Abstract

The transcriptional regulators for anthocyanin biosynthesis include members of proteins containing an R2R3-MYB domain, a bHLH (basic helix–loop–helix) domain and conserved WD40 repeats (WDRs). Spacial and temporal expression of the structural genes encoding the enzymes for anthocyanin biosynthesis is thought to be determined by combinations of the R2R3-MYB, bHLH and WDR factors and their interactions. While the wild-type Japanese morning glory (Ipomoea nil) exhibits blue flowers with colored stems and dark-brown seeds, the c mutants display white flowers with red stems and colored seeds, and the ca mutants exhibit white flowers with green stems and ivory seeds. Here, we characterize the tissue-specific expression of three MYB genes, three bHLH genes and two WDR genes in I. nil. We also show that the recessive c-1 and ca alleles are frameshift mutations caused by a 2 bp deletion and 7 bp insertions in the genes for the R2R3-MYB and WDR transcriptional regulators designated as InMYB1 and InWDR1, respectively. In addition to defects in flower, stem and seed pigmentations, the ca mutants were found to show reduced trichome formation in seeds but to produce leaf and stem trichomes and root hairs normally. Except for the gene for chalcone synthase E in the ca mutant, all structural genes tested were coordinately reduced in both c-1 and ca mutant flower limbs. However, slight but significant expression of the genes for chalcone synthase D, chalcone isomerase and flavanone 3-hydroxylase in the pathway for flavonol biosynthesis was detectable in c-1 and ca mutants, whereas no such residual expression could be observed in other genes involved in the later anthocyanin biosynthesis pathway. The biological roles of the C-1 and Ca genes in I. nil epidermal traits and their evolutionary implications are also discussed.

Introduction

The Japanese morning glory (Ipomoea nil) has an extensive history of genetic studies, and about one-third of >200 spontaneous mutants described exhibit mainly altered flower pigmentation (Imai 1938a, Imai 1938b, Hagiwara 1954, Hagiwara 1956, Iida et al. 1999). The wild-type I. nil plants display blue flowers with pigmented red stems and dark-brown seeds (Fig. 1A); the blue flowers contain a polyacylated pigment called Heavenly Blue anthocyanin (HBA) (Fig. 2A, Kondo et al. 1987, Lu et al. 1992b). Almost all structural genes that encode enzymes for the production of anthocyanidin 3-O-sophorosides, which are intermediates in HBA biosynthesis, have been characterized (Fig. 2B, Hoshino et al. 2001, Iida et al. 2004, Morita et al. 2005), and reddish flowers containing Wedding Bells anthocyanin (WBA) bloom in magenta mutants deficient in the gene for flavonoid 3′-hydroxylase (F3′H) (Fig. 2, Lu et al. 1992a, Hoshino et al. 2003). According to classical genetic studies (Imai 1938a, Hagiwara 1956), mutations conferring white flowers can be classified into four groups, a, c, ca and r, and flavonoids accumulating in the white flowers of these mutants have been described (Hagiwara 1954, Saito et al. 1994). White flowers with green stems and normal colored seeds are produced in mutants deficient in the A and R genes, some of which have been shown to be structural genes; the A-3 and R-1 genes encode dihydroflavonol 4-reductase (DFR) and chalcone synthase (CHS), respectively (Inagaki et al. 1994, Inagaki et al. 1996, Hoshino et al. 2001). The c mutants, represented by c-1, exhibit white flowers with red stems and colored seeds, whereas the ca mutants display white flowers with green stems and ivory or whitish seeds (Fig. 1B, C). The C-1 gene was presumed to encode a transcriptional regulator (Hoshino et al. 2001), because the expression of all structural genes examined was shown to be drastically reduced in the young flower buds of a c-1 mutant, 78WWc-1 (Abe et al. 1997, Fukada-Tanaka et al. 1997, Hoshino et al. 1997, Yamaguchi et al. 2001, Hoshino et al. 2003, Morita et al. 2005).

The transcriptional regulators for anthocyanin biosynthesis are known to include member of proteins containing an R2R3-MYB domain, a basic helix–loop–helix (bHLH) domain and WD40 repeats (WDRs); in addition, combinations of the R2R3-MYB, bHLH and WDR proteins and their interactions determine the set of genes to be expressed (Mol et al. 1998, Springob et al. 2003, Broun 2005, Koes et al. 2005, Ramsay and Glover 2005). For example, pairs of duplicated genes encoding transcriptional regulators in maize, R2R3-MYB proteins COLORED ALEURONE1 (C1) and PURPLE PLANT1 (Pl1), as well as bHLH factors RED1 (R1) and BOOSTER1 (B1), are involved in anthocyanin pigmentation; anthocyanin accumulation in the kernel aleurone is controlled by R1 and C1, whereas B1 and Pl1 are required for pigmentation in plant parts (Chandler et al. 1989, Ludwig and Wessler 1990, Cone et al. 1993). Among mutations in the Arabidopsis genes GLABRA3 (GL3), ENHANCER OF GLABRA3 (EGL3) and TRANSPARENT TESTA8 (TT8), which encode bHLH proteins, an egl3 mutant showed reduced anthocyanin pigmentation in the hypocotyls and cotyledons, and an egl3 and gl3 double mutant produced no observable purple anthocyanins without affecting their production of proanthocyanidins (or condensed tannins) in normal brown seed coats (Zhang et al. 2003), whereas tt8 mutants exhibited yellow seeds due to failure to express at least DFR and BAN (BANYULS) for proanthocyanidin biosynthesis in siliques and slightly reduced pigmentation in leaves and stems (Shirley et al. 1995, Nesi et al. 2000). Mutations in the genes for bHLH, petunia anthocyanin1 (an1) and Arabidopsis gl3 and egl3, as well as in the genes for WDR, petunia an11 and Arabidopsis transparent testa glabra1 (ttg1), have been reported to affect not only pigmentation but also other epidermal traits, such as trichome formation in leaves, root hair formation and cell morphology in seed coat cells (Walker et al. 1999, Payne et al. 2000, Spelt et al. 2002, Zhang et al. 2003, Ramsay and Glover 2005).

The structural genes for flower pigmentation in dicotyledonous plants can be divided into early biosynthetic genes (EBGs) and late biosynthetic genes (LBGs), while anthocyanin pigmentation in monocotyledonous maize appears to be coordinately regulated as a single module without division of EBGs and LBGs (Martin and Gerats 1993, Mol et al. 1998, Schwinn and Davies 2004). For example, EBGs and LBGs occur before and after the gene for flavanone 3-hydroxylase (F3H) in snapdragon and petunia, respectively. Since F3H is not required for flavone production but is necessary for flavonol production (Fig. 2B), EBGs in snapdragon and petunia are thought to be involved in the flavone and flavonol biosynthesis pathways, respectively. The spatial and temporal expression of the structural genes might be controlled by distinct sets of regulators (Quattrocchio et al. 1998, Ramsay and Glover 2005). Apart from the c-1 and ca mutations in I. nil, the mutable ivs allele in morning glory, Ipomoea tricolor, which confers pale-blue flowers with a few fine blue spots and ivory seeds with tiny dark-brown spots, was recently shown to be caused by an intragenic tandem duplication of a gene encoding a bHLH transcriptional regulator, while the wild-type plants produce blue flowers and dark-brown seeds (Park et al. 2004). In the common morning glory, Ipomoea purpurea, which displays dark-purple flowers, a recessive w mutant exhibiting white flowers with a pigmented spot in each ray (Clegg and Durbin 2003) was found to carry two 6 and 19 bp deletions causing a frameshift within an R2R3-MYB gene, IpMYB1 or IpMYB1/W (Chang et al. 2005).

Here, we first characterize the tissue-specific expression of three genes for putative R2R3-MYB transcriptional regulators, three genes for bHLH factors and two genes for WDR proteins from I. nil. Subsequently, we show that both C-1 and Ca genes encode transcriptional regulators, an R2R3-MYB protein and a WDR factor designated as InMYB1 and InWDR1, respectively. The recessive c-1 and ca alleles are frameshift mutations caused by a 2 bp deletion and 7 bp insertions, respectively. In addition to defects in flower, stem and seed pigmentations, we found that the ca mutations drastically reduce trichome formation in seeds but confer apparently normal trichomes in leaves and stems as well as root hairs. Using these c-1 and ca mutants, we reinvestigated the temporal expression profiles of the structural genes encoding enzymes for anthocyanin biosynthesis (Fig. 2B) in the flower limbs from young flower buds to open flowers. While most of the EBGs and LBGs were coordinately activated by C-1 and Ca proteins, EBGs involved in flavonol synthesis (Fig. 2B) were also expressed to a lesser extent independently of either C-1 or Ca activity.

Results

Characterization of the regulatory genes for anthocyanin pigmentation in I. nil flowers

To isolate cDNAs encoding transcriptional regulators for anthocyanin biosynthesis in flowers, we employed two wild-type I. nil cultivars, TKS and KK/ZSK-2, both of which display blue flowers (Morita et al. 2005), and searched for homologs of petunia AN2 for R2R3-MYB, petunia AN1 and JAF13, as well as snapdragon Delila, for bHLH, and petunia AN11 for WDR, whose mutations have been shown to affect flower pigmentation (Table 1). The cDNAs for InMYB1, InMYB2, InbHLH1 (or InDEL, Park et al. 2004), InbHLH2 (or InIVS), InbHLH3, InWDR1 and InWDR2 were obtained either from expressed sequence tags (ESTs) of TKS flowers or by screening a cDNA library from young flower buds of KK/ZSK-2. On the basis of the full-length cDNA sequences of the transcriptional regulators obtained, the lengths of their predicted amino acid sequences are shown in Table 1. Their assignments were supported by phylogenetic trees (Fig. 3A–C), which also include InMYB3 deduced from the genomic InMYB3 gene sequence (see Materials and Methods) and perilla regulators MYC-RP, MYC-F3G1 and PFWD (Yamazaki et al. 2003). It is clear that InbHLH1 and InbHLH3 are more closely related to petunia JAF13, whereas InbHLH2 is a relative of petunia AN1 (Quattrocchio et al. 1998, Spelt et al. 2000). Likewise, all InMYBs are closer relatives to petunia AN2 and Arabidopsis PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1), which are responsible for anthocyanin pigmentation (Quattrocchio et al. 1999, Borevitz et al. 2000, Tohge et al. 2005), and InWDR1 is closely related to petunia AN11 and Arabidopsis TTG1 (de Vetten et al. 1997, Walker et al. 1999). Alignment of InMYBs, AN2 and PAP1 showed highly conserved R2 and R3 DNA-binding domains (Fig. 4A), whereas identical amino acids were found in all regions of InWDR1, AN11 and TTG1 (Fig. 5A). We analyzed the tissue-specific expression of the isolated genes and could not detect any transcripts from InMYB3, whose cDNAs we were unable to obtain (Fig. 3D). The genes for InMYB1 and InbHLH2 were predominantly expressed in flower limbs and tubes, whereas those for InWDR1 and InWDR2 were expressed in every tissue tested.

