121(2):437–52.
tube growth in the tomato. New York: The University; 1935. 58. Spena A, Rotino GL, Bhajwani SS, Soh WY. Parthenocarpy. State of the art,
28. Rylski I. Effects of season on parthenocarpic and fertilized summer squash Current trends in the embryology of Angiosperms. Kluwer Academic
(Cucumis pepo L.). Exp Agric. 1974;10(01):39–44. Publishers; 2001. p. 435-450.
29. Sun C, Li Y, Zhao W, et al. Integration of hormonal and nutritional cues 59. Marcelis LFM, Baan Hofman-Eijer LR. Cell division and expansion in the
orchestrates progressive corolla opening. Plant Physiol. 2016;171(2):1209–29. cucumber fruit. J Hortic Sci. 1993;68(5):665–71.
30. Cholodny N. Beiträge zur hormonalen Theorie von Tropismen. Planta. 1928; 60. Huitrón MV, Diaz M, Diánez F, et al. Effect of 2, 4-D and CPPU on triploid
6(1):118–34. watermelon production and quality. Hortscience. 2007;42(3):559–64.
31. Went FW, Thimann KV. Phytohormones. NY: Macmillan; 1937. 61. Bangerth F, Schröder M. Strong synergistic effects of gibberellins with the
32. Rudich J, Baker LR, Sell HM. Parthenocarpy in Cucumis sativus L. as affected synthetic cytokinin N-(2-chloro-4-pyridyl)-N-phenylurea on parthenocarpic
by genetic parthenocarpy, thermo-photoperiod, and femaleness. J Am Soc fruit set and some other fruit characteristics of apple. Plant Growth Regul.
Hort Sci 1977;102(2):225-8. 1994;15(3):293–302.
33. Matlob AN, Kelly WC. Growth regulator activity and parthenocarpic fruit 62. Lewis DH, Burge GK, Hopping ME, et al. Cytokinins and fruit development in
production in snake melon and cucumber grown at high temperature. J the kiwifruit (Actinidia deliciosa). II. Effects of reduced pollination and CPPU
Am Soc Hortic Sci. 1975;100:406–9. application. Physiol Plant. 1996;98(1):187–95.
34. Kim IS, Okubo H, Fujieda K. Endogenous levels of IAA in relation to 63. NeSmith DS. Response of rabbiteye blueberry (Vaccinium ashei Reade) to the
parthenocarpy in cucumber (Cucumis sativus L.). Sci Hortic. 1992;52(1–2):1–8. growth regulators CPPU and gibberellic acid. Hortscience. 2002;37(4):666–8.
35. Huang S, Li R, Zhang Z, et al. The genome of the cucumber, Cucumis sativus 64. Boonkorkaew P, Hikosaka S, Sugiyama N. Effect of pollination on cell
L. Nat Genet. 2009;41(12):1275–81. division, cell enlargement, and endogenous hormones in fruit development
36. Hawthorn LR, Wellington R. Geneva, a greenhouse cucumber that develops in a gynoecious cucumber. Sci Hortic. 2008;116(1):1–7.
fruit without pollination. N Y State Agric Exp Station. 1930;2:3–11. 65. Fang JB, Tian LL, Li SH, et al. Influence of CPPU on the sink and source
37. Pike LM, Peterson CE. Inheritance of parthenocarpy in the cucumber of kiwifruit. Acta Horticulturae Sin. 2000;27(6):444–6. (in Chinese with
(Cucumis sativus L.). Euphytica. 1969;18:101–5. English abstract)
38. Kvasnikov BV, Rogova NT, Tarakanova SI, Ignatov SI. Methods of breeding 66. Zeng H, Yang W, Lu C, et al. Effect of CPPU on carbohydrate and
vegetable crops under the covered ground. Trudy Prikl Bot Genet Selek. endogenous hormone levels in young macadamia fruit. PLoS One. 2016;
1970;42:45–57. 11(7):e0158705.
39. Juldasheva L. Inheritance of the tendency towards parthenocarpy in 67. Ando K, Grumet R. Transcriptional profiling of rapidly growing
cucumbers. Byull Vsesoyuznogo Ordena Lenina Inst Rastenievodstva Imeni cucumber fruit by 454-pyrosequencing analysis. J Am Soc Hortic Sci.
NI Vavilova. 1973;32:58–9. 2010;135(4):291–302.
Li et al. BMC Genomics (2017) 18:896 Page 18 of 18

