Mechanism Analysis Of SlTpx And SlNTRB Genes In Tomato Under Nitrate Stress

Soil secondary salinization is one of the important obstacles restricting the sustainable development of facility vegetables.The study found that the salt composition of the facility soil is different from that of the coastal inland saline soil.The anion is mainly NO₃^-,and the cation is mainly Ca²⁺and K⁺.Nitric oxide（NO）is a signaling molecule that plays an important role in stress,and S-nitrosylation is the main way for NO to exert its biological activity.The thioredoxin（Trx）system regulates the redox state of plants.In this paper,the mechanisms of thioredoxin peroxidase（Sl Tpx）and NADPH dependent thioredoxin reductase B（Sl NTRB）,important components of tomato Trx system,involved in no alleviating nitrate stress were studied.The main results are as follows:1.The effects of exogenous application of Trx reductase inhibitor ANF and NO donor nitroprusside（SNP）on the physiological and biochemical properties of tomato were studied under nitrate treatment.Compared with the control group（CK）,tomato under nitrate treatment had fewer and shorter root systems,slower growth,and lower plant height.After applying SNP,the growth state of tomato plants was improved compared with nitrate stress.At this time,the contents of reactive oxygen species（ROS）and NO in the roots of the plants and the contents of MDA and H₂O₂in the leaves increased,and the activities of antioxidant enzymes increased..Compared with nitrate stress,the contents of ROS,NO,MDA and H₂O₂decreased.Except for the increase of POD activity,other enzyme activities of antioxidant enzymes were lower than those of nitrate treatment group.The expression levels of related genes were determined,and all the other genes were up-regulated except for NTRB gene expression.After exogenous application of ANF,the expression of NTRB was inhibited,and the growth of tomato seedlings was inhibited.The contents of ROS and NO in roots and MDA and H₂O₂in leaves were higher than those in the nitrate stress group,and the activities of antioxidant enzymes were significantly increased.The expressions of Sl Trx,Sl Tpx and Sl NTRB decreased,while the expression of Sl NR increased.When exogenously applied SNP and ANF co-treated,compared with tomato seedlings with ANF only,the phenotype was alleviated,the contents of ROS and NO in roots and MDA and H₂O₂in leaves were decreased,and the activities of antioxidant enzymes were increased.The expression levels of related genes were all up-regulated except for the decrease of NR.The above results indicated that exogenous application of SNP effectively alleviated nitrate stress and also alleviated the tomato seedlings inhibited by NTRB.2.Sl Tpx gene-edited and overexpressed tomato were obtained,and the resistance function of CRISPR-Cas9-Sltpx gene-edited tomato was analyzed under nitrate stress.After nitrate stress,the ROS content in CRISPR-Cas9-Sltpx tomato roots was higher than that in WT plants,and the NO content decreased.DAB and NBT histochemical staining showed that the content of O₂^-and H₂O₂in leaves was higher than that in WT plants.Compared with WT plants,the gene-edited plants had decreased antioxidant enzyme activities and increased MDA and H₂O₂contents after nitrate stress.The results showed that the nitrate stress resistance of gene-edited plants was lower than that of WT plants.3.Nitrate treatment was performed on Sl Tpx overexpressing tobacco obtained in the laboratory to analyze its resistance function.The germination rate of Sl Tpx overexpressed tobacco was tested in MS medium containing nitrate,and the germination rate of overexpressed tobacco was higher than that of WT tobacco.Compared with the wild type,the fluorescence intensity of ROS in overexpressed tobacco was weaker than that of WT,the fluorescence intensity of NO was higher than that of WT,and the activity of antioxidant enzymes was also higher than that of WT.The expression levels were all up-regulated compared to WT tobacco.It shows that the nitrate resistance of Sl Tpx overexpressing plants is improved.In addition,tobacco germination experiments under methyl viologen（MV）and H₂O₂stress were also carried out.The germination rate of overexpressed tobacco was higher than that of WT tobacco,indicating that the antioxidant capacity of overexpressed plants was improved.4.Nitrate treatment was performed on Sl NTRB-overexpressing tobacco obtained in the laboratory to analyze its resistance function.The germination rate of tobacco seeds overexpressing Sl NTRB was higher than that of WT seeds.Under nitrate stress,the content of ROS and H₂O₂in the roots of the overexpressed tobacco were lower than those of the WT tobacco,and the NADPH-dependent thioredoxin reductase activity and the ratios of GSH/GSSG and As A/DHA increased,and the gene expression levels of related genes were determined.,found that the gene expression levels were all up-regulated in overexpressed tobacco compared with WT plants.The results showed that Sl NTRB overexpressed transgenic tobacco had improved resistance under nitrate stress.5.The Sl Tpx protein was induced and purified by prokaryotic expression,and a series of in vitro experiments were performed on the Sl Tpx protein.The MFO experiment proved that Sl Tpx can protect DNA supercoil from the cleavage of harmful ROS;through the stress resistance experiments of different concentrations of heavy metals and H₂O₂,it was proved that Sl Tpx improved the antioxidative ability and antitoxicity of heavy metals in Escherichia coli.6.The Sl NTRB protein was induced and purified by prokaryotic expression,and it was found that the Sl NTRB protein could undergo S-nitrosylation modification.The key Cys sites that may undergo S-nitrosylation in Sl NTRB were predicted by GPS-SNO 1.0,and the proteins of three key Cys site mutants that may undergo S-nitrosylation were obtained and transformed into S-nitrosylation of the protein.Nitrosylation,the 346th position may be the key Cys site for S-nitrosylation of Sl NTRB.7.By screening the yeast two-hybrid library,the possible Sl Tpx interacting proteins were obtained,and the Bi LC experiment,yeast two-hybrid experiment and Co IP experiment proved that there is a protein interaction between Sl Tpx and Sl TFT4in tomato.At the same time,the above three experiments also verified the interaction between Sl NTRB and Sl TFT4 of tomato.From the physiological,biochemical and molecular levels,we revealed the mechanism of Sl Tpx and Sl NTRB in the response to nitrate stress in tomato,which could help to enrich our understanding of the mechanism of secondary salinization in vegetables.