The Mechanism Of Tomato ASA-GSH Cycle Key Enzymes Involved In Alleviating Nitrate Stress By NO

Soil secondary salinization is one of the main factors limiting the development of protected vegetable,which seriously affects the yield and quality of vegetables.The study showed that the anionic type in the greenhouse soil was mainly NO₃^-.NO is an important cell signaling molecule,which plays an important role in plant growth and development and stress response.On the one hand,NO can directly or indirectly stimulate multiple redox regulation?defense related?gene expression.On the other hand,under stress,NO mainly regulates enzyme activity through translational modification of target proteins of S-nitrosylation and nitrification.Ascorbic acid peroxidase?APX?,monodehydroascorbate reductase?MDHAR?and glutathione reductase?GR?are involved in the scavenging of reactive oxygen species in ascorbic acid-glutathione cycle?ASA-GSH?.The activity of these enzymes is regulated by NO mediated S-nitrosylation and nitrification.In this paper,the key enzymes?ASA-GSH,APX,MDHAR and GR?of tomato were involved in the mechanism of NO alleviating nitrate and the results were as follows:1.The effects of exogenous NO?SNP?on the key enzymes in ASA-GSH cycle were studied.Compared with the normal tomato seedlings,the content of H2O2 and malondialdehyde?MDA?in tomato seedlings increased significantly under the condition of 100 m M nitrate stress,but the content of exogenous SNP decreased significantly.Compared with the control?CK?,the expression of APX,GR and MDHAR genes in the leaves and roots of tomato seedlings increased significantly,and the expression of MDHAR protein and GR protein in root and leaf increased under nitrate stress.After adding exogenous NO,the gene expression and enzyme activity of APX in leaves decreased,and the APX gene and enzyme activity in the root increased,the activity of GR in the leaves decreased,the activity of GR in the roots changed little,and the activity of MDHAR in the leaves and roots decreased,while the viability of MDHAR in the leaves and roots was lower than that of CK in the treatment of 3 h,6 h and 1 D.In addition,under nitrate stress,the activity of SOD and POD in leaves and roots of tomato seedlings increased,and the activity of CAT in leaves and roots decreased and CAT activity in roots increased.Compared with nitrate and nitrate,the activity of SOD in tomato leaves and roots decreased,and the activity of SOD in tomato leaves and roots was decreased,and the activity of 3 D and 5d in tomato seedlings increased,and the roots of the tomato seedlings increased,and the roots of the tomato seedlings were 3 D and 5 d.The activity of POD also increased,and the CAT of 3 h,6 h and 1 D in roots increased,and the CAT activity of 6 h,1 D,3 D,5 d in leaves increased.Compared with CK,under nitrate stress,DHAR activity in leaves decreased,and DHAR activity in roots increased.Compared with nitrate stress,the activity of DHAR in leaves increased after nitrate treatment and SNP treatment,while the DHAR activity in roots was lower than that in CK and nitrate stress.The activity of 1D in roots increased.The above results indicated that APX,MDHAR and GR were involved in the process of NO alleviating nitrate stress in ASA-GSH cycle.2.The S-nitrosalytion level in tomato leaves and roots after nitrate treatment and NO treatment was studied and the target protein of S-nitrosalytion was identified.The results showed that the level of S-nitrosylation in leaves and roots increased after nitrate treatment.By the transformation of biotin and combining with MALDI-TOF-TOF,it was foundthattheproteinsintherootS-nitrosylationincluded phosphoenolpyruvate carboxylase1,aconitate hydratase,sucrose synthase,adenosylhomocystein- ase,s-adenosylmethionine synthase1,phosphoglycerate kinase,annexin,peptidyl-prolylcis-transisomerase,elongationfactor1-alphaand uncharacterized protein,the proteins of S-nitrosylation in the leaves includes ribulose bisphosphate carboxylase large chainthe long chain,oxygen-evolving enhancer protein 1 and uncharacterized protein.In the isolated S-nitrosalytion protein,MDHAR were found to be S-nitrosylated by Western blot.The Cys27,Cys181 and Cys379 in MDHAR were predicted to the probably site of S-nitrosylation by GPS-SNO 1 software.3.Using prokaryotic expression,the purified recombinant tomato cytosolic APX1 were obtained.The impact of S-nitrosoglutathione?GSNO?andperoxynitrite?ONOO^–?whichareknowntomediateprotein S-nitrosylation and nitrification processes,respectively,was analysed on the enzyme activity of APX.While peroxynitrite inhibits APX1 activity,GSNO enhances its enzymatic activity.The S-nitrosylation and nitrification sites of tomato cytoplasm APX1 were predicted by GPS-SNO 1 and GPS-YNO2 1 respectively.Cys4 may be S-nitrosylation site and Tyr5,Tyr12 and Tyr235 may be nitrification sites.4.We obtained tomato full-length c DNA sequences of ascorbic acid peroxidase,glutathione reductase and monodehydroascorbic acid reductase gene.Then overexpression vectors of p RI101-Sl APX,p RI101-Sl GR and p RI101-Sl MDHAR were constructed and transformed into tobacco leaf discs.The transgenic tobacco plants were analyzed by genomic PCR,q RT-PCR and western blot and the Sl APX,Sl GR and Sl MDHAR transgenic tobacco plants were obtained.