Effects of a dramatic composition change of high-molecular-weight glutenin subunits by gene editing on flour-processing quality in wheat

doi:10.1016/j.jia.2025.05.017

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Jinxin Hu¹^,²^,^3*, Jiahui Zhang^1*, Wanxin Wang^1*, Junxian Liu²^,^4*, Huali Tang¹, Yingxiu Wan⁵, Xiao Zhang⁶, Weihong Huang¹, Xi Li¹, Yueming Yan^2#, Xingguo Ye^1#, Ke Wang^1#

¹State Key Laboratory of Crop Gene Resources and Breeding/National Key Facility for Crop Gene Resources and Genetic Improvement/Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China

²College of Life Science, Capital Normal University, Beijing 100048, China

³College of Agronomy, Henan Agricultural University, Zhengzhou 450002, China

⁴College of Agronomy, China Agricultural University, Beijing 100193, China

⁵Crop Research Institute, Anhui Academy of Agricultural Sciences

⁶Institute of Agricultural Sciences for Lixiahe Region in Jiangsu

Highlights

1. A series of HMW-GS-edited mutants were generated using the CRISPR/Cas9 system.

2. The contribution of HMW-GSs to flour processing quality—including bread, noodles, and biscuits—was evaluated within the wheat variety CB037.

3. Despite reduced bread quality, the mutants demonstrated improved noodle traits and, in some cases, better biscuit quality.

Abstract
References

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摘要

小麦（Triticum aestivum L.）是全球最重要的粮食作物之一，其面粉可加工成多种食品。高分子量谷蛋白亚基（HMW-GSs）对面粉加工品质具有决定性作用。本研究利用CRISPR/Cas9系统创制了同时沉默1-4个HMW-GSs基因的8种小麦突变体。通过PCR-限制性内切酶（PCR-RE）分析和测序，在T₁代中鉴定到这些突变。T₂代突变体经十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）和超高效液相色谱（UPLC）验证，证实缺失1-4个HMW-GSs基因。表型分析表明，突变体在主要农艺性状和籽粒性状方面与野生型相当。然而，突变体中的谷蛋白大聚合物（GMP）含量明显低于野生型。透射电镜（TEM）观察显示，突变体籽粒胚乳中存在片状的GMP结构，表明HMW-GSs的缺失不影响GMP的形成。SDS-沉降值（SDS-SV）和面包烘焙试验表明，在CB037遗传背景中，各HMW-GSs对加工品质的贡献等级为1Dx5>1Dy12>1Ax1。有趣的是，虽然面包烘焙品质下降，但有些突变体的饼干和面条品质却提高了。其中dDx突变体制成的饼干最薄，直径最大，延展系数最高。这些HMW-GSs含量降低的突变体，可能为培育专用型饼干和面条小麦提供新策略。

Abstract

Wheat (Triticum aestivum L.) is one of the most important food crops globally, and its flour can be processed into a wide variety of foods. The high-molecular-weight glutenin subunits (HMW-GSs) play a crucial role in determining the flour-processing quality. In this study, we used the CRISPR/Cas9 system to generate eight types of wheat mutants with the silencing of one to four HMW-GS-encoding genes simultaneously. These mutations were identified in the T₁ generation by PCR-restriction enzyme (PCR-RE) analysis and sequencing. In the T₂ generation, mutants were confirmed to express one to four HMW-GSs by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and ultra-high-performance liquid chromatography (UPLC). Phenotypic analysis showed that the mutants were comparable to the wild-type (WT) in terms of major agronomic and grain traits. However, glutenin macropolymers (GMP) content in the mutants was significantly lower than in the WT. Transmission electron microscopy (TEM) revealed a flaky GMP structure in the mutant grain endosperms, indicating that the absence of HMW-GSs did not affect GMP formation. SDS-sedimentation volume (SDS-SV) and bread-baking tests revealed that the contribution of HMW-GSs to processing quality was ranked as 1Dx5>1Dy12>1Ax1 in the genetic background of CB037. Interestingly, although bread-baking quality deteriorated, the cookie-making and noodle quality of the mutants improved. The cookie made from the dDx mutant had the thinnest, largest diameter, and the highest spread factor. Mutants with reduced HMW-GS content may provide a new strategy for wheat breeding tailored for cookie and noodle production.

Keywords: wheat HMW-GSs CRISPR/Cas9 gluten quality GMP content

Online: 16 May 2025

Fund:

This research was supported by the grants from the Science and Technology Department of Ningxia in China (2022BBF02039), the National Natural Science Foundation of China (32472182, 32272180), and the Biological Breeding-National Science and Technology Major Project (2024ZD0407704).

About author: #Correspondence Ke Wang, Tel/Fax: 86-10-82105174, E-mail: wangke03@caas.cn; Xinguo Ye, Tel/Fax: 86-10-82105173, E-mail: yexingguo@caas.cn; Yueming Yan, Tel/Fax: 86-10-68902777, E-mail: yanym@cnu.edu.cn *These authors contributed equally to this article

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Jinxin Hu, Jiahui Zhang, Wanxin Wang, Junxian Liu, Huali Tang, Yingxiu Wan, Xiao Zhang, Weihong Huang, Xi Li, Yueming Yan, Xingguo Ye, Ke Wang. 2025. Effects of a dramatic composition change of high-molecular-weight glutenin subunits by gene editing on flour-processing quality in wheat. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2025.05.017

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