Transcriptome analysis reveals the regulation of PhAN2 on PhANS, PhRT and PhAN9 in petunia

Anthocyanins are the main pigments in the flowers of most plant species. Previous studies showed that petunia PhAN2, a R2R3-MYB transcription factor, exclusively controlled anthocyanin biosynthesis in the corollas by regulating dihydroflavonol 4-reductase (PhDFR), chalcone synthase J (PhCHSJ). Recently, we identified anthocyanin acyltransferase 1 (PhAAT1) and demonstrated that PhAN2 directly regulates the transcription of PhAAT1. However, it is unclear whether AN2 regulates the genes of other pathways and other structural genes of the anthocyanin pathway. In this study, PhAN2-silenced petunia plants were gained by virus-induced gene silencing (VIGS) and transcriptome analysis showed that a total of 2438 differential expression genes (DEGs), including 832 downregulated and 1606 upregulated genes, were identified. KEGG pathway analysis revealed that 9 pathways were associated with downregulated genes, while 18 pathways were linked to upregulated genes. Six DEGs of the anthocyanin pathway were identified and among them, besides PhDFR, PhCHSJ and PhAAT1, anthocyanidin synthase (PhANS), glutathione s-transferase (PhAN9), rhamnosyl transferase (PhRT) were downregulated. The assay of Y1H and dual luciferase-based transactivation confirmed that PhAN2 directly regulated the transcripts of PhANS, PhRT and PhAN9 in petunia. At last, VIGS-mediated PhANS, PhAN9, and PhRT silencing phenotypes were analyzed.