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Plant Cell. Apr 2006; 18(4): 831–851.
PMCID: PMC1425845

A Small Family of MYB-Regulatory Genes Controls Floral Pigmentation Intensity and Patterning in the Genus Antirrhinum[W]

Abstract

The Rosea1, Rosea2, and Venosa genes encode MYB-related transcription factors active in the flowers of Antirrhinum majus. Analysis of mutant phenotypes shows that these genes control the intensity and pattern of magenta anthocyanin pigmentation in flowers. Despite the structural similarity of these regulatory proteins, they influence the expression of target genes encoding the enzymes of anthocyanin biosynthesis with different specificities. Consequently, they are not equivalent biochemically in their activities. Different species of the genus Antirrhinum, native to Spain and Portugal, show striking differences in their patterns and intensities of floral pigmentation. Differences in anthocyanin pigmentation between at least six species are attributable to variations in the activity of the Rosea and Venosa loci. Set in the context of our understanding of the regulation of anthocyanin production in other genera, the activity of MYB-related genes is probably a primary cause of natural variation in anthocyanin pigmentation in plants.

INTRODUCTION

Some of the most striking features of insect-pollinated flowering plants are their highly distinctive patterns of floral pigmentation. Many color patterns provide honey guides that direct pollinators toward the reproductive organs and the source of nectar within the flowers (Waser and Price, 1983). In Antirrhinum majus, color patterning results from the localized production of yellow aurone pigment in the hinge region created by the ventral and lateral petals of the corolla. The yellow is surrounded by magenta anthocyanin, which is produced more or less evenly in both inner and outer epidermal layers of the corolla lobes and at somewhat lower levels in the epidermal layers of the corolla tube (Figures 1A and 1J) (Jackson et al., 1992). This arrangement of yellow (aurone) and magenta (cyanidin) pigments provides a visual target for prospective pollinating bumblebees and probably guides them to the mouth of the fused corolla (Harbourne and Smith, 1978; Penny, 1983; Lunau et al., 1996). Some natural isolates of A. majus have additional patterning, in the form of increased pigmentation in regions of the epidermis overlying the vascular strands of the petal (Baur, 1910a, 1910b; Kuckuck and Schick, 1930; Stubbe, 1966). This venal pattern of pigmentation predominates on the inner (adaxial) epidermis of the dorsal petal lobes but continues into the inner epidermis of the corolla tube and, in contrast with the UV light–absorbing flavonoids, probably provides additional visual guides for the bees once they enter the corolla tubes (Thompson et al., 1972; Lunau et al., 1996).

Figure 1.
Floral Phenotypes of Regulatory Mutants of A. majus.

Europe hosts 20 distinct species of Antirrhinum, which are native to Spain, Portugal, France, and Italy (Sutton, 1988). Considerable variations in color pattern exist between species. Most are acyanic, or very palely pigmented with anthocyanin, although several of the palely pigmented species exhibit the strong venal pattern of pigmentation also observed in some isolates of A. majus. Some species also have enlarged zones of aurone accumulation, rendering the corolla lobes entirely yellow. This diversity in floral pigmentation patterning is diagnostic of the different species of Antirrhinum and is often used in their classification (Sutton, 1988). Flower color intensity and patterning may be traits that contribute significantly to pollinator selection in Antirrhinum and possibly also to reproductive isolation and speciation (Hodges and Arnold, 1994; Oyama, 2002).

A number of loci control anthocyanin production in flowers of the model species, A. majus. Mutations that affect the activity of structural genes encoding the enzymes of anthocyanin biosynthesis are well characterized and include mutations in the genes encoding chalcone synthase (nivea; CHS) (Sommer and Saedler, 1986), flavanone 3-hydroxylase (incolorata; F3H) (Martin et al., 1991), dihydroflavonol 4-reductase (pallida; DFR) (Martin et al., 1985), anthocyanidin synthase (candica; ANS) (Martin et al., 1991), and flavonoid 3′-hydroxylase (eosinea; F3′H) (Stickland and Harrison, 1974). Generally, knockout mutations at these loci give rise to acyanic flowers or, in the case of eosinea, flowers that produce alternative types of anthocyanin. Some mutations of these genes can give rise to patterned alleles (Coen et al., 1986; Martin et al., 1987; Coen and Carpenter, 1988; Martin and Lister, 1989), but patterned phenotypes invariably result from alleles in which the regulation of the expression of the structural gene has been affected, either through changes to the regulatory motifs of the promoters of the structural genes or through likely production of small interfering RNAs (Coen et al., 1986; Martin et al., 1987; Coen and Carpenter, 1988; Almeida et al., 1989; Martin and Lister, 1989; Robbins et al., 1989; Bollmann et al.,1991; Della Vedova et al., 2005). A number of mutations that affect the activity of regulatory genes that control the expression of the structural genes of floral pigment production have also been described, including delila (del), Eluta (El), rosea (ros), Venosa (Ve), and mutabilis (mut) (Wheldale, 1907; Baur, 1910a, 1910b; Kuckuck and Schick, 1930; Stubbe, 1966). Mutations in the regulatory genes do not abolish pigmentation but change the pattern of pigmentation within the flowers: Delila affects pigmentation in the corolla tube; Mut affects pigmentation in the corolla lobes; Rosea affects the pattern and intensity of pigmentation in both lobes and tubes; and Venosa affects pigmentation of the epidermis overlying the veins in both lobes and tubes. Of the regulatory genes, only Delila has been characterized molecularly and shown to encode a basic helix-loop-helix (bHLH) transcription factor that is required for the activation of expression of the late biosynthetic genes (including F3H, DFR, AS, and UDP-glucose 3-O-flavonoid transferase [UFGT]) in the corolla tube (Martin et al., 1991; Goodrich et al., 1992; Martin and Gerats, 1993). The other regulatory loci also influence the levels of transcripts of the biosynthetic genes active late in the anthocyanin biosynthetic pathway for their control of pigment patterning (Martin et al., 1991; Schwinn, 1999).

In maize (Zea mays), it has been shown that two types of transcription factor, a MYB-related protein and a bHLH-containing protein, interact to activate genes in the anthocyanin biosynthetic pathway (Cone et al., 1986, 1993; Paz-Ares et al., 1986, 1987; Chandler et al., 1989; Ludwig et al., 1989; Consonni et al., 1992, 1993; Goff et al., 1992; Sainz et al., 1997b; Zimmermann et al., 2004). In different accessions of maize, the genes encoding these proteins have been variously amplified, such that genes encoding functionally equivalent proteins are active in different tissues of the vegetative and reproductive phases of growth (Chandler et al., 1989; Cone et al., 1993; Pilu et al., 2003). Some of the resultant variation in plant pigment patterning may be attributable to human selection for more exotic pigmentation forms, because such lines were highly prized by the indigenous peoples of Central America (Lonnig and Saedler, 1997). In maize, an additional gene, pac1, encodes a WD repeat protein that affects the levels of anthocyanin production in kernels (Carey et al., 2004). By analogy to the activity of the homologous protein TRANSPARENT TESTA GLABRA1 (TTG1) in Arabidopsis thaliana (Walker et al., 1999), it seems likely that WD repeat proteins stabilize the interaction between MYB and bHLH proteins and so promote the transcriptional activation of the structural genes of the anthocyanin biosynthetic pathway (Serna, 2004; Zimmermann et al., 2004; Broun, 2005; Ramsay and Glover, 2005; Lepiniec et al., 2006).

In A. majus, the molecular characterization of delila demonstrated that gene products similar to those in maize regulate anthocyanin production in dicotyledonous species (Goodrich et al., 1992). This has also been shown to be the case for flowers of other dicotyledonous species such as morning glory (Ipomoea purpurea, Ipomoea tricolor) (Park et al., 2004; Chang et al., 2005), in vegetative tissues of Perilla frutescens (Gong et al., 1999; Sompornpailin et al., 2002; Springob et al., 2003), and in Petunia, in which a MYB-related gene, AN2, is required for anthocyanin production in flowers (Quattrocchio, 1994; Quattrocchio et al., 1999) and a bHLH protein, AN1, interacts with AN2 to activate anthocyanin biosynthesis (Spelt et al., 2000). A second gene encodes another bHLH protein, JAF13, which also interacts with AN2, but JAF13 is not able to complement an1 mutants and so is unlikely to be involved directly in regulating the transcription of anthocyanin biosynthetic genes (Quattrocchio et al., 1998; Spelt et al., 2000). It has been suggested that AN2 activates the expression of AN1 (Spelt et al., 2000). The AN2 MYB protein is thought to interact with AN1 to activate anthocyanin biosynthetic gene expression in conjunction with AN11, a WD repeat protein structurally similar to pac1 and TTG1 (de Vetten et al., 1997). Mutants of AN2 still synthesize anthocyanins in the corolla tubes and the limb retains pale pigmentation, which has led to the suggestion that other genes encoding R2R3 MYB proteins structurally related to AN2 also contribute to the regulation of anthocyanin biosynthesis in Petunia flowers. One possible candidate is the AN4 gene (Spelt et al., 2000; Koes et al., 2005).

