isolation of a regulatory gene of anthocyanin …...isolation of a regulatory gene of anthocyanin...

Isolation of a Regulatory Gene of AnthocyaninBiosynthesis in Tuberous Roots of Purple-FleshedSweet Potato[OA]

Hironori Mano*, Fumiaki Ogasawara, Kazuhito Sato, Hiromi Higo, and Yuzo Minobe

Plant Genome Center, Tsukuba, Ibaraki 305–0856, Japan

Many transcriptional factors harboring the R2R3-MYB domain, basic helix-loop-helix domain, or WD40 repeats have beenidentified in various plant species as regulators of flavonoid biosynthesis in flowers, seeds, and fruits. However, the regulatoryelements of flavonoid biosynthesis in underground organs have not yet been elucidated. We isolated the novel MYB genes IbMYB1and IbMYB2s from purple-fleshed sweet potato (Ipomoea batatas L. Lam. cv Ayamurasaki). IbMYB1 was predominantly expressedin the purple flesh of tuberous roots but was not detected (or only scarcely) in other anthocyanin-containing tissues such asnontuberous roots, stems, leaves, or flowers. IbMYB1 was also expressed in the tuberous roots of other purple-fleshed cultivars butnot in those of orange-, yellow-, or white-fleshed cultivars. Although the orange- or yellow-fleshed cultivars containedanthocyanins in the skins of their tuberous roots, we could not detect IbMYB1 transcripts in these tissues. These results suggest thatIbMYB1 controls anthocyanin biosynthesis specifically in the flesh of tuberous roots. The results of transient and stabletransformation experiments indicated that expression of IbMYB1 alone was sufficient for induction of all structural anthocyaningenes 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 theworld’s seventh most important crop after wheat(Triticum aestivum), rice (Oryza sativa), maize (Zeamays), potato (Solanum tuberosum), barley (Hordeumvulgare), and cassava (Manihot esculenta Crantz.). Mostsweet potato cultivars, like the other major crops thatare grown and consumed, have white or yellow flesh.However, there are also some sweet potato cultivarswith orange flesh that contains carotenoids or withpurple flesh that contains anthocyanins.

Anthocyanins and related compounds such asproanthocyanidins protect the leaves from variousstressors such as strong light and heavy metals (forreview, see Gould, 2004), play a role in entomophily inflowers, and deter pathogens and predators of seeds(Shirley, 1998). The role of anthocyanins in under-ground organs (such as tuberous roots) that grow un-der dark conditions is not clear, but it is conceivablethat it is similar to that in seeds (e.g. protection frompathogens and predators and improvement of preser-vation and thus reproductive advantage), becausetubers, like seeds, play a reproductive role.

The reason why most sweet potato cultivars havewhite or yellow flesh may be because consumers pre-

fer pale foods, as is the case with other staples, andbecause the anthocyanins in the tuber are an obstacleto industrial starch production. Nevertheless, attentionis now being focused on the multiple physiologicalfunctions of the anthocyanins derived from purple-fleshed sweet potatoes, such as their strong antioxida-tive activity (Kano et al., 2005), antimutagenicity(Yoshimoto et al., 1999b), antihyperglycemic effect(Matsui et al., 2002), and hepatoprotective and antihy-pertensive effects (Suda et al., 2003). New purple-fleshed cultivars have been developed recently. Thecontent of anthocyanins in indigenous purple-fleshedcultivars 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 stableproduction of anthocyanins from tissue or cell cul-ture of various plant species, including sweet potato.For example, optimum conditions for the culture ofanthocyanin-producing sweet potato cells (Nishimakiand Nozue, 1985; Nozue et al., 1987) or Ayamurasakihairy roots (Nishiyama and Yamakawa, 2004) havebeen investigated. However, low anthocyanin contentand the light dependency of anthocyanin productionhave been noted as problems. Konczak-Islam et al.(2000) obtained a cell line that had high anthocyanincontent and was derived from the tuberous roots ofAyamurasaki. This line showed light-independent an-thocyanin production and may prove useful in theimprovement of anthocyanin productivity, althoughthe regulatory elements of anthocyanin biosynthesisare still unknown.

The mechanisms regulating flavonoid pigmentshave been studied in various plant species. Detailed

* Corresponding author; e-mail [email protected]; fax 81–29–839–4829.

The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Hironori Mano ([email protected]).

[OA] Open Access articles can be viewed online without a sub-scription.

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studies have been done in maize, Arabidopsis (Arabi-dopsis thaliana), and petunia (Petunia hybrida) by nu-merous mutation analyses, and many regulatory geneshave been identified that control the transcription offlavonoid structural genes. Transcriptional factors withMYB or basic helix-loop-helix (bHLH) domains and aWD40 protein are commonly identified in differentspecies (for review, see Broun, 2004; Koes et al., 2005).Although a model has been proposed that these reg-ulators interact with each other and make transcrip-tion complexes with the promoters of the structuralgenes, the partnership seems to be complex. For ex-ample, MYB domain C1 protein regulating the antho-cyanin pathway in maize needs a bHLH partner (B/R)to activate the flavonoid structural gene dihydroflavo-nol reductase (DFR) promoter, whereas MYB domainP protein regulating the phlobaphene pathway canactivate the promoter without a bHLH partner (Sainzet al., 1997). Moreover, other regulatory genes, such asTTG2, 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), andANL2, a homeodomain (HD) protein (Kubo et al.,1999), have been reported. Some of these genes areinvolved in various aspects of plant development be-sides flavonoid biosynthesis, including developmentof 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), acidifi-cation of vacuoles (AN1 and AN11), or mucilage for-mation (TTG2, TTG1, GL3, and EGL3).

The genes responsible for the color differencesamong cultivars have been revealed. One amino acidalternation in bHLH protein created the green formaeof perilla from red formae (Gong et al., 1999), and aretrotransposon-induced mutation on the promoterof the MYB gene created white-skinned grape (Vitisvinifera) cultivars from red-skinned ones (Kobayashiet al., 2004). In the case of rice, red or brown grain wasselected out by ancient breeders. Two loci, Rc and Rd,that are responsible for red seed color were identifiedby classical genetic analysis (Kato and Ishikawa, 1921),and recently they were revealed to encode the bHLHand DFR genes, respectively (Sweeney et al., 2006;Furukawa et al., 2007).

