Li-Wei Chiu2,Xiangjun Zhou,Sarah Burke,Xianli Wu,Ronald L.Prior,and Li Li*

Department of Plant Breeding and Genetics(L.-W.C.,X.Z.,S.B.,L.L.)and Robert W.Holley Center for Agriculture and Health,United States Department of Agriculture-Agricultural Research Service(L.L.), Cornell University,Ithaca,New York14853;and United States Department of Agriculture-Agricultural Research Service,Arkansas Children’s Nutrition Center,Little Rock,Arkansas72202(X.W.,R.L.P.)

Anthocyanins are responsible for the color of manyflowers,fruits,and vegetables.An interesting and unique Purple(Pr)gene mutation in cauliflower(Brassica oleracea var botrytis)confers an abnormal pattern of anthocyanin accumulation,giving the striking mutant phenotype of intense purple color in curds and a few other tissues.To unravel the nature of the Pr mutation in cauliflower,we isolated the Pr gene via a combination of candidate gene analysis andfine mapping.Pr encoded a R2R3MYB transcription factor that exhibited tissue-specific expression,consistent with an abnormal anthocyanin accumulation pattern in the mutant.Transgenic Arabidopsis(Arabidopsis thaliana)and cauliflower plants expressing the Pr-D allele recapitulated the mutant phenotype,confirming the isolation of the Pr gene.Up-regulation of Pr specifically activated a basic helix-loop-helix transcription factor and a subset of anthocyanin structural genes encodingflavonoid3’-hydroxylase,dihydroflavonol 4-reductase,and leucoanthocyanidin dioxygenase to confer ectopic accumulation of pigments in the purple cauliflower.Our results indicate that the genetic variation including a Harbinger DNA transposon insertion in the upstream regulatory region of the Pr-D allele is responsible for the up-regulation of the Pr gene in inducing phenotypic change in the plant.The successful isolation of Pr provides important information on the regulatory control of anthocyanin biosynthesis in Brassica vegetables,and offers a genetic resource for development of new varieties with enhanced health-promoting properties and visual appeal.

Vegetables and fruits are fundamental components of human diets.They not only are important sources of essential vitamins and minerals,but also contain a wide variety of secondary metabolites important to human health.Colored vegetables and fruits have gained an increasing interest as functional foods, owing to their high levels of plant pigments with po-tent nutritional and health-promoting effects.Among them,purple cauliflower(Brassica oleracea var botrytis)is a very eye-catching vegetable and available commer-cially.The purple coloration is due to the accumulation of anthocyanins.

Anthocyanins are a group offlavonoid compounds that fulfill important biological functions in protecting plants against various biotic and abiotic stresses.All of the anthocyanin biosynthetic pathway genes and nu-merous regulatory factors have been identified from studies of Arabidopsis(Arabidopsis thaliana),maize (Zea mays),petunia(Petunia hybrida),snapdragon(An-tirrhinum majus),and other plant species(Broun,2005; Dixon et al.,2005;Koes et al.,2005;Grotewold,2006). Transcriptional regulation of structural genes appears to be a major mechanism by which anthocyanin bio-synthesis is regulated in plants.R2R3MYB and basic helix-loop-helix(bHLH)transcription factors as well as WD40proteins represent the three major families of anthocyanin regulatory proteins(Paz-Ares et al.,1987; Chandler et al.,19;Ludwig and Wessler1990;de Vetten et al.,1997;Quattrocchio et al.,1999).They form regulatory complexes to activate expression of antho-cyanin structural genes(Goff et al.,1992;Grotewold et al.,2000).In Arabidopsis,several MYB proteins, including PAP1,PAP2,MYB113,MYB114,and MYBL2 (Borevitz et al.,2000;Dubos et al.,2008;Gonzalez et al., 2008;Matsui et al.,2008),three bHLH proteins of TT8, GL3,and EGL3(Nesi et al.,2000;Payne et al.,2000; Zhang et al.,2003),and a WD40repeat protein of TTG1 (Walker et al.,1999)are involved in anthocyanin biosynthesis.While the R2R3MYB proteins of PAP1, PAP2,MYB113,and MYB114cause tissue-specific an-thocyanin accumulation in Arabidopsis(Borevitz et al.,2000;Gonzalez et al.,2008),the R3-MYB protein, MYBL2,acts as an inhibitor of anthocyanin biosynthe-sis(Dubos et al.,2008;Matsui et al.,2008).TT8is required for the full transcriptional activation of late anthocyanin pathway genes(Nesi et al.,2000),and is partially functionally redundant with its closest ho-

1This work was supported by the U.S.Department of Agricul-ture-Agricultural Research Service base fund.

2Present address:Pioneer Hi-Bred International Inc,4010Point Eden Way,Hayward,CA94545.

*Corresponding author;e-mail ll37@cornell.edu.

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: Li Li(ll37@cornell.edu).

