A Preliminary Study Of Sulfhydryl Ion Transporters In Arabidopsis

In recent years,studies have found that hydrogen sulfide(H₂S)is the third gas signal molecule after CO and NO,and it plays important physiological functions in animals and plants.In the physiological state,the main form of H2S is HS^-,that is,HS^-as the conjugate base of H₂S,exists in a large number of organisms.H₂S itself is a highly lipophilic molecule that easily penetrates the phospholipid bilayer of cells,but HS^-requires mediating substances.Corresponding HS^-transporters have been found in the study of HS^-transport mechanisms in bacteria and animals,but there is still no report on HS^-transport in the field of plants.Then,how does HS^-transport across the membrane in plants to perform the physiological function of H₂S signaling?In this study,the model plant Arabidopsis thaliana was used as the experimental material,and the phylogenetic analysis and experimental verification were carried out to analyze and identify possible Arabidopsis HS^-transporters.This study first conducted bioinformatics analysis on the known major Arabidopsis anion channel families SLAC1/SLAH family,CLC family,NRT/PTR family,VDACs family,etc.Based on previous research reports such as high degree of similarity between HS^-and Cl^-,and there are already relevant information about HS^-transporters,we focused on the analysis of the sequence and structural characteristics of the Arabidopsis Cl^-transporters protein SLAC1 and SLAH3.Through the discussion of the analysis results,SLAC1 and SLAH3 in the SLAC1/SLAH family of Arabidopsis chronic anion channels were selected as the key research objects of this thesis,and the functions of SLAC1 and SLAH3 as potential Arabidopsis HS^-transporters were further verified.This article uses Arabidopsis wild-type(WT),chronic anion channel SLAC1 deletion mutant(slac1)and chronic anion channel SLAH3deletion mutant(slah3)as experimental materials,and based on the existing reports about research on HS^-transporter,we focus on the H₂S sensitivity of SLAC1 and SLAH3 proteins in Arabidopsis and their impact on HS^-transport.The main results are as follows:(1)The stomata density and pore diameter of the Arabidopsis ion channel protein SLAC1 and SLAH3 deletion mutants slac1 and slah3were significantly higher than that of WT,and the individual showed a significant decrease in drought resistance.After fumigated with exogenous physiological concentration of H₂S,the stomata pore diameter of WT was decreased,while the pore diameter of mutants slac1 and slah3increased instead.It shows that the transduction process of H₂S signal is mediated by SLAC1 and SLAH3 proteins,futher suggesting that these two proteins may be potential HS^-transporters.(2)After the fumigated with exogenous physiological concentration of H₂S,chemical and fluorescence methods are used to detect and observe showed that the H₂S content in WT vivo increases significantly,while the increase of H₂S content of slac1 and slah3 is not significant.It shows that the deletion of SLAC1 and SLAH3 proteins leads to a decrease of exogenous H₂S entering plant cells,which further confirms the possible HS^-transport function of SLAC1 and SLAH3 proteins.(3)The prokaryotic expression vector of SLAC1 and SLAH3 was successfully constructed to provide materials for the next induced expression and purification of protein,and laying foundation for the subsequent in vitro study of the corresponding protein function.At the same time,successfully constructed two gene over expression vectors in order to obtain the corresponding genetic materials OE-SLAC1 And OE-SLAH3,and identify the function of the corresponding protein in vivo.(4)Under normal conditions,the background content of H₂S in slac1and slah3 mutant plants is higher than that of WT.Analysis of the expression levels of the endogenous H₂S-producing enzyme encoding genes LCD and DES1,we found that the expression of LCD in slac1 was significantly down-regulated,and the expression of DES1 was significantly up-regulated;while the expression of LCD in slah3 was significantly up-regulated,and the expression of DES1 was not significant changes.In this view,the proteins SLAC1 and SLAH3 may be upstream of LCD and DES1,and they can feedback regulate the physiological functions of H₂S signals by affecting the production of H₂S in plants.In summary,SLAC1 and SLAH3 may be candidate proteins for HS^-transport.This have important theoretical significance for the in-depth study of the H₂S gas signal transduction mechanism of plants and the artificial synthesis of HS^-receptors.It can also guide the molecular design of resistant crops,the synthesis of H₂S sustained-release donors and drug carriers,with broadly application foreground.