Mechanism For The Establishment Of Wheat Processing Quality In Response To Nitrogen Levels

Wheat?Triticum aestivum L.?is one of the major cereal crops in the world.Protein and starch is the major component in the wheat endosperm.It provides an important source of vegetable nutrient and energy for a large number of the world's population.As an important agrotechnical practice,N application is arrested to serve the improvement of progressing quality and yield in wheat production.Basis for the effects of nitrogen?N?on the establishment of wheat processing quality are far from clear.Four common wheat cultivars with different quality parameters were investigated responses of grain storage proteins?GSPs?and starch to four N levels using physiological and RNA-seq technologies at two sites over two growing seasons.Besides,an extraction and measurement protocol of amino acid in plant was established using RP-HPLC.Based on the method,the correlation between the export and transformation status of amino acid after anthesis and the accumulation of GSPs was investigated.The main results were as follows.1.Grain protein content,sedimentation volume,gluten content,development time and stability as well as GSPs quantities remarkably increased with increasing N level,whereas gluten index significantly decreased.N level explained 4.2⁵9.2%and 10.4⁸0.0%variability in GSPs fractions and quality parameters,respectively.The remobilization of N absorbed pre-anthesis was significantly enhanced after N application.GSPs fractions and processing quality except GI were only highly and positively correlated with the amount of N remobilized from stem with sheath.N reassimilation in grain was remarkably strengthened by the elevated glutamine synthetase?GS?activity and the upregulation of GSr2.RNA-seq identified 2293 differentially expressed genes?DEGs?in grains at high N level during 10³5 days after anthesis,and 158 DEGs were detected at least two stages.Nineteen KEGG pathways and 94 GO terms were involved in GSPs biosynthesis.The abundances of GSPs transcripts basically increased with the upregulation of transcription factors,such as SPA,DOF,NAM,MADS-box29 and 58,MYB44 and 86.2.Contents of total starch,amylose and amylopection,ratio of amylose to amylopection,accounting for 34.1⁷6.9%variability explained by N levels,significantly decreased with increasing N levels in all cultivars,whereas proportion of B-type SGs increased and size of A-type SGs exerted firstly enlarged and then shortened.Peak 1,Trough 1,Final viscosity and Setback significantly increased by 7.0⁴6.1%at both sites during two growing seasons.Activities of AGPase and GBSS and theirs transcript levels of AGP-L and Wx-DI were inhibited by high N level,while SSS activity and expression level of Sbe1D were improved during middle and late filling stage.Beside,RNA-seq identified 14 KEGG pathways and 71 GO terms might be involved in starch granules deposition in grain.DEGs in carbohydrate metabolism were largely enriched in starch degradation,sugar transport and transcription factors.3.The content of free amino acid in leaves,developing and mature grains were significantly affected by the amount of N,and there existed difference between cultivars.Asp,Ala,Glu,Ser,Lys and Leu were the main amino acids in leaves,Glu,Ala,Asp,Ser,Gln,Met,Pro and Lys in developing grains,and Asp,Ala,Glu,Asn and Met in mature grains.With the increase of N application from 20 kg N/ha to 120 kg N/ha,contents of His,Thr,Tyr,Val,Met,Ile,Leu and Phe in flag leaf of the three cultivars significantly increased by 21.3³65.2%.Contents of Met,Ile,Leu and Phe in 2^nd leaf significantly increased by20.5¹95.6%.Contents of His,Met,Ile,Leu and Trp in developing grains significantly increased by 21.8¹348.7%.However,results of other amino acids varied depending on filling stages and cultivars.The content of total amino acid in leaves highly increased,whereas little difference weas observed in developing grains.Above changes were extremely obvious during the early 14 days after anthesis.The contents of amino acid in leaves were highly correlated with that in developing grains.Besides,the content of Asn decrease by increased nitrogen,while that of Trp increased.The composition of GSPs was significantly influenced by increased nitrogen,whereas no abvious difference was detected in total proteins in mature grain.High correlations were observed between contents of amino acid in developing grains and content of GSPs fractions,especially Trp,Lys,Cys,Tyr,Phe and Ile.4.Key points for extraction and analysis protocol of amino acid in fresh plant tissue were as follows.Fresh sample was ground and extracted 6 h by ddH₂O at-4 ^oC.Equal volume of 20%TCA with supernatant was added to precipitate protein.The supernatant passed through and purified by DOWEX 50W×8H⁺column after adjusting pH to 9.5.Amino acid was derived 90 min by 2,4-dinitrochlorobenzene in Na₂CO₃-NaHCO₃ buffer?pH=9.5?at90 ^oC in dark.Acetonitrile and 0.03 mol/L CH₃COOH-CH₃COONa buffer with 0.15%TEA as B and A mobile solution of HPLC,A and B gradually varied from 87%to 56%?v/v?and from 13%to 44%?v/v?in 40 min,respectively,at 40 ^oC and 360 nm.