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Plant Physiol. Mar 2007; 143(3): 1252–1268.
PMCID: PMC1820918

Isolation of a Regulatory Gene of Anthocyanin Biosynthesis in Tuberous Roots of Purple-Fleshed Sweet Potato[OA]

Abstract

Many transcriptional factors harboring the R2R3-MYB domain, basic helix-loop-helix domain, or WD40 repeats have been identified in various plant species as regulators of flavonoid biosynthesis in flowers, seeds, and fruits. However, the regulatory elements of flavonoid biosynthesis in underground organs have not yet been elucidated. We isolated the novel MYB genes IbMYB1 and IbMYB2s from purple-fleshed sweet potato (Ipomoea batatas L. Lam. cv Ayamurasaki). IbMYB1 was predominantly expressed in the purple flesh of tuberous roots but was not detected (or only scarcely) in other anthocyanin-containing tissues such as nontuberous roots, stems, leaves, or flowers. IbMYB1 was also expressed in the tuberous roots of other purple-fleshed cultivars but not in those of orange-, yellow-, or white-fleshed cultivars. Although the orange- or yellow-fleshed cultivars contained anthocyanins in the skins of their tuberous roots, we could not detect IbMYB1 transcripts in these tissues. These results suggest that IbMYB1 controls anthocyanin biosynthesis specifically in the flesh of tuberous roots. The results of transient and stable transformation experiments indicated that expression of IbMYB1 alone was sufficient for induction of all structural anthocyanin genes and anthocyanin accumulation in the flesh of tuberous roots, as well as in heterologous tissues or heterologous plant species.

Sweet potato (Ipomoea batatas L. Lam.) ranks as the world's seventh most important crop after wheat (Triticum aestivum), rice (Oryza sativa), maize (Zea mays), potato (Solanum tuberosum), barley (Hordeum vulgare), and cassava (Manihot esculenta Crantz.). Most sweet potato cultivars, like the other major crops that are grown and consumed, have white or yellow flesh. However, there are also some sweet potato cultivars with orange flesh that contains carotenoids or with purple flesh that contains anthocyanins.

Anthocyanins and related compounds such as proanthocyanidins protect the leaves from various stressors such as strong light and heavy metals (for review, see Gould, 2004), play a role in entomophily in flowers, and deter pathogens and predators of seeds (Shirley, 1998). The role of anthocyanins in underground organs (such as tuberous roots) that grow under dark conditions is not clear, but it is conceivable that it is similar to that in seeds (e.g. protection from pathogens and predators and improvement of preservation and thus reproductive advantage), because tubers, like seeds, play a reproductive role.

The reason why most sweet potato cultivars have white or yellow flesh may be because consumers prefer pale foods, as is the case with other staples, and because the anthocyanins in the tuber are an obstacle to industrial starch production. Nevertheless, attention is now being focused on the multiple physiological functions of the anthocyanins derived from purple-fleshed sweet potatoes, such as their strong antioxidative activity (Kano et al., 2005), antimutagenicity (Yoshimoto et al., 1999b), antihyperglycemic effect (Matsui et al., 2002), and hepatoprotective and antihypertensive effects (Suda et al., 2003). New purple-fleshed cultivars have been developed recently. The content of anthocyanins in indigenous purple-fleshed cultivars was low, and the development of a new purple-fleshed cultivar, Ayamurasaki (Yamakawa et al., 1997), enabled edible dye production from sweet potato. Trials have been done on the efficient and stable production of anthocyanins from tissue or cell culture of various plant species, including sweet potato. For example, optimum conditions for the culture of anthocyanin-producing sweet potato cells (Nishimaki and Nozue, 1985; Nozue et al., 1987) or Ayamurasaki hairy roots (Nishiyama and Yamakawa, 2004) have been investigated. However, low anthocyanin content and the light dependency of anthocyanin production have been noted as problems. Konczak-Islam et al. (2000) obtained a cell line that had high anthocyanin content and was derived from the tuberous roots of Ayamurasaki. This line showed light-independent anthocyanin production and may prove useful in the improvement of anthocyanin productivity, although the regulatory elements of anthocyanin biosynthesis are still unknown.

The mechanisms regulating flavonoid pigments have been studied in various plant species. Detailed studies have been done in maize, Arabidopsis (Arabidopsis thaliana), and petunia (Petunia hybrida) by numerous mutation analyses, and many regulatory genes have been identified that control the transcription of flavonoid structural genes. Transcriptional factors with MYB or basic helix-loop-helix (bHLH) domains and a WD40 protein are commonly identified in different species (for review, see Broun, 2004; Koes et al., 2005). Although a model has been proposed that these regulators interact with each other and make transcription complexes with the promoters of the structural genes, the partnership seems to be complex. For example, MYB domain C1 protein regulating the anthocyanin pathway in maize needs a bHLH partner (B/R) to activate the flavonoid structural gene dihydroflavonol reductase (DFR) promoter, whereas MYB domain P protein regulating the phlobaphene pathway can activate the promoter without a bHLH partner (Sainz et al., 1997). Moreover, other regulatory genes, such as TTG2, a WRKY transcription factor (Johnson et al., 2002), TT1, a zinc finger protein (Sagasser et al., 2002), TT16, a MADS domain protein (Nesi et al., 2002), and ANL2, a homeodomain (HD) protein (Kubo et al., 1999), have been reported. Some of these genes are involved in various aspects of plant development besides flavonoid biosynthesis, including development of the trichome and root hair (TTG2, WRKY; TTG1, WD40; GL3 and EGL3, bHLH; Zhang et al., 2003), root (ANL2, HD), and seed (TT1, zinc finger; TT16, MADS; AN1, bHLH; AN11, WD40; Spelt et al., 2002), acidification of vacuoles (AN1 and AN11), or mucilage formation (TTG2, TTG1, GL3, and EGL3).

