Proteomic and Phosphoproteomic Analyses Reveal Extensive Phosphorylation of Regulatory Proteins in Developing Rice Anthers

Anther development, particularly around the time of meiosis, is extremely crucial for plant sexual reproduction. Meanwhile, cell-to-cell communication between somatic (especial tapetum) cells and meiocytes are important for both somatic anther development and meiosis. To investigate possible molecular mechanisms involved in protein activities during anther development, we applied high-resolution mass spectrometry-based proteomic and phosphoproteomic analyses for developing rice (Oryza sativa) anthers around the time of meiosis (RAM). In total, we identified 4,984 proteins and 3,203 phosphoproteins with 8,973 unique phosphorylation sites (p-sites). Among those detected here, 1,544 phosphoproteins are currently absent in the Plant Protein Phosphorylation DataBase (P3DB), substantially enriching plant phosphorylation information. Mapman enrichment analysis showed that “DNA repair”, “transcription regulation” and “signalling” related proteins were over-represented in the phosphorylated proteins. Ten genetically identified rice meiotic proteins were detected to be phosphorylated at a total of 25 p-sites; moreover more than 400 meiotically expressed proteins were revealed to be phosphorylated and their phosphorylation sites were precisely assigned. 163 putative secretory proteins, possibly functioning in cell-to-cell communication, are also phosphorylated. Furthermore, we showed that DNA synthesis, RNA splicing and RNA-directed DNA methylation pathways are extensively affected by phosphorylation. In addition, our data support forty-six kinase-substrate pairs predicted by the rice Kinase-Protein Interaction Map, with SnRK1 substrates highly enriched. Taken together, our data revealed extensive protein phosphorylation during anther development, suggesting an important post-translational modification mechanism for protein activity.

花药发育，尤其是减数分裂（meiosis）前后的阶段，对植物有性生殖至关重要。与此同时，体细胞（尤其是绒毡层（tapetum）细胞）与性母细胞（meiocytes）之间的细胞间通讯，对花药体细胞发育与减数分裂过程均具有重要意义。为探究花药发育过程中蛋白质活性相关的潜在分子机制，本研究针对减数分裂前后（RAM）的水稻（Oryza sativa）发育花药，开展了基于高分辨率质谱的蛋白质组学与磷酸化蛋白质组学分析。本研究共鉴定得到4984种蛋白质与3203种磷酸化蛋白质，涵盖8973个独特的磷酸化位点（p-sites）。在本次检测到的磷酸化蛋白质中，有1544种目前未被收录于植物蛋白质磷酸化数据库（Plant Protein Phosphorylation Database, P3DB），极大丰富了植物磷酸化相关的研究数据。Mapman富集分析结果显示，与"DNA修复"、"转录调控"及"信号转导"相关的蛋白质在磷酸化蛋白质中显著富集。本研究检测到10种经遗传学验证的水稻减数分裂相关蛋白质，共计存在25个p-sites；此外，还发现了400余种减数分裂期表达的蛋白质发生磷酸化，并精准注释了其磷酸化位点。另有163种推测参与细胞间通讯的分泌型蛋白质，同样发生了磷酸化修饰。进一步分析表明，DNA合成、RNA剪接以及RNA指导的DNA甲基化通路均广泛受到磷酸化修饰的调控。此外，本研究数据验证了水稻激酶-蛋白质相互作用图谱所预测的46个激酶-底物相互作用对，其中SnRK1的底物显著富集。综上，本研究数据揭示了花药发育过程中广泛存在的蛋白质磷酸化修饰现象，表明磷酸化作为一种重要的翻译后修饰机制，对蛋白质活性发挥关键调控作用。