MADS-BOX PROTEIN3 acts cooperatively with AGAMOUS-like6 and MADS-BOX PROTEIN22 to suppress parthenocarpy in tomato.

Ovule fertilization initiates fruit set, the transition from a quiescent ovary to a growing ovary (fruit), yet how ovary growth is arrested before fertilization remains poorly understood. Previously, we have shown that the ovaries of the tomato (Solanum lycopersicum) AGAMOUS-like6 (SlAGL6) loss-of-function mutant (slagl6CR-sg1) develop normally until anthesis but nevertheless can set fruits independently of fertilization (parthenocarpy). Here, we demonstrate that SlAGL6 suppresses parthenocarpy specifically from the ovules, as reintroducing SlAGL6 into slagl6CR-sg1 using an ovule-specific promoter restored ovary arrest and eliminated parthenocarpy. A yeast 2-hybrid (Y2H) screen of a cDNA library derived from arrested and developing ovaries identified MADS-domain protein3 (SlMBP3) and MADS-domain protein22 (SlMBP22), both predominantly expressed in arrested ovary ovules, as high-confidence SlAGL6 interactors. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-mediated knockout of SlMBP3 and SlMBP22, followed by genetic interaction study involving slagl6CR-sg1, revealed that SlMBP3 acts cooperatively with SlAGL6 and SlMBP22 to suppress parthenocarpy. Protein interaction assays, together with AlphaFold-based structural predictions, revealed that SlMBP3 and SlMBP22 form homomeric and heteromeric complexes in planta, but only in the presence of SlAGL6, and that these complexes are predicted to adopt a Flower Quartet-like complex (FQC) structure. Taken together, our results establish a mechanistic framework for ovary arrest regulation, indicating that complexes containing SlAGL6, SlMBP22, and SlMBP3 operate within ovules to arrest ovary growth and prevent parthenocarpy.