Physiological And Molecular Mechanism Of Nitrogen Application In Regulating Maize Grain Development In The Rainfed Loess Plateau

The Loess Plateau is a typical semi-arid agricultural grain-producing area.Less rainfall and the incoordination of nutrients and rainfall often limited crop yields.With the wide application of film fully mulched and double furrow sowing technology,maize has become a new leading crop in this area.Optimizing the application of nitrogen（N）fertilizer to give the nutrients needed for crop growth was an important way to increase production.To understand the physiological and molecular mechanism of N fertilizer application in the development of maize（Zea mays L.）grains,we carried out study relying on the positioning experiment of N fertilizer application.The planting technology is the wide application of film fully mulched and double furrow sowing technology,and the experiment had four N fertilizer levels(N0:no N fertilizer,N1:100 kg·hm^-2,N2:200 kg·hm^-2,N3:N 300 kg·hm^-2)and two N times（T1:1/3base fertilizer+2/3 jointing fertilizer,T2:1/3 base fertilizer+1/3 jointing fertilizer+1/3large bell mouth fertilizer）.We analyzed the effects of N fertilizer application on maize grain yield,yield components,maize growth,and photosynthetic characteristics,and discussed the regulation of plant nutrient transfer,amino acid and starch metabolic enzymes and hormones in maize grain filling,and studied the changes in grain transcriptome during grain filling in maize.The main results were as follows:（1）Appropriate N fertilizer application improved the maize grain filling process,increased the grain weight,and yield.Through the equation simulation,we found that the yield tended to increase when it exceeds N2,but compared with N2,N3 did not significantly increase yield,and the simulation equation also showed that the optimal N application level is around N2.In this experimental design,at the same N rate,the N application period couldn’t significantly affect the yield.N2 increased grain yield and water use efficiency increased by2.8 and 2.5 times compared with N0,respectively.The N agronomic use efficiency was higher than N3 when the N application rate was N2.We simulated the effect of N fertilizer application on grain filling with a logistic equation,and the correlation coefficient of all simulation equations was R²>0.98.In the range of 0-200 kg N hm^-2,grain weight increment achieves maximum grain-filling rate(W_max),maximum grain-filling rate(G_max),active grain-filling period（P）,and average grain filling rate(G_ave)increased with N application.However,occurrence time of maximal grain-filling rate(T_max)decreased as the amount of N increased.If they exceeded N2,G_max,W_max,G_ave and P did not increase significantly.Correlation analysis showed that 100-grains weight was significantly positively correlated with W_max,G_max,and Gave.（2）Reasonable N fertilizer application promoted the growth of maize and the distribution of dry matter and N matter.N application significantly increased the plant height,leaf area index and dry matter accumulation of maize.When the N application rate exceeded N2,there was no significant difference in the indicators.The N application period had no significant effect on the plant height and leaf area index of maize.Compared with N0,N2 significantly increases the leaf area index,which was about 1.5-2 times higher than that of N0.N2 and N3not only increased dry matter accumulation,but also increased the distribution of dry matter in the grain,and the distribution ratio accounts for about 40-55%of the total dry matter accumulation.N2 and N3 significantly increased the N accumulation in various organs at the flowering and mature stages,and the proportion of post-flowering N uptake to the total N content in the mature stage was higher under N0 treatment.Under different N fertilizer,the total N transfer rate,N transfer rate of the stem,and N transfer rate of the leaves were 40.38-68.14%,16.85-71.20%,and 46.12-69.57%,respectively.The contribution of N re-transport to grain was higher in N2 and N3 than that of N0 in 2019 and 2020.（3）Appropriate N fertilizer application optimized the antioxidant system,which in turn improved the photosynthetic capacity and helped grain filling.In the range of 0-200 kg N hm^-2,the activities of superoxide dismutase,catalase,peroxidase and the soluble protein content increased with the increase of N application level,and there was no significant difference between N2 and N3.The N application period had no significant impact on the above indicators.Higher antioxidant enzymes delayed the decomposition of chlorophyll in leaves after flowering and maintain higher photosynthetic capacity.During the flowering period,the chlorophyll content of N2 was 1.78-2.04 times higher than that of N0.About 50 days after flowering,the chlorophyll content of N2 was 2.34-3.11 times higher than that of N0.Compared with N0,N2 and N3 increased the net photosynthetic rate and transpiration rate of leaves from flowering to about 50 days after flowering.