An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae
© Lin-Wang et al; licensee BioMed Central Ltd. 2010
Received: 8 November 2009
Accepted: 21 March 2010
Published: 21 March 2010
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.
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.
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.
The Rosaceae is an economically important group of cultivated plants, which includes fruit-producing genera such as Malus (apples), Pyrus (pears), Prunus (e.g. peach, plums, apricots), Fragaria (strawberries), and Rubus (raspberry, blackberry, boysenberry), as well as ornamental plants such as Rosa (rose). In these fruits and flowers, colour is a key quality trait and is often caused by anthocyanin. Anthocyanins are water-soluble pigments that belong to the flavonoid family of compounds giving red, blue and purple colours in a range of flowers, fruits, foliage, seeds and roots . Anthocyanins are involved in a wide range of functions, such as the attraction of pollinators, seed dispersal, protection against UV light damage, and pathogen attack [2–5]. Recently, research on anthocyanins has intensified because of their potential benefits to human health, including protection against cancer, inflammation, coronary heart diseases and other age-related diseases [6–11].
In plants, the structural genes of the flavonoid biosynthetic pathway are largely regulated at the level of transcription. In all species studied to date, the regulation of the expression of anthocyanin biosynthetic genes are through a complex of MYB transcription factors (TF), basic helix-loop-helix (bHLH) TFs and WD-repeat proteins (the MYB-bHLH-WD40 "MBW" complex; ). A model has been proposed for the activation of structural pigmentation genes, with regulators interacting with each other to form transcriptional complexes in conjunction with the promoters of structural genes . For example, the R2R3 MYB C1 protein, that regulates the anthocyanin pathway in maize, interacts with a bHLH TF (either of the genes termed B or R) to activate the promoter of dihydroflavonol reductase (DFR). In contrast, the R2R3 MYB P protein, which regulates the phlobaphene pathway in maize, can activate the same promoter without a bHLH TF .
MYB TFs can be classified into three subfamilies based on the number of highly conserved imperfect repeats in the DNA-binding domain including R3 MYB (MYB1R) with one repeat, R2R3 MYB with two repeats, and R1R2R3 MYB (MYB3R) with three repeats [15, 16]. Among these MYB transcription factors, R2R3-MYBs constitute the largest TF gene family in plants, with 126 R2R3 MYB genes identified in Arabidopsis . Those associated with up-regulation of the anthocyanin pathway are R2R3 MYBs. Over-expression of the AtPAP1 gene (AtMYB75, At1 g56650) results in the accumulation of anthocyanins in Arabidopsis . Several repressors of the phenylpropanoid pathway, and perhaps anthocyanins specifically, are also MYB TFs, including an R2R3 MYB repressor from strawberry FaMYB1 , Arabidopsis AtMYB6, 4, and 3 , Antirrhinum AmMYB308 , and a one repeat MYB in Arabidopsis, AtMYBL2 [22, 23]. How the repressor MYBs interact with the MBW transcriptional complex is beginning to be elucidated [22, 23].
Based on the phylogenetic relationship between Arabidopsis R2R3 MYB TFs and anthocyanin-related MYBs of other species, it appears that anthocyanin-regulating R2R3 MYBs fall into one or two clades [17, 24, 25]. Anthocyanin-regulating MYBs have been isolated from many species, including Arabidopsis AtMYB75 or PAP1, AtMYB90 or PAP2, AtMYB113 and AtMYB114 , Solanum lycopersicum ANT1 , Petunia hybrida AN2 , Capsicum annuum A , Vitis vinifera VvMYB1a , Zea mays P , Oryza saliva C1 , Ipomoea batatas IbMYB1 , Anitirrhinum majus ROSEA1, ROSEA2 and VENOSA , Gerbera hybrid GhMYB10 , Picea mariana MBF1 , Garcinia mangostana GmMYB10 , Malus × domestica MdMYB10, MdMYB1/MdMYBA [24, 38, 39], and Gentian GtMYB3 .
