a grapevine anthocyanin acyltransferase, transcriptionally … · in cv pinot noir, a red-berried...

A Grapevine Anthocyanin Acyltransferase,Transcriptionally Regulated by VvMYBA, Can ProduceMost Acylated Anthocyanins Present in Grape Skins1

Amy R. Rinaldo2, Erika Cavallini, Yong Jia, Sarah M.A. Moss, Debra A.J. McDavid, Lauren C. Hooper,Simon P. Robinson, Giovanni B. Tornielli, Sara Zenoni, Christopher M. Ford, Paul K. Boss, andAmanda R. Walker*

Commonwealth Scientific and Industrial Research Organization-Agriculture, Wine Innovation West, HartleyGrove, South Australia 5064, Australia (A.R.R., S.M.A.M., D.A.J.M., L.C.H., S.P.R., P.K.B., A.R.W.); Schoolof Agriculture, Food, and Wine, University of Adelaide, Adelaide, South Australia 5005, Australia (A.R.R.,Y.J., S.M.A.M., C.M.F.); and Department of Biotechnology, University of Verona, 37134 Verona, Italy(E.C., G.B.T., S.Z.)

ORCID IDs: 0000-0001-6450-2657 (A.R.R.); 0000-0002-0394-3966 (Y.J.); 0000-0002-0060-5486 (S.M.A.M.); 0000-0003-4812-6094 (S.P.R.);0000-0001-5027-0269 (G.B.T.); 0000-0002-0496-8161 (S.Z.); 0000-0003-0356-9342 (P.K.B.); 0000-0002-7596-8484 (A.R.W.).

Anthocyanins are flavonoid compounds responsible for red/purple colors in the leaves, fruit, and flowers of many plant species.They are produced through a multistep pathway that is controlled by MYB transcription factors. VvMYBA1 and VvMYBA2activate anthocyanin biosynthesis in grapevine (Vitis vinifera) and are nonfunctional in white grapevine cultivars. In this study,transgenic grapevines with altered VvMYBA gene expression were developed, and transcript analysis was carried out on berriesusing a microarray technique. The results showed that VvMYBA is a positive regulator of the later stages of anthocyanin synthesis,modification, and transport in cv Shiraz. One up-regulated gene, ANTHOCYANIN 3-O-GLUCOSIDE-699-O-ACYLTRANSFERASE(Vv3AT), encodes a BAHD acyltransferase protein (named after the first letter of the first four characterized proteins: BEAT [foracetyl CoA:benzylalcohol acetyltransferase], AHCT [for anthocyanin O-hydroxycinnamoyltransferase], HCBT [for anthranilateN-hydroxycinnamoyl/benzoyltransferase], and DAT [for deacetylvindoline 4-O-acetyltransferase]), belonging to a cladeseparate from most anthocyanin acyltransferases. Functional studies (in planta and in vitro) show that Vv3AT has a broadanthocyanin substrate specificity and can also utilize both aliphatic and aromatic acyl donors, a novel activity for this enzymefamily found in nature. In cv Pinot Noir, a red-berried grapevine mutant lacking acylated anthocyanins, Vv3AT contains anonsense mutation encoding a truncated protein that lacks two motifs required for BAHD protein activity. Promoter activationassays confirm that Vv3AT transcription is activated by VvMYBA1, which adds to the current understanding of the regulation ofthe BAHD gene family. The flexibility of Vv3AT to use both classes of acyl donors will be useful in the engineering ofanthocyanins in planta or in vitro.

Anthocyanins, a group of water-soluble flavonoidcompounds, are produced by almost all vascular plantsand have been shown to have a diverse range of bio-logical functions. They are major contributors to theorange, red, purple, and blue colors seen in the leaves,fruit, and flowers of many plant species and, hence,have important roles in attracting pollinators and seeddispersers (Schaefer et al., 2004). It has been suggestedthat they also act as protection agents against UV light(Markham, 1988) and are involved in plant stress re-sponses (Dixon and Paiva, 1995). Anthocyanins havepotent antioxidant capacity, providing numeroushealth-promoting properties, including cardiovasculardisease prevention and antiinflammatory, antimicro-bial, and anticarcinogenic activities (He and Giusti,2010).

