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First published online September 29, 2006; 10.1104/pp.106.088104 Plant Physiology 142:1216-1232 (2006) © 2006 American Society of Plant Biologists Light-Induced Expression of a MYB Gene Regulates Anthocyanin Biosynthesis in Red Apples1Commonwealth Scientific and Industrial Research Organization, Plant Industry, Adelaide Laboratory, Waite Campus, Urrbrae, SA 5064, Australia (A.M.T., F.W.J., J.B., S.P.R., A.R.W.); and Department of Agriculture and Food Western Australia, Manjimup Horticultural Research Institute, Manjimup, WA 6258, Australia (S.R.J.)
Anthocyanins are secondary metabolites found in higher plants that contribute to the colors of flowers and fruits. In apples (Malus domestica Borkh.), several steps of the anthocyanin pathway are coordinately regulated, suggesting control by common transcription factors. A gene encoding an R2R3 MYB transcription factor was isolated from apple (cv Cripps' Pink) and designated MdMYB1. Analysis of the deduced amino acid sequence suggests that this gene encodes an ortholog of anthocyanin regulators in other plants. The expression of MdMYB1 in both Arabidopsis (Arabidopsis thaliana) plants and cultured grape cells induced the ectopic synthesis of anthocyanin. In the grape (Vitis vinifera) cells MdMYB1 stimulated transcription from the promoters of two apple genes encoding anthocyanin biosynthetic enzymes. In ripening apple fruit the transcription of MdMYB1 was correlated with anthocyanin synthesis in red skin sectors of fruit. When dark-grown fruit were exposed to sunlight, MdMYB1 transcript levels increased over several days, correlating with anthocyanin synthesis in the skin. MdMYB1 gene transcripts were more abundant in red skin apple cultivars compared to non-red skin cultivars. Several polymorphisms were identified in the promoter of MdMYB1. A derived cleaved amplified polymorphic sequence marker designed to one of these polymorphisms segregated with the inheritance of skin color in progeny from a cross of an unnamed red skin selection (a sibling of Cripps' Pink) and the non-red skin cultivar Golden Delicious. We conclude that MdMYB1 coordinately regulates genes in the anthocyanin pathway and the expression level of this regulator is the genetic basis for apple skin color.
Anthocyanins are a class of secondary metabolites that contribute to the red, blue, and purple colors in a range of flowers and fruits. In flowers these pigments attract pollinators and in fruit skin they attract animals to aid in seed dispersal (Regan et al., 2001  ; Schaefer et al., 2004). Anthocyanins are also found in leaves, particularly during senescence (Feild et al., 2001), in stems, roots, and occasionally in fruit flesh and seeds.
Anthocyanins, condensed tannins (CTs), and flavonols are synthesized via the flavonoid pathway, a branch of the phenylpropanoid pathway. The flavonoid pathway consists of a number of enzymatic steps that each catalyzes a sequential reaction for flavonoid synthesis, as illustrated in Figure 1
. The pathway leads to synthesis of anthocyanins with branches for synthesis of flavonols (via flavonol synthase [FLS]) and synthesis of CTs (via leucoanthocyanidin reductase [LAR] and anthocyanidin reductase [ANR]). The genetics and biochemistry of this pathway have been characterized in petunia (Petunia hybrida), maize (Zea mays), snapdragon (Antirrhinum majus), and Arabidopsis (Arabidopsis thaliana; Winkel-Shirley, 2001
Two families of regulators, the bHLH and MYB proteins, are conserved in the regulation of the anthocyanin and CT pathways in all species analyzed to date (Koes et al., 2005 ). The bHLH (also called MYC) proteins may have overlapping regulatory targets (Zhang et al., 2003; Zimmermann et al., 2004), but the MYB proteins are the key components providing specificity for the subsets of genes activated. For example, AtPAP1 and AtPAP2, which regulate anthocyanin synthesis, and AtTT2, which regulates CT synthesis, are specific for regulation of these branches of the flavonoid pathway (Borevitz et al., 2000; Nesi et al., 2001).