As mentioned above, numerous reports have de-scribed the genes regulating flavonoid pigmentation inflowers, leaves, seeds, and fruits but not in under-ground organs such as tuberous roots. In sweet potato,no MYB, bHLH, or WD40 protein has been reportedthus far; instead, a MADS-box gene, IbMADS10, wasrecently isolated and suggested to be involved inanthocyanin pigmentation (Lalusin et al., 2006), al-though its involvement in the underground organ isunclear.

We report here the isolation of a new R2R3-typeMYB gene, IbMYB1, from a purple-fleshed sweet po-tato cDNA library and its predominant expression inthe tuberous roots of purple-fleshed cultivars. Tran-

sient and stable forced expression of the IbMYB1 generesulted in intense anthocyanin pigmentation in var-ious tissues. These results suggest that the IbMYB1gene is responsible for purple pigmentation in theflesh of tuberous roots of sweet potato.

RESULTS

Isolation of cDNA Clones with Tissue- andCultivar-Specific Expression

We constructed cDNA libraries from the tuber-ous roots of the purple-fleshed sweet potato cultivarAyamurasaki and the yellow-fleshed sweet potato culti-var Kokei-14, and we determined the sequences of 3,783randomly isolated cDNA clones from Ayamurasaki and2,804 from Kokei-14. The cDNA clones were clustered,and 25 sequence groups were selected according totheir frequency of appearance in the libraries and/orspecificity to the Ayamurasaki library. The 25 selectedgroups were subjected to reverse transcription (RT)-PCR analysis, and the results indicated that fivegroups, IT1, IT4, IT4785, IT666, and IT4097, wereexpressed in a tissue-specific manner (Fig. 1). WhereasIT948 was identical to the Actin gene and was ex-pressed equally in all tissues tested, IT1 and IT4 wereexpressed predominantly in tuberous-root tissue andIT4097 was expressed in the tuberous roots and thecalli. IT4785 and IT666 were expressed in a cultivar-specific manner; that is, they were expressed only in

Figure 1. Genomic PCR and RT-PCR performed on five clones isolatedfrom a cDNA library derived from sweet potato tuberous roots revealedtissue- and/or cultivar-specific expression. Amplified products (30cycles) were size fractionated on a 1.0% agarose gel. Names of clonesare indicated at left and identities of the clones at right. A, Ayamurasaki;K, Koukei-14.

IbMYB1, a Regulator of Sweet Potato Anthocyanin Biosynthesis

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Ayamurasaki, not in Kokei-14. IT4785 was homolo-gous to the flavonoid 3#-hydroxylase of Ipomoea tri-color, and IT666 showed partial identity to R2R3-typeMYB genes. We therefore acquired a novel MYB-likegene, IT666, with tissue- and cultivar-specific expres-sion. As there have been no reports of MYB-like genesin sweet potato, to our knowledge, we named thisIT666 gene IbMYB1. The IbMYB1 cDNA was predictedto have an open reading frame (ORF) of 750 bp, in-cluding 312 bp of the R2R3-type MYB-like region inthe first half; the latter half had no marked similarity(Fig. 2A).

Southern-blot analysis using IbMYB1 probes (MYBprobe for the putative R2R3 DNA binding domain andSpecific probe for the C-terminal region of IbMYB1)indicated that a number of MYB genes existed in sweetpotato (Fig. 2B). As there were few differences be-tween the blots achieved using the MYB and Specific

probes and only a small number of signals in the caseof the EcoRV and HindIII digests, one would expect asmall number of loci to encode several similar-typeMYB genes in the two cultivars of sweet potato. How-ever, although the band patterns were similar, thepresence of small variations indicated that a few of theMYB-like genes had structures that differed betweenAyamurasaki and Kokei-14.

Isolation of MYB Gene Family from Sweet Potato

Sequence analysis of 39 randomly isolated clones ofgenomic PCR products revealed the presence of atleast another four MYB members, named IbMYB2-1 toIbMYB2-4. Although the predicted ORF sequences ofIbMYB1 and IbMYB2 were quite similar, the predictedlengths of their second introns differed (Fig. 3). Thededuced amino acid sequence encoded by all of these

Figure 2. Schematic representation of IbMYB1 cDNA and Southern-blot analysis. A, Schematic representation of IbMYB1 cDNA withR2R3-MYB domain (shaded box). Positions of primers (arrows) used forprobe preparation are indicated. MYB and Specific probes wereprepared with F8/R13 and F9/gRp primer sets, respectively. B, Southern-blot analysis of cultivars Ayamurasaki and Kokei-14 using the MYBand Specific probes of IbMYB1. Twenty micrograms of genomic DNAwas digested with EcoRI (EI), EcoRV (EV), and HindIII (H), fragmentedon a 0.8% agarose gel, transferred to nylon membrane, and hybridizedwith MYB (left) and Specific (right) probes. Asterisks indicate differ-ences between Ayamurasaki and Kokei-14. Aya, Ayamurasaki; Ko,Koukei-14. Molecular marker sizes are indicated in kilobase pairs atleft.

Figure 3. Schematic representation of genomic organization of IbMYBgenes, with exons (white boxes) and introns (lines between exons).Positions of primers (arrows) used for RT-PCR analysis (Fig. 5) areindicated, and resulting amplification products (lines) are shown.Product sizes from amplification of genomic DNA and fully processedmRNAs (shown in parentheses) are given for each PCR reaction. FA/RA2 and FB/RB2 annealed specifically to the IbMYB1 and IbMYB2genes, respectively.