Wine grapes (Vitis vinifera) contain both 3-O-mono-glucoside and 3-O-acyl-monoglucoside anthocyaninsderived from five main anthocyanidin aglycones: del-phinidin, cyanidin, peonidin, petunidin, and malvidin.The structural genes involved in their production have

all been isolated and characterized (Fig. 1; for review,see He et al., 2010). The regulation of the flavonoidpathway is through the action of transcriptional com-plexes comprising three transcription factor (TF) fami-lies: a basic helix-loop-helix protein (bHLH), a Trp-Asprepeat protein (WDR or WD40), and an R2R3-MYBprotein. The MYB/bHLH/WDR complexes are thoughtto recognize and bind to responsive elements found inthe promoters of biosynthesis genes in the pathway,usually resulting in activation of the expression of thatgene (for review, see Matus et al., 2010). The MYB TFsdetermine the specificity of this complex and have beenshown to bind directly to the structural gene promoters(Sainz et al., 1997).

Anthocyanin synthesis in grapes is specifically regu-lated by transcriptional complexes containing eitherVvMYBA1 or VvMYBA2 through the activation ofURIDINE DIPHOSPHATE GLUCOSE-FLAVONOID3-O-GLUCOSYLTRANSFERASE (VvUFGT) transcrip-tion (Walker et al., 2007). VvUFGT catalyzes the finalstep of anthocyanin synthesis, where anthocyanidins

Plant Physiology�, November 2015, Vol. 169, pp. 1897–1916, www.plantphysiol.org � 2015 American Society of Plant Biologists. All Rights Reserved. 1897 www.plantphysiol.orgon September 24, 2020 - Published by Downloaded from

Copyright © 2015 American Society of Plant Biologists. All rights reserved.

http://orcid.org/0000-0001-6450-2657

http://orcid.org/0000-0002-0394-3966

http://orcid.org/0000-0002-0060-5486

http://orcid.org/0000-0003-4812-6094

http://orcid.org/0000-0001-5027-0269

http://orcid.org/0000-0002-0496-8161

http://orcid.org/0000-0003-0356-9342

http://orcid.org/0000-0002-7596-8484

http://www.plantphysiol.org

are glycosylated on the 3-hydroxyl group of the C ringof the flavylium molecule, to produce stable anthocy-anins (Ford et al., 1998). The VvMYBA1 and VvMYBA2coding sequences are almost identical in their R2R3domains, but VvMYBA2 has a 93-amino acid repeat ofthe activation domain (Walker et al., 2007). White-fruited grapevine cultivars have arisen due to the lackof a functional VvMYBA protein, caused by a retro-transposon insertion in the promoter of VvMYBA1(Kobayashi et al., 2004) and two nonconservative mu-tations in VvMYBA2 (Walker et al., 2007).

In grapevine, anthocyanins can be further modifiedby O-methyltransferases, which methylate hydroxylgroups at the 39 and 59 positions of the B ring(Fournier-Level et al., 2011), and acyltransferases,which produce 3-O-acetyl-, 3-O-coumaroyl-, and 3-O-caffeoyl-monoglucosides by attaching acyl groups tothe C699 position of the Glc moiety (Mazza, 1995;Nakayama et al., 2003). Acylated anthocyanins in var-ious flowering species are more stable compared withtheir nonacylated counterparts, most likely due to in-creased intramolecular stacking (Yonekura-Sakakibaraet al., 2008). Wines made from red-berried grapevinecultivars such as cv Ives or Veeport with high propor-tions of acylated anthocyanins can have greater colorstability when exposed to light compared with thosefrom red varieties with no acylated anthocyanins, suchas cv Pinot Noir (Van Buren et al., 1968; Smart, 1992).Despite the importance of red color stability to winequality and the correlation between poorly colored red

