Ectopic expression of CsMADS 24 , an AGAMOUS ortholog from cucumber , causes homeotic conversion of sepals into carpels in transgenic Arabidopsis plants

The floral homeotic C-function MADS gene AGAMOUS (AG) in Arabidopsis plays crucial roles in specifying stamen and carpel identities as well as determining floral meristem. However, there have been only a few studies of floral homeotic C-function genes in cucumber thus far. In the present study, CsMADS24, a putative AG ortholog from cucumber, was isolated and characterized. Sequence analysis and protein sequence alignment revealed that the deduced CsMADS24 protein contained the typical MIKC structure and the N-terminal extension, as well as two highly conserved AG motifs (I and II). Phylogenetic analysis showed that CsMADS24 fell into the clade of core eudicots, while being distant from the AG orthologs of basal eudicots, monocots and gymnosperms. Expression analysis by RT-PCR showed that CsMADS24 was exclusively expressed in female flower buds. In situ hybridization revealed that CsMADS24 expression was only detected in the carpels. Functional analyses indicated that the sepals were partly converted into carpelloid-like structures in 35S::35S::CsMADS24 transgenic plants. In addition, earlier flowering and delayed floral organ abscission during the development of siliques were also observed in transgenic Arabidopsis. Our findings demonstrate that the AG ortholog plays an exclusive role in carpel specification of cucumber, providing a basis for revealing the mechanisms of reproductive development in cucumber.


INTRODUCTION
Normal development of floral organs is the basis for plant breeding, as well as for key physiological processes for improving the yield of agricultural products.Research on floral organ development can be of great importance for plant improvement.The analysis of the homeotic floral mutants of Arabidopsis thaliana and Antirrhinum majus resulted in the formulation of a genetic model named as the ABCDE model, which explains how the functions of five classes of genes (A, B, C, D and E) are combined to specify four different floral organs [1][2][3][4].Nearly all of these genes encode MIKC-type MADS-box transcription factors, which contain a 60-amino acid MADS-box domain in the N-terminal region, a less conserved intervening region contributing to the DNA binding specificity and dimerization, a moderately conserved keratin-like domain involved in protein-protein interaction, and a highly variable C-terminal region [5][6][7].
In Arabidopsis, the C-class gene AGAMOUS (AG) plays a critical role in specifying stamen and carpel identities as well as determining floral meristem [8,9].The Arabidopsis ag mutant has normal sepals and petals, but there was a homeotic conversion of stamens into petals, and the carpals were replaced by another flower [9,10].Overexpression of AG induces a conversion of sepals into carpels and petals into stamens in the flower [9].To date, the AG orthologs have been identified and characterized from a diverse number of plant species, such as Lilium longiflorum [11], Fraxinus pennsylvanica [12], Hosta plantaginea [13], Magnolia wufengensis [14], Prunus lannesiana [15], Magnolia stellata [16], Carya illinoinensis [17] and Fraxinus nigra [18].Arabidopsis plants with ectopic expression of AG orthologs from these plants also displayed homeotic changes.Similar results were obtained for the overexpression of AG orthologs using transgenic approaches in other plant species as well, such as tobacco [19][20][21][22], tomato [23] and petunia [24].In addition, the endogenous AG gene function of specifying stamen and carpel identities in the Arabidopsis ag mutant could be partially or fully compensated by ectopic expression of AG orthologs from other plant species, such as MastAG [16], FaesAG [25] and TcAG [26].These findings indicate that AG orthologs can also be responsible for the formation of reproductive organs.Cucumber (Cucumis sativus L.) is an economically-and nutritionally-important vegetable crop cultivated worldwide and it is also a model system for flower development studies [27,28].Our previous study revealed that there is only one AG subfamily member (CsMADS24) in cucumber [5], but its roles in determining reproductive floral organs have not yet been elucidated.In this study, CsMADS24 was isolated and its sequence, expression pattern and the effect of overexpression in Arabidopsis were analyzed.The results showed that CsMADS24 may be a functional AG ortholog in cucumber.

Plant material and growth conditions
Cucumis sativus var.sativus line 9930 and Arabidopsis thaliana Col-0 were used in this study.Cucumber seeds were germinated and grown in trays containing a soil mixture (peat:sand:pumice, 1:1:1, v/v/v).The seedlings were adequately watered and grown at day/night temperatures of 24/18°C, respectively, with a 16 h photoperiod.Wild-type (WT) and transgenic Arabidopsis seeds were placed in Petri plates containing solid 1/2 Murashige and Skoog (MS) medium.After being kept at 4°C in darkness for 2 days, the plates were transferred in a growth chamber to the greenhouse under long-day conditions (16 h light/8 h dark) at 22°C for 10 days; the seedlings were then transplanted into pots with soil for further study.

