SlABF4 acts as a negative regulator of ethylene biosynthesis in tomato fruit ripening

In plants, ABF (ABA-responsive element binding factor)/AREB (ABA-responsive element binding) proteins regulate processes such as abiotic stress response, pathogen defense, and seed germination. However, the specific function and regulatory networks of ABF/AREB transcription factors during fruit ripening remain largely unknown. In this study, we found that the expression level of SlABF4 is abundant in tomato fruits and gradually decreases during the ripening process. Functional analysis confirmed that SlABF4 was localized in the cell nucleus and responded to ABA treatment. To further investigate the function of SlABF4 in fruit ripening, we generated overexpressing SlABF4 lines and CRISPR/Cas9-slabf4 knockout mutants. Compared with wild type (WT), the onset of fruit ripening in OE-SlABF4 lines was delayed and ethylene production reduced, while the slabf4-deficient mutants showed accelerated fruit ripening and increased ethylene release. Meanwhile, transcriptomic and qRT-PCR analyses indicated that the expression levels of several fruit ripening-related transcriptional factors and ethylene-associated genes were downregulated in OE-SlABF4 lines and upregulated in CR-slabf4 mutants. Importantly, yeast one-hybrid (Y1H), dual-luciferase reporter assays and electrophoretic mobility shift assays (EMSA) demonstrated that SlABF4 directly binds to the promoters of ethylene biosynthesis genes (SlACS2 and SlACS12) and represses their expression. Moreover, we found that SlABF4 can physically interact with two key ripening-related transcription factors (SlFUL1 and SlMADS1) to regulate tomato fruit ripening in vivo and in vitro, while SlABF4 inhibited the expression of SlACS2 to modulate ethylene biosynthesis. Together, these findings expand our insights into the negative regulatory role and molecular mechanism of SlABF4 in the fruit ripening process, which will provide support for precise breeding designed to mediate fruit ripening in tomato.