A Cucumber DELLA Homolog CsGAIP May Inhibit Staminate Development through Transcriptional Repression of B Class Floral Homeotic Genes

In hermaphroditic Arabidopsis, the phytohormone gibberellin (GA) stimulates stamen development by opposing the DELLA repression of B and C classes of floral homeotic genes. GA can promote male flower formation in cucumber (Cucumis sativus L.), a typical monoecious vegetable with unisexual flowers, and the molecular mechanism remains unknown. Here we characterized a DELLA homolog CsGAIP in cucumber, and we found that CsGAIP is highly expressed in stem and male flower buds. In situ hybridization showed that CsGAIP is greatly enriched in the stamen primordia, especially during the hermaphrodite stage of flower development. Further, CsGAIP protein is located in nucleus. CsGAIP can partially rescue the plant height, stamen development and fertility phenotypes of Arabidopsis rga-24/gai-t6 mutant, and ectopic expression of CsGAIP in wide-type Arabidopsis results in reduced number of stamens and decreased transcription of B class floral homeotic genes APETALA3 (AP3) and PISTILLATA (PI). Our data suggest that monoecious CsGAIP may inhibit staminate development through transcriptional repression of B class floral homeotic genes in Arabidopsis.


Introduction
Gibberellins (GAs) are one class of tetracyclic diterpenoid phytohormones that play essential roles in diverse aspects of plant growth and development, including seed germination, hypocotyl elongation, root growth, stem elongation, leaf expansion, trichome formation, floral induction, flower development, and fruit development [1], in which, floral induction and flower development are the most important events regulated by GA [2]. GA content has been shown to increase dramatically before anthesis in flowers of both monocotyledonous and dicotyledonous species, such as barely (Hordeum vulgare), rice (Oryza sativa), Mirabilis jalapa and Pharbitis, implying that GA may be required for flower opening [3]. GA treatment, however, has distinct, even opposite effects on flower development in different species. For example, GA application can promote staminate development in Arabidopsis, rice and tomato (Solanum lycopersicum), whereas stimulate pistillate development in castor bean (Ricinus communis), Hyoscyamus and maize (Zea mays) [3].
However, so far, most GA-regulated flower development studies were performed in hermaphroditic species, and rarely in monoecious plants. Cucumber (Cucumis sativus L.) is a typical monoecious vegetable with individual male and female flowers, and has been served as a model system for sex determination in planta [33]. GA can promote male flower formation in cucumber, and the molecular mechanism remains unknown. In this study, we found that cucumber homologs of GA signal transduction factors GID1, DELLA and GAMYB have much higher expression than those of GA synthesis genes during male flower development, and the cucumber DELLA homolog CsGAIP has the highest expression. We cloned the CsGAIP and characterized its spatial and temporal expression patterns. CsGAIP is mainly expressed in stems and male flower buds, and CsGAIP protein is located in nucleus. Ectopic expression of CsGAIP can partially rescue the phenotypes of rga-24/gai-t6 double mutant in Arabidopsis, and overexpression of CsGAIP in wild type resulted in reduced number of stamens and decreased transcription of B class floral homeotic genes. Our results suggested that CsGAIP inhibits stamen development through transcriptional repression of B class floral homeotic genes in Arabidopsis.

