Studies On Nitrogen Metabolism Characteristics And Mechanisms Of Low Nitrogen Response In ZmPEPC Transgenic Wheat

Phosphoenolpyruvate carboxylase(PEPC)is an important enzyme,with functions in the primary metabolism for carbon and nitrogen metabolism.In previous studies,the gene of key enzyme in the C4 pathway have been introduced into C3 crop,which showed significantly improved photosynthesis and yield,and enhanced drought and heat tolerance.In the present work,three types of homozygous ZmPEPC transgenic wheat lines(08T1-27,08T1-47,and 08T1-51)were independent transformants and have been stably inherited.We took three transgenic wheat lines and untransformed lines(WT,common wheat variety,Zhoumai 19)as the experimental material.We studied the molecular characteristics of transgenic wheat lines and evaluated nitrogen uptake and utilization of transgenic wheat lines under different fertility level in field.Afterwards,we analysed root morphology and nitrogen metabolism of transgenic wheat lines at seeding stage,also researched effects of ZmPEPC gene on nitrogen assimilation in transgenic wheat lines,and proteomic profiling of transgenic wheat leaves under low nitrogen treatment.These results gave us more information to discuss the mechanisms of the effects of overexpression ZmPEPC gene on nitrogen metabolism in transgenic wheat.The main results in this study are listed as follows:1.The results of PCR analysis showed that the exogenous ZmPEPC gene was integrated into the genome of transgenic wheat lines and have been stably inherited;Analysis the results of RT-PCR and qRT-PCR suggested that ZmPEPC gene expressed correctly in transcriptional level;The results of Western blotting indicated that ZmPEPC gene translated into the corresponding protein correctly.2.The mean of nitrogen content of shoot and grain in the plant of three transgenic wheat lines were 13.66%,11.34%and 17.28%higher than WT for flowering stage and mature stage under low fertility treatment,respectively;The mean of nitrogen concentration of shoot and grain in three transgenic wheat lines were 25.15%,15.54%and 7.55%higher than WT at flowering stage and mature stage under low fertility treatment,respectively;The mean of NRA,nitrogen content of shoot and grain per unit area at mature stage in three transgenic wheat lines under low fertility treatment were 14.98%,13.82%and 15.48%higher than WT,respectively,these results were similar to the changes trends of nitrogen content in the plant of transgenic wheat lines,there were not significant change in NHI,NUtE and NRE.The transgenic wheat lines increased grain yield by increasing 1000-grain weight compared with WT,grain yield and 1000-grain weight of three transgenic wheat lines were significantly increased under low fertility treatment,with mean that were 4.03%and 6.24%higher than WT,respectively.The results showed that nitrogen uptake was enhanced in transgenic wheat under low fertility treatment.3.The root surface area and total root length of three transgenic wheat lines were significantly increased than WT under low nitrogen treatment,with mean that were 43.92%and 36.39%higher than WT;Under low nitrogen treatment,different organ nitrogen concentration and content of three transgenic wheat lines were significantly increased,the magnitude of the increase was higher than that under high nitrogen treatment,the mean of whole plants nitrogen content in three transgenic wheat lines was 23.11%higher than WT;Root activity,NR and GS enzyme activity of root,and relative expression of primary nitrogen metabolism related gene(TaNR?TaGS1?TaGOGAT?TaGDH and TaAspAT)were significantly increased compare with WT under low nitrogen treatment.The results indicated that the transgenic wheat have better root morphology and root activity,increased primary nitrogen metabolism of root enzyme activity and gene expression,enhanced nitrogen assimilation under low nitrogen treatment.4.Increases in gene expression associated with Rubisco,Rubisco content,and carboxylase activity of Rubisco were found in leaves of ZmPEPC transgenic wheat lines subjected to low nitrogen treatment.The transgenic wheat lines expressing ZmPEPC exhibited up-regulation gene expression associated with the anaplerotic pathway for the TCA cycle,suggesting that more carbon skeleton material is being allocated for nitrogen assimilation under low nitrogen treatment.Furthermore,ZmPEPC overexpression in transgenic wheat lines induced the up-regulation of gene expression associated primary nitrogen metabolism,including TaNR,TaGS2,TaGOGAT,TaAspAT,and TaASNI,in addition the corresponding enzyme activity were increased.The average total free amino acid content in the transgenic wheat lines were 48.18%higher than WT,The levels of Asn,Gln,Asp,and Ser were also markedly enhanced.In addition,the results showed that nitrogen content,carbon content,and biomass of the transgenic wheat lines increased under low nitrogen treatment.These results indicated ZmPEPC overexpression in wheat could modulate carbon metabolism,significantly improve nitrogen assimilation,and enhance growth under low nitrogen conditions.Overexpression of the ZmPEPC gene displayed a positive effect on nitrogen metabolism.5.Differential expressed proteins(DEPs)of flag leaves at flowering stage were analyzed in the transgenic wheat lines and WT under low nitrogen treatment via using iTRAQ-based quantitative proteomic methods.We identified 218,298 and 106 DEPs with significantly altered by ZmPEPC responsive,nitrogen stress responsive and common responsive,respectively.All identified DEPs were analysed via KEGG pathway,which show that ZmPEPC responsive DEPs mainly involved in Glyoxylate and dicarboxylate metabolism,Carbon fixation in photosynthetic organisms,Photosynthesis,Nitrogen and Sulfur metabolism,Biosynthesis of amino acids.Sedoheptulose-1,7-bisphosphatase(SBPase),Malic enzyme,Ribulose 1,5-bisphosphate carboxylase,large subunit(rbcL),Chlorophyll a-b binding protein and Glutamine synthetase(GS)were induced by ZmPEPC expression;Nitrogen stress responsive DEPs mainly involved in Glyoxylate and dicarboxylate metabolism,Starch and sucrose metabolism,Amino sugar and nucleotide sugar metabolism,Carbon fixation in photosynthetic organisms,Photosynthesis,Biosynthesis of amino acids.Catalase(CAT),Sucrose synthase(SS),Malate dehydrogenases(MDH),Glyceraldehyde phosphate dehydrogenase(GAPDH),Photosystem II reaction center protein L and H were induced by nitrogen stress.The common responsive DEPs mainly involved in Starch and sucrose metabolism,Carbon fixation in photosynthetic organisms,Photosynthesis,Biosynthesis of amino acids,Robisom.The large subunit of Ribisco,Chlorophyll a-b binding protein,Fructose-1,6-bisphosphate aldolase(FBPase),Isocitrate dehydrogenases(ICDH)were induced by ZmPEPC expression and nitrogen stress.These indentified DEPs explained effects of overexpression ZmPEPC on physiological and biochemical changes,and provide new insights to further investigate overexpression ZmPEPC regulation metabolic pathway in transgenic wheat under low nitrogen treatment.