Responses Of Protein Quality Spatial Gradient In Wheat Grain To Nitrogen Topdressing Timing And The Underlying Physiological Mechanisms

Wheat is one of the main cereal crops in China,and it is one of the most popular food for humans.The wheat production has aimed to increase the yield rather than quality so far,and the proportion of mid-gluten wheat is high,resulting in bad quality of both strong-and weak-gluten wheat.Processing quality of wheat flour is determined by protein and wet gluten content to a large extent.Increase the protein content in strong and mid-gluten variety while decrease or maintain the protein content in weak-gluten variety without yield loss is the priority aim in wheat production.It has been observed that protein content largely varies within different layers of endosperm,and this spatial variation differently responds to nitrogen fertilization.Therefore,a research assumption was proposed accordingly:regulate the protein content in endosperm by modifying nitrogen topdressing timing to produce high-protein flour in strong and mid-gluten varieties;concentrate the protein content in outer layers to avoid overproof protein content in weak-gluten varietis.Protein synthesis in wheat grain is regulated by amino acid supplementary and synthesis ability.In this study,Yangmai 16 was grown in the field experiment.Nitrogen was topdressed according to the leaf age to study the response of protein quality and baking quality of grains to N topdressing timing.The results illustrated the transportation pathway and supplying capacity of amino acids and the synthesis capacity of proteins at different positions of the endosperms.The responses of these processes to nitrogen fertilization and the underlying regulatory mechanism was also illustrated.The main results are as follows:1.The quality of grain increased with delaying nitrogen topdressing timing,and the best topdressing stage is at the top first leaf.The contents of protein,protein components and gluten increased with delaying nitrogen topdressing timing,reaching the highest at top 1st leaf or anthesis.Topdressing N at jointing stage showed the highest yield.Therefore,three treatments,topdressed N at the emergence of the top fifth(TL5),top third(TL3)and top first leaf(TL1)were chosen to do the further analysis.Proteomic analysis of wheat flour was conducted by isobaric tag for relative and absolute quantitation(iTRAQ).Collectively,591 proteins classified into 17 molecular functions were identified.In comparison to TL3,significant changes in 50 and 56 proteins were observed in TL5 and TL1,respectively.Amongst the gluten proteins,gamma gliadins and high molecular weight glutenin subunits(HMW-GS)were promptly modified by N topdressing timing,resulting in higher ratio of gliadin/glutenin and HMW-GS/LMW-GS in TL1.In addition,the allergenic potential of the flour and the hardness of grain were altered when delaying N topdressing.2.Topdressing timing modified the grain quality by regulating the amino acid supplement.Delayed nitrogen topdressing up-regulated gene expression related to nitrogen metabolism and protease synthesis in the flag leaf,followed by more free amino acids transported to both the cavity and the endosperm from 7 days after anthesis(DAA)to 13 DAA in TL1.In consistence,TL1 enhanced the conversion between free amino acids in endosperm and upregulated the expression of genes encoding high molecular weight(HMW)and low molecular weight(LMW)subunits and protein disulfide isomerases-like(PDIL)proteins,indicating that the synthesis and folding of glutenin were upregulated by delayed nitrogen topdressing(TL1).As a consequense,the content of glutenin macropolymers(GMP)and glutenin increased with delaying nitrogen topdressing.The results highlight the relationship between N remobilization and final grain protein production and suggest that the nitrogen remobilization processes could be a potential target for improving the quality of wheat grain.3.Protein and baking quality of flour from outer layers of grain was modified promptly by nitrogen topdressing timing.The grains were pearled into nine fractions from the outer layer to the inner layer.The contents of albumin and globulin showed continuous decrement from P1 to P9.Gliadin,glutenin and gluten contents presented a unimodal curve and peaked at the 2nd(P2)or the 3rd(P3)layer from the outmost of grains.The spatial distributions of GMP,HMW-GS and LMW-GS were consistent with storage proteins,presenting as unimodal curves and peaking at P3 or P4 layer.Delaying application of topdressed nitrogen increased contents of gluten proteins in each fraction,especially for layers of the aleurone and the outer endosperm.No difference in gluten index was observed except of the P6 layer.Predating N application showed the opposite regulatory effects.Baking quality of bread made of flour from different pearling fractions was evaluated.The breads made of flour from the P4 fraction showed the best baking quality with the highest volume as well as the best texture and sensory evaluation.The bread made of flour from the P1 fraction showed poor quality because the bran contents were high,which affected the volume,appearance and taste.In addition,topdressing at the emergence of the topmost leaf of the main stem(the flag leaf,TL1)resulted in the highest bread-baking quality traits of volume,sensory score and texture profile analysis indexes with flour from P2 to P4 layers.4.The amino acid transportation pathway changed with grain development,resulting in the spatial distribution formation of protein in grain.During the early stage,free amino acids(FAA)in the cavity are mainly transported via aleurone-outer endosperm.Gradient FAA concentration from aleurone to inner endosperm provides the condition for passive transport.During the late stage,the primary transport pathway for FAA is via transfer cells-inner endosperm driven by amino acid transporter in transfer cells.At the same time,FAA is transported into aleurone directly without further transportation into endosperm due to low FAA concentration in the whole endosperm.The combined analysis of gene expression profiling and amino acids distribution in different endosperm layers was conducted to illustrate that protein gradient formation was restricted by amino acid supplement rather than transcription rate of gluten protein genes.Though the results showed significantly higher content of FAA in aleurone than in endosperm at 11 DAA,we could not conclude its correlation with final protein gradient because of slow protein accumulation before 11 DAA.After 11 DAA,little FAA was detected,making it impossible to compare the FAA content between layers.However,high expression of ASN1 in aleurone after 14 DAA indicated abundant substrate(Asn and Gln)were transported into aleurone,utilised for Asp and Glu synthesis and further used for protein synthesis.From this point of view,the FAA were vital for protein gradient formation.The results above mentioned revealed a precise distinction between different N topdressing timing and provided new perspectives for the application of N regime.The relationship between N remobilization and final grain protein production was highlighted and the result suggested that the nitrogen remobilization processes could be a potential target for improving the quality of wheat grain.The differences of gluten protein distribution in spatial resulting in unique processing quality of different pearling fractions also provide a novel view to improve the quality of bread by adding flour from different fractions of grains.Also,the results help to get a comprehensive insight into protein synthesis and amino acid transport pathways in endosperm and suggest targets for the enhancement of specialty wheat with high-qualified quality.