Identification of the c-1 and ca mutations

To examine whether the c-1 or ca mutation affecting flower pigmentation falls into one of these regulatory genes, we analyzed the expression of the genes for InMYB1, InbHLH1, InbHLH2 and InWDR1 because they were closely related to the known regulatory genes expressed in flower limbs. As Fig. 3E shows, the accumulation of the InMYB1 and InWDR1 transcripts was reduced the most in the c-1 and ca mutants, respectively, suggesting that they are candidate genes for the mutations. Although InbHLH2 expression was also drastically reduced in the ca mutant, the phenotype of the ca mutant appeared to be different from that of the I. tricolor ivs mutant that is deficient in the corresponding ItbHLH2 gene: the ca mutant in I. nil displays white flowers, whereas the ivs mutant exhibits palely pigmented flowers with colored rays (Park et al. 2004). Indeed, no sequence alterations in the InbHLH2 cDNA could be detected between the wild type and the ca mutant (data not shown). We sequenced the 0.86 kb InMYB1 cDNA of 78WWc-1 carrying the c-1 mutation and the 1.2 kb InWDR1 cDNA of NS/W1ca1 with the ca mutation and found that both cDNAs contained frameshift mutations: a 2 bp deletion in the highly conserved R3 MYB DNA binding domain of the R2R3-MYB protein (Stracke et al. 2001) in the c-1 mutant (Fig. 4D) and a 7 bp insertion GGAGTAC of the ca mutant in the middle of the coding region of InWDR1 (Fig. 5D).

To confirm that the c-1 mutation is the frameshift mutation caused by the 2 bp deletion in InMYB1, we analyzed 1.3 kb PCR fragments from the genomic InMYB1 sequence amplified with primers InMYB1-F0 and InMYB1-R0, which correspond to the ends of the InMYB1 coding region (Fig. 4C). Comparison of the genomic and cDNA sequences of the 1.3 kb fragments from two wild-type lines, TKS and KK/ZSK-2, as well as Q173 carrying the wild-type C-1 allele, revealed that their genomic sequences are identical to each other and that the InMYB1 gene consists of three exons (Fig. 4C). We also examined the 1.3 kb fragments from nine c-1 mutants having various genetic backgrounds and found that all of them contain the same 2 bp deletion (Table 2). Furthermore, the 2 bp deletion was co-segregated with c-1 among the selfed progeny of a hybrid between Q173 and 78WWc-1 with the c-1 allele; out of the 33 progeny, eight plants bearing the 2 bp deletion in the homozygous condition produced white flowers, and 19 heterozygous plants as well as six plants without the 2 bp deletion displayed pigmented flowers. Based on these results, we can conclude that the c-1 allele is a frameshift mutation caused by the 2 bp deletion within exon 2 of the InMYB1 or InMYB1/C-1 gene.

We also examined 1.2 kb PCR fragments amplified with primers InWDR1-F1 and InWDR1-R1 corresponding to the 5′- and 3′-untranslated regions (UTRs) of the InWDR1 cDNA adjacent to its coding region and found that InWDR1 comprises a single exon (Fig. 5C), which conforms to maize PALE ALEURONE COLOR1 (PAC1), petunia AN11 and Arabidopsis TTG1 (Fig. 3C, de Vetten et al. 1997, Walker et al. 1999, Carey et al. 2004). Sequencing of the 1.2 kb fragments from two wild-type lines, TKS and KK/ZSK-2, a Ca plant, Q65 and eight ca mutants having various genetic backgrounds revealed that all of the ca mutants tested carry either one of two 7 bp insertions at the identical site, which we designated as ca-1 and ca-2 alleles (Table 2, Fig. 5D). Only NS/W1ca1 and NS/W2ca3, whose flavonoid contents were reported earlier (Saito et al. 1994), were found to carry the ca-1 allele, and the remaining mutants had the ca-2 allele. Since the ca-2 allele coincides with a restriction site BsiWI (CGTACT), we employed the line Q931 with ca-2 for co-segregation analysis (Fig. 5E). Out of 192 selfed progeny of a hybrid between Q65 and Q931, only 50 plants containing the BsiWI site in the homozygous condition exhibited white flowers, and the remaining 97 heterozygous plants and 45 homozygous Ca plants displayed pigmented flowers. Of these, 142 plants were allowed to set seeds (33 Ca/Ca, 79 Ca/ca and 30 ca/ca). The phenotypes of their seed coats, which were strictly of maternal origin, conformed in full with their flower phenotypes. It is clear that the ca mutants carry frameshift mutations due to 7 bp insertions in the InWDR1 or InWDR1/Ca gene.

Characterization of the genomic InMYB1/C-1 and InWDR1/Ca gene regions

As Fig. 4B and 5B show, Southern blot analysis indicated that each of the InMYB1/C-1 and InWDR1/Ca genes carries a single copy in the I. nil genome. It was further supported by the facts that single bands were detected in Southern blot analysis for InMYB1/C-1 in the BamHI, HindIII, KpnI and XhoI digests, and for InWDR1/Ca in the EcoRI, KpnI and NheI digests (data not shown). We cloned a 12.9 kb HindIII fragment bearing InMYB1/C-1, and its partial sequencing showed that InMYB1/C-1 resides within the 5.4 kb segment (Fig. 4C). We thus sequenced the 5.4 kb segment from wild-type TKS and KK/ZSK-2 as well as Q173 containing the wild-type C-1 allele and 78WWc-1 bearing the c-1 allele, and no sequence alterations were detected, except for the 2 bp deletion in 78WWc-1. We also isolated a 9.4 kb HindIII fragment containing a novel InMYB3 gene. Although no InMYB3 cDNA is available, its putative exons could be assigned by comparing the genomic sequence of InMYB3 with those of InMYB1 and InMYB2 (Fig. 4E). The positions of two introns in all three InMYBs appeared to be identical to each other, and dinucleotide GC was commonly found instead of the GT consensus sequence at their donor sites of intron 2 (data not shown). We also obtained 9.2 kb XbaI–XhoI fragments for InWDR1 from TKS, KK/ZSK-2 and NS/W1ca1, and InWDR1 was found to reside between the genes encoding a caffeic acid 3-O-methyltransferase and a hairpin-induced family protein (Fig. 5C). Six single nucleotide polymorphisms (SNPs) were detected between KK/ZSK-2 and TKS, and no SNPs were observed between KK/ZSK-2 and NS/W1ca1 except for the 7 bp insertion corresponding to the ca-1 allele.

Temporal expression of the genes for anthocyanin pigmentation in flower limbs

Since the expression of several structural genes was known to be affected in 78WWc-1 (Abe et al. 1997, Fukada-Tanaka et al. 1997, Hoshino et al. 1997, Yamaguchi et al. 2001, Hoshino et al. 2003, Morita et al. 2005), a detailed investigation of the temporal expression of the genes for anthocyanin pigmentation in flower limbs was performed in TKS, 78WWc-1 and NS/W1ca1 (Fig. 6). In addition to the genes for anthocyanin biosynthesis in flower limbs (Fig. 2B), the analysis included CHS-E, which is responsible mainly for pigmentation in flower tubes (Fukada-Tanaka et al. 1997, Johzuka-Hisatomi et al. 1999, Durbin et al. 2000), GST, which encodes glutathione S-transferase, a homolog of petunia AN9 that is believed to be involved in escorting anthocynain pigments to the vacuole (Alfenito et al. 1998, Park et al. 2004), and NHX1 for the major vacuolar Na+/H+ exchanger that is responsible for blue flower coloration (Fukada-Tanaka et al. 2000, Yamaguchi et al. 2001). The expression of InMYB1 increased after about 60 h before flower opening (BFO), whereas that of InWDR1 was more abundant in the earlier stages before around 72 h BFO in the wild type and continued up to about 24 h BFO in the c-1 and ca mutants (Fig. 6). Thus, the InWDR1 expression in the wild type appears to precede the expression of most of the structural genes except for the gene for UDP-glucose:anthocyanidin 3-O-glucose-2′′-O-glucosyltransferase (3GGT), and the molecular mechanisms of this apparent discordance remain to be elucidated. Among the genes studied, the CHS-D expression in TKS appeared to be most closely coordinated with InMYB1 expression. Except for CHS-E and NHX1, the expression of all genes presented in Fig. 6B was drastically reduced in the c-1 and ca mutants. Interestingly, the expression of CHS-E was reduced in 78WWc-1 but not affected in NS/W1ca1, and earlier expression of CHS-E in the wild-type flower tubes of I. nil conformed to that of I. purpurea (Durbin et al. 2000). The expression profiles of NHX1 in TKS and 78WWc-1 confirmed and further strengthened the previous observation that the c-1 mutation did not reduce the NHX1 expression (Yamaguchi et al. 2001). No reduction of NHX1 expression was observed in the ca mutant, NS/W1ca1. Like NHX1, neither the c-1 nor the ca mutations affected the expression of NHX2 for a minor vacuolar Na+/H+ exchanger (data not shown), although its mRNA accumulation was scantier than that of NHX1 (Ohnishi et al. 2005).

