AtPRMT5-mediated AtLCD Methylation Increases H₂S Production To Improve Cd²⁺ Tolerance In Arabidopsis

Hydrogen sulfide（H₂S）is a recently characterized gasotransmitter that also regulates various important physiological processes.L-cysteine desulfhydrase（LCD）is a key enzyme of endogenous H₂S production.However,our understanding of the upstream regulatory mechanisms of endogenous H₂S production is limited in plant cells.It is still unknown whether the At LCD activity can be regulated at the post-translational level.Protein arginine methyltransferase（PRMT）,a highly conserved methyltransferase protein,regulates multiple aspects of the growth,development,and environment stress responses by methylating in arginine（Arg）in histones and some m RNA splicing-relating proteins in plants.In this study,we found that At PRMT5 methylation of At LCD increases its enzymatic activity,thereby strengthening the endogenous H₂S signal and ultimately improving plant tolerance to cadmium(Cd²⁺)stress in Arabidopsis thaliana.The main results are as follows:1.T-DNA insertion mutants atprmt5-1,atprmt5-2,atlcd plants were identified.At PRMT5-OE（overexpression）and At LCD-OE plants were constructed and obtained.Cd²⁺tolerance of these plant materials were analysed.Compared with wild type,atprmt5-1,atprmt5-2,atlcd plants showed Cd²⁺sensitivity,while At PRMT5-OE and At LCD-OE plants exhibited a higher tolerance to Cd²⁺.These results showed that At PRMT5 and At LCD played vital roles in response to Cd²⁺stress in Arabidopsis.2.H₂S content of At PRMT5-OE,atprmt5-1 and atprmt5-2 plants were determined by fluorescence probe and methylene blue methods.The results showed that the H₂S content of At PRMT5-OE plants exhibited obvious increases,whereas the H₂S content of atprmt5-1,atprmt5-2 mutants significantly decreases compared with the WT.Further analysis indicated that transcription and translation levels of At LCD in atprmt5-1,atprmt5-2 plants did not change.The activity of methyltransferase and L-Cys desulfhydrase showed that At PRMT5 methylated At LCD and increased its activity in vitro.3.It was identified by mass spectrometry that At PRMT5 mediated the methylation of Arg-83 of At LCD,a key enzyme of H₂S generation in vivo.The L-Cys desulfhydrase activity of At LCD^Arg83Alaprotein methylated by At PRMT5 was significantly lower than that of At LCD protein after methylation.Subsequently,At LCD^Arg83Alatransgenic plants were constructed and obtained for Cd²⁺tolerance analysis.Compared with that of At LCD-OE plants,the Cd²⁺tolerance of At LCD^Arg83Ala-OE plants was significantly reduced.These results indicated that Arg-83 of At LCD was the methylation modification site of At PRMT5,which significantly improved the At LCD activity.4.The bimolecular fluorescence complementary,protein immunoprecipitation,and pull down experiments showed that At PRMT5 interacted with At LCD in vitro and in vivo.5.The atprmt5-2 atlcd homozygous double-mutant was generated by crossing atprmt5-2 with atlcd.The Cd²⁺sensitivity of the atprmt5-2 atlcd double mutant was much like that of the atlcd single mutant in response to Cd²⁺stress.The content of Cd²⁺,malondialdehyde and the expression level of genes related to Cd²⁺detoxification in WT,atlcd,atprmt5-2,atprmt5-2 atlcd plants were detected.The content or expression pattern of these indicators in atprmt5-2 atlcd double mutant were more similar to that of the atlcd single mutant.These results suggested that At LCD acted downstream of At PRMT5.6.At PRMT5-OE/atlcd transgenic plants were constructed and obtained for Cd²⁺tolerance analysis.The results showed that the root length of At PRMT5-OE/atlcd plants were significantly inhibited compared with that of At PRMT5-OE plants.In addition,the growth of At PRMT5-OE plants was significantly inhibited when grown in Cd²⁺stress medium supplemented with either aminooxyacetic acid（AOA）or hypotaurine（HT）.H₂S can alleviate the inhibitory effects of Cd²⁺on seedlings roots tips in WT,atprmt5-1,atprmt5-2 plants.These results showed that H₂S played a critical role in the process of At PRMT5-mediated Cd²⁺tolerance.7.In addition to above studies,we also cloned and identified a new H₂S generating enzyme encoding gene At MST1 in plants,and analyzed its function.Two alternative splicing transcripts of At MST1 gene,At MST1.1 and At MST1.3,were amplified by PCR.Further analysis indicated that At MST1.1 and At MST1.3 were involved in response to different abiotic stresses.At MST1.1-OE,At MST1.3-OE,RNAi-At MST1 plants were constructed and obtained for drought tolerant analysis.The results showed that At MST1.1-OE,At MST1.3-OE plants exhibited a higher tolerance to drought stress,whereas RNAi-At MST1 plants had a drought-sensitive phenotype compared with WT.The enzyme activity of the recombinant proteins At MST1.1 and At MST1.3 were determined.It was found that the proteins had the ability to catalyze the H₂S production.In addition,the H₂S content and production rate of At MST1.1-OE,At MST1.3-OE plants exhibited obvious increased,while RNAi-At MST1 plants showed significantly decreased H₂S content and production rate compared with that of WT.At MST1.1 and At MST1.3 improve drought resistance by affecting stomatal movement.The above results confirmed that At MST1 was another unreported endogenous H₂S producing enzyme in plants.In conclusion,we found that At PRMT5 mediated protein methylation of At LCD at Arg-83 to improve At LCD enzyme activity,promote endogenous H₂S production,thereby enhancing Cd²⁺tolerance in Arabidopsis.The function of a new H₂S producing enzyme At MST1 was confirmed in plants.