We have characterized the activity of three genes encoding MYB-related transcription factors active in the flowers of A. majus. Analysis of mutant phenotypes showed that these genes contribute to the intensity and pattern of anthocyanin production in flowers. Despite the close structural similarity of these regulatory proteins, analysis of mutants showed that they influence the expression of target genes encoding the enzymes of anthocyanin biosynthesis with different specificities. Consequently, they are not precisely equivalent, biochemically, in their activities. Different species of the genus Antirrhinum, native to Spain and Portugal, show striking differences in their patterns and intensities of floral pigmentation. The differences in anthocyanin production between the six species investigated are attributable to variations in the activity of the Rosea and Venosa loci encoding these MYB-related regulatory proteins. Set in the context of our understanding of the regulation of anthocyanin production in other genera, the activity of MYB-related genes is probably a primary cause of natural variation in this trait in plants.

RESULTS

Phenotypes of the rosea and venosa Mutants Affecting Floral Pigmentation Intensity and Patterning

There are two extant mutant alleles of the rosea locus of A. majus called roseacolorata (roscol) (Figure 1B) and roseadorsea (rosdor) (Figure 1C) (Baur, 1910a; Stubbe, 1966). Wild-type (Ros+) flowers are almost completely self-colored, with high levels of red/magenta anthocyanin produced in both lobes and tubes of the corolla. Only the central region of the fused ventral and lateral petals is colored yellow, as a result of the accumulation of aurone in the absence of anthocyanin (Figure 1A). The vegetative parts of the plant also accumulate anthocyanin, including leaves (especially in the cells of the ventral [abaxial] epidermis) and stems.

Both rosea mutants have paler floral pigmentation. roscol has weak anthocyanin production restricted to the inner epidermis of the petals and a ring of pigment at the base of the corolla tube (Figure 1B). There is no anthocyanin pigment produced in the stems of the plant, although the abaxial surfaces of the leaves accumulate anthocyanin in field-grown plants. rosdor has flowers that have a weak ring of anthocyanin production toward the base of the tube and anthocyanin accumulation on the outer epidermis of the dorsal lobes (Figure 1C). The floral pigmentation in rosdor is very dependent on environmental conditions. At the higher temperatures (25°C) and lower light levels typical of greenhouses in the United Kingdom, no pigment is produced in the lobes (Figure 1I). Under the higher light but lower temperatures outside, field-grown rosdor plants have prominent pigmentation on their dorsal lobes (Figure 1C). The vegetative parts of rosdor plants are darkly pigmented, including stems and leaves. The mutations are fully recessive to the wild-type Ros+ allele. Furthermore, crosses between roscol and rosdor do not complement (Figures 1D to 1F). F1 plants have flowers with an intermediate phenotype between the two alleles, indicating that they carry mutations at the same locus.

The phenotype conferred by Venosa (Ve+) in A. majus involves the production of magenta anthocyanin pigment in tissue over the veins of the corolla (Figure 1G) (Baur, 1910a; Kuckuck and Schick, 1930; Stubbe, 1966). Pigment production is limited to the inner epidermis of the petal lobes and to the inner epidermis of the corolla tube (Figure 1H). The phenotype of Ve+ is not apparent in wild-type A. majus because the strong self-color masks the venal patterning. However, the phenotype is clear in the rosdor and roscol backgrounds (i.e., Ros+ is epistatic to Ve+). Unfortunately, the dominant Venosa+ (Ve+) accession, described by Stubbe (1966), has been lost from the Gatersleben germplasm collection. However, after outcrossing of another Gatersleben accession, decipiens, to stocks from the collection at the John Innes Centre, including roscol and rosdor, segregation of the Ve+ phenotype was apparent. Wild type accessions (John Innes Centre stocks 7 and 522) carry the recessive ve allele, as do the rosdor and roscol stock accessions. The Ve+ allele in the decipiens accession is very closely linked to the recessive decipiens allele, and we have recovered no recombinants in examination of >300 F2 progeny. The Ve+ phenotype segregates independently of Rosea; therefore, these two loci are unlinked.

Isolation of MYB-Related Genes Controlling Anthocyanin Biosynthesis from A. majus

To identify genes encoding MYB-related proteins controlling anthocyanin pigmentation in A. majus, oligonucleotide primers to the most conserved regions of MYB genes C1 (from maize) and AN2 (from Petunia), which are the regions encoding the recognition helices of the DNA binding domain (Figure 2A), were used for 3′ rapid amplification of cDNA ends (RACE) PCR amplification of cDNA from flowers of the wild type and rosea, Eluta, and mut mutants. cDNA fragments were amplified from wild-type flowers with an oligonucleotide encoding part of the first recognition helix and with one encoding part of the second recognition helix. One of the fragments amplified by the oligonucleotide from the first recognition helix in R2 (G1709) was not present when cDNA from flowers of rosdor or roscol were used (Figure 2B). This fragment was amplified from cDNA from flowers of the other mutants.

Figure 2.
Structure of the Rosea Locus in Wild-Type A. majus and in roscol and rosdor Mutants.

The cDNA fragment amplified by 3′ RACE from wild-type flower buds using the G1709 oligonucleotide as a primer was used to probe a cDNA library made from wild-type flowers of A. majus. Two cDNA clones (that hybridized under high-stringency washing conditions) were identified. These were subcloned into pBluescript SK+ and sequenced. Both cDNA clones contained the same open reading frame (ORF) but contained slightly different lengths of 5′ and 3′ untranslated region. Both contained the complete ORF encoding a MYB-related protein very similar to AN2. This gene product was named Rosea1 (Ros1) (see Supplemental Figure 1 online).

Genomic clones encoding the Ros1 gene were isolated by screening a genomic library from A. majus in λEMBL4 (a gift from Hans Sommer) with the Ros1 cDNA. Five positive clones were identified and mapped (Figure 2C). Two of the independent genomic clones contained the Ros1 coding sequence within three adjacent EcoRI fragments. They also contained a second region of homology with the Ros1 cDNA, situated 4.3 kb downstream of Ros1 within a 2.4-kb XbaI fragment (Figure 2D). The three other clones contained only the second region of homology defined by the 2.4-kb XbaI fragment (Figures 2C and 2D). The sequences of the three EcoRI fragments containing the genomic sequence for Ros1 were determined. The ORF was interrupted by two introns situated at equivalent positions to the introns in the C1 gene of maize (Paz-Ares et al., 1987) and AN2 from Petunia (Quattrocchio et al., 1999), the first of 231 bp and the second of 1498 bp (Figure 2D). There was a sequence, TATTT, located 115 bp upstream of the initiating ATG in Ros1 that approximates a plant TATA binding site (Molina and Grotewold, 2005). Farther upstream at −489 and −587 were two additional motifs (TATAA) that approximated better to the TATA binding consensus.

The second region of homology with the Ros1 cDNA (contained within the 2.4-kb XbaI fragment) in all five λEMBL4 clones was also sequenced (Figure 2C). This contained a sequence encoding the N-terminal region of a second MYB-related protein, distinct but structurally very similar to the N terminus of Ros1. This gene fragment was termed Rosea2 (Ros2). All five λ clones ended within the second intron of Ros2, and we were unable to identify any overlapping clones that contained the downstream ORF of Ros2. To determine whether this second gene was expressed and functional, we examined the expression of Ros2 in the wild type and ros mutant lines.

The Ros2 cDNA was amplified by 3′ RACE PCR (Frohmann et al., 1988) using an oligonucleotide primer to the sequence encoding the initiating ATG codon of the Ros2 protein in the genomic clone and cDNA made from flowers of roscol. A cDNA fragment was amplified that contained the entire Ros2 ORF, 3′ to the priming oligonucleotide (Figure 3B). Comparison with the partial genomic sequence of Ros2 confirmed that it contained at least two introns, the first two being in identical positions to those in Ros1, An2 from Petunia, and C1 and Pl from maize. The first intron in Ros2 was 111 bp, whereas the size of the second intron remains unknown but it is >9 kb, as deduced from the genomic sequences in the λEMBL4 clones available. A potential TATA box was identified 109 bp upstream of the first ATG in the Ros2 ORF (Figure 2D).

Figure 3.
Expression of Rosea Genes in Flowers of Wild-Type A. majus and rosea Mutants.

Further analysis of the 3′ end of the Ros2 gene in the wild type, roscol, and rosdor by PCR amplification using primers from the cDNA clone revealed that the sequences lying 3′ to the second intron in Ros2 were present in the genomes of both wild-type and roscol lines. There were eight single-nucleotide differences in this region between these two lines (Figure 2E), one of which caused a change in the encoded amino acid sequence (Lys-124 to Iso-124; Ros2 numbering). In rosdor, the equivalent region was highly rearranged; the sequence encoding the recognition helix of R3 of Ros2 was missing in the rosdor line (Figure 2F). The sequences farther downstream were present but rearranged, including six single-nucleotide differences between rosdor and roscol and the insertion of a transposon that interrupted the Ros2 ORF at codon 206 (Glu-206). The transposon had 11-bp terminal inverted repeats and had generated a 9-bp direct duplication in the Ros2 ORF upon insertion.

