Global landscape of protein phosphorylation during plant regeneration initiation in cotton (Gossypium hirsutum L.)

Phosphorylation is one of the most common post-translational modifications and is central to many cellular signaling events; however, little is currently known about the phosphorylation landscape during somatic embryogenesis (SE) for plant regeneration. Here, we systematically analyzed the phosphoproteomic profile of three typical developmentally staged cultures of SE, non-embryogenic calli (NEC), primary embryogenic calli (PEC), and globular embryos (GE), in cotton (Gossypium hirsutum L.), the pioneer crop for genetic biotechnology applications. Our data revealed a total of 6301 quantifiable phosphorylation sites in 2627 quantifiable phosphoproteins from 5548 modified peptides, of which 1105 phosphoproteins (2147 sites) were differentially phosphorylated. Functional enrichment analyses revealed that differentially regulated phosphoproteins (DRPPs) were significantly enriched in DNA mismatch repair and peroxisome during callus embryogenic differentiation (PEC vs. NEC) and somatic embryo initiation (GE vs. PEC), respectively. Notably, six dynamic trajectory patterns of DRPP enrichment were observed. In addition, preferentially activated DRPPs with specific phosphorylation patterns were identified at different developmental stages. These DRPPs were mainly involved in hormone-responsive and photosystem events during initiation of plant regeneration. Overall, this study identified a series of potential phosphoproteins responsible for SE trans-differentiation and plant regeneration, providing a valuable resource and molecular basis for understanding the regulatory pathways underlying cell totipotency at the post-translational modification level.

磷酸化是最为常见的翻译后修饰（post-translational modification）之一，在诸多细胞信号事件中发挥核心作用；然而目前学界对植物再生过程中体细胞胚发生（SE）阶段的磷酸化调控图谱仍知之甚少。本研究以遗传生物技术应用的模式作物棉花（陆地棉Gossypium hirsutum L.）为材料，系统分析了体细胞胚发生（SE）三个典型发育阶段的磷酸化蛋白质组谱，分别为非胚性愈伤组织（NEC）、初级胚性愈伤组织（PEC）与球形胚（GE）。本研究共从5548条修饰肽段中鉴定得到2627个可定量磷酸化蛋白质，涵盖6301个可定量磷酸化位点；其中1105个磷酸化蛋白质（对应2147个位点）存在磷酸化差异。功能富集分析显示，在愈伤组织胚性分化阶段（PEC vs. NEC）与体细胞胚起始阶段（GE vs. PEC），差异调控磷酸化蛋白质（differentially regulated phosphoproteins, DRPPs）分别显著富集于DNA错配修复通路与过氧化物酶体通路。值得注意的是，本研究共观察到6种差异调控磷酸化蛋白质富集的动态轨迹模式。此外，研究在不同发育阶段鉴定得到了具有特定磷酸化模式的优先激活差异调控磷酸化蛋白质。这些差异调控磷酸化蛋白质主要参与植物再生起始过程中的激素响应与光系统相关生物学事件。综上，本研究鉴定得到一系列参与体细胞胚发生转分化与植物再生的潜在磷酸化蛋白质，为从翻译后修饰层面解析细胞全能性的调控通路提供了宝贵的研究资源与分子理论基础。