68. Ando K, Carr KM, Grumet R. Transcriptome analyses of early cucumber fruit
growth identifies distinct gene modules associated with phases of
development. BMC Genomics. 2012;13(1):518.
69. Thimm O, Bläsing O, Gibon Y, et al. MAPMAN: a user-driven tool to display
genomics data sets onto diagrams of metabolic pathways and other
biological processes. Plant J. 2004;37(6):914–39.
70. Dreze M, Carvunis A-R, Charloteaux B, et al. Evidence for network evolution
in an Arabidopsis interactome map. Science. 2011;333:601–7.
71. Lü S, Zhao H, Des Marais DL, et al. Arabidopsis ECERIFERUM9 involvement
in cuticle formation and maintenance of plant water status. Plant Physiol.
2012;159(3):930–44.
72. Springer PS, Holding DR, Groover A, et al. The essential Mcm7 protein
PROLIFERA is localized to the nucleus of dividing cells during the G (1)
phase and is required maternally for early Arabidopsis development.
Development. 2000;127(9):1815–22.
73. De Jong M, Wolters-Arts M, Feron R, et al. The Solanum Lycopersicum auxin
response factor 7 (SlARF7) regulates auxin signaling during tomato fruit set
and development. Plant J. 2009;57(1):160–70.
74. Goetz M, Hooper LC, Johnson SD, et al. Expression of aberrant forms of
AUXIN RESPONSE FACTOR8 stimulates parthenocarpy in Arabidopsis and
tomato. Plant Physiol. 2007;145(2):351–66.
75. Ren Z, Li Z, Miao Q, et al. The auxin receptor homologue in Solanum
Lycopersicum stimulates tomato fruit set and leaf morphogenesis. J Exp
Bot. 2011;62(8):2815–26.
76. Dharmasiri N, Dharmasiri S, Weijers D, et al. Plant development is regulated
by a family of auxin receptor F box proteins. Dev Cell. 2005;9(1):109–19.
77. Kepinski S, Leyser O. The Arabidopsis F-box protein TIR1 is an auxin
receptor. Nature. 2005;435(7041):446–51.
78. Guo H, Ecker JR. Plant responses to ethylene gas are mediated by SCF
EBF1/EBF2-dependent proteolysis of EIN3 transcription factor. Cell. 2003;
115(6):667–77.
79. Li J, Li Z, Tang L, et al. A conserved phosphorylation site regulates the
transcriptional function of ETHYLENE-INSENSITIVE3-like1 in tomato. J Exp
Bot. 2012;63(1):427–39.
80. Key JL. Ribonucleic acid and protein synthesis as essential processes for cell
elongation. Plant Physiol. 1964;39(3):365.
81. Faurobert M, Mihr C, Bertin N, et al. Major proteome variations associated
with cherry tomato pericarp development and ripening. Plant Physiol. 2007;
143(3):1327–46.
82. Weiler EW, Jourdan PS, Conrad W. Levels of indole-3-acetic acid in intact
and decapitated coleoptiles as determined by a specific and highly
sensitive solid-phase enzyme immunoassay. Planta. 1981;153(6):561–71.
83. Omar AA, Song WY, Grosser JW. Introduction of Xa21, a Xanthomonas-
resistance gene from rice, into ‘Hamlin’ sweet orange [Citrus sinensis (L.)
Osbeck] using protoplast-GFP co-transformation or single plasmid
transformation. J Hortic Sci Biotechnol. 2007;82(6):914–23.
84. Bradford MM. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye
binding. Anal Biochem. 1976;72(1–2):248–54.
85. Wisniewski JR, Zougman A, Nagaraj N, et al. Universal sample preparation
method for proteome analysis. Nat Methods. 2009;6(5):359.
86. Wang Y, Zhang WZ, Song LF, et al. Transcriptome analyses show changes in
gene expression to accompany pollen germination and tube growth in
Arabidopsis. Plant Physiol. 2008;148(3):1201–11.
87. Rajjou L, Belghazi M, Huguet R, et al. Proteomic investigation of the effect
of salicylic acid on Arabidopsis seed germination and establishment of early
defense mechanisms. Plant Physiol. 2006;141(3):910–23.
88. Menges M, Hennig L, Gruissem W, et al. Cell cycle-regulated gene Submit your next manuscript to BioMed Central
expression in Arabidopsis. J Biol Chem. 2002;277(44):41987–2002.
89. Hajduch M, Hearne LB, Miernyk JA, et al. Systems analysis of seed filling in and we will help you at every step:
Arabidopsis: using general linear modeling to assess concordance of
• We accept pre-submission inquiries
transcript and protein expression. Plant Physiol. 2010;152(4):2078–87.
90. Hwang IS, Kim NH, Choi DS, et al. Overexpression of Xanthomonas • Our selector tool helps you to find the most relevant journal
campestris pv. Vesicatoria effector AvrBsT in Arabidopsis triggers plant cell • We provide round the clock customer support
death, disease and defense responses. Planta. 2012;236(4):1191–204.
• Convenient online submission
91. Irshad M, Canut H, Borderies G, et al. A new picture of cell wall protein
dynamics in elongating cells of Arabidopsis Thaliana: confirmed actors and • Thorough peer review
newcomers. BMC Plant Biol. 2008;8(1):94. • Inclusion in PubMed and all major indexing services
• Maximum visibility for your research

Submit your manuscript at

www.biomedcentral.com/submit