Genotypic Constitution of Wild-Type, roscol, and rosdor Lines at the Rosea Locus

Because Ros1 cDNA was not amplified using the G1709 oligonucleotide primer from cDNA derived from RNA from flowers of either rosdor or roscol, the possibility that the phenotypes conferred by rosea resulted from mutations in the Ros1 or Ros 2 gene was investigated at the molecular level. First, the linkage of the two genes to the ros mutations was analyzed in crosses between the wild type and roscol and the wild type and rosdor. The Ros1 gene was highly polymorphic between wild-type, roscol, and rosdor lines. Using EcoRI digests, 126 progeny were examined from F2 crosses between the wild type and roscol and 96 progeny were examined from crosses between the wild type and rosdor. The roscol and rosdor plants that segregated (scored phenotypically) in F2 were all homozygous for the parental versions of the Ros locus identified in the stock roscol and rosdor lines.

The expression of Ros1 in the wild type, roscol, and rosdor was examined by RNA gel blot analysis. A high level of transcript was detected in maturing petals of wild-type plants (Figure 3A). A considerably reduced level of transcript of a slightly smaller size accumulated in petals of the same age from roscol (Figure 3A). This transcript was amplified by RT-PCR and sequenced. It contained several single-nucleotide substitutions compared with wild-type Ros1, but the most significant difference was a 64-bp deletion that included the region encoding the first recognition helix (in R2) of the DNA binding domain (Figure 2E). This mutation introduced a stop codon 36 bp downstream of the deletion, indicating that a nonfunctional Ros1 product was produced, because the encoded protein would lack more than half of its DNA binding domain and the entirety of its C terminus. The 64-bp deletion included sequence encoding the recognition helix of R2, which explains why the original oligonucleotide, G1709, failed to amplify a cDNA product from roscol RNA by 3′ RACE PCR.

The rosdor line produced no Ros1 transcript detectable on RNA gel blots (Figure 3A). However, RT-PCR did amplify a Ros1 cDNA that was cloned and sequenced. This showed 11 differences from the sequence of Ros1 from wild-type lines, of which 8 affected the identity of the encoded amino acids (codon Glu-27 to Lys, Gly-116 to Arg, Leu-131 to Gln, Lys-174 to Thr, Thr-176 to Lys, Ala-183 to Val, Glu-190 to Asp, and Asp-191 to Glu). However, comparison with the sequences of Ros2 and Ve suggested that it was unlikely that any of these changes would contribute to the phenotype, implying that the primary change in rosdor is a change in the expression pattern of Ros1. To check this, we cloned the genomic DNA encoding the promoter and N terminus (up to the second intron) of Ros1 from the rosdor background. Within the 910-bp region of the Ros1 promoter (compared between the wild type and rosdor), there were nine single-nucleotide substitutions, two single-nucleotide additions, one single-nucleotide loss, and an insertion of 12 bp at 440 bp upstream of the initiating ATG codon. The biggest difference, however, was a deletion of 187 bp in the promoter (from −140 to −326), which is likely to have a significant impact on the transcription of Ros1.

The expression of the full-length Ros2 transcript was analyzed using RT-PCR. Using a 5′ oligonucleotide covering the initiating ATG of Ros2 and a reverse primer representing sequences in the 3′ untranslated region, a full-length transcript was detected in roscol flowers but not in wild-type or rosdor flowers (Figure 3B). These data showed that roscol expresses a full-length transcript of Ros2 at a very low level, but there is no expression of full-length Ros2 in the wild type or rosdor. Therefore, the wild type and rosdor were deemed to be ros2.

These data suggested that both roscol and rosdor are determined by mutations in the complex Rosea locus. roscol carries a loss-of-function allele of Ros1 but expresses Ros2. The Ros2 gene contributes the weak pigmentation restricted to the inner epidermis of the corolla lobes and the base of the tube seen in roscol, but it is not expressed in wild-type lines. rosdor has no Ros2 expression and so no pigment on the inner epidermis of the lobes. Ros1 expression is modified in rosdor, most likely as a result of changes to the promoter of Ros1, such that it induces pigmentation only in the outer epidermis of the dorsal petals and in a ring at the base of the tube under specific environmental conditions of high light and relatively low temperature. Its contribution to vegetative pigmentation compared with the wild type is relatively normal.

To confirm that Ros1 and Ros2 both control anthocyanin production, and that Ros1 and Ros2 could complement rosea mutants, we used particle bombardment of rosdor lines grown in the greenhouse, so that the corolla lobes were acyanic. Each cDNA was cloned between the cauliflower mosaic virus (CaMV) 35S promoter and the octopine synthase terminator and was bombarded into the inner epidermis of the dorsal petals of rosdor flowers, as well as a control plasmid containing β-glucuronidase (GUS) driven by the 35S promoter. In tissue bombarded with the control plasmid alone, patches of gold were visible after 2 d (Figure 4A). The gold particles were surrounded by tissue that had browned slightly as a result of physical damage from bombardment. No spots of magenta anthocyanin pigment were ever observed with the control plasmid (Figure 4A). However, histochemical GUS staining revealed that the control plasmid was active in the bombarded tissue (Figure 4B). Bombardment with the Ros1 cDNA resulted in the development of single magenta cells within 48 h of bombardment. On average, for Ros1 >100 spots were observed per replicate piece of petal (Figures 4C and 4D). Bombardment with Ros2 also gave rise to magenta spots, but at a lower frequency than in Ros1 (Figure 4D).

Figure 4.
Complementation of the Mutant Phenotype Conferred by rosdor by Particle Bombardment of Petal Lobe Tissue with the Rosea Genes.

These experiments showed that the Rosea locus is complex, consisting of two very closely linked genes, Ros1 and Ros2. Loss of function of Ros1 gives rise to the roscol phenotype, whereas modified and severely reduced expression of Ros1 and loss of function of Ros2 gives rise to the rosdor phenotype.

A Third Unlinked Gene Encoding a MYB-Related Protein Controls the Phenotype Conferred by Venosa

Because the Ve+ allele confers strong magenta pigmentation to the epidermal cells overlying the petal veins in ros mutants, but other structural and regulatory mutations are epistatic to Ve+, we reasoned that Venosa most likely encodes a MYB-related transcription factor that was functionally similar to Ros1 and Ros2. To identify such a gene, we digested genomic DNA from Ve+/ve heterozygotes and ve homozygotes with a range of restriction enzymes, separated the DNA by gel electrophoresis, and blotted it onto nitrocellulose filters. The DNA gel blots were probed with a fragment of cDNA encoding the Ros1 MYB DNA binding domain and were subsequently washed at low stringency. The EcoRI digest revealed a number of hybridizing bands, one of which (2.9 kb) segregated clearly with the phenotype conferred by Ve+ (Figure 5A). This fragment of genomic DNA was cloned from DNA from a Ve+/ve heterozygote in λNM1149 and subsequently was subcloned in pBluescript SK+ and sequenced. The genomic DNA fragment contained the sequence encoding the N-terminal region of a MYB-related transcription factor. This sequence was used to design a gene-specific primer to the sequence, including the initiating ATG. This oligonucleotide was used for 3′ RACE PCR on cDNA prepared from Ve+ and ve corolla tissue. A transcript was amplified only from Ve+ cDNA. This was cloned and sequenced. The Ve cDNA encoded an R2R3 MYB protein very similar to Ros1 and Ros2 (see Supplemental Figure 1 online). By comparison with the genomic DNA of the active allele, the gene was deduced to have at least two introns, the first of 480 bp and the second of 1450 bp. The 5′ upstream region contained a motif, TATAT, 75 bp upstream of the initiating ATG, and another, TTATTT, 101 bp upstream. Either of these may constitute the TATA box. The cDNA clone was used to map the Ve gene relative to the phenotype conferred by Ve+ in 179 segregating F2 individuals. No recombinants were found between the phenotype conferred by Ve+ and the 2.9-kb EcoRI fragment (Figure 5B).

Figure 5.
Identification of the Venosa Gene and Phylogenic Analysis of the MYB Proteins Encoded by Ros1, Ros2, and Ve.

RNA from flowers of ros lines with or without the Ve+ phenotype was examined for the expression of Ve by RNA gel blot analysis (Figure 5C). Ve transcript was detected in the lobes and to a lesser extent in the tubes of Ve+ lines, but no transcript was detected in ve lines, confirming the 3′ RACE results. Genomic DNA encoding the Ve gene from the ve allele was also cloned from the Ve+/ve heterozygote. The ve allele had several differences in sequence from the functional Ve+ allele, most significantly the replacement of a 1221-bp region of the gene from the middle of the first intron to the middle of the second intron with an unrelated sequence of 1958 bp (Figure 5D). This removed the second exon of the gene, a change likely to render the protein nonfunctional. The 1958-bp insertion sequence showed no significant similarity to sequences of known function on a search of the GenBank databases. However, it did include direct flanking repeats of 40 bp, suggestive of the sequence of a transposon. Other differences in sequence between the Ve+ and ve alleles were minor, consisting of 23 mostly single-nucleotide changes over the 3158-bp sequence of Ve analyzed (Figure 5D). None of these resulted in changes to the encoded amino acid residues.

