Multiple R2R3-MYB Transcription Factors Involved in the Regulation of Anthocyanin Accumulation in Peach Flower

Abstract

Anthocyanin accumulation is responsible for flower coloration in peach. Here, we report the identification and functional characterization of eight flavonoid-related R2R3-MYB transcription factors, designated PpMYB10.2, PpMYB9, PpMYBPA1, Peace, PpMYB17, PpMYB18, PpMYB19, and PpMYB20, respectively, in peach flower transcriptome. PpMYB10.2 and PpMYB9 are able to activate transcription of anthocyanin biosynthetic genes, whilst PpMYBPA1 and Peace have a strong activation on the promoters of proanthocyanin (PA) biosynthetic genes. PpMYB17-20 show a strong repressive effect on transcription of flavonoid pathway genes such as dihydroflavonol 4-reductase. These results indicate that anthocyanin accumulation in peach flower is coordinately regulated by a set of R2R3-MYB genes. In addition, PpMYB9 and PpMYB10.2 are closely related but separated into two groups, designated MYB9 and MYB10, respectively. PpMYB9 shows a strong activation on the PpUGT78A2 promoter, but with no effect on the promoter of PpUGT78B (commonly called PpUFGT in previous studies). In contrast, PpMYB10.2 is able to activate the PpUFGT promoter, but not for the PpUGT78A2 promoter. Unlike the MYB10 gene that is universally present in plants, the MYB9 gene is lost in most dicot species. Therefore, the PpMYB9 gene represents a novel group of anthocyanin-related MYB activators, which may have diverged in function from the MYB10 genes. Our study will aid in understanding the complex mechanism regulating floral pigmentation in peach and functional divergence of the R2R3-MYB gene family in plants.

Keywords: R2R3-MYB; anthocyanin; flower coloration; peach; proanthocyanin.

FIGURE 1

Heatmap of R2R3-MYBs in peach transcriptomes of different red-colored tissues. Green and red boxes indicate low- and high-expression levels, respectively. Red-colored leaf, flower, and fruit samples were collected from cv. Hongyetao, Hongbaihua, and Dahongpao, respectively.

FIGURE 2

Phylogenetic tree derived from amino acid sequences of R2R3-MYBs in peach and other dicot species. The peach R2R3-MYBs are highlighted with a solid black circle. Full amino acid sequences of R2R3-MYBs were aligned using Muscle software, and phylogenetic tree was constructed with MEGA software (version 6.0) using maximum likelihood method. Numbers near branches indicate bootstrap values that were calculated from 1000 replicate analyses. The scale bar represents 0.2 substitutions per site. The accession numbers are as follows: Arabidopsis thaliana AtMYB4 (AT4G38620), AtMYB6 (AT4G09460), AtPAP1 (AT1G56650), AtMYB123 (AT5G35550), AtMYB32 (AT4G34990); Vitis vinifera VvMYBA1 (AB097923), VvMYBPA2 (EU919682), VvMYBPA1 (AM259485), VvMYBC2-L1 (JX050227); Prunus persica PpMYB10.2 (EU155160), PpMYBPA1 (CV047374), Peace (AB897865), PpMYB9 (KT159232), PpMYB17 (KT159233), PpMYB18 (KT159234), PpMYB19 (KT159235), PpMYB20 (KT159236), PpMYB10.4 (KF999985), PpMYB10.1 (XM_007216468); Malus domestica MdMYB10 (DQ267897), MdMYB16 (HM122617); Fragaria ananassa FaMYB9 (JQ989281), FaMYB11 (JQ989282), FaMYB1 (AF401220); Oryza sativa OsMYB3 (D88619); Diospyros kaki DkMYB4 (AB503701); Trifolium arvense TaMYB14 (JN049641); Petunia x hybrid PhAN2 (ABO21074); Citrus sinensis CsRuby (NM_001288889); Lotus japonicus LjTT2a (AB300033); Antirrhinum majus AmMYB308 (P81393); Populus tremula x Populus tremuloides PtMYB182 (KP723392); Rosa hybrida RhMYB10(EU155164); Fragaria vesca FvMYB90 like(XM_004302074); Pyrus x bretschneideri PbMYB208 like (XM_009339740).

FIGURE 3

Diagram and alignment of eight R2R3-MYB transcription factors (TFs). (A), structural diagram of the eight R2R3-MYBs highly expressed in peach flower. Cross lines indicate R2 and R3 SANT domains. The black and grey boxes stand for C1 and C2 motif, respectively. (B,C), amino acid sequence alignment of R2R3 domain and C1/C2 motif, respectively, of the MYBs in peach and other dicots. Arrows highlight key amino acid residues that mediate promoter targets specificity for the anthocyanin and proanthocyanin (PA) pathway (Heppel et al., 2013).

FIGURE 4

Estimation of effect of different R2R3-MYBs with PpbHLH3 partner on activation of the promoter of anthocyanin or PA pathway genes using transient dual luciferase assay. Promoter activity was shown as the ratio of firefly Luciferase (Luc) activity to Renilla luciferase (Ren) activity. Different lowercase letters indicate the significant differences among treatments by Tukey’s test at P < 0.01. Error bars show SE of four biological replicates.

FIGURE 5

Validation of activation effect of four R2R3-MYBs on the PpDFR promoter using dual luciferase assay. Different lowercase letters indicate the significant differences among treatments by Tukey’s test at P < 0.001. Results represent the means of four biological replicates.

FIGURE 6

Transient color assay of the PpMYB9 activity in N. tabacum leaf. (A–C) indicate infiltration with PpMYB9+PpbHLH3, PpMYB9+PpbHLH3+proPpUGT78A2::PpUGT78A2, and proPpUGT78A2::PpUGT78A2, respectively. Photos were taken 2 weeks after infiltration.

FIGURE 7

Expression profiles of the eight R2R3-MYBs in flowers of cv. Mantianhong at different developmental stages. Different lowercase letters indicate the significant differences among treatments by Tukey’s test at P < 0.01. Error bars show SE of the means of three biological replicates.