For rosaceous species, MYBs that regulate the genes of the anthocyanin pathway have been examined in apple and strawberry. In apple (Malus × domestica) MYB10 was isolated from red-fleshed apple 'Red Field' , and showed a strong correlation between the expression of MYB10 and apple anthocyanin levels during fruit development. Transgenic apple lines constitutively expressing MYB10 produced highly pigmented shoots. Two more apple TFs, MYB1 and MYBA, were also reported to regulate genes in the anthocyanin pathway in red-skinned fruit [38, 39]. Both MYB1 and MYBA share identical sequences , while MYB10 and MYB1 genes are located at very similar positions on linkage group 9 of the apple genetic map . In strawberry (Fragaria × ananassa), the R2R3 MYB TF FaMYB1 plays a key role in down-regulating the biosynthesis of anthocyanins and flavonols .
In this current study, we used an allele-specific PCR primer approach to show that MdMYB1/MdMYBA/MdMYB10 are highly likely to be allelic in the apple genome. We then isolated genes with high sequence similarity to MYB10 from 20 species within the Rosaceae. Sequence and functional characterization of these genes provides insight into the evolution of this TF, within a plant family where higher levels of pigmentation has been selected for during the process of domestication. Expression analysis during the fruit development, and functional testing using transient assays and transgenic plants suggest that these R2R3 MYBs are responsible for controlling anthocyanin biosynthesis in these crops.
The MdMYB10/MdMYB1/MdMYBAgenes are likely to be allelic
While these end-point PCR amplifications are not quantitative, the fluorescence from ethidium bromide (EtBr) indicated that in those tissues where both 900- and 1000-bp fragments are amplified, these molecules are likely to be in equivalent molar quantity within the genome. This is based on the observation that when a mixture of diluted PCR products from the 900-bp and 1000-bp fragments are mixed in ratios of 3:1 or 1:3 respectively, the EtBr fluorescence of the end-point PCR amplifications reflects the corresponding molar ratios (Figure 1A). Furthermore, PCR analysis of the progeny from crosses made between the R1 homozygous Pacific Rose™ cultivar and the heterozygous R1R6 'Red Field' shows segregation of the homozygous R1 allele and the heterozygous R1 and R6 alleles (Figure 1B). If MYB1 and MYB10 were different genes, band intensity ratios of 3:1 would be possible but as only 1:1 ratios are observed, MYB1 and MYB10 are likely to be allelic, representing the R1 and R6 alleles.
Isolation of MYB10 homologues from the major rosaceous crop species
Anthocyanin activating R2R3 MYBs transcription factors
% similarity to AtMYB75 protein
% identity to AtMYB75
Solanum lycopersicum (tomato)
Vitis vinifera (grape)
Zea mays (Maize)
Oryza sativa (Rice)
Ipomoea batatas (Sweet potato)
Antirrhinum majus (snapdragon)
Malus domestica (apple)
Malus domestica (apple)
Malus sylvestris (crab apple)
Cydonia oblonga (quince)
Eriobotrya japonica (loquat)
Mespilus germanica (medlar)
Pyrus communis (Pear)
Pyrus pyrifolia (Nashi)
Pyrus × bretschneideri (Chinese pear)
Prunus armeniaca (Apricot)
Prunus insititia (Damson)
Prunus domestica (European plum)
Prunus avium (sweet cherry)
Prunus cerasus (sour cherry)
Prunus cerasifera (cherry plum)
Prunus dulcis (almond)
Prunus persica (peach)
Prunus salicina (Japanese plum)
Fragaria × ananassa (strawberry)
Fragaria vesca (strawberry)
Rosa hybrida (rose)
Rubus idaeus (red raspberry)
For both protein sequence and coding DNA sequence (CDS) of rosaceous MYBs, the percentage of identity to Arabidopsis AtMYB75 (PAP1, AT1G56650) varied from 58 to 64%, and 40 to 49%, respectively. The length of CDS and protein sequence was similar between each species analysed, but the length of genomic DNA (gDNA) sequence varied significantly from 1122 bp (Rosa hybrida) to 4055 bp (Malus × domestica, Table 1). This is due almost entirely to the variable length of intron 2, which ranges from 82 bp (AtMYB90) to 3000 bp (MdMYB1). A schematic of MYB10-like genes from rosaceous species is shown in Additional File 1. The large size of intron 2 in apple correlates with its higher DNA content than close relatives; apple has almost 2.5 times more DNA mass than pear http://www.kew.org/cval/homepage.html. Intron 2 of apple MYB10 is 2995 bp, compared with 487 bp in pear (Additional File 1B).
