Apple skin patterning is associated with differential expression of MYB10

Abstract

Background: Some apple (Malus × domestica Borkh.) varieties have attractive striping patterns, a quality attribute that is important for determining apple fruit market acceptance. Most apple cultivars (e.g. 'Royal Gala') produce fruit with a defined fruit pigment pattern, but in the case of 'Honeycrisp' apple, trees can produce fruits of two different kinds: striped and blushed. The causes of this phenomenon are unknown.

Results: Here we show that striped areas of 'Honeycrisp' and 'Royal Gala' are due to sectorial increases in anthocyanin concentration. Transcript levels of the major biosynthetic genes and MYB10, a transcription factor that upregulates apple anthocyanin production, correlated with increased anthocyanin concentration in stripes. However, nucleotide changes in the promoter and coding sequence of MYB10 do not correlate with skin pattern in 'Honeycrisp' and other cultivars differing in peel pigmentation patterns. A survey of methylation levels throughout the coding region of MYB10 and a 2.5 Kb region 5' of the ATG translation start site indicated that an area 900 bp long, starting 1400 bp upstream of the translation start site, is highly methylated. Cytosine methylation was present in all three contexts, with higher methylation levels observed for CHH and CHG (where H is A, C or T) than for CG. Comparisons of methylation levels of the MYB10 promoter in 'Honeycrisp' red and green stripes indicated that they correlate with peel phenotypes, with an enrichment of methylation observed in green stripes.

Conclusions: Differences in anthocyanin levels between red and green stripes can be explained by differential transcript accumulation of MYB10. Different levels of MYB10 transcript in red versus green stripes are inversely associated with methylation levels in the promoter region. Although observed methylation differences are modest, trends are consistent across years and differences are statistically significant. Methylation may be associated with the presence of a TRIM retrotransposon within the promoter region, but the presence of the TRIM element alone cannot explain the phenotypic variability observed in 'Honeycrisp'. We suggest that methylation in the MYB10 promoter is more variable in 'Honeycrisp' than in 'Royal Gala', leading to more variable color patterns in the peel of this cultivar.

Figure 1

Schematic representation of the flavonoid biosynthetic pathway in apple regulated by MYB10. Flavonoid intermediates (gray boxes) and end products (black boxes) are indicated. Enzymes required for each step are shown in bold uppercase letters (PAL, phenylalanine ammonia lyase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone-3β-hydroxylase; FLS, flavonol synthase; GT, unidentified enzyme encoding a glycosyl transferase for flavonol glycone synthesis; DFR, dihydroflavonol-4-reductase (denoted as DFR1 in the text); LAR, leucoanthocyanidin reductase; LDOX, leucoanthocyanidin dioxygenase; ANR, anthocyanidin reductase; UFGT, UDP-glycose:flavonoid-3-O-glycosyltransferase (adapted from [17]).

Figure 2

Different types of fruit peel pigment patterns in 'Honeycrisp' apple. Distribution of anthocyanin in apple peels of blushed A) and striped B) fruits of 'Honeycrisp', indicating regions classified as red or green stripes.

Figure 3

The levels of cyanidin-3-galactoside differ in red and green stripes of 'Honeycrisp' and 'Royal Gala'. HPLC traces at 520 nm of A) green and B) red stripes of 'Honeycrisp' and C) green and D) red stripes of 'Royal Gala'. Peak identification (observed molecular ion/major fragment, masses in Da): 1 - Cyanidin-3-galactoside (M⁺ = 449, 287); 2 - Cyanidin-3-glucoside (M⁺ = 449, 287); 3 - Cyanidin pentoside (M⁺ = 419, 287 most likely the arabinoside); 4 and 5 - Tentatively identified (ions were low intensity) as pelargonidin derivatives (M⁺ = 557, 395, 271 Da, implies presence of pelargonidin, hexoside sugar and an unidentified species; mass 124). Chromatograms are offset on the time axis by one minute for clarity.

Figure 4

Transcript levels of apple anthocyanin genes determined by real-time PCR. Values indicate the ratio between the normalized transcript levels (relative to actin) of structural genes (CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone-3β-hydroxylase; DFR, dihydroflavonol-4-reductase (denoted as DFR1 in the text); LDOX, leucoanthocyanidin dioxygenase; UFGT, UDP-glycose:flavonoid-3-O-glycosyltransferase) and transcription factors (MYB10, MYB17, bHLH3 and bHLH33) in red and green stripes of 'Honeycrisp' and 'Royal Gala' as indicated. Reactions were performed in triplicate. Error bars are SE.

Figure 5

Methylation levels across MYB10 in 'Honeycrisp' and 'Royal Gala'. Percent methylation in A) 'Honeycrisp' and B) 'Royal Gala' green and red stripes across the MYB10 locus (Genbank accession EU518249) (C), estimated using an assay combining McrBC digestions and real-time PCR amplification. Percent methylation indicates the proportion of copies cut by McrBC. Values on the X-axis indicate the location of the primers used relative to the ATG translation start site of MYB10. Panel C indicates the relative location of the TRIM element, the MYB10 promoter and three exons (1, 2, 3); this figure is not to scale. The blue triangles indicate the approximate positions of E-box motifs within the promoter region. The calculated % methylation for the -51 to 105 fragment in 'Honeycrisp' and the -2254 to -2098 in 'Royal Gala' were negative, therefore a value of 0 is indicated in the plot. Methylation in the -704 to -555 fragment in 'Royal Gala' could not be estimated given the extremely low template levels in the McrBC treated sample. The -1874 to -1681, -303 to -182, 146 to 257 and 494 to 710 fragments were not evaluated in 'Royal Gala' (N). Reactions were performed in triplicate and two or three independent digestions were used. Error bars are SE and stars indicate significant differences (p ≤ 0.05).

Figure 6

Methylation levels in two MYB10 promoter regions in 'Fireside' and 'Connel Red'. Percent methylation in a low (-2254 to -2098) and a high (-846 to -651) methylation region of the MYB10 promoter (GenBank accession EU518249) in 'Connel Red' and 'Fireside' peel DNA (2007 fruit samples). Percent methylation was calculated using an assay combining McrBC digestions and real-time PCR and indicates the proportion of copies cut by McrBC. The X-axis indicates nucleotide positions relative to the ATG translation start site. Reactions were performed in triplicate and two independent digestions were used.

Figure 7

Methylation levels in three MYB10 promoter regions in striped and blushed 'Honeycrisp' peels. Comparison of percent methylation in the highly methylated region (-1411 to -651) of the MYB10 promoter (GenBank accession EU518249) between red and green stripes, and red and green areas of blushed 'Honeycrisp' (2008 fruit samples). Percent methylation was calculated using an assay combining McrBC digestions and real-time PCR and indicates the proportion of copies cut by McrBC. The X-axis indicates nucleotide positions relative to the ATG translation start site. Reactions were performed in triplicate and two independent digestions were used. Error bars are SE and stars indicate significant differences (p ≤ 0.05).

Figure 8

Methylation levels in 'Honeycrisp' evaluated using bisulfite sequencing. Comparison of percent methylation in two regions (-1007 to -684 and -534 to -184) of the MYB10 promoter (GenBank accession EU518249) between red and green stripes (A) and among three methylation contexts (B). Percent methylation was calculated based on the cytosine methylation status of a number of clones after bisulfite conversion and sequencing. The X-axis indicates nucleotide positions relative to the ATG translation start site. E-box motifs are indicated with blue triangles in panel A. Values in panel B represent the average of green and red stripes.