TaWUS-like-5D affects grain weight and filling by inhibiting the expression of sucrose and trehalose metabolism-related genes in wheat grain endosperm.

Plant-specific WUSCHEL-related homeobox (Wox) transcription factors (TFs) are crucial for plant growth and development. However, the molecular mechanism of Wox-mediated regulation of thousand kernel weight (TKW) in crops remains elusive. In this research, we identified a major TKW-associated quantitative trait locus (QTL) on wheat chromosome 5DS by performing a genome-wide association study (GWAS) of a Chinese wheat mini-core collection (MCC) in four environments combined by bulked segregant analysis (BSA) and bulked segregant RNA-sequencing (BSR-seq) of wheat grains exhibiting a wide range of TKWs. The candidate TaWUS-like-5D was highly expressed in developing grains and was found to strongly negative influence grain TKW and wheat yield. Meanwhile, the RNAi lines, CRISPR/Cas9-edited single and double knockout mutants (AABBdd and AAbbdd), as well as the stop-gained aaBB Kronos mutants, exhibited a significant increase in grain size and TKW (P < 0.05 or P < 0.01) and a 10.0% increase in yield (P < 0.01). Further analyses indicated that TaWUS-like-5D regulates TKW by inhibiting the transcription of sucrose, hormone and trehalose metabolism-related genes, subsequently sharply decreasing starch synthesis in wheat grains. The results of this study provide a fundamental molecular basis for further elucidating the mechanism of Wox-mediated regulation of grain development in crops.