Portal de Periódicos da CAPES

Dissecting and Enhancing the Contributions of High-Molecular-Weight Glutenin Subunits to Dough Functionality and Bread Quality

2015; Elsevier BV; Volume: 8; Issue: 2 Linguagem: Inglês

10.1016/j.molp.2014.10.002

1674-2052

Yiwen Li, Xueli An, Ran Yang, Xiaomin Guo, Guidong Yue, Renchun Fan, Bin Li, Zhensheng Li, Kunpu Zhang, Zhenying Dong, Luyan Zhang, Jiankang Wang, Xu Jia, Hong‐Qing Ling, Aimin Zhang, Xiangqi Zhang, Daowen Wang,

Bioenergy crop production and management

Seed storage proteins (SSPs) are frequently important determinants of crop quality traits (Shewry and Casey, 1999Shewry P.R. Casey R. Seed Proteins. Kluwer Academic Publishers, Dordrecht1999Crossref Google Scholar). Dissecting and enhancing the genetic contributions of individual SSPs to their target traits are essential for effectively improving crop quality attributes. However, such a task is often difficult to accomplish, because SSPs are frequently expressed from multigene families and exhibit strong allelic variation. Consequently, detailed knowledge of the function of individual SSPs in crop quality trait is still limited. This scenario is well illustrated by high-molecular-weight glutenin subunits (HMW-GSs), a complex family of SSPs that are involved in wheat end-use quality through affecting dough functionality (Békés, 2012Békés F. New aspects of quality related wheat research: I. Challenges and achievements.Cereal Res. Commun. 2012; 40: 159-184Crossref Scopus (23) Google Scholar, Rasheed et al., 2014Rasheed A. Xia X. Yan Y. Appels R. Mahmood T. He Z. Wheat seed storage proteins: advances in molecular genetics, diversity and breeding applications.J. Cereal Sci. 2014; 60: 11-24Crossref Scopus (120) Google Scholar). In bread wheat, HMW-GSs are specified by Glu-A1, -B1, and -D1 homoeoloci (Payne, 1987Payne P.I. Genetics of wheat storage proteins and the effect of allelic variation on bread-making quality.Annu. Rev. Plant Physiol. 1987; 38: 141-153Crossref Google Scholar). Within each Glu-1 locus, there are two HMW-GS genes encoding one x- and one y-type subunit, respectively (Gu et al., 2006Gu Y.Q. Salse J. Coleman-Derr D. Dupin A. Crossman C. Lazo G.R. Huo N. Belcram H. Ravel C. Charmet G. et al.Types and rates of sequence evolution at the high-molecular-weight glutenin locus in hexaploid wheat and its ancestral genomes.Genetics. 2006; 174: 1493-1504Crossref PubMed Scopus (78) Google Scholar). Because of allelic variation and gene silencing, bread wheat varieties usually express three to five HMW-GSs and frequently differ in HMW-GS expression level and composition. Past studies have mainly investigated the genetic effects of Glu-A1, -B1, and -D1 on wheat end-use quality by comparing varieties differing in Glu-1 loci, analyzing deletion lines lacking one or more Glu-1 loci, and mapping quantitative trait loci (Lawrence et al., 1988Lawrence G.J. Macritchie F. Wrigley C.W. Dough and baking quality of wheat lines deficient in glutenin subunits controlled by the Glu-A1, Glu-B1 and Glu-D1 loci.J. Cereal Sci. 1988; 7: 109-112Crossref Scopus (195) Google Scholar, Rasheed et al., 2014Rasheed A. Xia X. Yan Y. Appels R. Mahmood T. He Z. Wheat seed storage proteins: advances in molecular genetics, diversity and breeding applications.J. Cereal Sci. 2014; 60: 11-24Crossref Scopus (120) Google Scholar). However, there is still no report of systematic mutagenesis analysis of individual HMW-GSs using well-defined mutants. Thus, the contributions of individual HMW-GSs to dough functionality and different end uses are still not well understood. Furthermore, the potential of generating superior HMW-GS alleles through artificial mutagenesis has not been explored. In order to dissect and enhance the contributions of individual HMW-GSs to different end uses, we chose to develop knockout and missense mutants for the complete set of five HMW-GSs (Ax1, Bx14, By15, Dx2, and Dy12) of the winter wheat variety Xiaoyan 54 (Li et al., 2004Li W. Wan Y. Liu Z. Liu K. Liu X. Li B. Li Z. Zhang X. Dong Y. Wang D. Molecular characterization of HMW glutenin subunit allele 1Bx14: further insights into the evolution of Glu-B1-1 alleles in wheat and related species.Theor. Appl. Genet. 