MADS gatekeepers: Ovule protein complexes ensure fertilization-dependent fruit set in tomato

Fruit set, the transition from an arrested ovary to a developing fruit, is typically triggered by ovule fertilization. However, the mechanisms underlying ovary growth arrest remain poorly understood. We have found that in tomatoes, ovaries of AGAMOUS-LIKE6 (SlAGL6) loss-of-function mutant (slagl6CR-sg1) develop normally until anthesis but, instead of arresting, continue to grow and set normal yet seedless fruits independent-of-fertilization (parthenocarpic fruits). This indicates that the MADS-domain transcription factor (MTF) SlAGL6 is pivotal for maintaining ovary arrest and suppress parthenocarpy. Restoring SlAGL6 expression specifically in ovules rescues the parthenocarpic phenotype of slagl6CR-sg1, suggesting that SlAGL6 functions within ovules to enforce ovary arrest. Transcriptome analysis of isolated slagl6CR-sg1 ovules suggests that SlAGL6 prevents parthenocarpy by regulating the transcription of a set of genes involved in the fertilization-induced reprogramming of ovules that underlies fruit set. Screening a yeast two-hybrid library of arrested ovaries using the SlAGL6 protein identified several ovule-expressed MADS transcription factors (MTFs) as candidate interactors (CIs). We demonstrated that SlAGL6 forms heterodimers with all in planta and acts as a scaffold, enabling CI2 and CI3 to interact and assemble into multimeric complexes. AlphaFold modeling predicts that these complexes adopt tetrameric Flower Quartet-like configurations. Genetic interaction study suggest redundant roles for SlAGL6-CI2-SlAGL6-CI2, SlAGL6-CI3-SlAGL6-CI3, and SlAGL6-CI2-SlAGL6-CI3 complexes in suppressing parthenocarpy. Moreover, it suggests the presence of CI3-based complexes that do not involve SlAGL6 but contribute to parthenocarpy suppression. Together, our findings reveal that SlAGL6, CI2, and CI3 function as subunits of Flower Quartet-like complexes in ovules to promote ovary arrest and prevent parthenocarpy.