[W]The online version of this article contains Web-only data.

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

www.plantphysiol.org/cgi/doi/10.1104/pp.110.1160

mologs,GL3and EGL3(Zhang et al.,2003).The WD40protein,TTG1,is known to physically interact with the MYB and bHLH transcription factors in controlling anthocyanin biosynthesis (Zhang et al.,2003).

R2R3MYB transcription factors have been impli-cated to play an important role for color difference in plant species.Activation of an R2R3MYB transcrip-tion factor confers anthocyanin production in a num-ber of anthocyanin-accumulating plants (Borevitz et al.,2000;Mathews et al.,2003).In contrast,loss of function of a R2R3MYB results in color loss in the normal anthocyanin-accumulating tissues.For exam-ple,a retrotransposon insertion in the promoter of Vv MYBA1and mutations in the adjacent Vv MYBA2inactivate their expression and convert red-skinned grape (Vitis vinifera )into white-skinned one (Walker et al.,2007).In Antirrhinum majus ,alteration of MYB -related gene expression controls floral pigmentation intensity and pattern (Schwinn et al.,2006).

Similarly,bHLH transcription factors have also been found to be responsible for color difference in plant species.Alteration of a bHLH transcription factor causes an increase in red pigment production in the aleurone of maize (Burr et al.,1996).Knockout of a bHLH function by a frame shift changes seed pericarp color from red into white in rice (Oryza sativa ;Sweeney et al.,2006).An insertion of a DNA transposon into a bHLH regulatory gene alters flower color of the com-mon morning glory (Ipomoea tricolor )into pale pig-mented flowers (Park et al.,2007).

Mutant analyses have facilitated gene discovery and elucidation of the regulatory control of anthocyanin biosynthesis.Although there are large numbers of researches on the underlying mechanisms controlling anthocyanin production in flowers,fruits,and model plants,only few have focused on vegetables.Purple cauliflower mutant represents an interesting mutation that confers profound anthocyanin production in the otherwise low-pigmented curds and seeds—the two tissues of agricultural importance.Thus,this mutant provides an excellent opportunity to reveal regulatory control of anthocyanin biosynthesis in vegetable crops.The cauliflower purple mutation was found to be controlled by a single,semidominant gene.We desig-

nate the symbol Pr -D for the Purple allele,and pr for its wild-type counterpart.Through a combination of can-didate gene analysis and fine mapping,we isolated the Pr gene and found that it encoded a R2R3MYB transcription factor.Comparison of Pr sequences from wild type and the mutant allele revealed allelic vari-ation including a Harbinger DNA transposon inser-tion in the upstream regulatory region of the Pr -D allele.Such an alteration caused an increased Pr gene transcription,which in turn up-regulated anthocyanin structural gene expression to produce the striking purple phenotype.The activation of the Pr gene,probably by introduction of new regulatory motifs in the promoter region,provides a way that anthocyanin transcriptional regulation can be switched on differ-ently in plants.

RESULTS

Phenotypic Characterization of the Purple Cauliflower Mutant

The cauliflower purple mutant plants grew and developed normally when compared to white cauli-flower under normal growth conditions in field and in greenhouse.The mutant exhibited a tissue-specific pattern of anthocyanin accumulation.Intense purple coloration was observed in curd and a few other tissues of the plant (Fig.1,A–D,F,and G),including young seedlings,very young leaves,and very young flower buds and siliques,as well as the endosperm of seeds,a tissue that is rarely reported to accumulate pigments in anthocyanin-accumulating mutants,e.g.in red cabbage (Brassica oleracea var capitata ;Yuan et al.,2009).The purple color was not observed in older leaves,stems,flower petals,and older siliques (Fig.1,B,E,and F).The purple phenotype appears to be associated primarily with very young tissues,curds,and seeds,rather than in flower petals,the most common anthocyanin-accumulating tissue.Under the same growth conditions,the wild-type cauliflower plants exhibited no purple hue in these tissues (Fig.1,

A–G).

Figure 1.Phenotypic comparison between wild type and the cauliflower Pr -D mutant.A,Y oung seedlings of 5-d-old plants.B,Y oung plants of 3-week-old.C,Curds of cauliflower plants grown in field.D,Y oung flower buds.E,Flowers.F,Y oung (inner)and old siliques of wild type (left)and mutant (right).G,Seed endosperms.

Anthocyanin Biosynthesis in Purple Cauliflower

Purple Mutant Predominantly Accumulates Cyanidin Glucosides

To examine the composition and content of antho-cyanins accumulated in young leaves,curds,and seeds of the purple mutant,we performed HPLC analysis and found high levels of anthocyanins in the purple tissues (Fig.2A).The curds accumulated ap-proximately 3.75mg cyanidin diglucoside equivalent g 21fresh weight,a level that was comparable with that found in blueberries (Vaccinium myrtillus ;Gao and Mazza,1994).In contrast,the samples of the wild-type control plants contained undetectable amounts of anthocyanins (data not shown),indicating that the anthocyanin pathway was biochemically quiescent under normal growth conditions.

