| The composition of high molecular weight glutenin subunit (HMW-GS) and low molecular weight glutenin subunit (LMW-GS) in wheat kernel (Triticum aestivum L.) affects the size of glutenin macropolymer (GMP), which is considered the important flour quality traits in wheat. Three wheat cultivars with different end-use quality were used to study the effects of water-nitrogen management (W×N) on relative HMW-GS and GMP contents. Under irrigation, increased N levels promoted the accumulation of H- and LMW-GS, GMP content and the proportion of the larger particle of GMP, with higher yield and better end-use quality. Under rainfed conditions, increased N fertilizer also increased protein content in the three cultivars. As opposed to the Gaocheng8901 (GC8901), the Shannong1391 (SN1391) and Taishan23 (TS23) got higher H- and LMW-GS contents and larger GMP particles, yields of both reduced. Irrigation increased the yield markedly, the N application significantly affected the glutenin, but the W×N interaction was not significant. On one hand, increased N levels improved the dough development time (DDT) by increasing the numbers of larger GMP particles and enhancing volume and surface area percentages on the other. It was suggested that the interaction was a complicated network and some individual effect might be important. When managing for wheat yield and quality, growers should first consider the water factor, then the N fertilizer factor to improve the end-use quality of wheat.Full-length HMW-GS Sequence Analysis of tthree wheat cultivarsThe HMW-GS pairs 1Dx5+1Dy10, 1Bx14+1By15 are recognized to positively correlate with bread-making quality. Both the reading frames (ORFs) of 1Ax and of 1Bx7 contain 4 cysteines, but the ORF of 1Ax forms 20 potentialα-helix structures while 1Bx7 forms 9 potentialα-helix structures. The ORF of 1Dx which forms 9 potentialα-helix structures contains 5 cysteines; the ORF of 1Bx14 which forms 11 potentialα-helix structures contains only 2 cysteines. The ORFs of 1Dy10, 1By8, 1By15 and 1Dy12 all contain 7 cysteines and form respectively 9, 11, 11 and 11 potentialα-helix structures. The DNA sequences showed they are homologous and would had the similar structure and function in wheat kernel. So the cysteine residue which plays important roles in forming the intra- and inter molecular structures of HMW-GS could not be used as the sole evaluation criterion for the merits of subunits. The proline, glycine and glutamine play important roles in forming advanced structure of glutenin, so the three amino acid residues would be the important reference standard for forming the visco-elasticity of quality dough. Meanwhile, the three amino acids have taken a large proportion in x- style HMW-GS. It means that x- style subunits contribute more for dough quality.Differences in Experimental FactorsAnalysis of variance for the contents of subunits, GMP content, the D4.3, D3.2, the single kernel weight, yield, grain protein content and the DDT made it possible to identify the sources of variation. Nitrogen (N) main effect and water×N (W×N) interaction failed to influence those traits except single kernel weight and grain protein content. The Student Newman and Keuls test (p≤0.05) showed that subunits in 1Bx chromosome, HMW-GS content and yield were significantly affected by water treatment. Although the N main effect as well as the W×N were not significant through three wheat cultivars, some individual effect was important to determine the quality and yield. It was suggested the interaction was a complicated network. Variations due to years and in interactions between year and nitrogen (Yr×N), year and water (Yr×W) treatment were not significant (p≤0.05). Since year was not a significant factor in the total reaction, some data from both years were averaged.RP-HPLC Analysis for GluteninConsidering the three cultivars, the 1Dx2 and 1Dx5 subunits accumulated to the highest level about 42% and 38% of total high molecular weight glutenin subunit (HMW-GS) content. The 1Bx7 and 1Dy12 subunits were present in a smaller amount, 31% and 26%, and the 1Ax1 and 1By8 subunits were the least abundant and nearly equal, 14% and 12%. The proportions of linked subunit pairs were nearly equal among different N levels, but the proportions of single subunits showed various under the two water treatments. As compared with irrigated regimen, rainfed regimen increased the mean proportion of 1Ax1 subunit by 10%, decreased 1Bx7 by 26.5%, increased 1By8 by 85.7%, whereas, the proportion of 1Dx5 and 1Dy10 remained stable in GC8901. The mean proportion of 1Ax1 in TS23 under irrigated regimen increased by 25%, 1Bx7 increased by 12.2% and 1By8 decreased by 38.7% compared with of under rain-fed regimen. Water treatment showed no significant effects on the subunit pairs in SN1391 with increased N application. Take all the subunit pairs into account, 1Bx7+1By8 seemed more sensitive to the environment and 1Dx2+1Dy12 always high expressed in middle and weak gluten wheat. We observed that under irrigation, both in GC8901 and TS23, the content of HMW-GS and H/L value increased with N increased, and the LMW-GS contents reached the maximums at 120 kg N ha-1 (N1). However, the trend was opposite in SN1391 under the same condition. Under rainfed regimen, the contents of H- and LMW-GS increased in three wheat cultivars with higher N application (N2, N3), especially in SN1391. However, the H- and LMW-GS contents of three cultivars decreased markedly under rainfed condition with low N application (N1).These results indicated that the ratios of the linked subunit pair's proportions to the mean proportion of different N levels were nearly identical. The two linked subunit pairs had certain complementary effects, the final proportions showed no significant relationship with W×N interaction and mainly determined by genotypes. Whereas, a significantly correlated was shown among single kernel weight, protein content, GMP content (r = -0.829**; 0.563*; 0.803**) and H/L. It was indicated that the grain kernel production interacts with H- and LMW-GS contents. Rainfed condition could increase the contents of H- and LMW-GS and H/L value in three wheat cultivars.Content of GMP ParticlesThe