The genes responsible for the color differences among cultivars have been revealed. One amino acid alternation in bHLH protein created the green formae of perilla from red formae (Gong et al., 1999), and a retrotransposon-induced mutation on the promoter of the MYB gene created white-skinned grape (Vitis vinifera) cultivars from red-skinned ones (Kobayashi et al., 2004). In the case of rice, red or brown grain was selected out by ancient breeders. Two loci, Rc and Rd, that are responsible for red seed color were identified by classical genetic analysis (Kato and Ishikawa, 1921), and recently they were revealed to encode the bHLH and DFR genes, respectively (Sweeney et al., 2006; Furukawa et al., 2007).

As mentioned above, numerous reports have described the genes regulating flavonoid pigmentation in flowers, leaves, seeds, and fruits but not in underground organs such as tuberous roots. In sweet potato, no MYB, bHLH, or WD40 protein has been reported thus far; instead, a MADS-box gene, IbMADS10, was recently isolated and suggested to be involved in anthocyanin pigmentation (Lalusin et al., 2006), although its involvement in the underground organ is unclear.

We report here the isolation of a new R2R3-type MYB gene, IbMYB1, from a purple-fleshed sweet potato cDNA library and its predominant expression in the tuberous roots of purple-fleshed cultivars. Transient and stable forced expression of the IbMYB1 gene resulted in intense anthocyanin pigmentation in various tissues. These results suggest that the IbMYB1 gene is responsible for purple pigmentation in the flesh of tuberous roots of sweet potato.

RESULTS

Isolation of cDNA Clones with Tissue- and Cultivar-Specific Expression

We constructed cDNA libraries from the tuberous roots of the purple-fleshed sweet potato cultivar Ayamurasaki and the yellow-fleshed sweet potato cultivar Kokei-14, and we determined the sequences of 3,783 randomly isolated cDNA clones from Ayamurasaki and 2,804 from Kokei-14. The cDNA clones were clustered, and 25 sequence groups were selected according to their frequency of appearance in the libraries and/or specificity to the Ayamurasaki library. The 25 selected groups were subjected to reverse transcription (RT)-PCR analysis, and the results indicated that five groups, IT1, IT4, IT4785, IT666, and IT4097, were expressed in a tissue-specific manner (Fig. 1). Whereas IT948 was identical to the Actin gene and was expressed equally in all tissues tested, IT1 and IT4 were expressed predominantly in tuberous-root tissue and IT4097 was expressed in the tuberous roots and the calli. IT4785 and IT666 were expressed in a cultivar-specific manner; that is, they were expressed only in Ayamurasaki, not in Kokei-14. IT4785 was homologous to the flavonoid 3′-hydroxylase of Ipomoea tricolor, and IT666 showed partial identity to R2R3-type MYB genes. We therefore acquired a novel MYB-like gene, IT666, with tissue- and cultivar-specific expression. As there have been no reports of MYB-like genes in sweet potato, to our knowledge, we named this IT666 gene IbMYB1. The IbMYB1 cDNA was predicted to have an open reading frame (ORF) of 750 bp, including 312 bp of the R2R3-type MYB-like region in the first half; the latter half had no marked similarity (Fig. 2A).

Figure 1.
Genomic PCR and RT-PCR performed on five clones isolated from a cDNA library derived from sweet potato tuberous roots revealed tissue- and/or cultivar-specific expression. Amplified products (30 cycles) were size fractionated on a 1.0% agarose gel. Names ...
Figure 2.
Schematic representation of IbMYB1 cDNA and Southern-blot analysis. A, Schematic representation of IbMYB1 cDNA with R2R3-MYB domain (shaded box). Positions of primers (arrows) used for probe preparation are indicated. MYB and Specific probes were prepared ...

Southern-blot analysis using IbMYB1 probes (MYB probe for the putative R2R3 DNA binding domain and Specific probe for the C-terminal region of IbMYB1) indicated that a number of MYB genes existed in sweet potato (Fig. 2B). As there were few differences between the blots achieved using the MYB and Specific probes and only a small number of signals in the case of the EcoRV and HindIII digests, one would expect a small number of loci to encode several similar-type MYB genes in the two cultivars of sweet potato. However, although the band patterns were similar, the presence of small variations indicated that a few of the MYB-like genes had structures that differed between Ayamurasaki and Kokei-14.

Isolation of MYB Gene Family from Sweet Potato

Sequence analysis of 39 randomly isolated clones of genomic PCR products revealed the presence of at least another four MYB members, named IbMYB2-1 to IbMYB2-4. Although the predicted ORF sequences of IbMYB1 and IbMYB2 were quite similar, the predicted lengths of their second introns differed (Fig. 3). The deduced amino acid sequence encoded by all of these members had a length of 249 amino acids, with more than 99% identity of amino acid sequences among the protein encoded by IbMYB2 members and around 93% identity between that encoded by IbMYB1 and IbMYB2 members. Clones belonging to IbMYB1, IbMYB2-1, IbMYB2-2, and IbMYB2-4 were found in both Ayamurasaki and Kokei-14, and no differences in the sequences of the areas tested were detected between the two cultivars. In contrast, IbMYB2-3 was found only in Kokei-14.

Figure 3.
Schematic representation of genomic organization of IbMYB genes, with exons (white boxes) and introns (lines between exons). Positions of primers (arrows) used for RT-PCR analysis (Fig. 5) are indicated, and resulting amplification products (lines) are ...