Several different groups of anthocyanins (e.g.del-phinidin,pelargonidin,and cyanidin)exist in the plant kingdom (Tanaka et al.,2008).To identify antho-cyanin composition in the mutant,anthocyanins in curd tissue were separated and analyzed using HPLC-electrospray ionization (ESI)-tandem mass spectrome-try (MS/MS;Wu and Prior,2005b).The purple mutant contained one major peak (Fig.2B),which was iden-tified as cyanidin 3-(coumaryl-caffeyl)glucoside-5-(malonyl)glucoside (Fig.2C).The other minor peaks were also identified as different forms of cyanidin glycosides based on the dominant ion pairs observed in the mass spectrometry data and anthocyanin struc-tures (Mazza and Miniati,1993;Wu and Prior,2005a).

The Purple Mutation Is Controlled by a Single Semidominant Gene

To examine whether the purple mutant was con-trolled by one or multiple genes,we crossed the purple mutant Graffiti to an inbred white cultivar Stovepipe,selfed the heterozygous F1plants,and generated a large F2population.A subpopulation of 102individ-uals was germinated in a greenhouse.Genotyping of these F2individuals was completed by visually exam-ining the absence and presence of light or dark purple color in very young leaves and curds of the progeny,followed by further confirmation of the individual genotype in some cases by visual examination of the color with 16F3individuals.The segregation ratio of these F2plants for white:light purple:dark purple was 31:53:18.x 2test showed a good fit with a 1:2:1ratio (P .0.05),which was consistent with the ratio ex-pected for the progeny derived from selfing a parent heterozygous at one locus.This result suggests that the purple phenotype is controlled by a single,semi-dominant gene,Pr .

Expression of Anthocyanin Biosynthetic and Regulatory Genes

To investigate whether Pr represented one of the biosynthetic genes that was significantly up-regulated in the Pr -D mutant,we examined the expression of anthocyanin pathway genes in curds and leaves of wild type and the Pr -D mutant by northern-blot anal-ysis.The cDNAs coding chalcone synthase (CHS ),chal-cone isomerase (CHI ),flavanone 3-hydroxylase (F3H ),flavonoid 3’-hydroxylase (F3’H ),dihydroflavonol 4-reduc-tase (DFR ),leucoanthocyanidin dioxygenase (LDOX ),and UDP-glucosyltransferase (UGT )were used as probes.While the early pathway genes were expressed at similar levels between wild type and the Pr -D mutant in both leaves and curds,the late pathway genes,BoF3’H ,BoDFR ,and BoLDOX ,were dramatically up-regulated in the mutant (Fig.3A).The co-up-regulation of three structural genes suggests that Pr is unlikely a mutation of one specific anthocyanin pathway gene.Many regulatory genes that control anthocyanin biosynthesis have been isolated from plant species (Paz-Ares et al.,1987;Chandler et al.,19;Ludwig and Wessler 1990;de Vetten et al.,1997;Quattrocchio et al.,1999).Since cauliflower genes typically share high coding sequence identity with Arabidopsis genes,the available sequences of anthocyanin regula-tory genes from Arabidopsis and the

significant

Figure 2.Analysis of anthocyanin content and composition in the Pr -D mutant.A,Total anthocyanin levels as cyanidin diglucoside equivalent in curds,leaves,and seeds.FW,Fresh weight.B,HPLC elution profile of an-thocyanins accumulated in curds.The absorbance was monitored at A 520nm.C,Major anthocyanins identified by HPLC-ESI-MS/MS in curds.

Chiu et al.

amounts of Brassica sequence information in the public domains provide direct resources for designing the gene-specific primers for some cauliflower homologs.In Arabidopsis,the R2R3MYB family proteins of PAP1,PAP2,MYB113,and MYB114,bHLH proteins of TT8and EGL3,and WD40protein of TTG1are known to regulate anthocyanin biosynthesis.To in-vestigate whether the Pr gene represented a mutation of one of the known regulatory genes,transcript levels of these homologous genes in wild type and the Pr -D mutant were examined by quantitative reverse tran-scription (qRT)-PCR using gene-specific primers (Yuan et al.,2009;Supplemental Table S1).As shown in Figure 3B,P AP -like MYB family genes,i.e.BoMYB2and BoMYB4,as well as BobHLH1,a homologous gene of Arabidopsis TT8(Nesi et al.,2000)exhibited differ-ential expression in both leaves and curds between wild type and the Pr -D mutant.