To confirm that Ve could regulate anthocyanin biosynthetic gene expression, the cDNA was cloned between the double CaMV 35S promoter and the octopine synthase terminator and was bombarded into rosdor petals (greenhouse-grown). A large number (>100 per replicate) of magenta single-cell spots were observed within 48 h, whereas the control plasmid gave no magenta spots (Figure 5E).

Ve, Ros1, and Ros2 Appear to Have Arisen by Gene Duplication in Antirrhinum

The deduced amino acid sequences of the Ros1, Ros2, and Ve peptides were compared with those of other MYB proteins identified as regulators of flavonoid biosynthesis: AN2 from Petunia (Quattrocchio et al., 1999), Vv MYBA1 and Vv MYBA2 from grape (Vitis vinifera) (Kobayashi et al., 2002), TT2 (At MYB123), PAP1 (At MYB75), PAP2 (At MYB90), At MYB113, and At MYB114 from Arabidopsis, and C1 and Pl from maize. GLABROUS1 (GL1), WERWOLF (WER), and At MYB23 from Arabidopsis (Stracke et al., 2001) were included as outgroups (Figure 5F; see Supplemental Figure 1 online). All of the anthocyanin-regulatory MYB proteins were very similar over their DNA binding domains, with 59 of 100 amino acids in the R2R3 MYB DNA binding domain conserved in all proteins. In R2R3 MYBs, the DNA binding domain forms two repeats of helix-helix-turn-helix structure. The third helix in each repeat is believed to interact with DNA to bind it in a sequence-specific manner. The greatest conservation of sequence between these proteins was in the third region of α helix in R2 and in the second region of α helix in R3 (see Supplemental Figure 1A online). Surprisingly, there were quite a number of amino acid substitutions within the third helix (the recognition helix) of R3 between Ros1, Ros2, and Ve, suggesting that the different proteins might have somewhat different binding site preferences (see Supplemental Figure 1 online).

Several amino acids within helix 1 and helix 2 of R3 have been identified by Grotewold et al. (2000) as important for interaction with bHLH proteins (Leu-76, Arg-79, Arg-82, Leu-83, Gly-94, and Arg-95; C1 numbering). All are conserved in Ros1, Ros2, and Ve. The extended interaction signature identified by Zimmermann et al. (2004) as [DE]Lx2[RK]x3Lx6Lx3R is also conserved and identical in the R3 regions of Ros1, Ros2, and Ve (DLivRlhkLlgnkwsLiagR; where uppercase letters indicate amino acids that are completely conserved, lowercase letters indicate alternative amino acids, and x indicates any amino acid). This implies that all three Antirrhinum proteins interact with bHLH proteins in their activation of anthocyanin biosynthesis, as has been shown for C1, Pl, and AN2.

The C-terminal domains of the Antirrhinum MYB regulators were much less well conserved, but one patch of conserved sequence was obvious: a more acidic region toward the C terminus of each protein that surrounds a core sequence of higher conservation (in dicot MYB proteins, it was Ww/lx+/−LL, where the annotation is as before and +/− indicates a charged amino acid). This motif is part of a longer motif identified by Kranz et al. (1998) from Arabidopsis MYB proteins defining R2R3 MYB subgroup 6. This short motif shows similarity to sequence in the KIX domain of animal c-MYB, which lies within the activation domain of that protein and interacts with the transcriptional coactivator, CREB. In C1 and Pl, there are also acidic regions at the C termini of the proteins structured around the sequence WLRC+T, which might represent the equivalent domain in the monocot proteins. One residue, Asn, which lies immediately N terminal to the WLRC+T motif, has been shown to contribute significantly to the ability of the C-terminal domain of C1 to activate transcription (Sainz et al., 1997a). The C-terminal domains of C1 and AN2 have also been shown to activate transcription in yeast one-hybrid assays (Goff et al., 1992; Sainz et al., 1997a; Quattrocchio et al., 1999).

Phylogenetic analysis of the anthocyanin MYB regulators Ros1, Ros2, and Ve was performed. A well-supported phylogeny was recovered that placed the Antirrhinum proteins as more closely related to one another than to their orthologs from other species (Figure 5F; see Supplemental Figure 1 online). This suggested that the three Antirrhinum genes were derived from relatively recent duplications of a common ancestral gene. The first duplication created the unlinked genes, the progenitor of Venosa and the progenitor of Rosea. The second duplication gave rise to Rosea1 and Rosea2 and occurred intrachromosomally. Similar duplication events appear to have also occurred recently in Arabidopsis to create MYB75, MYB90, MYB113, and MYB114, which are clustered on chromosome 1 (Stracke et al., 2001), in grape to create MYBA1 and MYBA2, which are closely linked (Kobayashi et al., 2002, 2004, 2005), in tomato (Lycopersicon esculentum) to create ANT1 and a very closely linked MYB-like gene (Mathews et al., 2003; De Jong et al., 2004), and in potato (Solanum tuberosum) to create the linked F locus controlling floral pigmentation and the D(I) locus controlling tuber color (De Jong et al., 2004); a duplication of the whole genome may have created C1 and Pl in maize (Cone et al., 1993).

Comparison of the Effects of Ros1, Ros2, and Ve on Target Gene Expression Reveals the Proteins to Have Similar but Distinct Biochemical Specificities

Although Ros1, Ros2, and Ve can all activate anthocyanin biosynthesis, we were interested to know whether they are truly functionally identical proteins or whether they have evolved distinct specificities in their regulatory properties. To this end, we examined the target genes of Ros1, Ros2, and Ve by comparing the levels of transcripts encoding the enzymes of anthocyanin biosynthesis in plants, both wild type and mutant, for the expression of each regulatory gene. To analyze the effects of Ros1, we compared the expression of the biosynthetic genes in flowers of the wild type and rosdor (Figure 6). No change in transcript levels was observed for CHS. A small decrease in the level of chalcone isomerase (CHI) transcript was observed in rosdor petals compared with wild-type petals. Much less transcript was observed in rosdor than in the wild type for F3H, although greater differences were observed for F3′H, DFR, and UFGT. Smaller but still significant reductions in transcript levels in rosdor compared with the wild type were observed for flavonol synthase (FLS), ANS, and AT, an anthocyanin permease from A. majus (GenBank accession number AJ796511) homologous with a tomato MATE transporter induced by a MYB transcription factor regulating anthocyanin production in tomato (Mathews et al., 2003; Bey et al., 2004). These data showed that Ros1 increases the transcript levels of the late biosynthetic genes (F3H, DFR, ANS, and UFGT) as well as F3′H, FLS, and AT, although not all of these target genes are equally dependent on Ros1 activity. F3′H, F3H, DFR, and UFGT were particularly dependent on Ros1 activity, because virtually no transcripts for these genes were detected in rosdor flowers. By contrast, ANS and AT were much less dependent on Ros1. Ros1 was not required for the accumulation of transcripts of the early biosynthetic gene CHS and had only a very minor influence on CHI. This is similar to the effect of Delila on the regulation of the anthocyanin biosynthetic pathway in Antirrhinum (Martin et al., 1991), supporting the view that Ros1 may interact with Delila to activate gene transcription.

Figure 6.
RNA Gel Blots of Poly(A)+ RNA from Petal Tissue of Wild-Type, roscol, rosdor, and rosdor Ve+ Plants Probed with cDNA Fragments of the Genes Encoding the Enzymes of the Anthocyanin Biosynthesis Pathway.

The influence of Ros2 on biosynthetic gene expression was assayed by comparing expression in roscol with expression in rosdor (Figure 6). rosdor does not express Ros2 and has virtually undetectable levels of expression of Ros1 (Figure 3A). Consequently, comparison of expression of the biosynthetic genes in roscol and rosdor revealed the influence of Ros2 on biosynthetic gene transcript levels. No differences were observed in transcript levels of CHS. A small increase in transcript levels of CHI was observed in roscol compared with rosdor. However, significantly lower levels of F3′H transcripts were observed in rosdor compared with roscol, suggesting that Ros2 regulates the expression of F3′H and possibly, in contrast with Ros1, contributes to the activation of CHI.

The influence of Ve on anthocyanin biosynthetic gene expression was assayed by comparing transcript levels in Ve+ and ve lines in the rosdor background (Figure 6). No difference was detected in the levels of CHS transcripts. The presence of an active Ve+ allele did increase the transcript levels of CHI, F3H, F3′H, FLS, ANS, UFGT, and AT. Surprisingly, no difference could be detected in the levels of DFR transcript between Ve+ and ve lines; virtually no transcript was detected in either line. These data showed that Ve does increase the transcript levels of most of the biosynthetic genes and is not required for the expression of the early gene, CHS. However, they also indicate that Ve does not activate DFR expression nearly as effectively as Ros1. These data imply that there are subtle but reproducible differences in the abilities of Ve, Ros1, and Ros2 to activate the transcription of different target genes. This could reflect differences in target site specificity affecting the affinity with which each activator binds to the target promoters of the different biosynthetic genes. Such specificity might also involve differences in the efficacy of the complexes that each MYB protein makes with the bHLH and WD repeat proteins.