Cloning of the CsMADS24 gene
Total RNA was extracted using Plant RNA Purity Reagent (Invitrogen, USA) from the cucumber inflorescences.Single-stranded cDNA was synthesized by priming with the oligo(dT) using M-MLV reverse transcriptase (TaKaRa, Japan).The ORF sequence of CsMADS24 was amplified by semiquantitative reverse transcription PCR (RT-PCR) with the CsMADS24 specific primers CsMADS24-1F (5ʹ-ATGAGTTGTTATGAGGAAG-3ʹ) and Cs-MADS24-1R (5ʹ-TTACACAAGTTGAAGAGAG-3ʹ) using the following procedure: 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 1 min, and then a final extension at 72°C for 10 min.The PCR product was cloned into the pMD18-T vector (TaKaRa, Japan) and sequenced.
Multiple sequence alignments were performed on the protein sequences using Clustal Omega (https:// www.ebi.ac.uk/Tools/msa/clustalo/) with the default parameters and alignments adjusted manually by GeneDoc.A neighbor-joining (NJ) phylogenetic tree was constructed on the basis of a comparative analysis of amino acid sequences of CsMADS24 and the AG members from different plant species using the MEGA 5.0 software, which was supported by bootstrapping based on 1000 replicates.The accession numbers of these proteins are listed in Supplementary Table S1.

In situ hybridization
The cucumber floral development could be divided into 12 stages, from meristem initiation (stage 1) to anthesis (stage 12); the male and female flower buds became morphologically distinguishable from each other at stage 6 [27].Flower buds collected from cucumbers grown in the greenhouse at stage 6 were fixed, embedded, sectioned, and hybridized with digoxigenin-labeled probes as described previously [28].Sense and antisense RNA probes were amplified by PCR using the Digoxigenin RNA labeling kit (Roche) with the following primers: CsMADS24-3F (5ʹ-GTGAAATTGGAGAAAGGAAT-3ʹ) and Cs-MADS24-3R (5ʹ-GATAGGGTGATTATGATGGG-3ʹ).

Transformation of Arabidopsis and analysis of transgenic plants
The CsMADS24 ORF in pMD18-T vector was cleaved using Pst I and Xba I, and subcloned into binary vector pHB under the control of a double cauliflower mosaic virus 35S promoter in the sense orientation.The resulting 35S::35S::CsMADS24 construct was induced into Agrobacterium tumefaciens strain GV3101.The transformed bacteria were used to obtain transgenic Arabidopsis plants by the floral dip procedure [30].Transgenic lines were checked by germinating the seeds on 1/2 MS medium containing 50 mg/L hygromycin for 10 days at 22°C.Putative hygromycin-resistant transformants were further confirmed by RT-PCR analysis using CsMADS24-2F and CsMADS24-2R.AtTubulin4 was used as an internal control with the following primers: AtTubulin4-F (5ʹ-GCGAACAGTTCACAGCTATGTTCA-3ʹ) and At-Tubulin4-R (5ʹ-GAGGGAGCCATTGACAACATCTT-3ʹ).T 2 and T 3 homozygous transgenic plants were used for phenotypic evaluation.

Cloning and sequence analysis of the CsMADS24 gene from cucumber
The CsMADS24 gene was cloned from C. sativus by RT-PCR using primers designed on the basis of a previous report [5].The ORF of CsMADS24 is 765 bp in length and encodes a peptide of 254 amino acid residues with an estimated molecular weight of 28.98 kDa, a pI of 9.03 and an average hydropathy index (GRAVY) of -0.833.NCBI Conserved Domain Database (CDD) analysis demonstrated that the MADS and K domains were present in the CsMADS24 protein (Fig. 1A).A BLAST search of CsMADS24 against GeneBank showed that it was identical to CUS1 (100% amino acid identity) [31] and has only one non-conserved amino acid substitution of CAG2 [32].GSDS analysis performed by comparing the sequences of ORF and genomic DNA suggested that CsMADS24 harbored 7 exons and 6 introns (Fig. 1B).Sequence analysis via SOPMA showed that the secondary structure of CsMADS24 includes 56.30% alpha helix, 11.81% extended strand, 4.72% beta turn and 27.17% random coil sequences (Fig. 1C).In addition, ProtComp and CELLO analyses suggested that CsMADS24 was localized to the nucleus.
To determine the evolutionary relationship between CsMADS24 and other AG orthologs, we constructed a phylogenetic tree based on their amino acid sequences.As shown in Fig. 3, CsMADS24 fell into the clade of core dicots, and was the most closely related to Cm-MADS01 from Cucumis melo [35].

