Study On The Mechanism Of Hydrogen Sulfide Regulating Submergence Tolerance And Growth And Development Of Arabidopsis

Flooding is one of the frequent natural disasters in the world,which seriously endangers agricultural production and food security of people.Hydrogen sulfide（H₂S）,as the third kind of gas signal molecule,plays important roles in plant growth and development and responses to various abiotic stresses.However,the mechanism by which endogenous H₂S regulates plant growth and response to submergence remains unclear.In addition,autophagy is also an important protective mechanism in plants,but the functional relationship between H₂S and autophagy during the submergence response is also unclear.In this study,we explored the molecular mechanisms by which endogenous H₂S regulates plant growth and development and response to submergence,and the functional relationship between H₂S and autophagy under submergence via phenotypic observation,physiological and biochemical detection and transcriptome sequencing（RNA-Seq）,using the Arabidopsis wild-type Col-0,the mutant or transgenic line with reduced content of H₂S,des1（L-cysteine desulfhydrase 1）,35S::GFP-OASA1（O-Acetylserine thiol lyase A1）/des1,the autophagy-related（ATG）mutants or transgenic lines atg5,atg7,atg4a/4b,GFP-ATG8e,and the mutants or transgenic lines related to H₂S and autophagy des1/atg5,des1/GFP-ATG8e as materials.The main results are as follows.1.It was found that exogenous H₂S pretreatment significantly improved the survival rate of leaves and plants and the cell membrane integrity,and decreased the cell death of leaves and roots in wild type（WT）after submergence compared to the plants without H₂S pretreatment.Meanwhile,exogenous H₂S pretreatment significantly increased the endogenous H₂S content,indicating that exogenous H₂S pretreatment enhanced the submergence tolerance in Arabidopsis by increasing the content of endogenous H₂S and alleviating the cell death.2.Analyses of RNA-Seq and western blotting showed that the expression of H₂S synthesis-related gene DES1 was down-regulated,and the H₂S metabolism-related gene OASA1 and OASA1 protein were up-regulated under submergence.Determination of H₂S content confirmed that submergence caused a decrease in endogenous H₂S content and the mutation of DES1 and overexpression of OASA1further decreased the endogenous H₂S level,showing that submergence reduced the content of endogenous H₂S via the regulation of DES1 and OASA1 expression.3.Moreover,both des1 and 35S::GFP-OASA1/des1 showed sensitive to submergence compared with the WT.And the cell death of 35S::GFP-OASA1/des1was the most severe.After 4 or 7 days of reoxygenation,the survival number of leaves and survival rate of plants in WT were 5.14 and 84.71%,respectively,which were significantly higher than those of des1 mutant（2.95 and 44%）and35S::GFP-OASA1/des1-#56/#61（0.67/0.67 and 5%/15%）,suggesting that endogenous H₂S plays an essential role in the plant response to submergence.4.RNA-Seq analysis showed that the differentially expressed genes（DEGs）down-regulated in both Col-0 and 35S::GFP-OASA1/des1-#56 were mainly involved in“oxidation-reduction process”,“metabolism”and“hormone response”.And the DEGs with the opposite expression pattern in 35S::GFP-OASA1/des1-#56 and Col-0were mainly involved in“hypoxia response”,“signal transduction”,“transcription regulation”and“cytokinin-activated signaling pathway”.q RT-PCR analysis showed that the expression levels of LBD41（LOB DOMAIN-CONTAINING PROTEIN 41）,WIP3（WOUND-INDUCED POLYPEPTIDE 3）,TEM1（TEMPRANILLO 1）,CAT2（CATALASE 2）and ERF9（ERF DOMAIN PROTEIN 9）in35S::GFP-OASA1/des1-#56 were significantly lower than those in WT at 24 h of submergence.Therefore,endogenous H₂S modulate the submergence response of Arabidopsis mainly by regulating the expression of genes related to“hypoxia response”,“phytohormone signal transduction”,“redox process”and“transcription regulation”,etc.5.Detection of DNA ladder and TUNEL staining confirmed