Phylogeny of Sweet Potato R2R3-Type MYB Factors

The sequences of the IbMYB1 and IbMYB2 genes were compared with those encoding other R2R3-type MYB factors in various plant species: VvmybA1 (Kobayashi et al., 2004) and VlmybA1-1 (Kobayashi et al., 2002) from grape; Ipmyb1 (Chang et al., 2005), InMYB1, InMYB2, and InMYB3 (Morita et al., 2006) from morning glory (Ipomoea nil); AN2 (Quattrocchio et al., 1999) from petunia; ANT1 (Mathews et al., 2003) from tomato (Lycopersicon esculentum); PAP1, PAP2 (Borevitz et al., 2000), and TT2 (Nesi et al., 2001) from Arabidopsis; ROSEA1 (Schwinn et al., 2006) from snapdragon (Antirrhinum majus); GMYB10 (Elomaa et al., 2003) from gerbera (Gerbera hybrida); C1 (Paz-Ares et al., 1987) and Pl (Cone et al., 1993) from maize; and FaMYB1 (Aharoni et al., 2001) from strawberry (Fragaria spp.). The R2R3-binding domain of the deduced amino acid sequence encoded by the IbMYB1 and IbMYB2 genes showed high sequence similarity with those encoded by other MYB genes of other plant species (Fig. 4A), and splice-site locations and intron phases were conserved with the majority of the Arabidopsis (77 of 130) and rice (45 of 85) MYB genes (Fig. 4B; Jiang et al., 2004). We performed phylogenetic analysis of a selected set of R2R3-type MYB factors from various plant species. Using the neighbor-joining method of Saitou and Nei (1987), phylogenetic trees were produced of the aligned amino acid sequences of R2R3 DNA-binding domains (Fig. 4C). The result indicated that the IbMYB1 and IbMYB2 genes were located in the N09 subgroup comprising Arabidopsis PAP1 and PAP2 and petunia AN2 (Jiang et al., 2004). The predicted C-terminal regions of the IbMYB1 and IbMYB2 genes showed similarity to those of the InMYB2 and InMYB3 genes, but there was very limited sequence identity in the case of the other MYB proteins outside the R2R3 domain, except for the presence of a KPRPR(S/T) F-like motif (boxed in Fig. 4, B and D), which was previously reported for the subgroup of R2R3 MYB genes comprising petunia AN2 and Arabidopsis AtMYB75 (PAP1), AtMYB90 (PAP2), and AtMYB113 (Stracke et al., 2001).

Figure 4.
IbMYB genes show features of an R2R3-MYB-binding domain protein. A, Amino acid comparison of the R2 and R3 DNA-binding regions of IbMYB1 and IbMYB2-1 with selected set of R2R3-type MYB factors from various plant species shows high sequence conservation ...

Expression Analysis of IbMYB1 and IbMYB2s

The expression patterns of IbMYB1 and IbMYB2 were analyzed by RT-PCR using specific primers: FA/RA2 for IbMYB1 and FB/RB2 for IbMYB2 members. As shown in Figure 5, when genomic DNAs were used as templates for the reaction, the band patterns were consistent with the predictions from Figure 3: that is, a single band of about 800 bp was found in IbMYB1 and multiple bands of about 900 to 1,250 bp in IbMYB2s. Whereas IbMYB1 mRNA accumulated predominantly in the tuberous roots of Ayamurasaki and slightly in the calli, no signals of IbMYB2 members were detected in these tissues. Two transcripts of different sizes detected in the IbMYB1 mRNA amplification may indicate alternative splicing, as the smaller transcript was consistent in size with mature mRNA and the larger transcript with retention of the second intron. Whereas the first intron of IbMYB1 was under the GT-AG rule, the second intron had GC-AG at the ends; this may have been the cause of the alternative splicing. However, it is not clear whether the larger transcript of the IbMYB1 gene has any functions; many MYB genes of Arabidopsis and rice undergo alternative splicing and are suggested to have multiple biological processes, as some of these splice variants are differentially regulated (Li et al., 2006).

Figure 5.
Expression analysis of IbMYB1 and IbMYB2s. Genomic PCR and RT-PCR were performed using the FA/RA2 and FB/RB2 primer sets indicated in Figure 3. Amplified products (28 cycles) were size fractionated on a 1.0% agarose gel. Amplification of Actin was used ...

Tissue-Specific Expression of Genes Involved in Anthocyanin Biosynthesis and Accumulation

In Ayamurasaki, various tissues besides the tuberous roots, such as the nontuberous roots, stems (ST), young leaves (YL), stressed leaves (RL), flower buds (FB), and open flowers (FL), accumulate anthocyanins and exhibit pigmentation. Anthocyanins were extracted from each tissue and quantified (Fig. 6A), and expression analysis of the genes involved in anthocyanin biosynthesis and accumulation was performed at the same time (Fig. 6B).

Figure 6.
Anthocyanin accumulation and gene expression in Ayamurasaki tissues. A, Photometric determination of anthocyanin content in methanolic extracts of various tissues of Ayamurasaki. N (number of samples) is indicated below. Error bars represent sd. B, RT-PCR ...

The IbMYB1, IbMYB2, and IbMADS10 genes were candidates for regulators of anthocyanin biosynthesis; CHS, CHI, F3H, DFR, ANS, and 3GT (encoding chalcone synthase, chalcone isomerase, flavanone-3-hydroxylase, DFR, anthocyanidin synthase, and flavonoid 3-glucosyl-transferase, respectively) were genes involved in parts of the anthocyanin biosynthesis pathway; VP24 (encoding vacuolar protein) was considered to be involved in anthocyanin vacuolar transport and/or trapping (Nozue et al., 1997; Xu et al., 2001).

The structural anthocyanin genes (CHS to 3GT) were expressed in pigmented tissues, with the exception of flesh of stored (i.e. stored for few months before use) tuberous roots (sTF) and FL (Fig. 6B). IbMYB1 gene expression was detected mainly in flesh of developing (i.e. used just after harvest) tuberous roots (dTF), less in peeled outer layer (less than 1 mm thick) including the skins of developing and stored tuberous roots (dTS and sTS), and a little in sTF, red and white nontuberous roots (RR and WR), and ST. Slight expression of the IbMYB2 genes was detected in dTF and dTS. The IbMADS10 gene was detected a little in dTF, sTF, RR, WR, and FB. Expression of the VP24 gene was detected in all tissues tested except for expanded green leaves (EL).

