Genetic basis of sex determination and differentiation is not well studied in C. grandis. Identification and investigation of sex-linked genes would lead to better understanding of dioecy in plants and this can be achieved by whole genome sequencing approach. However, sex-determining genes are most likely linked to non-recombining regions of Y-chromosome, which are difficult to assemble from sequence data [40]. An alternative approach is to use comparative transcriptomics to identify sex-biased genes that could play a role in sex differentiation and determination [24]. Further, the presence of mutations and SNPs in sex-biased genes can provide insights regarding the evolution of dioecy. Using this approach, we have assembled and annotated a de novo transcriptome from the flower buds of dioecious, gynomonoecious and AgNO 3 treated female C. grandis. We have identified differentially expressed genes which might be playing a role in stamen arrest of female flowers. Also, we have analysed the genes that were differentially expressed upon AgNO 3 treatment on female plants promoting stamen development. Finally, we have compared middle-staged male (bearing fertile pollens) and GyM-H buds (bearing sterile pollens) to study the genes involved in pollen maturation and fertility.

Differential expression of stamen developmental genes and arrest of stamen growth in female flowers

At the early stages (stages 3–4) of flower development in female C. grandis, both carpel and stamen organs are initiated simultaneously. However, stamen growth gets arrested during the course of development (stages 4–5) resulting in a female flower with rudimentary stamens. In contrast, no sign of carpel primordia was observed during the histological study of flower development in male C. grandis as described in our previous report [14]. The molecular players involved in stamen initiation and development process are well characterized in the hermaphrodite plant Arabidopsis. In order to identify the stage at which stamen growth gets arrested, Coccinia grandis homologs of Arabidopsis stamen development genes were identified from the de novo-assembled transcriptome. Among genes involved in stamen initiation, Pistillata (CgPI, TRINITY_DN71631_c0_g1_i1) was found to be expressed in a male-biased fashion (Additional file 8: Table S5). Pistillata has been shown to specify stamen identity in Arabidopsis [41] (Table 4). Further, EXCESS MICROSPOROCYTES 1 (EMS1) has been shown to interact with TAPETUM DETERMINANT 1 (TPD1) regulating specification of reproductive as well as somatic cells in Arabidopsis anthers [42]. Differential expression analyses revealed that homologs of both EMS1 (TRINITY_DN106236_c0_g4_i1) and TPD1 (TRINITY_DN116795_c2_g1_i3) were enriched in male flowers compared to female flowers (Table 4; Additional file 8: Table S5; Fig. 7). DYSFUNCTIONAL TAPETUM 1 (DYT1) plays an important role in tapetum development by regulating the expression of DEFECTIVE IN TAPETAL DEVELOPMENT AND FUNCTION 1 (TDF1) in Arabidopsis [43]. Also, DYT1 is known to interact with Basic helix-loop-helix protein 89 (bHLH89) which is highly expressed in anthers and required for normal anther development and male fertility [44]. TDF1 homolog (TRINITY_DN97604_c1_g7_i1) as well as bHLH89 homolog (TRINITY_DN85771_c0_g1_i1) showed male-biased expression in C. grandis (Table 4; Additional file 8: Table S5). Differential regulation of these genes related to stamen development explains the possible cause for early stamen arrest in female flowers of C. grandis.

Table 4 Digital Expression profile for genes in anther developmental pathway Full size table

According to recent reports from monoecious cucurbits like melon, cucumber, and watermelon, ethylene plays a major role in sex determination by inhibiting stamen development process [45,46,47,48]. We found that compared to male, GO:0009723 (response to ethylene) was enriched in female buds indicating a potential role of ethylene in sex determination of C. grandis (Additional file 9: Table S6).