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Figure 4. IbMYB genes show features of an R2R3-MYB-binding domain protein. A, Amino acid comparison of the R2 and R3DNA-binding regions of IbMYB1 and IbMYB2-1 with selected set of R2R3-type MYB factors from various plant species showshigh sequence conservation (amino acids identical with those of IbMYB1 are marked in gray). Previously published MYB geneamino acid sequences were retrieved from the DNA data bank of Japan/EMBL/GenBank databases (grape VvmybA1[BAD18977], grape VlmybA1-1 [BAC07537], morning glory Ipmyb1 [AAY54243], morning glory InMYB1 [BAE94389],morning glory InMYB2 [BAE94709], morning glory InMYB3 [BAE94710], petunia AN2 [V26 allele; AAF66727], tomato ANT1[AAQ55181], Arabidopsis PAP1 [AAG42001], Arabidopsis PAP2 [AAG42002], snapdragon ROSEA1 [ABB83826], gerberaGMYB10 [CAD87010], maize Pl [AAA19819], maize C1 [AAA33482], Arabidopsis TT2 [CAC40021], and strawberry FaMYB1[AAK84064]). The amino acid residues shown to be required for interaction of maize C1 with bHLH cofactor R (L77, R80, R83,and L84) are marked with asterisks. B, Deduced amino acid sequence of IbMYB1. The R2R3-binding domain is shaded in gray.Underlined sequence is aligned in Figure 4D. Boxed sequence is KPRPR(S/T) F-like motif. Splice-site locations of two introns aremarked with asterisks. C, Phylogenetic analysis of a selected set of R2R3-type MYB factors from various plant species.Phylogenetic trees were produced by the neighbor-joining method of Saitou and Nei (1987) for the aligned amino acidsequences of R2R3 DNA-binding domains. For construction of the trees, we used only the R2R3 MYB domain sequence (104





members had a length of 249 amino acids, with morethan 99% identity of amino acid sequences amongthe protein encoded by IbMYB2 members and around93% identity between that encoded by IbMYB1and IbMYB2 members. Clones belonging to IbMYB1,IbMYB2-1, IbMYB2-2, and IbMYB2-4 were found inboth Ayamurasaki and Kokei-14, and no differencesin the sequences of the areas tested were detected be-tween the two cultivars. In contrast, IbMYB2-3 was foundonly in Kokei-14.

Phylogeny of Sweet Potato R2R3-Type MYB Factors

The sequences of the IbMYB1 and IbMYB2 geneswere compared with those encoding other R2R3-type MYB factors in various plant species: VvmybA1(Kobayashi et al., 2004) and VlmybA1-1 (Kobayashiet al., 2002) from grape; Ipmyb1 (Chang et al., 2005),InMYB1, InMYB2, and InMYB3 (Morita et al., 2006)from morning glory (Ipomoea nil); AN2 (Quattrocchioet 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) frommaize; and FaMYB1 (Aharoni et al., 2001) from straw-berry (Fragaria spp.). The R2R3-binding domain of thededuced amino acid sequence encoded by the IbMYB1and IbMYB2 genes showed high sequence similaritywith those encoded by other MYB genes of other plantspecies (Fig. 4A), and splice-site locations and intronphases were conserved with the majority of the Arabi-dopsis (77 of 130) and rice (45 of 85) MYB genes (Fig.4B; Jiang et al., 2004). We performed phylogeneticanalysis of a selected set of R2R3-type MYB factorsfrom various plant species. Using the neighbor-joiningmethod of Saitou and Nei (1987), phylogenetic treeswere produced of the aligned amino acid sequences ofR2R3 DNA-binding domains (Fig. 4C). The result indi-cated that the IbMYB1 and IbMYB2 genes were locatedin the N09 subgroup comprising Arabidopsis PAP1 andPAP2 and petunia AN2 (Jiang et al., 2004). The pre-dicted C-terminal regions of the IbMYB1 and IbMYB2genes showed similarity to those of the InMYB2 andInMYB3 genes, but there was very limited sequenceidentity in the case of the other MYB proteins out-side the R2R3 domain, except for the presence of aKPRPR(S/T) F-like motif (boxed in Fig. 4, B and D),which was previously reported for the subgroupof R2R3 MYB genes comprising petunia AN2 and

Arabidopsis AtMYB75 (PAP1), AtMYB90 (PAP2), andAtMYB113 (Stracke et al., 2001).

Expression Analysis of IbMYB1 and IbMYB2s

The expression patterns of IbMYB1 and IbMYB2were analyzed by RT-PCR using specific primers: FA/RA2 for IbMYB1 and FB/RB2 for IbMYB2 members. Asshown in Figure 5, when genomic DNAs were used astemplates for the reaction, the band patterns wereconsistent with the predictions from Figure 3: that is, asingle band of about 800 bp was found in IbMYB1 andmultiple bands of about 900 to 1,250 bp in IbMYB2s.Whereas IbMYB1 mRNA accumulated predominantlyin the tuberous roots of Ayamurasaki and slightly inthe calli, no signals of IbMYB2 members were detectedin these tissues. Two transcripts of different sizes de-tected in the IbMYB1 mRNA amplification may indi-cate alternative splicing, as the smaller transcript wasconsistent in size with mature mRNA and the largertranscript with retention of the second intron. Whereasthe first intron of IbMYB1 was under the GT-AG rule,the second intron had GC-AG at the ends; this mayhave been the cause of the alternative splicing. How-ever, it is not clear whether the larger transcript of theIbMYB1 gene has any functions; many MYB genes ofArabidopsis and rice undergo alternative splicing andare suggested to have multiple biological processes, assome of these splice variants are differentially regu-lated (Li et al., 2006).

Tissue-Specific Expression of Genes Involved inAnthocyanin Biosynthesis and Accumulation

In Ayamurasaki, various tissues besides the tuber-ous roots, such as the nontuberous roots, stems (ST),young leaves (YL), stressed leaves (RL), flower buds(FB), and open flowers (FL), accumulate anthocyaninsand exhibit pigmentation. Anthocyanins were ex-tracted from each tissue and quantified (Fig. 6A), andexpression analysis of the genes involved in antho-cyanin biosynthesis and accumulation was performedat the same time (Fig. 6B).

