HSFs调控自噬提高番茄花粉耐热性的分子机制

Pollen grain development is highly sensitive to temperature stresses in plants. High temperature can cause oxidation and denaturation of proteins that subsequently damage cellular structure and inhibit pollen germination in pollen cells. However, the mechanisms that control pollen thermotolerance is still unclear. The applicants have discovered that the expression of autophagy-related genes (ATGs) and formation of autophagy were induced to degrade the denatured proteins under heat stress in tomato plants, and heat shock elements (HSE)were detected in the promoters of these ATGs. Meanwhile, pollen viability was also decreased in tomato autophagy mutants. Therefore, the applicants hypothesize that HSFs may also induce expression of autophagy-related genes and formation of autophagy to degrade denatured proteins in tomato pollen and increase pollen viability under temperature stresses. Thus, the present project is planned to utilize thermotolerant tomato cultivars, and transgenic tomato plants as materials to investigate the expression of relative genes, accumulation of denatured proteins and formation of autophagy in different tomato genotypes. The project is focused on the mode of action on the involvement of HSFAs-regulated heat shock elements of ATGs and autophagy in degradation of denatured proteins in tomato pollen, and the relationship between autophagy and HSPs in protein degradation. It is expected to provide a theoretic basis to our current knowledge of the mechanisms of tomato temperature tolerance and technical guidance for thermotolerance breeding of tomato, and it has potentially economic and social benefits.

植物花粉对高温极为敏感，高温会造成花粉细胞蛋白变性，进而造成花粉结构破坏，活力下降，萌发受到抑制。然而关于花粉耐热调控机制尚不清楚。申请人前期研究发现高温下番茄自噬基因ATGs被诱导表达促进自噬体形成降解高温产生的变性蛋白，且ATGs基因中存在热激转录因子HSFs结合元件HSE；同时自噬基因缺失后番茄花粉活力显著下降。在此基础上，申请人提出高温下HSFs通过调控ATGs表达和自噬体产生降解变性蛋白，提高番茄花粉耐热性的学术思想。本项目拟以蔬菜主栽作物番茄为材料，利用已获得的番茄不同温敏型和转基因材料，研究高温下不同耐热性材料中相关基因表达、变性蛋白与自噬体数目差异；阐明HSFs通过结合ATGs启动子上热激元件调控自噬体产生提高番茄花粉耐热性的信号途径；并明确自噬降解变性蛋白与热激蛋白的关系，有望揭示番茄高温抗性机制，为创制耐热番茄新种质并提高番茄产量品质提供导向，具有潜在的经济和社会效益。

植物花粉对高温极为敏感，高温会造成花粉细胞蛋白变性，破坏花粉结构，导致花粉活力下降，萌发受到抑制。然而关于花粉耐热调控机制尚不清楚。申请人前期研究发现高温下番茄自噬基因ATGs被诱导表达促进自噬体形成降解高温产生的变性蛋白，同时自噬基因缺失后番茄花粉活力显著下降。在此基础上，申请人对HSFs调控自噬提高番茄花粉耐热性的分子机制展开了研究，总计发表论文18篇，授权发明专利1件。发现（1）HsfA1a 对番茄在热胁迫下的花粉活力至关重要。HsfA1a可以影响热胁迫下花药中抗氧化酶的表达和活性来减轻 ROS 积累，调控HSP的表达和积累来增强蛋白质修复能力，并影响泛素化蛋白质的降解能力。（2）HsfA1a可以影响花粉中自噬体的形成，并直接和ATG10基因的启动子结合调控其转录，从而调控热胁迫下番茄花药的自噬水平，增强热胁迫下对变形蛋白的降解能力，提高花粉耐热性。（3）HsfA1a可以作为上游调控信号诱导褪黑素的合成，HsfA1a能够直接结合到褪黑素合成基因COMT1 基因的启动子上诱导其转录，而沉默 HsfA1a 则抑制了褪黑素合成基因COMT1 的表达并降低褪黑素的含量（4）油菜素内酯BR合成和信号转导提高番茄花粉生殖能力和种子产量，并且ROS参与BRs对绒毡层PCD发生和降解的促进作用。（5）BRs通过激活RBOH1基因表达诱导ROS产生促进绒毡层降解，BZR1通过与RBOH1启动子的结合调控RBOH1的基因表达，并且RBOH1-RNAi导致绒毡层中的ROS积累显著减少，种子产量降低。（6）发现miR171b基因的过表达和敲除影响了番茄花粉活力和萌发率。以上研究结果系统阐明了HsfA1a调控番茄花粉耐热性的机制，进一步完善了热休克反应（HSR）的调控网络，明确了热胁迫下番茄花粉中泛素-蛋白酶体系统和自噬协同维持蛋白质稳态的重要作用。加深了对于 BRs 和 ROS 作为信号分子对花粉发育和绒毡层降解过程的调控以及两者互作的分子机制的理解，这些研究结果都为花粉的耐热性研究的潜在应用提供了重要信息，同时有助于运用分子和生理生化等手段提高蔬菜的耐热性和产量，对保障我国的蔬菜均衡供应和农业经济效益均具有重要的现实意义。