Shoumin Zhen,Xiong Deng,Xuexin Xu, Nnnn Liu, Dong Zhu,Zhimin Wng,*,Yueming Yn,b,**

aCollege of Life Science,Capital Normal University,Beijing,China

bHubei Collaborative Innovation Center for Grain Industry(HCICGI),Yangtze University, 434025 Jingzhou,China

cInstitute of Scientific and Technical Information of China,Beijing 100038,China

dCollege of Agronomy and Biotechnology,China Agricultural University,100081 Beijing,China

Keywords:Bread wheat High nitrogen Gliadins Glutenins RP-UPLC 2D-DIGE

ABSTRACT Nitrogen (N), a macronutrient essential for plant growth and development, is needed for biosynthesis of protein and starch,which affect grain yield and quality.Application of high-N fertilizer increases plant growth, grain yield, and flour quality. In this study, we performed the first comparative analysis of gliadin and glutenin subproteomes during kernel development in the elite Chinese wheat cultivar Zhongmai 175 under high-N conditions by reversed-phase ultra-performance liquid chromatography and twodimensional difference gel electrophoresis (2D-DIGE). Application of high-N fertilizer led to significant increases in gluten macropolymer content,total gliadin and glutenin content,and the accumulation of individual storage protein components. Of 126 differentially accumulated proteins(DAPs)induced by high-N conditions,24 gliadins,12 high-molecularweight glutenins, and 27 low-molecular-weight glutenins were significantly upregulated.DAPs during five kernel developmental stages displayed multiple patterns of accumulation.In particular, gliadins and glutenins showed respectively five and six accumulation patterns. The accumulation of storage proteins under high-N conditions may lead to improved dough properties and bread quality.

1. Introduction

Wheat(Triticum aestivum L.)is one of the three most important grain crops and is a staple food and major source of protein worldwide. Indeed, wheat provides ＞20% of the calories consumed worldwide [1]. Mature wheat kernels contain three major components that are important for human nutrition: starch, proteins, and cell-wall polysaccharides [2].Starch is the major storage component in the endosperm of wheat kernels, and increased starch content is largely responsible for increases in grain yield [2]. The amount of N fertilizer applied and the timing of its application strongly influence wheat yield and quality. Generally, application of high-N fertilizer increases storage protein content, endosperm protein body quantity, and flour-processing quality by altering the expression levels of genes that encode components of protein biosynthesis pathways[3].

The proteins in wheat kernels are classified as albumins,globulins,gliadins,and glutenins.Albumins and globulins are more abundant in the essential amino acids lysine, tryptophan, and methionine. Gliadins, the major components of wheat storage proteins, are classified into α/β-, γ-, and ωgliadins [4]. Gliadins play important roles in determining the extensibility of gluten dough. Glutenins, which comprise high- and low-molecular-weight glutenin subunits (HMW-GS and LMW-GS),account for 40%of all wheat grain proteins[5].Although HMW-GS are minor endosperm proteins and comprise 10% of the total seed-storage proteins [6,7], they play key roles in breadmaking and other flour-processing operations owing to the formation of networks in dough caused by gluten polymerization. LMW-GS content is closely associated with the extensibility and strength of dough[8-12].In particular, HMW-GS form disulfide bonds with LMW-GS and gliadins, resulting in the generation of glutenin macropolymers (GMPs), which are insoluble in sodium dodecyl sulfate(SDS)and strongly influence bread quality[13].

Storage proteins are encoded by multiple gene families and exhibit extensive allelic variation [14,15]. Numerous glutenin and gliadin genes are located in a few major chromosomal regions, suggesting that these genes are physically closely linked or clustered [16]. Genetic studies have confirmed that all ω-gliadins, most γ-gliadins, and a few β-gliadins are encoded by the Gli-1 loci on the short arms of homoeologous group-1 chromosomes, tightly linked to the LMW-GSencoding Glu-3 loci. The production of α-gliadins, most βgliadins,and some γ-gliadins is controlled by genes in the Gli-2 loci on homoeologous chromosomes 6A, 6B, and 6D [17,18].It is estimated that Triticum aestivum cv. Chinese Spring contains 15-40 γ-gliadin genes [19]. HMW-GS are encoded by two tightly linked genes (x- and y-type) at the Glu-A1, Glu-B1,and Glu-D1 loci on the long arms of chromosomes 1A,1B,and 1D,whereas LMW-GS are encoded by genes at the Glu-3 loci on the short arms of chromosomes 1A, 1B, and 1D. The copy numbers of LMW-GS genes in hexaploid wheat reportedly vary from 10 to 15 [20] or from 35 to 40 [19]. These proteins contain regions of highly repetitive sequences, which interfere with their separation, identification, and quantitation. Various methods have been developed to separate and characterize wheat storage proteins, including size-exclusion and reversed-phase high-performance liquid chromatography (SE-HPLC and RP-HPLC) [21,22],reversed-phase ultra-performance liquid chromatography(RP-UPLC) [23-25], high-performance capillary electrophoresis (HPCE) [26], two-dimensional electrophoresis (2-DE)[27,28], and mass spectrometry (MS) [29]. These methods have facilitated investigation of the gliadin and glutenin subproteomes.

