<i>CmPLL16a</i> and <i>CmPLL16b</i> Regulate Cell Wall Remodelling and Somatic Embryogenesis in <i>Castanea mollissima</i>

Chinese chestnut (Castanea mollissima [C. mollissima] Blume), an important nut crop with recalcitrant seeds and contributor to forest ecosystem services, faces challenges due to seed abortion, which seriously influences yield. In this study, stark microstructural and ultrastructural differences were observed between fertile and abortive seeds, with the abortive seeds exhibiting abnormal cell wall shapes and irregular thickening. Immunohistochemical labelling revealed that the content of demethylesterified homogalacturonan (HG) pectin in the cell wall of abortive seeds was significantly lower than fertile seeds. Through a genome-wide analysis, a total of 16 Pectate Lyase-Like (PLL) genes were identified in C. mollissima. Based on the qRT-PCR analysis and subcellular localization observation, CmPLL16a and CmPLL16b were found to be highly expressed in abortive seeds and localized to the cell wall. Silencing of CmPLL16a and CmPLL16b led to an increase in demethylesterified HG pectin concentrations, thereby enhancing somatic embryogenesis in the RNAi lines. In contrast, overexpression of CmPLL16a and CmPLL16b resulted in reduced demethylesterified HG pectin concentrations and decreased embryo production in calli. This study pioneers gene knockdown in Chinese chestnut somatic embryos to analyze gene function, achieving a key technical breakthrough. These findings reveal CmPLLs' roles and provide genetic targets for enhancing somatic embryogenesis in C. mollissima.