Expression pattern of CsMADS24 during flower development
Our previous results also showed that CsMADS24 was expressed in flowers while no transcript was detected in roots, shoots and leaves [5].To further examine the spatial expression pattern of CsMADS24 during flower development, we investigated its expression at five different developmental stages of male and female flowers.These floral developmental stages were sorted according to their corolla length, which ranged from 2.5±1 mm in FF1/MF1 to 20±2 mm in FF5/MF5 [29].In female flowers, the transcripts of CsMADS24 were first detected at the FF2 stage, increasing at the FF3 stage and finally decreasing at stages FF4 and FF5 (Fig. 4A).However, in male flower buds, no transcript was detected in any of the five developmental stages (Fig. 4B).
To dissect the detailed expression patterns of Cs-MADS24 during cucumber flower development, in situ hybridization was performed.As shown in Fig. 4C, CsMADS24 RNA was detected in flower buds at stage 6 exclusively in the carpel.

Phenotypes of CsMADS24 overexpression in Arabidopsis
To gain further insight into the function of Cs-MADS24, we transformed the model species Arabidopsis with CsMADS24, which is under the control of two cauliflower mosaic virus 35S promoters (Fig. 5A).Among 64 35S::35S::CsMADS24 independent transgenic plants, 23 exhibited significantly earlier   flowering phenotypes.The significantly earlier flowering transgenic Arabidopsis plants were identified by RT-PCR analysis, and three transgenic lines (OE1, OE2 and OE3) were selected for further analysis (Fig. 5B).
The phenotypes of 35S::35S::CsMADS24 Arabidopsis transgenic plants at different flower developmental stages were investigated using the method of a previous study [36].The results showed that the transgenic plants flowered significantly earlier than WT plants (Fig. 6A,  B).Usually, the flower buds of WT plants were open after stage 12; however, those of transgenic plants were prematurely open, sometimes before stage 10 (Fig. 6A, D).The sepals of transgenic plants were homeotically transformed into carpelloid-like structures with stigmatic papillae (Fig. 6C).At stage 10, the stigmatic papillae showed a relatively earlier and more rapid extension out of the sepals in transgenic plants than in WT plants (Fig. 6D).In WT plants, stigmatic papillae did not appear until stage 11, and at this time the flower buds were still enclosed by sepals (Fig. 6E).In addition, delayed floral organ abscission during the development of siliques was observed in transgenic plants (Fig. 6F), while in WT plants it was normal (Fig. 6G).