Arabidopsis has been an excellent model for delineating this fundamental basis of flavonoid synthesis and gene regulation (Winkel-Shirley, 2001
In the fruit of many crops, such as apple (Malus domestica), citrus (Citrus sinensis), peach (Prunus persica), pear (Pyrus communis), strawberry (Fragaria spp.), Vaccinium sp. such as cranberry (Vaccinium macrocarpon) and bilberry (Vaccinium myrtillus), eggplant (Solanum melongena), and lychee (Litchi chinensis), multiple flavonoid structural genes are coregulated for anthocyanin synthesis or in many of these fruit anthocyanin synthesis is enhanced by sunlight (Tyas et al., 1998
In horticultural plants, color is an important consideration in consumer choice. In fruits such as apple, grape, cherry (Prunus avium), strawberry, and others there has been much interest in breeding varieties bearing fruit with altered color, hues, patterns, or total anthocyanin content. In apples, red skin fruit are preferred to other fruit because consumers associate better-colored apples with better taste, ripeness, and flavor (King and Cliff, 2002
In apple skin, sunlight is the most important external factor regulating anthocyanin synthesis (Saure, 1990 In this study, we report the isolation of a light-induced gene that encodes a MYB regulator of anthocyanin synthesis in apple fruit skin. This gene, called MdMYB1, was characterized in terms of the correlation of its expression with anthocyanin synthesis and function in two heterologous transgene assays. A derived cleaved amplified polymorphic sequence (dCAPS) marker for an allele of MdMYB1 showed segregation with skin color. This marker will be a useful tool for apple breeding programs. Our analysis has delineated the regulation of anthocyanin synthesis in apple fruit skin and will further our understanding of flavonoid regulation in other crops.
Flavonoid Synthesis and Gene Expression in the Skin of Apple Cultivars Levels of anthocyanins, CTs, and flavonols accumulated in apple fruit skin were measured in several cultivars collected when the fruit was ripening (Fig. 2 ). Skin from the fruit of the non-red skin cultivars Golden Delicious, Granny Smith, Grandspur, Firm Gold, Shuzaka, and Einscheimer did not contain any detectable anthocyanins (Fig. 2A). Anthocyanin levels ranged from approximately 15 ng mg–1 to approximately 165 ng mg–1 in the red skin cultivars, an unnamed selection with red skin (US), Cripps' Red, Gala, Galaxy, and Hi Early. There was significant variation in the levels of flavonols (Fig. 2B) and CTs (Fig. 2C) among the different cultivars but no clear correlation with color. The non-red skin cultivars accumulated flavonols and CTs in a similar range to that measured in the red skin cultivars.
The transcript levels of genes that encode the enzymes of the flavonoid pathway (Fig. 1) were measured in the skin of non-red and red skin apple cultivars by real-time PCR. Transcripts of the early genes of the pathway, required for both anthocyanin and CT synthesis (MdCHS, MdF3H, and MdDFR), were typically 5- to 20-fold higher in red skin cultivars than non-red skin cultivars (Fig. 3A ). Transcripts of the late genes of the pathway (MdLDOX and MdUFGT), of which the latter gene is specific for anthocyanin synthesis, ranged from 25- to 180-fold higher in red skin cultivars compared to non-red skin cultivars (Fig. 3B). The exceptions were Granny Smith and Grandspur, which had higher transcript levels of MdUFGT than other non-red skin cultivars although transcript levels were still well below that of the red skin cultivars. Transcripts of the MdCHI gene, which is also required for both anthocyanin and CT synthesis, did not have the same pattern as that observed for the other genes and its expression was at similar levels in all the cultivars analyzed (Fig. 3C). Transcripts of MdFLS, which is specific for the synthesis of flavonols, and MdLAR1 and MdANR, which are specific for the synthesis of CTs, were at similar levels in non-red and red skin cultivars (Fig. 3, D and E).
