基于CRISPR/Cas9敲除阐明AtMST1通过H2S合成调控拟南芥耐盐性OA

Hydrogen sulfide(H2S)is an important gaseous signaling molecule in plants,and its biosynthesis relies on various endogenous enzymes.The enzyme 3-mercaptopyruvate sulfurtransferase(MST)has been established as an H2S generator in animal systems,and a similar enzymatic activity has been reported for Arabidopsis MST1.To further validate the H2S-producing function of AtMST1 in plants and explore its role in salt stress response,CRISPR/Cas9-mediated gene editing to target the AtMST1 gene was employed in Arabidopsis thaliana.By designing four target sites and constructing the gene editing vector,homozygous atmst1 mutants following Agrobacterium-mediated transformation were successfully obtained.Genotyping analysis revealed that the mutant carried a single T-nucleotide insertion at the target site,resulting in a frameshift mutation and premature termination of translation.Compared with the wild type,the atmst1 mutant showed a significant reduction in the intensity of H2S-specific fluorescent signals,as well as in both H2S content and production rate.Salt stress treatment resulted in a clear salt-sensitive phenotype and greater reactive oxygen species accumulation in the roots of atmst1 seedlings.In summary,this study successfully created an AtMST1 loss-of-function mutant,providing genetic evidence for its crucial role in endogenous H2S synthesis in plants and revealing its physiological function in positively regulating salt tolerance in Arabidopsis by modulating H2S levels.