The IbMYB1, IbMYB2, and IbMADS10 genes werecandidates for regulators of anthocyanin biosynthe-sis; CHS, CHI, F3H, DFR, ANS, and 3GT (encodingchalcone synthase, chalcone isomerase, flavanone-3-hydroxylase, DFR, anthocyanidin synthase, andflavonoid 3-glucosyl-transferase, respectively) weregenes involved in parts of the anthocyanin biosynthe-sis pathway; VP24 (encoding vacuolar protein) wasconsidered to be involved in anthocyanin vacuolar

Figure 4. (Continued.)amino acid residues; Fig. 4A) of each selected MYB-related protein. The matrix of sequence similarities was calculated by theClustal program (ClustalW 1.83; Thompson et al., 1994) and subjected to neighbor-joining analysis to generate branchingpatterns. N09, N08, and N14 are the subgroups clustered by Jiang et al. (2004). D, Amino acid comparison of C-terminal region.KPRPR(S/T) F-like motif is boxed. Genes harboring KPRPR(S/T) F-like motif correspond to the genes of the N09 subgroup inFigure 4C.

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transport and/or trapping (Nozue et al., 1997; Xuet al., 2001).

The structural anthocyanin genes (CHS to 3GT) wereexpressed in pigmented tissues, with the exception offlesh of stored (i.e. stored for few months before use)tuberous roots (sTF) and FL (Fig. 6B). IbMYB1 geneexpression was detected mainly in flesh of developing(i.e. used just after harvest) tuberous roots (dTF), lessin peeled outer layer (less than 1 mm thick) includingthe skins of developing and stored tuberous roots (dTSand sTS), and a little in sTF, red and white nontuber-ous roots (RR and WR), and ST. Slight expressionof 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 wasdetected in all tissues tested except for expanded greenleaves (EL).

Anthocyanin Accumulation and Gene Expressionin Seven Sweet Potato Cultivars

The relationship between anthocyanin accumula-tion and gene expression was examined in seven sweetpotato cultivars: three purple fleshed, one orange fleshed(containing carotenoids), two yellow fleshed, and onewhite fleshed (Fig. 7A). Among various sweet potatocultivars, the color and/or color intensity of the flesh

seems to be not related to those of other tissues, such asthe tuber skins, flowers, leaves, and stems. For exam-ple, the flesh colors were not related to the tuber skincolors or young leaf colors (Fig. 7A). The family treesof the cultivars used are shown in Figure 7B. The

Figure 5. Expression analysis of IbMYB1 and IbMYB2s. Genomic PCRand RT-PCR were performed using the FA/RA2 and FB/RB2 primer setsindicated in Figure 3. Amplified products (28 cycles) were size frac-tionated on a 1.0% agarose gel. Amplification of Actin was used as acontrol. A, Ayamurasaki; K, Koukei-14. Molecular marker sizes areindicated in kilobase pairs at left.

Figure 6. Anthocyanin accumulation and gene expression in Ayamur-asaki tissues. A, Photometric determination of anthocyanin content inmethanolic extracts of various tissues of Ayamurasaki. N (number ofsamples) is indicated below. Error bars represent SD. B, RT-PCR analysisof genes hypothesized to be involved in anthocyanin biosynthesis andaccumulation. Amplified products (30 cycles) were size fractionated ona 1.0% agarose gel. Actin was used as a control. See text for abbre-viation descriptions.





literature-based relative anthocyanin contents of ma-ture tuberous roots of the purple-fleshed cultivars arealso presented in Figure 7B, and our measurements arepresented in Figure 8A. Anthocyanins in the flesh oftuberous roots were detected only in the three purple-fleshed cultivars, but in the outer layer of the tuberousroots, they were also detected in orange- and yellow-fleshed cultivars (Fig. 8A).

All tested genes were detected in almost everycultivar tested by genomic PCR (Fig. 8B), with excep-tions 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 geneswere expressed predominantly in purple-fleshed cul-tivars (Fig. 8C). Expression of IbMYB2 was scarcelydetected in each cultivar. Expression of IbMADS10 andVP24 was not associated with anthocyanin content. Inthe peeled outer layer containing the skin, the expres-sion patterns of the genes encoding the transcriptionfactors were similar to those in the flesh. The structuralanthocyanin genes were expressed in the outer layersof the six red-skinned cultivars.

Transient Expression Analysis of IbMYB1

Using a particle gun, the IbMYB1 gene was tran-siently expressed in sweet potato leaves, tuberous roots,and calli under the control of the cauliflower mosaicvirus (CaMV)-derived 35S promoter or sweet potato-derived IT394 promoter (pIT394). The constructs usedfor the bombardment are shown in Figure 9. The greenfluorescent protein (GFP) gene was used as the markerof bombarded cells. Anthocyanin pigmentation wasobserved 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 GFPgene (Fig. 10, A and C), whereas no pigmentation wasdetected when the IbMYB1 antisense fragment wasintroduced with GFP (Fig. 10, B and D). The GFP fluo-rescence in most leaf cells diffused to neighboring cells,whereas the anthocyanin pigment stayed in the bom-barded cells. Detection of anthocyanin pigmentationwas delayed until 1 or 2 d after the detection of GFPfluorescence. The pigmentation deepened over morethan 1 week and then remained even after the GFP

Figure 7. Seven sweet potato culti-vars were tested: three purple-fleshedcultivars, one orange-fleshed cul-tivar, and three yellow or white-fleshed cultivars. A, Appearance offlesh or skin of mature tuberous rootsor young developing tuberous rootsor adaxial side of young leaf of theseven cultivars. AM, Ayamurasaki;MM, Murasakimasari; PS, PurpleSweet Lord; AK, Ayakomachi; KK,Kokei-14; BA, Beniazuma; JW, JoyWhite. B, Family trees of the sweetpotato cultivars used in the experi-ments. Purple- or orange-fleshed cul-tivars are indicated by purple ororange text. The cultivars usedare indicated by framed rectangles.Numbers in parentheses afterpurple-fleshed cultivar names arerelative anthocyanin contents oftheir tuberous roots. (Yamakawaet al., 1995, 1997, 1999; Kumagaiet al., 2002; Tamiya et al., 2003).

