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.

Genetic variation contributes to the phenotypic diversity of plants. Most red pear cultivars that accumulate anthocyanins originated from somatic variation and are termed as bud sports. ‘Hongzaosu’ is a bud sport derived from the green pear ‘Zaosu’. A new genetic map was constructed from a population derived from the cross of ‘Yuluxiang’ and ‘Hongzaosu’, from which PpBBX24 was identified as a crucial gene controlling anthocyanin accumulation. Genetic and phylogenetic evidences revealed that PpBBX24 is a repressor of anthocyanin biosynthesis in pear. A 14 bp deletion was detected in the third exon of PpBBX24 in ‘Hongzaosu’, resulting in premature termination of protein translation. The truncated PpBBX24 protein was a positive regulator of anthocyanin biosynthesis by activating the transcription of PpCHS and PpUFGT. Three independent variations in the BBX24 coding region that led to premature termination of translation were detected in other pear bud sports and the progeny of a bud sport that all accumulate anthocyanins. The genome of ‘Hongzaosu’ was assembled using both long-read and short-read sequences. By combining genomic and transcriptomic data, allele-specific expression of PpMYB110a was observed in the fruit skin of ‘Hongzaosu’, which was likely caused by a large variation in the promoter. This work enhances understanding of coloration in red pears and provides a novel genomic resource for further studies on ‘Hongzaosu’ pear.