Differences In Activities And Gene Expression Of Key Enzymes Involved In Carbon-Nitrogen Metabolism And Response To Nitrogen Fertilizer In Wheat With Different NUE

Nitrogen（N）is one of the essential nutrient elements for wheat growth and development.Rational N fertilization can improve crop yield and quality,but excessive N application can reduce nitrogen use efficiency（NUE）and cause a series of environmental pollution issues.Therefore,breeding wheat varieties with improved NUE is one of the main ways to promote agricultural sustainable development.NUE is a complex quantitative trait controlled by heredity,involving the coordination of many genes.There are significant physiological and genetic differences in NUE among different wheat genotypes,and it is of great importance to explore the differences in physiological,biochemical and genetic mechanisms among different genotypes of wheat for breeding high-NUE varieties.In this study,eight pairs of near-isogenic lines（NILs）of wheat were used to analyze the differences in NUE at the seedling and maturity stage and their correlations with morphological,physiological,and yield traits,proposed traits for screening high-and low-NUE materials,and evaluated the characteristics of NUE in paired materials.On this basis,a pair of wheat NILs（1Y,high-NUE;1W,low-NUE）with great differences in N efficiency were used for transcriptome sequencing and metabolome profiling analysis to clarify the related genetic and physiological mechanisms under low-nitrogen（0 g N pot-1）and normal N conditions（1.6 g N pot-1）.The main results are as follows:1.The effects of nitrogen（N）on 10 traits of 8 pairs of wheat near-isogenic lines（NILs）were quite different.The relative dry weight of stem and leaf,the relative ratio of root to shoot,the relative N accumulation in stem and leaf,and the relative N accumulation in the plant could be used as the screening indexes of the N efficiency in wheat at the seedling stage.According to the comprehensive evaluation value of N efficiency,the genotypes of each pair of NILs were divided into high-NUE（Y）and low-NUE（W）genotypes.The comparative analysis of grain yield and N utilization rate at the maturity stage showed that the high-NUE material（Y）had a higher grain yield and N utilization rate than the low-NUE material（W）in each pair of NILs,and the classification results at the two stages were consistent.2.Under two N levels,there were differences in the activities of key enzymes of N metabolism between high-and low-NUE genotypes in each pair of NILs,and the activities of nitrate reductase（NR）,glutamine synthase（GS）,and glutamate synthase（GOGAT）in flag leaves of wheat with high-NUE were higher than those of low-NUE genotype at grain filling stage when N was applied at the same rate.In terms of gene expression,the expression levels of many genes associated with N metabolism（TaNR、TaGS、TaGOGAT）were generally higher in high-NUE materials than those in low-NUE materials,indicating that the higher expression levels of the genes involved in N metabolism could improve key N metabolism enzyme activities and promote the N metabolism in leaves.3.Based on the analysis of transcriptomic data,a total of 7023 differentially expressed genes（DEGs）were identified in the N1₁Y vs.N1₁W comparison under the condition of N application,including 4738 upregulated genes and 2285 downregulated genes.GO analysis showed that these DEGs were significantly enriched in "DNA-binding transcription factor activity","catalytic activity",and "protein kinase activity",indicating that N1₁Y had strong cellular catalytic activity and enzyme activity.KEGG enrichment analysis showed that these DEGs were involved in "starch and sucrose metabolism","phenylalanine,tyrosine,tryptophan biosynthesis",and other metabolic processes.Given the above analysis,the plant regulated N efficiency through coordinated regulation of C and N metabolism processes.In the N1₁Y vs.N1₁W comparison,a total of 20 DEGs were involved in N metabolism,of which 13 DEGs were upregulated in the N 1₁ Y leaves,including those encoding six nitrate transporter（NRT）,four ammonium transporter（AMT）,two glutamine synthase（GS）,and one glutamate synthase（GOGAT）,indicating that 1Y had higher capability of N metabolism and promoted the activity of N metabolism enzymes to a certain extent.Most of the genes involved in photosynthesis（photosystem I,photosystem II and photosynthetic antenna proteins）and respiration（glycolysis,tricarboxylic acid cycle,and pentose phosphate pathway）were also upregulated in N1₁Y,which could provide more energy,carbon skeletons,and NADPH for N metabolism.In addition,the number of upregulated genes