CRISPR/Cas9 multiplex editing of four TaSK41 genes increases grain size and weight and reveals a TaSK41–TaSnRK1β1 module associated with carbohydrate metabolism in wheat

Grain size and weight contribute to wheat (Triticum aestivum L.) yield, yet how Glycogen synthase kinase 3 (GSK3)/SHAGGY-like kinase signaling interfaces with carbohydrate metabolism during grain development remains poorly understood. In this study, we characterized four wheat TaSK41 gene copies (TaSK41-1A, −4A, −5B, and −5D), which were preferentially expressed in young spikes and developing grains, particularly in the pericarp during early development. TaSK41-5B localizes to both the cytoplasm and nucleus. Using CRISPR/Cas9-mediated multiplex genome editing, we generated two independent quadruple mutant lines (task41-cr1 and task41-cr2) in the wheat cultivar ‘Fielder’ with all four TaSK41 copies simultaneously disrupted. The quadruple mutants exhibited a greater number of grains per spike, increased thousand-grain weight, larger grain size, and enhanced starch accumulation. The larger grains in the mutant lines were associated with increased cell proliferation in the outer pericarp and higher levels of auxin (IAA and IBA) in developing grains. TaSK41 physically interacted with TaSnRK1β1, the β regulatory subunit of SNF1-related protein kinase 1 (SnRK1) and promoted its phosphorylation in vivo, supporting that the TaSK41–TaSnRK1β1 module is associated with carbohydrate metabolism. Transcriptomic profiling revealed coordinated changes in genes related to phytohormone signaling, cell-wall remodeling, and starch/sucrose metabolism in developing grains of task41 mutants. Moreover, haplotype association analysis revealed that TaSK41-5B-HapI was significantly associated with higher thousand-grain weight across 233 hexaploid wheat accessions. These results demonstrate that TaSK41 acts as a negative regulator of wheat grain size and weight and provide genetic and haplotype resources for yield improvement.