Ca²⁺ mediated PbCML46-PbbHLH96 module negatively regulates lignin biosynthesis in pear fruit

Stone cell formation, resulting from aberrant lignin deposition in parenchyma cells, is a key determinant of pear fruit quality. Although exogenous calcium application is known to inhibit lignin biosynthesis and stone cell development, the underlying molecular mechanism involving calcium sensor proteins remains poorly understood. This study aimed to elucidate the molecular pathway by which calcium signaling modulates lignin biosynthesis. Our findings demonstrate that the calmodulin-like protein PbCML46 significantly suppresses lignin accumulation. This function was validated through complementary approaches, including transient injection in pear fruit and stable overexpression in pear calli. A yeast two-hybrid screen revealed that PbCML46 specifically interacts with the bHLH transcription factor PbbHLH96, an interaction further confirmed by pull-down and luciferase complementation imaging (LCI) assays. Further yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA), and luciferase (LUC) reporter assays analysis showed that PbbHLH96 directly binds to the MYC cis-element in the PbCAD6 promoter and functions as a transcriptional repressor. Dual-luciferase reporter (DLR) and in vivo co-expression assays indicated that PbCML46 markedly enhances the repressive activity of PbbHLH96 on the PbCAD6 promoter, an effect strictly dependent on PbbHLH96. In conclusion, this study reveals a Ca²⁺-initiated regulatory cascade in which calcium signaling promotes the interaction between the sensor PbCML46 and the transcriptional repressor PbbHLH96, leading to cooperative suppression of the key lignin biosynthetic gene PbCAD6 and thereby negatively regulating lignin deposition in pear stone cells. These findings provide novel mechanistic insights into how calcium signaling improves fruit quality.