Anthocyanins in the Pr -D mutant exhibited tissue-and development-specific accumulation (Fig.1).To examine whether the specific anthocyanin accumula-tion pattern was correlated with gene expression pat-tern,we analyzed transcript levels of BoCHS and the differentially expressed genes in different tissues of wild-type and the Pr -D plants.Similar level of BoCHS

transcript was observed in tissues between wild type and the Pr -D mutant.In contrast,the transcripts of BoF3’H ,BoDFR ,and BoLDOX ,as well as BoMYB2and BobHLH1accumulated highly in very young leaves,curds,very young flower buds,and young siliques in the mutant.The expression patterns of these genes were consistent with the tissue-specific anthocyanin accumulation pattern in the Pr -D mutant.

Identification of a MYB Gene That Cosegregates with Pr -D

To test whether BobHLH1or one of the PAP -like MYB family genes represented Pr ,association map-ping was carried out to investigate whether any of them cosegregated with the Pr -D locus.To develop markers for association mapping,full genomic frag-ments of BobHLH1and four BoMYB genes including promoter sequences were isolated through genomic DNA walking from both wild type and the Pr -D mutant.Comparison of the DNA sequences between wild type and mutant allele revealed that there were no polymorphisms for BobHLH1and BoMYB1,sug-gesting that they were unlikely the gene representing Pr .Based on different insertion/deletion or

single

Figure 3.Expression of anthocyanin structural and regulatory genes in wild type and the Pr -D mutant.A,northern-blot analysis of transcript levels of anthocyanin pathway genes in curds and leaves.BoAct serves as equal loading control.P ,Pr -D mutant;W,wild-type control.B,qRT-PCR analysis of anthocyanin regulatory genes in curds and leaves.The identities of the amplified prod-ucts were confirmed by sequencing.Expression of genes in wild-type leaves and curds were set to 1.WL,Wild-type leaves;PL,Pr -D leaves;WC,wild-type curds;PC,Pr -D curds.BoWD40,BobHLH1,and BobHLH2are cauliflower homologs of Arab-idopsis TTG1,TT8,and EGL3,respectively.C,qRT-PCR analysis of transcript levels of BoCHS and the differentially expressed genes during different developmental stages.Expression of genes in wild-type (WT)young leaves was set to 1.YL,Y oung leaves;OL,old leaves;C,curds;YFB,young flower buds;P ,petals;YS,young siliques;OS,old siliques;R,roots.qRT-PCR re-sults represent mean values +SD from three biological replicates with three technical repli-cates for each.

Anthocyanin Biosynthesis in Purple Cauliflower

nucleotide polymorphisms in BoMYB2,BoMYB3,and BoMYB4,PCR-based markers were developed (Sup-plemental Table S1).These three MYB genes were found to cosegregate with the Pr -D locus in a mapping population of 102F2plants.

To define the gene that represented Pr ,the three BoMYB genes were mapped in a large mapping pop-ulation of 1,8F2individuals.Two and seven recom-binant events were detected for BoMYB3and BoMYB4marker,respectively.In contrast,no recombinant event was observed for BoMYB2marker,indicating that BoMYB2cosegregated with the Pr -D locus and most likely the Pr gene.A high-resolution genetic map for this region is shown in Figure 4A.

Structural Analysis of Pr

The Pr gene from both wild-type and mutant plants contained an open reading frame of 744bp that en-coded a protein of 247amino acids with an estimated molecular mass of 27.99kD.Pr encoded BoMYB2that was predicted to be a nuclear protein (WoLF PSORT,wolfpsort.org)and contained R2and R3MYB repeat domains with the signature motif of KPRPR[S/T]F,specific for MYB proteins that activate anthocyanin biosynthesis (Stracke et al.,2001;Fig.4B).Noticeably,the KPRPR[S/T]F motif overlaps with a TAS4-siR81target site identified in Arabidopsis P AP1and P AP2(Rajagopalan et al.,2006),suggesting a possible

addi-

Figure 4.High-resolution genetic map of the Pr region,MYB sequence align-ment,and phylogenetic tree of BoMYB homologs.A,Linkage map of BoMYB2,BoMYB3,BoMYB4,and the Pr -D locus in a mapping population of 1,8F2individuals.B,Sequence alignment of cauliflower and Arabidopsis R2R3MYB proteins.R2and R3repeat do-mains as well as the conserved KPRPR [S/T]F motif are indicated.C,Phyloge-netic tree of BoMYBs and R2R3MYBs from other plant species.Numbers along branches indicate bootstrap sup-port determined from 1,000trials.The length of the branch lines indicates the extent of divergence.The GenBank accession numbers of these proteins are provided in Supplemental Table S2.

Chiu et al.