Isolation of MdMYB1
Anthocyanin synthesis in apple fruit skin is induced by sunlight (Saure, 1990 One or more transcription factors specific to the anthocyanin pathway should be induced when bags are removed from dark-grown fruit and anthocyanin synthesis is induced by sunlight. To exploit this possibility we prepared cDNA from Cripps' Pink fruit skin in which the anthocyanin pathway had been strongly induced by light and used it as a template for PCR to isolate anthocyanin MYB regulators. Degenerate primers were designed from the conserved regions in the R2R3 domain of MYB transcription factors from other plant species that had been functionally characterized to regulate anthocyanin synthesis. A 246 bp cDNA was isolated that encoded a peptide with approximately 80% sequence identity to the R2R3 region of the petunia MYB transcription factor PhAN2. The 5‘ and 3’ ends of the cDNA were isolated by RACE PCR. The 848 bp full-length cDNA contained a coding region for a deduced amino acid sequence of 243 residues in length and this protein was designated as MdMYB1. A phylogenetic analysis of the R2R3 region of this deduced amino acid sequence places MdMYB1 in a cluster of MYB proteins that include: Gerbera hybrida GMYB10, snapdragon AmVENOSA, AmROSEA1, and AmROSEA2, pepper CaA, tomato (Lycopersicon esculentum) LeANT1, petunia PhAN2, grape VvMYBA1 and VvMYBA2, and Arabidopsis AtPAP1 and AtPAP2 (Fig. 4A ). These MYBs have all been characterized as anthocyanin regulators. The cluster that contains maize ZmPL and ZmC1, which are also specific for regulation of anthocyanin synthesis, and the Arabidopsis AtTT2, which regulates CT synthesis, are more distantly related, as are MYBs that regulate other processes.
An alignment of the deduced amino acid sequences of some of these proteins shows the high sequence homology within the R2R3 domain at the amino terminus (Fig. 4B). All of the proteins contain the motif [D/E]Lx2[R/K]x3Lx6Lx3R in the R3 domain for interactions with R-like bHLH proteins (Zimmermann et al., 2004 ). Within the highly conserved R2 domain it is interesting to note that MdMYB1 and the anthocyanin regulators (with the exception of ZmC1) have an Arg at position 39 when a Gly residue is found at this position in ZmC1 and AtTT2 and in 88% of Arabidopsis R2R3 MYBs (Stracke et al., 2001). It is not clear if this residue is critical for the specificity of the anthocyanin MYBs; however, it may be an interesting target for mutagenesis.
There is little homology in the sequence carboxyl terminal to the R2R3 domain; an exception is the motif KPRPR[S/T]F defined by Stracke et al. (2001)
Transcript levels of MdMYB1 were determined by real-time PCR using gene-specific primers designed to the region encoding the carboxyl domain. When bags were removed from dark-grown Cripps' Red fruit and the fruit reexposed to sunlight, MdMYB1 transcripts increased by approximately 20-fold after 1 d and then increased to approximately 25-fold higher after 6 d before transcript levels declined again (Fig. 5A
). This is similar to the pattern observed for MdUFGT transcript levels and correlates with anthocyanin synthesis when bagged fruit were uncovered (Takos et al., 2006a
Transcript levels of MdMYB1 in Cripps' Red fruit were approximately 10-fold higher in red skin sectors than in flesh, seeds, flower buds, or young fruit (Fig. 5B). Skin sector samples were taken at 48 and 147 d after full bloom (DAFB), which we found in a previous study coincided with two distinct phases of anthocyanin synthesis in Cripps' Red fruit (Takos et al., 2006b ). At 48 DAFB MdMYB1 transcript levels were approximately 24-fold higher in red skin than green skin sectors and at 147 DAFB were approximately 4.5-fold higher. Transcript levels of MdMYB1 were 15- to 50-fold higher in red skin cultivars than in non-red skin cultivars (Fig. 5C). This correlates with the levels of anthocyanin measured in these cultivars (Fig. 2A) and transcripts of the genes required for anthocyanin synthesis (Fig. 3, A and B). The exceptions were Granny Smith and Grandspur, which had 5-fold higher levels of MdMYB1 transcripts than the other non-red skin cultivars. The data shows a positive correlation between MdMYB1 expression levels and anthocyanin synthesis in response to light, tissue specificity, and genetic background.
The function of the MdMYB1 protein was tested in planta by introducing MdMYB1-1 cDNA under the transcriptional control of the cauliflower mosaic virus 35S promoter into two heterologous systems: Arabidopsis plants and cultured grape cells. Arabidopsis plants, transformed with 35S:MdMYB1-1 cDNA, developed seeds with spots of red pigmentation at the funicular end, indicating anthocyanin synthesis (Fig. 6A ). For comparison, plants were also transformed with 35S:AtPAP1 cDNA, the endogenous Arabidopsis MYB anthocyanin regulator. These plants developed a stronger phenotype than for plants transformed with 35S:MdMYB1-1 cDNA as the red pigmentation in seeds was more intense and not restricted to the funicular end (Fig. 6B). For both constructs the phenotype was best observed in immature seeds (less than 10 d after anthesis) before cells of the seed coat die and compress (about 15 d after anthesis) and the seed coat darkens. Seeds of the wild-type plants (ecotype Columbia) had no pigmentation (Fig. 6C) and no pigmentation was observed in any other plant structures for either the wild-type or transformed plants. The data shows that MdMYB1 can function to induce anthocyanin synthesis in Arabidopsis.