We also noticed that most of the structural genes tended to be more expressed before dawn than before sunset (more at 72, 48 and 24 h BFO than at 60 and 36 h BFO), suggesting that they are also partially subjected to a circadian clock, which was shown in Arabidopsis to orchestrate the coordinate expression of genes for phenylpropanoid biosynthesis to peak before dawn (Harmer et al. 2000). Although most of the structural genes were coordinately reduced in both c-1 and ca mutants, close inspection of the results indicated that slight but significant expression of EBGs involved in flavonol synthesis, CHS-D, CHI (chalcone isomerase) and F3H (Fig. 2B), was detectable, whereas no such residual expression was observed in LBGs (Fig. 6B). To quantify the expression of the individual genes in the c-1 and ca mutants, we employed the cDNA for the γ-subunit of the mitochondrial F1F0-ATP synthase of I. nil as a probe for an internal control and compared the relative intensities of the expressed transcripts in each gene at 36 h BFO (Fig. 6C). The accumulation of the CHS-D, CHI and F3H transcripts in c-1 and ca mutants was 10–20% of the wild-type levels, whereas that of the F3H, DFR-B, ANS (anthocyanidin synthase), 3GT (UDP-glucose:flavonoid 3-O-glucosyltransferase), 3GGT and GST transcripts in c-1 and ca mutants was <1% compared with that in the wild type. These features were further confirmed by real-time reverse transcription–PCR (RT–PCR) analysis; the expression levels of DFR-B and 3GT were 0.2–1.6% compared with those of CHS-D and F3H in both c-1 and ca mutants, whereas those of CHS-D, F3H, DFR-B and 3GT in wild type were comparable (data not shown). These results imply that EBGs are not only controlled coordinately with LBGs but also activated to a lesser extent by another regulation system in the absence of either C-1 or Ca activity. For the CHS-E gene in flower tubes, only about 5% of the wild-type level of expression was detected in 78WWc-1, while the ca mutation did not affect its mRNA accumulation (Fig. 6C).

Trichome formation in seeds

Since it is known that mutations in Arabidopsis TTG1 and petunia AN11 affect not only anthocyanin and/or proanthocyanidin pigmentation but also leaf trichome and root hair formation in Arabidopsis and the morphology of seed coat cells in petunia (Walker et al. 1999, Spelt et al. 2002, Zhang et al. 2003), we examined whether the ca mutation in InWDR1 also confers similar deficiencies. Although trichome formation in leaves and stems as well as root hair formation appeared to be normal in the ca mutant (Fig. 1G, I, K), we found that the wild-type seed coats produced trichomes (Fig. 1A, D) and that the seed trichomes were smaller in size and remarkably fewer in number than those of the wild type (Fig. 1C, E). No apparent morphological alterations were observed in trichomeless cells between the wild-type and ca mutant seed coats (data not shown). The seed trichome formation in the c-1 mutant was found to be normal (Fig. 1B). Thus, seed trichome formation is a new epidermis trait detected in I. nil that is controlled by the WDR transcriptional regulator, InWDR1.

Discussion

We have isolated and characterized the genes for three R2R3-MYB, three bHLH and two WDR transcriptional regulators (Fig. 3, Table 1). In accordance with the notion that duplication and subsequent divergence of each group of transcriptional regulators results in subfunctionalization, such as tissue-specific expression, neo-functionalization or non-functionalization (Heim et al. 2003, Carey et al. 2004, Grotewold 2005), all three InMYBs were found to carry three exons with identical intron positions and the dinucleotide GC instead of the GT consensus sequence at the donor site of intron 2, and InMYB1 was found to be expressed in flower limbs and tubes, and InMYB2 in sepals, petioles and stems, whereas no expression of InMYB3 was detected in any of the tissues tested (Fig. 3D, 4). Diversified tissue-specific expression could also be seen in three InbHLHs, whereas both InWDRs were expressed in all tissues examined. Anthocyanin biosynthesis in different tissues is thought to be determined by combinations of the R2R3-MYB, bHLH and WDR factors and their interactions, which control the set of structural genes to be expressed (Mol et al. 1998, Broun 2005, Koes et al. 2005, Ramsay and Glover 2005). Preliminary results indicated that a major anthocyanin in red stems of the wild-type plant TKS, which exhibits blue flowers containing HBA, is a much simpler anthocyanin, peonidin 3-O-caffeoylsophoroside (Fig. 2A, Y. Morita unpublished), and pigmentation in red stems and blue flowers occurs in the subepidermal and epidermal layers, respectively (Inagaki et al. 1996), suggesting that anthocyanin biosynthesis in stems and flowers is controlled by different sets of transcriptional regulators. Consistent with this notion, the c-1 mutants deficient in InMYB1, which is expressed only in flower limbs and tubes (Fig. 3D), exhibit white flowers with pigmented red stems (Fig. 1B).

While an I. nil variety producing white flowers and whitish seeds was documented by Gen-nai Hiraga (1763), the first genetically studied I. nil mutations conferring white flowers were only c and r, and the allele ca (originally designated as ca) was later identified as a third mutation that confers white flowers with yellow tubes, green stems and ivory seeds (Hagiwara 1929). The name of the gene Ca appeared to be given because it was thought to activate the function of the C gene. We showed that the c-1 and ca alleles are frameshift mutations caused by a 2 bp deletion within exon 2 of InMYB1/C-1 and 7 bp insertions GGAGTAC or TCCGTAC at the same position in InWDR1/Ca, respectively (Fig. 4D, 5D). In the 2 bp deletion, dinucleotide AG was removed from a simple repeat sequence, AGAGAG. Regarding the 7 bp insertions, we presumed that they are footprints generated by independent excisions of a DNA transposon in the Tpn1 family because the wild-type sequence, TAC, had changed into TAC(GGAG/TCCG)TAC and because elements of the Tpn1 family, which belong to the En/Spm or CACTA superfamily and generate 3 bp target site duplications, are thought to act as major mutagens for the generation of various spontaneous mutations in I. nil (Iida et al. 1999, Hoshino et al. 2001, Iida et al. 2004, Kawasaki and Nitasaka 2004). We also described previously that two dusky alleles are frameshift mutations due to conversion of the wild-type sequence GAT into GAT(G/C)GAT (4 bp insertions) at the identical position, which we speculated to be footprints generated by independent excisions of a Tpn1-related element (Morita et al. 2005). The accumulation of InMYB1 mRNAs was reduced in the c-1 mutant, whereas that of InWDR1 and InbHLH2 mRNAs decreased in the ca mutant (Fig. 3E). Even though the reduction of InMYB1 and InWDR1 can be explained by nonsense-mediated decay (Belostotsky and Rose 2005), it is not clear why the InbHLH2 mRNAs were reduced in the ca mutant containing the frameshift mutation within the InWDR1 gene. It might be a consequence of a certain interaction and/or a regulatory network between these genes, since AN4 for R2R3-MYB was shown to be required for the expression of AN1 for bHLH in petunia anthers (Spelt et al. 2000) and since up-regulation of TT8 for bHLH was noted in a PAP1 (for R2R3-MYB) overexpresser of Arabidopsis (Tohge et al. 2005). It is highly likely that the transcriptional regulators InMYB1/C-1 and InWDR1/Ca operate coordinately with InbHLH2/InIVS to activate the structural genes for anthocyanin biosynthesis because the ivs mutant of I. tricolor that is deficient in the ItbHLH2/ItIVS function exhibits palely pigmented flowers with colored rays, slightly reduced coloration in stems and ivory-colored seeds (Park et al. 2004). Whether the pale pigmentation with colored rays in the ivs mutant is due to the partial complementation effects of the bHLH1 gene that is also expressed in flowers (Fig. 3D) remains to be elucidated. Except for CHS-E and NHX1, the expression of all genes in Fig. 6B was reduced in both c-1 and ca mutants, and the CHS-E expression in flower buds was reduced only in the c-1 plant. The CHS-D and CHS-E genes are predominantly expressed and responsible for pigmentation in flower limbs and tubes, respectively (Johzuka-Hisatomi et al. 1999, A. Hoshino unpublished), and CHS-E in the tubes appears to be independent of InWDR1. It remains to be studied whether CHS-E in flower tubes is controlled by InWDR2, which was shown to be expressed in flower tubes (Fig. 3D).

The structural genes for flower pigmentation in dicotyledonous plants, such as snapdragon and petunia, are thought to be divided into EBGs and LBGs, whereas those in monocotyledonous maize are coordinately regulated as a single module without division of EBGs and LBGs (Martin and Gerats 1993, Mol et al. 1998, Schwinn and Davies 2004). A similar division after F3H was also observed in the proanthocyanidin biosynthetic genes in Arabidopsis seeds (Nesi et al. 2001, Debeaujon et al. 2003). Regarding the flowers in I. nil, most of the structural genes were found to be coordinately reduced in both c-1 and ca mutants, but about 10–20% of the wild-type levels of expression of EBGs (CHS-D, CHI, and F3H) were certainly detectable (Fig. 6B, C). The results imply that EBGs for flavonol biosynthesis (Fig. 2B) are subjected to dual controls: they are mainly controlled coordinately with other LBGs, but they are also controlled by a secondary regulation system, which appears to operate to a lesser extent independently of either C-1 or Ca activity. Indeed, the accumulation of flavonols was reported in the flowers of both c-1 and ca mutants (Saito et al. 1994). Since MYB12 in Arabidopsis was recently reported to be a flavonol-specific transcriptional regulator (Mehrtens et al. 2005), it is conceivable that a similar MYB protein also activates EBGs for flavonol biosynthesis.

Although both I. nil ca and petunia an11 mutations in the WDR genes confer white flowers and ivory seeds (Fig. 1), morphological alterations in the seed coat epidermis are different: the I. nil ca mutation affects seed trichome formation (Fig. 1C, E), whereas the petunia an11 allele confers more fragile seed coats with an increased number of smaller cells (Spelt et al. 2002). The effects of vacuolar pH in flowers were also different: vacuolar alkalinization for blue flower pigmentation occurs during flower opening in I. nil, and the expression of both InNHX1 and InNHX2 that is responsible for increasing the vacuolar pH is not controlled by InWDR1 (Fig. 6B, Yamaguchi et al. 2001); on the other hand, acidification in the vacuole takes place in petunia, and the an11 mutants failed to decrease the vacuolar pH (Spelt et al. 2002). Both genera Ipomoea and Petunia belong to the order Salanales and are proposed to have diverged from a common ancestor about 70 million years ago based on the comparison of their CHS genes (Durbin et al. 2000). It is tempting to speculate that the observed differences, particularly the controlling vacuolar pH, are partly attributable to a consequence of evolutionary processes for flower pigmentation designed to attract pollinators, i.e. blue flower pigmentation in I. nil, and that duplication and subsequent divergence of transcriptional regulators must have played important roles in such evolutionary processes.

Materials and Methods

Plant materials

All I. nil lines used are listed in Table 2. Plants were usually grown in pollinator-free greenhouses and occasionally grown in fields (Hoshino et al. 2003).

General nucleic acid procedures

General nucleic acid procedures, including the preparation of DNAs from young leaves and RNAs from flower limbs and tubes, Southern and Northern blot analyses, and DNA sequencing analysis, were performed as described previously (Johzuka-Hisatomi et al. 1999, Hoshino et al. 2003, Park et al. 2004, Morita et al. 2005). I. nil cDNAs used for hybridization probes, CHS-D, CHS-E, CHI, F3H, F3H, DFR-B, ANS, 3GT, 3GGT, NHX1, GST and bHLH2 were described previously (Abe et al. 1997, Fukada-Tanaka et al. 1997, Hoshino et al. 1997, Yamaguchi et al. 2001, Hoshino et al. 2001, Hoshino et al. 2003, Park et al. 2004, Morita et al. 2005).