2004; 109: 1093-1104Crossref PubMed Scopus (52) Google Scholar). By screening an ethyl methanesulfonate–induced mutant population, knockout mutants were identified for all five subunits and missense mutants for Ax1, Dx2, and Dy12 (Figure 1A and Supplemental Table 1). Because Ax1 and Bx14 are frequently used in international wheat quality breeding programs (Oury et al., 2010Oury F.X. Chiron H. Faye A. Gardet O. Giraud A. Heumez E. Rolland B. Rousset M. Trottet M. Charmet G. et al.The prediction of bread wheat quality: joint use of the phenotypic information brought by technological tests and the genetic information brought by HMW and LMW glutenin subunits.Euphytica. 2010; 171: 87-109Crossref Scopus (42) Google Scholar, Rasheed et al., 2014Rasheed A. Xia X. Yan Y. Appels R. Mahmood T. He Z. Wheat seed storage proteins: advances in molecular genetics, diversity and breeding applications.J. Cereal Sci. 2014; 60: 11-24Crossref Scopus (120) Google Scholar), we comparatively analyzed two knockout mutants of Ax1 (ma1-1 and ma1-2), two knockout mutants of Bx14 (mb14-1 and mb14-2), one double mutant (ma1-1mb14-1) lacking both Ax1 and Bx14, and one missense mutant of Ax1 (ma1-3) to reveal the contributions of the two subunits to dough functionality and bread quality. For ma1-3, the single amino acid substitution (G330E) in Ax1 was confirmed to be the cause of the reduced electrophoretic mobility of the mutant Ax1 protein by bacterial expression (Supplemental Figure 1). After minimizing background mutations through six rounds of backcrossing, the selected mutants were planted in the field in three crop seasons and evaluated with Xiaoyan 54 as wild-type (WT) control. The six mutants did not differ substantially from the WT control in the days to heading, flowering, and harvesting. No significant differences in plant height and yield-related traits were found (Supplemental Table 2). Compared with the WT control, the content of HMW-GSs was significantly decreased in ma1-1 (−11.13%), ma1-2 (−13.41%), mb14-1 (−15.41%), mb14-2 (−16.69%), and ma1-1mb14-1 (−32.95%) but did not change substantially in ma1-3 (Supplemental Table 3). Six major parameters related to dough functionality and bread quality were compared among the WT control and the six mutants, and the resulting data are listed in Supplemental Table 4. Development and stability times, indicating dough strength (Ross and Bettge, 2009Ross A.S. Bettge A.D. Passing the test on wheat end-use quality.in: Carver B.F. Wheat Science and Trade. Wiley-Blackwell, Iowa2009: 455-493Crossref Scopus (35) Google Scholar), were significantly lower in the five knockout mutants than in the WT control, with the decrease being much larger for ma1-1mb14-1. By contrast, the two parameters were both substantially increased in ma1-3 compared with the WT control. Maximum resistance, also reflecting dough strength (Ross and Bettge, 2009Ross A.S. Bettge A.D. Passing the test on wheat end-use quality.in: Carver B.F. Wheat Science and Trade. Wiley-Blackwell, Iowa2009: 455-493Crossref Scopus (35) Google Scholar), was reduced in the five knockout mutants, with the reduction being highest for ma1-1mb14-1, intermediate for ma1-1 and ma1-2, and lowest for mb14-1 and mb14-2. However, the maximum resistance value of ma1-3 dough was nearly twice that of the WT control. Relative to the WT control, mb14-1, mb14-2, and ma1-1mb14-1 showed significantly reduced dough extensibility, whereas ma1-1, ma1-2, and ma1-3 did not exhibit substantial changes in this parameter. The seven genotypes also differed in tensile energy, which is jointly determined by dough strength and extensibility (Ross and Bettge, 2009Ross A.S. Bettge A.D. Passing the test on wheat end-use quality.in: Carver B.F. Wheat Science and Trade. Wiley-Blackwell, Iowa2009: 455-493Crossref Scopus (35) Google Scholar). Compared with the WT control, this parameter was decreased in ma1-1, ma1-2, mb14-1, and mb14-2, more strongly reduced in ma1-1mb14-1, but substantially increased in ma1-3. Loaf volume, a key indicator of breadmaking quality (Ross and Bettge, 2009Ross A.S. Bettge A.D. Passing the test on wheat end-use quality.in: Carver B.F. Wheat Science and Trade. Wiley-Blackwell, Iowa2009: 455-493Crossref Scopus (35) Google Scholar), was significantly reduced in the five knockout mutants but substantially increased in ma1-3 (Figure 1B). The mean loaf volume of ma1-3 (894.11 ml) was