Anthocyanin Accumulation and Gene Expression in Seven Sweet Potato Cultivars

The relationship between anthocyanin accumulation and gene expression was examined in seven sweet potato cultivars: three purple fleshed, one orange fleshed (containing carotenoids), two yellow fleshed, and one white fleshed (Fig. 7A). Among various sweet potato cultivars, the color and/or color intensity of the flesh seems to be not related to those of other tissues, such as the tuber skins, flowers, leaves, and stems. For example, the flesh colors were not related to the tuber skin colors or young leaf colors (Fig. 7A). The family trees of the cultivars used are shown in Figure 7B. The literature-based relative anthocyanin contents of mature tuberous roots of the purple-fleshed cultivars are also presented in Figure 7B, and our measurements are presented in Figure 8A. Anthocyanins in the flesh of tuberous roots were detected only in the three purple-fleshed cultivars, but in the outer layer of the tuberous roots, they were also detected in orange- and yellow-fleshed cultivars (Fig. 8A).

Figure 7.
Seven sweet potato cultivars were tested: three purple-fleshed cultivars, one orange-fleshed cultivar, and three yellow or white-fleshed cultivars. A, Appearance of flesh or skin of mature tuberous roots or young developing tuberous roots or adaxial side ...
Figure 8.
Anthocyanin accumulation and gene expression in different sweet potato cultivars. A, Anthocyanin contents of mature tuberous roots (including skin and flesh, grown in the field, longer than 15 cm) and flesh or peeled outer layer (less than 1 mm thick) ...

All tested genes were detected in almost every cultivar tested by genomic PCR (Fig. 8B), with exceptions being IbMADS10 in Ayakomachi and Joy White. By RT-PCR, we found that, in the flesh of small dTF, the IbMYB1 gene and the structural anthocyanin genes were expressed predominantly in purple-fleshed cultivars (Fig. 8C). Expression of IbMYB2 was scarcely detected in each cultivar. Expression of IbMADS10 and VP24 was not associated with anthocyanin content. In the peeled outer layer containing the skin, the expression patterns of the genes encoding the transcription factors were similar to those in the flesh. The structural anthocyanin genes were expressed in the outer layers of the six red-skinned cultivars.

Transient Expression Analysis of IbMYB1

Using a particle gun, the IbMYB1 gene was transiently expressed in sweet potato leaves, tuberous roots, and calli under the control of the cauliflower mosaic virus (CaMV)-derived 35S promoter or sweet potato-derived IT394 promoter (pIT394). The constructs used for the bombardment are shown in Figure 9. The green fluorescent protein (GFP) gene was used as the marker of bombarded cells. Anthocyanin pigmentation was observed in the bombarded cells of leaves (Fig. 10A), tuberous roots (Fig. 10C), and calli (data not shown) when the IbMYB1 gene was introduced with the GFP gene (Fig. 10, A and C), whereas no pigmentation was detected when the IbMYB1 antisense fragment was introduced with GFP (Fig. 10, B and D). The GFP fluorescence in most leaf cells diffused to neighboring cells, whereas the anthocyanin pigment stayed in the bombarded cells. Detection of anthocyanin pigmentation was delayed until 1 or 2 d after the detection of GFP fluorescence. The pigmentation deepened over more than 1 week and then remained even after the GFP fluorescence had disappeared. The speed of accumulation of GFP and anthocyanin was slower in the tuberous roots than in the leaves. Differences in promoter (CaMV 35S or pIT394) or the cultivar of bombarded cells (Ayamurasaki or Kokei-14) did not affect these results (data not shown).

Figure 9.
Constructs used for transient expression analysis and for stable transformation. Plasmid vector names of pBluescriptII background are represented. Names of binary vector (pPZP2H-lac) background are represented in parentheses.
Figure 10.
Transient expression analysis of IbMYB1. GFP (pHM160) and IbMYB1 (pHM196) or IbMYB1 antisense (pHM197) were cobombarded into sweet potato leaves (A and B) and sections of tuberous roots (C and D). A and C, Cobombardment of pHM160 and pHM196. B and D, ...

Overexpression of IbMYB1 in Transgenic Plants

Forced expression analysis of the IbMYB1 gene was also performed in sweet potato calli and heterologous plants (Arabidopsis and rice) with stable transformation. In Arabidopsis, overexpression of IbMYB1 induced ectopic pigmentation in seedlings (Fig. 11A), roots (Fig. 11B), flowers, leaves, and stems (Fig. 11C), and even in seeds (Fig. 11D). However, most of the plants with severe pigmentation were sickly and could not survive without the addition of Suc to the culture medium. In rice, we could not produce a pigmented plant. In sweet potato, overexpression of IbMYB1 induced strong pigmentation in transgenic calli (Fig. 12A). We obtained 17 independent pigmented transgenic calli by transformation of about 3 g of embryogenic calli of Kokei-14. Ten of the 17 lines of pigmented transgenic calli grew well and were subjected to anthocyanin quantification and gene expression analysis. Sometimes untransformed calli of Kokei-14 or Ayamurasaki show patchy pigmentation (Fig. 12A), but we could not obtain calli as severely and uniformly pigmented as the pHM158 (35S::IbMYB1) transformants when the calli were not transformed or were transformed with other constructs, such as pHM150b (35S::GFP) or pHM159 (35S::anti-IbMYB1; data not shown). The amount of anthocyanin in the transgenic calli was increased by nearly 200 times that in the nontransgenic Kokei-14 calli (Fig. 12B) and was comparable to that in the tuberous roots of purple-fleshed cultivars (Fig. 8A). The growth rates of the transgenic callus lines were inversely related to the levels of anthocyanin accumulation (Fig. 12C) and were much higher than those of nontransgenic calli (Fig. 12B). We also assessed the influence of light on anthocyanin accumulation and growth rate. There was a tendency for anthocyanin accumulation to increase and growth rate to decrease in calli grown in light, and light-exposed nontransgenic calli of Ayamurasaki showed significantly higher rates of anthocyanin accumulation than did dark-exposed calli of this cultivar (Fig. 12D).