Functional Complementation of the Purple Mutant Phenotype in Arabidopsis and Cauliflower

To confirm the function of Pr,genomic fragments of the Pr gene from both wild-type and mutant plants were introduced into wild-type Arabidopsis and cau-liflower.Over40independent transgenic Arabidopsis lines were generated for each construct.Like the vector-only control(Fig.5A,1–6),the transgenic Arab-idopsis expressing the wild-type pr allele under the control of endogenous promoter(pr pro:pr)exhibited no or very low levels of pigmentation(Fig.5C,1–6).In contrast,the Pr-D(Pr pro:Pr-D)transformants showed tissue-specific anthocyanin accumulation(Fig.5B, 1–6).Purple pigments accumulated mainly in young tissues of Arabidopsis,such as young leaves and youngflower buds,and in seeds,but not in old leaves,flower petals,or mature siliques.A similar pattern of anthocyanin accumulation was also observed in trans-genic cauliflower plants expressing the Pr-D allele (Fig.5G,1–6).Ectopic expression of the Pr-D allele under the control of endogenous promoter in both transgenic Arabidopsis and cauliflower induced tis-sue-specific anthocyanin accumulation,which showed the same anthocyanin accumulation pattern as in the Pr-D mutant.Thus,these results further confirm the isolation of the Pr gene.

Both wild-type and mutant Pr genes under the control of cauliflower mosaic virus(CaMV)35S (35S pro:pr and35S pro:Pr-D)were also introduced into wild-type Arabidopsis and cauliflower.Overexpres-sion of either wild-type or mutant gene resulted in the production of dark-purple transgenic plants with high levels of anthocyanin accumulation in Arabidopsis (Fig.5,D and E,1–6).Pigments accumulated in the

entire transgenic plants that included leaves,roots,flower buds,flower petals,siliques,and seeds.Simi-larly,when these gene constructs were introduced into wild-type cauliflower plants,anthocyanins appeared in the entire transgenic plants(Fig.5I,1–6and J,1and 2).Interestingly,the young top curd tissues of some overexpression lines were white(Fig.5,I and J,2)with the below curd tissues purple(Fig.5I,4).These results suggest that both wild-type and mutant genes encode functional proteins.Pr Specifically Regulates a bHLH Transcription Factor and a Subset of Genes Involved in

Anthocyanin Production

A number of late anthocyanin pathway genes and a transcription factor BobHLH1along with Pr were expressed highly in the Pr-D mutant(Fig.3).To investigate whether expression of Pr in transgenic cauliflower caused such specific activation of gene expression,transcript levels of anthocyanin

biosyn-Figure5.Complementation of the purple phenotype in wild-type Arabidopsis and cauliflower.Top images:Arabidopsis transgenic lines containing empty vector(VC),the Pr-D allele with endogenous pro-moter(Pr pro:Pr-D),the wild-type pr allele with endogenous promoter (pr pro:pr),the Pr-D allele with CaMV35S promoter(35S pro:Pr-D),and the wild-type pr allele with CaMV35S promoter(35S pro:pr).The sections from top represent7-d-old seedlings,3-week-old plants, youngflower buds,flowers,older siliques,and seeds.Scale bars for seed images:0.1mm.Bottom images:Cauliflower transgenic lines containing the samefive constructs as top images.The different sections from top represent T0young plants,young curds,flowers,part of more mature curds,siliques,and seeds.

Anthocyanin Biosynthesis in Purple Cauliflower

thetic genes and a number of regulatory genes in various transgenic lines were analyzed.The late path-way genes,BoF3’H ,BoDFR ,BoLDOX ,and the transcrip-tion factor BobHLH1were expressed highly in the Pr pro :Pr -D transformants as well as in 35S pro :Pr -D and 35S pro :pr transgenic lines in comparison with vector-only control and wild-type pr pro :pr lines (Fig.6).The early pathway genes showed no dramatic up-regulation.These results clearly demonstrate that Pr specifically activates BobHLH1and the late pathway genes in controlling anthocyanin biosynthesis in cauliflower.

Allelic Variation at Upstream Regulatory Region of the Pr -D Allele Enhances Promoter Activity

Pr was expressed highly in the mutant and shared 99.2%nucleotide sequence identity in the coding re-gion with the wild-type gene.Thus,we hypothesized that the mutation in the promoter region controlled Pr expression in regulating anthocyanin accumulation.Analysis of the promoter sequences between the wild-type and Pr -D alleles revealed that the first 2370-bp sequences were nearly identical except five nucleotide differences and one extra TATA box at the wild-type promoter (Supplemental Fig.S1).A Harbinger DNA transposon insertion (giri:www.girinst.org)was found at 2373bp of the Pr -D mutant allele.To test whether the sequence rearrangement due to the mu-tation altered promoter activity,comparable lengths of promoter sequences from wild-type and Pr -D allele were fused to GUS gene (Fig.7A)and transformed into Arabidopsis.Interestingly,although the promoter sequences between the 2378/2373regions of Pr -D and pr were nearly identical,the mutant Pr -D 2373:GUS transformants exhibited significantly higher GUS ac-tivity than the wild-type pr 2378:GUS transformants

(P ,0.01).Similarly,transformants expressing Pr -D 21079:GUS that included part of the transposon se-quence also yielded significantly higher GUS activity than transgenic lines containing wild-type pr 21061:GUS construct (Fig.7B).These results indicate that the allelic alteration at the upstream regulatory region of the mutant allele enhances the promoter activity of the Pr gene in activating its expression.