Results
Cucumber DELLA homolog GAIP may have prominent role during male flower development GA has been shown to promote male flower development in cucumber [3], but the underlining mechanism remains elusive. As the first step to uncover this mystery, we explored the expression patterns of cucumber homologs of GA biosynthesis genes CPS, KS, KO, KAO and GA signal transduction factors GID1, DELLA and GAMYB during different stages of male flower development. Using the sequence information in Arabidopsis, we performed BLAST analysis in Cucumber Genome Database [34], and defined the best hit as the corresponding cucumber homolog and the relative unique region of each gene was designed for quantitative real-time RT-PCR (qRT-PCR) analyses.
The developmental process of cucumber male flower can be divided into 12 stages [35], in which, five stages including hermaphrodite stage (stage 5), microsporocyte stage (stage 9), meiosis stage (stage 10), uninuclear pollen stage (stage 11) and mature pollen stage (stage 12) were identified based on morphological indications [35,36] ( Figure 1A) and the lengths of cucumber male floral buds for each stage was calculated (Table 1). Then, RNA samples were collected from at least three independent male flower buds and qRT-PCR was performed using these samples. As shown in Figure 1B, GA signal transduction factors GID1, GAIP (the best hit for DELLA homolog) and GAMYB have much higher expression than those of GA synthesis genes CPS, KS, KO, KAO during cucumber male flower development. In which, GAIP has the highest expression among all, particularly in the hermaphrodite stage (stage 5), for example, expression of GAIP is more than 20 fold and 6 fold higher than GA synthesis genes and other GA signal transduction factors, respectively. Further, expression of GAIP decreases as the male flower develop, suggesting that cucumber GAIP may play a key role during male flower development and promote male determination in the hermaphrodite stage.
Cloning and phylogenetic analysis of cucumber DELLA homolog CsGAIP Through BLAST analysis, we found four DELLA homologs in cucumber, CsGAIP (Csa021618), CsGAI1 (Csa015919), CsGAI2 (Csa008181) and CsGAI3 (Csa015258), in which CsGAIP has the highest similarity to DELLAs in Arabidopsis, so we chose CsGAIP for further analysis in this study. CsGAIP was cloned using cDNA derived from cucumber leaves through PCR technology. The fulllength CsGAIP cDNA consists of 1761 bp and encodes 587 amino acids. Consistent with the five DELLA genes of Arabidopsis, CsGAIP gene also has no introns [13,14]. Previous studies showed that DELLA proteins belong to a GRAS subfamily that contains two highly conserved domains named as DELLA and VHYNP in their N-terminal regions [14,22,37]. Sequence alignment of the Nterminal 150 amino acid residues of CsGAIP using ClustalW indicated that CsGAIP also has the DELLA and VHYNP domains, which may be essential for GID1-DELLA interaction [11,[38][39][40][41][42] (Figure 2A). Full-length CsGAIP is 89.25%, 64.72%, 64.91%, 53.28%, 51.96%, 52.53%, 52.9% identical to CmGAIP, AtRGA, AtGAI, ZmD8, TaRHT1, HvSLN1, OsSLR1, respectively. To understand the evolutional relationship between CsGAIP and other DELLA proteins, we constructed phylogenetic tree using the neighbor-joining (NJ) method [43] (Figure 2B), cucumber CsGAIP, CsGAI2 and CsGAI3 are placed in the same cluster as other DELLA homologs, while CsGAI1 is distantly related, suggesting that CsGAIP, CsGAI2 and CsGAI3 are more likely to be the DELLA homologs in cucumber. Phylogenetic tree divides DELLA homologs into two clades: dicotyledon (green line) such as Arabidopsis, cucumber, pumpkin (Cucurbita maxima), lettuce (Lactuca sativa), pea (Pisum sativum), bean (Phaseolus vulgaris), and monocotyledon (red line) such as maize, rice, barley and wheat. Within dicotyledon clade, CsGAIP and CmGAIP, which belong to the cucurbitaceae family with unisexual flowers, fall into the same clade that is distinct from those of CsGAI2, CsGAI3 and other DELLA homologs in hermaphroditic species, such as Arabidopsis, lettuce, pea and bean, implying that CsGAIP may be involved in the unisexual flower development in cucumber.