Figure 11.
Overexpression of IbMYB1 in Arabidopsis under the control of the CaMV 35S promoter. Five-day-old seedlings (A), a root (B), flowers, leaves, and stems (C), and seeds (D) of Arabidopsis plants transformed with the construct pHM158 (CaMV35S::IbMYB1 ...
Figure 12.
Overexpression of IbMYB1 in sweet potato calli under the control of the CaMV 35S promoter. A, Kokei-14-derived calli transformed with the construct pHM158 (CaMV35S::IbMYB1) turned a deep purple. The calli of 10 independent transgenic lines and ...

The IbMYB1 gene and the structural anthocyanin genes were predominantly expressed in the pigmented transgenic calli and pigmented untransformed Ayamurasaki calli (Fig. 13). Scant expression of the IbMYB2 genes and the IbMADS10 gene was detected in some transgenic or nontransgenic calli. The VP24 transcripts were decreased when the calli (Ayamurasaki, Kokei-14, and pHM158-2) were grown in the dark, but the expression pattern was not correlated with the anthocyanin content. Whereas two transcripts of different sizes were detected by RT-PCR analysis of IbMYB1 in untransformed Ayamurasaki tissues (Figs. 5, ,6,6, and and13),13), a single band (of a size consistent with introduced IbMYB1 cDNA and the same size as the smaller transcript detected in untransformed tissues) was detected in pHM158 transgenic calli (Fig. 13).

Figure 13.
RT-PCR analysis of the genes hypothesized to be involved in anthocyanin biosynthesis and accumulation was performed in sweet potato calli. Amplified products (30 cycles for IbMYB1, IbMYB2s, and IbMADS10; 28 cycles for the rest of the genes) were size ...

DISCUSSION

The pathways of biosynthesis of flavonoid pigments have been investigated well, perhaps because of the color that they add to plants. Their regulatory genes in aerial parts such as flowers, leaves, seeds, and fruits have been identified in various plant species, whereas little is known about their regulation in underground organs such as tuberous roots. Although the role of anthocyanins in underground organs is not clear, the fact that many sweet potato cultivars with not only purple flesh but also orange, yellow, or white flesh retain anthocyanins in the skin of their tuberous roots suggests that they play an important role.

Sweet potato accumulates anthocyanins in various tissues such as the flowers, leaves, stems, and nontuberous roots, as well as the tuberous roots (Fig. 6A). The IbMYB1 gene was expressed predominantly in tuberous roots, whereas the expression of IbMYB2 genes was scarcely detected (Fig. 6B). WR was the only tissue that showed expression of the IbMYB1 gene in the absence of anthocyanin detection (Fig. 6). The fact that the expression levels of structural anthocyanin genes were similar to that in RR indicates that anthocyanin biosynthesis was already active in WR but anthocyanin had not accumulated to levels high enough to detect. There was no tissue that expressed the IbMYB1 gene without structural anthocyanin gene expression. We sometimes could not detect IbMYB1 expression in tuberous roots when the samples had been stored for a few months (e.g. sTF in Fig. 6B; also in sTS in some cases) or in seed tubers (data not shown). These tissues may have been losing their anthocyanin biosynthesis activity, because they also showed diminished expression of the structural anthocyanin genes. In any case, a striking correlation of expression in the tuberous roots was observed between IbMYB1 and the structural anthocyanin genes.

We did not detect IbMYB1 expression in flowers and leaves despite the anthocyanin accumulation and the strong expression of structural anthocyanin genes (YL, RL, and FB in Fig. 6). IbMYB1 expression was also not detected in the skins of tuberous roots of non-purple-fleshed cultivars (Ayakomachi, Kokei-14, and Beniazuma in Fig. 8C). These results suggest that IbMYB1 acts specifically in the flesh of tuberous roots and that other regulatory genes are active in the other tissues. Contamination of the outer layer samples with flesh tissue may be the reason why IbMYB1 was detected in the outer layers of the purple-fleshed tuberous roots (dTS and sTS in Fig. 6; Ayamurasaki, Murasakimasari, and Purple Sweet Lord in Fig. 8C). The difference in anthocyanin composition of the outer layer and the inner portion of Ayamurasaki tuberous root (Yoshimoto et al., 1999a) may reflect a difference in the genes responsible for the regulation of anthocyanin biosynthesis. The fact that, among various sweet potato cultivars, the color and/or color intensity of the flesh were not related to those of other tissues (Fig. 7A) also supports these ideas.

The results of bombardment of the IbMYB1 gene into leaves (Fig. 10A) indicated the existence of mechanisms of anthocyanin biosynthesis common to different tissues. However, Ayamurasaki leaves display a complex change in color; they are red when they are young, turn green as they develop, and turn red again with stress (YL, EL, and RL in Fig. 6A). Therefore, we can assume that multiple regulatory genes and a degradation mechanism are active in these leaves.

This is, to our knowledge, the first report of the identification of MYB genes in sweet potato, and other transcription factors related to anthocyanin biosynthesis, such as the bHLH and WD40 genes, are still unknown in sweet potato. However, several members of the MYB, bHLH, and WD40 gene families have been isolated from morning glory and their differential expression pattern reported; InMYB1 is expressed in the flower and InMYB2 is expressed in the petiole, stem, and root, but expression of InMYB3 has not been detected (Morita et al., 2006). According to phylogenic analyses (Fig. 4C), the InMYB2 and InMYB3 genes are more closely related to IbMYB1 and IbMYB2 than to InMYB1. These results indicate that the MYB genes working in flowers and the MYB genes working in roots were divided before the division of morning glory and sweet potato.

A number of MADS-box genes have been isolated from sweet potato, and one of them, IbMADS10, has been suggested to be involved in anthocyanin biosynthesis (Lalusin et al., 2006). However, the level of correlation of this gene with anthocyanin accumulation or expression of the structural anthocyanin genes was not high in our experiments. It is possible that another transcription factor for IbMADS10 activity, such as a bHLH transcription factor that needs an MYB transcription factor to activate it, is involved.