Wheat storage proteins gradually accumulate during grain development,a process in which many genes participate[30].Environmental factors affect the composition and relative proportions of gliadins and glutenins, strongly influencing flour quality [31,32]. Gluten strength is influenced by N application, and increased N supply is leads to increased quantities of all gliadin and glutenin-containing protein components [33]. N and S fertilizers applied by foliar spray at anthesis modulate simultaneously the synthesis of storage proteins and the degree of polymerization,in turn influencing the mixing properties of dough [34]. Size-exclusion fast protein liquid chromatography (SE-FPLC) and proteomics analyses showed that the application of fertilizer containing both N and S affected the proportions of HMW polymeric proteins, resulting in changes in protein composition and quality [35]. N treatment increases the content of storage proteins, the quantity of endosperm protein bodies, and processing quality by altering the expression levels of genes involved in the production of storage proteins [3]. Variable N and S supplies modulated the expression of genes involved in transport and metabolism, altering the concentration of free amino acids [36]. Metabolomic analysis during kernel development showed that application of high-N fertilizer promoted kernel development and increased dough quality [37]. A comparative proteomic analysis of developing wheat kernels revealed that application of high-N fertilizer increased the biosynthesis of storage proteins and starch [38]. However,most studies have focused mainly on the effects of N or S on grain yield, nutrition quality, and protein composition. Indepth proteomic studies of the subproteomes of gliadins and glutenins under high-N conditions and their relationships with gluten quality are lacking.

In this study,we used RP-UPLC and 2D-DIGE to perform the first comparative analysis of gliadin and glutenin subproteomes during wheat grain development under high-N conditions.We assessed the effect of high-N fertilizer on the synthesis and accumulation of storage proteins and their components and investigated the molecular mechanisms that modulate wheat quality under high-N conditions.

2. Materials and methods

2.1. Plant materials, field trials, and high-N treatment

The elite Chinese winter wheat cultivar Zhongmai 175, with high yield and N use efficiency [39] and cultivated widely in the north of China, was used in this study. Field trials were performed during the 2016-2017 growing season. The field experimental design included a high N-fertilizer group,receiving 240 kg ha-1N fertilizer (urea) and a control group with 180 kg ha-1, based on recent studies [38,39]. Each group contained three biological replicates and each plot occupied 20 m2.Plants were marked at 15,20,25,30 days post anthesis(DPA) and maturity periods and samples were collected following our recent study[38].

2.2. GMP content determined by SE-HPLC

GMPs were extracted and separated by SE-HPLC (Agilent,California, USA) following a recent report [27]. An Agilent Bio Sec-5 column (Agilent, California, USA) with 5 μm diameter was used and 0.05 mol L-1phosphate buffer saline(PBS)with 0.1%SDS was used as the mobile phase.

2.3. Gliadin and glutenin accumulation determination

Gliadins and glutenins were extracted and separated by RPUPLC following standard methods [23,25]. RP-UPLC was performed on an Acquity UPLC instrument (Waters Corp,Milford, USA), using a Waters 300SB C18 column (1.7 μm)(Waters Corp,Milford,USA).

2.4. Storage protein extraction, 2D-DIGE, and image analysis

Kernel gliadins and glutenins were extracted and protein concentration was determined with a 2-D Quant Kit (General Electric Company,Fairfield,USA)based on a reported method[27].

