Solanum lycopersicum CLASS-II KNOX genes regulate fruit anatomy via gibberellin-dependent and independent pathways.

The pericarp is the predominant tissue determining the structural characteristics of most fruits. However, the molecular and genetic mechanisms controlling pericarp development remain only partially understood. Previous studies have identified that CLASS-II KNOX genes regulate fruit size, shape, and maturation in Arabidopsis thaliana and Solanum lycopersicum. Here we characterized the roles of the S. lycopersicum CLASS-II KNOX (TKN-II) genes in pericarp development via a detailed histological, anatomical, and karyotypical analysis of TKN-II gene clade mRNA-knockdown (35S:amiR-TKN-II) fruits. We identify that 35S:amiR-TKN-II pericarps contain more cells around their equatorial perimeter and fewer cell layers than the control. In addition, the cell sizes but not the ploidy levels of these pericarps were dramatically reduced. Further, we demonstrate that fruit shape and pericarp layer number phenotypes of the 35S:amiR-TKN-II fruits can be overridden by the procera mutant, known to induce a constitutive response to the plant hormone gibberellin. However, neither the procera mutation nor exogenous gibberellin application can fully rescue the reduced pericarp width and cell size phenotype of 35S:amiR-TKN-II pericarps. Our findings establish that TKN-II genes regulate tomato fruit anatomy, acting via gibberellin to control fruit shape but utilizing a gibberellin-independent pathway to control the size of pericarp cells.

CLASS-II KNOX genes coordinate spatial and temporal ripening in tomato.

Fruits can be divided into dry and fleshy types. Dry fruits mature through senescence and fleshy fruits through ripening. Previous studies have indicated that partially common molecular networks could govern fruit maturation in these different fruit types. However, the nature of such networks remains obscure. CLASS-II KNOX genes were shown to regulate the senescence of the Arabidopsis (Arabidopsis thaliana) dry fruits, the siliques, but their roles in fleshy-fruit development are unknown. Here, we investigated the roles of the tomato (Solanum lycopersicum) CLASS-II KNOX (TKN-II) genes in fleshy fruit ripening using knockout alleles of individual genes and an artificial microRNA line (35S:amiR-TKN-II) simultaneously targeting all genes. 35S:amiR-TKN-II plants, as well as a subset of tkn-II single and double mutants, have smaller fruits. Strikingly, the 35S:amiR-TKN-II and tknII3 tknII7/+ fruits showed early ripening of the locular domain while their pericarp ripening was stalled. Further examination of the ripening marker-gene RIPENING INHIBITOR (RIN) expression and 35S:amiR-TKN-II rin-1 mutant fruits suggested that TKN-II genes arrest RIN activity at the locular domain and promote it in the pericarp. These findings imply that CLASS-II KNOX genes redundantly coordinate maturation in both dry and fleshy fruits. In tomato, these genes also control spatial patterns of fruit ripening, utilizing differential regulation of RIN activity at different fruit domains.