Ros1 and Ve Show Differences from Ros2 in the Specificity of Their Interaction with the bHLH Regulator Mut

We examined the interactions of the different MYB transcription factors with bHLH proteins through genetic analyses. The Delila gene product is a bHLH protein that is required for anthocyanin biosynthesis in the corolla tubes. It is also expressed in the lobes, as demonstrated by in situ hybridization, and it is active in the lobes (Almeida et al., 1989; Goodrich et al., 1992). A second gene, Mut, is required for anthocyanin biosynthesis in the lobes if Delila is nonfunctional. This encodes another bHLH protein (P. Piazza, C. Tonelli, and C. Martin, unpublished data). The specificity of Ros1, Ros2, and Ve interactions with these two bHLH proteins was tested by making double and triple mutants.

The line mutant for mut and del is acyanic. Ve+ is not visible in this background, suggesting that Ros1, Ros2, and Ve must interact with either Del or Mut to activate anthocyanin biosynthesis. When rosdor is combined with del, the tubes are unpigmented, but there is little effect on pigmentation in the lobes, indicating that Del interacts with Ros1 in the tubes but that in the lobes Ros1 interacts with Mut to direct anthocyanin biosynthesis. This confirms the phenotype of del alone, in which the tubes are acyanic but the lobes (principally the product of Ros1 and Mut interaction) have wild-type levels of pigmentation.

When roscol (in which only Ros2 of the MYB regulators is expressed) was combined with del, the double mutant was completely acyanic, despite Ros2 being active in the lobes (cf. Figures 7A and 7B). This indicates that in the lobes, Ros2 can interact with Del but not with Mut. This could be because Ros1 activates the expression of Mut, because in Petunia the AN2 MYB protein has been reported to activate the expression of AN1 (encoding a bHLH protein) in leaves (although not in flowers) (Spelt et al., 2000). However, although when roscol Ve was combined with del the background pigmentation in the corolla lobes of these plants was missing (acyanic), the venal pattern, determined by Ve, remained distinct (cf. Figures 7C and 7D). These results indicated that although Ros2 interacts effectively only with Del in the lobes, Ve (like Ros1) can interact effectively with Mut. These interactions are summarized in Figure 7E. Because our analysis of target gene activation showed that the three MYB proteins, Ros1, Ros2, and Ve, had differential specificities for each of the target genes, the ineffectual interaction between Ros2 and Mut may reflect the loss of activity of these proteins on a single target promoter, or their lack of activity on all target promoters. These possibilities cannot be resolved through genetic analysis, which measures only the accumulation of the end product, anthocyanin, and not the activation of the individual biosynthetic genes.

Figure 7.
Interaction of MYB Proteins with bHLH Proteins Revealed by Mutant Analysis.

Variation in Anthocyanin Patterns in Flowers of Different Antirrhinum Species Results Principally from Variation in the Activity of the MYB-Related Genes

Many different species of Antirrhinum have been described, some native to North America and some native to Europe. On the European branch of the genus, whose members are diploid, A. majus is one of several species that have strong, self-colored anthocyanin pigmentation of the corolla, others being Antirrhinum australe and Antirrhinum barrelieri (Figures 8E and 8F). Other closely related species include Antirrhinum latifolium, Antirrhinum graniticum, Antirrhinum molle, Antirrhinum mollissimum, and Antirrhinum meonanthemum. These species vary in their floral pigmentation and typically all have a very pale or no background anthocyanin accumulation in their corolla lobes. The floral pigmentation phenotypes of accessions of these species are shown in Figure 8 and tabulated in Table 1. We analyzed two wild accessions of A. majus subsp majus originating from Toulouse (Figures 8A and 8B) and Barcelona (Figures 8C and 8D), which both had medium-intensity anthocyanin accumulation in the corolla with a weak venal pattern superimposed. Two A. latifolium accessions had no background anthocyanin pigmentation but a clear venal pattern of accumulation (Figures 8G and 8H). In addition to the restricted anthocyanin pattern, both A. latifolium accessions showed strong aurone production all over their corolla lobes, giving them a pale yellow color typical of A. latifolium (Stubbe, 1966). Four other accessions were analyzed. These were A. graniticum, which had very weak anthocyanin accumulation in the inner epidermis of the corolla lobes but no evidence of venal patterning (Figure 8I); A. molle, which had no background pigmentation to the corolla lobes but a clear venal patterning of anthocyanin accumulation (Figure 8K); A. mollissimum, which had a weak background pigmentation and strong venal patterning in its floral pigmentation (Figure 8L); and A. meonanthemum, which had no background pigmentation to the corolla lobes but a clear venal patterning of anthocyanin accumulation and strong aurone production all over the corolla lobes, giving flowers a pale yellow color (Figure 8J) (Oyama and Baum, 2004). All of these accessions conformed to the general descriptions of floral pigmentation for the appropriate species (Sutton, 1988).

Figure 8.
Floral Phenotypes of Accessions of Different Species within the Genus Antirrhinum.
Table 1.
Origins of Species Accessions, Floral Phenotypes, and Inferred Genotypes from Genetic Analysis

Despite strong self-incompatibility in some of these accessions (A. graniticum, A. meonanthemum, and A. barrelieri), all can cross-hybridize with A. majus. Therefore, we were able to test the genetic basis for the weak pigmentation in A. graniticum, A. molle, A. mollissimum, A. meonanthemum, and A. latifolium by crossing them to the different mutants of A. majus. Although crosses of either of the wild accessions of A. majus to rosdor gave F1 progeny with fully pigmented flowers, none of the accessions of the other five species tested was able to complement the rosea mutants (neither rosdor nor roscol), indicating that, in all, the genetic basis for the low levels of anthocyanin was attributable to the reduced activity of the Rosea locus compared with its activity in A. majus. Crosses of A. graniticum, A. molle, and A. mollissimum to wild-type A. majus gave rise to fully self-colored F1 progeny. In the F2 of these species crosses, the pale pigmentation phenotype segregated 1:3, indicating that variation at a single recessive locus was responsible for the pale pigmentation. The lack of complementation in these species crosses to the rosdor line demonstrated this locus to be Rosea. Crosses of A. graniticum, A. molle, and A. mollissimum to mut del double mutants of A. majus gave rise to fully self-colored progeny, indicating that the activity of the Mut and Del genes does not limit pigmentation in these species (see Supplemental Figure 2A online).

Crosses of A. meonanthemum and A. latifolium to the mut del double mutant or to wild-type A. majus gave darker pigmentation to the lobes of flowers of the F1 progeny than that in A. meonanthemum and A. latifolium, but pigmentation was paler than in wild-type A. majus, especially over the outer edges of the corolla lobes (see Supplemental Figures 2B and 2C online). This phenotype matched exactly that of the Eluta mutant of A. majus (Baur, 1910a; Stubbe, 1966) (Figures 1J to 1L), which is a semidominant diluter of anthocyanin pigment in flowers. In the F2 of these species crossed to wild-type A. majus, pale pigmentation, the diluted pigmentation phenotype, and full-red pigmentation segregated 1:2:1, respectively, indicating that the pale pigmentation in these species was attributable to a semidominant allele at a single locus. The fact that in F2 populations of A. meonanthemum and A. latifolium (Marseilles) crossed to rosdor, no full-red plants segregated (30 and 96 progeny examined, respectively) indicated that both A. meonanthemum and A. latifolium carry semidominant alleles of the Rosea locus that dictate their pale floral pigmentation. These alleles are likely to be very similar in their activity to the Eluta mutant of A. majus. Indeed, Eluta has been reported to be very closely linked to rosea by Stubbe (1966). In F2 populations of Eluta crossed to rosdor or roscol, we have never observed full-red recombinants, supporting the idea that Eluta is a semidominant allele of the Rosea locus. We have indicated the semidominance of the pigment-diluting Rosea alleles in these species with the term RosEl.

Crosses of the species accessions to rosdor suggested that A. majus subsp majus from both Barcelona and Toulouse was polymorphic for an active Ve+ allele (Figures 8A to 8D). Crosses between these accessions and rosdor Ve revealed that the venal patterning of pigmentation could be attributed to the activity of the Ve locus, because no unpatterned progeny segregated in the F2 population (see Supplemental Table 1 online). Similar analyses with the species showed that all A. molle, A. mollissimum, A. meonanthemum, and A. latifolium individuals were homozygous for active Ve+ alleles. The deduced genotypes of the species analyzed for the loci affecting floral pigmentation are listed in Table 1.

These data revealed that the loci controlling anthocyanin pigmentation patterning in different Antirrhinum species principally encoded MYB-related transcription factors. DNA gel blots revealed that all three genes, Ros1, Ros2, and Ve, are present in the genomes of each of the accessions tested. The differences in background pigmentation patterns between the different species (most readily observed in the F2 segregants from crosses to wild-type A. majus, in which genetic background is homogenized, allowing for easier comparisons) suggest that differences in the activity of the Rosea locus is the single most important determinant of pigmentation intensity in the species tested. In addition, the other MYB-related gene, Venosa, contributes significantly to pigmentation in A. latifolium, A. molle, A. meonanthemum, and A. mollissimum and is also significant and variable in wild accessions of A. majus (see Supplemental Table 1 online).

