Physiological And Molecular Mechanisms Of Nitrate-dependent Alleviation Of Ammonium Toxicity In Wheat(Triticum Aestivum L.)

Ammonium（NH₄⁺） is one of the most important nutrients required by plants.Appropriate amount of NH₄⁺promotes plant growth and increase crop yield.However,a high concentration of NH₄⁺as the sole nitrogen source suppresses plant growth and production.Wheat（Triticum aestivum L.）is the raw material of many foods and this crop is very important to human life.Therefore,it is important to study the mechanism of NH₄⁺toxicity and alleviation.In this study,wheat plants were cultivated in 1/2 Hoagland hydroponic solution,containing 7.5 mmol/L NO₃^-as control（CK）,containing 7.5 mmol/L NH₄⁺（HA）or 7.5 mmol/L NH₄⁺plus 1.0 mmol/L NO₃^-（AN）as treatments.We studied the physiological and molecular mechanisms of the NO₃^--dependent alleviation of NH₄⁺toxicity.The main results are summarized as follows:1)Physiological mechanism of NO₃^--dependent alleviation of NH₄⁺toxicityCompared with the control,the HA treatment significantly decreased root growth and the activity of enzymes and the concentrations of most intermediates from the tricarboxylic acid（TCA）cycle,increased the activity of plasma membrane H⁺-ATPase and the rate of H⁺efflux along roots,caused solution acidification,In addition,HA treatment increased the activity of electron transport chain complexes I-IV,the increased contents of root protein-bound carbonyls and malondialdehyde,and thus induced ultrastructural disruption and reduced the viability of root cells.Compared with the HA treatment,the AN treatment increased root growth,the activity of anaplerotic enzymes and the concentrations of most intermediates from the TCA cycle.Furthermore,AN treatment decreased the net rate of H⁺efflux,retarded medium acidification,decreased protein carbonylation and lipid peroxidation in roots and relieved ultrastructure disruption and increased the viability of root cells.2)Transcriptome analysis of wheat seedling root under different N treatmentsUnder NH₄⁺ stress,the differentially expressed genes（DEGs）encoding the enzymes of glucolysis（EMP）,lactic fermentation and alcoholic fermentation were significantly up-regulated.The DEGs encoding the enzymes of TCA cycle and ATP synthase were down-regulated.The DEGs encoding aquaporin were also down-regulated.Compared with the HA treatment,the number of up-regulated DEGs encoding the enzymes of EMP,lactic fermentation and alcoholic fermentation were significantly reduced under AN treatment.The number of down-regulated DEGs encoding the anaplerotic enzymes of TCA cycle,ATP synthase and aquaporin were decreased,and AN treatment increased ATP synthesis and stress resistance in plants.3)Comparative analysis of root proteomics in wheat seedlings exposed to different N treatmentsUnder high NH₄⁺ stress,a total of 251 differentially expressed proteins（DEPs）were identified.And under AN treatment,178 DEPs were identified.In order to further analyze the biological functions of DEPs,the KEGG was classified and statistically analyzed.According to the KEGG enrichment analysis,these DEPs were mainly mapped to matabolic pathways of biosynthesis of secondary metabolites,carbon metabolism and glycometabollism.The up-regulated DEPs encoding the enzymes of EMP and fermentation under AN treatment were significantly decreased compared with the HA treatment.Taken together,these results indicate that NO₃^--dependent alleviation of NH₄⁺toxicity is through supplementing carbon skeletons,alleviating rhizospheric acidification,reducing the production of ROS and oxidative damage,and increasing aerobic respiration metabolism.