Light-Induced Basic/Helix-Loop-Helix64 Enhances Anthocyanin Biosynthesis and Undergoes CONSTITUTIVELY PHOTOMORPHOGENIC1-Mediated Degradation in Pear

Light is indispensable for the anthocyanin accumulation of red pear (Pyrus pyrifolia). Anthocyanin biosynthesis is catalyzed by a series of enzymes encoded by structural genes, which are regulated by a MYB-basic/helix-loop-helix-WD repeat (MYB-bHLH-WDR [MBW]) complex. The bHLH proteins of subgroup (SG) IIIf are believed to be involved in the regulation of anthocyanin accumulation. In this study, we revealed that pear PpbHLH64, which belongs to SGIIIb, positively regulates anthocyanin biosynthesis and is regulated by light at the transcriptional and posttranslational levels. Specifically, an exposure to light induced PpbHLH64 expression and anthocyanin accumulation in pear fruit and calli. Under light conditions, pear calli overexpressing PpbHLH64 exhibited enhanced red coloration, whereas the anthocyanin accumulation decreased in the PpbHLH64-RNA interference calli. Additionally, the transient overexpression of PpbHLH64 in pear fruit peel increased anthocyanin accumulation, whereas the virus-induced gene silencing of PpbHLH64 had the opposite effect. Further analyses indicated that PpbHLH64 is a transcriptional activator that directly binds to the promoter of UDP-GLUCOSE:FLAVONOID 3-O-GLYCOSYLTRANFERASE to upregulate expression. Moreover, PpbHLH64 interacted with PpMYB10, but not with PpMYB114, to form an MBW complex that significantly induces the accumulation of anthocyanins. Furthermore, PpbHLH64 was targeted by CONSTITUTIVE PHOTOMORPHOGENIC1 in darkness for subsequent degradation by the 26S proteasome. A genetic analysis indicated that PpbHLH64 functions downstream of B-BOX18, a component of the light signal transduction pathway. However, we were unable to detect the direct interaction between PpbHLH64 and PpBBX18. The characterization of PpbHLH64 in this study highlights the importance of SGIIIb bHLH proteins for light-induced anthocyanin accumulation.