DAPs at 25 DPA between high N-fertilizer and control were identified by 2D-DIGE. The experimental design for 2D-DIGE analysis, protein separation, and image analysis were based on previous studies[40,41].Protein samples were labeled with Cy2, Cy3, and Cy5, separately. To facilitate tandem mass spectrometry analysis, 2-DE was used to separate DAP spots identified by 2D-DIGE and protein dynamic accumulated patterns during kernel development were determined[42,43].

2.5. Protein identification using MALDI-TOF/TOF-MS

Protein identification was based on our previous study [42],MS/MS spectra were obtained using ABI 4800 Proteomics Analyzer MALDI-TOF/TOF (Applied Biosystems, Carlsbad,USA). The protein score C. I.% and total ion score C. I.% were both set to ＞95% and the significance threshold P ＜0.05 for MS/MS was based on a previous report[44].

2.6. Statistical analysis

Principal component analysis(PCA)is a method that finds the main variances and reveals hidden structure present in a dataset. PCA was performed using SPSS 19.0 software (IBM,Armonk, USA). Abundance changes of gliadin and glutenin spots were determined by ImageMaster 2D Platinum Software Version 7.0(Amersham Biosciences,General Electric Company,Fairfield, USA). The DAP spots were subjected to hierarchical clustering analysis by Cluster 3.0 software(Stanford University,Palo Alto, USA) following Eisen et al. [45]. Euclidean distance was used as a measure of similarity and the hierarchical clusters were assembled using the complete linkage clustering method. The clustering results were visualized with TreeView software(Oracle,Beijing,China).

3. Results

3.1. Kernel development and GMP changes under high-N conditions

Changes in kernel morphology and weight in the high-N and control groups were similar during five developmental stages(Fig. 1-A, B). Kernel fresh weight did not differ significantly between these two groups.Application of high-N fertilizer led to a slight increase in kernel fresh weight, which increased rapidly from 20 to 30 DPA in both groups(Fig.1-B).Kernel GMP content determined by SE-HPLC gradually increased in both groups, and application of high-N fertilizer resulted in a significant increase in the GMP content,particularly at 30 DPA(18.75%) and at maturity (11.95%) (Fig. 1-C). Our results showed that application of high-N fertilizer increased the GMP content of grain and thus may improve mixing properties and breadmaking quality under high-N conditions.

3.2. Accumulation patterns of gliadins and glutenins in developing kernels under high-N conditions

In general, storage proteins gradually accumulated from 15 DPA to maturity in both control and high-N groups.However,the accumulation patterns in the high-N group differed significantly from those in the CK group(Figs.2,3, 4).

By RP-UPLC, α/β-, γ-, and ω-gliadins were eluted at 5-10,10-15,and 0-5 min,respectively(Fig.2-A).All of these gliadins gradually accumulated after 15 DPA, and their levels peaked at grain maturity. Accumulation was rapid from 25 DPA to maturity(Fig.2-B).The changes in their quantities are shown in Fig.2-C-E.The levels of accumulation of total,α/β-,γ-,and ω-gliadins were greater in the high-N than in the control group,particularly at 30 DPA and at grain maturity.

The patterns of accumulation of total glutenins,HMW-GS,and LMW-GS by RP-UPLC are shown in Figs. 3 and 4. The patterns of accumulation of total glutenins, HMW-GS, and LMW-GS in the control and high-N groups were similar to those of gliadins during grain development, with rapid accumulation from 25 DPA to maturity (Fig. 3-A). Under high-N conditions,the accumulation of total glutenins markedly increased, particularly at 25 and 30 DPA (Fig. 3-B). The total content of HMW-GS and LMW-GS and that of individual HMW-GSs showed a similar trend (Fig. 4-A, B). In particular,the levels of the four HMW-GSs (1Bx7 + 1By9 and 1Dx2 + 1 Dy12)showed a highly increase at grain maturity under high-N conditions(Fig.4-C-F).

Fig.2-Gliadin accumulation during kernel development in Zhongmai 175 under high-N and normal conditions as revealed by RP-UPLC.(A)RP-UPLC separation.(B)Total gliadins.(C)α/β-gliadins.(D)γ-gliadins.(E)ω-gliadins.Error bars indicate standard error of the mean of three biological replicates.Significant differences from CK group by independent Student's t-tests:*P ＜0.05, **P ＜0.01.

Fig.3-Glutenin accumulation during kernel development of Zhongmai 175 under high-N and normal conditions as revealed by RP-UPLC.(A)RP-UPLC separation.(B)Total glutenins.HMW-GS(including 1Bx7,1By9,1Dx2,and 1Dy12)are indicated in(A).Error bars indicate standard error of the mean of three biological replicates. Significant differences from the CK group by independent Student's t-tests:*P ＜0.05,**P ＜0.01.

