Flowering plants show three major sexual systems viz. hermaphroditism, monoecy and dioecy. Around 90% of the angiosperm species are hermaphroditic bearing perfect flowers having both male as well as female reproductive sex organs [1]. Monoecy exists at a frequency of ~ 5% in angiosperms, wherein unisexual male and female flowers are produced on the same individual plant. Remaining ~ 5% angiosperm species are dioecious, having separate unisexual plants bearing either only male or female flowers [2,3,4]. Dioecious species show patchy phylogenetic distribution and are reported in around three-fourth of the angiosperm families. This indicates that the evolution of dioecy has occurred multiple times in different families independently and hence, the molecular mechanisms of sex determination might vary between distant dioecious species and is a matter of great research interest [5,6,7]. Out of ~ 14,600 known dioecious species in 200 families, plant sex chromosomes have been reported in just around 40 species till date [8]. The mechanism of sex determination in plants can be complex and it may also involve environmental factors apart from genetic factors [9].

Coccinia grandis (L.) Voigt, is a dioecious member of Cucurbitaceae, a family known for its diverse sexual systems [10]. In general, C. grandis is not widely used as a model system to understand sex expression and modification. Commonly known as ivy gourd, C. grandis is also used as a vegetable. Male and female unisexual flowers are borne on separate plants. Similar to Silene latifolia (Caryophyllaceae), the presence of large Y-chromosome in male plant determines the sex [11,12,13]. The chromosome constitution of male and female C. grandis plants found to be 22A + XY and 22A + XX, respectively [14,15,16]. The male flower is characterized by the presence of three convoluted (bithecous) stamens and it has no carpel, whereas the female flower possess three rudimentary stamens that surround the three fused carpels having an inferior ovary [17, 18].

Two possible explanations can be put forward with respect to unisexual flower development. (1) Primordia for both male and female sex organs are initiated during the early stages of flower development and one of them gets aborted during the later stages (eg: S. latifolia). (2) The flower buds are unisexual right from the inception with the primordia initiation for only one of the two sex organs (eg: Thalictrum dioicum) [19, 20]. In some species, inappropriate sex organs are retained in the rudimentary form instead of getting aborted (eg: Rumex acetosa) [21]. Previously, we have demonstrated that application of AgNO 3 on female C. grandis plant modifies the sex expression by inducing stamen development leading to formation of hermaphrodite flowers (such flowers will be referred to as Ag-H hereafter) [18]. Ag+ ion is a known inhibitor of ethylene response [22]. Binding of Ag+ ion to the ethylene receptor locks the conformation such that it remains continuously active and represses the ethylene responses [23]. Silver compounds (AgNO 3 and Ag 2 S 2 O 3 ) have masculinizing effect on monoecious plants (Cucumis sativus) as well as female plants of dioecious species (S. latifolia and Cannabis sativa) [24,25,26]. However, the molecular mechanism of AgNO 3 -mediated induction of stamen development remains unknown till date [26] .

At present, our knowledge about the sex determination mechanisms in plants is fairly limited. Major limitation for studying mechanisms of sex determination in plants is that majority of the dioecious plants are non-model organisms without the availability of genome sequence. Hence, the rate at which sex-linked genes are identified from dioecious plant species is very low [27]. However, the advent of NGS (next-generation sequencing) technologies has enabled the high-throughput identification of sex-biased genes from dioecious plant species in recent times [27]. Also, advanced proteomic approaches may lead to the identification of novel sex-linked proteins that can eventually expand our understanding in evolutionary, developmental and molecular mechanism(s) associated with sex determination and modification.

Muyle, et al. [27] took an RNA-Seq approach to identify sex-biased gene expression in S. latifolia and demonstrated the dosage compensation in plants for the first time. Similarly, other transcriptome studies in this plant have helped in understanding the Y chromosome degeneration and identification of new sex-linked genes [28, 29]. Another study in persimmon (Diospyros lotus) showed that OGI (Oppressor of MeGI), a Y-chromosome-encoded small RNA governs pollen fertility by targeting a homeodomain transcription factor MeGI in a dose-dependent manner [30]. Comparative de novo transcriptomics approach taken in Asparagus resulted in identification of genes involved in pollen microspore and tapetum development that were expressed specifically in male flowers [31]. Similar transcriptomics studies have also been carried out in papaya and cucumber that has led to better understanding of sex determination [32,33,34]. It can be noted that recently, we have carried out a de novo transcriptome profiling from male, female, GyM-H and Ag-H flower buds of C. grandis and identified many sex-biased genes that can provide crucial insights in to stamen arrest, pollen fertility and sex modification mechanism [35].

By using transcriptomics over genomics, it is possible to capture differential gene regulations that may arise due to changes in environmental cues/signals or based on the sex of the plant as in case of dioecious species. However, transcriptomics has a disadvantage that the mRNA abundance may not always accurately reflect protein levels, which are the end-products in the realization of hereditary information carrying out various structural and functional duties [36, 37]. Moreover, only proteomics can capture the post-translational modifications, which are very well-known to control protein functions. However, proteomics study generally depends on the availability of genomic/transcriptomic data, which is limited for most of the dioecious species. Hence, there are only few studies, which have employed proteomics approach in order to understand sex determination and differentiation in dioecious plants [38]. One such study carried out in Asparagus identified differentially accumulated proteins in the form of spots on 2-D gels from flowers of male and female plants [39]. Another study in Pistacia vera purified a 27 kDa glycoprotein specific to female inflorescence [40]. Proteomic approaches has also been attempted to identify sex-linked proteins in Ginkgo biloba. A 28 kDa protein specific to male and 36 kDa as well as 92 kDa proteins specific to female inflorescences have been identified [38]. Recently, Manzano, et al. [41] showed that overexpression of aerolysin-like protein from the dioecious plant R. acetosa induces male sterility in transgenic tobacco.