Expression pattern of DELLA homologs in cucumber
To characterize the spatial distribution of DELLA homologs transcripts, qRT-PCR was performed in various cucumber tissues including roots, stems, leaves, male flower buds, female flower buds and fruits. As shown in Figure 3, expressions of CsGAIP and CsGAI2 are much higher than those of CsGAI1 and CsGAI3 in all the tissues we examined, and that CsGAIP and CsGAI2 display similar expression patterns, which are predominantly expressed in stems and male flower buds. CsGAI3 transcript is more enriched in roots as compared to other tissues, while CsGAI1 shows equivalent expression in all the tissues we tested. Among all the four DELLA homologs, CsGAIP displays the highest expression especially in stems and male flower buds, implicating that CsGAIP may play important roles in stem and male flower development.
Next, we examined the expression pattern of CsGAIP during male flower development of cucumber by in situ hybridization ( Figure 4). CsGAIP RNA was found throughout in the inflorescence meristem (im) and floral meristem (fm) ( Figure 4A), as well as in the vascular strands (arrow in Figure 4A) in stage 1 male flowers [35].
During stages 2-6 (hermaphrodite stage), CsGAIP is expressed in the developing sepals, petals, stamens and carpels, with the strongest expression in stamen primordia (arrows in Figure 4B-E). As the male flower further develop, microsporocytes initiate in stage 9, uninuclear pollen appear in stage 11 and mature pollen form by stage 12, and CsGAIP is expressed mainly in the microsporocytes ( Figure 4F), anther wall and pollen grains ( Figure 4G-J), despite the signal is weaker than those in hermaphrodite stage. This data is consistent with the higher expression in hermaphrodite stage as detected by qRT-PCR ( Figure 1B). As negative controls, CsGAIP sense probe hybridizations show no signals in male flowers of stage 1, stage 6, stage 9 and stage 12 ( Figure 4K-N).

Subcellular localization of CsGAIP
In Arabidopsis, the DELLA proteins RGA and GAI have been shown to contain putative nuclear localization signal (NLS) and  localize in nucleus [14]. Sequence alignment of the N-terminal 200-300 amino acid residues of CsGAIP with AtRGA and AtGAI showed that CsGAIP also has a putative NLS ( Figure 5A). Subcellular localization of CsGAIP in cucumber protoplasts indicated that CsGAIP locates in nucleus as well ( Figure 5B, top row), and the same result was found in epidermal cells of onion (Allium cepa) ( Figure 5C, top row). As a control, signals of 35S:GFP were detected throughout the cell ( Figure 5B and C, bottom row).
CsGAIP can partially rescue rga-24/gai-t6 double mutant in Arabidopsis To explore the function of CsGAIP, we ectopically expressed the full-length CsGAIP cDNA under the control of 35S promoter of Cauliflower mosaic virus (CaMV) in Arabidopsis rga-24/gai-t6 double mutant, and 13 independent transgenic lines were obtained. Previous study reported that rga-24/gai-t6 double mutant displayed higher plant height, reduced number of pollens, shorter filaments and thus decreased seed numbers per silique [21]. As showed in Figure 6 and Table 2, all the transgenic lines display partial rescue of the rga-24/gai-t6 phenotypes. The average plant height of rga-24/ gai-t6 plants is 38% taller than that of Ler, while in the transgenic lines, the average plant height is only 8% taller than that of Ler ( Fig. 6A; Table 2), suggesting that CsGAIP can greatly rescue the plant height phenotype in Arabidopsis. Further, flowers in the CsGAIP transgenic plants display increased filaments length and amount of pollen as compared to those in rga-24/gai-t6 (Fig. 6B, C). Consequently, the silique length and the seed number per silique increase in the transgenic plants ( Figure 6D-I). For example, there are around 8 seeds per silique in the rga-24/gai-t6 plant, while ectopic expression of CsGAIP in rga-24/gai-t6 background results 43 seeds/silique, which is close to that in Ler (56 seeds/silique) ( Table 2). These data suggested that cucumber CsGAIP can partially replace the function of RGA and GAI in Arabidopsis with respect to plant height, stamen development and plant fertility.
CsGAIP suppresses stamen development by downregulating floral homeotic genes AP3 and PI in Arabidopsis We further explore the function of CsGAIP by overexpression of CsGAIP in Arabidopsis wide-type Ler, and 25 independent transgenic lines were obtained. As shown in Figure 7A, ectopic expression of CsGAIP in Arabidopsis led to reduced number of stamens. In contrast to the six stamens in Ler flowers, the flowers in 35S::CsGAIP plants only display 4.660.5 stamens ( Table 3). Given that the floral homeotic genes, including APETALA1 (AP1), APETALA2 (AP2), APETALA3 (AP3), PISTILLATA (PI) and AGAMOUS (AG), are involved in floral patterning in Arabidopsis [44], and that B genes (AP3 and PI) and C gene (AG) are downregulated by RGA activity [27], we examined the expression of floral homeotic genes in 35S::CsGAIP plants by qRT-PCR and semi-quantitative RT-PCR. We found that the expression of A class (AP1 and AP2) and C class of gene (AG) were not substantially changed in the transgenic plants, but transcripts of B function genes (AP3 and PI) were significantly decreased (Fig. 7B). For example, the transcripts of AP3 and PI in the 35S::CsGAIP plants were reduced by around 80% and 50% respectively as compared to those in the Ler background. These data suggested that CsGAIP can suppress the expression of B function genes in Arabidopsis, which may be the cause for reduced number of stamens as observed in the ectopic expression lines.

