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.

Stone cells are a remarkable trait of pear (Pyrus spp.) fruit, with their abundance directly affecting fruit quality and varying significantly among cultivars. While plant hormones influence fruit development, their role in stone cell formation remains poorly understood. Here, we conducted a co-expression network analysis using RNA-seq data from 206 pear (Pyrus pyrifolia), linking hormone biosynthesis genes with known regulators of stone cell formation. This analysis identified that gibberellin (GA) 2-oxidase 1-1 (GA2ox1-1), a GA oxidase gene whose expression correlates significantly with stone cell content across cultivars, increases during key developmental stages, and localizes specifically to stone cells, as revealed by RNA in situ hybridization. Functional validation showed that expression of GA2ox1-1 in pear fruit, callus, and Arabidopsis (Arabidopsis thaliana) resulted in significantly lower lignin content and markedly lower active GA levels. To elucidate the GA-mediated regulation of stone cell lignification, we identified the DELLA protein REPRESSOR of ga1-3 Like 3 (RGL3), a key component of the GA signaling pathway. RGL3 interacts with myeloblastosis transcription factor MYB169, a known positive regulator of lignin biosynthesis, and suppresses MYB169-mediated activation of lignin biosynthesis genes, thereby negatively regulating lignin biosynthesis. However, in the presence of GA, RGL3 inhibition was relieved, leading to MYB169 activation, upregulation of lignin biosynthesis genes, and enhanced lignin deposition. These findings clarify the role of GA in stone cell formation and reveal a regulatory mechanism involving the RGL3–MYB169 module. This work provides insights into modulating stone cell content to improve pear fruit quality.