Sequence signatures specific for anthocyanin-related MYBs
The large gene family of R2R3 MYB proteins was examined using conserved regions of homology. Over 172 proteins were included; all Arabidopsis R2R3 MYBs, 38 other dicot anthocyanin-promoting MYBs, including apple MYB8, MYB9 and MYB11 (GenBank DQ267899, DQ267900, and DQ074463 respectively), strawberry anthocyanin repressor MYB1, as well as anthocyanin-related MYBs from four monocots and one gymnosperm. All the MYBs associated with promoting anthocyanin biosynthesis from dicot species cluster within the same clade as PAP1 and other Arabidopsis MYBs of this subgroup (Figure 3A). Monocot sequences, such as C1 and P, as well as the gymnosperm Picea mariana MBF1, cluster outside this group, suggesting that this clade is dicot-specific. The function of promoting anthocyanin biosynthesis for this subgroup may therefore have evolved after the divergence between dicots and monocots.
Outside of the DNA-interacting R2R3 domain, most R2R3 MYB proteins have a long C-terminal sequence. In this region of Arabidopsis anthocyanin-promoting MYBs, the motif KPRPR [S/T]F has been identified (Box B in Figure 2) , which is not present in other R2R3 MYBs. When anthocyanin-promoting MYB sequences from other species are aligned, this C-terminus consensus motif was still identifiable but with slight variations (Figure 2) to become [R/K]Px [P/A/R]xx [F/Y]. Within the subfamilies Maloideae and Amygdeloideae, there was over 70% similarity of C-terminus. An 18 amino acid deletion occurred in the C-terminus of both almond and peach (Figure 2) which is within exon 3, indicating that this is not a mis-prediction of an exon-intron boundary. However, this deletion did not disrupt the activity of peach MYB10 (see next section). Other anthocyanin-related MYBs are known to repress the biosynthetic pathway (e.g., FaMYB1, AtMYB3, AtMYBL2). These contain C-terminal motifs such as the ERF-associated amphiphilic repression (EAR) motif or the TLLLFR motif [22, 23]. Such motifs were not found in any of the MYB10-like predicted proteins identified in this study.
A conserved amino acid signature ([D/E]Lx2 [R/K]x3Lx6Lx3R) (the locations indicated by the arrows in Figure 2) has been shown to be functionally important for the interaction between MYB and R/B-like bHLH proteins . All rosaceous MYB sequences, as well as anthocyanin-related dicot MYBs and PmMBF1 and C1 had this signature. However, other R2R3 MYB TFs also have this signature (e.g., Arabidopsis MYBs TT2  and AtMYBL2 ). Therefore, the presence of this motif is not indicative of the candidate MYB being within the anthocyanin-promoting clade, but rather suggests that these MYBs require an interacting bHLH partner.
Functional assay of rosaceous MYB activity
Transient luciferase assays in the tobacco species Nicotiana benthamiana have been used to assay MYB activity against the Arabidopsis DFR-promoter (dihydro flavanoid reductase; At5g42800, [24, 46]). Full length cDNAs of apple (MYB10), wild and cultivated strawberry (Fv and FaMYB10), rose (RhMYB10) and raspberry (RiMYB10), and genomic DNA of pear, European plum, cherry-plum, cherry, apricot, and peach (PcMYB10, PdmMYB10, PcfMYB10, PavMYB10, ParMYB10, and PprMYB10, respectively) were cloned into the transient expression vector pGreen II 0024 62K  and transfected into Agrobacterium. These TFs were then co-infected into N. benthamiana leaves with AtDFR-LUC in a second Agrobacterium strain, with or without a bHLH co-factor in a third Agrobacterium strain. Trans-activation was assayed 3 days later as a change in LUC/REN ratio.
