Fruit firmness is an important trait for characterizing the quality and value of apple. It is also serves as an indicator of fruit maturity, as it is a complex trait regulated by multiple genes. Re-sequencing techniques can be employed to elucidate variations in such complex fruit traits. Here, the whole genomes of 294 F₁ hybrids of ‘Fuji’ and ‘Cripp's Pink’ were re-sequenced, and a high-density bin-map was constructed using 5,014 bin markers with a total map distance of 2,213.23 cM and an average map distance of 0.44 cM. Quantitative trait loci (QTLs) of traits related to fruit were mapped, and an A-T allele variant identified in the coding region of MdNAC5 was found to potentially regulate fruit firmness and ripening. The overexpression of MdNAC5^A resulted in higher production of methionine and 1-aminocyclopropanecarboxylic acid compared to MdNAC5^T, leading to reduced fruit firmness and accelerated ripening in apples and tomatoes. Furthermore, the activities of MdNAC5^A and MdNAC5^T were enhanced through their differential binding to the promoter regions of MdACS1 and MdERF3. Spatial variations in MdNAC5^A and MdNAC5^T caused changes in MdACS1 expression following their interaction with MdERF3. Ultimately, utilizing different MdNAC5 alleles offers a strategy to manipulate fruit firmness in apple breeding.

Sunburn in apple peel significantly affects fruit appearance and reduces its commercial value. Previous research has shown that apple peel reduces sunburn by increasing the accumulation of proanthocyanidins (PAs) and other protective compounds. However, the precise molecular regulatory mechanism remains unclear. In this study, we systematically investigated MdANR, a key gene involved in PAs biosynthesis. We found that MdANR expression in apple peel is responsive to temperature and light fluctuations, with higher expression levels observed under increased temperature and light exposure. Functional analysis revealed that MdANR overexpression in apple peel and callus enhanced resistance to high-temperature and -light-intensity stress, accompanied by a corresponding increase in PAs and chlorogenic acid contents. In addition, we demonstrated that MdMYBR9 can activate MdANR promoter activity and promote its expression through yeast one-hybrid, dual-luciferase, and electrophoretic mobility transfer experiments. The results indicated that MdMYBR9 was an upstream regulator of MdANR. Based on these findings, this study proposes the MdMYBR9-MdANR-PAs regulatory model for apple sunburn resistance, providing a molecular framework for enhancing sunburn tolerance in apple breeding programs.