Studies Of The S-nitrosylation Of BR Co-receptor BAK1 Enhancing The Salt Tolerance Of Tomato Seedlings

Salt stress is one of the major environmental stresses that remarkably inhibits crop productivity and causes multiple reactions from gene expression to cell metabolism in plants,thus affecting plant growth and development.Nitric oxide(NO)and brassinolides(BR)play a key role in regulating various physiological processes in plants.NO could exert its biological functions through NO-dependent posttranslational modification,protein S-nitrosylation.However,it is not clear whether NO regulates BR signaling through protein S-nitrosylation in response to salt stress.Therefore,in this study,tomato seedlings(Solanum lycopersicum cv.Micro-Tom)were used as experimental materials to study the key role and response mechanism of S-nitrosylation of BR co-receptor protein BAK1(SERK3A,SERK3B)during NO enhancing the salt tolerance of tomato seedlings through physiological and biochemical methods,genetics and molecular biology.This study revealed the role of protein S-nitrosoylation as a post-translational modification in response to salt stress,providing theoretical basis for the mechanism of NO and BR in regulating plant growth and development under adverse conditions.The main results are as follows:1.BR was involved in NO enhancing the salt tolerance of tomato seedlings.We found that 1μM BR significantly promoted the growth and development of tomato seedlings under salt stress.Therefore,0.1μM BR was selected for following study.Our results found that BR was involved in NO-induced the growth and development of tomato seedlings under salt stress.In addition,NO significantly increased the endogenous BR content,up-regulated the expression of BR synthesis-related genes and key genes in BR signaling pathway during salt stress,implying that NO positively regulated BR biosynthesis and signal transduction in tomato seedlings.We also found that BR induced endogenous NO production through NR pathway in tomato under salt stress.2.BR was involved in the NO-induced S-nitrosylation under salt stress.The results showed that the co-treatment of exogenous BR and NO increased the content of endogenous SNO and NO,enhanced the activity of GSNOR,up-regulated the expression of GSNOR and enhanced the abundance of protein S-nitrosylation,whereas this trend was reversed after adding BR scavenger Brz.These results suggested that BR exerts key role in the S-nitrosylation regulation induced by NO under salt stress.3.BR co-receptor protein BAK1(SERK3A,SERK3B)can be S-nitrosylated.Both SERK3 A and SERK3 B were predicted to undergo S-nitrosylation.By biotin switch assay,western blot combined with LC-MS/MS,it was found that BAK1 could undergo S-nitrosylation in vitro and in vivo,and SERK3 B was S-nitrosylated at cysteine residue 137.Importantly,the S-nitrosylation of BAK1 was inhibited under salt stress,which was reversely enhanced by exogenous NO.Moreover,the BAK1S-nitrosylation in GSNOR silenced plants was significantly enhanced.These results indicated that BR co-receptor protein BAK1 can undergo S-nitrosylation modification,and NO can enhance the S-nitrosylation of BAK1 under salt stress.4.S-nitrosylateted BAK1 was involved in NO-enhanced salt tolerance of tomato seedlings.SERK3 A and SERK3 B silenced plants were obtained by VIGs.The results showed that the silencing of SERK3 A and SERK3 B significantly affected the growth of seedlings,the content of endogenous BR,the expression of BR synthesis-related genes and key genes in signaling pathway.And the content of endogenous NO and the abundance of S-nitrosylated proteins were significantly reduced.Further,the total S-nitrosylation level,abundance of BAK1 S-nitrosylation and salt tolerance of silenced plants were further enhanced after adding GSNO under salt stress.In addition,slserk3 a and slserk3 b knockout mutants were obtained using by CRISPR/Cas9.Compared with WT,the growth of slserk3 a and slserk3 b seedlings was inhibited,and the content of BR,the expression of BR synthesis-related genes and key genes in signaling pathway were significantly decreased.Importantly,the endogenous NO content and total S-nitrosylated protein abundance of slserk3 a and slserk3 b mutants were significantly reduced compared with WT.Under salt stress,the endogenous S-nitrosylation level,the S-nitrosoylation abundance of BAK1 and the salt tolerance of seedlings in WT,slserk3 a and slserk3 b plants were further enhanced when GSNO was added.In addition,endogenous S-nitrosylation levels and S-nitrosylated protein abundance were enhanced in SERK3 A and SERK3 B overexpression strains of tobacco.These results su ggested that BAK1 S-nitrosylation plays an important regulatory role in NO-enhanced the salt tolerance of tomato seedlings.5.BAK1 S-nitrosylation affects BAK1 subcellular localization and BAK1-BRI1 interaction.Subcellular localization results showed that SERK3 A and SERK3 B were located on the cell membrane.Under salt stress,after induction of exogenous NO,the fusion proteins of SERK3A-GFP and SERK3B-GFP showed GFP signals in the nucleus and cell membrane.Additionally,SERK3 A and SERK3 B could interact wi th BR another receptor protein,BRI1,by yeast two-hybrid,luciferase complementary and bimolecular fluorescence complementation assay.When exogenous Na Cl is applied,the interaction between SERK3 A and SERK3 B and BRI1 could be weakened,while exogenous NO reversely strengthened their interaction.These results implied that NO-induced S-nitrosylation of BAK1 may regulate the subcellular localization of BAK1 and protein interaction of BAK1-BRI1 under salt stress.