Identification Of Key Genes For Nitrogen Efficiency Differences In Wheat Varieties And Analysis Of Ureide Permease Gene Family

In the current study,two related wheat cultivars ZM27 and AK58 were grown in the field environment,exposed to high,moderate and low N supply and analyzed during reproductive growth to(i)analysis phenotype and N efficiency of the two wheat cultivars to varying N nutrition,(ii)identify molecular mechanisms in flag leaves triggering differences in yield performance and NUE and(iii)to understand if and how seed/grain development is controlled by source leaf physiological processes and the subsequent partitioning of N and C assimilates from source to sink.Furthermore several ureide permase(UPS)genes were differentially expressed between the two cultivars and during the process of their adaptation to low and high N.In order to study whether UPS can play a role in N utilization,UPS gene family members were identified and their expression profiles were analyzed in wheat.TaUPS1-D.2 was transformed to Arabidopsis,and its function in improving yield and NUE was analyzed.The main conclusions are as follows:1.Wheat cultivar Zhoumai 27(ZM27)outperformed Aikang 58(AK58)and showed improved plant height,canopy leaf area index,flag leaf surface,seed development and grain yield,as well as enhanced NUE due to both increased N uptake and utilization efficiency.2.Further,transcriptome and proteome analyses of flag leaves,a major source of N and C for grain development,showed that the inherent differences in NUE between ZM27 and AK58 are controlled by source N and C metabolism and transport processes.Such as genes involved in carbon assimilation and transport including several subunits in photosystem I and II,ferredoxin NADP+oxidoreductase(FNR),Rubisco,sucrose phosphate synthase(SPS)and sucrose synthase(SS),sucrose transporters SUT2、SUT4,and genes involved in N assimilation and transport such as glutamate dehydrogenase(GDH)as well as amino acid permease 6(AAP6-2A and AAP6-2D),amino acid transporter ANT1-like(ANT1L),peptide transporter PTR1 and oligopeptide transporter 4-like(OPT4),all up-regulated in ZM27 compared to AK58.Among them,FNR,Rubisco,SUT2 also up-regulated in ZM27 compared to AK58 at protein level.This was also in line with increased net photosynthetic rates of flag leaves,source-to-sink allocation of N/C assimilates and grain protein/starch yield in Nefficient ZM27 plants.3.There are not only differences in N efficiency,but also different adaptive mechanisms for low and high N between the two wheat varieties.The adaptation of the two varieties to low and high N included the changes of related genes or proteins in photosynthesis,response to stimulation and amino acid metabolism,which indicated that the genes/proteins related to these pathways or biological processes were sensitive to N level.In addition,several UPS genes were differentially expressed between the two N efficient varieties and during the adaptation of the two varieties to low and high N.4.UPS mediates the transport of ureides in plants,ureides includes allantoin and allantoic acid.It is the important form of N transport in N-fixing legumes and non-N-fixing plants such as Arabidopsis,rice and wheat.Few information about UPS in wheat.The UPS gene family was identified in Wheat(Triticum aestivum L.)for the first time.Total 13 UPS genes were identified and divided into three groups: TaUPS1,TaUPS2.1 and TaUPS2.2.TaUPS2.1 group genes are highly expressed,TaUPS1 group genes are expressed unevenly,and TaUPS2.2 group genes are almost no-expression in various tissues and growth stages.TaUPSs are co-expressed with WRKY transcription factor,polyamine transporter,NRT1/PTR family Ta NPF8.10,Ta NPF8.4,Ta NPF7.8,potassium transporter,Lasparaginase,and aspartic protease.5.Analysis of the natural variation of TaUPSs in wheat in four different historical years showed that there were 19,8,3 allelic variations of TaUPS2.2-A,TaUPS1-B and TaUPS1-A.The frequency of allele variation of TaUPS1-B of chr7B: 647752667 and chr7B: 647752935 decreased with the advance of breeding years,which indicated that the two loci were selected artificially by breeders in the breeding process.The SNP variation of TaUPS1-B gene can be divided into six haplotypes.Among them,the proportion of cultivated and modern varieties is more in haplotype I,while the proportion of modern varieties is the least in haplotype II,III,IV and V,which indicates that these haplotypes are artificially selected in breeding.The number of tillers,the number of siliques of tillers,the number of all siliques,the calculated yield,and the dry weight are higher in overexpressing TaUPS1-D.2(At UPS1pro: TaUPS1-D.2: WT)Arabidopsis plants compared to wild-type(WT)plants under normal N supply.Overall,this study supports that N and C metabolism and transport processes are key players in wheat N use efficiency,irrespective of the N supply.Candidate genes were identified that may individually or concurrently regulate NUE and that are promising targets for maximizing NUE in crop plants,in both high and low N environments.In addition,several TaUPS genes were differentially expressed between the two N efficiency varieties and during their adaptation to low and high N levels.In this study,heterologous expression of TaUPS1-D.2 increased the yield and NUE in Arabidopsis.Therefore,TaUPS may be used as one of the key candidate genes to improve wheat NUE.