CLASS-II KNOX genes coordinate spatial and temporal patterns of the tomato ripening

Ripening is a complex developmental change of a mature organ, the fruit. In plants like a tomato, it involves softening, pigmentation, and biosynthesis of metabolites beneficial for the human diet. Examination of the transcriptional changes towards ripening suggests that redundant uncharacterized factors may be involved in the coordination of the ripening switch. Previous studies have demonstrated that Arabidopsis CLASS-II KNOX genes play a significant role in controlling the maturation of siliques and their transition to senescence. Here we examined the combined role of all four tomato CLASS-II KNOX genes in the maturation and ripening of fleshy fruits using an artificial microRNA targeting them simultaneously. As expected, the knockdown plants (35S::amiR-TKN-CL-II) exhibited leaves with increased complexity, reminiscent of the leaf phenotype of plants overexpressing CLASS- I KNOX, which antagonize CLASS-II KNOX gene functions. The fruits of 35S::amiR-TKN-CL-II plants were notably smaller than the control. While their internal gel/placenta tissue softened and accumulated the typical pigmentation, the pericarp color break took place ten days later than control, and eventually, it turned yellow instead of red. Additionally, the pericarp of 35S::amiR-TKN-CL-II fruits remained significantly firmer than control even after three weeks of shelf storage. Strikingly, the 35S::amiR-TKN-CL-II fruits showed early ethylene release and respiration peak, but these were correlated only with liquefaction and pigmentation of the internal tissues. Our findings suggest that CLASS-II KNOX genes are required to coordinate the spatial and temporal patterns of tomato fruit ripening.