Isolation of cDNAs and genomic DNAs for the InMYB genes

To obtain InMYB1 genes and cDNAs, we screened the IpMYB1 cDNA from an I. purpurea flower-bud cDNA library (Hoshino et al. 2003) under the low-stringency hybridization condition (Hisatomi et al. 1997) using an IpMYB cDNA fragment provided by Dr. M. D. Rausher as a probe. Out of about 700 positive clones from 1.0×107 recombinant phages, nine strongly hybridized clones containing IpMYB1 were obtained, and one of them carrying the longest 1.1 kb cDNA was used further. Using the IpMYB1 cDNA as a probe, we first tried to clone a 12.9 kb HindIII fragment bearing InMYB1/C-1 into the λZAP Express vector (Stratagene, La Jolla, CA, USA) and obtained a 9.4 kb HindIII fragment containing a novel gene, InMYB3, by screening a genomic DNA library prepared form KK/ZSK-2 under the same low-stringency hybridization condition. Subsequently, we succeeded in cloning a 12.9 kb HindIII fragment bearing InMYB1/C-1 into the λDASH II vector (Stratagene) from TKS and KK/ZSK-2 and recloned the genomic fragments into the pZErO-2 cloning vector (Invitrogen, Carlsbad, CA, USA) for further characterization. To examine the InMYB1 genes of Q173 and 78WWc-1 containing the C-1 and c-1 alleles, respectively, the 5.4 kb regions containing the mutated InMYB1 genes were prepared by the three overlapping PCR-amplified fragments with appropriate primers (Fig. 4C, Table 3) and sequenced without cloning.

By comparing IpMYB1 cDNA with the wild-type genomic InMYB1 sequence, we designed primers InMYB1-F0 and InMYB1-R0 (Table 3) as putative 5′ and 3′ends of the InMYB1 coding region, respectively, and examined whether InMYB1 cDNAs could be obtained by RT–PCR amplification; RT–PCR-amplified fragments from TKS were cloned into the pGEM-T Easy vector (Promega, Madison, WI, USA) for further analysis. The obtained 0.86 kb cDNAs were well fitted with the expected InMYB1 cDNAs, confirming that the designed primers are useful to isolate InMYB1 cDNA fragments containing the InMYB1 coding region. We thus determined the sequences of InMYB1 cDNA fragments obtained from two C-1 plants, KK/ZSK-2 and Q173, as well as 78WWc-1, by RT–PCR amplification with primers InMYB1-F0 and InMYB1-R0. Subsequently, we also prepared 1.3 kb PCR-amplified fragments of the genomic InMYB1 coding regions with two introns of eight c-1 mutants having various genetic backgrounds (Table 2; all available c-1 mutants, except for 78WWc-1) using primers InMYB1-F0 and InMYB1-R0, and cloned them into the pGEM-T Easy vector. To obtain the InMYB1 cDNA containing the entire coding sequence, RT–PCR amplification with primers InMYB1-F1 and InMYB1-R1 (Table 3), which correspond to the 5′- and 3′-UTRs adjacent to the coding region, respectively, was performed using RNAs prepared from flower buds of TKS; the resulting InMYB1 cDNA was confirmed by DNA sequencing.

In the meantime, we also screened >56,000 ESTs from TKS flowers including sepals, petioles, anthers and stigma (A. Hoshino et al. unpublished), the majority of which have been deposited in the DDBJ database, and found one cDNA for InMYB1 and another cDNA for InMYB2.

Isolation of cDNAs for the InbHLH genes

To isolate the InbHLH1 (or InDEL, Park et al. 2004) cDNA from a KK/ZSK-2 flower-bud cDNA library, we screened 4.3×105 recombinant phages under standard hybridization conditions (Hisatomi et al. 1997) using the Ipomoea batatas bHLH (IbbHLH) cDNA as a probe, and five clones containing InbHLH1 were obtained. IbbHLH was isolated by Dr. T. Hattori as a homolog of the snapdragon Delila cDNA (T. Hattori, personal communication). We also obtained eight InbHLH2 cDNAs and three InbHLH3 cDNAs from the 56,000 ESTs prepared from TKS flowers.

Isolation of cDNAs and genomic DNAs for the InWDR genes

During isolation of Ip3GGT cDNAs from flower buds of I. purpurea (Morita et al. 2005), we happened to isolate a clone apparently containing an IpWDR factor, which we later designated IpWDR2. Subsequently, using the cDNA fragment as a probe, we obtained InWDR1 and InWDR2 from a flower-bud cDNA library of KK/ZSK-2 and IpWDR1 from the I. purpurea flower-bud cDNA library under the low-stringency hybridization condition (Hisatomi et al. 1997) using the IpWDR2 cDNA as a probe. Out of about 80 positive clones from 3.0×106 recombinant phages, we obtained 10 weakly and five strongly hybridized clones containing InWDR1 and InWDR2 cDNAs, respectively. In addition, we also isolated five InWDR1 and two InWDR2 cDNA clones from the 56,000 ESTs prepared from TKS flowers.

To determine the genomic sequences of the 1.2 kb fragments of InWDR1 from TKS, KK/ZSK-2 and Q65 as well as eight ca mutants having various genetic backgrounds (Table 2), PCR-amplified fragments with primers InWDR1-F1 and InWDR1-R1 (Table 3) were cloned into the pGEM-T Easy and subsequently sequenced. For cloning of the InWDR1 gene, 9.2 kb XbaI–XhoI fragments from TKS, KK/ZSK-2 and NS/W1ca1 were cloned into the λZAP Express vector for further characterization.

RT–PCR and real-time RT–PCR analyses

For spacial expression of the regulatory genes by RT–PCR analysis, total RNAs were prepared from various tissues. First-strand cDNAs were synthesized using SuperScript III reverse transcriptase (Invitrogen), and AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA, USA) was used for subsequent PCR amplification with appropriate primers (Table 3). The expression level was estimated semi-quantitatively by altering the cycles of PCR amplification.

For real-time PCR analysis, the expression levels of CHS-D, F3H, DFR-B and 3GT as well as the gene for the γ-subunit of the mitochondrial F1F0-ATP synthase as an internal control were measured with ABI PRISM 7000 using SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA). Thermal cycling conditions used for PCR amplification with appropriate primers (Table 3) were 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 68°C for 1 min.

Co-segregation analysis

The genotypes of the InMYB1 genes in the 33 selfed progeny of a hybrid between the C-1 line Q173 and 78WWc-1 were determined by directly sequencing the PCR fragments amplified with the primers InMYB1-F1 and InMYB1-R1 (Fig. 5E), and the sequences of the PCR fragments from homozygous and heterozygous plants gave unique or mixed sequences (due to the 2 bp deletion of the c-1 allele), respectively. Because the ca-2 allele carried by Q931 coincides with the BsiWI restriction site due to the 7 bp insertion, the genotypes of 192 selfed progeny of a hybrid between Q65 with the wild-type Ca gene and Q931 were analyzed by cleaving the PCR fragments amplified with primers InWDR1-F1 and InWDR1-R1 with BsiWI. Both the pigmentation in flowers and seeds and the altered morphology of trichomes in the seeds were scored.

Acknowledgments

We thank Miwako Matsumoto, Yuki Okumura and Kyoko Ikegaya for technical assistance, Mitsumasa Okada and Hiroyuki Satoh for their encouragement, support and advice, Norio Saito for providing the NS/W1ca1 and NS/W2ca3 seeds and for valuable discussions, Yasuyo Johzuka-Hisatomi and Eisho Nishino for discussions, and Mark D. Rausher and Tsukaho Hattori for providing probes for the isolation of InMYB1 and InbHLH1 cDNAs, respectively. This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

The nucleotide sequences reported in this paper have been submitted to the DDBJ database under accession numbers AB232769 (IpMYB1 mRNA), AB232770 (InMYB1/C-1 mRNA), AB232771 (InMYB1/C-1 genomic DNA of TKS), AB232772 (InMYB1/C-1 genomic DNA of KK/ZSK-2), AB232773 (InMYB1/C-1 genomic DNA from a c-1 mutant line 78WWc-1), AB234211 (InMYB2 mRNA), AB234212 (InMYB3 genomic DNA), AB232774 (InbHLH1/InDEL mRNA), AB232775 (InbHLH2/InIVS mRNA), AB232776 (InbHLH3 mRNA), AB232777 (IpWDR1 mRNA), AB232778 (IpWDR2 mRNA), AB232779 (InWDR1/Ca mRNA), AB232780 (InWDR2 mRNA), AB232781 (InWDR1/Ca genomic DNA of KK/ZSK-2), AB232782 (InWDR1/Ca genomic DNA of TKS) and AB232783 (InWDR1/Ca genomic DNA from a ca-1 mutant line NS/W1ca1).

Fig. 1 Flower and seed phenotypes of TKS (A), 78WWc-1 (B) and NS/W1ca1 (C). The top, middle and bottom panels show flower and stem pigmentation, seed color and trichome formation in the seed coat, respectively. Bars = 500 µm. Scanning electron microscope (SEM) photographs of seed epidermis in TKS (D) and NS/W1ca1 (E). Bars = 100 µm. Microscopic photographs of a stem in TKS (F) and NS/W1ca1 (G) and leaf surface in TKS (H) and NS/W1ca1 (I). Bars = 2.0 mm. Microscopic photographs of root hairs in TKS (J) and NS/W1ca1 (K). Bars = 200 µm. Microscopic and SEM photographs were taken using a digital microscope VHX-100 and a digital SEM VE-8800, respectively (Keyence, Osaka, Japan).

Fig. 1 Flower and seed phenotypes of TKS (A), 78WWc-1 (B) and NS/W1ca1 (C). The top, middle and bottom panels show flower and stem pigmentation, seed color and trichome formation in the seed coat, respectively. Bars = 500 µm. Scanning electron microscope (SEM) photographs of seed epidermis in TKS (D) and NS/W1ca1 (E). Bars = 100 µm. Microscopic photographs of a stem in TKS (F) and NS/W1ca1 (G) and leaf surface in TKS (H) and NS/W1ca1 (I). Bars = 2.0 mm. Microscopic photographs of root hairs in TKS (J) and NS/W1ca1 (K). Bars = 200 µm. Microscopic and SEM photographs were taken using a digital microscope VHX-100 and a digital SEM VE-8800, respectively (Keyence, Osaka, Japan).

