H₂S Signal And ETF/ETFQO System Co-regulate The Mechanism Of Drought Resistance In Plants

Among many abiotic stresses,drought stress has a huge impact on crop yield,and leaf senescence caused by drought stress will influence the growth and development of plants greatly.During hydrogen sulfide(H₂S)was confirmed as the third gasotransmitter,its role in plants was gradually revealed including the development of plants and the resistance of various biotic and abiotic stresses.Leaf senescence caused by drought stress in plants must be accompanied by changes in energy.Previous studies have shown that H₂S could delay leaf senescence caused by drought stress by regulating energy metabolism.Mitochondria are the important source of energy,in which H₂S could delay leaf senescence by acting as a regulator of energy production.However,the underlying mechanism of H₂S signaling in the process of plant senescence is still unclear,and the mode of H₂S action in the electron transport chain is still unknown.In this study,the Arabidopsis thaliana WT,endogenous H₂S producing enzyme deletion mutant(lcd/des1),T-DNA insertion mutant(etf)of the ETF gene and OE-ETF overexpression plants were used as experimental materials.From different aspects such as the regulation of transcription level expression,the change of alternative splicing mode of electron-transfer flavoprotein:ubiquinone oxidoreductase(ETFQO)and the effect of protein post-translational level S-sulfhydryl modification,we will deeply explore the mechanism of H₂S signal on the ETF/ETFQO system,an important link in the branch of the electron transport chain.The main results are as follows:(1)Firstly,the gene expression patterns of gene ETF and ETFQO were carried out.Both ETF and ETFQO had the highest expression levels in 4-week old rosette leaves,and then decreased after bolting;the expression levels of the two genes in various tissues were different,and there was the highest expression level in flowers.However,the mutant etf has obvious phenotypic differences in the growth stage compared with WT,so 4-week old rosette leaves were used in the following experiments.(2)In normal growth conditions,compared with WT seedlings,etf mutant grew slowly with fewer leaves and smaller leaves.However,there was no significant different phenotype between OE-ETF and WT.Under drought stress conditions,the expression of ETF and ETFQO genes increased significantly,and the expression of drought-related genes in etf mutants declined.However,their stomatal aperture and chlorophyll content were not significantly different from those of WT.It implied that the ETF/ETFQO system played an important role in the process of plant resistance to drought,but without regulating stomatal movement.(3)At the physiological level,under drought conditions,there was more leaf water content in OE-ETF than in WT and etf with significant differences;exogenous application of H₂S partially alleviated the lack of water content in WT and etf,indicating that ETF plays an important role in the process of drought resistance and water retention induced by H₂S signals.Correspondingly,the production rate and content of H₂S in etf mutants decreased,which was consistent with the results of the above water content.(4)At the transcription level,exogenous H₂S can significantly increased the expression of genes ETF and ETFQO under drought stress;in etf mutants,the expression of genes ETF and ETFQO gradually increased with the drought time,and their expression levels also increased significantly after exogenous H₂S was applied.After the endogenous H₂S was deleted,the expression of gene ETF was significantly down-regulated,but the expression of gene ETFQO was significantly up-regulated.These results indicated that endogenous and exogenous H₂S signals had a regulatory effect at the transcription level of genes ETF and ETFQO.In etf mutants,the expression of H₂S producing enzyme genes decreased,so ETF also affected the production of H₂S in plants,which was consistent with the above-mentioned results of the rate of H₂S production in etf mutants.(5)Transcriptome sequencing of different materials under different conditions showed that the H₂S signal under PEG stress induced changes in the alternative splicing mode of the ETFQO gene,resulting in intron retention.(6)Prokaryotic expression and purification of ETF protein,and in vitro S-sulfhydration detection using the biotin switch method.The results showed that,compared with the control,the ETF protein after H₂S treatment did not have a significant modification signal.In summary,ETF/ETFQO as an important branched-chain respiratory pathway,plays an important role in the resistance to drought in Arabidopsis.On the one hand,in addition to regulating the transcription and expression of genes ETF and ETFQO,H₂S signals was mainly regulated by alternative splicing of ETFQO genes.Different transcripts may perform different functions in the process of drought resistance in plants.On the other hand,ETF had an impact on the expression of endogenous H₂S producing enzyme genes and the content and production rate of H₂S.