番茄细胞壁相关激酶对寡聚半乳糖醛酸诱导果实乙烯生成的调控机制研究

Pectin degradation affect fruit texture and quality, and its degraded products oligogalacturonic acids (OGs) act as a signal to interact with its receptor wall associated kinases (WAKs), activating many plant development and immune responses. However, few works were carried out on the function of OGs/WAK pathway and its signal transduction in fruits. Our previous study found OGs promoted tomato fruit ripening through the regulation of expression of aminocyclopropane-1-carboxylic acid synthase2 (ACS2) at the transcriptional and post-translational levels. To further investigate the effect of WAK on the OGs-induced ethylene production and signal transduction, OGs treated WAK overexpressed or knocked out fruits were sampled to measure ethylene production, phosphorylated LeACS2 expression level and the activities of protein kinase and phosphatase. WAK conserved kinase domain was used to screen type 2C protein phosphatases (PP2C) by Yeast Two-Hybrid Analysis, then BiFC assay was used to test their interaction in vivo. To establish the interaction network of components regulating the phosphorylated LeACS2 in OGs/ WAK pathway, in vitro phosphorylation and dephosphorylation assay were carried out with different combination of recombinant purified WAK, PP2C, MAPK and ACS2 proteins. The project will be helpful for enriching our understanding on the function of cell wall pectin degradation products on the regulation of ethylene production and tomato fruits ripening.

果胶降解影响果实品质。其降解产物寡聚半乳糖醛酸（OGs）作为信号物质通过与受体细胞壁相关激酶（WAKs）结合，影响植物的发育及胁迫反应，但在果实中，OGs/WAK作用机制及信号传递却少有研究。前期我们发现，OGs通过ACS2磷酸化诱导番茄果实乙烯生成，促进果实成熟，但OGs是如何通过WAK及下游组分调控ACS的磷酸化分子机制有待深入研究。本项目以番茄ACS2磷酸化调控为研究点，首先获得WAK过表达及WAK缺失果实，验证WAK在OGs诱导的乙烯生成、ACS2磷酸化及果实成熟中作用；再以WAK蛋白激酶域为诱饵，酵母双杂交技术筛选与WAK互作的PP2C蛋白；最后，结合体内CO-IP、BiCF互作、体外磷酸化分析，揭示出OGs/WAK信号传递中WAK/PP2C/MAPK/ACS之间蛋白互作对乙烯生成调控的分子机制。本项目对于完善细胞壁降解产物在果实成熟衰老中分子作用机制具有重要意义。

植物细胞壁果胶组分代谢变化，影响并调控细胞壁中相关受体蛋白（Wall associated kinase/kinase-like）WAKs/WAKLs的基因表达及蛋白活性，引发细胞壁信号传递，在调控植物的生长发育及胁迫反应过程中发挥重要作用。本研究首先利用不同浓度果胶降解产物寡聚半乳糖醛酸（OGs）处理番茄黄化幼苗，利用SlACS2 反义材料、VIGS沉默的番茄MPK3材料，结合抑制剂及体外磷酸化实验，明确了SlMPK3可直接磷酸化SlACS2,SlACS2、SlMPK3是参与OGs介导乙烯生成过程主要组分；利用Hidden Markov Model (HMM) 模型在番茄基因组中共鉴定到11个WAKs和18个WAKLs 成员, 并系统分析了家族成员生信及组织表达特性；利用病毒介导的基因沉默（VIGS）技术筛选影响番茄果实成熟的WAKs/WAKLs成员，SlWAK1、SlWAKL2和SlWAKL4沉默植株表现出果实成熟延缓、种子发育缺陷的表型。随后，获得了SlWAKL2基因沉默和过表达的稳定转基因材料，发现SlWAKL2影响到果实大小、种子发育、表皮光泽度、果实组织中维管束发育，并正调控番茄幼苗的OGs反应；最后利用酵母文库筛选到多个与WAKL2的互作蛋白，其中WAKL2可以体内体外磷酸化转录因子bHLH3，WAKL2对bHLH3磷酸化影响到其定位、稳定性和转录活性，进而调控靶基因表达。该项目完善了OGs对植物生长发育的网络调控信号，填补了番茄WAKs/WAKLs对果实发育调控分子机制的空白。