As previously reported  a patch of foliar anthocyanin production can be induced in Nicotiana tabacum leaves by co-expression of MdMYB10 with MdbHLH3. Induction of anthocyanin biosynthesis in transient assays by rosaceous MYB10s was tested and found to be dependent on the co-expression of the bHLH proteins from Arabidopsis or apple. Patches of anthocyanin were most apparent with PdmMYB10 and PprMYB10 when AtbHLH2 was included as a partner (Figure 5B).
Expression of rosaceous MYB10 TFs correlate with anthocyanin biosynthesis
Under stressful conditions (high light), the petals of F. vesca flowers became pigmented (Figure 7D). While FvMYB1 showed little change in these petals, the transcript of FvMYB10 from this tissue showed a large increase in accumulation compared with the petals that were not exposed to high light and were unpigmented. This is further evidence that MYB10 in strawberry is involved in regulating anthocyanin accumulation.
Transformation of MYB10 into the crop of origin results in elevation of anthocyanin biosynthesis
The plant MYB family
The MYB TF superfamily illustrates how a relatively small family in animal genomes (3 members of this TF type in the human genome by BLAST match) controlling cell division and differentiation has become the most abundant TF group in plants  with diverse functions in hormone response , growth , epidermal cell fate and formation of trichomes , stomatal movements and development ; , seed development , response to drought  and cold [56, 57], pathogen-response [58, 59], light-sensing responses [60, 61], sugar-related responses , modulation of secondary metabolites such as glucosinolates [63, 64] and phenylpropanoids . MYB proteins have a conserved N-terminal DNA binding domain of 100-160 residues, depending on the number of R repeats, with each repeat containing a helix-helix-turn-helix structure. Within this N-terminal region are key residues important for trans-activation efficiency , residues that regulate and specify DNA binding , and interactions with bHLHs . We have identified in this study several residues shared by anthocyanin-promoting MYBs, from diverse species, that may be important in their function (Figure 4).
Consensus motifs in the C-terminus of MYBs, important for function are just beginning to be elucidated. One such example is the case of the C2 EAR motif repressor clade. AtMYB4 has the motif NLELRISLPDDV, which is essential for its repressive activity against the CH4 promoter . This motif (pdLNLD/ELxiG/S) is also conserved in a number of R2R3 MYB proteins belonging to subgroup 4 which includes AtMYB4, AtMYB6, AtMYB7 and AtMYB32, and Antirrhinum AmMYB308 and AmMYB330, which have very similar effects to AtMYB4 when over-expressed in tobacco . FaMYB1 also has such a motif . In anthocyanin-promoting MYBs, the motif KPRPR[S/T]F was identified . By analysing more MYBs of this clade we found variation in this C-terminal motif (Figure 2), but enough conservation to suggest it could be used as an identifier.
MYBs involved in regulation of phenylpropanoid levels
The phenylpropanoids include flavonoids, anthocyanins, and proanthocyanidins. The accumulation of these compounds in plants and plant organs is central to such quality parameters as colour, human health, bitterness and astringency, as well as plant response to biotic and abiotic stress. R2R3 MYBs are responsible for controlling different aspects of the phenylpropanoid pathway in a wide range of different plant species. These include flavonol-specific MYBs , proanthocyanidin-specific MYBs , inhibitors of branch points  and R2R3 MYBs specifically controlling the anthocyanin biosynthetic pathway genes as well as anthocyanin conjugation, transport into the vacuole , and acidification of this compartment to affect fruit/flower/foliage colour .