Discussion
Cucumber (Cucumis sativus L.) is a monoecious species with individual male and female flowers. During the early stage of flower development, both stamen primordia and carpel primordia are initiated, male or female flower is generated upon the arrestment of carpel or stamen development, respectively [33,35]. Due to the agricultural importance, extensive studies have been performed in the mechanism of female flower formation, while the molecular regulation of male flower generation is largely unknown [45][46][47][48][49][50][51]. GA can regulate flower development in both hermaphroditic and monoecious species [3]. In Arabidopsis, GA promotes stamen development by antagonizing the function of DELLA proteins [24]. In monoecious cucumber, how GA stimulates male flower development remain elusive. Here we found that the cucumber DELLA homolog may play important roles during male flower development in cucumber ( Figure 1, Table 1), and we cloned this DELLA homolog CsGAIP ( Figure 2) and investigated the expression pattern and subcellular localization (Figure 3-5). Further, we explored the function of CsGAIP through ectopic overexpression of CsGAIP in Arabidopsis (Figure 6 and 7, Table 2 and 3). Our data suggested that monoecious CsGAIP may repress staminate development through transcriptional downregulation of B class floral homeotic genes in Arabidopsis.

CsGAIP may be the homolog for both RGA and GAI in cucumber
In Arabidopsis, DELLA family has five members: RGA, GAI, RGL1, RGL2, and RGL3 [20], which coordinately function in stem  [21,[23][24][25][26]. In cucumber, there are four putative DELLA genes, CsGAIP (Csa021618), CsGAI1 (Csa015919), CsGAI2 (Csa008181) and CsGAI3 (Csa015258), with CsGAIP, CsGAI2 and CsGAI3 closely relate to RGA and GAI, and CsGAI1 likely to be the homolog for RGL1-3 ( Figure 2, data not shown). Phylogenetic analyses indicate that CsGAIP but not CsGAI2 or CsGAI3 falls into the same clade as RGA and GAI ( Figure 2). Further, similar to those of RGA and GAI, CsGAIP has a NLS domain and localizes to nucleus ( Figure 5), suggesting that CsGAIP may be the homolog for both RGA and GAI. RGA and GAI have been shown to be negative regulators for stem elongation and stamen development, in which RGA inhibits stamen development via repressing floral homeotic genes AP3, PI, and AG [21,24,27]. Similarly, CsGAIP is highly expressed in stem and male flower buds, and ectopic expression of CsGAIP can partially rescue the plant height, stamen development and fertility phenotypes of rga-24/gai-t6 double mutant (Figure 6), and that overexpression of CsGAIP in wide-type Arabidopsis leads to decreased transcription of AP3 and PI (Figure 7). These data suggested that CsGAIP may functions as the homolog of both RGA and GAI in cucumber.

