SlMYB73 acts as a negative regulator of fruit ripening by recruiting histone deacetylase HDA3 in tomato

Ripening is a complex process involving various physiological changes determining fruit nutritional value and quality. Studies have demonstrated that multiple transcription factors and hormones establish a network to regulate fruit ripening. However, the negative regulators of fruit ripening and their underlying molecular mechanisms remain largely elusive in tomato. Here, we characterize an ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif-containing transcription factor SlMYB73 that negatively regulates the onset of tomato fruit ripening. Initial analysis revealed that SlMYB73 exhibited a reduced expression during fruit ripening and responded negatively to exogenous ethylene in tomato. The knockout mutants of SlMYB73 generated using the CRISPR/Cas9 system showed accelerated ethylene production and enhanced carotenoid accumulation, resulting in premature ripening. Further molecular and biochemical approaches demonstrated that SlMYB73 directly represses the expression of ripening-related genes, including ACO1, ACO3, ACS2, ACS4, and PSY1. In vitro and in vivo analyses demonstrated the physical interaction of SlMYB73 with histone deacetylases HDA3/6. Dual-luciferase reporter assay showed that co-expression of SlMYB73 and HDA3 enhanced the transcriptional repression of ripening-related genes. ChIP assay demonstrated significantly higher H3K9ac levels of targeting ripening-related genes in SlMYB73-CR fruits. Furthermore, expression of SlMYB73 was directly inhibited by Auxin Responsive Factor 2A (SlARF2A), which positively regulates tomato fruit ripening. Thus, our study revealed that SlMYB73 negatively regulates fruit ripening in tomato by recruiting HDA3 and mediates the crosstalk between ethylene and ABA/auxin by acting downstream of SlARF2A.