Cultured grape cells bombarded with gold particles coated with MdMYB1-1 cDNA and the bHLH partner AtEGL3, both driven by the cauliflower mosaic virus 35S promoter, were induced to synthesize anthocyanin (Fig. 6D). No pigmented cells were observed when transformed with AtEGL3 cDNA alone. The MdMYB1-1 and MdMYB1-3 cDNAs, which encoded either an Arg or Ser at position 191, respectively (Fig. 4B), were tested for their efficacy to induce expression from the promoters of apple flavonoid structural genes in the grape cell assay. Both these MdMYB1 cDNAs were able to induce an approximately 25-fold and approximately 10-fold increase in luciferase (LUC) enzyme activity when cobombarded with constructs containing the MdDFR and MdUFGT promoters, respectively, fused to the firefly LUC gene (Fig. 6E). Similar numbers of pigmented cells were also observed after bombardment with both MdMYB1 cDNAs (data not shown). These results show that MdMYB1 can activate expression of two apple anthocyanin pathway genes and indicate that either an Arg or a Ser at position 191 does not affect MdMYB1 function.
Genomic fragments encompassing approximately 6 kb of MdMYB1 and spanning from approximately 2 kb of the promoter to the stop codon were isolated from genomic DNA of Golden Delicious and the red skin siblings Cripps' Pink and Cripps' Red. These siblings are progeny of a cross of the red skin cultivar Lady Williams with the non-red skin cultivar Golden Delicious. Sequence analysis revealed the presence of three MdMYB1 alleles in these cultivars (Fig. 7A ). The MdMYB1-1 allele was inherited by both Cripps' Pink and Cripps' Red but was not present in the parent Golden Delicious. Therefore, MdMYB1-1 must have been inherited from the red skin parent Lady Williams and thus was an excellent candidate single dominant gene controlling anthocyanin synthesis in apple skin.
The genomic organization of MdMYB1-1 consists of three exons and two introns (Fig. 7B). The R2 domain is split over exons 1 and 2, while the R3 domain is split over exons 2 and 3. This is similar to the structure of other R2R3 MYBs (Jiang et al., 2004 ). Intron 1 is 323 bp in length while intron 2 is 3 kb in length. Southern blotting under stringent conditions using a DNA probe made to the region encoding the carboxyl-terminal domain indicated there is a single copy of MdMYB1 in the genome of Lady Williams, Golden Delicious, Cripps' Red, and Cripps' Pink (data not shown). The other two alleles of MdMYB1 have the same basic gene structure as MdMYB1-1; one of these was designated MdMYB1-2 and the other corresponded to MdMYB1-3 that was functional in the grape cell bombardment (Fig. 6E). A comparison of the nucleotide sequences of the three alleles shows that they are identical in exons 1 and 2 but in MdMYB1-1 and MdMYB1-2 there are a small number of nucleotide differences in the promoter and a single nucleotide difference in intron 2 (Table I ). The MdMYB1-3 allele is more different from the other alleles with many nucleotide differences in the promoter and introns and a nucleotide change in exon 3 that results in the Arg-to-Ser change at position 191 of the deduced amino acid sequence (Fig. 4B).
MdMYB1-1 Cosegregation with Skin Color A single nucleotide polymorphism (SNP) in the promoter of the MdMYB1-1 allele is different from the SNP in the MdMYB1-2 and MdMYB1-3 alleles. This SNP was used as the basis for designing primers for a dCAPS PCR assay to test the segregation of MdMYB1 alleles with the inheritance of fruit skin color. The forward primer was designed with the SNP incorporated in the primer sequence and its location in the promoter is shown in Figure 7B. After amplification, PCR products that contained the MdMYB1-1 promoter sequence were restricted by the enzyme BstEII to a size of 263 bp, while products that did not contain this sequence, such as derived from the MdMYB1-2 and MdMYB1-3 alleles, were not digested and were 291 bp in size. The 263 bp PCR fragment was detected in all of the red skin cultivars we tested, indicating they contained the MdMYB1-1 allele (Fig. 8A ). Most of these cultivars appeared to be heterozygous for MdMYB1-1 as an undigested PCR product of 291 bp was also detected, indicating the presence of another allele. For two of the red skin cultivars, Fuji and Hi Early, only the 263 bp PCR fragment was detected, which indicates they could be homozygous for MdMYB1-1 or that they contain an allele whose sequence could either not be amplified or distinguished from MdMYB1-1 by the dCAPS assay. The latter possibility seems most likely since Fuji is heterozygous for skin color inheritance as when it is crossed with a non-red skin cultivar it produces progeny with a 1:1 segregation of skin color.
