An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae

Abstract

Background: The control of plant anthocyanin accumulation is via transcriptional regulation of the genes encoding the biosynthetic enzymes. A key activator appears to be an R2R3 MYB transcription factor. In apple fruit, skin anthocyanin levels are controlled by a gene called MYBA or MYB1, while the gene determining fruit flesh and foliage anthocyanin has been termed MYB10. In order to further understand tissue-specific anthocyanin regulation we have isolated orthologous MYB genes from all the commercially important rosaceous species.

Results: We use gene specific primers to show that the three MYB activators of apple anthocyanin (MYB10/MYB1/MYBA) are likely alleles of each other. MYB transcription factors, with high sequence identity to the apple gene were isolated from across the rosaceous family (e.g. apples, pears, plums, cherries, peaches, raspberries, rose, strawberry). Key identifying amino acid residues were found in both the DNA-binding and C-terminal domains of these MYBs. The expression of these MYB10 genes correlates with fruit and flower anthocyanin levels. Their function was tested in tobacco and strawberry. In tobacco, these MYBs were shown to induce the anthocyanin pathway when co-expressed with bHLHs, while over-expression of strawberry and apple genes in the crop of origin elevates anthocyanins.

Conclusions: This family-wide study of rosaceous R2R3 MYBs provides insight into the evolution of this plant trait. It has implications for the development of new coloured fruit and flowers, as well as aiding the understanding of temporal-spatial colour change.

Figure 1

Analysis of apple MYB10/MYB1 in diverse apple cultivars. (A) Homozygous R1 MYB10 Pacific Rose™ (1), 'Royal Gala' (2), 'Granny Smith' (3); Heterozygous 'Red Field' OP (4), Niedzwetzkyana (5), 'Roberts Crab' (6); Homozygous R6 MYB10 Malus sieversii 01P22 (7), Malus sieversii 629319 (8); Mixture of diluted (1 to 10⁶) PCR products R1:R6 (3:1) (9), R1:R6 (1:3) (10); no template control (11). (B) Analysis of apple MYB10/MYB1 in Pacific Rose™ (lane 1) × 'Red Field' (lane 2) & segregation of progeny (lanes 3 to18). Lane 19 is no template control.

Figure 2

Protein sequence alignment of rosaceous MYB10 and known anthocyanin MYB regulators from other species. Arrows indicate specific residues that contribute to a motif implicated in bHLH co-factor interaction in Arabidopsis [44]. Box (A) a conserved motif [A/S/G]NDV in the R2R3 domain for dicot anthocyanin-promoting MYBs. Box (B) a C-terminal-conserved motif KPRPR [S/T]F for Arabidopsis anthocyanin-promoting MYBs [17].

Figure 3

Phylogenetic relationships between Arabidopsis MYB transcription factors and anthocyanin-related MYBs of rosaceous and other species. Rosaceous MYB10s cluster next to PAP1 (AtMYB75) and PAP2 (AtMYB90), within the anthocyanin MYB regulator subgroup (A). A Phylogeny of MYB10 from all the major rosaceous species and known anthocyanin MYB regulators from other species (B). Sequences were aligned using Clustal W (opening = 15, extension = 0.3) in Vector NTI 9.0. Phylogenetic and molecular evolutionary analysis was conducted using MEGA version 3.1 [80] [using minimum evolution phylogeny test and 1000 bootstrap replicates].

Figure 4

Analysis of R2R3 DNA binding domains of anthocyanin-promoting MYBs. Alignment (A) of three consensus amino-acid sequences from 22 rosaceous MYB10s, 38 dicot anthocyanin-promoting MYBs, and the other 134 proteins included in Figure 3A. To obtain three consensus sequences, the sequences in each of three groups were aligned using AlignX (opening = 15, extension = 0.3) in Vector NTI 9.0, and residue fraction for consensus was set to 0.9 for the alignments of 22 rosaceous MYB10s and 38 dicot anthocyanin-promoting MYBs, and 0.3 for the alignment of the other 134 proteins. (B) Frequency of residues at position 90 to 93 of the R2R3 domain covering 168 MYB TFs of Arabidopsis, rosaceous species, and other dicot sequences.

Figure 5

Transient activation of anthocyanic responses by rosaceous MYB10s and bHLH transcription factors. (A) Activation of the Arabidopsis DFR promoter by MYB10 and bHLH transcription factors. Error bars are the SE for eight replicate reactions. (B) Patches of anthocyanin production in tobacco leaves by PdmMYB10 (i), PprMYB10, but not by the negative control MdMYB8 (iii).

Figure 6

Normalized quantitative Real-Time of the expression of cherry PavMYB10. Expression of PavMYB10 n the developmental series from sweet cherry 'Rainier' and 'Stella'. (A) Fruit sampled and (B) qPCR expression of PavMYB10, CHS and LDOX using 'Rainier' green fruitlet as a calibrator. Error bars are the SE for three replicate reactions.

Figure 7

Normalized qPCR data of the expression of strawberry MYB10 and MYB1. qPCR expression of Fragaria MYB10 and MYB1 in a fruit developmental series from both cultivated and wild strawberry (A). Fv stage 1 was set as a calibrator. Error bars are the SE for three replicate reactions. (B) Six developmental stages of cultivated strawberry. (C) Six developmental stages of wild strawberry. (D) White and red petals of wild strawberry.

Figure 8

Transformation of strawberry with 35S:FaMYB10 elevates anthocyanin synthesis. Cultivated strawberry was transformed with 35S:FaMYB10. Visible reddening was seen in leaves (A; WT on right) and roots (B; WT on right), and in flowers (C; WT on right). Fruits showed red seeds and elevated anthocyanin (D; transgenic top photos, WT below). Extracted pigment was anthocyanin and increased in all lines (E). Error bars are the SE for four replicate extracts per line.