more than 16% higher than that of the WT control (769.35 ml). Using the phenotypic data described above, the relative genetic contributions of Ax1 and Bx14 to six major dough functionality and bread quality parameters were statistically analyzed (Supplemental Table 5). Pair-wise comparison of P values showed that the single knockout mutants of Ax1 and Bx14 differed significantly only in maximum resistance and extensibility. However, the double knockout differed from the Ax1 single knockout in five of the six parameters examined and from the Bx14 single knockout in all six parameters. These comparisons indicate that there are significant differences in the contributions of Ax1 and Bx14 to dough functionality and bread quality, and that the contributions from the two subunits together are different from those by either alone. The percentage of phenotypic variance explained (PVE, %) by the two subunits for the six dough functionality and bread quality parameters was computed using two-way analysis of variance with interaction (Supplemental Table 6). In general, the PVE by the additive effect of Ax1 was higher than that of Bx14 for most of the parameters examined except for dough extensibility. The additive effect of Ax1 on dough extensibility was relatively small and statistically insignificant. The additive effects of Bx14 on the six parameters were all significant, particularly the one on dough extensibility, which explained 48.07% of the variation of the parameter. A significant epistatic effect by the interaction between Ax1 and Bx14 was detected only on dough extensibility, with the PVE being 10.02%. Therefore, we suggest that Ax1 contributes more strongly to dough strength and bread quality than Bx14, but Bx14 exerts a greater influence on dough extensibility (Figure 1C). The two subunits act mainly in an additive manner, although they also interact epistatically on dough extensibility (Figure 1C). The PVE by this epistatic interaction (10.02%) is smaller than that by the additive effect of Bx14 on extensibility (Supplemental Table 6), and its contribution to bread quality requires further investigation (Figure 1C). The total additive effects of Ax1 and Bx14 explained 44.09%–75.07% of the phenotypic variations of the six parameters examined (Supplemental Table 6), indicating that the two subunits are among the major contributors to dough functionality and bread quality. Consistent with the substantial contributions of WT Ax1 to dough strength and bread quality, the novel mutant ma1-3 exhibits a significant improvement in dough strength and increases bread volume by more than 16%. A single amino acid substitution (G330E) in Ax1 is responsible for the observed improvement, suggesting that artificial mutagenesis can generate a superior HMW-GS allele. As the major agronomic characteristics of ma1-3 are not compromised (see earlier discussion), this mutant may be used immediately in international wheat quality improvement research. Collectively, our results show clearly that individual HMW-GSs differ substantially in their contributions to dough functionality and bread quality, and that such contributions can be reliably dissected and enhanced using appropriate mutants. Therefore, it is now proper to extend this type of research to other HMW-GSs and additional end uses. The approach practiced here may also be applicable for studying and enhancing the functions of other SSPs, such as low-molecular-weight glutenin subunits, which have been found to interact with HMW-GSs and to affect wheat dough functionality and end-use traits (Zhang et al., 2012Zhang X. Jin H. Zhang Y. Liu D. Li G. Xia X. He Z. Zhang A. Composition and functional analysis of low-molecular-weight glutenin alleles with Aroona near-isogenic lines of bread wheat.BMC Plant Biol. 2012; 12: 243Crossref PubMed Scopus (72) Google Scholar, Rasheed et al., 2014Rasheed A. Xia X. Yan Y. Appels R. Mahmood T. He Z. Wheat seed storage proteins: advances in molecular genetics, diversity and breeding applications.J. Cereal Sci. 2014; 60: 11-24Crossref Scopus (120) Google Scholar). This work was supported by Ministry of Science and Technology of China (2012AA10A308), National Natural Science Foundation of China (30930059 and 31300280), and Ministry of Agriculture of China (2013ZX08009003-004).