CsGAIP may inhibit male tendency during sex determination of cucumber flowers
In cucumber, exogenous GA treatment can promote male flower formation [52,53]. In this study, we found that CsGAIP is predominantly expressed in the male specific organs during flower development of cucumber, particularly in stamen primordia (Figure 1, 3 and 4). Ectopic expression of CsGAIP results in transcriptional repression of B class floral homeotic genes AP3 and PI in Arabidopsis. Therefore, we propose that CsGAIP may function as a major repressor for GA-induced male flower tendency. During the hermaphrodite stage, there may be equal activities for male-promoting and female-promoting factors, male and female flowers are produced by random with similar chance. Exogenous GA application may promote the interaction between GA receptors and CsGAIP, which may lead to rapid proteolysis of CsGAIP protein through the SCF SLY1/GID2 ubiquitin-proteasome pathway. Such CsGAIP degradation can stimulate the transcription of B class floral homeotic genes and thus promote staminate (male flower) development. Genetic transformation in cucumber upon CsGAIP RNA interference or overexpression would shed light on the molecular function of CsGAIP during sex determination of cucumber flowers. Meanwhile, F (CsACS1G) and M (CsACS2) genes have been demonstrated to regulate unisexual flower development in cucumber, specifically, F gene promotes female flower development [50,54,55], and M gene inhibits stamen development in floral buds [47][48][49]. It would be interesting to dissect the interactions, if any, between CsGAIP, F and M during sex determination in future studies. In addition, in monoecious maize, GA causes feminization instead of staminate production [56], implying that distinct mechanisms may be involved in the GA-mediated flower development in different species.
Unisexual CsGAIP displays conserved as well as divergent functions with its bisexual homologs Loss of function of RGA and GAI in Arabidopsis results in higher plant height and earlier flowering [21], while lack of DELLA homologs REDUCED HEIGHT and DWARF8 leads to dwarfism in wheat and maize, respectively [37,[57][58][59], indicating that DELLA homologs have conserved role in stem elongation, but the specific role maybe even opposite in different species. In this study, CsGAIP is highly expressed in cucumber stems ( Figure 3) and that CsGAIP can rescue the plant height phenotype of rga-24/gai-t6 (Fig. 6, Table 2), suggesting that CsGAIP may also function as a suppressor for stem elongation as those of Arabidopsis RGA and GAI. Similarly, transcripts of CsGAIP are enriched in stamen primordia, and ectopic expression of CsGAIP can rescue the stamen development and plant fertility phenotypes in rga-24/gai-t6 ( Figure 6, Table 2), and lead to reduced number of stamens and decreased expression of B function genes AP3 and PI upon ectopic expression in Ler (Figure 7, Table 3), supporting that CsGAIP has a conserved role in flower development, specifically, inhibits staminate development via repressing B class of floral homeotic genes. However, unlike the down-regulating of both B and C function genes upon RGA induction in Arabidopsis [27], the transcription of C class gene AG remains unchanged upon ectopic expression of CsGAIP ( Figure 7B), similarly, flowering time appeared to be undisturbed upon overexpression of CsGAIP in Arabidopsis (data not shown), suggesting that monoecious CsGAIP has divergent functions from RGA and GAI in hermaphroditic Arabidopsis.
Given that Arabidopsis DELLAs have specific as well as partially overlapping roles, it would be interesting to explore the specificity of the function for each DELLA homologue in cucumber. The four cucumber DELLAs display distinct expression patterns (Figure 3), in which CsGAI1 has low transcript accumulation in all the tissues we examined, CsGAI3 is predominantly expressed in roots, whereas CsGAIP and CsGAI2 are highly enriched in stems and male flower buds, suggesting that CsGAIP and CsGAI2 may play important and probably partially redundant roles in stem and male flower development in cucumber. However, for elucidating the functional similarities and differences among these four DELLAs, cucumber transformation, a currently difficult technique, is the best way to uncover the mystery in future studies. In addition, given that DELLA can regulate the cross-talks between GA and other signaling pathways through protein-protein interactions with regulatory factors such as PIF3/PIF4 (PHYTO-CHROME-INTERACTING FACTOR 3/4), SCL3 (SCARE-CROW-LIKE 3), ALC (ALCATRAZ) and JAZs (JA ZIM-domain proteins) [18,60], identifying the DELLA interacting proteins will greatly advance our knowledge of the diverse functions of DELLA homologs in cucumber development.

