Flower bud proteome reveals modulation of sex-biased proteins potentially associated with sex expression and modification in dioecious Coccinia grandis.

BACKGROUND: Dioecy is an important sexual system wherein, male and female flowers are borne on separate unisexual plants. Knowledge of sex-related differences can enhance our understanding in molecular and developmental processes leading to unisexual flower development. Coccinia grandis is a dioecious species belonging to Cucurbitaceae, a family well-known for diverse sexual forms. Male and female plants have 22A + XY and 22A + XX chromosomes, respectively. Previously, we have reported a gynomonoecious form (22A + XX) of C. grandis bearing morphologically hermaphrodite flowers (GyM-H) and female flowers (GyM-F). Also, we have showed that foliar spray of AgNO3 on female plant induces morphologically hermaphrodite bud development (Ag-H) despite the absence of Y-chromosome. RESULTS: To identify sex-related differences, total proteomes from male, female, GyM-H and Ag-H flower buds at early and middle stages of development were analysed by label-free proteomics. Protein search against the cucumber protein sequences (Phytozome) as well as in silico translated C. grandis flower bud transcriptome database, resulted in the identification of 2426 and 3385 proteins (FDR ≤ 1%), respectively. The latter database was chosen for further analysis as it led to the detection of higher number of proteins. Identified proteins were annotated using BLAST2GO pipeline. SWATH-MS-based comparative abundance analysis between Female_Early_vs_Male_Early, Ag_Early_vs_Female_Early, GyM-H_Middle_vs_Male_Middle and Ag_Middle_vs_ Male_Middle led to the identification of 650, 1108, 905 and 805 differentially expressed proteins, respectively, at fold change ≥1.5 and P ≤ 0.05. Ethylene biosynthesis-related candidates as highlighted in protein interaction network were upregulated in female buds compared to male buds. AgNO3 treatment on female plant induced proteins related to pollen development in Ag-H buds. Additionally, a few proteins governing pollen germination and tube growth were highly enriched in male buds compared to Ag-H and GyM-H buds. CONCLUSION: Overall, current proteomic analysis provides insights in the identification of key proteins governing dioecy and unisexual flower development in cucurbitaceae, the second largest horticultural family in terms of economic importance. Also, our results suggest that the ethylene-mediated stamen inhibition might be conserved in dioecious C. grandis similar to its monoecious cucurbit relatives. Further, male-biased proteins associated with pollen germination and tube growth identified here can help in understanding pollen fertility.

Genome-wide transcriptome analysis reveals small RNA profiles involved in early stages of stolon-to-tuber transitions in potato under photoperiodic conditions.

BACKGROUND: Small RNAs (sRNAs), especially miRNAs, act as crucial regulators of plant growth and development. Two other sRNA groups, trans-acting short-interfering RNAs (tasiRNAs) or phased siRNAs (phasiRNAs), are also emerging as potential regulators of plant development. Stolon-to-tuber transition in potato is an important developmental phase governed by many environmental, biochemical and hormonal cues. Among different environmental factors, photoperiod has a major influence on tuberization. Several mobile signals, mRNAs, proteins and transcription factors have been widely studied for their role in tuber formation in potato, however, no information is yet available that describes the molecular signals governing the early stages of stolon transitions or cell-fate changes at the stolon tip before it matures to potato. Stolon could be an interesting model for studying below ground organ development and we hypothesize that small RNAs might be involved in regulation of stolon-to-tuber transition process in potato. Also, there is no literature that describes the phased siRNAs in potato development. RESULTS: We performed sRNA profiling of early stolon stages (4, 7 and 10 d) under long-day (LD; 16 h light, 8 h dark) and short-day (SD; 8 h light, 16 h dark) photoperiodic conditions. Altogether, 7 (out of 324) conserved and 12 (out of 311) novel miRNAs showed differential expression in early stolon stages under SD vs LD photoperiodic conditions. Key target genes (StGRAS, StTCP2/4 and StPTB6) exhibited differential expression in early stolon stages under SD vs LD photoperiodic conditions, indicative of their potential role in tuberization. Out of 830 TAS-like loci identified, 24 were cleaved by miRNAs to generate 190 phased siRNAs. Some of them targeted crucial tuberization genes such as StPTB1, POTH1 and StCDPKs. Two conserved TAS loci, referred as StTAS3 and StTAS5, which share close conservation with members of the Solanaceae family, were identified in our analysis. One TAS-like locus (StTm2) was validated for phased siRNA generation and one of its siRNA was predicted to cleave an important tuber marker gene StGA2ox1. CONCLUSION: Our study suggests that sRNAs and their selective target genes could be associated with the regulation of early stages of stolon-to-tuber transitions in a photoperiod-dependent manner in potato.

