The TaMYB44–TaMYB1 module regulates grain amylose biosynthesis and flour viscosity in wheat

Starch content and composition in wheat endosperm are critical determinants of wheat yield and quality. Amylose content is a key parameter used to characterize starch viscosity in wheat flour, which influences pasting time, peak viscosity, and, consequently, the eating quality of flour-based foods. However, the transcriptional regulatory mechanisms governing amylose biosynthesis in wheat remain poorly understood. Here, yeast one-hybrid (Y1H) screening identified a MYB transcription factor, TaMYB44-4D, that binds to the TaWx-7A promoter. Electrophoretic mobility shift assays (EMSAs), Y1H assays, and transactivation assays demonstrated that TaMYB44-4D binds to the TaWx-7A promoter and represses its transcriptional activity. Knockout (KO) of TaMYB44-A/B/D simultaneously increases amylose content, total starch content, swelling power, and rapid viscosity analyzer parameters, whereas overexpression of TaMYB44-4D reduces these traits. Our results indicate that TaMYB44 functions as a transcriptional repressor of starch biosynthesis. We further confirmed that TaMYB44 interacts with TaMYB1; similar phenotypes were observed in tamyb44-KO and tamyb1-KO lines, as well as in TaMYB44-4D-overexpression (OE) and TaMYB1-6B-OE lines, suggesting that the two factors act as co-repressors. A single-nucleotide polymorphism (T/C) identified in the TaMYB44-D promoter among various wheat varieties is associated with differential TaMYB44 expression levels. Phenotype–genotype association analysis in a natural wheat population revealed that varieties with the CC haplotype exhibit higher amylose content than those with the TT haplotype. In conclusion, our findings provide important insights into the transcriptional regulation of amylose biosynthesis and offer a genetic framework for the improvement of wheat starch quality.