甲烷通过调控谷胱甘肽稳态提高苜蓿镉耐性的分子机理

Although stress-induced production and emission of methane (CH4) in crops is well-known as a universal phenomenon, its metabolic pathway(s) and biological functions remain elusive. Our recent results showed that cadmium in low concentration could induce the production of CH4 in alfalfa seedlings. Meanwhile, the maintenance of glutathione (GSH) homeostasis by exogenous applying of CH4 may attribute to the above mentioned enhanced cadmium tolerance. To investigate intrinsic mechanism, in this project, we firstly investigated the possible involvement of early reactive oxygen species (ROS) burst in the cadmium-induced CH4 biosynthesis in alfalfa seedlings, and its contribution in the enhanced cadmium tolerance. Subsequently, silencing γ-ECS (encoding γ-glutamylcysteine synthetase responsible for GSH biosynthesis) expression by CRISPR-Cas9 was constructed. By exogenous applying the inhibitor of γ-ECS (BSO) and GSH, the differences in antioxidant response of wild-type and mutant were compared, and the relationship between GSH homeostasis and CH4-induced cadmium tolerance were further clarified. To identify proteins undergoing thiolation regulated by cadmium, the combination of in vivo and in vitro labeling methods utilizing biotinylated, oxidized glutathione (GSSG-biotin) was developed to isolate alfalfa proteins/protein complexes that can be reversibly glutathionylated. Combining with prokaryotic expression, point mutation, immunoassay, and enzyme activity and kinetic parameter determination, we would focus on two reported modified proteins, dehydroascorbate reductase and glutathione S-transferase, to explore the key proteins which might be involved in CH4-mediated cadmium tolerance in terms of S-glutathionylation. Together, these results would provide the novel knowledge related to the biological roles of methane in plants.

胁迫诱导作物产生并释放甲烷（CH4）是一个普遍现象，但是不清楚其代谢途径以及生理功能。最近我们发现，低浓度镉诱导苜蓿幼苗产生CH4，外源CH4提高苜蓿的耐镉性可能与其能维持谷胱甘肽（GSH）的稳态有关。为了探查相关作用机制，本课题首先探查镉诱导的早期活性氧迸发是否介导CH4的生物合成；构建催化GSH合成的关键基因（γ-ECS）的CRISPR突变体苜蓿，结合γ-ECS酶的抑制剂BSO和外源GSH处理，比较野生型和突变体抗氧化防护以及耐镉性的差异，明确GSH稳态与CH4介导的耐镉性关系；采用生物素标记法和质谱筛查受镉诱导的谷胱甘肽化修饰蛋白，以初步推测的修饰蛋白脱氢抗坏血酸还原酶和谷胱甘肽S转移酶为研究重点，结合原核表达、半胱氨酸点突变表达产物、免疫分析以及酶活和动力学参数测定，从谷胱甘肽化修饰的角度探查CH4提高耐镉性的关键蛋白和机理。上述结果将尝试提供CH4植物学效应的新知识。

胁迫诱导作物产生并释放甲烷是一个普遍现象，但是不清楚其生理功能。我们已经发现，低浓度镉诱导苜蓿幼苗产生甲烷，外源甲烷提高苜蓿的耐镉性可能与其能维持谷胱甘肽（GSH）的稳态有关。为了探查相关作用机制，本课题进一步发现：1）甲烷通过调节重金属转运蛋白以及重建GSH和氧化还原稳态来降低苜蓿幼苗Cd的积累，γ-谷氨酰半胱氨酸合成酶（γ-ECS）是甲烷调控GSH稳态的重要靶点；2）内源一氧化氮（NO）可以介导甲烷缓解苜蓿的镉毒害，其相关的机理包括重建GSH和氧化还原稳态；3）甲烷缓解镉胁迫引起的紫花苜蓿根生长抑制和细胞死亡的过程也可以通过依赖于L-半胱氨酸脱巯基酶的内源硫化氢介导，其相关机制包括减少镉的积累和吸收，同时也与重建GSH稳态有关；4）构建的过表达内源甲烷的拟南芥转基因株系可以通过ABA信号转导来提高其对盐害和干旱的耐性，进一步研究发现拟南芥转基因株系的耐镉性也显著得到提高；5）甲烷可以通过改变苜蓿和转基因拟南芥幼苗根部细胞壁结构来降低镉的积累，包括改变共价结合型果胶（CSP）和离子可溶性果胶（ISP）含量及去甲基化水平，从而增加了细胞壁镉的积累，阻止了镉进入植物细胞，其相关机理与钙信号以及依赖于NADPH氧化酶的过氧化氢信号的互作有关。上述结果提示维持GSH稳态和改变细胞壁结构是甲烷缓解镉毒害的主要中心环节。总之，本课题的相关发现不仅尝试提供甲烷植物学效应的新知识，而且也为合理利用农田栽培过程中产生的甲烷提出新的思路。