（4）The optimization of the filling process under N fertilizer was regulated by amino acid and starch metabolic enzymes and hormones.Under different N fertilization rate,the sucrose content of grains at the filling stage increased first and then decreased after flowering.N0 and N1 delayed the peak time of sucrose and inhibited the accumulation of sucrose.Starch accumulated faster during flowering at 28 days and slowed down in the later period.N fertilizer significantly affected the activity of amino acid and starch metabolic enzymes in grains.N2 and N3 significantly increased the activities of sucrose phosphate synthase and sucrose synthase and reached the maximum value at 14 days after flowering.The activities of ADPG pyrophosphorylase and starch synthase were significantly reduced under N0,and starch synthase decreased significantly after 21 days after flowering.Glutamine synthetase and glutamate synthetase have a similar trend of change,and the enzymes reached the maximum at 21 days after flowering,then decreased.N2 and N3 significantly increased their activity compared to N0.The N application period had no significant effect on amino acid and starch metabolic enzymes.The contents of auxin（IAA）,abscisic acid（ABA）,and cytokinin（CTK）in the grains at the filling stage increased first and then decreased,and there was no significant difference in the time of N application for various hormones.Compared with N0,N2 and N3 significantly increased the contents of IAA and CTK,but the difference between N2 and N3 was not significant.The changing trend of ABA content in 2019 and 2020was similar,reaching a maximum value at 21 days after flowering,and the change of ABA in2019 was smaller than that in 2020 at 21 days after flowering.The content of gibberellin（GA）continued to decrease with the progress of the filling period,and N2 and N3 significantly increased the content of GA compared with N0.Regression analysis showed that the contents of starch and protein had significant effects on grains at 0-35 days after flowering,while the contents of starch and protein were affected by their metabolic enzymes;there was a significant positive correlation between hormones and grain weight from 0 to 35 days after flowering.（5）Transcriptome analysis found that 3160 genes were differentially expressed under the N0and N2（N2T1）.These differentially expressed genes were mainly enriched in carbon metabolism,amino acid metabolism and secondary metabolism.Through KEGG analysis,in carbon metabolism,N0 up-regulated genes enriched in“glycolysis/gluconeogenesis”and“tricarboxylic acid cycle”（TCA cycle）,while the genes involved in“starch and sucrose metabolism”were down-regulated.In the absence of N,the genes involved in“phenylalanine,tyrosine and tryptophan synthesis”were down-regulated,while the genes involved in“degradation of glutamate and aspartate”were up-regulated,resulting in increasing amino acid content in maize grains.At the same time,the metabolism of these amino acids provided substrates for the synthesis of hormones and secondary metabolites.Notably,in secondary metabolism,N0 up-regulated genes for“phenylpropanoid metabolism”and“flavonoid biosynthesis”,enhancing the synthesis of secondary metabolites such as lignin and flavonoids.Furthermore,under different N conditions,lnc RNAs affected genes mainly through trans-regulatory functions.These results showed that N0 was not conducive to the accumulation of sucrose,starch,and amino acids,at the same time,it promoted the degradation of amino acids,and provided substrates for the synthesis of purine,pyrimidine,and secondary metabolites,finally inhibited grain development.To sum up,reasonable N fertilizer application optimized the antioxidant system in maize,delayed leaf senescence,thereby increasing the net photosynthetic rate and transpiration rate after flowering,and promoting the accumulation of dry matter;higher N accumulation,transportation and distribution in the vegetative organs promoted the grain filling;at the same time,it promoted the hormone balance in grains,improved the activity of amino acid and starch related enzymes in the grain,promoted the synthesis of starch and protein,and finally increased the grain weight.In the case of N deficiency,more resources were allocated to the secondary metabolite synthesis pathway,which inhibited the synthesis of amino acids and starch in the grain,and inhibited the development of the grain.