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fluorescence had disappeared. The speed of accumu-lation of GFP and anthocyanin was slower in the tu-berous roots than in the leaves. Differences in promoter(CaMV 35S or pIT394) or the cultivar of bombardedcells (Ayamurasaki or Kokei-14) did not affect these re-sults (data not shown).

Overexpression of IbMYB1 in Transgenic Plants

Forced expression analysis of the IbMYB1 gene wasalso performed in sweet potato calli and heterologousplants (Arabidopsis and rice) with stable transforma-tion. In Arabidopsis, overexpression of IbMYB1 induced

ectopic pigmentation in seedlings (Fig. 11A), roots (Fig.11B), flowers, leaves, and stems (Fig. 11C), and even inseeds (Fig. 11D). However, most of the plants withsevere pigmentation were sickly and could not survivewithout the addition of Suc to the culture medium. Inrice, we could not produce a pigmented plant. In sweetpotato, overexpression of IbMYB1 induced strong pig-mentation in transgenic calli (Fig. 12A). We obtained17 independent pigmented transgenic calli by trans-formation of about 3 g of embryogenic calli of Kokei-14.Ten of the 17 lines of pigmented transgenic calli grewwell and were subjected to anthocyanin quantificationand gene expression analysis. Sometimes untransformed

Figure 8. Anthocyanin accumulationand gene expression in different sweetpotato cultivars. A, Anthocyanin con-tents of mature tuberous roots (includingskin and flesh, grown in the field, longerthan 15 cm) and flesh or peeled outerlayer (less than 1 mm thick) including theskin of young developing tuberous roots(grown in the greenhouse, smaller than10 cm) of each cultivar were deter-mined. Three samples were tested in allexperiments. Error bars represent SD. B,Genomic PCR of 10 sweet potato culti-vars. About 30 ng of genomic DNA wassubjected to PCR. Amplified products(40 cycles) were size fractionated on1.0% agarose gel. Flesh colors of tuber-ous roots are indicated above the culti-var names. C, RT-PCR of flesh or outerlayer (including the skin) of young de-veloping tuberous roots of seven culti-vars. Amplified products (30 cycles forIbMYB1, IbMYB2s, and IbMADS10; 28cycles for the rest of the genes) were sizefractionated on 1.0% agarose gel. Actinwas used as a control. Flesh or skin colorsof tuberous roots are indicated abovethe cultivar names. AM, Ayamurasaki;MM, Murasakimasari; PS, Purple SweetLord; TM, Tanegashimamurasaki; AK,Ayakomachi; HK, Hamakomachi; SR,Sunny Red; KK, Kokei-14; BA, Beniazuma;JW, Joy White.





calli of Kokei-14 or Ayamurasaki show patchy pig-mentation (Fig. 12A), but we could not obtain calli asseverely and uniformly pigmented as the pHM158(35STIbMYB1) transformants when the calli were nottransformed or were transformed with other con-structs, such as pHM150b (35STGFP) or pHM159(35STanti-IbMYB1; data not shown). The amount ofanthocyanin in the transgenic calli was increased bynearly 200 times that in the nontransgenic Kokei-14calli (Fig. 12B) and was comparable to that in the tu-berous roots of purple-fleshed cultivars (Fig. 8A). Thegrowth rates of the transgenic callus lines were in-versely related to the levels of anthocyanin accumula-tion (Fig. 12C) and were much higher than those ofnontransgenic calli (Fig. 12B). We also assessed the in-fluence of light on anthocyanin accumulation andgrowth rate. There was a tendency for anthocyanin ac-cumulation to increase and growth rate to decrease incalli grown in light, and light-exposed nontransgeniccalli of Ayamurasaki showed significantly higher ratesof anthocyanin accumulation than did dark-exposedcalli of this cultivar (Fig. 12D).

The IbMYB1 gene and the structural anthocyanin geneswere predominantly expressed in the pigmented trans-genic calli and pigmented untransformed Ayamurasakicalli (Fig. 13). Scant expression of the IbMYB2 genesand the IbMADS10 gene was detected in some trans-genic or nontransgenic calli. The VP24 transcripts weredecreased when the calli (Ayamurasaki, Kokei-14, andpHM158-2) were grown in the dark, but the expressionpattern was not correlated with the anthocyanin con-tent. Whereas two transcripts of different sizes weredetected by RT-PCR analysis of IbMYB1 in untrans-formed Ayamurasaki tissues (Figs. 5, 6, and 13), a singleband (of a size consistent with introduced IbMYB1cDNA and the same size as the smaller transcriptdetected in untransformed tissues) was detected inpHM158 transgenic calli (Fig. 13).

DISCUSSION

The pathways of biosynthesis of flavonoid pigmentshave been investigated well, perhaps because of thecolor that they add to plants. Their regulatory genes inaerial parts such as flowers, leaves, seeds, and fruitshave been identified in various plant species, whereaslittle is known about their regulation in undergroundorgans such as tuberous roots. Although the role ofanthocyanins in underground organs is not clear, thefact that many sweet potato cultivars with not onlypurple flesh but also orange, yellow, or white fleshretain anthocyanins in the skin of their tuberous rootssuggests that they play an important role.

