Reversible phosphorylation plays a crucial role in regulating protein activities and functions. Sexual reproduction directly affects yield of most agricultural crops. As the male reproductive organ, anther generates microspores (pollen), delivering gametes (sperms) to complete double fertilization in higher plants. Here, we took the advantage of Nano UHPLC-MS/MS to analyze maize (Zea mays, B73) early anthers at proteomic and phosphoproteomic levels, to explore the protein and phosphorylation modification regulatory networks controlling maize anther development. Our proteomic analysis identified 3 016 unique peptides, belonging to 1 032 maize proteins. MapMan analysis revealed variously potential proteins associated with maize anther development, such as receptor-like kinases (GRMZM2G082823_P01 and GRMZM5G805485_P01). Using phospho-peptides enriched by TiO2 affinity chromatography, our phosphoproteomic analysis detected 257 phospho-peptides from 210 phosphoproteins, discovering 223 phosphosites. Compared to the 86 maize phosphoproteins collected in the Plant Protein Phosphorylation Data Base (P3DB), we found that 203 phosphoproteins and 218 phosphosites were not revealed before. Further bioinformatics analysis revealed that phosphorylation of 14-3-3 proteins, kinases, phosphatases, transcription factors, cell cycle and chromatin structure related proteins might play important roles in regulating normal anther development in maize. Our findings not only enlarged the maize phosphoproteome data, but also provided information for analyzing the molecular mechanism controlling maize anther development at genetic and biochemical levels.
蛋白质磷酸化修饰是调控其功能的一种重要方式。植物有性生殖过程在农作物产量形成和物种繁衍过程中起着重要作用。作为植物雄性生殖器官的花药，其正常生长发育对于保证形成功能性配子 (花粉) 以及完成双受精过程至关重要。本研究以重要农作物玉米 (B73) 为材料，利用Nano UHPLC-MS/MS质谱技术对玉米早期发育的花药在蛋白质组和磷酸化蛋白质组水平进行全面分析，以探究玉米花药发育过程中的蛋白调控网络和磷酸化修饰调控网络。在蛋白质组学分析中，共鉴定到了3 016个多肽，匹配到1 032个蛋白质上。通过MapMan分析，预测到了一些和花药发育相关的蛋白质，例如受体激酶 (GRMZM2G082823_P01、GRMZM5G805485_P01等)。另外，在磷酸化蛋白质组学研究中，通过对TiO2亲和层析富集到的磷酸化多肽进行质谱分析，检测到了257个磷酸化多肽，匹配到 210个蛋白质上。我们的数据揭示了玉米花药发育过程中的 223个磷酸化位点。与已发现的玉米中的86个磷酸化蛋白质 (植物蛋白磷酸化数据库 (P3DB)：http://www.p3db.org/organism.php) 相比，其中203个磷酸化蛋白和218个磷酸化位点为首次揭示。进一步生物信息学分析表明：磷酸化在14-3-3蛋白质、激酶、磷酸酶、转录因子、细胞周期和染色质结构相关的蛋白质介导的玉米早期花药发育过程中起着重要的调控作用。总之，本研究首次在蛋白质组学和磷酸化蛋白质组学水平研究了玉米早期花药发育的蛋白质调控网络，不仅丰富了玉米蛋白质和磷酸化修饰蛋白质数据库，并为利用遗传学和生物化学手段深入研究玉米花药发育的分子调控机理提供了基础。.
Keywords: kinase; maize anther; phosphorylation; proteome; regulatory network.