Fig. 2 Structures of anthocyanins (A) and simplified flavonoid biosynthesis pathways (B). Major anthocyanins in blue flowers, reddish flowers and red stems contain Heavenly Blue anthocyanin (HBA), Wedding Bells anthocyanin (WBA) and peonidin 3-O-caffeoylsophoroside. Conversion of flavanones to flavones is catalyzed by flavone synthase (FNS), and that of dihydroflavonols to flavonols is mediated by flavonol synthase (FLS). Both C-1 and Ca control the expression of structural genes for anthocyanin biosynthesis, and a putative secondary regulation system for flavonol biosynthesis, as indicated by an asterisk, controls CHS-D, CHI and F3H.

Fig. 2 Structures of anthocyanins (A) and simplified flavonoid biosynthesis pathways (B). Major anthocyanins in blue flowers, reddish flowers and red stems contain Heavenly Blue anthocyanin (HBA), Wedding Bells anthocyanin (WBA) and peonidin 3-O-caffeoylsophoroside. Conversion of flavanones to flavones is catalyzed by flavone synthase (FNS), and that of dihydroflavonols to flavonols is mediated by flavonol synthase (FLS). Both C-1 and Ca control the expression of structural genes for anthocyanin biosynthesis, and a putative secondary regulation system for flavonol biosynthesis, as indicated by an asterisk, controls CHS-D, CHI and F3H.

Fig. 3 Genes encoding transcriptional regulators for anthocyanin biosynthesis. Phylogenetic trees for three groups of transcriptional regulators, R2R3-MYB transcription factors (A), bHLH transcription regulators (B) and WDR proteins (C). The entire amino acid sequences were aligned with CLUSTALW, and the tree was constructed as described before (Fukada-Tanaka et al. 1997). The bootstrap values out of 100 retrials are indicated at each branch, and the scale shows the 0.1 amino acid substitution per site. The numerals given in parentheses indicate the ratio (%) of identical amino acid residues to the transcriptional regulators of I. nil represented in bold. The abbreviation shown in front of each protein indicates the plant species: In, Ipomoea nil; Ip, I. purpurea; It, I. tricolor; At, Arabidopsis thaliana; Am, Antirrhinum majus; Le, Lycopersicon esculentum; Pf, Perilla frutescens; Ph, Petunia hybrida; Vv, Vitis vinifera; Zm, Zea mays. R2R3-MYB transcription factors: AtPAP1 (AAG42001), AtPAP2 (AAG42002), AtTT2 (CAC40021), LeANT1 (AAQ55181), PhAN2 (AAF66727), VvMYBA1 (BAD18977), VvMYBA2 (BAD18978), ZmC1 (AAK09327), ZmPl (T03972). bHLH transcriptional regulators: IpbHLH2/IpIVS (BAD18982), ItbHLH2/ItIVS (BAD18984), AmDEL (AAA32663), AtEGL3 (Q9CAD0), AtGL3 (NP_680372), AtTT8 (Q9FT81), PfMYC-F3G1 (BAC56998), PfMYC-RP (BAA75513), PhAN1 (AAG25928), PhJAF13 (AAC39455), ZmIN1 (AAB03841), ZmLC (AAA33504), ZmB-Peru (CAA405449). WDR proteins: AtAN11 (AAC18912), AtTTG1 (CAB45372), PFWD (BAB58883), PhAN11 (AAC18914), ZmPAC1 (AAM76742), ZmMP1 (AAR01949). (D) Semi-quantitative RT–PCR analysis for the spatial expression of the regulatory genes in the wild-type TKS line. The constitutively expressed gene for the mitochondrial F1F0-ATP synthase γ-subunit (γ-sub) in I. nil (AB194067) was used as an internal control. The numerals in parenthesis indicate the cycles of PCR amplification. (E) Northern blot analysis of the genes encoding transcriptional regulators. Total RNAs (10 µg) prepared from flower limbs at 36 h before flower opening were separated and hybridized with appropriate cDNA probes from I. nil or I. purpurea, and γ-sub was used as an internal control. The intensity of each band was measured using the software NIH Image (version 1.62; W. Rasband, NIH, Bethesda, MD, USA); the expression of each gene is represented by the ratio between the wild-type and c-1 plants and that between the wild-type and ca plants after normalizing to the intensity of the internal control.

Fig. 3 Genes encoding transcriptional regulators for anthocyanin biosynthesis. Phylogenetic trees for three groups of transcriptional regulators, R2R3-MYB transcription factors (A), bHLH transcription regulators (B) and WDR proteins (C). The entire amino acid sequences were aligned with CLUSTALW, and the tree was constructed as described before (Fukada-Tanaka et al. 1997). The bootstrap values out of 100 retrials are indicated at each branch, and the scale shows the 0.1 amino acid substitution per site. The numerals given in parentheses indicate the ratio (%) of identical amino acid residues to the transcriptional regulators of I. nil represented in bold. The abbreviation shown in front of each protein indicates the plant species: In, Ipomoea nil; Ip, I. purpurea; It, I. tricolor; At, Arabidopsis thaliana; Am, Antirrhinum majus; Le, Lycopersicon esculentum; Pf, Perilla frutescens; Ph, Petunia hybrida; Vv, Vitis vinifera; Zm, Zea mays. R2R3-MYB transcription factors: AtPAP1 (AAG42001), AtPAP2 (AAG42002), AtTT2 (CAC40021), LeANT1 (AAQ55181), PhAN2 (AAF66727), VvMYBA1 (BAD18977), VvMYBA2 (BAD18978), ZmC1 (AAK09327), ZmPl (T03972). bHLH transcriptional regulators: IpbHLH2/IpIVS (BAD18982), ItbHLH2/ItIVS (BAD18984), AmDEL (AAA32663), AtEGL3 (Q9CAD0), AtGL3 (NP_680372), AtTT8 (Q9FT81), PfMYC-F3G1 (BAC56998), PfMYC-RP (BAA75513), PhAN1 (AAG25928), PhJAF13 (AAC39455), ZmIN1 (AAB03841), ZmLC (AAA33504), ZmB-Peru (CAA405449). WDR proteins: AtAN11 (AAC18912), AtTTG1 (CAB45372), PFWD (BAB58883), PhAN11 (AAC18914), ZmPAC1 (AAM76742), ZmMP1 (AAR01949). (D) Semi-quantitative RT–PCR analysis for the spatial expression of the regulatory genes in the wild-type TKS line. The constitutively expressed gene for the mitochondrial F1F0-ATP synthase γ-subunit (γ-sub) in I. nil (AB194067) was used as an internal control. The numerals in parenthesis indicate the cycles of PCR amplification. (E) Northern blot analysis of the genes encoding transcriptional regulators. Total RNAs (10 µg) prepared from flower limbs at 36 h before flower opening were separated and hybridized with appropriate cDNA probes from I. nil or I. purpurea, and γ-sub was used as an internal control. The intensity of each band was measured using the software NIH Image (version 1.62; W. Rasband, NIH, Bethesda, MD, USA); the expression of each gene is represented by the ratio between the wild-type and c-1 plants and that between the wild-type and ca plants after normalizing to the intensity of the internal control.

Fig. 4 Structure of the InMYB genes. (A) Multiple alignments of the deduced amino acid sequences of InMYB1, InMYB2 and InMYB3 with petunia An2 (PhAN2) and Arabidopsis PAP1 (AtPAP1). Asterisks (*) under the sequences and hyphens (-) indicate identical amino acids and gaps in the sequences for the alignment, respectively, and the highly conserved R2 and R3 DNA-binding domain is underlined. (B) Southern blot analysis of the InMYB1 gene. Genomic DNA (5 µg) of KK/ZSK-2 (C-1) or 78WWc-1 (c-1) was digested with BglII and HindIII, and InMYB1 cDNA was used as a probe. (C) Structure of the 12.9 kb HindIII fragment containing InMYB1. The gray and white boxes indicate the coding and the untranslated regions, respectively, and the horizontal arrow indicates the direction of the gene. The thick line indicates the 5.4 kb sequenced segment. The horizontal lines and the flanking small arrowheads under the map represent PCR fragments and primers, respectively. (D) Partial sequences of the wild-type and c-1 InMYB1 genes. The position of the 2 bp deletion is indicated by the bold characters AG in C-1 and the hyphens in c-1. Numbers above and below the sequences indicate the positions of nucleotides in the coding region and those of amino acid residues, respectively. The open and filled arrowheads in (A), (C) and (D) point to the positions of introns and the 2 bp deletion in the c-1 allele, respectively. (E) Structure of the 9.4 kb HindIII fragment containing InMYB3.

Fig. 4 Structure of the InMYB genes. (A) Multiple alignments of the deduced amino acid sequences of InMYB1, InMYB2 and InMYB3 with petunia An2 (PhAN2) and Arabidopsis PAP1 (AtPAP1). Asterisks (*) under the sequences and hyphens (-) indicate identical amino acids and gaps in the sequences for the alignment, respectively, and the highly conserved R2 and R3 DNA-binding domain is underlined. (B) Southern blot analysis of the InMYB1 gene. Genomic DNA (5 µg) of KK/ZSK-2 (C-1) or 78WWc-1 (c-1) was digested with BglII and HindIII, and InMYB1 cDNA was used as a probe. (C) Structure of the 12.9 kb HindIII fragment containing InMYB1. The gray and white boxes indicate the coding and the untranslated regions, respectively, and the horizontal arrow indicates the direction of the gene. The thick line indicates the 5.4 kb sequenced segment. The horizontal lines and the flanking small arrowheads under the map represent PCR fragments and primers, respectively. (D) Partial sequences of the wild-type and c-1 InMYB1 genes. The position of the 2 bp deletion is indicated by the bold characters AG in C-1 and the hyphens in c-1. Numbers above and below the sequences indicate the positions of nucleotides in the coding region and those of amino acid residues, respectively. The open and filled arrowheads in (A), (C) and (D) point to the positions of introns and the 2 bp deletion in the c-1 allele, respectively. (E) Structure of the 9.4 kb HindIII fragment containing InMYB3.

