Effects Of Nickel Ions On Root Physiological Characteristics Of Tomato Seedlings At Low Nitrogen Level

Nitrogen（N）is one of the key factors that limits growth and development of tomatoes（Solanum lycopersicum L.）,but China has been facing nitrogen shortage,low utilization efficiency,and overuse of nitrogen fertilizer in soil,which have negative impacts on the healthy development of the tomato industry.In recent years,adding exogenous substances to alleviate the harm caused by low nitrogen stress on crops has become an effective way to overcome the lack of nitrogen in the environment.Previous studies have shown that a certain concentration of nickel ions can increase plant nitrogen assimilation and nitrogen metabolism,and promote plant growth and development.However,it is not clear how nickel ions specifically regulate the root system of tomato seedlings to cope with low nitrogen stress.This experiment mainly investigates the impact of nickel ions on the root system of tomato seedlings under low nitrogen stress from the perspectives of the relationship between nickel ions and the antioxidant system,the relationship between carbon and nitrogen metabolism,and the relationship between related genes,in order to provide a theoretical basis for the improvement of tomato’s low nitrogen tolerance by nickel ions in practical production.In this study,tomato variety’Micro Tom’was cultured under hydroponic conditions,with different nitrogen levels(7.66 and 0.383mmol·L^-1)and different Ni²⁺levels(0 and 0.1 mg·L^-1Ni SO₄·6H₂O)applied to analyze the effects of exogenous Ni²⁺on the growth and development of tomato seedlings,antioxidant system,carbon and nitrogen metabolism under low nitrogen conditions.The differentially expressed genes in the carbon and nitrogen metabolism pathway were screened,and their cloning,physicochemical property analysis and relative expression level determination were carried out,and the activities and product contents of key enzymes in the pathway were determined.The identified key genes of carbon and nitrogen metabolism were subjected to gene family member identification,bioinformatics analysis,determination of gene expression levels,and construction of overexpression vectors for subcellular localization.The main research results are as follows:1.The effects of nickel ions on the growth,development,and antioxidant capacity of tomato seedlings under low nitrogen conditionsThe results show that low nitrogen inhibits the growth and development of tomato seedlings,significantly increases the content of hydrogen peroxide（H₂O₂）,superoxide anion（O₂^-）,and malondialdehyde（MDA）,indicating that the root system of tomato seedlings is subject to oxidative damage.However,Ni²⁺treatment can increase the activity of superoxide dismutase（SOD）,peroxidase（POD）,catalase（CAT）,ascorbate peroxidase（APX）,monodehydroascorbate reductase（MDHAR）,dehydroascorbate reductase（DHAR）,glutathione reductase（GR）,glutathione peroxidase（GPX）,glutathione S-transferase（GST）,and thioredoxin peroxidase（TPX）,increase the content of non-enzymatic antioxidants ascorbic acid（ASA）and reduced glutathione（GSH）,and improve the efficiency of the ASA-GSH cycle,significantly reducing the H₂O₂and MDA content,alleviating the oxidative damage caused by low nitrogen stress to the root system of tomato seedlings,and improving the biomass and root activity of tomato seedlings.Ni²⁺treatment promotes the activity of antioxidant enzymes in the root system of tomato seedlings to increase the content of antioxidants,reduce oxidative damage caused by low nitrogen stress,and improve the tolerance of tomato seedlings to low nitrogen levels,thereby maintaining the normal growth and development of tomato seedlings.2.The effects of nickel ions on the carbon and nitrogen metabolism physiology and transcriptional response of tomato roots under low nitrogen conditionsThe results show that low nitrogen stress inhibits the activity of nitrogen assimilation-related enzymes,while Ni²⁺treatment can significantly alleviate this process and increase the content of nitrate（NO₃^-）,ammonium ion（NH₄⁺）,and total amino acids.Through transcriptome analysis of roots,3,738 differentially expressed genes（DEGs）were identified.The DEGs related to carbon and nitrogen metabolism were down-regulated under low nitrogen treatment,while after Ni²⁺treatment,the DEGs related to carbon and nitrogen metabolism such as PK,PDHB,GAPDH,NR,Ni R,GS,and GOGAT were significantly up-regulated.The results indicate that Ni²⁺can regulate the carbon and nitrogen metabolic pathways of tomato roots to alleviate the effects of low nitrogen stress.3.Identification of the tomato NRT2 gene familyHierarchical clustering and KEGG analysis of differentially expressed genes in the tomato root transcriptome treated with different nitrogen and nickel concentrations found that the NRT1,NRT2,and chloride channel（CLC）protein families,especially the NRT2 gene family,positively responded to changes in nitrogen levels.Therefore,the NRT2gene family of tomato was identified by gene family member identification,bioinformatics analysis,fluorescent quantification,and construction of overexpression vectors for subcellular localization.The results showed that six tomato NRT2 gene family members were identified,and four genes were predicted to be expressed in the cell membrane,while Sl NRT2.2 was predicted to be expressed in the endoplasmic reticulum.In addition,Sl NRT2.1 could be expressed not only in the vacuoles but also in the lysosomes.A total of 8-10 conserved motifs,including a conserved PLN00028 signal peptide,were identified in the Sl NRT2 protein.Transcriptional element analysis showed that the NRT2 gene family may be regulated by light and respond to various plant hormones and growth regulators.The relative expression levels of the six tomato NRT2 gene family members were determined,and the expression of NRT2 genes in tomato seedling roots was significantly promoted by treatment with 0.1 mg·L^-1Ni²⁺under low nitrogen stress,thereby improving the adaptation of tomato to low nitrogen stress.Among them,the expression of Sl NRT2.5 gene was the most significant.Furthermore,subcellular localization analysis of the Sl NRT2.5 gene overexpression vector showed that Sl NRT2.5 could be expressed in the cell membrane,which explains well the function of the NRT2 gene as a high-affinity transport protein for nitrogen transport in the cell membrane.