encoding transcription factors（TFs）was about two time of downregulated genes in the leaves of N1₁ Y,which suggested that the TFs may play key roles in the regulation of the genes related to N metabolism.4.According to the results of transcription profile analysis,a total of 3290 DEGs were identified in the comparison of N0₁Y vs.N0₁W under the condition of no N application,including 2123 upregulated genes and 1167 downregulated genes.There were 379 GO terms significantly enriched among these genes,including "amino acid metabolic process","response to nitrogen compound","catalytic activity",and so on.Meanwhile,six metabolic pathways were identified to be significantly enriched,including "glutathione metabolism",and"starch and sucrose metabolism".In the NO₁Y vs.N0₁W comparison,21 DEGs were associated with carbon and nitrogen metabolic pathways.Additionally,4 genes involved in nitrogen metabolism and 10 genes associated with carbon metabolism were up-regulated in the N0₁Y leaves,which ensured its higher N utilization efficiency under low N conditions.5.Compared with no N application treatment,a total of 7669 and 7800 DEGs were identified after N fertilizer application in the leaves of 1Y（N1₁Y vs.N0₁Y）and 1W（N1₁W vs.N0₁W）,respectively.The differential expression analysis showed that the number and magnitude of upregulated genes encoding GS and GOGAT were higher（5 genes,1.12-2.07 times）in 1Y than those in 1W（3 genes,1.09-1.42 times）,suggesting that the higher expression of N assimilation genes promoted the enhancement of the N metabolism pathway in 1Y.Moreover,the number of upregulated genes encoding photosystem I,photosystem II and photosynthetic antenna proteins（36 genes）was higher in 1Y than that in 1W（13 genes）,indicating that 1Y had higher capacity of photosynthetic rate.Meanwhile,the genes encoding the key enzymes involved in glycolysis pathway and TCA cycle were upregulated（1.06-4.47 fold）in 1Y leaves,which provided more energy for N metabolism.The gene encoding 6phosphogluconate dehydrogenase（6PGD）was only upregulated in 1Y but not changed in 1W.The results indicated that the enhancement of the pentose phosphate pathway（PPP）in the leaves of 1Y could provide more NADPH for N metabolism.6.Transcriptional differences in response to low N stress were analyzed between 1Y and 1W,and these N-responsive genes were divided into two categories:frontloaded genes（DEGs had higher expression levels before low N stress）and relatively upregulated genes（DEGs had higher expression levels after low N stress）.A total of 146 N-responsive genes were identified in 1Y,including 103 frontloaded genes（amino acid transporter genes,peroxidase genes,cytochrome P450 genes,mitogen-activated protein kinase genes,and transcription factors）,43 relatively upregulated genes（glutamate dehydrogenase gene,glutamate receptor gene,malate synthase gene and so on）.A total of 95 N-responsive genes were identified in 1W,including 45 frontloaded genes（nitrate transporter gene,protein phosphatase 2C gene,cytochrome P450 gene,and peroxidase gene）,50 relatively upregulated genes（phenylalanine ammonia-lyase gene,UDP-glycosyltransferase gene,1-aminocyclopropane-1-carboxylate synthase gene,and ERF transcription factor）.Hence,in comparison with 1 W,1 Y had more frontloaded genes.Based on the classification of frontloaded genes and relatively upregulated genes,it was speculated that 1Y and 1W showed different gene expression patterns and physiological characteristics in response to low N stress,and there were two potential response mechanisms.7.Based on the metabolome data analysis,a total of 26 metabolites related to carbon and nitrogen metabolism were detected in the N1₁Y vs.N0₁Y comparison,including 15 upregulated metabolites and 11 downregulated metabolites.Correspondingly,a total of 15 carbon and nitrogen metabolites were detected in the N1₁W vs.N0₁W comparison,including 6 upregulated metabolites and 9 downregulated metabolites.Among them,the contents of most amino acid metabolites（phenylalanine,tyrosine,γ-aminobutyric acid,proline,and aspartic acid）increased significantly in both 1Y and 1W leaves,but the increase rate in 1Y was significantly higher than that in 1W,indicating that the metabolism of amino acids in 1Y leaves was enhanced,which was helpful to improve the ability of efficient absorption and utilization of nitrogen in wheat.The relative contents of sucrose,lactose,and fructose related to carbon metabolism were higher in 1Y than in 1W,which could improve the stability of the cell membrane.In addition,the enhancement of the TCA cycle in 1Y can provide sufficient energy and metabolic intermediates for other metabolisms.