The Mechanism Of Tomato Thioredoxin(SlTrxh) Involved In The Process Of NO Alleviating Nitrate Stress

Secondary soil salinization is a one of the main problems in the soil of the China’s greenhouse,which affects the growth and quality of vegetables and is closely related to the sustainable development of agriculture.NO₃^-is the most abundant negative ion accumulated in this kind of soil,causing secondary salinization.NO is a messenger molecule,which plays important physiological roles in plant growth,development and in response to stress.NO plays roles by post-translational modification of the target protein of S-nitrosylation.The thioredoxin（Trx）system（i.e.Trx and Trx reductase（NTR））is the main denitrosation system in plants and participates in NO-mediated protein nitration and denitrolation oxidation in vivo redox regulation.This study focused on the mechanism of tomato thioredoxin（SlTrxh）involved in the process of NO alleviating nitrate stress.The results obtained are as follows:1.The effects of exogenous SNP（NO donor）nitrate treatment on the phenotype,antioxidant system and SlTrx expression of tomato seedlings were studied.Compared with the control,after one week of nitrate treatment,the root system of tomato seedlings was underdeveloped,the plants were short,and the biomass decreased.The growth state of tomato was better with 100μM SNP solution,which was the closest to the growth of control seedlings.Compared with the control,the ROS,MDA and NO contents of tomato roots increased significantly under 150 mM nitrate stress.The addition of 100μM SNPs can effectively alleviate the oxidative damage of tomatoes by decreasing the ROS,MDA and NO contents.The activity of antioxidant enzymes（SOD,POD,CAT）increased significantly under stress.With the addition of 100μM SNP,the antioxidant enzyme activity of CAT and POD increased significantly.The expression levels of SlTrxh and SlTpx also increased after exogenous SNP application under nitrate stress.The S-nitrosylated protein increased after nitrate stress,and the protein expression of S-nitrosylated SlTrxh and SlNTRB also increased.The expression of S-nitrosylated SlTrxh protein increased after adding SNP under nitrate stress.The above results indicate that the thioredoxin（Trx）system is involved in the process of NO alleviating nitrate stress.2.Tomato SlTrxh,SlTpx and SlNTRB proteins were obtained through prokaryotic expressed,purified and the denitrosylated function were assayed.GPS-SNO 1.0software was used to predict the key S-nitrosylation site and the result showed that position 54 Cys might be the main S-nitrosylation site in tomato Trxh sequence.We then mutated Cys54 to Ser and obtained the SlTrxC54S mutant protein and its insulin reduction activity reduced.NO donor（GSNO）caused the S-nitrosylation of BSA,and it is verified that the complete thioredoxin system can make it denitrosylation,while the SlTrxC54S lost the denitrosylation activity.3.The overexpressing SlTrxh,SlTpx and SlNTRB transgenic tobacco were obtained and the the function of SlTrxh transgenic tobacco plants were analysed.The germination rate,root length were lower,H₂O₂,and O₂^-contents were higher in the WT plants than the SlTrxh transgenic tobacco plants seeds under nitrate stress.The overexpressed SlTrxh tobacco plants showed better growth performance,higher ratio of ASA/DHA and GSH/GSSG,NTR activities and lower MDA and H₂O₂ contents than that of WT.Methyl viologen（MV）treatment further confirmed the increased oxidative tolerance of tobacco overexpressing SlTrxh than the WT plants.4.The SlTrxh protein has interaction with the GSNO reductase（GSNOR）through yeast two-hybrid assay,pull-down experiment and co-immunoprecipitation assayes.From the physiological,biochemical and molecular levels,the mechanism by which SlTrxh participates in tomato response to nitrate stress is revealed,which helps to enrich our understanding of the mechanism of vegetables responding to secondary salinization.