Plant materials and growth conditions
A monoecious cucumber (Cucumis sativus L.) line 3461 was used in this study. The plants were grown in a growth chamber under 16 h/8 h and 25uC/18uC in day/night, respectively. Upon two true-leaf stage, plants were transferred to a greenhouse in the experimental field of China Agricultural University in Beijing. The Arabidopsis mutant rga-24/gai-t6 (Landsberg background) was pro-vided by Sun's lab [21], and Ler was used as wild type control. Arabidopsis seeds were germinated on Murashige-Skoog (MS) medium, which contains 1% sucrose and 0.2% phytagar at 4uC for 3 days and then moved to 22uC under a regime of 16 h light/ 8 h dark. Seedlings were transferred to soil 7-10 d after germination.
Cloning of CsGAIP, sequence alignment and phylogenetic analysis Total RNA was extracted from cucumber leaves using the Promega's SV Total RNA Isolation System, and cDNA was synthesized using MultiScribe reverse transcriptase (Applied Biosystems). The cDNA was amplified with primers CsGAIP-F (59-ATGAAGAGGGAGCATCACCATCTTC-39) and CsGAIP-R (59-TCACTTAGCGACCACCGGGTT-39) at 95uC for 5 min; 30 cycles of 95uC for 30 s, 54uC for 30 s, and 72uC for 2.5 min; and then 72uC for 10 min. The amino acid sequence of related DELLA proteins were obtained from National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) or the Arabidopsis Information Resource (http://www.arabidopsis.org), and protein alignment of CsGAIP and related DELLAs was performed using ClustalW in the MEGA5 software package, and the boxes were drawn using the BoxShade web site (http://www. ch.embnet.org/software/BOX_form.html). The phylogenetic tree was constructed using the neighbor-joining (NJ) method [43] through MEGA5 software using the bootstrap analysis with1000 replications.

Gene expression analysis
Total RNA was extracted using Promega's SV Total RNA Isolation System, and cDNA was synthesized using MultiScribe reverse transcriptase (Applied Biosystems). Quantitative real-time RT-PCR (qRT-PCR) was performed using SYBR Premix Ex Taq from TaKaRa   . The primers of AP1, AP2, AP3, PI and AG for semi-quantitative PCR were performed as previously reported [27].

In situ hybridization
Shoot apex of 10-day-old seedling and male flower buds from 45-day-old cucumbers grown in the greenhouse were fixed and hybridized as described [61]. Digoxigenin-labeled probes were generated through PCR amplification of cDNA using gene specific primers containing SP6 and T7 RNA polymerase-binding sites. SP6 and T7 RNA polymerase were used for the synthesis of sense and antisense probes, respectively. The primers of cucumber CsGAIP in situ probes were as follow: 59-GATTTAGGTGACAC-TATAGAATGCTATCCGATGCCTAATTTTGCGA-39 (bold Table 2. CsGAIP can rescue the plant height and fertility of rga-24/gai-t6 in Arabidopsis.

Subcellular localization in cucumber protoplasts and onion epidermal cells
For transient expression in cucumber protoplasts and onion epidermal cells, the full length coding region of CsGAIP were cloned using primers 59-ACGCGTCGACATGAAGAGGGAG-CATCACCATCTTC-39 (Sal I site in bold) and 59-CGGGATCCCTTAGCGACCACCGGGTTGTT-39 (BamH I site in bold), and then inserted into the pEZS-NL vector (with GFP protein driven by 35S promoter) to generate 35S:GFP-CsGAIP, and the empty pEZS-NL vector was used a control. The constructs were introduced into cucumber protoplasts using Huang's method [62]. The onion epidermal layers were prepared and bombarded, as previously described [63], with gold particles containing the plasmid using a Bio-Rad PDS-1000/He particle delivery system. After bombardment, the onion epidermises were placed on MS medium and incubated in darkness at 22uC for 24 h. Fluorescence signals were detected using Olympus BX 51 fluorescence microscopy.

Ectopic expression of CsGAIP in Arabidopsis
To make the CsGAIP overexpression construct, full length CsGAIP cDNA were cloned using primers 59-GGACTAGTAT-GAAGAGGGAGCATCACCATCTTC-39 (Spe I site in bold) and 59-GACTGCCACG TGTCACTTAGCGACCACC-GGGTT-39 (Pml I site in bold), and inserted into the pCAMBIA1305.1 vector with 35S promoter. The construct was then introduced into Agrobacterium by electroporation and transformed into Ler or rga-24/gai-t6 plants as described [64]. The transgenic plants were screened on MS medium with 25 mg/ L hygromycin.