contents of GMP in three wheat cultivars were ordered as follows: GC8901 > TS23 > SN1391. The contents of GMP all increased as N rates increased under irrigated regimen. But under rainfed regimen, the content of GMP in GC8901 showed difference with the N level, and reached the maximum at 240 kg N ha-1 (N2); the GMP contents of TS23 and SN1391 both were markedly increased by 6-15.4% and 30.9-82% with no N applied (N0), and tended to decline as increased N application, while the differences of contents between N application of 240 kg N ha-1 (N2) and 360 kg N ha-1 (N3) were not significant. The results indicated that increasing N level could promote the accumulation of GMP in three wheat cultivars under irrigated regimen. Rainfed condition significantly promoted the accumulation of GMP in middle and weak gluten wheat, but the content remained stable with N rates.GMP Particle DistributionThe surface area percent of GMP particles at < 12μm, 12-110μm and > 110μm in three cultivars took up 72.3-89.4%, 9.5-21.5% and 1.1-10.1%. Under irrigation, the surface area percentages of GMP particles at 12-110μm and < 110μm in GC8901 decreased first and then markedly increased with N increased, whereas that of < 12μm decreased markedly when compared with those under rainfed regimen. However, those in SN1391 showed a character of opposite extremes. Those at < 12μm in TS23 decreased with N increased, and those of 12-110μm and > 110μm increased under irrigation regimen. While under rainfed regimen, the GMP surface area percentage of TS23 reached the maximum value with no N applied (N0). The irrigation was beneficial to increasing the surface area percent of GMP particles as increased N application, and sight water stress could markedly promote those in weak and middle gluten wheat.The data in Table 4 indicated that D4.3 and D3.2 were completely in line with the surface area percentages of GMP particles at > 110μm. Dispersed GMP of three cultivars under W×N interaction displayed that rainfed regimen and lower N level could promote the volume of particles in middle and weak gluten wheat. Strong gluten wheat was more adapted to plenteous irrigation and higher N application. And interestingly, the D4.3 under rain-fed regimen of TS23 and SN1391 were bigger than that of in GC8901, but the content of GMP in TS23 and SN1391was less.Irrigated and rainfed conditions had different influence on percent number of GMP particles in three wheat cultivars. Under irrigation, the number percentage of GC8901 and TS23 at < 4.5μm increased, while those of 4.5-12μm and > 12μm decreased when compared with those under rainfed condition. Increased N level contributed to increase the GMP particles numbers of GC8901 and TS23 at >12μm. The increased N level increased number percentage of GMP particles in SN1391 at <4.5μm, decreased that of 4.5-12μm, whereas that of > 12μm remained the same. The GMP particles number percentage responses of SN1391 to water treatment were similar to those to N application. The number distribution revealed that increased N level could increase the numbers large GMP particles in strong and middle gluten wheat. While in weak gluten wheat, N effect and W×N interaction showed no influence on the numbers of larger GMP particles.Observations of GMP ParticlesTo further analyze the effects of W×N interaction on glutenin particle, GMP dispersions of three wheat cultivars were examined for particulate structures obtained. The SEM figures showed that large GMP particle was made from the polymerization of small GMP particles, the smaller GMP particles were made of H- and LMW-GS in various proportions. We also estimated that GC8901 got a highest H/L ratio ranged from 0.36-0.47 and the highest D4.3, TS23 got a middle H/L ratio ranged from 0.26-0.33 and smaller D4.3, SN1391 got the least H/L ratio ranged from 0.20-0.25 and least D4.3. It indicated that D4.3 was mainly determined by genotypes, but there was no evidence to confirm their proportions of H- and LMW-GS in this paper. The D4.3 under irrigated regimen showed strong relation to protein content, GMP content, single kernel weight and H/L value (r=0.822**; 0.755**; -0.806**; 0.698*), but little relation of D4.3 to these traits was found under rainfed regimen. The D4.3 also showed no significantly correlated with above traits (r=0.234; 0.404; -0.189; 0.279) under W×N interaction. Water treatment influenced the accumulation and the size of glutenin particles than any other factors when the genotypes were chosen.Analysis of Water-Nitrogen Interaction on Single Kernel Weight, Protein content, Yield and DDTIncreasing N fertilizer application increased protein content and average single kernel weight, no matter whether irrigated or not. Compared with irrigated regimens, the protein percentage of GC8901 was decreased by 4.8-22.9%, while in TS23 and SN1391, the flour protein percentage sharply increased by 45.6% and 36.7% at 360 kg N ha-1 (N3) under rain-fed regimens. Across the two years, mean grain yields of three cultivars ranged from 6.9-9.9 mt ha-1 with irrigation, Rainfed condition led to decline by 2.0-38.2% of the yield. Dough Development Time (DDT) was recognized as an indicator of dough strength and baking quality. we observed that DDT had a similar tendency to GMP content, and there was a strong relation between them (r = 0.739**). Taking the two water treatments together, there was no significant correlation between DDT and D4.3 (r = 0.190), although D3.2 and D4.3 increased under rainfed condition. However, a stronger correlation was observed between DDT and H/L value (r = 0.803**). Apparently, the amounts of H- and LMW-GS were more important than GMP content for DDT.We suggest that when the genotypes were chosen, the irrigation and N level contributed more to the yield and DDT, when the N application was higher than 240 kg N ha-1, the increasing amount of grain yield was not significant, and the effects were often mixed. It might be helpful to explain that wheat tended to earn higher biological mass and yield under well irrigated conditions with sufficient fertilizer, while increasing single kernel weight and keeping more protein in storage under rainfed regimen.
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