DISCUSSION

In Antirrhinum majus, a small family of MYB-related proteins controls the pattern and intensity of floral pigmentation. Members of this family are closely related structurally to MYB proteins known to regulate anthocyanin production in other plant species, including maize (Paz-Ares et al., 1987; Cone et al., 1993) Petunia (Quattrocchio et al., 1999), grape (Kobayashi et al., 2002, 2004, 2005), pepper (Capsicum annuum) (Borovsky et al., 2004), morning glory (Chang et al., 2005), tomato (Mathews et al., 2003; De Jong et al., 2004), potato (De Jong et al., 2004), and Arabidopsis (Borevitz et al., 2000). Like other species, these different genes appear to have been derived by gene duplication and subfunctionalization, although our data from the investigation of mutant alleles suggests that the Ros1, Ros2, and Ve proteins are not functionally equivalent. These analyses were complicated by the different expression levels and patterns of the Ros1, Ros2, and Ve genes, but analysis of the transcript levels of potential target genes in lines defective in Ros1, Ros2, and Ve activity showed F3H, F3′H, FLS, DFR, and UFGT to be highly dependent on Ros1 for induction, whereas ANS and AT were less dependent and CHI was very much less dependent on Ros1 for induction. The only gene that was significantly induced by Ros2 was F3′H, although CHI transcript levels were also increased to a small extent by this protein. Ve induced the expression of CHI, F3H, F3′H, FLS, ANS, UFGT, and AT, but its induction of F3H, F3′H, and UFGT was stronger than that for ANS and AT and much stronger than that for CHI. Remarkably, Ve did not detectably activate DFR expression/transcript levels. These data suggest that each of these MYB proteins has distinct biochemical specificity in terms of its ability to activate transcription from different target promoters. This might reflect differences in their DNA binding affinities. There are a number of amino acid differences in the recognition helices of these three MYB proteins that could influence their DNA binding affinities and sequence motif recognition (see Supplemental Figure 1 online). Alternatively, differences in specificity might be attributable to differences in target promoter architecture, which could make binding or activation easier by one MYB protein relative to another, or to differences in the ability of the MYB regulators to interact with target promoters with different chromatin structures. However, although understanding of this specificity must await comparative biochemical characterization of the MYB proteins, we conclude that these regulatory proteins have diverged functionally as well as in their expression patterns, unlike the situation in maize, in which C1 and Pl appear to be functionally equivalent (Cone et al., 1993).

Given the observed specificity for target gene activation by the three MYB proteins, it was surprising that all could complement the rosdor mutation and produce pigmented cells after particle bombardment of petals (Figures 4C, 4E, and and5D).5D). In addition, Ve can complement the loss of Ros1 activity in the roscol line to give regions of highly pigmented epidermal tissue (Figure 7C), and it can complement the loss of both Ros1 and Ros2 activity in rosdor to give highly pigmented epidermal tissue in the regions overlying the veins on the inner epidermis of the petal lobes (Figure 1G). An explanation for this is that our complementation assays are based on the ability of cells to synthesize anthocyanins, an ability that is determined principally by the degree of activation of the biosynthetic step limiting the flux to anthocyanin accumulation in flowers. Although DFR was not detectably induced by Ve on RNA gel blots, 40 cycles of RT-PCR amplification of cDNA revealed low levels of DFR transcript in both rosdor and roscol flowers (see Supplemental Figure 3 online). This may be enough to allow anthocyanin biosynthesis if other steps, rate-limiting during flower development, are also induced. All of the data available for Antirrhinum flowers suggest that F3H catalyzes the step with greatest influence on the accumulation of anthocyanins (Martin et al., 1991). Consequently, the differing abilities of the MYB proteins to induce anthocyanin biosynthesis in Antirrhinum flowers are probably tied most closely to their relative abilities to induce F3H gene expression.

roscol is a loss-of-function mutant of Ros1 but expresses Ros2, although the latter gene is not expressed in wild-type A. majus. The basis for the absence of expression of Ros2 in the wild type was not absolutely clear from molecular analysis, but it is probably related to the exceptionally large second intron in this gene. Ros2 is expressed at very low levels in roscol flowers: transcript could only be detected by PCR amplification. The low steady state levels of Ros2 transcript in roscol lines may be associated with inefficiency in splicing this large intron (which is of unknown size but is >9 kb), which is unusually large for plant genes. In wild-type A. majus lines, problems associated with the efficiency of splicing of the Ros2 primary transcript (e.g., increases in intron size) might have escaped detection in our analyses but could result in the abolition of mature Ros2 transcript production.

In rosdor, the Ros2 gene is undoubtedly nonfunctional as a result of point mutations, deletions, and insertion of a transposon sequence within the ORF. In rosdor, the promoter region of Ros1 is highly modified compared with that in the wild type, suggesting significant differences in the control of expression of Ros1 in the mutant line. This was confirmed by RNA gel blot and RT-PCR analyses. Both roscol and rosdor, therefore, show multiple sequence differences in both Ros genes compared with wild-type A. majus, and it is difficult to consider them as simple mutations (Figures 2D to 2F). Lines homozygous for either allele were identified very early in the history of plant genetics, being described by Baur (1910) in his original article on the genetics of floral pigmentation in A. majus. At that time, chemical mutagens were not in use, and it is likely that mutants were selected initially as natural variants. Given the ability of different Antirrhinum species to form fertile hybrids with A. majus, it seems quite likely that these rosea mutants were identified in natural populations and may have resulted from introgressions of the Rosea locus from other species. This idea is supported by phylogenetic analysis of the sequence of the third intron of the gene encoding nitrate reductase (NIA; considered to be under neutral selection) and the cDNA sequence of DFR (a single gene [pallida] that is linked to the Rosea locus) from the roscol line, other A. majus accessions, and other Antirrhinum species. The NIA and DFR sequences from roscol cluster with all of the other A. majus accessions, whereas the Ros1 gene sequence from roscol clusters with Ros1 from Antirrhinum siculum, and that from rosdor clusters with Ros1 from A. molle and A. meonanthemum (Venail, 2005).

This small family of MYB-related genes controls the pattern and intensity of pigmentation of flowers. Neither the roscol nor the rosdor mutation eliminates pigmentation of the vegetative tissues, although the stem tissue of the roscol mutant lacks anthocyanin pigmentation when plants are grown in the field. This suggests that Ros1 is responsible for the induction of anthocyanin biosynthesis in stem tissues as well as inducing the strong production of magenta cyanidin in flowers. In roscol plants, the abaxial (ventral) epidermis of the leaves is pigmented when plants are grown in the field, suggesting that color production there is either under the control of Ros2 or under the control of another MYB-like gene that is not involved in floral pigmentation. The small family of MYB-like genes controlling floral pigmentation in A. majus appears to have resulted from successive duplications, one producing the ancestral versions of Ve and Ros1/Ros2, and the second, which occurred intrachromosomally, giving rise to Ros1 and Ros2. Similar recent intrachromosomal amplifications have occurred for the orthologous family of genes in Arabidopsis (PAP1, PAP2, MYB113, and MYB114) (Stracke et al., 2001), in grape (MYBA1 and MYBA2) (Kobayashi et al., 2004, 2005), and in potato and tomato (De Jong et al., 2004). In Petunia, AN2 is reportedly structurally very similar to AN4, which regulates pigment production in anthers (Spelt et al., 2000; Koes et al., 2005), although these duplicate genes are not linked. What is perhaps even more remarkable is that variation in these MYB-like genes forms the basis for all of the variations in color pattern and intensity between different species that have been investigated. This has already been demonstrated for the difference between the white flowers of Petunia axillaris and the purple flowers of Petunia integrifolia, in which loss of activity at the AN2 locus underpins the acyanic coloring of P. axillaris (Quattrocchio et al., 1999).

