拟南芥PP1新调节亚基PP1R3调控ABA信号的分子机理

Protein phosphatase 1 (PP1) holoenzyme, consisting of a catalytic subunit (PP1c) and regulatory subunits (PP1r), is involved in many important life processes in plants. However, researches on the genetic function of PP1 are relatively retarded. Therefore, it is very important to identify new PP1r and elucidate how holoenzyme controls life processes through specifically regulating its substrates in plants. In previous work, we successfully identified a new regulatory subunit of PP1 in Arabidopsis, PP1R3, and found MYBx was a candidate substrate of PP1R3-TOPPs holoenzyme. We also identified a potential kinase of MYBx and found the phosphorylation sites of MYBx by mass spectrometry (MS). Physiological analyses showed that all those components are involved in ABA responses. Based on these preliminary results, we intend to deeply study the detailed mechanism of PP1R3-TOPPs holoenzyme in controlling ABA signaling by focusing on the regulation of phosphorylation modification of MYBx. Using strategies of MS identification, physiological and genetic analyses, protein phosphorylation experiments, site-directed mutagenesis of protein phosphorylation sites to mimic phosphorylation or dephosphorylation status, and CHIP-Seq, we plan to clarify the mechanism of how PP1R3-TOPPs control ABA biosynthesis or signal transduction via modulating the phosphorylation modification of MYBx, and reveal the molecular mechanism of PP1 in regulating stress responses in plants. Meanwhile, we propose to study the function mechanism of these related homologous genes in stress adaptation in rice and maize, which will provide strategies for PP1 utilization in improving crop adaptation to stress conditions.

蛋白磷酸酶1（PP1）全酶由催化亚基（PP1c）和调节亚基（PP1r）组成，参与植物许多重要的生命活动过程。但是其遗传功能研究比较滞后，因此鉴定新的PP1r，研究全酶如何通过特异性调节底物来调控植物生命活动具有非常重要的意义。本项目前期工作鉴定到一个拟南芥PP1的新调节亚基PP1R3，发现其全酶的可能底物是MYBx，并鉴定到MYBx的激酶及磷酸化位点，这些组分都参与了ABA响应。在此基础上，以MYBx磷酸化修饰调控为中心，深入研究PP1R3-TOPPs全酶在ABA信号调控中的详细机理。通过质谱鉴定、生理与遗传分析、蛋白磷酸化实验、模拟磷酸化位点突变、CHIP-Seq等技术，阐明PP1R3-TOPPs通过对MYBx的磷酸化修饰如何调节ABA合成或信号转导的机理，揭示PP1调控植物响应逆境的分子机制；同时研究水稻和玉米中相关同源基因在逆境适应中的作用机理，为PP1在农作物抗逆中的应用奠定基础。

蛋白磷酸酶1 (PP1) 全酶由催化亚基 (PP1c) 和调节亚基 (PP1r) 组成，参与调节许多重要的生命活动过程。但是植物中PP1的功能研究比较滞后，特别是对调节亚基的认识更是有限。因此鉴定新的PP1r，研究PP1如何通过特异性调节底物来调控植物生命活动意义重大。本项目首先鉴定到拟南芥PP1 (也称TOPPs)的新调节亚基PP1R3，证明PP1R3可以抑制TOPPs的酶活性，促进TOPPs的核定位：发现2个pp1r3 T-DNA插入突变体对ABA和盐更敏感，在pp1r3-2和TOPP七突topp-7m中，大量与ABA合成、响应及转运相关的基因表达量变化一致，表明PP1R3和TOPPs可能处于同一通路调节ABA及逆境胁迫响应；且PP1R3-TOPPs全酶可能独立于ABI1发挥调节ABA信号的作用。其次发现PP1R3-TOPPs的下游蛋白MYBx，证明MYBx通过抑制ABA响应基因ABIx和ABIz的转录负调节ABA信号通路；鉴定到调控MYBx降解的E3泛素连接酶ME3I；而ME3I又与TOPP1/4互作，推测TOPP可能也通过ME3I调节了ABA介导的MYBx的稳定性。最后鉴定了玉米中ZmTOPPs在逆境胁迫中的功能，发现ZmTOPPa-OE超表达株系与zmtoppb/c双突变体均表现出抗旱性增强的表型，表明ZmTOPPs参与了玉米逆境胁迫响应，且家族成员的功能有差异，为TOPP在农作物抗逆中的应用奠定了基础。.总之，本项目发现PP1R3-TOPPs全酶调节ABA信号；鉴定到PP1R3-TOPPs下游蛋白MYBx，其通过抑制ABIx和ABIz的转录负调控ABA信号；TOPP可能通过ME3I调控MYBx的稳定性；此外，发现玉米中部分ZmTOPPs可提高植株抗旱能力。深入揭示了TOPPs介导的蛋白去磷酸化在调控植物逆境胁迫反应中的重要功能及作用机理，为挖掘农业生产中有利用价值的抗逆基因打下了基础，完全实现了研究目标。.在本项目资助下，在国际学术期刊上发表SCI论文6篇：包括1篇The Plant Cell, 1篇New Phytologist和1篇Plant Physiology。培养毕业研究生19名（硕士13名、博士6名），博士后1名，青年教师3名，有力支持了兰州大学生物学一流学科的建设。初步建成了具有影响力的植物蛋白磷酸酶研究中心。

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