As there is a strong interest in red-skinned pears, the molecular mechanism of anthocyanin regulation in red-skinned pears has been widely investigated; however, little is known about the molecular mechanism of anthocyanin regulation in red-fleshed pears due to limited availability of such germplasm, primarily found in European pears (Pyrus communis).  In this study, based on transcriptomic analysis in red-fleshed and white-fleshed pears, we identified an ethylene response factor (ERF) from P. communis, PcERF5, of which expression level in fruit flesh was significantly correlated with anthocyanin content.  We then verified the function of PcERF5 in regulating anthocyanin accumulation by genetic transformation in both pear skin and apple calli.  PcERF5 regulated anthocyanin biosynthesis by different regulatory pathways.  On the one hand, PcERF5 can activate the transcription of flavonoid biosynthetic genes (PcDFR, PcANS and PcUFGT) and two key transcription factors encoding genes PcMYB10 and PcMYB114.  On the other hand, PcERF5 interacted with PcMYB10 to form the ERF5-MYB10 protein complex that enhanced the transcriptional activation of PcERF5 on its target genes.  Our results suggested that PcERF5 functioned as a transcriptional activator in regulating anthocyanin biosynthesis, which provides new insights into the regulatory mechanism of anthocyanin biosynthesis.  This new knowledge will provide guidance for molecular breeding of red-fleshed pear.

The red coloring of pear fruits is mainly caused by anthocyanin accumulation.  Red sport, represented by the green pear cultivar ‘Bartlett’ (BL) and the red-skinned derivative ‘Max Red Bartlett’ (MRB), is an ideal material for studying the molecular mechanism of anthocyanin accumulation in pear.  Genetic analysis has previously revealed a quantitative trait locus (QTL) associated with red skin color in MRB.  However, the key gene in the QTL and the associated regulatory mechanism remain unknown.  In the present study, transcriptomic and methylomic analyses were performed using pear skin for comparisons between BL and MRB.  These analyses revealed differential PcHY5 DNA methylation levels between the two cultivars; MRB had lower PcHY5 methylation than BL during fruit development, and PcHY5 was more highly expressed in MRB than in BL.  These results indicated that PcHY5 is involved in the variations in skin color between BL and MRB.  We further used dual luciferase assays to verify that PcHY5 activates the promoters of the anthocyanin biosynthesis and transport genes PcUFGT, PcGST, PcMYB10 and PcMYB114, confirming that PcHY5 not only regulates anthocyanin biosynthesis but also anthocyanin transport.  Furthermore, we analyzed a key differentially methylated site between MRB and BL, and found that it was located in an intronic region of PcHY5.  The lower methylation levels in this PcHY5 intron in MRB were associated with red fruit color during development, whereas the higher methylation levels at the same site in BL were associated with green fruit color.  Based on the differential expression and methylation patterns in PcHY5 and gene functional verification, we hypothesize that PcHY5, which is regulated by methylation levels, affects anthocyanin biosynthesis and transport to cause the variations in skin color between BL and MRB.