In Arabidopsis, one of the R2R3 anthocyanin-related clades is made up of AtMYB75 (PAP1, At1g56650; , AtMYB90 (AtPAP2, At1g66390), AtMYB113 (At1g66370), and AtMYB114 (At1g66380). As three of these MYBs occur in order on chromosome 1, they may have arisen by tandem duplications of AtMYB75. Over-expression of PAP1 , AtMYB113 and AtMYB114  all result in elevated anthocyanin levels. By examining homologues of PAP1 in other species, we have identified residues that predict MYBs involved in anthocyanin regulation. This anthocyanin-promoting clade is apparently absent in the rice genome and other monocots and gymnosperms, suggesting recent divergence of these MYBs.
In apple, three MYB genes have been independently isolated, all of which control anthocyanin levels and show a very high degree of sequence similarity; MYB10, MYB1 and MYBA. It has been suggested that MYB1 and MYBA are alleles arising from the different varieties from which they were cloned . The MYB10 sequence is more diverse. It is difficult, from sequence analysis alone, to distinguish between recently duplicated gene paralogues and allelic variation between different varieties. By designing PCR primers to a region of sequence common to both MYB10 and MYB1, we were able to distinguish between the MYB10 allele from 'Red Field', which produces a 1000-bp amplification product and other MYB10 alleles (from 'Royal Gala' amongst others) and the MYB1 alleles from Pacific Rose™ and 'Royal Gala' that produce a 900-bp fragment. If MYB1 and MYB10 were paralogues then, in the varieties that only amplified a 900-bp fragment, this fragment would be the product of four alleles (i.e. two from each of the parologous genes). However, if MYB10 and MYB1 are allelic, the 900-bp fragment would only be produced by two alleles, one from each of the MYB10/MYB1 alleles of the parents. Accordingly, in red-fleshed varieties which are heterozygous for the promoter polymorphism, such as 'Robert's Crab', one of the MYB10 alleles produces a 1000-bp fragment, and the other allele a 900-bp fragment. In homozygous Malus sieversii 01P22 there is only the 1000-bp fragment. In addition to this, if MYB1 was a paralogue, a further two 900-bp products would be contributed from the MYB1 alleles. As we do not see DNA fluorescence consistent with a 1:3 amplification of the 1000- and 900-bp fragments, we propose that MYB10 and MYB1/MYBA are alleles. The future availability of whole genome sequence for apple will aid a conclusion on the allelic structure of this gene.
Identification of anthocyanin-promoting MYB10 genes in rosaceous crops
Using degenerate PCR based on the MYB10 sequence, we have been able to isolate 20 MYB10-like genes from a range of rosaceous species. Analysis of the genomic DNA of these species predicts that all the genes contain two introns in positions consistent with the intron location of other MYB genes . Almost all of the variation in gene size is due to alterations in the predicted length of intron 2 (Table 1). Aligning intron 2 of Malus MYB10 (crab and domesticated apple; ~3000 bp) and Pyrus MYB10 (European and Asian pears; ~500 bp) revealed a high degree of similarity except within a region of intron 2 where there appears to have been a 2500-bp insertion. This does not contain inverted repeats or sequence signatures that are indicative of mobile genetic elements such as transposons or helitrons . This insertion could be the result of a local genome rearrangement that took place after the speciation of apple and pear, but before the divergence of apple and crab apples.
Botanical classification of the Rosaceae has recently been undertaken using 88 species analysed for a combination of phenotypic and molecular marker . Using the nuclear encoded genes, polygalacturonase inhibitor protein (PGIP) and polyphenol oxidase (PPO), a weak or conflicting phylogenic resolution was produced. We have complemented this analysis, on a smaller dataset, by adding additional information relating to a single copy nuclear gene, where orthology has been inferred by both sequence and functional characterisation. The phylogenic placements are in broad agreement with the Pyrinae sub-tribe and Amygdaloideae tribe described .
