Effects Of Nitrogen Rates On Nitrogen Assimilation,Remobilization To Wheat Grain And The Underlying Physiological And Molecular Mechanisms In The Semi-Arid Loess Plateau Zechariah Effah

To examine the effects of nitrogen fertilization rates on nitrogen assimilation,remobilization and its underlying physiological and molecular mechanisms to the grain of spring wheat in the Semi–arid Loess Plateau,the present study was carried out based on a long-term nitrogen fertilization rates field experiment on wheat located at the Rainfed Agricultural Experimental Station（35°28′N,104°44′E）of the Gansu Agricultural University,Dingxi,Gansu Province,P.R.of China.The field experiment was initiated in 2003;however,this thesis reports the experimental data for the 2019,2020 and 2021 cropping seasons.The experiment was laid in a randomized complete block design with three replications,the treatments consisted of five N fertilizer（urea）application rates:0,52.5,105,157.5,and 210 kg ha^-1,designated N1,N2,N3,N4,and N5,respectively.The study’s key findings are summarized as follows:1.N fertilization significantly increased photosynthetic parameters,N metabolite enzymes and N accumulation in various organs at all growth stages which directly affected grain yield.N3produced optimum total dry matter(5407 kg ha^-1),1000 grain weight（39.7 g）,grain yield(2.64 t ha^-1),and protein content（13.97%）.When the N rate was increased from 105 to 210 kg ha^-1,wheat grain yield did not differ significantly.Grain yield and 1000 grain weight（TGW）were consistently and positively associated with Pn,gs,and Tr at all the post-anthesis developmental stages.Higher N rates（N3 to N5）maintained higher photosynthetic capacity and lower intercellular CO₂ content which influenced dry matter accumulation and grain yield by improving the photosynthetic process.The nitrate reductase（NR）,glutamine synthetase（GS）,and glutamate dehydrogenase（GDH）activities of the flag leaves were lower in control（N1）than the N application（N2–N5）at all studied stages from anthesis to maturity.At anthesis,N3 enhanced NR and GS activity by 66%and 43%,respectively,compared to N1,resulting in higher grain yield and protein content.In general,nitrogen fertilization up to the optimal rate（N3）resulted in the highest yield components,photosynthetic activities,and N metabolic enzyme activities,while increasing the nitrogen dose had a negative impact on all yield,physiological,and quality indicators.2.Long term and excessive nitrogen fertilization resulted in lower NUE values at maturity,an indication that excessive and continuous application of N fertilizer reduces plants’ability to utilize nutrients under field conditions.N fertilizer rate had a significant effect on N uptake by grain and straw,signifying enhanced biomass yield and N concentration of the N treatments when compared to the control.Wheat N uptake was between 7.88–29.27 kg ha^–1 for straw and 41.85–95.27 kg ha^–1 for grain,indicating that different fertilizer rates affect nitrogen usage in spring wheat and that increasing nitrogen rates decreased plant N uptake and reduced nitrogen use efficiency.Increasing N application rates improved both grain(17.91–19.66 g kg^-1)and straw(3.12–5.29 g kg^-1)N concentration of spring wheat in our present field experiment,with the best effect observed in N3in both growing seasons 2019 and 2020.Wheat’s total grain N derived from ¹⁵N fertilizer（44.19–73.84%）increased as the N application rate increased.Total straw N derived from fertilizer ranged between 7.14-15.80%.Wheat grain N derived from soil N ranged between 21.43-28.57%,while that of straw N derived from soil N ranged between 6.78-13.47%.The ear maintained a higher percentage of total plant nitrogen than leaves and stems during the grain filling stage（14DAA）indicating that the ear can serve as both a nitrogen source and a sink during the grain filling process.Increasing the rate of applied N increased post-anthesis N losses in spring wheat.However,N3compared to the other treatments（N2,N4,and N5）recorded the least N loss,indicating that at this rate the plant can remobilize a greater portion of the applied N.3.N application caused reprogramming of plant hormone signal transduction,photosynthesis and several other