Fig. 5 Structure of the InWDR1 gene. (A) Multiple alignments of the deduced amino acid sequences of InWDR1 with petunia AN11 (PhAN11) and Arabidopsis TTG1 (AtTTG1). (B) Southern blot analysis of the InWDR1 gene. Genomic DNA (5 µg) of KK/ZSK-2 (Ca) and NS/W1ca1 (ca-1) was digested with ApaI, SalI and XbaI, and InWDR1 cDNA was used as a probe. (C) Structure of the 9.2 kb XbaI–XhoI fragment containing InWDR1. The structures of two putative genes flanking InWDR1 are also shown. Small dots above the physical map indicate the position of single nucleotide polymorphisms found between KK/ZSK-2 and TKS. (D) Partial sequences of the wild-type, ca-1 and ca-2 InWDR1 genes. The arrowheads in (A), (C) and (D) indicate the position of the 7 bp insertion sequences in the ca mutants. The sequences of the 7 bp insertions, GGAGTAC and TCCGTAC, in the ca mutants are shown in bold. The putative TAC target site duplications and a BsiWI site, CGTACG, are indicated by underlining and overlining, respectively. (E) PCR analysis of the 7 bp insertion in the ca-2 allele. To distinguish the Ca and ca-2 alleles, the 1.2 kb fragments amplified with primers InWDR1-F1 and InWDR1-R1 were digested with BsiWI. The 1.2 kb fragments from the ca-2 allele containing the BsiWI site produced 0.7 and 0.5 kb fragments, whereas those from the Ca allele remained intact. Other symbols are as in Fig. 4.

Fig. 5 Structure of the InWDR1 gene. (A) Multiple alignments of the deduced amino acid sequences of InWDR1 with petunia AN11 (PhAN11) and Arabidopsis TTG1 (AtTTG1). (B) Southern blot analysis of the InWDR1 gene. Genomic DNA (5 µg) of KK/ZSK-2 (Ca) and NS/W1ca1 (ca-1) was digested with ApaI, SalI and XbaI, and InWDR1 cDNA was used as a probe. (C) Structure of the 9.2 kb XbaI–XhoI fragment containing InWDR1. The structures of two putative genes flanking InWDR1 are also shown. Small dots above the physical map indicate the position of single nucleotide polymorphisms found between KK/ZSK-2 and TKS. (D) Partial sequences of the wild-type, ca-1 and ca-2 InWDR1 genes. The arrowheads in (A), (C) and (D) indicate the position of the 7 bp insertion sequences in the ca mutants. The sequences of the 7 bp insertions, GGAGTAC and TCCGTAC, in the ca mutants are shown in bold. The putative TAC target site duplications and a BsiWI site, CGTACG, are indicated by underlining and overlining, respectively. (E) PCR analysis of the 7 bp insertion in the ca-2 allele. To distinguish the Ca and ca-2 alleles, the 1.2 kb fragments amplified with primers InWDR1-F1 and InWDR1-R1 were digested with BsiWI. The 1.2 kb fragments from the ca-2 allele containing the BsiWI site produced 0.7 and 0.5 kb fragments, whereas those from the Ca allele remained intact. Other symbols are as in Fig. 4.

Fig. 6 Temporal expression of the genes for anthocyanin pigmentation. (A) Flower development in the wild-type line TKS. The numerals below the photographs indicate the time (h) before flower opening (BFO). In TKS, flower pigmentation occurs by 60 h BFO. Bar = 10 mm. (B) Northern blot analysis of the genes for flower coloration. Total RNAs (10 µg) prepared at the indicated time were hybridized with entire cDNA sequences as probes except for CHS-D and CHS-E, for which PCR fragments containing their unique 3′-UTRs were used as gene-specific probes. The ethidium bromide-stained rRNA bands are shown as loading controls. (C) Quantitative analysis of the gene expression for anthocyanin pigmentation at 36 h BFO. Quantitative analysis was performed as described in Fig. 3E. RNA samples for CHS-E were prepared from flower tubes; all others were from flower limbs.

Fig. 6 Temporal expression of the genes for anthocyanin pigmentation. (A) Flower development in the wild-type line TKS. The numerals below the photographs indicate the time (h) before flower opening (BFO). In TKS, flower pigmentation occurs by 60 h BFO. Bar = 10 mm. (B) Northern blot analysis of the genes for flower coloration. Total RNAs (10 µg) prepared at the indicated time were hybridized with entire cDNA sequences as probes except for CHS-D and CHS-E, for which PCR fragments containing their unique 3′-UTRs were used as gene-specific probes. The ethidium bromide-stained rRNA bands are shown as loading controls. (C) Quantitative analysis of the gene expression for anthocyanin pigmentation at 36 h BFO. Quantitative analysis was performed as described in Fig. 3E. RNA samples for CHS-E were prepared from flower tubes; all others were from flower limbs.

Table 1

Transcriptional regulator genes of I. nil and their homologs

Protein type Gene Homologous gene     
  Petunia Arabidopsis Perilla Snapdragon Maize 
R2R3-MYB InMYB1/C-1 (269), InMYB2 (261), InMYB3 (251) AN2 PAP1, PAP2 –  –  – 
 —  – TT2  –  – C1, Pl 
bHLH InbHLH1/InDEL (625), InbHLH3 (607) JAF13 GL3, EGL3 Myc-rp Delila B-Peru, Lc 
 InbHLH2/InIVS (669) AN1 TT8 Myc-F3G1  – In1 
WDR InWDR1/Ca (343) AN11 TTG1 PFWD  – PAC1 
 InWDR2 (346)  – AtAN11   – MP1 
Protein type Gene Homologous gene     
  Petunia Arabidopsis Perilla Snapdragon Maize 
R2R3-MYB InMYB1/C-1 (269), InMYB2 (261), InMYB3 (251) AN2 PAP1, PAP2 –  –  – 
 —  – TT2  –  – C1, Pl 
bHLH InbHLH1/InDEL (625), InbHLH3 (607) JAF13 GL3, EGL3 Myc-rp Delila B-Peru, Lc 
 InbHLH2/InIVS (669) AN1 TT8 Myc-F3G1  – In1 
WDR InWDR1/Ca (343) AN11 TTG1 PFWD  – PAC1 
 InWDR2 (346)  – AtAN11   – MP1 

Numbers given in parentheses indicate the number of predicted amino acid residues in each I. nil gene. Hyphens (–) show that the corresponding gene has not been characterized in the species. For the transcriptional regulators encoded by the genes described, see Fig. 3.

Table 2

I. nil lines used in this study

Genotype Line 
Wild-type KK/ZSK-2, TKS 
C-1 Q173  
c-1  78WWc-1, YM/W1c-1, YM/W2c-1, YM/W3c-1, Q410, Q421, Q431, Q501, Q681 
Ca Q65 
ca-1 NS/W1ca1, NS/W2ca3 
ca-2 Q599, Q646, Q871, Q905, Q923, Q931 
Genotype Line 
Wild-type KK/ZSK-2, TKS 
C-1 Q173  
c-1  78WWc-1, YM/W1c-1, YM/W2c-1, YM/W3c-1, Q410, Q421, Q431, Q501, Q681 
Ca Q65 
ca-1 NS/W1ca1, NS/W2ca3 
ca-2 Q599, Q646, Q871, Q905, Q923, Q931 

All lines, including KK/ZSK-2 (Inagaki et al. 1994), TKS (Kawasaki and Nitasaka 2004) and Q173 (Hoshino et al. 2003), were from our collections, and 78WWc-1 (Hoshino et al. 1997), NS/W1ca-1 (Saito et al. 1994) and NS/W2ca-3 (Saito et al. 1994) are also designated as Q260, Q263 and Q264, respectively. Phenotypes of the above lines can be found in the morning glory web site at Kyusyu University (http://mg.biology.kyushu-u.ac.jp/index.html). YM/W1c-1 and YM/W2c-1 also carry s and we alleles for the Star and Weeping genes, respectively (Imai 1938a, Hagiwara 1956, Kitazawa et al. 2005).

