Decoding the temporal regulome of wheat starch: Wx allelic hierarchy unveils novel negative regulators

The temporal regulation of the three homoeologous Wx genes, which direct amylose biosynthesis in wheat, remains unclear, hindering a systems-level understanding of this pathway. Using near-isogenic lines (NILs), we delineated their allele-specific regulatory hierarchy during grain filling. Amylose content decreased in a strict gene dosage-dependent manner, accompanied by a compensatory increase in the amylopectin fraction from 15 days after pollination (DAP). Loss-of-function Wx alleles significantly altered starch granule morphology, increasing the proportion of smaller B-type granules during later stages, with the triple-deletion line (BBB) producing markedly smaller and irregular B-type granules. These deletions also reduced granule nano-lamellar thickness and remodeled internal starch architecture, changes that were linked to higher proportion of short-chain amylopectin. These structural changes led to altered pasting properties, including reduced viscosity and a broader gelatinization temperature range. Integrated transcriptomic profiling revealed that Wx deficiency influenced central metabolic pathways, including carbohydrate and starch-sucrose metabolism. This analysis identified a glycoside hydrolase family 9 protein (TaGHF9) and a domain of unknown function-containing protein (TaDUF) as novel negative regulators of amylose biosynthesis, whose function was validated using EMS-induced mutants. Our study establishes the temporal effects of Wx alleles, unveiling a multi-level regulatory network and precision targets for improving wheat starch quality.