Fig.4-Accumulation of total HMW-GS(A)and LMW-GS(B)and of individual HMW-GS 1Bx7(C),1By9(D),1Dx2(E),and 1Dy12(F)during kernel development in Zhongmai 175 under high-N and normal conditions as revealed by RP-UPLC.Error bars indicate standard error of the mean of three biological replicates. Significant differences compared to the CK group by independent Student's t-tests: *P ＜0.05,**P ＜0.01.

3.3. Identification of DAPs and principal component analysis(PCA)

DAPs in gliadins and glutenins at 25 DPA were determined by 2D-DIGE (Table S1, Fig. 5), and changes in their accumulation at five kernel developmental stages were analyzed by 2-DE to assess the effect of high-N conditions on the grain subproteomes (Figs. S1, S2, Tables S2, S3). The analysis resulted in identification of 46 gliadin (Fig. 5-A) and 80 glutenin (including 18 HMW-GS and 62 LMW-GS, Fig. 5-B)DAPs across the five developmental stages(Tables 1,2,Fig.5-A).

A PCA was performed to estimate the contributions of the gliadin and glutenin DAPs to total variance and to identify proteins responsible for these contributions during kernel development under normal and high-N conditions (Fig. 6).

Fig.5-2-DIGE map of the gliadin and glutenin components of Zhongmai 175 under high-N conditions.DAPs of(A)gliadins and(B)glutenins are marked on the 2-DIGE maps.IPG strips(pH 6-11, 18 cm)were used to separate the DAPs.

The gliadin subproteome was fully separated in the PCA image (Fig. 6-A). The first principal component (PC1), which had the greatest variance (35.51%) across the dataset, separated the samples according to kernel developmental stage.Three clusters were included in PC1: 15-25 DPA, 30 DPA, and maturity. This clustering is in accord with the three distinct phases of wheat kernel development: cell division and differentiation, grain filling, and desiccation/maturation [46].Thus, differences in the proteome among the developmental periods were greater than those induced by application of high-N fertilizer. In the high-N group, more significant differences in the gliadin subproteome occurred at 15-30 DPA and maturity (Fig. 5-A). The gliadin DAP spots 16, 30, 43,and 50 made greater contributions to total variance under the high-N conditions (Fig.6-B).

PCA of the glutenin subproteome revealed that PC1 accounted for the greatest variance (64.69%) among the developmental periods. Application of high-N fertilizer induced greater differences in the glutenin subproteome at 25-30 DPA than during the other developmental stages(Fig.6-C). Glutenin DAPs 24, 80, 97, 98, 104, and 159 made greater contributions than the other DAPs to total variance (Fig.6-D).Moreover, DAPs 97, 98, 104, and 159 were identified as HMWGS(Table 2),suggesting that they are important determinants of bread quality under high-N conditions.

3.4. Effect of application of high-N fertilizer on gliadin and glutenin subproteomes

The levels of accumulation of 46 gliadin DAPs from 15 DPA to grain maturity are shown in Table S2, and the patterns of changes in their accumulation are displayed as heat maps in Fig. 7-A. Application of high-N fertilizer markedly altered the gliadin subproteome during the five stages of grain development. The DPAs displayed five patterns of accumulation (I to V). Pattern I comprised nine DAPs (19.6%) that showed a down-up-down accumulation trend, with peak accumulation at 30 DPA. Pattern II comprised seven DAPs (15.2%) that displayed an up-down accumulation trend, with a peak at 20 or 25 DPA. Pattern III comprised six DAPs (13%) that displayeda decreasing accumulation trend, with peak accumulation at 15, 20, or 25 DPA. Pattern IV comprised five DAPs(10.9%)that showed a down-up-down pattern of accumulation. Pattern V comprised 19 proteins (41.3%) that were upregulated, with a peak at 30 DPA or maturity.

Table 1-Accumulation patterns of gliadin protein spots during kernel development in response to high N fertilization.

Table 2-Accumulation patterns of glutenin protein spots during kernel development in response to high N fertilization.

