TaqW-6BL, a novel gene positively regulates grain WE-AX content in wheat

The TaqW-6BL gene, which encodes a TAXI-type inhibitor, influences the water-extractable arabinoxylan content in wheat grains. Water-extractable arabinoxylans (WE-AX) are major dietary fiber components and play a crucial role in wheat end-use quality, especially its nutritional quality. In this study, TaqW-6BL was cloned from parental varieties of a recombinant inbred line (RIL) population, in which a missense mutation (+ 296 bp, T/C) was detected and found to cause a methionine (Met) to threonine (Thr) substitution. Overexpression of TaqW-6BL positively regulated grain WE-AX content, thousand kernel weight (TKW) and mean grain perimeter (MP), with increases of 30.80%, 14.59% and 14.53%, respectively. Conversely, CRISPR/Cas9-mediated knockout of TaqW-6BL led to significant decreases in WE-AX content, TKW and MP. Cytological observations revealed that the aleurone layer of overexpression lines partially proliferated into two layers, whereas knockout lines showed disordered cell arrangement and abnormal morphology. Subcellular localization analysis confirmed that TaqW-6BL protein localizes in the endoplasmic reticulum (ER). The relative expression level and WE-AX content of the favorable haplotype (TaqW-6BL^Avocet) were significantly higher than those of the unfavorable haplotype (TaqW-6BL^Chilero) at 14 days and 21 days after flowering (DAF) (P < 0.01). Meanwhile, we developed a functional cleaved amplified polymorphic sequence (CAPS) marker CAPS-6B-296. The wheat varieties with favorable haplotype TaqW-6BL^Avocet accounted for 68% of 160 wheat varieties. Therefore, our findings provide a valuable gene and a reliable functional marker for nutritional quality breeding in wheat.