that the cell death caused by submergence was a process of programmed cell death（PCD）.q RT-PCR analysis and western blotting showed that submergence induced the expression of ATG8a/b/h and the processing of GFP-ATG8e.And the atg mutants,including atg5,atg7 and atg4a/4b,were all sensitive to submergence.Moreover,although the survival rates of atg5 and atg7 in the H₂S pretreatment group were partially improved compared to the un-pretreated plants,they were still significantly lower than that of WT.In addition,the number of surviving leaves and plant survival rates of des1/atg5after submergence were significantly lower than those of des1 and atg5 single mutants,indicating that endogenous H₂S and autophagy co-regulate the PCD induced by submergence,and H₂S pretreatment partly depended on autophagy to enhance the submergence tolerance in Arabidopsis.6.RNA-Seq and q RT-PCR analyses showed that the ATG genes in H₂S-pretreated WT were down-regulated under normal conditions,but almost all of them were up-regulated after 24 h of submergence compared with the unpretreated group.The statistics of number of autophagosomes and western blotting in the GFP-ATG8e seedlings showed that the number of autophagosomes and the ratio of free GFP/GFP-ATG8e in the H₂S pretreatment group was less than the unpretreated group under normal conditions,while they were more after submergence.These results indicated that H₂S negatively regulated autophagy under normal conditions,but positively regulates the autophagy process under submergence by regulating the expression of ATG genes.7.Statistics of the number of autophagosomes in the root cells of GFP-ATG8e and des1/GFP-ATG8e seedlings showed that des1/GFP-ATG8e exhibited more autophagosomes than that of GFP-ATG8e under normal conditions,but less than that of GFP-ATG8e under submergence for 3-6 h.q RT-PCR analysis showed that ATG8c and ATG18a genes were down-regulated in 35S::GFP-OASA1/des1-#56 after submergence compared with WT,which were contrary to the expression under normal conditions.This genetically indicated that H₂S pretreatment promoted the process of autophagy via positive regulation of the expression of ATG genes to enhance the submergence tolerance of Arabidopsis.8.The phenotypic observations of Col-0,des1 and 35S::GFP-OASA1/des1 under normal conditions showed that des1 mutant is similar to Col-0 in phenotype.However,the main root length was shorter,the apical angle was larger,and the cotyledons were curved downward in 35S::GFP-OASA1/des1 compared with the Col-0 seedlings.35S::GFP-OASA1/des1 plants were dwarfed,with shriveled leaves and delayed bolting during the growth stage.In the reproductive phase,larger flowers,defection in the pattern of 4 long and 2 short stamens,shortened siliques,seed abortion and delayed senescence were found in 35S::GFP-OASA1/des1 plants.RNA-Seq analysis in 35S::GFP-OASA1/des1-#56 and Col-0 under normal conditions showed that the DEGs up-regulated or down-regulated in 35S::GFP-OASA1/des1-#56 were mainly involved in“transcriptional regulation”,“ribosome production”,“auxin transport”,“phase transition regulation in plants”and“pollen recognition”,compared with Col-0.Therefore,it can be seen that H₂S affects the seedling growth,leaf development,reproduction and senescence of Arabidopsis by regulating the expression of genes related to the above pathways.In summary,H₂S pretreatment increased the content of endogenous H₂S.And endogenous H₂S regulated the expression of genes related to“hypoxia response”,“plant hormone signal transduction”,“transcription regulation”,meanwhile,it positive regulated the process of autophagy under submergence to enhance the response capability of plants to submergence stress.In addition,H₂S also affects plant normal growth and development by regulating the expression of genes associated with“transcriptional regulation”,“ribosome production”,“auxin transport”,“plant phase transition regulation”and“pollen recognition”under normal conditions.