Among the 46 DAPs, 24 were upregulated compared with the control group at one or more of the five kernel developmental periods (Table 1, red color), suggesting increased gliadin content and improved bread quality. MALDI-TOF/TOF-MS analysis of these DAPs resulted in identification with high confidence of 13 proteins, including 3 gliadins(Table S4).

The HMW-GS identified in Zhongmai 175 included Null at Glu-A1, 1Bx7 + 1By9 at Glu-B1, and 1Dx2 + 1Dy12 at Glu-D1.Resolution by 2-DE separated 1Bx7, 1By9, and 1Dy12 into respectively five (104, 106, 145, 159, and 390), five (91, 98, 141,158, and 358), and eight (86, 88, 89, 90, 93, 94, 97, and 157)protein spots. Thus, totals of 18 HMW-GS and 62 LMW-GS spots were induced by application of high-N fertilizer(Table 2,Fig. 7-B). Analysis of the LMW-GS DAPs by MALDI-TOF/TOFMS resulted in identification with high confidence of 15 proteins (Table S5). Among the glutenin DAPs identified, 12(66.67%) HMW-GS and 27 (43.55%) LMW-GS were upregulated during kernel development compared with the control group.

The accumulation levels of 80 glutenin DAPs are shown in Table S3 and displayed as a heat map in Fig.7-B.The heat map revealed six accumulation patterns (I to VI) under high-N conditions. Pattern I comprised nine DAPs (11.25%) that showed an up-down trend during grain development, with a peak at 20 or 25 DPA.Pattern II comprised seven DAPs(8.75%)that showed a down-up-down trend,with peaks at 20,25,and 30 DPA.Pattern III comprised seven DAPs(8.75%)that showed an up-down-up-down trend with peaks at 15 and 20 DPA.Pattern IV comprised 40 DAPs (50%) that were upregulated and showed peaks at 30 DPA and maturity. Pattern V comprised 9 DAPs (11.25%) that showed a down-up trend,and pattern VI comprised 8 DAPs(10%)that were upregulated and showed a peak at maturity. These results indicate that application of high-N fertilizer increased glutenin accumulation, particularly at the middle and late stages of kernel development.

4. Discussion

4.1. Influence of high-N fertilizer on the accumulation of gliadin and glutenin components

Application of N fertilizer increases wheat yield and quality.Study of the response of glutenin polymerization to increased N fertilization in various wheat cultivars have shown that N treatment increased not only grain yield, protein content,sedimentation, and gluten content but also insoluble GMP,soluble glutenin polymer,and total glutenin concentration in flour, and the size distribution of glutenin polymers [47].Application of N fertilizer increased the synthesis and accumulation of HMW-GS, but its effect on HMW-GS expression differed among the types of wheat. Specifically, N fertilization showed a greater effect on medium-gluten than on strong-gluten wheat [48]. RP-HPLC indicated that the contents of total protein and of protein components increased and then decreased as the amount of N applied increased.The contents of protein components were higher, and processing quality was improved by application of 240 kg ha-1N fertilizer(urea), but the synthesis and accumulation of HMW-GS were reduced by application of excess N fertilizer [49].Transcriptomic and proteomic analyses and light microscopy showed that N treatment increased the contents of storage proteins,quantity of endosperm protein bodies,and processing quality by altering the expression levels of genes involved in the production of storage proteins [3]. The expression of a novel family of γ-gliadin genes was reportedly regulated by N supply in developing wheat kernels [50]. Increasing the level of N fertilizer increased the ω-gliadin transcript levels and the proportions of ω-5 gliadins[51].In the present study,RP-UPLC and 2D-DIGE analyses of the gliadin and glutenin subproteomes revealed that application of a large quantity of high-N fertilizer (240 kg ha-1urea) highly increased the accumulation of gliadin and glutenin components and the contents of storage proteins(Tables 1,2,Figs.2,3),suggesting improved dough viscoelasticity and bread quality.