Transient activation of the anthocyanin pathway by rosaceous MYB10s requires a bHLH
Within the R2R3 domain of all 20 MYB10s there were several key motifs suggesting an association with a bHLH partner. Several anthocyanin-promoting MYBs have been assayed in heterologous systems; for example, tobacco [24, 39], Arabidopsis  or Antirrhinum petal cells  and this trans-activation is often enhanced by the co-infiltration of an appropriate bHLH gene [24, 37, 42]. We used transient assay of rosaceous MYB genes, with the DFR promoter from Arabidopsis and bHLH genes from either apple or Arabidopsis. All of the isolated MYB TFs were able to trans-activate the DFR promoter in the presence of at least one of the four bHLHs tested. Only the apple MYB10 gene responded equally to either apple or Arabidopsis bHLH genes. This may indicate a degree of specificity that exists for the apple bHLH gene and its association with apple MYB gene and target promoter. However, the degree of trans-activation, and interaction with bHLH partners, varies greatly amongst the rosaceous MYBs genes tested (Figure 5). In particular, plum and apricot showed trans-activation values in excess of five times that of the 35S-promoter. Such high trans-activation potential may be due to more effective interaction of plum and cherry MYBs with tobacco transcription factors endogenous within the transient assay, or could point to an enhanced ability of these MYBs to promote high levels of anthocyanin. Further analysis of these MYB TFs in homologous systems is required, and techniques such as yeast-2-hybrid used to probe which protein residues are responsible for strong or weak interactions.
MYB10 expression is strongly associated with anthocyanin production in fruits
During fruit development, in both strawberry and cherry, the transcript level of MYB10 was up-regulated. A correlation between transcript and anthocyanin production has already been reported in apple [24, 38, 39]. In a cherry cultivar which has lower anthocyanin levels at maturity the expression of MYB10 transcript was lower than in a dark-fruited cultivar. It remains for these genes to be mapped in crops segregating for different pigmentation levels. However, for apple, MYB10/MYB1/MYBA is the major gene in a crossed population segregating for red flesh  and red skin .
Transformation of strawberry with FaMYB10 resulted in plants with elevated root, foliar and fruit anthocyanin levels (Figure 8). These levels were not as high as previously reported in 35S:MdMYB10 apple transformants , due perhaps to other partners in the MYB/bHLH/WD40 complex. It has been shown recently that tomato fruits, with elevated anthocyanins due to over-expression of MYB and bHLH members of the MBW complex, are responsible for promoting human health attributes [6, 76].
The Rosaceae family-wide characterisation of MYBs provides insight into the evolution of this TF and has implications for the understanding of temporal-spatial colour change. Our identification of this set of MYBs will aid development of new rosaceous fruit and flowers by allowing the testing of co-segregation of MYB alleles with pigment phenotypes in Rosaceae, which are both common and highly sought after (e.g., rose, plum, cherry, peach). If these candidate genes do segregate for anthocyanin levels, gene-based marker-assisted selection or even cisgenics could be used in breeding programmes. This approach has worked for apple  and there is preliminary evidence that PavMYB10 co-locates with a QTL for fruit and flesh colour in cherry (A. Iezzoni and J. Bushakra, pers. comm.) and PprMYB10 co-locates with anther colour segregating in the peach reference map (J. Bushakra and P. Arus, pers. comm.). This group of transcription factors therefore becomes useful as a breeding and biotechnological tool.
Isolation of rosaceous transcription factors
Fruit and leaf samples of 20 rosaceous species were collected as follows: crab apple (Malus sylvestris), sweet cherry (Prunus avium), sour cherry (Prunus cerasus), almond (Prunus dulcis), peach (Prunus persica), Japanese plum (Prunus salicina) and rose (Rosa hybrida) from Auckland Botanic Gardens (Auckland, New Zealand); quince (Cydonia oblonga), loquat (Eriobotrya japonica), medlar (Mespilus germanica), pear (Pyrus communis), apricot (Prunus armeniaca), Damson (Prunus insititia), European plum (Prunus domestics), cherry-plum (Prunus cerasifera) from local gardens (Auckland, New Zealand); strawberry (Fragaria × ananassa and Fragaria vesca) from Plant & Food Research greenhouse (Auckland, New Zealand); red raspberry 'Latham' (Rubus idaeus), pear 'Nashi' (Pyrus pyrifolia), and Chinese pear 'Yali' (Pyrus × bretschneideri) from various Plant & Food Research orchards.
