GA2ox1-1 modulates bioactive gibberellin levels to regulate the RGL3-MYB169 pathway in pear stone cell formation

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.