Clade II SHAGGY-like kinases regulate fruit shape in tomato by modulating SlMAP65-1 stability.

Fruit shape is a key agronomic trait influencing harvest efficiency, post-harvest handling, and market value. Therefore, creating diverse fruit morphologies and elucidating their regulatory networks are major objectives in tomato breeding. Currently, although multiple genes regulating fruit shape (such as SUN, OVATE, FAS, etc.) have been identified, the molecular network governing tomato fruit shape remains incomplete. SHAGGY-like kinases (SKs)/Glycogen synthase kinase3 (GSK3) are evolutionarily conserved regulators of plant development and have been implicated in diverse processes. However, the biological functions of this family in tomato fruit development remain to be systematically elucidated. In this study, we performed systematic CRISPR/Cas9 mutagenesis of all nine members of the tomato SHAGGY-like kinases family (SlSKs), generating higher order mutants across different clades. Phenotypic screening revealed that SlSKs clade II (SlSK2s) members (SlSK21/SlSK22/SlSK23) redundantly regulate fruit shape by acting as negative regulators, controlling cell expansion in the columella and modulating ovary wall cell division patterns during ovary development. Proteomics analysis revealed a significant reduction of the microtubule-associated protein SlMAP65-1 in the slsk2s mutant lines. Our results further confirmed that SlMAP65-1 interacts with SlSK21, SlSK22, and SlSK23, respectively, in planta. Phosphorylation assays demonstrated that SlSK23 directly phosphorylates SlMAP65-1 at Ser504 and Ser532, thereby enhancing its protein stability. These results shed new light on the molecular mechanism by which SlSK2s modulate fruit shape in tomato.