Ca2+ suppresses stone cell through PuNAC21–PuDof2.5 module that 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 (<i>PuPRX42</i>-like) promoter, Ca²⁺ reduced pear fruit stone cell production dependent on PuNAC21 positively regulating <i>PuPRX42</i>-like expression. Furthermore, PuNAC21 directly regulated the expression of <i>PuDof2.5</i>, a TF involved in lignin biosynthesis by binding to <i>PuPRX42</i>-like and caffeoyl-CoA-O-methyltransferase 1(<i>PuCCoAOMT1</i>) promoters. Moreover, PuNAC21 interacted with PuDof2.5 to form a transcriptional regulatory module, lowering the transcription of <i>PuPRX42</i>-like and <i>PuCCoAOMT1</i> 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.