Here, we have shown that it is differences in the activity of the three MYB-like genes controlling floral pigmentation that underpin the flower color and pattern differences in at least six species within the genus Antirrhinum. The number of genes that could contribute to variations in color patterning and intensity is large: all of the structural genes committed to flavonoid biosynthesis, which in Antirrhinum amounts to 10 (CHS, CHI, F3H, F3′H, DFR, ANS, UFGT, RT, GST, and AT), and the genes encoding bHLH proteins involved in regulating floral pigmentation (Del and Mut) (Stubbe, 1966; Goodrich et al., 1992; P. Piazza, C. Tonelli, and C. Martin, unpublished data). Although Stubbe (1966) reported that A. latifolium differs from A. majus in the activity of the incolorata locus (which encodes F3H), we did not find any evidence for this in the two accessions of A. latifolium (from Marseilles and Pyrea) we examined. This difference is likely to be attributable to differences between the individual accessions that were studied in each case. It was surprising that all of the variation in pigmentation color and pattern that we examined in the genus Antirrhinum could be attributed to variation in the activity of the three MYB genes, Ros1, Ros2, and Ve. One possible explanation is that the full-red color of A. majus flowers was derived from an ancestor with very pale floral pigmentation, and all of the other palely pigmented members of the genus Antirrhinum might carry the same ancestral, low-activity ros allele. Investigation of this possibility would require a robust molecular phylogeny, which is not currently available for the European species, largely because the high degree of sequence similarity among species for the markers investigated to date makes resolution difficult, and also because there are significant levels of gene flow between sympatric populations as a result of the cross-fertility of the species (Oyama and Baum, 2004; Vargas et al., 2004; Mateu-Andres and de Paco, 2005; Venail, 2005). However, such an explanation of why color variation derives from variations in the activity of the MYB genes in the genus Antirrhinum is unlikely to be correct, because each species accession carries at least one distinct Ros allele, as judged by restriction fragment length polymorphisms with several different enzymes (Venail, 2005), and, although palely pigmented, the patterns of anthocyanin production in A. graniticum, A. molle, A. mollissimum, A. latifolium, and A. meonanthemum are quite distinct, when examined closely. Indeed, the ros alleles in A. graniticum, A. molle, and A. mollissimum are fully recessive to Ros+ from A. majus, whereas the ros alleles from A. latifolium and A. meonanthemum are semidominant to Ros+. We have found that there is also significant variation at the Ve locus, with A. graniticum lacking Ve activity and two independent A. majus accessions being polymorphic for Ve activity.

The fact that variation in anthocyanin production in flowers is attributable to variation in MYB gene activity in both Antirrhinum and Petunia (Quattrocchio et al., 1999), in berry skin color in grape (Kobayashi et al., 2004, 2005), and in tuber color in potato (De Jong et al., 2004) suggests that the same route for generating pattern and color diversity has been followed independently on a number of occasions. In contrast with these results, examination of the sequences encoding the variable C-terminal domain of an equivalent protein, myb1, in different Ipomoea species suggested neutral variation, leading to the conclusion that rapid evolution of Ipomoea myb1 has not contributed to differences in floral hue and color patterning among Ipomoea species (Chang et al., 2005). However, that study did not investigate possible differences in the level or patterning of Ipomoea myb1 expression in relation to differences in pigmentation. In addition, the Ipomoea species selected for comparison in this study differed principally in the type of anthocyanin they accumulate, a trait less likely to be determined by regulatory gene activity than pigmentation intensity and patterning. Interestingly, an earlier investigation concluded that most of the differences in color between species in the genus Ipomoea were attributable to differences in the expression (i.e., the regulation) of the anthocyanin biosynthetic genes (Durbin et al., 2003), suggesting that variation in regulatory gene activity is central to variation in color intensity and patterning within this genus as well.

Is there something special about the activity of the MYB proteins in the regulation of anthocyanin biosynthesis that makes them particularly suitable for the generation of diversity? Clearly, gene duplication and divergence in the patterns of expression of the encoded regulatory proteins offer a rapid means of generating differences in patterns and intensity of pigmentation, which require a significant number of enzymes for their synthesis. However, the evolutionary emphasis on variation in the regulation of MYB gene activity remains puzzling.

In Arabidopsis, a complex of MYB, bHLH, and WD repeat proteins regulates not only anthocyanin biosynthesis but also condensed tannin biosynthesis, trichome initiation, and non-root-hair cell specification (Zhang et al., 2003; Serna, 2004; Broun, 2005; Ramsay and Glover, 2005; Lepiniec et al., 2006). A similar complex probably also regulates seed coat mucilage production. In these cases, the WD repeat protein (TTG1) is the same in all of the different functional complexes, and the bHLH proteins are flexible in their participation, GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3) being thought to be involved in non-root-hair cell specification, trichome initiation, and anthocyanin production (Payne et al., 2000; Zhang et al., 2003), and TT8 being involved in both condensed tannin and anthocyanin production (Nesi et al., 2000; Zhang et al., 2003). A similar diversification of functions has been described for the AN1 bHLH protein in Petunia, which controls both anthocyanin formation and seed coat morphology (Spelt et al., 2002). Functional specificity is provided in all of these complexes by the participation of specific MYB proteins, although, in the case of non-root-hair cell specification and trichome initiation, the MYB proteins involved, WER and GL1, are functionally interchangeable (Lee and Schiefelbein, 2001) and specificity must be provided by the cellular context in which the proteins are normally active. The specific roles of MYB proteins in multifunctional MYB-bHLH-WD repeat complexes may mean that it is their activity that dictates the net activity of the regulatory complex in controlling any specific target pathway. If the activity of the MYB-related proteins is generally the component that defines the intensity and/or pattern of anthocyanin production, plants may be particularly sensitive to the dosage of the genes encoding these proteins. Such sensitivity might favor repeated, selective duplication of these genes to generate variation in the intensity and patterning of color in plant tissues.

METHODS

Plant Material

Antirrhinum majus mutants roscol and rosdor were obtained as standard genetic stocks from the germplasm collection at the Institut für Kulturpflanzenforschung in Gatersleben, Germany. These stocks were crossed to a wild-type, full-red stock (JI:522), and the mutants were reselected in the F2 generation. The lines carrying these mutations were maintained subsequently by self-pollination for more than five generations. Line JI:522 is a wild-type revertant from an unstable nivearecurrens mutant (Martin et al., 1991). The dominant Ve+ allele of A. majus was identified in F2 populations from crosses between the decipiens mutant of A. majus (from the germplasm collection at the Institut für Kulturpflanzenforschung) and roscol and rosdor. Ve+ is very tightly linked to the decipiens mutant allele, and no recombinants between the two loci have been found. Because the decipiens mutation affects the development of petals, the Ve+ allele has been maintained as a heterozygote (Ve+/ve) in both roscol and rosdor backgrounds. Comparisons of biosynthetic gene transcript levels were made on RNA from flowers pooled from individuals of the same phenotype segregating in F2 populations of the wild type × roscol and rosdor Ve × rosdor.

The origins of the different Antirrhinum species accessions used in this work were A. majus subsp majus var majus (Toulouse and Barcelona) and A. latifolium (Marseilles and Pyrea), all from the germplasm collection at the Institut für Kulturpflanzenforschung, and A. graniticum, A. molle, A. mollissimum, A. meonanthemum, A. barrelieri, and A. australe, which were collected by R.K. Oyama in Spain, vouchers of which are deposited at the Herbarium of the Arnold Arboretum of Harvard University.

RNA Extraction and Gel Electrophoresis

Total RNA was extracted by freezing tissue in liquid nitrogen and then grinding it to a fine powder, which was then added to extraction buffer (150 mM LiCl, 50 mM Tris-HCl, pH 9, 5 mM EDTA, pH 8.0, and 5% [w/v] SDS). Two phenol–chloroform extractions were performed on the extract, followed by a chloroform extraction. The RNA in the resulting aqueous phase was precipitated overnight at 4°C by the addition of 8 M LiCl to a final concentration of 2 M LiCl. The precipitate was resuspended in TE buffer (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA). Two further rounds of precipitation in 2 M LiCl were performed. Samples were redissolved in TE buffer. Poly(A)+ RNA was isolated using poly(A)+ RNA purification kits (Amersham Pharmacia Biotech). RNA gel electrophoresis was performed as described by Martin et al. (1991).

First-Strand cDNA Synthesis

First-strand cDNA was made using a cDNA synthesis kit (Amersham) according to the manufacturer's instructions, except that the cDNA was primed with the dT17 adaptor sequence (Frohmann et al., 1988). Ten micrograms of total RNA was used for each reaction, and the cDNA was diluted to a final volume of 1 mL with sterile, distilled water. Samples of 10 μL (~200 ng of cDNA template) were used for 3′ RACE PCR.

3′ RACE PCR

Reactions were conducted in a 100-μL final volume including 200 ng of template cDNA, 1× AmpliTaq buffer (Perkin-Elmer), 50 μM deoxynucleotide triphosphates, 50 nM of each primer, and 0.5 μL (2.5 units) of AmpliTaq polymerase (Perkin-Elmer). The primers used for Ros1 were G1709 (forward, 5′-AAAAGCTGCAGACTTAGGTGGTTGAATTATCTAAAGCC-3′) and the adaptor sequence (reverse, 5′-GACTCGAGCGACATCGAT-3′) (Frohmann et al., 1988), and those used for Ros2 were K17 (forward, 5′-TAGTGCATATGCTAAACGCAATGC-3′) and the adaptor sequence (reverse, 5′-GACTCGAGCGACATCGAT-3′) (Frohmann et al., 1988). The PCR conditions were 40 cycles of 94°C for 1 s, 94°C for 40 s, 55°C for 2 min, 55°C for 1 s, 72°C for 1 min, and 72°C for 3 min, followed by 10 min of final extension at 72°C.

RT-PCR Amplification of DFR cDNA

cDNA was amplified by the method of Frohmann et al. (1988) using the primers DFR-F (5′-ATGAGTCCCACTTCACTAAATACGAGTTCGGAAAC-3′) and DFR-R (5′-CTAGATTCTGCCATCAGTATGATCGTTTGCAATGTC-3′) for 40 cycles. DNA was transferred by blotting to nitrocellulose filters and hybridized with an EcoRI-BamHI fragment of genomic DNA from the Pallida (DFR) locus of A. majus (Coen et al., 1986).