Table 3

Primers used for PCR and/or RT–PCR amplifications

Gene Primer Sequence 
InMYB1 InMYB1-F0 5′-ATGGTTAATTCTTCTGCAAGG-3′ 
 InMYB1-F1 a 5′-CGTAAGAATTTACGTCAGCCTGCC-3′ 
 InMYB1-F2 5′-GAAATTACATTCTCCTCCGTGGTCC-3′ 
 InMYB1-F3 5′-CACTCGGGTTCAATATCTTGTC-3′ 
 InMYB1-R0 5′-GTCACATCAATCGGAAAGTC-3′ 
 InMYB1–R1 a 5′-CATAATATCCACCGATGTGGATGCT-3′ 
 InMYB1-R2 5′-AAATCTTGTCTGCCACGAGTC-3′ 
 InMYB1-R3 5′-ACAAAGATTTGAACCCTTGACCTCAC-3′ 
InMYB2 InMYB2-F1 a 5′-ATGGTTAATTCATCATCTGCATG-3′ 
 InMYB2-R1 a 5′-CGGTAACGTGTGTCCTTGGAT-3′ 
InMYB3 InMYB3-F1 a 5′-GTATAACAAAGTTTACGATACCCTGCC-3′ 
 InMYB3-R1 a 5′-ATCTAAAGATTCCATGTAGACTTTATTAGC-3′ 
InbHLH1 InbHLH1-F1 a 5′-AGCCACAACCAGCTACCTGCACC-3′ 
 InbHLH1-R1 a 5′-AGCTTGCACTGTTTTCCTCG-3′ 
InbHLH2 InbHLH2-F1 a 5′-GTCAAGCAGTTGCGCCGGAG-3′ 
 InbHLH2-R1 a 5′-CTGAGGAATTATACTATGAATTGC-3′ 
InbHLH3 InbHLH3-F1 a 5′-ATGCCAAGAACCAGAACTCGT-3′ 
 InbHLH3-R1 a 5′-CCTGCACGATTCTCATCAACA-3′ 
InWDR1 InWDR1-F1 a 5′-CGGCGATTCAACGCTCCGAG-3′ 
 InWDR1-R1 a 5′-AGATAACCAATTACAGACACATACC-3′ 
InWDR2 InWDR2-F1 a 5′-TAGTAGTGAGTGAGCAGGTGC-3′ 
 InWDR2-R1 a 5′-AGTGACACACTCACAACTACAG-3′ 
CHS-D CHS-D-F2 b 5′-TGAGCGAGTACGGGAACATGTC-3′ 
 CHS-D-R1 b 5′-GGCCGGACTTAAGCTGGGACG-3′ 
F3H F3H-F1 b 5′-TGCGGACCATCAAGCGGTGGTG-3′ 
 F3H-R2 b 5′-CCGCCTTTATCATTTGCTGCTCC-3′ 
DFR-B DFR-B-F1 b 5′-CTAGAGGACATGTACAGAGGAGC-3′ 
 DFR-B-R2 b 5′-CAATGGCAGTGGGTTCTTCTTCC-3′ 
3GT 3GT-F1 b 5′-GAAGGCCTTGAATGTTGTTCTGG-3′ 
 3GT-R1 b 5′-CTCTCCTTTTGGTGCATGCATGC-3′ 
γ-Subunit JMFS029C1-3F a 5′-ATCAAGCCGTTAAGAGCAAACCC-3′ 
 JMFS029C1-4R a 5′-TAGTCTGAATAGCTCGAAGCTT-3′ 
 ATPase-F3 b 5′-GGATGTCTGCCATGGACAGCTC-3′ 
 ATPase-R1 b 5′-TCAGCCCTCCAATGCTGATGCTC-3′ 
Gene Primer Sequence 
InMYB1 InMYB1-F0 5′-ATGGTTAATTCTTCTGCAAGG-3′ 
 InMYB1-F1 a 5′-CGTAAGAATTTACGTCAGCCTGCC-3′ 
 InMYB1-F2 5′-GAAATTACATTCTCCTCCGTGGTCC-3′ 
 InMYB1-F3 5′-CACTCGGGTTCAATATCTTGTC-3′ 
 InMYB1-R0 5′-GTCACATCAATCGGAAAGTC-3′ 
 InMYB1–R1 a 5′-CATAATATCCACCGATGTGGATGCT-3′ 
 InMYB1-R2 5′-AAATCTTGTCTGCCACGAGTC-3′ 
 InMYB1-R3 5′-ACAAAGATTTGAACCCTTGACCTCAC-3′ 
InMYB2 InMYB2-F1 a 5′-ATGGTTAATTCATCATCTGCATG-3′ 
 InMYB2-R1 a 5′-CGGTAACGTGTGTCCTTGGAT-3′ 
InMYB3 InMYB3-F1 a 5′-GTATAACAAAGTTTACGATACCCTGCC-3′ 
 InMYB3-R1 a 5′-ATCTAAAGATTCCATGTAGACTTTATTAGC-3′ 
InbHLH1 InbHLH1-F1 a 5′-AGCCACAACCAGCTACCTGCACC-3′ 
 InbHLH1-R1 a 5′-AGCTTGCACTGTTTTCCTCG-3′ 
InbHLH2 InbHLH2-F1 a 5′-GTCAAGCAGTTGCGCCGGAG-3′ 
 InbHLH2-R1 a 5′-CTGAGGAATTATACTATGAATTGC-3′ 
InbHLH3 InbHLH3-F1 a 5′-ATGCCAAGAACCAGAACTCGT-3′ 
 InbHLH3-R1 a 5′-CCTGCACGATTCTCATCAACA-3′ 
InWDR1 InWDR1-F1 a 5′-CGGCGATTCAACGCTCCGAG-3′ 
 InWDR1-R1 a 5′-AGATAACCAATTACAGACACATACC-3′ 
InWDR2 InWDR2-F1 a 5′-TAGTAGTGAGTGAGCAGGTGC-3′ 
 InWDR2-R1 a 5′-AGTGACACACTCACAACTACAG-3′ 
CHS-D CHS-D-F2 b 5′-TGAGCGAGTACGGGAACATGTC-3′ 
 CHS-D-R1 b 5′-GGCCGGACTTAAGCTGGGACG-3′ 
F3H F3H-F1 b 5′-TGCGGACCATCAAGCGGTGGTG-3′ 
 F3H-R2 b 5′-CCGCCTTTATCATTTGCTGCTCC-3′ 
DFR-B DFR-B-F1 b 5′-CTAGAGGACATGTACAGAGGAGC-3′ 
 DFR-B-R2 b 5′-CAATGGCAGTGGGTTCTTCTTCC-3′ 
3GT 3GT-F1 b 5′-GAAGGCCTTGAATGTTGTTCTGG-3′ 
 3GT-R1 b 5′-CTCTCCTTTTGGTGCATGCATGC-3′ 
γ-Subunit JMFS029C1-3F a 5′-ATCAAGCCGTTAAGAGCAAACCC-3′ 
 JMFS029C1-4R a 5′-TAGTCTGAATAGCTCGAAGCTT-3′ 
 ATPase-F3 b 5′-GGATGTCTGCCATGGACAGCTC-3′ 
 ATPase-R1 b 5′-TCAGCCCTCCAATGCTGATGCTC-3′ 

a Primers used for RT–PCR analysis in Fig. 3D.

b Primers used for real-time RT–PCR analysis.

References

Abe, Y., Hoshino, A. and Iida, S. (
1997
) Appearance of flower variegation in the mutable speckled line of the Japanese morning glory is controlled by two genetic elements.
Genes Genet. Syst.
 
72
:
57
–62.
Alfenito, M.R., Souer, E., Goodman, C.D., Buell, R., Mol, J., Koes, R. and Walbot, V. (
1998
) Functional complementation of anthocyanin sequestration in the vacuole by widely divergent glutathione S-transferases.
Plant Cell
 
10
:
1135
–1149.
Belostotsky, D.A. and Rose, A.B. (
2005
) Plant gene expression in the age of systems biology: integrating transcriptional and post-transcriptional events.
Trends Plant Sci.
 
10
:
347
–353.
Borevitz, J.O., Xia, Y., Blount, J., Dixon, R.A. and Lamb, C. (
2000
) Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis.
Plant Cell
 
12
:
2383
–2393.
Broun, P. (
2005
) Transcriptional control of flavonoid biosynthesis: a complex network of conserved regulators involved in multiple aspects of differentiation in Arabidopsis.
Curr. Opin. Plant Biol.
 
8
:
272
–279.
Carey, C.C., Strahle, J.T., Selinger, D.A. and Chandler, V.L. (
2004
) Mutations in the pale aleurone color1 regulatory gene of the Zea mays anthocyanin pathway have distinct phenotypes relative to the functionally similar TRANSPARENT TESTA GLABRA1 gene in Arabidopsis thaliana.
Plant Cell
 
16
:
450
–464.
Chandler, V.L., Radicella, J.P., Robbins, T.P., Chen, J. and Turks, D. (
1989
) Two regulatory genes of the maize anthocyanin pathway are homologous: isolation of B utilizing R genomic sequences.
Plant Cell
 
1
:
1175
–1183.
Chang, S.M., Lu, Y. and Rausher, M.D. (
2005
) Neutral evolution of the nonbinding region of the anthocyanin regulatory gene Ipmyb1 in Ipomoea.
Genetics
 
170
:
1967
–1978.
Clegg, M.T. and Durbin, M.L. (
2003
) Tracing floral adaptations from ecology to molecules.
Nat. Rev. Genet.
 
4
:
206
–215.
Cone, K.C., Cocciolone, S.M., Burr, F.A. and Burr, B. (
1993
) Maize anthocyanin regulatory gene pl is a duplicate of c1 that functions in the plant.
Plant Cell
 
5
:
1795
–1805.
de Vetten, N., Quattrocchio, F., Mol, J. and Koes, R. (
1997
) The an11 locus controlling flower pigmentation in petunia encodes a novel WD-repeat protein conserved in yeast, plants, and animals.
Genes Dev.
 
11
:
1422
–1434.
Debeaujon, I., Nesi, N., Perez, P., Devic, M., Grandjean, O., Caboche, M. and Lepiniec, L. (
2003
) Proanthocyanidin-accumulating cells in Arabidopsis testa: regulation of differentiation and role in seed development.
Plant Cell
 
15
:
2514
–2531.
Durbin, M.L., McCaig, B. and Clegg, M.T. (
2000
) Molecular evolution of the chalcone synthase multigene family in the morning glory genome.
Plant Mol. Biol.
 
42
:
79
–92.
Fukada-Tanaka, S., Hoshino, A., Hisatomi, Y., Habu, Y., Hasebe, M. and Iida, S. (
1997
) Identification of new chalcone synthase genes for flower pigmentation in the Japanese and common morning glories.
Plant Cell Physiol.
 
38
:
754
–758.
Fukada-Tanaka, S., Inagaki, Y., Yamaguchi, T., Saito, N. and Iida, S. (
2000
) Colour-enhancing protein in blue petals.
Nature
 
407
:
581
.
Grotewold, E. (
2005
) Plant metabolic diversity: a regulatory perspective.
Trends Plant Sci.
 
10
:
57
–62.
Hagiwara, T. (
1929
) On the role of the factors C and R in the production of the flower colours in Pharbitis nil.
Bot. Mag.
 
43
:
643
–656.
Hagiwara, T. (
1954
) Recent genetics on the flower-colour of Japanese morning glory, with reference to biochemical studies.
Bull. Res. Coll. Agric. Vet. Sci. Nihon Univ.
 
3
:
1
–15.
Hagiwara, T. (
1956
) Genes and chromosome maps in the Japanese morning glory.
Bull. Res. Coll. Agric. Vet. Sci. Nihon Univ.
 
5
:
35
–56.
Harmer, S.L., Hogenesch, J.B., Straume, M., Chang, H.S., Han, B., Zhu, T., Wang, X., Kreps, J.A. and Kay, S.A. (
2000
) Orchestrated transcription of key pathways in Arabidopsis by the circadian clock.
Science
 
290
:
2110
–2113.
Heim, M.A., Jakoby, M., Werber, M., Martin, C., Weisshaar, B. and Bailey, P.C. (
2003
) The basic helix–loop–helix transcription factor family in plants: a genome-wide study of protein structure and functional diversity.
Mol. Biol. Evol.
 
20
:
735
–747.
Hiraga, G. (
1763
) Butsurui-Hinshitsu. Shouraikan, Edo. (In Japanese.)
Hisatomi, Y., Hanada, K. and Iida, S. (
1997
) The retrotransposon RTip1 is integrated into a novel type of minisatellite, MiniSip1, in the genome of the common morning glory and carries another new type of minisatellite, MiniSip2.
Theor. Appl. Genet.
 
95
:
1049
–1056.
Hoshino, A., Abe, Y., Saito, N., Inagaki, Y. and Iida, S. (
1997
) The gene encoding flavanone 3-hydroxylase is expressed normally in the pale yellow flowers of the Japanese morning glory carrying the speckled mutation which produce neither flavonol nor anthocyanin but accumulate chalcone, aurone and flavanone.
Plant Cell Physiol.
 