4.2. The molecular mechanisms of high-N fertilizer effects on the synthesis and accumulation of wheat storage proteins

The molecular mechanisms by which high-N fertilizers modulate the accumulation and polymerization of grain storage proteins (GSP) have been investigated. A study [36] involving measurements of GSP, targeted metabolites, and transcript contents under a variety of N and S conditions showed that allometric allocation of N and S to GSPs was regulated at the transcriptional level and that several transcription factors(e.g.,HMG, AP2-EREBP, MYBS3, FUSCA3, and MCB1) were involved.Furthermore, changes in the expression of genes involved in transport and metabolism modulated the concentrations of free amino acids in response to plant nutritional status, leading to altered GSP accumulation. A study [52] of the nuclear and albumin-globulin subproteomes of einkorn wheat found that post-anthesis N supply resulted in the activation of amino acid metabolism at the expense of carbohydrate metabolism and transport processes, including nucleocytoplasmic transit. The abundance of a cysteine synthase increased considerably under high-N conditions, altering the balance between S-containing(methionine and cysteine) and other amino acids. Proteins participating in transport, e.g., importin, which mediates the translocation of nuclear localization sequence(NLS)-containing proteins from the cytoplasm to the nucleus, were more abundant under high-N conditions [53]. Communication between the cytoplasm and nucleus is important for plant growth,development, and environmental adaptation. The levels of several DNA-binding nuclear proteins were increased by N and S supply,suggesting that transcription is activated under high-N and -S conditions [54]. High N availability reportedly [55]prompted participation of glutamine in biological processes.Peptidyl-prolylcis-transisomerase (PPIase) was SUMOylated by small ubiquitin-related modifier 1, and this interaction was promoted under high-N conditions to facilitate protein polymerization. Luminal-binding protein 2 in the endoplasmic reticulum played a similar role to PPIase in protein aggregation under high-N conditions[55].Growth regulating factor 4(GRF4),a transcriptional regulator of multiple genes involved in N metabolism, increased carbon and N assimilation,biomass, leaf and stem width, grain yield, and N-use efficiency[56].

Fig.8- Putative pathway by which high-N fertilizer application increases wheat storage protein synthesis and accumulation and bread quality.PDC,pyruvate dehydrogenase complex;SPS,sucrose phosphate synthase;PGAM,2,3-bisphosphoglycerateindependent phosphoglycerate mutase;AGPase,ADP glucose pyrophosphorylase;MDH,malate dehydrogenase;Glu,Glutamic acid;Gln:Glutamine;Ala,Alanine;Fru,Fructose;Suc,Sucrose.Red arrows indicate upregulation of transcription factors and increase in protein phosphorylation levels and metabolites.

Our recent studies of proteomics, phosphoproteomics, and metabolomics have provided new evidence for understanding the molecular mechanisms by which high N affects the synthesis of wheat storage proteins and bread quality. Phosphorylation synergistically modulated the activities of key enzymes and proteins involved in the response to application of N fertilizer and accelerated starch and protein biosynthesis and accumulation[57].Application of high-N fertilizer increased the accumulation of metabolites involved in starch and storage protein synthesis[37].The levels of several proteins responsive to high-N conditions involved in N and protein metabolism and protein folding were greatly increased by application of high-N fertilizer,increasing grain yield and the bread quality[38].

Based on the results reported here and previously, we propose mechanisms by which application of high-N fertilizer modulates the synthesis and accumulation of wheat storage proteins and bread quality (Fig. 8). First, moderately high N fertilization induces the expression of transcription factors with N-responsive elements, such as GRF4 and MYBS3,accelerating the uptake and assimilation of ammonium and organic N. Second, proteins or enzymes such as PGAM and MDH involved in protein synthesis are phosphorylated under high-N conditions,increasing their activities and accelerating storage protein synthesis and accumulation. Third, high N speeds up plant N metabolism and increases the levels of various metabolites, such as amino acids, which are substrates for storage-protein synthesis.These synergistic effects result in upregulation of storage protein components and increased gliadin and glutenin content, ultimately improving dough viscoelasticity and breadmaking quality.

5. Conclusions

Proteomic analysis by RP-UPLC and 2D-DIGE during grain development in the elite Chinese wheat cultivar Zhongmai 175 revealed that application of high-N fertilizer induced changes in the gliadin and glutenin subproteomes.The contents of gliadin and glutenins and of the components of storage proteins increased markedly under high-N conditions. The levels of 24 gliadins, 12 HMW-GS, and 27 LMW-GS were significantly increased by application of high-N fertilizer. These results enhance our understanding of the accumulation and polymerization mechanisms of storage proteins driven by application of high-N fertilizers, also providing a theoretical basis for the genetic and agronomic improvement of wheat quality.

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2019.06.002.

Declaration of Competing Interest

All the authors have no conflict of interest.

Acknowledgments

This research was financially supported by the National Key Research and Development Program of China(2016YFD0100502)and the National Natural Science Foundation of China(31171773).