Large-Scale DNA Extractions

Twenty to thirty grams of young leaves were collected, frozen in liquid nitrogen, and used directly or stored at −70°C. The frozen material was ground to a fine powder and transferred to DNA extraction buffer (0.1 M EDTA, 3× SSC [1× SSC is 0.15 M NaCl and 0.015 M sodium citrate], 0.1 M sodium diethyldithiocarbamate, and 1% SDS). Samples were then placed at 37°C for 5 to 10 min to let the material thaw. The homogenate was extracted with phenol:chloroform (1:1) two times, and then once with chloroform alone. DNA was precipitated by the addition of two volumes of 90% ethanol to the aqueous phase. The pellet was resuspended in 9 mL of TE buffer, pH 8, and exactly 1 g/mL cesium chloride was added to the resuspended DNA solution. The gradients were centrifuged at 65,000 rpm at 15°C for 16 to 20 h, after which time the tubes were visualized under UV light and the DNA, visible as a single fluorescent band, was removed with a needle and hypodermic syringe. The ethidium bromide was removed with salt-saturated isopropanol. The DNA was precipitated with two volumes of 100% ethanol. The pellets were washed with 70% ethanol and resuspended in 500 μL of TE buffer, pH 8.

PCR Amplification of Genomic DNA

Reactions were performed in a total volume of 100 μL, containing 5 μL (~200 ng) of genomic DNA as a template, 50 nM primer, 250 μM deoxynucleotide triphosphates, 1× AmpliTaq buffer (Perkin-Elmer), and 0.5 μL (2.5 units) of AmpliTaq polymerase (Perkin-Elmer). Thirty-five cycles of amplification were performed under the following conditions: 94°C for 30 s, 50°C for 30 s, and 72°C for 1.5 min, followed by 10 min of final extension at 72°C. The enzyme AmpliTaq and its buffer were obtained from Perkin-Elmer. The primers used for Ros2 were J19 (forward, 5′-CCGAGCTTCGGACCTTCAATGGATTG-3′), J21 (forward, 5′-CCACTTTTATGCGTCACTACACATGTCATAT-3′), and J22 (reverse, 5′-CTATGTTTGCAAACGTTTATGGTTG-3′).

Oligolabeling of DNA Probes

The templates used for the synthesis of the DNA probes were PCR fragments amplified from plasmids containing the clones of Ros1, Ros2, Ve, CHS, CHI, F3H, F3′H, FLS, DFR, ANS, UFGT, and AT cDNAs cloned in pBluescript SK+ (Stratagene) or pGEM-T easy (Promega), or restriction fragments from the same plasmids. Radioactively labeled probes were produced using the rediprime II random prime labeling kit (Amersham Pharmacia Biotech), and newly synthesized DNA was made radioactive by replacing nonradioactive dCTP with [α-32P]dCTP. Reactions were fractionated on a drip column of Sephadex G-50 in TE buffer to separate the labeled DNA from the free nucleotides and boiled to denature the DNA before hybridization.

Transfer of Nucleic Acids to Membranes

DNA gel blotting followed the procedure of Maniatis et al. (1982). Twenty microliters of 3′ RACE PCR product or 5 to 10 μg of digested genomic DNA was loaded onto agarose gels in TBE buffer (0.09 M Tris-HCl, 0.09 M boric acid, and 2 mM EDTA) and separated by electrophoresis. After staining with ethidium bromide and photography over a UV light transilluminator, gels were treated with 0.25 M HCl to depurinate the DNA, then with denaturation buffer (0.5 M NaOH and 1.5 M NaCl), and finally with neutralization buffer (1 M Tris-HCl, pH 8.0, and 1.5 M NaCl). Gels were blotted onto nitrocellulose membranes (Protran BA 85; Schleicher and Schuell) overnight. RNA gels were blotted onto nitrocellulose directly in 10× SSC overnight. Filters were then baked in a vacuum oven at 80°C for 2 h before use.

Hybridization of Filters

Filters were prehybridized at 65°C for 2 h in prehybridization solution (6× SSC, 0.2% polyvinylpyrrolidone [molecular weight 40,000], 0.2% Ficoll [molecular weight 40,000], and 0.1% [w/v] SDS), supplemented with 50 μg/mL sonicated and boiled salmon sperm DNA, and hybridized overnight with denatured, radioactive probes at 65°C (high stringency) or 55°C (low stringency) in hybridization solution (3× SSC, 0.02% polyvinylpyrrolidone [molecular weight 40,000], 0.02% Ficoll [molecular weight 40,000], and 0.1% [w/v] SDS), supplemented with 50 μg/mL denatured salmon sperm DNA. They were then washed twice at 55°C for 2 h in low-stringency solution (3× SSC and 0.5% SDS) for low-stringency washes or twice for 20 min in high-stringency solution (0.1× SSC and 0.5% SDS) at 65°C for high-stringency washes. Filters were air-dried, wrapped in Saran wrap plastic film, and exposed to x-ray film (Kodak Biomax MS with Biomax imaging screens) at −70°C.

Particle Bombardment Experiments

A. majus plants of the genotype rosdor were grown in the greenhouse, conditions under which anthocyanin did not form on the petal lobes. Bombardment was conducted using a particle inflow helium gun based on the design of Vain et al. (1993), but modified to use a polycarbonate desiccator (Nalgene) as the chamber. The lobe tissue from young, just-opened flowers was dissected and sterilized in 10% bleach containing one drop of Tween 20 per 100 mL, for between 10 and 15 min, and rinsed in sterilized, distilled water. Plasmid DNA (20 μg) was precipitated onto 5 mg gold particles (1.0 μm diameter) through the addition of 50 μL of 2.5 M CaCl2 and 20 μL of 100 mM spermidine. After precipitation, 90 μL of supernatant was discarded. Gold particles were prepared immediately before use.

For bombardment, petal tissue was placed on an empty Petri dish or on 0.5× Murashige and Skoog (1962) medium plus 7.5% agar (MS medium) in a Petri dish, within the desiccator in the gun range of 120 to 160 mm. Tissue was bombarded with 4 μL of gold suspension using a 50-ms burst of helium at a pressure of 600 kPa within a vacuum of −95 kPa. Each sample was bombarded between one and three times. After bombardment, tissue was incubated on MS medium at 20°C in a 16-h-light/8-h-dark photoperiod with 35 mmol·m−2·s−1 cool-white fluorescent light. Tissue was observed for anthocyanin production after 2 d.

Histochemical staining for GUS activity was performed by incubating petal tissue in 50 mM phosphate buffer, pH 7.0, containing 0.35 mg/mL 5-bromo-4-chloro-3-indolyl-β-glucuronic acid substrate. Tissue was incubated in the dark at 37°C for 24 h before examination for staining.

Phylogenetic Methods

Protein sequences were manually aligned using MacClade 4.08 (D.R. Maddison and W.P Maddison, Sinauer Associates). Phylogenetic analysis was performed with PAUP* 4.0b10 (Swofford, 2001), using only the MYB domain of each protein (see Supplemental Figure 1 online) (Kranz et al., 1998). An optimal tree according to the distance criterion (minimum evolution; mean character difference) was obtained with a heuristic search (tree bisection reconnection). One thousand bootstrapped data sets were used to estimate the confidence of each tree clade.

Supplemental Data

The following materials are available in the online version of this article.

  • Supplemental Figure 1. Alignment of Sequences of MYB-Related Transcription Factors Controlling Anthocyanin Biosynthesis in Different Plant Species.
  • Supplemental Figure 2. Phenotypes from Genetic Analysis of A. graniticum, A. meonanthemum, and A. latifolium.
  • Supplemental Figure 3. Expression of the Gene Encoding DFR in Flowers with Different Phenotypes Shown by Saturating RT-PCR.
  • Supplemental Table 1. Genetic Analysis of Flower Color and Pattern in the Genus Antirrhinum.

Supplementary Material

[Supplemental Data]

Acknowledgments

We thank Rosemary Carpenter for providing seed of the species accessions from the Institut für Kulturpflanzenforschung and for advice on growth, selfing, and crossing these lines; Emma Conklin for technical assistance in part of this work; Hans Sommer for the Antirrhinum genomic library; Rachael Lewis (John Innes Centre) for researching many of the more difficult references; the New Zealand Institute for Crop and Food Research for sponsoring K.S.; the Marsden Fund, New Zealand for supporting K.S., Y.S., and C.M.; the Foundation for Research, Science, and Technology, New Zealand for supporting K.S. and K.D.; the European Union FP5 program for sponsorship through the Profood project (QLK1-CT-2001-01080); the Core Strategic Grant from the Biotechnology and Biological Science Research Council to the John Innes Centre for supporting C.M. and S.M.; and the Marshall Sherfield Foundation for support of R.O. during the course of this work.

Notes

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.plantcell.org) is: Cathie Martin (cathie.martin/at/bbsrc.ac.uk).

[W]Online version contains Web-only data.

Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.105.039255.

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