AgNO 3 treatment on female plant releases the stamen inhibition

Female plants of C. grandis bear flowers with fused carpels and rudimentary stamens. Earlier, we have shown that foliar spray of 35 mM AgNO 3 on the female plant of C. grandis promotes further development of the rudimentary stamens [14]. In the current study, gene expression profiles for early-staged Ag-H flower buds were compared with female buds (Table 3; Fig. 5c, g). Ag+ ions are known to inhibit responses to ethylene, a gaseous plant hormone [17]. Also, silver compounds have been shown to induce maleness by promoting stamen development in many monoecious and dioecious species [19,20,21]. No other inhibitors of ethylene biosynthesis or signaling could induce the stamen development in Silene latifolia, suggesting that ethylene signaling might not be the only pathway that gets affected upon application of silver thiosulphate [21]. In contrast to Silene latifolia, AVG (aminoethoxyvinylglycine), an inhibitor of ethylene-biosynthesis has been shown to induce male flowers in gynoecious muskmelon similar to silver compounds [49]. Considering the role of 1-aminocyclopropane-1-carboxylate synthase (ACS, an enzyme involved in ethylene biosynthesis) in sex determination of many other members of Cucurbitaceae, an ethylene-mediated effect of AgNO 3 seems more likely to be involved in the modification of sex in C. grandis [50].

In our study, GO:0009723 (response to ethylene) and GO:0009873 (ethylene-activated signaling pathway) were enriched in female buds compared to Ag-H buds (Additional file 9: Table S6). Transcripts for genes such as Ethylene-responsive transcription factors, ERF5 (TRINITY_DN102355_c3_g13_i1), ERF17 (TRINITY_DN80749_c0_g6_i1), EF109 (TRINITY_DN87049_c0_g1_i1), EF102 (TRINITY_DN90257_c1_g2_i1), ERF99 (TRINITY_DN93821_c0_g1_i2), ERF60 (TRINITY_DN93262_c1_g6_i2) and ERF78 (TRINITY_DN98503_c3_g1_i1) were downregulated in Ag-H buds indicating impaired ethylene signalling (Additional file 8: Table S5). Additionally, qRT-PCR based expression pattern analysis for ERF5, ERF17 and EF102 genes clearly showed the suppression of ethylene responses by AgNO 3 (Fig. 9) .

Downregulation of ethylene signaling in Ag-H buds was correlated with the promotion of stamen growth. GO:0048655 (anther wall tapetum morphogenesis), GO:0048657 (anther wall tapetum cell differentiation), GO:0048658 (anther wall tapetum development) were seen to be enriched in early-staged Ag-H buds compared to female buds (Additional file 9: Table S6). C. grandis homologs of MS1, MMD1 (TRINITY_DN109512_c4_g3_i1, TRINITY_DN108927_c0_g6_i1), ZAT3 (TRINITY_DN108658_c0_g2_i1) and AMS (TRINITY_DN116105_c0_g2_i1) genes which play important roles in tapetum and pollen development of Arabidopsis flowers were upregulated upon AgNO 3 treatment indicating promotion of stamen growth [51,52,53,54,55,56] (Additional file 8: Table S5; Fig. 7). MYB35 (TRINITY_DN92649_c0_g7_i1), which was proposed as a putative sex-determining gene in Asparagus was also found to be upregulated in Ag-H buds [57] (Additional file 8: Table S5). Apart from that, gene ontology terms related to pollen wall assembly (GO:0010208), pollen exine formation (GO:0010584), sporopollenin biosynthetic process (GO:0080110), pollen development (GO:0009555) and pollen sperm cell differentiation (GO:0048235) were also enriched in Ag-H buds (Additional file 9: Table S6). Further, we noticed that Ethylene-responsive transcription factors (ERFs) were not affected in GyM-H buds as compared to female buds suggesting that stamen development in GyM-H flower buds might be regulated by some other mechanism evading ethylene signaling inhibition.

