从硫巯基化修饰的角度探索硫化氢诱导番茄侧根发生的分子机理

S-sulfhydration of protein cysteine residues is a novel molecular mechanism underlying ample physiological effects of hydrogen sulfide (H2S) in animals. Our recent results showed that H2S and hydrogen peroxide (H2O2) may be respectively involved in auxin-induced tomato lateral root formation, and in vitro assay indicated that NADPH oxidase and ascorbate peroxidase (APX) might be the possible targets of H2S-mediated S-sulfhydration. To further assess the intrinsic mechanism, in this project, firstly, biotin switch method was used to screen the protein(s) modified by S-sulfhydration during H2S-triggered tomato lateral root formation. Subsequently, silencing expression of RBOH1 and APX2, two lateral root formation-related genes, was constructed by CRISPR-Cas9 approach. By exogenous applying the scavenger of H2S (HT) and its donor, the blocker of S-sulfhydration (DTT), and corresponding enzymatic inhibitor, the differences in lateral root formation and S-sulfhydration between wild type and mutant plants were compared. Combining with prokaryotic expression, immunoassay, and LC/MS/MS, the modified cysteine site(s) by S-sulfhydration were explored. To further clarify the possibility of S-sulfhydration involved in H2S-induced lateral root formation, site mutation in the possible modified cysteine site(s) was constructed and transformed, and the effects of S-sulfhydration on enzyme activity and lateral root formation were investigated. Together, these results not only elucidated the molecular mechanism underlying tomato lateral rooting mediated by H2S via S-sulfhydrated H2O2 metabolizing proteins, but also provided the novel mechanism related to the biological roles of H2S in plants.

对靶蛋白半胱氨酸残基的硫巯基化修饰（R-SSH）是一种新发现的动物硫化氢（H2S）生理效应的分子机制。我们发现，H2S以及过氧化氢（H2O2）分别介导生长素诱导的番茄侧根发生，体外实验表明H2S对NADPH氧化酶和抗坏血酸过氧化物酶（APX）可能具有硫巯基化修饰。为了探索相关机制，本课题采用生物素标记法筛查H2S诱导番茄侧根发生过程中的硫巯基化修饰蛋白；以与侧根发生有关的RBOH1和APX2为重点，分别构建CRISPR突变体番茄，结合H2S及其清除剂、阻断硫巯基化修饰的DTT以及酶抑制剂处理，比较野生型和突变体硫巯基化修饰以及侧根发生的差异；结合原核表达、免疫分析和LC/MS/MS明确硫巯基化修饰的半胱氨酸位点；构建硫巯基化位点的点突变载体并转化番茄，分析硫巯基化修饰对酶活和侧根发生的影响，明确硫巯基化修饰H2O2代谢酶参与H2S诱导侧根发生的机制。上述结果将提供H2S植物学效应的新机制。

生长素诱导的硫化氢是一种新发现的诱导植物侧根发生的重要信号分子，但是一直不清楚其分子机制。为了探查相关作用机制，本课题进一步发现：1）硫化氢诱导内源过氧化氢的积累与番茄侧根发生有关，硫化氢促进的番茄侧根发生能被过氧化氢清除剂DMTU和合成抑制剂DPI所逆转，过氧化氢是硫化氢诱导番茄侧根发生过程中所必需的，其中细胞周期循环基因、miRNAs及靶基因可能是其下游靶基因；2）硫化氢促进番茄幼苗根部谷胱甘肽的生物合成，但谷胱甘肽不改变硫化氢的含量，硫化氢促进的番茄侧根形成能被谷胱甘肽合成抑制剂BSO所阻断，依赖于γ-谷氨酰半胱氨酸合成酶（γ-ECS）的谷胱甘肽生物合成可能部分参与硫化氢的生物学效应，细胞周期循环基因、miRNAs及靶基因和生长素信号相关基因可能也是被其调控的下游靶基因，进一步采用拟南芥γ-ECS缺失突变体atcad2证明谷胱甘肽参与硫化氢诱导的植物侧根发生；3）硫化氢增强了番茄硫巯基化修饰的水平，硫化氢清除剂HT或合成抑制剂PAG单独处理降低了其修饰水平，而外源硫化氢的施加可缓解HT和PAG的抑制作用，番茄3-磷酸甘油醛脱氢酶基因（GAPC2）原核表达产物可以发生外源硫巯基化修饰，推测硫巯基化修饰可能参与番茄侧根发生的信号转导。上述结果尝试提供植物硫化氢诱导根形态建成分子机制的新知识。