Sweet potato accumulates anthocyanins in varioustissues such as the flowers, leaves, stems, and non-tuberous roots, as well as the tuberous roots (Fig. 6A).The IbMYB1 gene was expressed predominantly intuberous roots, whereas the expression of IbMYB2genes was scarcely detected (Fig. 6B). WR was the onlytissue that showed expression of the IbMYB1 gene inthe absence of anthocyanin detection (Fig. 6). The factthat the expression levels of structural anthocyaningenes were similar to that in RR indicates that antho-cyanin biosynthesis was already active in WR but an-thocyanin had not accumulated to levels high enoughto detect. There was no tissue that expressed the IbMYB1gene without structural anthocyanin gene expression.We sometimes could not detect IbMYB1 expression intuberous roots when the samples had been stored for afew months (e.g. sTF in Fig. 6B; also in sTS in somecases) or in seed tubers (data not shown). These tissuesmay have been losing their anthocyanin biosynthesisactivity, because they also showed diminished expres-sion of the structural anthocyanin genes. In any case, astriking correlation of expression in the tuberous rootswas observed between IbMYB1 and the structural an-thocyanin genes.

We did not detect IbMYB1 expression in flowers andleaves despite the anthocyanin accumulation and thestrong expression of structural anthocyanin genes (YL,RL, and FB in Fig. 6). IbMYB1 expression was also notdetected in the skins of tuberous roots of non-purple-fleshed cultivars (Ayakomachi, Kokei-14, and Beniazumain Fig. 8C). These results suggest that IbMYB1 actsspecifically in the flesh of tuberous roots and that otherregulatory genes are active in the other tissues. Con-tamination of the outer layer samples with flesh tissuemay be the reason why IbMYB1 was detected in theouter layers of the purple-fleshed tuberous roots (dTSand sTS in Fig. 6; Ayamurasaki, Murasakimasari, andPurple Sweet Lord in Fig. 8C). The difference inanthocyanin composition of the outer layer and the in-ner portion of Ayamurasaki tuberous root (Yoshimotoet al., 1999a) may reflect a difference in the genesresponsible for the regulation of anthocyanin biosyn-thesis. The fact that, among various sweet potatocultivars, the color and/or color intensity of the fleshwere not related to those of other tissues (Fig. 7A) alsosupports these ideas.

Figure 9. Constructs used for transient expression analysis and forstable transformation. Plasmid vector names of pBluescriptII back-ground are represented. Names of binary vector (pPZP2H-lac) back-ground are represented in parentheses.

Mano et al.




The results of bombardment of the IbMYB1 geneinto leaves (Fig. 10A) indicated the existence of mech-anisms of anthocyanin biosynthesis common to dif-ferent tissues. However, Ayamurasaki leaves display acomplex change in color; they are red when they areyoung, turn green as they develop, and turn red againwith stress (YL, EL, and RL in Fig. 6A). Therefore, wecan assume that multiple regulatory genes and a deg-radation mechanism are active in these leaves.

This is, to our knowledge, the first report of theidentification of MYB genes in sweet potato, and othertranscription factors related to anthocyanin biosynthe-sis, such as the bHLH and WD40 genes, are still un-known in sweet potato. However, several members ofthe MYB, bHLH, and WD40 gene families have beenisolated from morning glory and their differentialexpression pattern reported; InMYB1 is expressed inthe flower and InMYB2 is expressed in the petiole,

Figure 10. Transient expression analy-sis of IbMYB1. GFP (pHM160) andIbMYB1 (pHM196) or IbMYB1 anti-sense (pHM197) were cobombardedinto sweet potato leaves (A and B) andsections of tuberous roots (C and D). Aand C, Cobombardment of pHM160and pHM196. B and D, Cobombard-ment of pHM160 and pHM197. Eachsection contains bright-field images(top) and fluorescent images (bottom).Times after bombardment are indicatedabove. Exposure times were identicalfor each bright-field image (0.2 s) andfluorescent image (2.0 s). Magnifica-tions were identical for all images.Scale bar in bottom left corner repre-sents 100 mm.





stem, and root, but expression of InMYB3 has not beendetected (Morita et al., 2006). According to phylogenicanalyses (Fig. 4C), the InMYB2 and InMYB3 genes aremore closely related to IbMYB1 and IbMYB2 than toInMYB1. These results indicate that the MYB genesworking in flowers and the MYB genes working inroots were divided before the division of morningglory and sweet potato.

A number of MADS-box genes have been isolatedfrom sweet potato, and one of them, IbMADS10, hasbeen suggested to be involved in anthocyanin biosyn-thesis (Lalusin et al., 2006). However, the level of cor-relation of this gene with anthocyanin accumulation orexpression of the structural anthocyanin genes was nothigh in our experiments. It is possible that another tran-scription factor for IbMADS10 activity, such as a bHLHtranscription factor that needs an MYB transcriptionfactor to activate it, is involved.

Sometimes red-pigmented cells appear in sweetpotato calli grown under light conditions (Fig. 12A).Nishimaki and Nozue (1985) used the yellow-fleshedcultivar Kintoki to establish cell lines (ALD and ALND)that accumulate anthocyanins. The ALD cell line pro-duced anthocyanins in the light but not in darkness,and the ALND line produced anthocyanins under bothlight and dark conditions. However, the content of an-thocyanins in the dark was one-fourth that in the light(Nozue et al., 1997). On the other hand, our transgeniccalli lines, made by forced expression of the IbMYB1gene in the yellow-fleshed cultivar Kokei-14, accu-mulated anthocyanins in a light-independent mannerat high levels comparable to those in the tuberousroots of purple-fleshed cultivars (Figs. 8A and 12B).These features are similar to those of cell lines selectedand established from Ayamurasaki tuberous roots(Konczak-Islam et al., 2000). It is possible that accu-mulation of anthocyanin in calli in a light-dependent

manner is caused by the activation of regulator genesthat act in the aerial parts and light-independent accu-mulation is caused by the activation of regulator genesthat act in the underground organs.

Light induced anthocyanin accumulation inAyamurasaki calli (Fig. 12D); sometimes IbMYB1 ex-pression was detected in this tissue (Fig. 13) and some-times not. Unlike cell lines, the callus samples may nothave been homogeneous and may have included di-verse cells pigmented by the activation of variousregulatory genes.

