拟南芥MPK3/MPK6磷酸化WRKY转录因子调控花粉发育过程中能量代谢的分子机制研究

Energy metabolism leading to the accumulation of storage compounds plays vital roles in pollen development. However, the underlying regulatory mechanism is far from clear. We found that two redundant WRKY transcription factors, WRKY2 and WRKY34, which are highly expressed during flowering stage, are downstream of MPK3 and MPK6, two mitogen-activated protein kinases involved in plant growth, development, and response to stress/pathogen infection. WRKY34 is phosphorylated by MPK3 and MPK6 in vivo in a spatiotemporal dependent manner between bicellular and tricellullar stages during pollen development. In mature wrky2 wrky34 pollen, the number of lipid bodies is greatly reduced, which is associated with ruptured and weak pollen grains. As a result, we believe that the MPK3/MPK6-WRKY34/WRKY2 is a key signaling pathway in pollen development, and propose that this module regulates the metabolism of storage compounds in pollen development. In this project, we will use pollen specific ChIP-SEQ and translatome analyses to identify the target genes of WRKY34/WRKY2. Functional analyses of these target genes will reveal how MPK3/MPK6-WRKY34/WRK2 regulates pollen development. In addition, we will identify the MAPKK(s) and MAPKKK(s) upstream of MPK3/MPK6 in this pathway. Through these efforts, we will be able to reveal a complete signaling pathway and the underlying molecular mechanism that regulates the spatiotemporal specific biosynthesis of storage compounds during pollen development. Understanding of pollen storage compound biosynthesis, which is critical to the pollen fitness and viability, could benefit crop production in agriculture.

能量物质代谢在花粉发育过程中起着至关重要的作用，但是相关的信号通路及调控机制了解并不多。在前期研究中，我们发现两个存在功能冗余的转录因子WRKY34和WRKY2在拟南芥花中特异表达，遗传及生化分析表明WRKY34和WRKY2位于 MPK3/MPK6下游。成熟wrky2 wrky34 花粉内脂质体数目明显减少。我们认为MPK3/MPK6-WRKY34/WRKY2是花粉发育的关键信号通路之一，并推测该信号通路调控花粉中脂类等相关能量物质代谢。本项目拟在前期研究基础上，通过花粉细胞特异的染色质免疫沉淀测序以及核糖体结合mRNA翻译组学技术对该通路的下游靶基因及调控机制进行深入分析，同时还将寻找该通路上游的MAPKK(s)及MAPKKK(s)，以期揭示一条完整的调控花粉中能量物质代谢的信号通路，并阐明该通路对花粉发育的具体调节机制。本课题的研究将会对作物的花粉研究提供重要的理论支持。

能量物质代谢在花粉发育过程中起着至关重要的作用。前期研究表明功能冗余的转录因子WRKY34和WRKY2位于 MPK3/MPK6下游，调控了花粉中脂肪酸等能量物质的合成，但是机制不明。本项目旨在此基础上深入研究该MAPK-WRKY途径调控花粉发育中脂肪合成与积累的机制。我们的研究结果表明GPT1是WRKY2/WRKY34下游的一个关键靶基因，在花粉脂肪合成与积累过程中有着重要的生理功能。GPT1编码一个位于质体膜上的葡萄糖-6-磷酸/磷酸转运体，该蛋白的主要功能是将细胞质中的葡萄糖-6-磷酸转运进入质体内以用于淀粉或者脂肪酸的生物合成。GPT1在花粉发育过程的TCP和MP时期表达量达到最高，其时空表达紧随WRKY2/WRKY34被MPK3/MPK6磷酸化过程之后。由GPT1自身启动子驱动表达的GPT1-eYFP在wrky2-/- wrky34-/-背景中相比在野生型花粉中荧光信号明显减弱，表明GPT1的表达依赖于WRKY2和WRKY34。由花粉特异性启动子LAT52启动的GPT1可以显著回复wrky2 wrky34双突变体花粉的表型，表明GPT1是WRKY2/WRKY34下游的重要功能基因。此外，研究发现MKK4/MKK5-MPK3/MPK6信号途径的缺失阻碍了GPT1的表达，脂肪的积累，以及花粉的正常发育。这些结果说明MKK4/MKK5-MPK3/MPK6-WRKY2/WRKY34途径通过时空调节GPT1的表达调控了花粉中脂肪合成和积累，从而调控花粉的发育。我们的结果为作物的花粉发育，以及植物的种子发育、胚胎发育过程中能量物质代谢过程有重要的理论参考价值。在该项目支持下，共发表SCI论文3篇。