For most of the non-red skin cultivars only the 291 bp PCR product was detected, indicating they do not contain the MdMYB1-1 allele. In the cases of Granny Smith and Grandspur, which are genetically related, the dCAPS assay indicated the presence of the MdMYB1-1 allele, yet these are non-red skin cultivars. Sequence analysis of this allele confirmed that, while it had the same sequence as MdMYB1-1 in the region where the dCAPS primers were designed, there were numerous other differences compared to the MdMYB1-1 promoter. Therefore these cultivars contain at least one unidentified allele of MdMYB1. The segregation of MdMYB1-1 with color inheritance was tested in progeny from the cross of an unnamed red skin selection (Red Selection, from a cross of Lady Williams by Golden Delicious and a sibling of Cripps' Pink and Cripps' Red) and Golden Delicious. The utility of the dCAPS assay for predicting skin color inheritance is shown in a small number of the progeny, which is typical of the results we obtained for a total of 136 progeny (Fig. 8B). Apples in which the 263 bp PCR fragment was detected, indicating the presence of MdMYB1-1, had varying levels of pigmentation perhaps reflecting the degree of exposure to sunlight. Apples in which only the 291 bp PCR fragment was detected had none or very poor pigmentation. In total, 93 progeny were scored for red skin color and in all of these the MdMYB1-1 allele was detected by the dCAPS assay, while for the 43 progeny scored as non-red the MdMYB1-1 allele was not detected (Table II ). The MdMYB1-1 allele shows segregation with red skin color inheritance, indicating it is probably the color locus for apple fruit skin in these cultivars.
Activity of the Flavonoid Pathway in Apple Cultivars
To determine the nature of the genetic basis of apple fruit skin color we analyzed flavonoid synthesis and structural gene transcription in several non-red and red-skin cultivars. We found that non-red skin cultivars, despite being deficient in anthocyanin synthesis, were still able to synthesize CTs and flavonols in fruit skin to a similar level as found in red skin cultivars. Our results are in agreement with the previous analysis of flavonoid accumulation in the fruit skin of several apple cultivars at ripening (Lata et al., 2005
Using real-time PCR we determined that transcript levels of MdCHS, MdF3H, MdDFR, MdLDOX, and MdUFGT genes required for anthocyanin synthesis were lower in fruit skin of non-red than red skin cultivars. This result is in agreement with the enzyme activity data of Lister et al. (1996)
Since transcription of at least five structural flavonoid genes was affected, the genetic basis of apple skin color is probably due to the activity of a common regulator of these genes. This would be analogous to AtPAP1, which is a specific regulator of the anthocyanin pathway in Arabidopsis (Borevitz et al., 2000
The MdCHI gene was the only anthocyanin pathway gene that did not seem to have reduced expression in non-red skin apple cultivars at fruit ripening. This suggests that MdCHI is not coregulated with the other pathway steps. However, both Lister et al. (1996)
We isolated a gene that encoded an R2R3 MYB family transcription factor we named MdMYB1. Phylogenetic analysis placed MdMYB1 in a group of MYB proteins that have been functionally characterized to regulate anthocyanin synthesis and separate from MYBs that regulate other processes.