However, the determinant of growth rate was notclear; the growth rates of most of the transgenic calliwere much higher than those of the nontransgenic calli(Fig. 12B). It is conceivable that the accumulated an-thocyanins protected the callus from the inhibitoryeffects of light and thus promoted their propagation.On the other hand, the growth rates of the transgeniccallus lines were inversely related to the levels ofanthocyanin accumulation (Fig. 12C). It is possible thatthe growth rates of some transgenic lines were so fastthat anthocyanin accumulation did not reach satura-tion in these lines. The fact that the growth rates of alltransgenic lines exceeded 2.0 (i.e. the weight of the callidoubled in 2 weeks; Fig. 12B), whereas the anthocya-nin contents of the leaf or tuber cells increased formore than 1 week (Fig. 10, A and C), may support thisassumption.

The fact that overexpression of the IbMYB1 geneinduces ectopic pigmentation not only in sweet potatobut also in heterologous Arabidopsis (Fig. 11) suggestsa common regulatory mechanism. On the other hand,the fact that most of the transgenic Arabidopsis plantswith severe pigmentation were sickly and unable tosurvive without Suc in the culture medium suggests adifference among species. Quattrocchio et al. (1998) andGong et al. (1999) also found common and different

Figure 11. Overexpression of IbMYB1 in Arabidopsisunder the control of the CaMV 35S promoter. Five-day-old seedlings (A), a root (B), flowers, leaves, andstems (C), and seeds (D) of Arabidopsis plants trans-formed with the construct pHM158 (CaMV35ST

IbMYB1) accumulated ectopic pigmentation.

Mano et al.




Figure 12. Overexpression of IbMYB1 in sweet potato calli under the control of the CaMV 35S promoter. A, Kokei-14-derivedcalli transformed with the construct pHM158 (CaMV35STIbMYB1) turned a deep purple. The calli of 10 independent transgeniclines and untransformed calli derived from Ayamurasaki (Aya) and Kokei-14 (Ko) were grown under light conditions. One of thetransgenic callus lines (pHM158-2) and the untransformed calli were also grown under dark conditions. B, Growth rate andanthocyanin content of each callus were determined. The fresh weight (FW) of each callus after culture for 2 weeks was dividedby the initial fresh weight (FWi) to determine the growth rate. 1 to 15, pHM158-1 to 15 transgenic lines; D, grown in darkconditions; L, grown under light conditions. N (number of samples) is indicated below. Error bars represent SD. C, Relationship





actions of anthocyanin regulatory genes in heterogonousplants; ectopic expression of petunia AN2 and JAF13induced CHS gene expression in heterologous maizebut not in petunia. Both bHLH alleles of perilla, MYC-RP and MYC-GP, induced anthocyanin accumulationin heterologous tobacco (Nicotiana tabacum) plants,whereas only MYC-RP acted as a regulator in perilla.

The biosynthesis of anthocyanins occurs in the cy-tosol, whereas the end products accumulate in thevacuole. This may be the reason why the anthocyaninpigment induced by bombardment with IbMYB1 re-mained in the bombarded cells, whereas the GFP fluo-rescence diffused to other cells. It is proposed thatglutathione-S-transferase-like proteins such as maizeBZ2 (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 pro-tein (MRP)-type transporter ZmMRP3 (Goodman et al.,2004) or the Arabidopsis multidrug and toxic com-pound extrusion transporter TT12 (Debeaujon et al.,2001). In sweet potato, it has been suggested that VP24precursor protein is involved in vacuolar transportand/or accumulation of anthocyanin (Nozue et al.,1997; Xu et al., 2001).

Transient and stable forced expression analysis ofIbMYB1 suggested that IbMYB1 induces not only struc-tural anthocyanin genes but also anthocyanin trans-porters such as ZmMRP3 that are expressed under thecontrol of the regulators of anthocyanin biosynthesis,R and C1 (Goodman et al., 2004). We examined whetherIbMYB1 induced VP24 expression (Fig. 13). The VP24gene was expressed not only in transgenic callibut also in unpigmented nontransgenic light-grownKokei-14 callus, but it was scarcely detected in dark-grown pHM158-2 callus and light-grown pHM158-12callus. These results deny the inductivity of VP24 geneexpression by IbMYB1. We also examined the expres-sion pattern of VP24 in sweet potato tissues (Figs. 6Band 8C). The relatively abundant expression in oldertissues (i.e. more abundant in sTF, sTS, and FL than indTF, dTS, and FB in Fig. 6B) suggests that VP24 acts inthe retention of anthocyanin rather than in transportand/or accumulation. There may be other IbMYB1-inducible genes that are involved in anthocyanin trans-port and accumulation in sweet potato.

However, the IbMYB1 transcript was not detected inthe tissues of Kokei-14, and there were no differencesin base sequences of the IbMYB1 coding region be-tween Ayamurasaki and Kokei-14. The fact that theoverexpression of IbMYB1 in Kokei-14 induced a largeamount of anthocyanin pigmentation suggests that mu-tation(s) on the promoter region of the IbMYB1 genecaused alteration in the flesh color of sweet potato,

as in the case for grape skin color; the insertion of a ret-rotransposon into the 5#-flanking region of VvmybA1was associated with loss of pigmentation in white-skinned cultivars, whereas the coding sequences wereidentical with those of red-skinned cultivars (Kobayashiet al., 2004). Alternatively, mutation(s) on regulator(s)of the IbMYB1 gene may have caused the alteration offlesh color, although the regulators of the anthocyaninregulatory genes are unknown.

All three purple-fleshed cultivars expressed theIbMYB1 gene in their tuberous roots (Fig. 8C). Presum-ably, they inherited the active promoter of the IbMYB1gene from a common ancestor, Yamagawamurasaki(Fig. 7B). However, their contents of anthocyanin differ

Figure 13. RT-PCR analysis of the genes hypothesized to be involved inanthocyanin biosynthesis and accumulation was performed in sweetpotato calli. Amplified products (30 cycles for IbMYB1, IbMYB2s, andIbMADS10; 28 cycles for the rest of the genes) were size fractionated on1.0% agarose gel. Actin was used as a control.