Messenger RNA (mRNA) was isolated using an adapted method  from pigmented fruit or flower tissue, and genomic DNA (gDNA) was isolated (DNeasy Plant Mini Kit, Qiagen) from young leaves or flower buds. MYB10s of pear (Pyrus communis) and cherry-plum (Prunus cerasifera) were successfully obtained by applying various primers based on MdMYB10 in cDNA or gDNA PCR amplification. With more primers based on PcMYB10 and PcfMYB10, MYB10s from two subfamilies, Maloideae and Amygdaloideae, were completed by overlapping PCR fragments. For the subfamily Rosoideae, which includes Fragaria, Rubus and Rosa, degenerate primers, designed to the consensus DNA sequence of R2R3 binding domain were used in 5' and 3' GeneRace (GeneRacer Kit, Invitrogen). The complete sequence for MYB10 was compiled from overlapping fragments and full length clones were isolated using gene-specific primers designed to the 5' and 3' UTR regions. Phylogenic trees were generated using MEGA 3.1, a minimum evolution phylogeny test and 1000 bootstrap replicates.
Dual luciferase assay of transiently transformed Nicotiana benthamiana leaves
The promoter of Arabidopsis DFR (TT3, AT5g42800) was isolated from genomic Arabidopsis DNA and cloned into pGreenII 0800-LUC vector . MYB10 cDNA or gDNA full length sequence from 10 selected rosaceous species was cloned into pGreen II 62-SK 0029 binary vectors .
Nicotiana benthamiana plants were grown under glasshouse conditions until about 5 cm in height. Approximately 150 μl of Agrobacterium culture was infiltrated at four points into a young leaf. Three days after inoculation, 3-mm leaf discs (4 technical replicates from each plant) were cut with a hole-puncher, placed into wells of a 96-well-plate containing 50 μl of PBS (phosphate buffered saline) in each well, and gently crushed with the hole-puncher. The measurement and analysis was carried out using an Orion Microplate Luminometer (Berthold Detection System), using the manufacturer's recommended conditions.
PCR expression analysis
Strawberry fruits from Fragaria × ananassa and Fragaria vesca were collected at six time points during fruit development: stage 1, pre-opened bud; stage 2, fully open flower; stage 3, petal drop; stage 4, expanding fruitlet; stage 5, expanded fruit; stage 6, red-ripe fruit, from plants grown under glasshouse conditions, using natural light with daylight extension to 16 h. In one instance, Fragaria vesca was grown under constant lighting, inducing red pigmented petals. RNA was isolated  from fruit (six samples from the same plant, skin and cortex combined), and red and white petals. First strand cDNA synthesis was carried out by using oligo dT according to the manufacturer's instructions (SuperScript III, Invitrogen). As the identity between cDNA sequences of Fragaria × ananassa and Fragaria vesca is as high as 95%, a set of qPCR primers was designed for both species using Vector NTI to a stringent set of criteria, enabling application under universal reaction conditions.
To eliminate gDNA contamination, both forward and reverse primers were designed to span an intron/exon boundary. The strawberry actin primers were based on the actin sequence of Fragaria × ananassa (Genebank number AB116565). The reverse primer of actin was also designed to span an intron.
Sweet cherry (Prunus avium) 'Stella' and 'Rainier' were collected at three time points during fruit development: stage 1, green fruitlet; stage 2, expanding fruit; stage 3, mature fruit, from a Plant & Food Research orchard (Clyde, New Zealand). Actin primers were based on the actin sequence of closely related sour cherry Prunus cerasus (Genebank number EE488162). The method of RNA extraction and the principles of primer design were the same as strawberry.