De novo transcriptome assembly from flower buds of dioecious, gynomonoecious and chemically masculinized female Coccinia grandis reveals genes associated with sex expression and modification.

BACKGROUND: Coccinia grandis (ivy gourd), is a dioecious member of Cucurbitaceae having heteromorphic sex chromosomes. Chromosome constitution of male and female plants of C. grandis is 22A + XY and 22A + XX respectively. Earlier we showed that a unique gynomonoecious form of C. grandis (22A + XX) also exists in nature bearing morphologically hermaphrodite flowers (GyM-H). Additionally, application of silver nitrate (AgNO3) on female plants induces stamen development leading to the formation of morphologically hermaphrodite flowers (Ag-H) despite the absence of Y-chromosome. Due to the unavailability of genome sequence and the slow pace at which sex-linked genes are identified, sex expression and modification in C. grandis are not well understood. RESULTS: We have carried out a comprehensive RNA-Seq study from early-staged male, female, GyM-H, and Ag-H as well as middle-staged male and GyM-H flower buds. A de novo transcriptome was assembled using Trinity and annotated by BLAST2GO and Trinotate pipelines. The assembled transcriptome consisted of 467,233 'Trinity Transcripts' clustering into 378,860 'Trinity Genes'. Female_Early_vs_Male_Early, Ag_Early_vs_Female_Early, and GyM-H_Middle_vs_Male_Middle comparisons exhibited 35,694, 3574, and 14,954 differentially expressed transcripts respectively. Further, qRT-PCR analysis of selected candidate genes validated digital gene expression profiling results. Interestingly, ethylene response-related genes were found to be upregulated in female buds compared to male buds. Also, we observed that AgNO3 treatment suppressed ethylene responses in Ag-H flowers by downregulation of ethylene-responsive transcription factors leading to stamen development. Further, GO terms related to stamen development were enriched in early-staged male, GyM-H, and Ag-H buds compared to female buds supporting the fact that stamen growth gets arrested in female flowers. CONCLUSIONS: Suppression of ethylene responses in both male and Ag-H compared to female buds suggests a probable role of ethylene in stamen suppression similar to monoecious cucurbits such as melon and cucumber. Also, pollen fertility associated GO terms were depleted in middle-staged GyM-H buds compared to male buds indicating the necessity of Y-chromosome for pollen fertility. Overall, this study would enable identification of new sex-biased genes for further investigation of stamen arrest, pollen fertility, and AgNO3-mediated sex modification.

Development of a Virus-Induced Gene Silencing System for Dioecious Coccinia grandis.

Coccinia grandis is an interesting model system to understand dioecy in Cucurbitaceae family. Recent transcriptomics and proteomics studies carried out to understand the sex expression in C. grandis have resulted in identification of many candidate sex-biased genes. In absence of an efficient genetic transformation protocol for C. grandis, virus-induced gene silencing (VIGS) would be a powerful tool to enable gene functional analysis. In current study, we explored the apple latent spherical virus (ALSV) for gene knockdown in C. grandis. The viral infection was achieved through mechanical inoculation of ALSV-infected Chenopodium quinoa leaf extract onto the cotyledons of C. grandis. ALSV-VIGS mediated knockdown of CgPDS gene was successfully achieved in C. grandis by mechanical inoculation method resulting in characteristic photobleaching. Subsequently, we developed agroinfiltration compatible vectors for direct infection of C. grandis and shortened the time-frame by skipping viral propagation in C. quinoa. Typical yellow-leaf phenotype was observed in C. grandis plants agroinfiltrated with ALSV-CgSU constructs, indicating robust silencing of CgSU gene. In addition, we improved the infection efficiency of ALSV by co-infiltration of P19 viral silencing suppressor. These results suggest that ALSV-VIGS is suitable for characterization of gene function in dioecious C. grandis and it can help us understand the mechanism of sex expression.