Sometimes red-pigmented cells appear in sweet potato calli grown under light conditions (Fig. 12A). Nishimaki and Nozue (1985) used the yellow-fleshed cultivar Kintoki to establish cell lines (ALD and ALND) that accumulate anthocyanins. The ALD cell line produced anthocyanins in the light but not in darkness, and the ALND line produced anthocyanins under both light and dark conditions. However, the content of anthocyanins in the dark was one-fourth that in the light (Nozue et al., 1997). On the other hand, our transgenic calli lines, made by forced expression of the IbMYB1 gene in the yellow-fleshed cultivar Kokei-14, accumulated anthocyanins in a light-independent manner at high levels comparable to those in the tuberous roots of purple-fleshed cultivars (Figs. 8A and 12B). These features are similar to those of cell lines selected and established from Ayamurasaki tuberous roots (Konczak-Islam et al., 2000). It is possible that accumulation of anthocyanin in calli in a light-dependent manner is caused by the activation of regulator genes that act in the aerial parts and light-independent accumulation is caused by the activation of regulator genes that act in the underground organs.

Light induced anthocyanin accumulation in Ayamurasaki calli (Fig. 12D); sometimes IbMYB1 expression was detected in this tissue (Fig. 13) and sometimes not. Unlike cell lines, the callus samples may not have been homogeneous and may have included diverse cells pigmented by the activation of various regulatory genes.

However, the determinant of growth rate was not clear; the growth rates of most of the transgenic calli were much higher than those of the nontransgenic calli (Fig. 12B). It is conceivable that the accumulated anthocyanins protected the callus from the inhibitory effects of light and thus promoted their propagation. On the other hand, the growth rates of the transgenic callus lines were inversely related to the levels of anthocyanin accumulation (Fig. 12C). It is possible that the growth rates of some transgenic lines were so fast that anthocyanin accumulation did not reach saturation in these lines. The fact that the growth rates of all transgenic lines exceeded 2.0 (i.e. the weight of the calli doubled in 2 weeks; Fig. 12B), whereas the anthocyanin contents of the leaf or tuber cells increased for more than 1 week (Fig. 10, A and C), may support this assumption.

The fact that overexpression of the IbMYB1 gene induces ectopic pigmentation not only in sweet potato but also in heterologous Arabidopsis (Fig. 11) suggests a common regulatory mechanism. On the other hand, the fact that most of the transgenic Arabidopsis plants with severe pigmentation were sickly and unable to survive without Suc in the culture medium suggests a difference among species. Quattrocchio et al. (1998) and Gong et al. (1999) also found common and different actions of anthocyanin regulatory genes in heterogonous plants; ectopic expression of petunia AN2 and JAF13 induced CHS gene expression in heterologous maize but not in petunia. Both bHLH alleles of perilla, MYC-RP and MYC-GP, induced anthocyanin accumulation in heterologous tobacco (Nicotiana tabacum) plants, whereas only MYC-RP acted as a regulator in perilla.

The biosynthesis of anthocyanins occurs in the cytosol, whereas the end products accumulate in the vacuole. This may be the reason why the anthocyanin pigment induced by bombardment with IbMYB1 remained in the bombarded cells, whereas the GFP fluorescence diffused to other cells. It is proposed that glutathione-S-transferase-like proteins such as maize BZ2 (Marrs et al., 1995), petunia AN9 (Mueller et al., 2000), and Arabidopsis TT19 (Kitamura et al., 2004) deliver their flavonoid substrates to the transporter, such as the maize multidrug resistance-associated protein (MRP)-type transporter ZmMRP3 (Goodman et al., 2004) or the Arabidopsis multidrug and toxic compound extrusion transporter TT12 (Debeaujon et al., 2001). In sweet potato, it has been suggested that VP24 precursor protein is involved in vacuolar transport and/or accumulation of anthocyanin (Nozue et al., 1997; Xu et al., 2001).

Transient and stable forced expression analysis of IbMYB1 suggested that IbMYB1 induces not only structural anthocyanin genes but also anthocyanin transporters such as ZmMRP3 that are expressed under the control of the regulators of anthocyanin biosynthesis, R and C1 (Goodman et al., 2004). We examined whether IbMYB1 induced VP24 expression (Fig. 13). The VP24 gene was expressed not only in transgenic calli but also in unpigmented nontransgenic light-grown Kokei-14 callus, but it was scarcely detected in dark-grown pHM158-2 callus and light-grown pHM158-12 callus. These results deny the inductivity of VP24 gene expression by IbMYB1. We also examined the expression pattern of VP24 in sweet potato tissues (Figs. 6B and and8C).8C). The relatively abundant expression in older tissues (i.e. more abundant in sTF, sTS, and FL than in dTF, dTS, and FB in Fig. 6B) suggests that VP24 acts in the retention of anthocyanin rather than in transport and/or accumulation. There may be other IbMYB1-inducible genes that are involved in anthocyanin transport and accumulation in sweet potato.

However, the IbMYB1 transcript was not detected in the tissues of Kokei-14, and there were no differences in base sequences of the IbMYB1 coding region between Ayamurasaki and Kokei-14. The fact that the overexpression of IbMYB1 in Kokei-14 induced a large amount of anthocyanin pigmentation suggests that mutation(s) on the promoter region of the IbMYB1 gene caused alteration in the flesh color of sweet potato, as in the case for grape skin color; the insertion of a retrotransposon into the 5′-flanking region of VvmybA1 was associated with loss of pigmentation in white-skinned cultivars, whereas the coding sequences were identical with those of red-skinned cultivars (Kobayashi et al., 2004). Alternatively, mutation(s) on regulator(s) of the IbMYB1 gene may have caused the alteration of flesh color, although the regulators of the anthocyanin regulatory genes are unknown.

All three purple-fleshed cultivars expressed the IbMYB1 gene in their tuberous roots (Fig. 8C). Presumably, they inherited the active promoter of the IbMYB1 gene from a common ancestor, Yamagawamurasaki (Fig. 7B). However, their contents of anthocyanin differ considerably (Fig. 7B). Interaction of IbMYB1 with other transcriptional factors may affect anthocyanin biosynthesis activity.