Transcripts governing pollen fertility are depleted in GyM-H and Ag-H flower buds

C. grandis is one of the few species in which the presence of heteromorphic sex chromosomes is reported. The large Y-chromosome present in males might play a major role in sex determination. The GyM form of C. grandis included in the current study does not have Y-chromosome [14]. GyM-H flowers still develop full-sized stamens despite lacking Y-chromosome. Similarly, AgNO 3 treatment induces stamen development in female plants having XX sex chromosomes. However, the pollens from GyM-H and Ag-H flowers buds were found to be sterile unlike the pollens from male buds [14]. Differential expression analysis revealed that gene ontology terms for pollen tube (GO:0090406), pollen germination (GO:0009846), regulation of pollen tube growth (GO:0080092), pollen tube growth (GO:0009860) and microsporogenesis (GO:0009556) were enriched in middle-staged male buds compared to middle-staged GyM-H buds (Additional file 9: Table S6).

GAUTE plays an important role in pollen tube wall biosynthesis in Arabidopsis [58]. TRINITY_DN111340_c1_g1_i6, which showed similarity with GAUTE was enriched in male buds compared to GyM-H buds. Unlike most other plant cell walls, pollen tube wall does not contain callose or cellulose. Pectin methylesterases (PMEs) have been shown to play a very important role in the growth of pollen tubes [59,60,61]. PME4 (TRINITY_DN14239_c0_g1_i1), PME37 (TRINITY_DN3663_c0_g1_i1) and PPME1 (TRINITY_DN66415_c0_g1_i1, TRINITY_DN71598_c0_g2_i1) were downregulated in GyM-H buds compared to male buds (Additional file 8: Table S5). This could be a possible cause for pollens from GyM-H not forming pollen tubes. H Zhan, Y Zhong, Z Yang and H Xia [62] has shown that IPMKB (Inositol polyphosphate multikinase beta) is an important factor for pollen development. We have found that TRINITY_DN96290_c0_g3_i2 transcript matching to Arabidopsis IPMKB (AtIpk2beta) was downregulated in GyM-H compared to male buds. Earlier, several reports have demonstrated that MALE STERILITY 1 (MS1) gene of Arabidopsis expresses in tapetal cells and plays an important role in pollen maturation [51, 54, 55]. C. grandis homolog of MS1, TRINITY_DN109512_c4_g3_i1 was expressed in a male-biased manner (Additional file 8: Table S5; Fig. 7). Similarly, homologs of genes important for pollen tube growth such as CSLD1 (TRINITY_DN92683_c0_g1_i1), CDPKO (TRINITY_DN93671_c0_g1_i3), NRX1 (TRINITY_DN106708_c1_g2_i3), PTR52 (TRINITY_DN112735_c0_g14_i3; TRINITY_DN112735_c0_g3_i1), TAF6 (TRINITY_DN96231_c1_g1_i2) and CALS5 (TRINITY_DN113564_c1_g1_i1) were enriched in male [63,64,65,66,67,68] (Fig. 8, Additional file 8: Table S5). Genes involved in pollen exine formation such as FACR2/MS2 (TRINITY_DN74585_c1_g5_i3), EA6 (TRINITY_DN76274_c1_g1_i1), C70A2/DEX2 (TRINITY_DN99059_c0_g1_i1) were also upregulated in male [69,70,71]. EMS1 (TRINITY_DN89942_c0_g7_i1), SERK1 (TRINITY_DN108624_c1_g7_i5), JASON (TRINITY_DN83440_c0_g1_i1), RPK2 (TRINITY_DN113423_c0_g1_i4), which are essential for microsporogenesis and pollen maturation were observed to be expressed at significantly higher levels in middle-staged male buds compared to GyM-H buds. [72,73,74,75] (Additional file 8: Table S5; Fig. 7).

Expression profiling for homologs of MS1, EMS1, DYT1, PME4, PME37, PPME1, CSLD1, CDPKO, and PTR52 was studied by qRT-PCR for all the tissue samples including middle-staged Ag-H buds (Figs. 7 and 8; Table 4). Transcripts for all these homologs were downregulated in Ag-H buds and GyM-H buds, suggesting a male-biased expression pattern implicating the reason for pollen sterility in Ag-H and GyM-H buds.