Consistent with a role in regulating anthocyanin synthesis, MdMYB1 gene transcripts increased within 1 d when bags were removed from apple fruit exposing skin to sunlight. This increase in MdMYB1 transcripts was accompanied by the accumulation of anthocyanin and flavonoid structural gene transcripts in the skin (Takos et al., 2006a
The preferential expression of MdMYB1 in red skin sectors of apple fruit is consistent with the model of a specific regulator of the anthocyanin branch of the pathway. This is because CT synthesis and MdLAR and MdANR transcripts are distributed almost equally between red and green skin sectors of the same fruit (Takos et al., 2006b
We demonstrated that MdMYB1 can function as an anthocyanin regulator by introducing the coding region of the cDNA into Arabidopsis plants and cultured grape cells. In both these systems anthocyanin synthesis was induced (Fig. 6, A–D). The Arabidopsis seed coat is rich in CTs, but when the AtANR gene is disrupted anthocyanins can accumulate (Devic et al., 1999
We have shown that MdMYB1 can activate transcription from the apple MdDFR and MdUFGT promoters when cobombarded into grape cells (Fig. 6E). The promoters of MdDFR and MdUFGT contain putative light regulatory units, consisting of an ACGT element and MYB-recognition element (MRE), or MRE-like sequences, which have been identified by Hartmann et al. (2005)
We isolated and sequenced three alleles of MdMYB1 in cultivars descended from Lady Williams crossed with Golden Delicious (Fig. 7A). Using a dCAPS PCR assay we found that one of these alleles, MdMYB1-1, segregated with skin color in most of the cultivars we tested. The exceptions were the non-red skin cultivars Granny Smith and Grandspur; however, these cultivars contain a different unidentified allele of MdMYB1 that is presumably nonfunctional, probably due to low expression (Fig. 5C). Our data is in agreement with the random-amplified polymorphic DNA markers developed by Cheng et al. (1996)
The perfect segregation of the MdMYB1-1 allele with color in 136 progeny of Red Selection (a sibling of Cripps' Pink) backcrossed to Golden Delicious demonstrated that this allele is tightly linked to the color locus in these cultivars. However, the segregation ratio of greater than 2:1 of red-skinned to green-skinned progeny is different from the expected 1:1 ratio. There are a number of possible reasons why this has occurred. Apples have a self incompatibility system that might reduce pollination by one allele since the parents are genetically related (Schneider et al., 2005
The two other alleles sequenced, MdMYB1-2 and MdMYB1-3, encode, respectively, an identical protein to MdMYB1-1 and a protein with an Arg-to-Ser exchange in the C-terminal domain (Fig. 4B), but this change does not perturb functional activity of the MdMYB1 cDNA in the grape cell assay (Fig. 6E). Therefore it seems likely these alleles are deficient in regulating anthocyanin synthesis due to altered gene regulation, as evidenced by the poor expression in non-red skin cultivars (Fig. 5C). This would explain why non-red skin cultivars can still develop a faint blush under high light (Reay, 1999
The MdMYB1-1 dCAPS marker could be a useful tool for apple breeding programs as it would allow the prediction of fruit skin color formation long before immature trees are able to bare fruit. This would be of even greater significance if MdMYB1 also controls later steps of pigment formation that could affect skin hue. For example, overexpression of the MYBs LeANT1 and AtPAP1 in tomato and Arabidopsis, respectively, induces genes involved in flavonoid modification and transport into vacuoles (Mathews et al., 2003
There has been extensive testing of growth conditions to improve anthocyanin synthesis in orchard-grown apples, including chemical sprays and plant hormones, the use of reflecting films on the canopy floor to alter light conditions and manipulation of the crop load, the canopy, and temperature conditions (Arakawa, 1988
In apple fruit the flavonoid pathway is spatially and temporally regulated but it is also responsive to light. We have identified a MYB regulatory gene, MdMYB1, that has an expression pattern correlating with that of the structural genes during fruit development and in response to light and coincides with anthocyanin synthesis. The MYB regulator can activate the promoters of two of these structural genes in a transient assay and one allele of this gene segregates with skin color in progeny of a cross of two apples with different-colored skins. This suggests that MdMYB1 regulates structural genes across the flavonoid pathway in apple fruit, determining skin color and providing the potential to modulate fruit color by altering expression of this regulatory gene. It will be of great interest to determine how the response to light is also mediated through this key regulator in apple fruit.