DISCUSSION

The Pr gene of cauliflower confers anthocyanin production in otherwise anthocyanin nonaccumulat-ing tissues,such as curds and seed endosperms,turning them purple.Here we report the successful isolation of the Pr gene via a combination of candidate gene analysis and fine mapping.The Pr gene was found to encode a R2R3MYB protein.R2R3MYB transcription factors belong to a large protein family (Stracke et al.,2001;Allan et al.,2008).In Arabidopsis,MYB transcription factor subgroup that activates an-thocyanin biosynthesis includes four genes,P AP1,MYB113,MYB114,and P AP2,with the last three local-ized in tandem on chromosome one,an arrangement also seen in grape (Walker et al.,2007).A total of

four

Figure 6.Expression of anthocyanin structural and regulatory genes in transgenic cauliflower.qRT-PCR analysis of transcript levels of selected genes in young leaves of cauliflower transformants containing different Pr gene constructs.The transcript levels of genes in vector control lines were set to 1.Results reported represent mean values 6SD from three biological replicate lines with three technical replicates for

each.Figure 7.Analysis of promoter activity of the Pr gene.A,GUS constructs containing different lengths of promoter sequences.The promoter regions were chosen based on transposon insertion site at 2373bp and the transposon transposase site at 21,079bp.B,GUS activity of 4-week-old stable Arabidopsis transgenic lines containing different GUS constructs.GUS activity was measured from at least four independent transgenic lines with three repeats and expressed as m mol 4-methylumbelliferone min 21mg 21protein.Error bars indicate SD (n .4plants).The letters above the bars indicate significant differences (P ,0.01).

Chiu et al.

Pr and BobHLH1as well as a number of late pathway genes including BoF3’H,BoDFR,and BoLDOX,were expressed highly in the Pr-D mutant.Introduction of the Pr-D allele into wild-type cauliflower specifically increased the transcript levels of BobHLH1and the same set late structural genes in the transformants.The up-regulation of BobHLH1in both the Pr-D mutant and the Pr-D transformants strongly suggests that Pr regulates the expression of BobHLH1.Although ex-pression of MdMYB10does not elevate a bHLH tran-scription factor expression in apple(Malus domestica; Espley et al.,2007),P AP1has been shown to regulate the expression of TT8in Arabidopsis(Baudry et al., 2006).Similarly,AN2and AN4,the genes encoding MYB transcription factors,activate the bHLH tran-scription factor of AN1in petunia(Spelt et al.,2000).Pr and BobHLH1likely work together to coordinately regulate several transcripts of anthocyanin late path-way genes in conferring anthocyanin accumulation in the Pr-D mutant and transformants.

Despite the fact that MYB and bHLH transcription factors share similar functions among plants,they exhibit species-specific differences in activating part or the entire set of anthocyanin pathway genes.For example,in maize the C1/P1family of MYB proteins and the R/B family of bHLH proteins activate the entire set of anthocyanin structural genes(Chandler et al.,19;Ludwig and Wessler1990;Cone et al.,1993; Grotewold et al.,1994).In petunia and Arabidopsis, these two families of proteins control a subset of structural genes(Quattrocchio et al.,1993;Spelt et al.,2000;Gonzalez et al.,2008).Like the later cases, BoMYB2and BobHLH1regulated a subset of antho-cyanin structural genes in inducing tissue-specific expression in the Pr-D mutant.However,how Pr exhibits its unique tissue-specific regulation of antho-cyanin accumulation remains to be determined.

The activity of MYB-like genes has been suggested to be the primary cause of natural variation in antho-cyanin pigmentation in plants(Quattrocchio et al., 1999;Schwinn et al.,2006).Increased expression of R2R3MYB transcription factors was found to be responsible for anthocyanin production in a number of anthocyanin-accumulating mutants.For example, the constitutive up-regulation of P AP1,ANT1,and MdMYB10causes anthocyanin accumulation through-out the plant in pap1-D Arabidopsis(Borevitz et al., 2000),antl tomato(Solanum lycopersicum;Mathews et al.,2003),and red-fleshed apple(Espley et al., 2007).While overexpression of P AP1and ANT1MYB transcription factors is due to activation-tagged inser-tions in their promoter sequences(Borevitz et al.,2000; Mathews et al.,2003),the high transcript level of MdMYB10was recently found to be due to the forma-tion of a minisatellite-like structure comprising multi-ple repeats of a promoter segment that generates a novel autoregulatory motif(Espley et al.,2009).Unlike these reported anthocyanin-accumulating mutants, the Pr-D mutant appears to display a different mech-anism in activating Pr expression.

