Biology Division, Indian Institute of Science Education and Research (IISER), Pune, 411008, India.
IPS2, INRA, CNRS, University Paris Sud, University of Evry, University of Paris Diderot, University of Paris Saclay, Batiment 630, 91405, Orsay, France.
BMC Plant Biol. 2019 Jul 23;19(1):330. doi: 10.1186/s12870-019-1937-1.
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 AgNO on female plant induces morphologically hermaphrodite bud development (Ag-H) despite the absence of Y-chromosome.
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. AgNO 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.
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.
雌雄异株是一种重要的性系统，其中雄性和雌性花分别着生于单性的雌雄异株植物上。对性别相关差异的了解可以增强我们对导致单性花发育的分子和发育过程的理解。Coccinia grandis 是葫芦科的一种雌雄异株物种，该科以多种性形式而闻名。雄性和雌性植物分别具有 22A+XY 和 22A+XX 染色体。以前，我们曾报道过 C. grandis 的一种雌雄同体形式（22A+XX），其具有形态上的两性花（GyM-H）和雌花（GyM-F）。此外，我们已经表明，尽管没有 Y 染色体，但在雌性植株上喷洒 AgNO3 会诱导形态上的两性芽发育（Ag-H）。
为了鉴定性别相关差异，我们通过无标记蛋白质组学分析了早期和中期发育的雄性、雌性、GyM-H 和 Ag-H 花芽的总蛋白质组。与黄瓜蛋白质序列（Phytozome）进行蛋白质搜索以及在计算机翻译的 C. grandis 花芽转录组数据库中进行搜索，分别鉴定出 2426 和 3385 种蛋白质（FDR≤1%）。选择后者数据库进行进一步分析，因为它可以检测到更多的蛋白质。使用 BLAST2GO 管道对鉴定出的蛋白质进行注释。在 Female_Early_vs_Male_Early、Ag_Early_vs_Female_Early、GyM-H_Middle_vs_Male_Middle 和 Ag_Middle_vs_Male_Middle 之间进行的基于 SWATH-MS 的比较丰度分析分别导致鉴定出 650、1108、905 和 805 种差异表达蛋白，其 fold change≥1.5 和 P≤0.05。蛋白质相互作用网络中突出显示的乙烯生物合成相关候选物在雌性芽中比在雄性芽中上调。在雌性植株上用 AgNO3 处理会诱导 Ag-H 芽中与花粉发育有关的蛋白质。此外，与 Ag-H 和 GyM-H 芽相比，雄性芽中高度富集了几个控制花粉萌发和管生长的蛋白质。
总的来说，目前的蛋白质组学分析提供了在鉴定葫芦科雌雄异株和单性花发育的关键蛋白质方面的见解，葫芦科是经济重要性方面第二大园艺科。此外，我们的结果表明，乙烯介导的雄蕊抑制可能在雌雄异株的 C. grandis 中保守，类似于其单性的葫芦科亲缘关系。此外，这里鉴定的与花粉萌发和管生长相关的雄性偏向蛋白有助于理解花粉活力。
