TaDEP1 regulates grain size and abiotic stress tolerance by interacting with TaMyb309 in bread wheat.

UNLABELLED: TaDEP1, a heterotrimeric G protein γ subunit gene, was characterized as a positive regulator of grain size and thousand-grain weight (TGW) in bread wheat. Association analysis in 492 global wheat accessions and validation in a recombinant inbred line population identified a missense SNP (C/G) in TaDEP1-5 A that causes a cysteine to tryptophan substitution, with the TaDEP1-5 A-C allele significantly associated with increased grain width and TGW. This favorable allele has been preferentially selected during breeding, increasing from 15.10% in landraces to 45.20% in modern cultivars. CRISPR/Cas9 knockout of all three TaDEP1 homoeologs reduced grain length (12.25-13.16%), grain width (15.01-18.25%), and TGW (42.31-45.67%). Notably, tadep1 mutants exhibited enhanced tolerance to salt, drought, alkali, and heat stress, revealing a trade-off where TaDEP1 promotes yield but represses stress tolerance. Yeast two-hybrid and luciferase complementation assays identified TaMyb309, a grain-specific MYB transcription factor, as a TaDEP1 interactor. BSMV-mediated CRISPR/Cas9 mutants of TaMyb309 showed increased grain size and weight, indicating it acts as a negative regulator antagonistic to TaDEP1. Our findings establish TaDEP1 as a key regulator balancing grain yield and stress adaptation, possibly through interaction with TaMyb309. A functional KASP marker (KS17) developed for the elite TaDEP1-5 A-C haplotype provides a valuable tool for marker-assisted selection in wheat breeding. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at https://doi.org/10.1007/s11032-026-01710-4.