Wheat Glu-A1a encoded 1Ax1 subunit enhances gluten physicochemical properties and molecular structures that confer superior breadmaking quality

Wheat gluten proteins serve as the largest protein molecules in nature and play key roles in breadmaking quality formation. In this study, we used a pair of Glu-A1 allelic variation lines to perform a comprehensive investigation on the effects of Glu-A1a encoded 1Ax1 subunit on gluten physicochemical properties, molecular structures and breadmaking quality. The results showed that the presence of the 1Ax1 subunit significantly increased gluten content, leading to marked improvement of dough rheological properties. Meanwhile, gluten physicochemical properties such as foaming ability and foaming stability, oil/water-holding capacity, emulsifying activity, di-sulfide bond content, and gluten degradation temperature were significantly improved. A confocal laser scanning microscope analysis revealed that the 1Ax1 subunit drastically enhanced gluten microstructure. Gluten sec-ondary structure analysis by Fourier transform infrared spectroscopy and laser scanning microscope-Raman spectroscopy indicated that 1Ax1 subunit significantly promoted beta-turn and beta-sheet content and reduced alpha-helix content. Three-dimensional structure analysis by AlphaFold2 revealed a similar structural feature of 1Ax1 with the superior quality subunit 1Ax2*. Correlation and principal component analyses demonstrated that alpha-helix and beta-sheet content had a significant correlation with dough rheological properties, gluten physico-chemical properties and breadmaking quality. Our results showed that 1Ax1 subunit positively affected gluten molecular structure and quality formation.