Apple Fruit Samples
Samples of fruit skin, flesh, seeds, and early fruit development stages of apple (Malus domestica Borkh. cv Cripps' Red) were collected at the Primary Industries and Resources South Australia Site at Lenswood in the Adelaide Hills, South Australia from November, 2002 until April, 2003 as described by Takos et al. (2006b)
Anthocyanins and flavonols were extracted from 0.1 g of finely ground plant material in 1 mL 1% (v/v) HCL-methanol for 1 h at room temperature on a rotating wheel in darkness. Samples were clarified by centrifugation at 13,000g for 15 min at room temperature and 10 µL of supernatant was analyzed by HPLC, as described by Takos et al. (2006a)
CTs were extracted from 0.1 g of finely ground plant material in 1 mL 70% (v/v) acetone. The extract was processed in a tannin-protein binding assay as according to Downey and Adams (2005)
Total RNA was isolated from 1 g apple tissue by a hot borate method (Wan and Wilkins, 1994 First-strand cDNA was synthesized from 4 µg total RNA except for flesh samples where 2 µg was used due to an inhibition of synthesis at higher concentrations. The reaction was primed by oligo dT(18) and synthesis of cDNA catalyzed by SuperScriptIII RNase H-Reverse Transcriptase with RNAseOUT (Invitrogen) included in the reaction to inhibit RNA degradation.
For real-time PCR, cDNA was diluted 1:20 with water and 5 µL placed into a reaction containing 266 nM of each primer and Absolute QPCR SYBR Green buffer (ABgene). Primers for flavonoid structural genes and thermocycling conditions are described in Takos et al. (2006b)
First-strand cDNA was synthesized as described above from 4 µg total RNA extracted from dark-grown Cripps' Pink fruit that had been exposed to sunlight for 2 d. Degenerate PCR primers for isolation of MYB genes were: forward, 5'-TGYATHRAYAARTAYGGIGARGGIAARTGG-3' and reverse, 5'-GTRTTCCARTARTTYTTIACRTCRTTNGC-3'. Standard PCR conditions were used for Platinum Taq DNA Polymerase (Invitrogen) with the addition of betaine to 1 M. Thermocycling conditions were: initial denaturation of 94°C for 2 min; 35 cycles of 94°C for 30 s, 45°C for 30 s, 72°C for 1 min; and final extension of 72°C for 5 min. A single 246 bp PCR product was ligated to pDrive (QIAGEN) and several clones sequenced. A single sequence was obtained and designated MdMYB1. Nested primers were designed for 3' and 5' RACE PCR. The cDNA template primed with oligo dT(18) described above was used for the 3' RACE PCR while for 5' RACE PCR fresh first-strand cDNA was made from the Cripps' Pink total RNA sample above using a GeneRacer kit (Invitrogen). Using the sequence information obtained by RACE PCR primers were designed to amplify a PCR fragment containing the full-length coding sequence of MdMYB1 and were: forward, 5'-GCGGTACCGGTAGCAGGCAAAAGAATAGCTAAGC-3' and reverse, 5'-GCGGATCCCACATTTACAAGCAAGGAAAATA-3'. An 848 bp PCR fragment was obtained from the Cripps' Pink cDNA template used above in a reaction catalyzed by PfuTurbo (Stratagene) following the manufacturer's reaction conditions. The PCR fragment was tailed with dATP using a QIAGEN A-Addition kit and was ligated to pDrive (QIAGEN). Several clones were sequenced and two MdMYB1 cDNAs, which differed in nucleotide 573 of the coding sequence resulting in an Arg (MdMYB1-1) or Ser (MdMYB1-3) in position 191 of the deduced amino acid sequence, were identified.
The full-length coding region sequence MdMYB1-1 or AtPAP1 was ligated to the binary vector pART27 or pBART27, a derivative of pART27, respectively (Gleave, 1992
Two MdMYB1 ORFs, MdMYB1-1 and MdMYB1-3, were ligated to the pART7 vector (Gleave, 1992
Primers for the PCR of the full-length MdMYB1 cDNA (above) were also used to amplify the full-length genomic sequence from Cripps Pink, Cripps' Red, and Golden Delicious genomic DNA templates. Fragments, approximately 4 kb in length, were amplified using Platinum Taq DNA Polymerase High Fidelity (Invitrogen) following the manufacturer's recommended reaction conditions. The PCR products were ligated to pDrive (QIAGEN) and several clones sequenced for each reaction. An approximately 2 kB fragment of the promoter sequence of MdMYB1 was isolated with a Universal GenomeWalker kit (BD Biosciences) as described above. Primers specific to each MdMYB1 allele, based upon differences in intron 1, were then used to amplify the promoter sequence that corresponded to each allele of MdMYB1 with Platinum Taq DNA Polymerase High Fidelity (Invitrogen) following the manufacturer's conditions. The PCR products were ligated to pDrive (QIAGEN) and several clones sequenced for each reaction.
