An ancient duplication of apple MYB transcription factors is responsible for novel red fruit-flesh phenotypes

Abstract

Anthocyanin accumulation is coordinated in plants by a number of conserved transcription factors. In apple (Malus × domestica), an R2R3 MYB transcription factor has been shown to control fruit flesh and foliage anthocyanin pigmentation (MYB10) and fruit skin color (MYB1). However, the pattern of expression and allelic variation at these loci does not explain all anthocyanin-related apple phenotypes. One such example is an open-pollinated seedling of cv Sangrado that has green foliage and develops red flesh in the fruit cortex late in maturity. We used methods that combine plant breeding, molecular biology, and genomics to identify duplicated MYB transcription factors that could control this phenotype. We then demonstrated that the red-flesh cortex phenotype is associated with enhanced expression of MYB110a, a paralog of MYB10. Functional characterization of MYB110a showed that it was able to up-regulate anthocyanin biosynthesis in tobacco (Nicotiana tabacum). The chromosomal location of MYB110a is consistent with a whole-genome duplication event that occurred during the evolution of apple within the Maloideae family. Both MYB10 and MYB110a have conserved function in some cultivars, but they differ in their expression pattern and response to fruit maturity.

Figure 1.

Comparison of type 1 and type 2 red flesh in apple. A, Type 2 red-flesh apple such as cv Sangrado OP is characterized by red pigmentation in the fruit cortex, white fruit core, and green foliage. B, Type 1 red-flesh apple such as cv Redfield OP is characterized by red pigmentation in the fruit core, fruit cortex, and foliage. C, Phenotype of type 2 red flesh in an F2 population observed in cv Sciros × NZSelectionT051 (cv Sciros × Sangrado OP). Tree numbers are as follows: i, R04T009; ii, NZSelectionT051; iii, R08T068; iv, R03T120; v, R06T062; vi, R08T069; vii, R05T060.

Figure 2.

Fine-mapping for type 2 red flesh in the QTL validation populations. A, QTL analysis using a Kruskal-Wallis test (K*) using the percentage of red pigmentation in the fruit cortex and 163 SNP markers scored on 450 F1 individuals from the QTL validation population. Significance levels are as follows: P < 0.0001: K* = 23.59 for markers segregating < ab×aa > (marker heterozygous for one parent and homozygous for the other; degrees of freedom [d.f.] = 1) and K* = 19.13 for markers segregating < ab×ab > (marker heterozygous for both parents of the progeny; degrees of freedom = 2). B, Multiple QTL mapping analysis at the bottom of LG 17 in the QTL validation populations for percentage of red coloration in the fruit cortex (solid line) and intensity (dotted line).

Figure 3.

Schematic representation of the three R2R3 MYB anthocyanin-activating homeologous genes in the apple genome. MYB10 controlling type 1 red flesh and skin color is located at the bottom of chromosome 9 in contig MDC13323_319, and MYB110a (GenBank accession no. JN711473) and MYB110b (GenBank accession no. JN711474) are located on homeologous apple chromosome 17 on contigs MDC013323_290 and MDC035405_20, respectively. According to the apple genome primary assembly, MYB110a and MYB110b are in opposite orientations, with their putative start codons facing each other at an estimated distance of 60 kb. The MYB110b second intron is larger than the second intron of MYB10 and MYB110a and is truncated in its third exon (premature stop codons shown as black dots), based on the protein translation. A 272-bp insertion is also present in exon 3.

Figure 4.

Sequence characterization of MYB10, MYB110a, and MYB110b. A, Phylogenetic analysis of anthocyanin-related MYBs in plant species. B, Amino acid sequence alignment of MYB110a, MYB110b, MYB10, and MYB1. The amino acid sequence of MYB110a was translated from sequences obtained from the apple cv Sangrado OP (SG), Royal Gala (RG; parent of cv Sciros), and Golden Delicious (GD). The R2 and R3 MYB motifs are indicated. Arrowheads indicate residues needed for the interaction with the bHLH partner, box A is a domain characteristic of anthocyanin-regulating MYBs, and box B indicates a conserved C-terminal motif.

Figure 5.

HPLC analysis of anthocyanin profiles in the cortex of progeny of an F2 cross of cv Sciros × Sangrado OP. A, Total anthocyanin (µg g⁻¹ fresh weight [FW]) for progeny (same trees as numbered in Fig. 1C) and cv Redfield cortex (RFc), cv Sciros cortex (ScRc), and cv Sciros peel (ScRp). B, Liquid chromatography-quadrupole-time of flight-mass spectrometry traces from pooled fruit of three representative progeny, i, ii, and iii (as in Fig. 1). Letters besides peaks represent different cyanidin glycosides: a, cyanidin 3-galactoside; b, cyanidin 3-arabinoside; c, cyanidin 3-pentoside1; d, cyanidin 3-pentoside2. [See online article for color version of this figure.]

Figure 6.

qPCR analysis of transcript abundance of LDOX (A), MYB110a (B), and MYB10 (C) in the cortex of progeny of an F2 cross of cv Sciros × NZSelectionT051 (numbered as in Fig. 1), Redfield cortex (RFc), Sciros cortex (ScRc), and Sciros peel (ScRp). Ratio is relative to three apple reference genes, Act, EF1α, and GAPDH. NZSelectionT051 (ii) was set as a calibrator for LDOX and MYB110a, and cv Sciros peel (ScRp) was set as a calibrator for MYB10. Primers are shown in Supplemental Table S2. Data are presented as means ± se of four biological replicates.

Figure 7.

Transient assays demonstrate the function of MYB110a as a regulator of anthocyanin biosynthesis. A, Leaves of N. benthamiana were infiltrated with the apple CHS promoter-LUC fusions on their own or coinfiltrated with 35S:MYB110a, with or without 35S:bHLH3 and 35S:WD40. As a positive control, 35S:MYB10 was similarly infiltrated. Luminescence of luciferase and renilla was measured 3 d later and expressed as a ratio of luciferase to renilla. Data are presented as means of four replicate reactions. B, Red coloration around the infiltration site in the leaves of tobacco 8 d after transient transformation with 35S:MYB110a showing the adaxial leaf surface (i), 35S:MYB110a abaxial surface (ii), and 35S:MYB10 adaxial leaf surface (iii). All three patches were coinfiltrated with 35S:bHLH3. No coloration was observed with infiltration of 35S:bHLH3 alone.

Figure 8.

Nucleotide sequence of the 5′ UTR and proximal promoter region of MYB110a. The positions of seven SNPs (black triangles) and one insertion-deletion (black diamond), cis-regulatory motifs, and the start codon (boxed) are indicated.