Transcriptome Co-Expression Network Analysis Identifies Key Genes and Regulators of Sweet Cherry Anthocyanin Biosynthesis

Anthocyanin is the key factor that results in the attractive color of sweet cherry fruits. However, information regarding sweet cherry coloration and the potential mechanisms underlying anthocyanin biosynthesis is limited. In this study, we found that the anthocyanin accumulation varied in sweet cherry flesh and peel, while the anthocyanin content increased sharply in the dark red (DR) stage. Correlations between anthocyanin concentrations and RNA sequencing (RNA-seq), constructed with Weighted Gene Co-Expression Network Analysis (WGCNA), indicated that two structural genes (Pac4CL2, PacANS) and 11 transcription factors (PacbHLH13/74, PacDIV, PacERF109/115, PacGATA8, PacGT2, PacGTE10, PacMYB308, PacPosF21, and PacWRKY7) had similar expression patterns with the changes in anthocyanin content. Additionally, real-time PCR verified all of these gene expression patterns and revealed that PacANS exhibited the highest transcription level. In order to search for potential regulators for anthocyanin biosynthesis, a dual-luciferase assay was performed to investigate the regulatory activities of 11 transcription factors on the PacANS promoter. The results revealed that two novelty bHLHs, PacbHLH13 and PacbHLH74, can trans-activate the PacANS promoter and they might be the candidate genes for regulating anthocyanin synthesis in sweet cherry fruits. The present findings provide a novel viewpoint with regard to anthocyanin biosynthesis mechanisms and the regulatory transcriptional network of fruit coloration in sweet cherries.