Study On The Endogenous H₂S-producing Enzyme Activity And Function Of AtMST In Arabidopsis

In recent years,the research of hydrogen sulfide(H₂S)is more and more in-depth.In mammalian cells,it participates in many important physiological processes.In plant cells,H₂S is widely invivoed in many physiological functions,such as plant growth and development and response to biotic and abiotic stress.In particular,the important role of H₂S in response to drought stress has attracted much attention.In animal cells,there have been three enzymes involved in the production of H₂S,including cystathionine?-lyase,cystathionine?-synthase and mercaptopyruvate Sulfurtransferase.Many enzymes that produce endogenous hydrogen sulfide also have been identified in plant cells,such as Sulfite reductase(Si R),Cysteine Desulphydrases(CDes),Cysteine Desulfurases,?-Cyano-Alanine Synthase,CAS and O-acetylserinelyase(OASTL).However,it has not been reported whether MST in plants has endogenous H₂S production activity.PPrevious functional studies of the gene mainly focused on dehydrogenation,seed germination and embryo development.Early studies have suggested that Arabidopsis AtMST protein has the activity of catalyzing the production of H₂S by way of vitro protein expression and enzyme activity determinationn in our lab.But there still is lack of vivo evidence.The obtain of more genetic material are necessary for further research.In this study,two gene copies of the Arabidopsis AtMST gene,AtMST.1(AT1G79230.1)and AtMST.2(AT1G79230.2),were cloned firstly.And the endogenous H₂S production activity was analyzed,in transgenic plant and wild type(WT).In addition,We also analyzed effects of endogenous H₂S in transgenic plants on drought resistance compared with that in WT.1.The expression of Arabidopsis AtMST gene has obvious developmental stage specificity and tissue specificity.The expression level of AtMST increases in seedling age,reaches a peak at 30 days and then begins to decline with the extension of time.The expression of AtMST in rosette leaves and pods is higher than in Root,stem and cauline leaf.2.The AtMST.1 and AtMST.2 were amplified successfully,their open reading frames were 1140bp and 849bp respectively.The plant overexpression vector XF246-OE-AtMST,gene editing vector CRISPR-AtMST and interference vector RNAi-AtMST were constructed.The transgenic materials obtained by agrobacterium-mediated genetic transformation.3.H₂S content and prodution rate were determined in the various genetic engineering genetically modified materials.Compared with that in WT,the H₂S content and production in OE-AtMST.1 and OE-AtMST.2 plants were increased,while the H₂S content and production in RNAi-AtMST plants were decreased.The fluorescence probe method was used to observe the H₂S content in the stomata of different genetic materials and in the root tips of seedlings.The fluorescence intensity of stomata and root tips in OE-AtMST.1 and OE-AtMST.2 plants is higher than that in WT plants,while the fluorescence intensity of RNAi-AtMST mutants is weaker,and the difference is significant.4.The expression of drought-related genes DREB2A,DREB2B and RD29A were detected in various genetic materials before and after drought stress.The results showed that before stress,the expression levels of DREB2A and DREB2B in OE-AtMST.1 plants were significantly higher than those in WT before stress;the expression levels of DREB2B and RD29A in OE-AtMST.2 plants were significantly up-regulated;mutant RNAi-AtMST DREB2A and DREB2B.The expression of RD29A and RD29A was significantly reduced.The expression of RD29A,DREB2A and DREB2B was significantly reduced in RNAi-AtMST plants.After drought stress,the expression of DREB2A,DREB2B and RD29A in OE-AtMST.1,OE-AtMST.2 and RNAi-AtMST plants also showed similar expression changes.However,the difference was greater than before control group.5.The determination of the physiological indicators in WT,OE-AtMST.1,OE-AtMST.2 and RNAi-AtMST plants before and after drought stress showed that:the POD,SOD,CAT,soluble sugar in OE-AtMST.1,OE-AtMST.2 and RNAi-AtMST plants were not significantly different from those in WT under control group;After drought stress,the three antioxidant enzyme activities and soluble sugars in OE-AtMST.1 and OE-AtMST.2 plants all showed up-regulated trend,and RNAi-AtMST plants showed down-regulated trend.The above data indicated that AtMST in Arabidopsis can catalyze the production of endogenous H₂S in plant cells.The morphological,molecular and physiological levels showed that AtMST can significantly improve the ability of Arabidopsis to respond to drought stress.This research is not only the discovery of new functions of plant MST genes,but also a new expansion of the members of plant endogenous H₂S-producing enzymes.