Hydrogen Sulfide Mediated Persulfidation Regulates SnRK2.6 Activity And Promotes ABA-induced Stomatal Closure

Abscisic acid?ABA?is an important hormone that regulates plant growth and development,enhances plant tolerance to stress.In the ABA signaling pathway,sucurose nonfermenting 1?SNF1?-related protein kinase 2s?SnRK2s?family proteins are the key components of ABA signaling,of which Open Stomata 1?OST1?/SnRK2.6 regulates ABA-dependent stomatal closure.Hydrogen sulfide?H₂S?is a gaseous small molecule signaling substance that is involved in regulating a variety of plant physiological processes,including promoting stomatal closure,affecting root development,enhancing photosynthesis and alleviating drought stress.H₂S can mediate post-translational modification of proteins that is named persulfidation.Persulfidation is the main way that H₂S regulates protein activity.Recently,some reports suggested that H₂S is involved in ABA-induced stomatal closure.Our study demonstrated that persulfidation exists in SnRK2.6 protein.Therefore,our subject used the post-translational modification of SnRK2.6 protein as the entry point to reveal a novel regulatory mechanism of ABA signaling in which H₂S persulfidates SnRK2.6to promote ABA-induced stomatal closure.The obtained results as follows:?1?The recombinant SnRK2.6 protein was obtained by prokaryotic expression in vitro.Our study carried out liquid chromatography?LC?-tandem mass spectrometry?LC-MS?analysis of the recombinant SnRK2.6 protein.The mass of the cysteine?Cys131?and Cys137 increased,suggesting that the two residues had gained a persulfidation modification.A 3D model of SnRK2.6 protein was constructed by using the protein structure information of SnRK2.6 and LC-MS analysis data.These data indicated that Cys131 and Cys137 exist in the SnRK2.6 surface and close to Ser175 which is a key phosphorylation site of SnRK2.6.?2?Modified biotin switch method?MBSM?proved that sodium sulfide?Na HS,a H₂S donor?treatment increased the persulfidation of SnRK2.6 in a dose-dependent manner.These suggested that H₂S can regulate SnRK2.6 persulfidation modification level in vitro and vivo.?3?Kinase activity was detected by phosphorylated antibodies.Our study found that Na HS treatment can induce SnRK2.6 enzyme activity in a dose-dependent manner,suggesting that H₂S can promote the activity of SnRK2.6 protein kinase.?4?Our study separately mutated Cys131 and Cys137 to a Ser?S?in SnRK2.6 and assessed.The activity of mutant proteins(SnRK2.6^C137S,SnRK2.6^C131S,and SnRK^{2.6C131SC137S})were analyzed by phosphorylated antibodies.Data indicated that Cys131 and Cys137mutation inhibited the activity of SnRK2.6.The double mutant protein SnRK2.6^C131SC137Shad the most significant decrease in kinase activity,followed by SnRK2.6^C137S.SnRK2.6^C131Sactivity was slightly down-regulated.These results suggested that Cys137 is the critical regulatory site for persulfidation of SnRK2.6.?5?The effect of persulfidation modification on the interaction between SnRK2.6 and the downstream transcription factor ABA response element-binding factor2?ABF2?was tested by Bimolecular Fluorescence Complementation?Bi FC?experiments.Our study found that Na HS treatment significantly enhanced the Bi FC fluorescence.However,the Bi FC fluorescence decreased in the SnRK2.6 mutant proteins.The effect of persulfidation on the interaction between SnRK2.6 and ABF2 was also verified by Pull-down experiments.Data indicated that the persulfidation enhances the interaction between SnRK2.6 and ABF2,and the protein mutation weakens the interaction between SnRK2.6 and substrate.?6?Our subject isolated a series of persulfidation site mutation genetic lines recombined with native promoters and GFP protein in ost1-3,including ost1-3/SnRK2.6^WT-GFP,ost1-3/SnRK2.6^C131S-GFP,ost1-3/SnRK2.6^C137-GFPand ost1-3/SnRK2.6^{C131SC137S-GFP}.The stomatal conductance,leaf water loss rate,drought tolerance,and H₂S content of guard cell were tested at physiological levels.ABA increased the H₂S content in guard cells,and then H₂S promoted ABA-induced stomatal closure.In addition,H₂S also improved the drought resistance of plants.The ost1-3/SnRK2.6^C131S-GFP,ost1-3/SnRK2.6^C137-GFPand ost1-3/SnRK2.6^{C131SC137S-GFP}point mutation genetic lines had the reduced sensitivity to ABA-induced stomatal closure,resulted in increasing leaf water loss rate and decreasing drought resistance.These results suggested that ABA-induced H₂S promotes the persulfidation of SnRK2.6 and ABA-induced stomatal closure.?7?A series of persulfidation site mutation genetic lines were used as materials,as described in?6?.The calcium ion fluorescence probe?Fluo-3AM?and non-invasive micro-test technique?NMT?were used to detect the cytosolic calcium[Ca²⁺]_cytcontent and Ca²⁺flux in guard cells.Our study found that H₂S and ABA increased the Ca²⁺content in guard cells and promoted the influx of Ca²⁺in guard cells.The ost1-3/SnRK2.6^C131S-GFP,ost1-3/SnRK2.6^C137-GFPand ost1-3/SnRK2.6^{C131SC137S-GFP}point mutation genetic lines were less sensitive to ABA and H₂S-induced Ca²⁺influx.That showed that the persulfidation modification of SnRK2.6 promoted Ca²⁺influx in guard cells,and the SnRK2.6 mutant proteins weakened Ca²⁺influx in guard cells.These results demonstrated that H₂S regulates ABA-induced Ca²⁺signaling by SnRK2.6 via persulfidation of SnRK2.6 in guard cells.