| The wheat mutant materials with high protein content were screened by screening the EMS（Ethyl methane sulfonate）wheat mutant library with stable inheritance.Characterization of nitrogen accumulation and transportation in plants,grain quality,and their responses to nitrogen fertilizer application rates would provide useful information for improving wheat grain quality and nitrogen fertilizer utilization efficiency.Using field and pot experiments,the grain quality and the characteristics of nitrogen accumulation and transportation in plants of wheat mutant materials with high protein content under different nitrogen fertilizer application rates were studied.The pot experiment was carried out with two N application rates,high-nitrogen(HN,N 5.78 g·pot-1)and low-nitrogen(LN,N 2.04 g·pot-1),by using eight wheat mutant materials（numbered: 401,467,512,883,1118,1236,1306 and 1443）and the control Zhoumai22.The field experiment was carried out with two N application rates,N210(N 210 kg·hm-2)and N105(N 105 kg·hm-2),by using two wheat mutant materials（401 and 883）,and one control（Zhoumai22）.The main results were as follows:1.Screening of Wheat Mutant Materials with High Protein ContentThe grain protein and wet gluten content of 963 EMS-induced wheat mutants were analyzed.The results showed that the variation range of grain protein was from 12.5% to 23.0%,and wet gluten content was from 25.6% to 42.0%.Among them,the grain protein contents of 35 materials were higher than 20.5%,accounting for 3.63%;The wet gluten contents of 21 materials were higher than 41%,accounting for 2.18%.A total of 40 wheat mutant materials with high grain protein and wet gluten content were further used to analyze their responses to different nitrogen fertilizer application levels.The results showed that,the grain protein content ranged from 14.73% to 24.71%,and from 14.73% to 25.21% under the non-nitrogen application treatment,and the normal nitrogen application group in the Zhengzhou site,respectively.Correspondingly,the grain weight of a 1-meter single row ranged from 80.12 g to 498.4 g,and from 120.1 g to 472.24 g,and the physiological nitrogen utilization efficiency ranged from 20.54% to 40.60%,and from 23.62% to 40.38%,respectively.In Yuanyang site,the protein content ranged from 15.16% to 25.24%,and from 14.37% to 27.49% under the non-nitrogen application level,and the normal nitrogen application rate,respectively.Correspondingly,the grain weight of a 1-meter single row ranged from 93.57 g to 602.76 g,and from 61.64 g to 428.96 g,and the physiological nitrogen utilization efficiency ranged from 19.16% to 37.23% and from 17.25% to 39.74%,respectively.Based on the above results,eight wheat mutants with high grain protein content while balancing grain weight and nitrogen efficiency were screened out.There are No.401,467,512,883,1118,1236,1306,and 1443.2.Characterization of Nitrogen Accumulation and Transportation in Wheat Mutant Materials with High Protein ContentIn the pot experiment,the protein content of the tested wheat mutant materials under the LN and HN condition ranged from 16.07% to 22.19%,and from 17.19% to 23.0%,respectively.Under the LN condition,the total amount of nitrogen accumulation in panicles of different wheat mutant materials at the flowering stage was higher than that of the control（except 883）,whereas their nitrogen accumulation per stem was higher than that of the control under the HN condition.The nitrogen accumulation per stem at the maturity stage showed no significant difference between 401,883,1118,1443 and the control under the LN condition,whereas no significant difference between 401,512,883,1118,1236,1443 and the control was observed under the HN condition.The distribution ratio of nitrogen in the grain at the maturity stage was 86.50% and 86.49%,for 883 and 1118 under the LN condition,respectively,which was 3.56% and 3.54% higher than that of the control.The translocation amount of N from the pre-anthesis accumulation and the contribution rate of pre-anthesis accumulation N to grains of 401,1118,and 1236 were higher than those of the control under both nitrogen application rates.3.Analysis of Protein Components and Amino Acid Components of Wheat Mutant Materials with High Protein ContentThe grain protein and its components content,the total amino acid（TAA）,and essential amino acid（EAA）content of wheat mutant materials 401 and 883 were significantly higher than those of the control Zhoumai22.The average EAA content of 401 and 883 was 21.72% and 22.89% higher than that of the control,respectively.Correspondingly,the TAA content was 13.72% and 16.38% higher than that of the control.The protein components of grain increased with the increase of nitrogen fertilizer,but the increase of glutenin was less than that of gliadin.The ratio of glutenin to gliadin of 401 and 883 was lower than that of the control.The essential amino acid index（EAAI）and protein digestibility modified amino acid fraction（PDCAAS）of 401 and 883 were higher than that of the control,and EAAI and PDCAAS showed an increasing trend with the nitrogen fertilizer application rate increasing.4.Analysis of Differentially Expressed Proteins between High Protein Content Wheat Mutant Materials and ControlProteomic analysis showed that there were 664 differentially expressed proteins between 401 and Zhoumai22,of which 380 were up-regulated and 284 were down-regulated.There were 699 differentially expressed proteins between 883 and Zhoumai22,of which 407 were up-regulated and 292 were down-regulated.These differentially expressed proteins mainly include nutrient reservoir activity proteins（5.51～17.53%）,signal transduction-related proteins（3.61～5.51%）,nitrogen metabolism-related proteins（10.31～14.17%）,carbohydrate metabolic-related proteins（9.79～15.75%）,transportation-related proteins（0.79～3.09%）,stress defense-related proteins（7.87～12.37%）,nucleus-related proteins（5.67～10.24%）,other proteins（6.70～14.96%）and undefined proteins（25.20～30.93%）.These results suggest that multiple metabolic pathways may be involved in the regulation of grain storage protein synthesis.Especially,the up-regulation of some endopeptidase inhibitor proteins（A0A3B6JN50,Q6QAX7）and regulatory proteins（A0A3B6PDY8）may be the reason for the high protein content of wheat mutant material 401.The up-regulation of nitrogen metabolism-related proteins（A0A3B5Y0N0）and signal transduction-related proteins（A0A3B6SJQ7）may contribute to the increase of protein content in 883.Wheat mutant materials 401 and 883 with high protein and high amino acid content were screened out in this study,which may involve multiple metabolic pathways to regulate the synthesis of grain storage proteins,thus increasing their grain protein content. |
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