ERF4 affects fruit firmness through TPL4 by reducing ethylene production.

The firmness of fleshy fruit crops has a significant effect on their quality, consumer preference, shelf life and transportability. In a combined quantitative trait locus and genome-wide association studies study of apple fruit texture, we identified a mutation (C-G) in the ethylene response factor-associated amphiphilic repression (EAR) motif in the coding region of the apple ETHYLENE RESPONSE FACTOR4 (ERF4) gene. Chromatin immunoprecipitation sequencing showed that ERF4 binds to the promoter of ERF3, which is involved in regulation of ethylene biosynthesis. The EAR mutation in ERF4 results in reduced repression of ERF3 expression, which is turn promotes ethylene production and loss of fruit firmness. ERF4 acts as a transcriptional repressor whose activity is modulated by a TOPLESS co-repressor 4 (TPL4)-binding EAR repression motif. Biolayer interferometry analysis showed that the mutation in the EAR motif causes a reduction in the interaction with TPL4. Suppression of ERF4 or TPL4 promoted fruit ripening and ethylene production. Taken together, our results provide insights into how ERF4 allelic variation underlies an important fruit quality trait.