The coding regions of Pr-D and pr shared99.2% sequence identity and both of them encoded func-tional proteins.Thus,the sequence variation in the promoter region of Pr is likely responsible for the activation of Pr in controlling anthocyanin biosynthe-sis.Indeed,the promoter from wild type and the Pr-D alleles showed different capacity in activating GUS expression.For example,although the promoter of the 2378/2373regions of Pr-D and pr shared nearly identical sequences,significantly higher GUS activity was observed in the transformants expressing the mutant Pr-D2373:GUS than the wild-type pr2373:GUS. Detailed examination of the Pr-D2373/pr2373sequences revealed that an insertion of two nucleotides in Pr-D2373range generated a new regulatory motif,the E-box(5#-CANNTG-3#),which is a cis-acting element with binding consensus site for bHLH proteins (Toledo-Ortiz et al.,2003;Supplemental Fig.S1).Fur-ther,both Pr-D2373and wild-type pr21061sequences contained one E-box and the transformants expressing these constructs exhibited similar promoter activity. The Pr-D21079sequence harbored two additional E-boxes and gave significantly higher promoter activ-ity than wild-type pr21061sequence(Fig.7).The trans-poson insertion in the Pr-D allele introduced additional E-box cis-acting elements,which likely provide more binding sites for bHLH transcription factors to activate Pr expression,as suggested in a recent study of the DNA transposon mPing in rice (Naito et al.,2009).E-boxes are identified in the pro-moter region of anthocyanin structural genes and have been shown to be required for bHLH transcription factor binding in enhancing gene expression(Shirley et al.,1992;Hartmann et al.,2005).Future experiments will help clarify the involvement of E-box amplification in activating Pr expression.Nevertheless,our results reported here clearly indicate that the mutation at the upstream promoter region in the Pr-D allele resulted in activating Pr gene transcription to confer the pheno-typic change in cauliflower.Pr exerts profound effect on anthocyanin production in plants.The successful clon-ing of Pr and the discovery of it as primary determinant of color in cauliflower have implications for breeding vegetable crops with enhanced health-promoting prop-erties and visual appeal.

Anthocyanin Biosynthesis in Purple CauliflowerMATERIALS AND METHODS

Plant Materials and Growth Conditions

Purple cauliflower(Brassica oleracea var botrytis)arose from a spontaneous mutation found in a cauliflowerfield about20years ago.A commercial purple cauliflower cultivar Graffiti(Harris Seeds)and a white cultivar Stovepipe as wild-type control were used in this study.Arabidopsis(Arabidopsis thaliana) ecotype Columbia was used for genetic transformation.Cauliflower plants were grown either in a greenhouse under14-h-light/10-h-dark photoperiod at 23°C or in afield.Arabidopsis plants were grown in a growth room under 14-h-light/10-h-dark photoperiod at23°C.

HPLC-ESI-MS/MS Analysis of Anthocyanins Anthocyanins in Graffiti was extracted and analyzed following the method as described by Wu and Prior(2005b).Freeze-dried curd sample (0.5g)was ground into powder and extracted twice in total25mL methanol/water/acetic acid(85:15:0.5).The extract was diluted2-fold. Aliquot(10m L)was injected into a Zorbax stablebond analytical SB-C18 column(4.63250mm,5m m,Agilent Technologies)and separated using 5%formic acid(A)and100%methanol(B)as mobile phases on an HP1100 series HPLC.Low-resolution electrospray mass spectrometry as de-scribed(Wu and Prior,2005b)was performed to identify the major anthocyanin peaks in the purple curd sample.Quantification was carried out based on peak areas and a calibration curve generated with a commercial standard of cyanidin3,5-diglucoside chloride(INDOFINE Chemical Company).

Nucleic Acid Analysis

Genomic DNA was isolated as described previously(Lu et al.,2006).Total RNA from purple and white cauliflower tissues was extracted using Trizol reagent following the manufacturer’s instruction(Invitrogen).mRNA was isolated from total RNA using PolyATtract mRNA isolation system IV (Promega).

For northern-blot analysis,mRNA(2m g)samples were used.Prehybrid-ization and hybridization with P32-labeled probes were performed as de-scribed previously(Li et al.,2001).Probes used included CHS(U21762),CHI (U204),F3H(U14735),and LDOX(YAY780)from Arabidopsis,as well as F3’H,DFR,UGT,and Actin amplified from white cauliflower DNA using primers as listed in Supplemental Table S1.

qRT-PCR analysis was conducted according to the requirements and guidelines described by Udvardi et al.(2008).The cDNAs were synthesized from DNase I treated total RNA(5m g)using Superscript III reverse transcriptase(Invitrogen).qRT-PCR was carried out using SYBR Green PCR master mix following the manufacturer’s instruction(Applied Biosys-tems)in an Applied Biosystems7900HT fast real-time PCR system.Gene-specific primers,which were confirmed to produce specific gene products by sequencing(Yuan et al.,2009),are listed in Supplemental Table S1.The relative transcript levels were calculated as described previously(Lyi et al., 2007).

