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

Highlights 1 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. 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 T 1 generation by PCR-restriction enzyme (PCR-RE) analysis and sequencing. In the T 2 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.