MATERIALS AND METHODS

Plant Materials

Sweet potato (Ipomoea batatas) L. Lam. (cultivars Ayamurasaki, Kokei-14, Murasakimasari, Purple Sweet Lord, Ayakomachi, Beniazuma, and Joy White) plants were grown in a greenhouse at 26°C under natural light. Sweet potato calli were induced from the meristems of apical and axillary buds on media (Murashige and Skoog Plant Salt mixture [Wako], 100 mg L−1 myo-inositol, 0.4 mg L−1 thiamine-HCl, 3% [w/v] Suc, 0.05% [w/v] MES, pH 5.7, 1 mg L−1 4-fluorophenoxyacetic acid, 0.8% [w/v] agarose) in growth chambers at 26°C in the dark, and transformed calli were cultured on selection media (Murashige and Skoog Plant Salt mixture [Wako], 100 mg L−1 myo-inositol, 0.4 mg L−1 thiamine-HCl, 3% [w/v] Suc, 0.05% [w/v] MES, pH 5.7, 1 mg L−1 4-fluorophenoxyacetic acid, 25 mg L−1 hygromycin B, 500 mg L−1 carbenicillin, 0.8% [w/v] agarose) in growth chambers at 26°C under 16 h of light (around 40 μmol m−2 s−1) and 8 h of dark. Arabidopsis (Arabidopsis thaliana) ecotype Columbia plants were grown in growth chambers at 22°C under continuous light.

RNA and DNA Preparation

RNA was isolated from various tissues and calli of sweet potato. After the samples had been ground into powder in mortars with liquid nitrogen, they were washed with buffer solution (0.1 m HEPES, 0.05 m l-ascorbic acid, 0.4% [v/v] polyvinyl pyrrolidone [Mr 40,000], and 2% [v/v] β-mercaptoethanol) four times. RNA was isolated according to the protocol of the National Science Foundation's Potato Genome Project (RNA isolation using phenol protocol; http://www.tigr.org/tdb/potato/microarray_SOPs.shtml). DNA was isolated from about 0.5 g of young leaves of sweet potato. After the samples had been ground into powder in mortars with liquid nitrogen, they were washed with buffer solution as for the RNA preparation, and then a PhytoPure plant DNA extraction kit (Amersham Biosciences) was used for DNA isolation in accordance with the manufacturer's instructions.

Construction of cDNA Libraries from Sweet Potato

cDNA libraries were constructed from tuberous roots of sweet potato using cDNA Synthesis kit (Stratagene), size fractioned with Size Sep400 Spun Columns (Amersham Biosciences), and introduced into vector pBluescript II SK+ (Stratagene) digested with XhoI and EcoRI. From the randomly isolated cDNA clones, we determined the sequences of 3,783 cDNA clones from Ayamurasaki and 2,804 cDNA clones from Kokei-14.

RT-PCR and Genomic PCR

For RT-PCR, first-strand cDNA was synthesized from 500 ng of total RNA with Superscript III (Invitrogen) in accordance with the manufacturer's instructions. Oligo(dT) 20 was used as the primer. Twenty microliters of the RT product was acquired from each reaction. One microliter of RT product was subjected to each PCR amplification. PCR was performed using Platinum Pfx DNA Polymerase (Invitrogen) and primers designed for each gene. The PCR conditions were 5 min at 95°C, followed by 25, 28, 30, or 35 cycles of 30 s at 94°C, 30 s at 56°C, and 2 min at 68°C, followed by 7 min at 68°C. At least two independent repeats of the RT-PCR experiments were performed, and typical images of electrophoresis are presented. We could not detect any signals when the reactions were performed under the same conditions without RT. For the amplification of genomic DNA, the PCR conditions were 5 min at 95°C, followed by 30 or 40 cycles of 30 s at 94°C, 30 s at 56°C, and 5 min at 68°C, followed by 7 min at 68°C, using PCR SuperMix High Fidelity (Invitrogen). The primers used for the PCR are listed in Table I.

Table I.
Primers used in RT-PCR experiments

Southern Hybridization

Southern-blot analysis of IbMYB1 was performed using the MYB and Specific probes. Twenty micrograms of genomic DNA of Ayamurasaki and Kokei-14 was digested with EcoRI, EcoRV, and HindIII, fragmented on a 0.8% agarose gel, transferred to nylon membrane (Boehringer Mannheim), and hybridized with the MYB and Specific probes of IbMYB1 (Fig. 2A). The MYB and Specific probes were prepared by PCR using primers 666-F8 (5′-GTGAGAAAAGGTTCATGGTCC-3′) and 666-R13 (5′-CTTCTTCTGAAGATGGGTGTTC-3′), and 666-F9 (5′-GTGTCTGCCATGGCTTCTTCAA-3′) and 666-gRp (5′-TGGCTGCAGATTACATTCTCAAATTTAATCGTACA-3′), respectively. The probes were labeled and detected using AlkPhos Direct Labeling and Detection system (Amersham Biosciences) and Hyperfilm ECL (Amersham Biosciences) in accordance with the manufacturer's instructions. Membranes were hybridized at 55°C overnight and washed twice for 10 min at 60°C.

Isolation of Genome Fragments of IbMYB1

The IbMYB1 genome fragments of Ayamurasaki and Kokei-14 were amplified using the primers 666-Fb2 (5′-ATGGATCCTAAGAATTTCCGACACCCTTC-3′) and 666-R (5′-CGGTGTTTTCCGTGATTTCT-3′) or 666-F3 (5′-CTCATTCTGCGCCTCCATAG-3′) and 666-R14 (5′-GGTGGCAATAGAATTTAAATCAAG-3′) and then cloned into vector pCR2.1-TOPO using a TOPO TA Cloning kit (Invitrogen). The clones were randomly isolated and sequenced, and the sequences of 39 clones were determined.