biosynthetic pathways in regulating leaf senescence and N remobilization,providing valuable information for gene functional and pathway engineering to improve NUE by manipulating leaf senescence and N remobilization.The prominent pathway identified was plant hormone signal transduction（map04075）.A total of 4485 DEGs comprising 2090 and 2395 down-and up-regulated genes,respectively,were detected in no N＿vs＿low N whereas a total of 4627genes were differentially expressed in no N relative to high N with 2183 down-regulated and 2444up-regulated signifying that plants respond actively to changes in their environment including variations in nutrient availability by eliciting significant transcriptional reprogramming to sustain and complete their lifecycle.The genes detected in the carotenoid biosynthetic pathway initiated by ABA（PYR/PYL,PP2C,Sn RK2 and ABF）expressed higher in either no or lower N than high N.This study detected 47 TF families regulating gene expression.Predominant among these TFs were,AUX/IAA,b HLH,AP2/ERF,b ZIP,MYB,NAC and WRKY,which have been revealed and confirmed to participate in leaf senescence and N remobilization.Of these,WRKY,NAC,AP2/ERF and b ZIP TF genes were mostly expressed higher in either no or low N groups,suggesting that they are positive regulators of leaf senescence.The differential application of N also altered several pathways;including plant hormone signal transduction,mitogen-activated protein kinase signaling pathway-plant,photosynthesis,phenylpropanoid biosynthesis and ATP-binding cassette transporters.Jasmonic acid,abscisic acid,salicylic acid and brassinosteroid related genes promoted leaf senescence in No N or low N,whereas auxin,gibberellin acid and cytokinins genes inhibited leaf senescence in high N.Contrary to the reports that Auxin,GA and cytokinins related genes are inhibitors of leaf senescence,the present study found inconsistent expression of these in either of the three（no,low and high N）situations.4.Stressful plants adopt the production of secondary metabolites such as flavonols,anthocyanins and catechins as survival mechanisms and that under no N,tend to spend much on energy for survival and maintenance of their physiological role.Two compounds（Pipecolic acid and Acarbose）accumulated higher in low N than in no N,suggesting their possible involvement in the delayed senescence and higher N remobilization in low N compared to no N increasing the photosynthetic capacity after anthesis,and promoting the accumulation of dry matter thereby increasing grain yield and its attributes.Astragalin/Kaempferol 3-O-glucoside,a flavonoid compound,accumulated nearly 60%higher in no N condition than in high N condition.In addition,1-O-Caffeoylglucose a well-known high-energy glucose ester potentially utilized as a donor molecule during the formation of diverse hydroxycinnamic acid O-esters in plants,accumulated79.50%and 387.91%higher in no N condition plants than low N and high N respectively.In conclusion,N fertilizer application increased the grain yield,dry matter accumulation and protein content of spring wheat in the semi-arid loess plateau.The plants fertilized with higher N rates had higher NR,GS,and GDH activities in the flag leave,demonstrating that these enzymes are closely linked with N metabolism in wheat plant,which might be a reason for higher grain yield and protein content.The genes detected in the carotenoid biosynthetic pathway initiated by ABA（PYR/PYL,PP2C,Sn RK2,and ABF）expressed higher in either no or low N than high N.These pathways could be targeted for possible pathway engineering to delay leaf senescence in low N conditions to minimize N application in wheat while preventing or reducing the environmental damages.Acarbose negatively correlated with Diethanolamine,4-Methoxyphenyl beta-D-glucopyranoside and Rhoifolin.These strong negative correlated compounds could be targeted to manipulate Acarbose and Rhoifolin to increase yield under no N condition and regulate leaf senescence.The N3(105 kg h^-1)produced the highest grain N content at maturity,which affects grain protein content.Therefore,increasing N rates beyond this optimum quantity only promotes vegetation development and may not increase grain yield in the study area.