H₂S Regulates The Activity Of BIN2 By Persulfidation In Arabidopsis Thaliana

Post-translational modifications（PTMs）play an important role in regulating the activity and function of plant proteins,and are critical for regulating plant growth and development as well as stress response.In Arabidopsis（Arabidopsis thaliana）,BIN2（Brassinosteroid insensitive 2）is involved in the Brassinosteroids（BRs）signaling pathway as a negative regulator,which regulates plant growth and development and stress tolerance.Hydrogen sulfide（H₂S）is an endogenous gas signaling molecule in plants that regulates protein structure and function through S-sulfhydration modification of proteins,which in turn affects plant growth and development,hormone physiology and stress response processes.The present investigations have demonstrated that the activity and function of BIN2 are regulated by various PTMs such as S-nitrosylation and reactive oxygen species（ROS）,while the mechanism of H₂S-mediated S-sulfhydration modification on the kinase activity and function of BIN2 remains unclear.We demonstrated the presence of S-sulfhydration modification of BIN2 by liquid chromatograph-tandem mass spectrometer（LC-MS/MS）,and the modification site of BIN2was clarified by secondary mass spectrometry;S-sulfhydration modification of BIN2verified by in vitro sulfhydration assay;the phosphorylation experiments in vitro shown that the autophosphorylation of BIN2 and the level of substrate phosphorylation are influenced by H₂S.Bimolecular fluorescence complementation（BIFC）was also performed to demonstrate that H₂S affects the interaction between BIN2 and BES1.H₂S signaling was proved by constructing transgenic lines to maintain chlorophyll content of leaves through S-sulfhydration modification of BIN2 protein as well as to improve salt resistance of the plants.Finally,the mechanism of H₂S-mediated S-sulfhydration modification regulating BIN2 activity in Arabidopsis is comprehensively described.The main results are as follows:1.pColdI-SUMO-BIN2 vector was successfully constructed for in vitro expression of recombinant protein BIN2.The presence of S-sulfhydration modification of BIN2 was demonstrated by LC-MS/MS,and the modification site of BIN2 was clarified by secondary mass spectrometry.2.In vitro sulfhydration assay of BIN2 protein was performed by modified biotin switch method（MBSM）to verify the persulfidation of BIN2.The results indicated that Na HS,an exogenous donor of H₂S,significantly increased the S-sulfhydration modification level of BIN2,and the level increased with increasing Na HS concentration;the modification site of BIN2 was mutated from cysteine（Cys/C）to alanine（Ala/A）to mimic the de-S-sulfhydration modification,which was noted as BIN2^CSSH-A.The in vitro sulfhydration assay showed that Na HS no longer affected the level of S-sulfhydration modification of BIN2^CSSH-A compared to the control.This suggests that BIN2 is modified by S-sulfhydration and BIN2^CSSH-A is the key modification site.3.The effect of S-sulfhydration modification on the kinase activity of BIN2 protein was determined by in vitro kinase reaction assay.The results revealed that H₂S markedly inhibited the autophosphorylation activity of BIN2;the in vitro kinase reaction assay on BIN2^CSSH-A indicated that the autophosphorylation activity of BIN2^CSSH-A was no more influenced by H₂S.It suggests that the S-sulfhydration modification inhibited the autophosphorylation activity of BIN2.4.pColdI-SUMO-BES1 vector was constructed and the exogenous recombinant protein BES1（99aa-267aa）was expressed,and the effect of H₂S on the phosphorylation of BES1,the natural substrate of BIN2,was determined.The results showed that H₂S decreased the autophosphorylation of BIN2 and the phosphorylation level of BES1;while in the reaction system of BIN2^CSSH-A and BES1,H₂S did have no effect on the autophosphorylation of BIN2and the phosphorylation level of BES1.It indicates that the S-sulfhydration modification reduces the phosphorylation level of BES1.BIN2 interacts with BES1,and the impact of H₂S on the interactions between BIN2 and BES1 was investigated with BIFC experiments,the results display that H₂S obviously weakened the interactions between them,but the interactions between BIN2^CSSH-A and BES1 were not affected by H₂S.It illustrates that the S-sulfhydration modification weakened the interaction between BIN2 and BES1.5.In this study,BIN2 gene complementary lines(bin2-3bil1bil2/Native-BIN2^WT)and S-sulfhydration site-directed mutant lines(bin2-3bil1bil2/Native-BIN2^CSSH-A)were constructed in the background of Arabidopsis bin2-3bil1bil2 mutant plants,and screened to obtain homozygous genetic plants.The leaves of the plants were subjected to nutrient starvation in vitro and the physiological status was characterized by chlorophyll content.The results displayed that nutrient starvation resulted in yellowing of leaves with low chlorophyll content,while H₂S treatment was able to maintain the green color of leaves,and Col（wild type）,bin2-1（gain of function mutant）,and bin2-3bil1bil2/Native-BIN2^WT possessed higher chlorophyll content compared to the control;however,in bin2-3bil1bil2/Native-BIN2^CSSH-A,there was no remarkable difference in chlorophyll content between the control and H₂S-treated groups.It implies that H₂S maintains chlorophyll content through S-sulfhydration modification of BIN2 protein.Salt treatment of the plants revealed that excessive salt caused growth inhibition and a considerable reduction in the primary root length（PRL）of the plants,which was rescued by H₂S.H₂S was effective in restoring the growth status of the salt-stressed plants,H₂S caused Col,WS（wild type）,bin2-1,bin2-3bil1bil2/Native-BIN2^WT to have significantly longer PRL than those of salt-stressed plants.In contrast,the inhibited PRL of bin2-3bil1bil2/Native-BIN2^CSSH-A had no notable recovery effect in the presence of H₂S.It indicates that H₂S improves the salt tolerance of plants through S-sulfhydration modification of BIN2.Observation of lateral root growth of the plants further revealed that the lateral root density（LRD）of bin2-1 was greater than that of the wild type,while H₂S increased the LRD of plants.Nevertheless,plant lateral root growth and development are regulated by extremely complex mechanisms,and hence their molecular mechanisms require more researches.