PCR walking on genomic DNA was performed using the Universal GenomeWalker kit following the manufacturer’s instruction as described previously(Li and Garvin,2003).Wild-type and mutant cauliflower ge-nomic DNA(2.5m g)were digested with Hin cII,Rsa I,Sma I,Stu I,Swa I,Dra I, Eco RV,Pvu II,Sca I,and Ssp I,respectively,and ligated to the GenomeWalker adaptors to produce Genome walking libraries.PCR products of secondary nested reactions were cloned into pCR2.1vector(Invitrogen)and se-quenced.

Association Mapping of the Candidate Genes DNA of the parents and1,8F2individuals of the mapping population were extracted(Lu et al.,2006).To design PCR-based markers,the candidate genes from both mutant and wild-type plants were isolated.Based on insertions and deletions or single nucleotide polymorphisms in the two alleles of these genes,primer sets for BoMYB2,BoMYB3,and BoMYB4 markers were developed(Supplemental Table S1).BoMYB2m and BoMYB4m were codominant markers and BoMYB3m was dominant marker.Genetic linkage map was generated using MapMaker(www.broadinstitute.org/ ftp/distribution/software/mapmaker3).Sequence Analysis

DNA and protein sequences were analyzed using the program of DNAStar (Lasergene).Multiple sequence alignments were produced by Vector NTI (Invitrogen)or CLC sequence viewer using default setting.The phylogenic tree was calculated by DNAStar and visualized by Treeview version1.6.6 (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html).

Plasmid Construction and Plant Transformation To create the constructs for phenotypic complementation,genomic DNA of Pr including1,755or1,061bp of promoter sequences for the mutant and wild-type allele,respectively,were amplified.Despite of extensive efforts,the upstream above1,061bp promoter sequence for wild-type allele could not be obtained due to highly repeated sequence and was not found from public sequence databases.The amplified products were cloned into pCAMBIA1300 vector(CAMBIA)with a nopaline synthase terminator to produce pr pro:pr and Pr pro:Pr-D constructs.To generate the overexpression constructs,comparable genomic DNA from start to stop codon of both Pr alleles were amplified and inserted into pCAMBIA1300S containing CaMV35S promoter(Zhou et al., 2009)to produce35S pro:pr and35S pro:Pr-D.To create the GUS constructs,region of wild-type pr promoter(2378and21061bp),and Pr-D promoter(2373and 21,079)were amplified and inserted into pSG506vector(kindly provided by Dr.Susheng Gan,Cornell University).The promoter fragments were selected based on transposable element insertion site.The fragments were then subcloned into pCAMBIA1300to produce various promoter-GUS constructs. All constructs were verified by sequencing.The constructs and vector-only controls were electroporated into Agrobacterium tumefaciens strain GV3101, and transformed into wild-type Arabidopsis using aflower-dipping method (Clough and Bent,1998),and into cauliflower using A.tumefaciens-mediated tissue culture method(Lu et al.,2006).

GUS Activity Analysis

Quantitative analysis of GUS activity in transformants expressing different GUS constructs was carried out usingfluorometric assay(Blazquez,2007) with slight modification.Four-week-old Arabidopsis leaf tissues(25mg)from at least four independent transgenic lines for each construct were extracted independently in100m L GUS extraction buffer.Total proteins in the crude extract were quantified using the RC DC protein assay kit(Bio-Rad).Extract (5m L)was then added to450m L GUS extraction buffer containing1m M 4-methylumbelliferyl b-D-glucuronide and incubated at37°C.Aliquot of the reaction mixture(20m L)was added into180m L stop solution(1M sodium carbonate)every10min.Fluorescence was then measured usingfluorometer Fluorolite1000(DYNEX technologies)at excitation wavelength of365nm and emission wavelength of450nm.The analysis was repeated three times. The mean values for each construct were compared.

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers GU219986(BoMYB Pr-D),GU219987 (BoMYB2pr),GU219985(BoMYB1),GU219988(BoMYB3),GU2199 (BoMYB4),and GU219990(BobHLH1).

Supplemental Data

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

Supplemental Figure S1.Nucleotide sequence comparison of the pro-moter region at2378/2373bp of the wild type and mutant Pr gene.

Supplemental Table S1.List of primers used in this study.

Supplemental Table S2.List of the GenBank accession numbers of the proteins used in the phylogenic tree of Figure4C. ACKNOWLEDGMENTS

We are especially grateful to Leon Kochian and Jim Giovannoni for valuable comments.We thank the Li lab members for helpful discussion and technical advice.We also thank Susheng Gan for providing the pSG506

Chiu et al.vector,Don Reed for providingfield space and assistance in growing cauliflower,and Chrissa McFarlane for her excellent technical assistance. Received August7,2010;accepted September19,2010;published September 20,2010.

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