Ca²⁺ suppresses stone cell through PuNAC21-PuDof2.5 module regulates lignin biosynthesis in pear fruits

Lignin deposition in stone cells is a critical factor that limits pear fruit quality, affecting their market value. Calcium ions (Ca ²⁺) play an essential role in lignin biosynthesis during fruit stone cell production. However, the genetic mechanisms underlying the Ca ²⁺ regulated lignin synthesis in stone cell formation are not fully understood. In this study, we identified an NAC transcription factor (TF) PuNAC21, which is repressed by CaCl₂ treatment. PuNAC21 bound directly to the lignin biosynthesis gene peroxidase 42-like (PuPRX42-like) promoter, Ca²⁺ reduced pear fruit stone cell production dependent on PuNAC21 positively regulating PuPRX42-like expression. Furthermore, PuNAC21 directly regulated the expression of PuDof2.5, a TF involved in lignin biosynthesis by binding to PuPRX42-like and caffeoyl-CoA-O-methyltransferase 1(PuCCoAOMT1) promoters. Moreover, PuNAC21 interacted with PuDof2.5 to form a transcriptional regulatory module, lowering the transcription of PuPRX42-like and PuCCoAOMT1 after Ca²⁺ treatment, which contributed to decrease pear stone cells production. Our results revealed Ca²⁺-induced PuNAC21-PuDof2.5-PuPRX42-like/PuCCoAOMT1 regulatory module inhibited lignin biosynthesis, giving important insights into reducing the stone cell content in pears via molecular breeding.