Transcriptome-based analysis reveals a role for PpCDF5 in the promotion of anthocyanin accumulation at a low nighttime temperature

For red pear, the anthocyanin content is a crucial factor determining the fruit skin color, which affects consumer preferences. Low overnight temperatures promote anthocyanin accumulation, but the molecular mechanism responsible is unclear. In this study, ‘Hongzaosu’ pear (Pyrus pyrifolia × Pyrus communis) fruit were treated with a low nighttime temperature (LNT, 16 ℃) or a warm nighttime temperature (WNT, 26 ℃), with sampling conducted within two diurnal cycles. The results showed that LNT promoted anthocyanin accumulation in the fruit skin. The structural anthocyanin biosynthetic genes PpCHS, PpF3H, and PpUFGT exhibited a rhythmic increase in expression at night under LNT. To examine the underlying mechanism, RNA sequencing was conducted using pear calli exposed to LNT and WNT for different durations (24, 48, 72, or 96 h). Transcriptome analysis revealed 285 differentially expressed genes (DEGs) common to all pairwise comparisons of LNT- and WNT-treated calli of ‘Clapp's Favorite’ (P. communis) at the sampling time points. KEGG pathway and gene ontology enrichment analyses indicated that the common DEGs were enriched in secondary metabolic processes and phenylpropanoid metabolic processes, which are associated with anthocyanin biosynthesis. The transcription factor PpCDF5, which was responsive to LNT, was selected for further study. Dual-luciferase assays showed that PpCDF5 activated the transcription of anthocyanin biosynthetic genes PpMYB10, PpCHS, PpF3H, PpDFR, PpANS, and PpUFGT. The yeast one-hybrid and EMSA assays demonstrated that PpCDF5 directly binds to the PpF3H promoter, which contains an AAAG motif. Overexpression of PpCDF5 in pear calli and transient overexpression in pear fruit both increased anthocyanin accumulation. The results indicate that PpCDF5 is involved in LNT-induced anthocyanin biosynthesis in pear fruit and provide insights into the molecular regulation of commercial fruit coloration.