DISCUSSION
Cucumber is one of the economically-important vegetable crops cultivated worldwide, and ABCDE model genes may play very important roles in flower development.However, only a number of floral homeotic genes from cucumber have been investigated in detail [28,31,37,38].In this study, an AG homologous gene, CsMADS24, was isolated from cucumber developing flowers.
The predicted protein encoded by CsMADS24 has a typical MIKC-type domain, suggesting that it is a MADS-box gene, and CsMADS24 was found to have high identities in deduced amino acid sequences with the AG orthologs from core eudicot species, particularly with CmMADS01 from C. melo.In addition, CsMADS24 contains an N-terminal extension ahead of the MADS-box, which is usually present in the AG ortholog proteins from core eudicot species but absent in those from basal eudicot species and monocot species [25,39].The N-terminal extension peptide seems to have no specific function since some AG orthologs lacking this extension are also functionally active in vitro, such as LLAG1 [11], OsMADS3 [33], HpAG [13], MAwuAG [14], PrAG1 [21], AcAG [40], and CiAG [17].Moreover, CsMADS24 also harbors two highly conserved AG motifs (I and II), which are specific to C-class proteins [39].These results indicate that CsMADS24 is an AG homolog and may function in regulating flower organ development like other AG-like genes.
Our previous study revealed that CsMADS24 is only expressed in flowers [5].In the present study, RT-PCR results showed that CsMADS24 is specifically expressed in female flowers and that the expression is much higher at certain specific stages of female flower development.The spatial expression pattern of Cs-MADS24 is in line with that of CUS1 and CAG2 [31,32].Compared with AG from A. thaliana and AG orthologs from other plant species, CsMADS24 showed a higher specificity of expression.In situ hybridization revealed that CsMADS24 RNA exclusively accumulated in the carpels, while no signal was detected in the stamens, which is different from the case of AG orthologs that have been identified so far, implying that CsMADS24 may only be required for specifying carpel identity.
The function of CsMADS24 was further investigated by its ectopic expression in Arabidopsis to examine whether CsMADS24 plays roles in specifying stamen and carpel identities.As a result, ectopic expression of CsMADS24 significantly promoted early flowering in transgenic plants.A similar early flowering phenotype was observed in transgenic Arabidopsis with ectopic expression of AG orthologs from different plant species, such as MAwuAG [14], PrseAG [15], FnAG [18], and FaesAG [25].In addition, the 35S::35S::CsMADS24 transgenic Arabidopsis plants displayed a homeotic transition of sepals into carpelloid-like structures with earlier appearance of stigmatic papillae, suggesting that ectopic expression of CsMADS24 is sufficient to convert sepals into carpels, but insufficient to convert petals into stamens.These results indicate that 35S::35S::CsMADS24 transgenic Arabidopsis plants only had partially similar phenotypes compared to the Arabidopsis plants with AG ectopic expression [41].Similar phenotypes were also reported previously for the ectopic expression of AG orthologs from several plant species.For example, MastAG from the ancestral angiosperm Magnolia stellata also plays a major role in carpel identity, and it can substitute for the endogenous AG gene of A. thaliana that specifies carpel identity, but fails to rescue stamen development [16].Ectopic expression of pMADS3 was sufficient to convert petals into anthers, but failed to convert sepals into carpels [4].In 35S::FAR Arabidopsis plants, the second whorl organs were stamenoid-like structures, but the first whorl organs showed almost no obvious phenotypic changes [42].The ectopic expression of MAwuAG can convert the petals into stamenoid structures in Arabidopsis, but is insufficient to convert sepals in the first whorls into carpels [14].These findings indicate that the establishment of complete C-function may require the joint effects of AG and other genes to specify the stamen and carpel identity in these plants.For example, OsMADS3 plays a more crucial role in regulating stamen identity, while OsMADS58 is required for carpel morphogenesis and meristem termination [33].As AG orthologs from Cyclamen persicum, CpAG1 plays a more predominant role in stamen formation and CpAG2 mainly functions in carpel formation and termination of meristematic activity [43].In addition, the transgenic Arabidopsis displayed delayed floral organ abscission during the development of siliques.Premature fruit shattering along the dehiscence zone was also observed in transgenic Arabidopsis plants with ectopic expression of TrAG and TrSHP [44] and MAwuAG [14].These results indicate that CsMADS24, which might also have undergone neofunctionalization like MAwuAG [14], may have a function in regulating the development of floral reproductive organs in cucumber in a unique manner.We speculate that the differences in the functions of AG orthologs between cucumber and Arabidopsis may be due to the fact that Arabidopsis is androgynous, while cucumber is a monoecious plant with distinct male and female flowers on a single plant.

CONCLUSIONS
An AG ortholog named as CsMADS24 was isolated from C. sativus and its function in flower development was analyzed.RT-PCR, in situ hybridization and transgenic analysis in Arabidopsis were performed to investigate the function of CsMADS24.The 35S::35S::CsMADS24 transgenic Arabidopsis plants exhibited a homeotic transition of sepals into carpelloid-like structures, but the petals were not converted into stamenoid structures.These results indicate that CsMADS24 plays an important role in regulating the reproductive organ identity of carpels in cucumber.

Fig. 1 .
Fig. 1.Characterization of the CsMADS24 gene and deduced CsMADS24 protein.A -Amino acid sequence-based CDD search of the CsMADS24 protein.B -Exon-intron structure of the CsMADS24 gene by GSDS.Exons and introns are indicated by dark green boxes and black lines, respectively.C -Secondary structure of CsMADS24.The alpha helix, extended strand, beta turn, and random coil residues are represented in the order from the longest to the shortest.

Fig. 3 .
Fig. 3. Phylogenetic analysis of AG orthologs from different plant species.The accession numbers of AG proteins used for phylogenetic tree analysis are shown in TableS1.CsMADS24 from C. sativus is bold.

Fig. 4 .
Fig. 4. Expression patterns of CsMADS24 during different flower developmental stages in cucumber.A, B -Expression analyses of CsMADS24 by RT-PCR using CsAct3 as an internal control in female (A) and male (B) flowers at different developmental stages.C -mRNA in situ hybridization of CsMADS24 in flower buds at stage 6.D -Negative control using the sense probe in flower buds at stage 6. S, sepal; P, petal; St, stamen; Ca, carpel.Scale bars = 100 μm.