38
:
970
–974.
Hoshino, A., Johzuka-Hisatomi, Y. and Iida, S. (
2001
) Gene duplication and mobile genetic elements in the morning glories.
Gene
 
265
:
1
–10.
Hoshino, A., Morita, Y., Choi, J.D., Saito, N., Toki, K., Tanaka, Y. and Iida, S. (
2003
) Spontaneous mutations of the flavonoid 3′-hydroxylase gene conferring reddish flowers in the three morning glory species.
Plant Cell Physiol.
 
44
:
990
–1001.
Iida, S., Hoshino, A., Johzuka-Hisatomi, Y., Habu, Y. and Inagaki, Y. (
1999
) Floricultural traits and transposable elements in the Japanese and common morning glories.
Ann. N. Y. Acad. Sci.
 
870
:
265
–274.
Iida, S., Morita, Y., Choi, J.D., Park, K.I. and Hoshino, A. (
2004
) Genetics and epigenetics in flower pigmentation associated with transposable elements in morning glories.
Adv. Biophys.
 
38
:
141
–159.
Imai, Y. (
1938
) The genes of the Japanese morning glory.
Jpn. J. Genet.
 
14
:
24
–33.
Imai, Y. (
1938
) Genetic literature of the Japanese morning glory.
Jpn. J. Genet.
 
14
:
91
–96.
Inagaki, Y., Hisatomi, Y. and Iida, S. (
1996
) Somatic mutations caused by excision of the transposable element, Tpn1, from the DFR gene for pigmentation in sub-epidermal layer of periclinally chimeric flowers of Japanese morning glory and their germinal transmission to their progeny.
Theor. Appl. Genet.
 
92
:
499
–504.
Inagaki, Y., Hisatomi, Y., Suzuki, T., Kasahara, K. and Iida, S. (
1994
) Isolation of a Suppressor-Mutator/Enhancer-like transposable element, Tpn1, from Japanese morning glory bearing variegated flowers.
Plant Cell
 
6
:
375
–383.
Johzuka-Hisatomi, Y., Hoshino, A., Mori, T., Habu, Y. and Iida, S. (
1999
) Characterization of the chalcone synthase genes expressed in flowers of the common and Japanese morning glories.
Genes Genet. Syst.
 
74
:
141
–147.
Kawasaki, S. and Nitasaka, E. (
2004
) Characterization of Tpn1 family in the Japanese morning glory: En/Spm-related transposable elements capturing host genes.
Plant Cell Physiol.
 
45
:
933
–944.
Kitazawa, D., Hatakeda, Y., Kamada, M., Fujii, N., Miyazawa, Y., et al. (
2005
) Shoot circumnutation and winding movements require gravisensing cells.
Proc. Natl Acad. Sci. USA
 
102
:
18742
–18747.
Koes, R., Verweij, W. and Quattrocchio, F. (
2005
) Flavonoids: a colorful model for the regulation and evolution of biochemical pathways.
Trends Plant Sci.
 
10
:
236
–242.
Kondo, T., Kawai, T., Tamura, H. and Goto, T. (
1987
) Structure determination of Heavenly blue anthocyanin, a complex monomeric anthocyanin from the morning glory Ipomoea tricolor, by means of the negative NOE method.
Tetrahedron Lett.
 
28
:
2273
–2276.
Lu, T.S., Saito, N., Yokoi, M., Shigihara, A. and Honoda, T. (
1992
) Acylated pelargonidin glycosides in the red-purple flowers of Pharbitis nil.
Phytochemistry
 
31
:
289
–295.
Lu, T.S., Saito, N., Yokoi, M., Shigihara, A. and Honoda, T. (
1992
) Acylated peonidin glycosides in the violet-blue cultivars of Pharbitis nil.
Phytochemistry
 
31
:
659
–663.
Ludwig, S.R. and Wessler, S.R. (
1990
) Maize R gene family: tissue-specific helix–loop–helix proteins.
Cell
 
62
:
849
–851.
Martin, C. and Gerats, T. (
1993
) Control of pigment biosynthesis genes during petal development.
Plant Cell
 
5
:
1253
–1264.
Mehrtens, F., Kranz, H., Bednarek, P. and Weisshaar, B. (
2005
) The Arabidopsis transcription factor MYB12 is a flavonol-specific regulator of phenylpropanoid biosynthesis.
Plant Physiol.
 
138
:
1083
–1096.
Mol, J., Grotewold, E. and Koes, R. (
1998
) How genes paint flowers and seeds.
Trends Plant Sci.
 
3
:
212
–217.
Morita, Y., Hoshino, A., Kikuchi, Y., Okuhara, H., Ono, E., et al. (
2005
) Japanese morning glory dusky mutants displaying reddish-brown or purplish-grey flowers are deficient in a novel glycosylation enzyme for anthocyanin biosynthesis, UDP-glucose:anthocyanidin 3-O-glucoside-2′′-O-glucosyltransferase, due to 4-bp insertions in the gene.
Plant J.
 
42
:
353
–363.
Nesi, N., Debeaujon, I., Jond, C., Pelletier, G., Caboche, M. and Lepiniec, L. (
2000
) The TT8 gene encodes a basic helix–loop–helix domain protein required for expression of DFR and BAN genes in Arabidopsis siliques.
Plant Cell
 
12
:
1863
–1878.
Nesi, N., Jond, C., Debeaujon, I., Caboche, M. and Lepiniec, L. (
2001
) The Arabidopsis TT2 gene encodes an R2R3 MYB domain protein that acts as a key determinant for proanthocyanidin accumulation in developing seed.
Plant Cell
 
13
:
2099
–2114.
Ohnishi, M., Fukada-Tanaka, S., Hoshino, A., Takada, J., Inagaki, Y. and Iida, S. (
2005
) Characterization of a novel Na+/H+ antiporter gene InNHX2 and comparison of InNHX2 with InNHX1, which is responsible for blue flower coloration by increasing the vacuolar pH in the Japanese morning glory.
Plant Cell Physiol.
 
46
:
259
–267.
Park, K.I., Choi, J.D., Hoshino, A., Morita, Y. and Iida, S. (
2004
) An intragenic tandem duplication in a transcriptional regulatory gene for anthocyanin biosynthesis confers pale-colored flowers and seeds with fine spots in Ipomoea tricolor.
Plant J.
 
38
:
840
–849.
Payne, C.T., Zhang, F. and Lloyd, A.M. (
2000
) GL3 encodes a bHLH protein that regulates trichome development in Arabidopsis through interaction with GL1 and TTG1.
Genetics
 
156
:
1349
–1362.
Quattrocchio, F., Wing, J.F., van der Woude, K., Mol, J.N.M. and Koes, R. (
1998
) Analysis of bHLH and MYB domain proteins: species-specific regulatory differences are caused by divergent evolution of target anthocyanin genes.
Plant J.
 
13
:
475
–488.
Quattrocchio, F., Wing, J., van der Woude, K., Souer, E., de Vetten, N., Mol, J. and Koes, R. (
1999
) Molecular analysis of the anthocyanin2 gene of petunia and its role in the evolution of flower color.
Plant Cell
 
11
:
1433
–1444.
Ramsay, N.A. and Glover, B.J. (
2005
) MYB–bHLH–WD40 protein complex and the evolution of cellular diversity.
Trends Plant Sci.
 
10
:
63
–70.
Saito, N., Cheng, J., Ichimura, M., Yokoi, M., Abe, Y. and Honda, T. (
1994
) Flavonoids in the acyanic flowers of Pharbitis nil.
Phytochemistry
 
35
:
687
–691.
Schwinn, K.E. and Davies, K.M. (
2004
) Flavonoids. In Plant Pigments and their Manipulation. Edited by Davies, K. pp.
92
–149. Blackwell Publishing Ltd, Oxford.
Shirley, B.W., Kubasek, W.L., Storz, G., Bruggemann, E., Koornneef, M., Ausubel, F.M. and Goodman, H.M. (
1995
) Analysis of Arabidopsis mutants deficient in flavonoid biosynthesis.
Plant J.
 
8
:
659
–671.
Spelt, C., Quattrocchio, F., Mol, J.N.M. and Koes, R. (
2000
) anthocyanin1 of petunia encodes a basic helix–loop–helix protein that directly activates transcription of structural anthocyanin genes.
Plant Cell
 
12
:
1619
–1631.
Spelt, C., Quattrocchio, F., Mol, J. and Koes, R. (
2002
) ANTHOCYANIN1 of petunia controls pigment synthesis, vacuolar pH, and seed coat development by genetically distinct mechanisms.
Plant Cell
 
14
:
2121
–2135.
Springob, K., Nakajima, J., Yamazaki, M. and Saito, K. (
2003
) Recent advances in the biosynthesis and accumulation of anthocyanins.
Nat. Prod. Rep.
 
20
:
288
–303.
Stracke, R., Werber, M. and Weisshaar, B. (
2001
) The R2R3-MYB gene family in Arabidopsis thaliana.
Curr. Opin. Plant Biol.
 
4
:
447
–456.
Tohge, T., Nishiyama, Y., Yokota Hirai, M., Yano, M., Nakajima, J., et al. (
2005
) Functional genomics by integrated analysis of metabolome and transcriptome of Arabidopsis plants over-expressing an MYB transcription factor.
Plant J.
 
42
:
218
–235.
Walker, A.R., Davison, P.A., Bolognesi-Winfield, A.C., James, C.M., Srinivasan, N., Blundell, T.L., Esch, J.J., Marks, M.D. and Gray, J.C. (
1999
) The TRANSPARENT TESTA GLABRA1 locus, which regulates trichome differentiation and anthocyanin biosynthesis in Arabidopsis, encodes a WD40 repeat protein.
Plant Cell
 
11
:
1337
–1349.
Yamaguchi, T., Fukada-Tanaka, S., Inagaki, Y., Saito, N., Yonekura-Sakakibara, K., Tanaka, Y., Kusumi, T. and Iida, S. (
2001
) Genes encoding the vacuolar Na+/H+ exchanger and flower coloration.
Plant Cell Physiol.
 
42
:
451
–461.
Yamazaki, M., Makita, Y., Springob, K. and Saito, K. (
2003
) Regulatory mechanisms for anthocyanin biosynthesis in chemotypes of Perilla frutescens var. crispa.
Biochem. Eng. J.
 
14
:
191
–197.
Zhang, F., Gonzalez, A., Zhao, M., Payne, C.T. and Lloyd, A. (
2003
) A network of redundant bHLH proteins functions in all TTG1-dependent pathways of Arabidopsis.
Development
 
130
:
4859
–4869.