Abstract: Lignified stone cells are a unique feature of pear fruit, significantly affecting fruit texture. Even though some research efforts have already been made, the stone cell formation mechanism is complex, with many aspects yet to be elucidated. Here, through a genome-wide association analysis of stone cell traits, we identified PbrMADS1, a member of the SEPALLATA3 (SEP3) subfamily, as a candidate gene specifically expressed in stone cells during early fruit development. Functional studies confirmed that PbrMADS1 promotes stone cell formation; however, it does not directly activate lignin-related genes. Instead, PbrMADS1 interacts with PbrMYB169, enhancing PbrMYB169's binding to AC elements and amplifying downstream gene activation. Notably, homologous MADS1 and MYB169 proteins from closely related species such as apple and loquat do not form a similar complex. Sequence analysis revealed that the protein sequence of PbrMADS1 contains methionine (M) at the 63^rd amino acid position, while apple and loquat homologs carry threonine (T) at the same site. Substituting M with T (PbrMADS1^M63T) weakened its interaction with PbrMYB169 and impaired its function in regulating stone cell formation. This study offers new insights into MADS gene-mediated stone cell formation and highlights functional divergence within the SEP3 subfamily among apple tribe species of the Rosaceae family.

Key words: functional divergence, PbrMADS1–PbrMYB169 complex, pear, stone cells