The PbMYB16–PbMYB308 module regulates stone cell lignification by activating the metacaspase PbMC1 in pear

Stone cells, which possess heavily lignified secondary cell walls, are a critical determinant of pear fruit texture and quality. Programmed cell death is known to accompany stone cell formation, yet the molecular link between programmed cell death signaling and lignin biosynthesis remains largely unexplored. Among the developmental stages examined (25, 45, and 65 DAFB), both programmed cell death signals and lignified stone cell area were most pronounced at 45 DAFB in pear fruitlets. Transcriptome profiling of the stone-cell-cluster-enriched and flesh-derived fractions at this stage identified a metacaspase gene, PbMC1, which is highly expressed in the stone-cell-cluster-enriched fraction. Functional analyses using transient overexpression, virus-induced gene silencing, and stable transformation demonstrated that PbMC1 positively regulates lignin biosynthesis, and its overexpression also enhances programmed cell death signals. Through yeast one-hybrid screening, we identified a MYB transcription factor, PbMYB16, that directly binds the PbMC1 promoter and activates its transcription. Targeted knockout of PbMYB16 reduced both PbMC1 expression and lignin content. Moreover, PbMYB16 transcription was rapidly induced by exogenous H₂O₂. Yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays revealed that PbMYB16 interacts with PbMYB308. Dual-luciferase reporter assays showed that PbMYB308 suppresses PbMYB16-mediated activation of PbMC1. Further functional experiments confirmed that PbMYB308 attenuates the lignin-promoting function of PbMYB16 through this physical interaction. Our findings uncover a PbMYB16/PbMYB308–PbMC1 regulatory module that connects programmed cell death signaling to stone cell lignification, offering new insights into pear fruit texture development and potential targets for quality improvement.