PpBBX32 and PpZAT5 modulate temperature-dependent and tissue-specific anthocyanin accumulation in peach fruit

Anthocyanins are important compounds for fruit quality and nutrition. The R2R3 MYB transcription factor PpMYB10.1 is known to be critical for regulating anthocyanin accumulation in peach. However, regulatory factors upstream of PpMYB10.1 which control temperature-dependent, cultivar-contrasted and tissue-specific anthocyanin accumulation remain to be determined. In this study, differential anthocyanin accumulation in the outer flesh near the peel (OF) of peach [Prunus persica (L.) Batsch] was observed between cultivars 'Zhonghuashoutao' and 'Dongxuemi', as well as among different storage temperatures and different fruit tissues of 'Zhonghuashoutao'. By cross-comparisons of RNA-Seq data of samples with differential anthocyanin accumulation, transcription factor genes PpBBX32 and PpZAT5 were identified. These were functionally characterized as two positive regulators for anthocyanin accumulation via transient expression and genetic transformation. Various interaction assays revealed that both PpBBX32 and PpZAT5 can directly activate the PpMYB10.1 promoter and meanwhile interact at protein level as a PpZAT5-PpBBX32-PpMYB10.1 complex. Furthermore, the results of in silico analysis and exogenous application of methyl jasmonate (MeJA) indicated that MeJA favored anthocyanin accumulation, while it was also found that anthocyanin accumulation as well as PpBBX32 and PpZAT5 expression correlated significantly with endogenous JA and JA-Ile in different fruit tissues. In summary, PpBBX32 and PpZAT5 are upstream activators of PpMYB10.1, allowing JAs to take part in temperature-dependent and tissue-specific anthocyanin accumulation by modulating their expression. This work enriches the knowledge of the transcriptional regulatory mechanisms for differential anthocyanin accumulation under internal and external factors.