Primers for dCAPS PCR were: forward, 5'-CCTGAACACGTGGGAACCGGCCCGTTGGTAAC-3' and reverse, 5'-GTGAAGGTTGTCTTTATTAGTGACGTG-3'. The approximately 250 bp PCR product was amplified with Platinum Taq DNA Polymerase (Invitrogen) following the manufacturer's conditions in a reaction of 50 µL. Thermocycling conditions were: initial denaturation of 94°C for 2 min; 35 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 30 s; and final extension of 72°C for 5 min. After PCR, 15 µL of the reaction was digested with 10 units of BstEII (Promega) at 65°C for 4 h. Digested DNA fragments were resolved on a 3% (w/v) NuSieve GTG Agarose gel (FMC BioProducts).
Sequences referred to in this article are deposited at the National Center for Biotechnology Information under the following accession numbers: apple, MdMYB1-1 (DQ886414), MdMYB1-2 (DQ886415), MdMYB1-3 (DQ886416), promoter of MdDFR (DQ886412), promoter of MdUFGT (DQ886413); gerbera hybrid, GMYB10 (CAD87010); tomato (Lycopersicon esculentum), LeANT1 (AAQ55181); petunia (Petunia hybrida), PhAN2 (AAF66727), PhPH4 (AAY52377), and PhODO1 (AAV98200); capsicum (Capsicum annuum) CaA (CAE75745); Arabidopsis, AtPAP1 (ABB03879), AtTT2 (Q9FJA2), AtMYB12 (ABB03913), AtWER (AAF18939), and AtGL1 (AAC97387); strawberry (Fragaria spp.), FaMYB1 (AAK84064); grapevine, VvMYBA1 (BAD18977), VvMYBA2 (AB097924), and VvMYB5a (AAS68190); maize (Zea mays), ZmC1 (AAA33482) and ZmPl (AAA19821); snapdragon (Antirrhinum majus), AmMIXTA (CAA55725), AmROSEA1 (ABB83826), AmROSEA2 (ABB83827), and AmVENOSA (ABB83828); carrot (Daucus carota), DcMYB1 (BAE54312); and tobacco (Nicotiana tabacum), NtMYB2 (BAA88222).
A similar gene, MdMYB10 (DQ267896–DQ267898), has been isolated from apple and shown to regulate anthocyanin production (Espley RV, Hellens RP, Putterill J, Stevenson DE, Kutty-Amma S, Allan AC [2006] Red colouration in apple fruit is due to the activity of MYB transcription factor, MdMYB10. Plant J [in press]).
We thank Commonwealth Scientific and Industrial Research Organization colleagues Nicole Cordon for help with HPLC analysis, and Karin Sefton and Debra McDavid for excellent technical assistance; staff from Primary Industries and Resources South Australia for allowing us to sample apple fruits at Lenswood in the Adelaide Hills; Eva Decker, the University of Freiburg, for luciferase expression vectors; Mark Downey, Department of Primary Industries Victoria, for developing methods of analysis of flavonoids; and the Department of Agriculture and Food Western Australia and Horticulture Australia Ltd. for a grant to support this work. Received August 9, 2006; accepted September 7, 2006; published October 13, 2006.
1 This work was supported by a grant from the Department of Agriculture and Food Western Australia and Horticulture Australia Ltd. 
2 Present address: Heidelberger Institut fuer Pflanzenwissenschaften, Im Neuenheimer Feld 360, D–69120 Heidelberg, Germany. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Amanda R. Walker (mandy.walker{at}csiro.au). www.plantphysiol.org/cgi/doi/10.1104/pp.106.088104 * Corresponding author; e-mail mandy.walker{at}csiro.au; fax 61–8–8303–8601.
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ABSTRACT
RESULTS
-hydroxylase; DFR, dihydroflavonol-4-reductase; LDOX, leucoanthocyanidin dioxygenase. Enzymes specific for various flavonoid compounds: UFGT, UDP-glycose:flavonoid-3-O-glycosyltransferase for anthocyanin synthesis. The unidentified enzyme encoding a glycosyl transferase for flavonol glycone synthesis is referred to as GT. The flavononoid-3'-hydroxylase enzyme has been omitted for clarity but it may hydroxylate several of the intermediates from the CHI step onwards.