Plasmid Construction

The CaMV 35S promoter was isolated from pBI221 (BD Biosciences CLONTECH) by PCR using primers that added the XbaI site at the 5′ end and the BamHI site at the 3′ end; i.e., 35S-F340x (5′-TGGTCTAGAGACTTTTCAACAAAGGGTAAT-3′) and 35S-Rb (5′-CGGGATCCTCTCCAAATGAAATGAACTTCC-3′). pHM144 was constructed by insertion of the CaMV 35S promoter into the XbaI and BamHI sites of the plasmid pblue-sGFP(S65T)-NOS SK, kindly provided by Dr. Y. Niwa of the University of Shizuoka. The IbMYB1 ORF region was isolated from the cDNA clone IT666 by PCR using primers that added the BamHI site at the 5′ end and the NotI site at the 3′ end; i.e. 666-Fb2 (5′-ATGGATCCTAAGAATTTCCGACACCCTTC-3′) and 666-Rn (5′-ATGCGGCCGCTTAGCTTAACAGTTCTGAC-3′). The IbMYB1 antisense fragment was isolated from cDNA clone IT666 by PCR using primers that added the BamHI site at the 5′ end and the NotI site at the 3′ end; i.e. 666-Rb (5′-TAGGATCCTAACGACGGTGTTTTCCG-3′) and 666-Fn2 (5′-ATGCGGCCGCTAAGAATTTCCGACACCCTT-3′). pHM156 and pHM157 were made by ligation of the IbMYB1 ORF and the IbMYB1 antisense fragment, respectively, with the ScaI-BamHI fragment of 1.4 kb and the ScaI-NotI fragment of 2.2 kb from pHM144.

The IT394 promoter (Mano et al., 2006) was isolated from the sweet potato genome by PCR using primers that added the SacII site and removed the SacI site at the 5′ end and that added the BamHI site at the 3′ end: 394F07s (5′-TGGCCGCGGATGTTGAcCTCTTTCATTTTGAACCAA-3′) and 394Rb (5′-CGGGATCCTTTTCACACAGAAGAGAGAAAGACAAGA-3′). pHM160, pHM196, and pHM197 were constructed by insertion of the IT394 promoter into the SacII and BamHI sites of the plasmid pblue-sGFP(S65T)-NOS SK, pHM156, and pHM157, respectively.

The CaMV 35S promoter, IbMYB1 ORF, and Nos terminator sequence and the CaMV 35S promoter, IbMYB1 antisense fragment, and Nos terminator sequence were excised from pHM156 and pHM157, respectively, using XbaI and KpnI and then cloned to the XbaI and KpnI site of the binary vector pPZP2H-lac, kindly provided by Dr. M. Yano of the National Institute of Agrobiological Sciences, Tsukuba, Japan (Fuse et al., 2001). The resulting binary vectors were named pHM158 and pHM159, respectively.

Transient Expression

Transient expression analysis was carried out as described (Higo et al., 2005). The same amount of plasmids pHM144 (35S: GFP) and pHM156 (35S::IbMYB1) or pHM157 (35S::anti IbMYB1), and pHM160 (pIT394::GFP) and pHM196 (pIT394::IbMYB1) or pHM197 (pIT394::anti IbMYB1) were bombarded into sweet potato leaves (Kokei-14 and Ayamurasaki), calli (Kokei-14 and Ayamurasaki), and sections of tuberous root (Kokei-14). The bombarded tissues were cultured on medium (Murashige and Skoog Plant Salt mixture [Wako], 100 mg L−1 myo-inositol, 0.4 mg L−1 thiamine-HCl, 0.05% [w/v] MES, pH 5.7, 3% [w/v] Suc, 0.1% [v/v] Plant Preservative mixture [Plant Cell Technology], 0.6% [w/v] agarose) in growth chambers at 26°C under 16 h of light (photon flux density around 40 μmol m−2 s−1) and 8 h of dark for leaves and continuous dark for calli and tuberous roots. Bombarded tissues were observed under a microscope (IX70, Olympus), and the fluorescence of GFP was observed through a filter set (excitation wavelength, 460–490 nm; emission, 515–550 nm; dichroic, 505 nm).

Stable Transformation

The binary vector pHM158 was transformed with Agrobacterium tumefaciens strain EHA101 by the freeze-thaw method (Holsters et al., 1978). Using this Agrobacterium, transformation of the sweet potato calli was performed in accordance with the method of Otani et al. (1998). Arabidopsis was transformed by the floral dip method (Clough and Bent, 1998).

Anthocyanin Quantification

Extraction and quantification of anthocyanins was performed in accordance with the protocols of Mehrtens et al. (2005), with minor modifications. One milliliter of acidic methanol (1% [w/v] HCl) was added to 0.3 g of fresh plant tissue. Samples were incubated for 18 h at 21°C under moderate shaking (95 rpm). After centrifugation (21,500g, room temperature, 3 min), 0.4 mL of the supernatant was added to 0.6 mL of acidic methanol. Absorption of the extracts at wavelengths of 530 and 657 nm was determined photometrically (DU 640 Spectrophotometer, Beckman Instruments). When the absorption value exceeded 2.5, extracts diluted with acidic methanol were used for the measurements. Quantitation of anthocyanins was performed using the following equation: Q (anthocyanins) = (A530 − 0.25 A657) × M−1, where Q (anthocyanins) is the concentration of anthocyanins, A530 and A657 are the absorptions at the wavelengths indicated, and M is the fresh weight (in grams) of the plant tissue used for extraction. The numbers of samples used for the measurements are indicated in each figure. Error bars indicate the sds of the average anthocyanin contents.

Acknowledgments

The authors are grateful to Dr. Yasuo Niwa of the University of Shizuoka for providing the vector pblue-sGFP(S65T)-NOS SK; Dr. Masahiro Yano of the National Institute of Agrobiological Sciences for the binary vector pPZP2H-lac; the National Institute of Crop Science for the sweet potato; Mr. Yuichi Minesaki of Hitachi Software Engineering for his help with the cDNA clustering analysis; and Mrs. N. Kawaguchi, Mrs. K. Kawajiri, and Mrs. K. Takahashi for their assistance.

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.plantphysiol.org) is: Hironori Mano (pj.oc.andcgp@onamh).

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