Regulation Mode of RaMYB1 of Ranunculus asiaticus L. in Anthocyanin Biosynthesis Responding to Temperature Decrease

Abstract

Color is of great importance for the testing and breeding of plant varieties, and is known to be varied along with temperature changes. However, at the transcription level, the exact binding sites and corresponding regulation properties in the pathway of anthocyaninn biosynthesis in vivo are rarely reported. In this study, variety ‘Doppio Pandora-purple’ of Ranunculus asiaticus L. whose flower color is sensitive to the decrease in temperature was used. In the results, 27 genes involved in anthocyanin biosynthesis were differentially expressed under cold stress, and 7 MYB genes were up-regulated; the most obviously up-regulated MYB (RaMYB1) gene belongs to R2R3-MYB type. Promoter sequences of the 27 genes were concluded from transcriptome sequences and genome walking, for the purpose of sequence alignment with ChIP-seq/DAP-seq sequences. 7 genes were determined as target genes of RaMYB1 in cold-induced transcription regulation, including 2 PAL (phenylalanine ammonia lyase), 2 CHS (chalcone synthase), 1 F3H (flavanone 3-hydroxylase), 1 DFR (dihydroflavonol 4-reductase), 1 F3’H (flavanoid 3’-hydroxylase). ChIP-PCR indicated that, the binding of RaMYB1 to DFR can be induced by temperature decrease, while the bindings to other 6 genes were constitutive and can be strengthened at 5 °C decrease. Regulation properties of RaMYB1 to the 7 genes were determined as enhancement due to their up-regulation as well as the enhancer in C terminal of RaMYB1. Regulation mode of RaMYB1 was mapped accordingly and validated via HPLC-based anthocyanin content, gene expression level and coloration. The regulation mode can illustrate the binding sites and enhancement property of RaMYB1 in the regulation of anthocyanin biosynthesis under cold stress. It can be applied in color-oriented temperature monitoring and in molecular breeding. The way to establishing the regulation mode may be interest for other non-model plant genera with little molecular background.