Identification And Functional Analysis Of Key NRT Genes In Influencing Nitrogen Use Efficiency In Wheat(Triticum Aestivum L.)

Wheat(Triticum aestivum L.)is an important staple food crop for more than one third of the world's population.With the increase of population,high-yield and high-quality wheat production is of great significance for realizing the sustainable development of agriculture and ensuring the food security.Nitrogen(N)fertilizer plays a pivotal role in wheat production and contributes about 30%to the yield increase.Nevertheless,the N fertilizer use efficiency is only about 33-50%for wheat,and the overuse of N fertilizer has led to higher agricultural input and a series of environmental risks.Therefore,how to improve the N use efficiency(NUE)of wheat has become an important issue.Breeding wheat varieties with high NUE is a pivotal approach to solve this problem.Breeding for improved NUE cannot be achieved without a full understanding of the molecular mechanisms underlying N uptake and utilization by wheat.In the present study,a meta-analysis was firstly conducted and showed that transporter genes are more efficient in improving yield and NUE parameters of crops,compared with other gene types.Thus,the gene families that may encode nitrate transporter(NRT)in the wheat genome were analyzed in depth.Meanwhile,a candidate gene association study was conducted to identify the target gene Ta NRT2.1-6B,which significantly affected NUE in wheat.Furthermore,the nitrate transport function of Ta NRT2.1-6B was verified via a heterologous expression system.We also characterized the function of Ta NRT2.1-6B in regulating plant N uptake and utilization by complement test in Arabidopsis mutant and overexpression in wheat.The main results are as follows:1.The meta-analysis showed that genetic transformations increased yield,shoot biomass,N uptake efficiency(NUp E),and partial factor productivity of N(PFPN)in the crops(rice,maize,and wheat)with an increase of 16.7%,10.1%,16.2%and 9.5%,respectively,but decreased shoot N utilization efficiency(SNUE)and grain N utilization efficiency(GNUE)by 6.6%and 5.0%.Compared with other gene types,transporter genes improved yield and NUE parameters more efficiently,increasing grain yield,NUp E and PFPN by 36.7%,46.1%and 32.4%,respectively.In addition,the effect sizes for some NUE parameters varied according to crop species and experimental conditions.The largest yield increase was rice(20.4%).In pot experiments,the yield and NUp E was increased by 36.7%and 26.8%,respectively,which was significantly greater than that in field experiments.However,the effect sizes were not significantly different between overexpression and ectopic expression methods.Most effect sizes did not correlate with gene overexpression levels.2.The NPF(Nitrate transporter 1 and peptide transporter family)and NRT2 gene families in the wheat genome were deeply analyzed,and 331 NPF genes and 46 NRT2 genes were identified in the wheat genome.The NPF genes were mainly distributed on the 2-,3-and7-chromosome groups and in the R2b region of chromosomes.The NRT2 genes were mainly distributed on the 6-chromosome group and in the R1 region.Tandem duplication was the main driver of the expansion of the NRT2 genes.Forty-one and 91 NPF genes in the wheat genome were collinear with the NPF genes in the Arabidopsis and rice genomes,respectively.Six NRT2 genes in the wheat genome were collinear with the NRT2 genes in the rice genome.Multiple transcription factor families such as b HLH,WRKY,MYB,NAC,b ZIP and GRAS were coexpressed with NPF or NRT2 genes in wheat indicated by weighted gene coexpression network analysis(WGCNA).The selected two NPFs and one NRT2 could transport NO₃^-under either 0.5 m M or 10 m M nitrate concentrations via a Xenopus laevis oocytes heterologous expression system.Natural variations in NPF genes and NRT2 genes resulted in differences in the NUE of wheat.Polymorphisms in NPF and NRT2 genes mainly accumulate by random drift rather than selection.3.Candidate gene association study was performed based on gene single nucleotide polymorphism(SNP)of 46 NRT2 genes in a panel composed of 291 wheat cultivars and six NUE related traits.Four NRT2 genes were significantly associated with NUE related traits.Traes CS6B02G044300(Ta NRT2.1-6B)was highlighted because it significantly associated with multiple NUE traits simultaneously and the different haplotypes of Ta NRT2.1-6B significantly affected the grain yield and NUE related traits of wheat.Ta NRT2.1-6B was located on the plasma membrane and can take up nitrate under 0.25 m M and 10 m M nitrate concentrations in the Xenopus laevis oocytes assay.Nitrate uptake kinetics tests showed that Ta NRT2.1-6B is a dual-affinity nitrate transporter and the Km value of Ta NRT2.1-6B was0.36±0.13 m M and 20.52±3.26 m M for the low-and high-affinity NO₃^-uptake,respectively.Overexpression of Ta NRT2.1-6B in Arabidopsis atnrt1.1 mutant improved the root growth and¹⁵N accumulation.The overexpression of Ta NRT2.1-6B in wheat increased root growth,¹⁵N accumulation and influx at seedling stage,and also increased grain yield,root dry weight,shoot dry weight,root N accumulation and grain N accumulation under both N-limited and sufficient conditions.Under the N-limited condition,the overexpression lines had higher N harvest index,grain N use efficiency and shoot N use efficiency.4.The transient expression of Ta NRT2.1-6B promoter fragments in tobacco showed that-160 bp upstream fragment had key elements to affect promoter activity.There might be silence elements within-1000 bp to-3000 bp that affected the promoter activity.Reverse chromatin Immunoprecipitation(Ch IP)and yeast one-hybrid assays indicated that the proteins such as histone,methylase,protein kinase,TCP transcription factor,b HLH transcription factor and GRAS transcription factor,may affect the expression of Ta NRT2.1-6B.In conclusion,transporter genes are more efficient at improving yield and NUE related traits of crops than other type of genes.A total of 331 NPF genes and 46 NRT2 genes were identified in the wheat genome.Natural variations in NPF genes and NRT2 genes resulted in differences in yield and NUE related traits of wheat.Ta NRT2.1-6B is a dual-affinity nitrate transporter in wheat and the overexpression of the gene improved yield and NUE related traits of wheat.The GRAS transcription factor Traes CS2D02G198200 is a potential unrecognized transcription factor regulating the expression of Ta NRT2.1-6B genes in wheat.