Natural regulatory variations of MdIAA14b links auxin signalling to fruit cell density and firmness in apple.

Fruit firmness is a major determinant of apple quality and shelf life. It is known to be regulated by ethylene signalling, however, the function of auxin in it is not known. Here, we identified MdIAA14b, an auxin-responsive gene, as a key regulator of apple fruit firmness. MdIAA14b showed allele-specific expression (ASE) across multiple apple cultivars and developmental stages as revealed by RNA-seq analysis. Relative to the low expression allele, the promoter of the high expression allele contained two insertions of 9 bp and 1636 bp. The 9-bp insertion generated an ARF-binding site that enabled activation by the transcription factor MdARF1. The 1636-bp insert, identified as a transposable element (TE), disrupted and reduced the expression of a long non-coding RNA (lncRNA_iaa14b), which exhibited an inverse expression pattern relative to MdIAA14b. Apple cultivars homozygous for the high-expression allele displayed increased cell density, smaller cell size, and firmer fruit, whereas those homozygous for the low-expression allele had larger cells and softer fruit. MdIAA14b overexpression in tomato increased cell number and enhanced fruit firmness. Furthermore, MdIAA14b overexpression altered the expression of auxin-responsive genes, including members of ARF, SAUR, and GH3 families, which may relate to increased cell division. Together, these findings reveal that naturally occurring cis-regulatory variations control MdIAA14b expression and, consequently, apple fruit firmness. This work provides a molecular framework for improving fruit texture and highlights the role of cis-regulatory variation in allele-specific gene regulation in perennial fruit crops.