Ppbbx24-del mutant positively regulates light-induced anthocyanin accumulation in the ‘Red Zaosu’ pear (Pyrus pyrifolia White Pear Group)

Red fruit peel is one of pear's most valuable economic traits and is mainly determined by anthocyanins. Many pear cultivars with a red peel originated from bud sports; however, little is known about the genetic mechanisms underlying this trait. We have previously identified a mutant PpBBX24 containing a 14-nucleotide deletion in the coding region (Ppbbx24-del) as the only known variant associated with the red coloration of the mutant ‘Red Zaosu’ pear (Pyrus pyrifolia White Pear Group). Herein, we analyzed the role of the mutant gene in red coloration and its mechanism of action. The results showed that light promoted red peel coloration in the ‘Red Zaosu’ pear, and Ppbbx24-del positively affected light-induced anthocyanin biosynthesis, while normal PpBBX24 had the opposite effects. Transient and stable transformation experiments confirmed that Ppbbx24-del could promote anthocyanin accumulation in pear fruit peels, calli, and tobacco flowers. Due to the loss of nuclear localization sequence (NLS) and viral protein (VP) domains, Ppbbx24-del co-localized in the nucleus and cytoplasm, whereas PpBBX24 localized only in the nucleus. Real-time PCR and transcriptome analyses indicated that PpMYB10 and PpHY5 are highly expressed in the ‘Red Zaosu’ pear. In yeast one-hybrid and dual-luciferase assays, Ppbbx24-del and PpHY5 independently promoted the expression of PpCHS, PpCHI, and PpMYB10 by binding to their promoters; however, PpBBX24 did not affect the expression of these genes. Additionally, we found that Ppbbx24-del and PpHY5 had additive effects on the expression of PpCHS, PpCHI, and PpMYB10, as they promote the expression of anthocyanin synthesis genes separately. The co-expression of PpBBX24 and PpHY5 inhibited the activation of downstream genes by PpHY5, which was attributed to the interaction between the two loci. In conclusion, our results clarify the molecular mechanism by which mutant Ppbbx24-del and PpBBX24 exert opposite effects in regulating anthocyanin accumulation in pear. These findings lay an important theoretical foundation for using Ppbbx24-del to create red pear cultivars.