Quantitative real time PCR (qPCR) DNA amplification and analysis was carried out using the LightCycler System (Roche LightCycler 1.5, Roche), with LightCycler software version 4. The LightCycler FastStart SYBR Green Master Mix (Roche) was used, and the 10 μl of total reaction volume applied in all the reactions following the manufacturer's method. qPCR conditions were 5 min at 95°C, followed by 40 cycles of 5 s at 95°C, 5 s at 60°C, and 10 s at 72°C, followed by 65°C to 95°C melting curve detection. The qPCR efficiency of each gene was obtained by analysing the standard curve of a cDNA serial dilution of that gene. The expression was normalized to Fragaria × ananassa actin and Prunus cerasus actin with Fragaria vesca stage 1 flower bud and Prunus avium 'Rainier' stage 1 fruitlet acting as calibrator with a nominal value of 1. Actin was selected as a reference gene because of its consistent transcript level throughout fruits and leaves. To confirm the amplification of the expected DNA sequence, qPCR amplicons were sequenced.
Endpoint PCR analysis used in the apple MYB allele study was carried out using Platinum Taq (Invitrogen). Reaction conditions were 95°C, 5 min followed by 35 cycles of 30 s at 95°C, 30 s at 55°C, and 60 s at 72°C. PCR products were separated on 1% agarose gels and stained with ethidium bromide. Primer sequences are listed in Additional File 4.
Growth of Strawberry plants and Generation of 35S:FaMYB10 Fragaria × ananassaplants
Strawberry plants of Fragaria × ananassa and Fragaria vesca were grown under controlled conditions (23°C day, 15°C night) in a short day room (8 h day, 16 h night) for 3 months, then plants were moved to long day conditions (16 h day, 8 h night, 25°C day, 15°C night) to encourage flowering.
Surface sterilized seeds were germinated on 1/2 MS basal salt and vitamins (Duchefa) + 3% sucrose + 0.7% agar (Germantown) (pH 5.7) medium. Seedlings were sub-cultured onto fresh medium every four weeks. Young leaves excised from in vitro grown shoots were cut into ~1 × 2 mm leaf strips. Transformation was via an adapted protocol (from  with Agrobacterium tumefaciens strain EHA105 , harbouring the binary plasmid pGreen II 0029 62-sk  containing the NOS/NPT II for kanamycin resistance, and a CaMV 35s promoter-driven full length FaMYB10 cDNA.
HPLC measurement of strawberry fruits
Two mature strawberry fruits were taken from each of three plants representing two transgenic lines and a wild-type control. The fruits were freeze-dried for at least 24 h. The dried tissue was then pulverized, resuspended in ethanol: distilled water: formic acid (80:20:1) with the ratio of 5 mL solvent to 1 g of original fresh fruit weight, extracted at room temperature for 3 h in the dark, centrifuged at 3500 rpm for 10 min. A 1-ml aliquot of the supernatant was analyzed for anthocyanin components by HPLC. The HPLC system consisted of a Waters Alliance Separation Module (model 2690) and a photodiode array detector (model 996) under the control of Chromeolen® (Dionex, USA) software. The separation column used was a Zorbax Rapid Resolution SB-C18 4.6 × 150 mm (Agilent, USA) with a binary solvent program (A = formic acid/MQ water (5:95); B = acetonitrile) that started at 95% A 5% B at injection, changed to 80%A 20%B at 9 minutes; 20%A 80%B at 18 minutes and held for 2 minutes before returning to 95%A 5%B ready for the next sample injection. Total flow rate was 0.8 mL/min and sample injection volumes were 5 μL. Anthocyanin components were detected at 530 nm, and peaks indentified by retention time with authentic standards, and previous reports of strawberry anthocyanins.
We wish to thank Mary Petley, Eric Walton, and Jill MacLaren, of Plant & Food Research for assistance with collection of plant material, and Jill Bushakra, David Chagné, and William Laing of Plant & Food Research for assistance with the manuscript, and Niels Nieuwenhuizen, Tim Holmes, and Minna Pesonen for technical assistance. This research was funded by a grant from the New Zealand FRST contract "HortGenomics CO6X0207".
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