Figure 12. (Continued.)between growth rates and anthocyanin content of transgenic callus lines. D, Influence of light conditions on growth rates andanthocyanin content. The anthocyanin content of Ayamurasaki calli was increased significantly by light.

Mano et al.




considerably (Fig. 7B). Interaction of IbMYB1 withother transcriptional factors may affect anthocyaninbiosynthesis 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 L21 myo-

inositol, 0.4 mg L21 thiamine-HCl, 3% [w/v] Suc, 0.05% [w/v] MES, pH 5.7,

1 mg L21 4-fluorophenoxyacetic acid, 0.8% [w/v] agarose) in growth cham-

bers at 26�C in the dark, and transformed calli were cultured on selection

media (Murashige and Skoog Plant Salt mixture [Wako], 100 mg L21 myo-

inositol, 0.4 mg L21 thiamine-HCl, 3% [w/v] Suc, 0.05% [w/v] MES, pH 5.7,

1 mg L21 4-fluorophenoxyacetic acid, 25 mg L21 hygromycin B, 500 mg L21

carbenicillin, 0.8% [w/v] agarose) in growth chambers at 26�C under 16 h of

light (around 40 mmol m22 s21) 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] b-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 isola-

ted 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

SK1 (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.

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#-GTG-

AGAAAAGGTTCATGGTCC-3#) and 666-R13 (5#-CTTCTTCTGAAGATG-

GGTGTTC-3#), and 666-F9 (5#-GTGTCTGCCATGGCTTCTTCAA-3#) and

666-gRp (5#-TGGCTGCAGATTACATTCTCAAATTTAATCGTACA-3#), respec-

tively. 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#-ATGGATCCTAAGAATTTCCGA-

CACCCTTC-3#) and 666-R (5#-CGGTGTTTTCCGTGATTTCT-3#) or 666-F3

(5#-CTCATTCTGCGCCTCCATAG-3#) and 666-R14 (5#-GGTGGCAATAGAA-

Table I. Primers used in RT-PCR experiments

Primer Name Forward Primer Reverse Primer

IT1-F/R 5#-CGGAACGTTCTTAGAAAAGCTG-3# 5#-GGCCAGAAGAAGGTGTACGA-3#IT4-F/R 5#-CCATACCAGCTCGGATTTGT-3# 5#-TGGATGCCAACCTTAACTCC-3#IT4785-F/R 5#-GAAACCGCACCAGTCGATAG-3# 5#-GCGAAGACGAGGTCTTGATAG-3#IT666-F/R 5#-GCGAATTTAGTCCCGATGAA-3# 5#-CGGTGTTTTCCGTGATTTCT-3#IT4097-F/R 5#-CATGCTCTCCAAACAATGGA-3# 5#-GTCTGCAGCAAGAAGGTTCC-3#IT948-F/R 5#-TGTGGAATTCGAAAGCTGAG-3# 5#-CCAAACAACACAACAATCCAA-3#IbMYB1-FA/RA2 5#-TATGGTCGGGATCGTCTTCG-3# 5#-TTCTGAAGATGGGTGTTCCAT-3#IbMYB2s-FB/RB2 5#-TATGGTCGGAATCGTCTTCC-3# 5#-TTCTGAAGATGGGTGTTCCAG-3#Act-F/R 5#-GACTACCATGTTCCCCGGTA-3# 5#-TTGTATGCCACGAGCATCTT-3#CHS-F/R 5#-GGACTACCAGCTCACCAAGC-3# 5#-GTCCTCCACTTGGTCCAGAA-3#CHI-F/R 5#-GTTAAGTGGAACGGGAAAAG-3# 5#-GAGACGACCGTTTGTGGAAT-3#F3H-F/R 5#-CGAGATTCCGGTGATATCGT-3# 5#-GGGGCATTTTGGGTAGAAAT-3#DFR-F/R 5#-TCCTGGGAACACAAAGAAGG-3# 5#-GAGCTTCGCAGAGATCATCC-3#ANS-F/R 5#-ATTTTCGCGGAGGAAAAGAT-3# 5#-CTTCCTTCTCCAGCCTTCCT-3#3GT-F/R 5#-AAGTATCGATCGGCGAAATG-3# 5#-CACGATATGGCCTCCAGAGT-3#IbMADS10-F/R 5#-CGAACTGAAGCGGATTGAGAAC-3# 5#-CCAGCTGTTGCTCTAAACTCTG-3#VP24-F/R 5#-CTTGACACTGCCCTCCAGTATG-3# 5#-ACGAGCAAGCTCCAACATAACA-3#





TTTAAATCAAG-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#-TGGTCTAGAGACTTTT-

CAACAAAGGGTAAT-3#) and 35S-Rb (5#-CGGGATCCTCTCCAAATGAAA-

TGAACTTCC-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#-ATGCGG-

CCGCTAAGAATTTCCGACACCCTT-3#). pHM156 and pHM157 were made

by ligation of the IbMYB1 ORF and the IbMYB1 antisense fragment, respec-

tively, 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

(35STIbMYB1) or pHM157 (35STanti IbMYB1), and pHM160 (pIT394TGFP)

and pHM196 (pIT394TIbMYB1) or pHM197 (pIT394Tanti 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 L21 myo-inositol, 0.4 mg L21 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 mmol m22 s21) 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 fluores-

cence 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 tume-

faciens 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 trans-

formed by the floral dip method (Clough and Bent, 1998).

Anthocyanin Quantification

Extraction and quantification of anthocyanins was performed in accor-

dance 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 fol-

lowing equation: Q (anthocyanins) 5 (A530 2 0.25 A657) 3 M21, 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.

Accession Numbers

Accession numbers for the genes isolated in this article are as follows:

AB258984 (IbMYB1 cDNA), AB258985 (IbMYB1 genome), AB258986 (IbMYB2-1

genome), AB258987 (IbMYB2-2 genome), AB258988 (IbMYB2-3 genome), and

AB258989 (IbMYB2-4 genome).

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.

Received December 8, 2006; accepted December 20, 2006; published January 5,

2007.

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