wines and cultivars containing no acylated anthocya-nins, anthocyanin acyltransferases from grapevine havenot yet been identified. A recent study by Costantiniet al. (2015) identified a number of quantitative trait loci(QTLs) associated with variation in acylated anthocy-anin levels in F1 progeny from a cv Syrah 3 cv PinotNoir cross. The strongest candidate genes within theseQTLs included those belonging to the BAHD acyltrans-ferase family (named after the first letter of the firstfour characterized proteins: BEAT [for acetyl CoA:benzylalcohol acetyltransferase], AHCT [for anthocyaninO-hydroxycinnamoyltransferase], HCBT [for anthranilateN-hydroxycinnamoyl/benzoyltransferase], and DAT[for deacetylvindoline 4-O-acetyltransferase]; St-Pierreand Luca, 2000) and the Serine carboxypeptidase-likeacyltransferase family. Yet, no QTL was found to de-scribe the presence/absence of acylation in berries,probably due to the genotype of the parents and thelack of segregation for this in the F1 population.

In a number of species, the expression of flavonoidpathway MYB TF transgenes, or the overexpression orsilencing of endogenous genes, has enabled detailedstudies of their regulatory roles in this pathway. Thishas also highlighted substantial differences in the fla-vonoid regulatory network between different species,particularly between monocots and dicots, and withinreproductive organs (for review, see Petroni andTonelli, 2011). Transcriptomic analyses of these trans-genic plants have often led to the discovery of novelgenes associated with anthocyanin synthesis, modifi-cation (including the identification of anthocyaninacyltransferases), or transport. For example, Tohgeet al. (2005) used microarrays to identify putativeacyltransferases, later characterized as anthocyaninacyltransferases by Luo et al. (2007), in an activation-tagged PAP1D line of Arabidopsis (Arabidopsis thaliana)in which PRODUCTIONOF ANTHOCYANIN PIGMENT1(AtPAP1) was overexpressed. Butelli et al. (2008) alsofound putative anthocyanin acyltransferases that wereup-regulated in transgenic tomato (Solanum lycopersi-cum) expressing the MYB TF transgene ROSEA1 fromAntirrhinum majus flowers. Cutanda-Perez et al. (2009)ectopically expressed the VlMYBA1 gene from Vitislabruscana in grapevine hairy root tissue and analyzedgene expression changes to the transcriptome of theseroots. They concluded that VlMYBA1 regulated a nar-row set of genes involved in anthocyanin biosynthesisand identified novel genes associated with anthocyanintransport. The regulatory effect of VvMYBA on thetranscriptome in berries of grapevine, where it is nat-urally expressed, has not yet been studied due to thecomplicated and lengthy protocols required to producestably transformed vines.

In this study, transgenic grapevines with alteredVvMYBA gene expression were generated. Over-expression of VvMYBA1 in the white-berried cv Char-donnay resulted in pigmented fruit, while silencing theVvMYBA1 and VvMYBA2 genes in the pigmented cvShiraz produced berries with lightly colored or whitephenotypes. When microarray techniques were used

1 This work was supported by the Grape and Wine Research andDevelopment Corporation (scholarship no. GWR Ph0903 to A.R.R.and grant no. CSP 0602 to S.P.R. and A.R.W.), by the Office of theChief Executive Science Team, Commonwealth Scientific and Indus-trial Research Organization, by an Australian Postgraduate Awardthrough the University of Adelaide (to A.R.R.), and by Common-wealth Scientific and Industrial Research Organization core funding.

2 Present address: Commonwealth Scientific and Industrial Re-search Organization-Agriculture, Crace Laboratories, 44 BellendenStreet, Crace, Australian Capital Territory 2911, Australia.

* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Amanda R. Walker ([email protected]).

E.C., S.Z., and G.B.T. designed and performed the microarrayexperiment, analyzed the results, and wrote the relevant sectionof the article; Y.J. and S.M.A.M. performed the protein modelingand alignment experiments, respectively, writing the relevant sec-tions of the article; A.R.W. and D.A.J.M. performed the grapevinetransformation; L.C.H. and A.R.W. characterized the transgenicgrapevines with guidance from S.P.R.; A.R.R. planned and per-formed most of the remaining experiments (with help from L.C.H.,A.R.W., and S.P.R. for the extra experiments for resubmission) underthe supervision of A.R.W., P.K.B., and C.M.F.; P.K.B. suggested fo-cusing on the acyltransferase based on preliminary results from otherexperiments and provided expert discussions on many points in theproject; C.M.F. provided expert advice, particularly for studying en-zyme kinetics; A.R.R. wrote many drafts of the article, which werethen edited by A.R.W., C.M.F., P.K.B., and S.P.R.

www.plantphysiol.org/cgi/doi/10.1104/pp.15.01255

1898 Plant Physiol. Vol. 169, 2015

Rinaldo et al.

www.plantphysiol.orgon September 24, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.

mailto:[email protected]


mailto:[email protected]

http://www.plantphysiol.org/cgi/doi/10.1104/pp.15.01255


to analyze the transcriptomes of the transgenic berries,transcription of a gene putatively encoding for a mem-ber of the BAHD protein family correlated with theexpression of VvMYBA. Members of the BAHD genefamily encode acyltransferases that utilize CoA thioes-ters as their donor substrate both in planta (Fujiwaraet al., 1998) and/or in vitro (Yonekura-Sakakibaraet al., 2000; Yabuya et al., 2001; Suzuki et al., 2004b;D’Auria et al., 2007). Here, the characterization of thefunction of this BAHD gene from grapevine, identifiedthrough the microarray analysis of the transcriptome ofberries with altered VvMYBA expression, is presented.Gene expression analyses, stable plant transformations,and recombinant protein assays were used to demon-strate that the gene encodes an anthocyanin acyl-transferase, ANTHOCYANIN 3-O-GLUCOSIDE-699-O-ACYLTRANSFERASE (Vv3AT), which is capable ofproducing the common acylated anthocyanins found ingrape berries. Vv3AT belongs to a BAHD protein claderesponsible for a range of functions, most of which donot involve the modification of anthocyanins, andcan use a broad range of acyl acceptor and donor

substrates, which include both aliphatic and aromaticacyl-CoA thioesters.

RESULTS

Altering VvMYBA Gene Expression Alters Berry Color inTransgenic Grapevines

A VvMYBA1 gene construct, under the control ofeither its own promoter (VvMYBA1pr:VvMYBA) or a35S constitutive promoter from the Cauliflower mosaicvirus (35S:VvMYBA1), was used to stably transformembryogenic callus of the white-berried cv Chardon-nay to produce mature transgenic vines. Comparedwith cv Chardonnay control berries (Fig. 2A), thetransgenic berries expressing the VvMYBA1pr:VvMYBA construct had red/purple pigmentation intheir skin (Fig. 2B) that developed at veraison (whenanthocyanin accumulation normally begins as berriesripen). The total anthocyanin concentration in theseberries was approximately 30% of that detected inpurple/black berries of cv Shiraz (Supplemental Fig. S1).

Figure 1. Schematic of the anthocyanin biosynthesis pathway in grapevine.Metabolites are boxed,with genes coding for the enzymescatalyzing each biochemical reaction in italics. C4H, Cinnamate 4-hydroxylase; CHI, chalcone isomerase; 4CL, 4-coumaroyl-CoAligase;DFR, dihydroflavonol 4-reductase; F3H, flavanone-3-hydroxylase; F39H, flavonoid 39-hydroxylase; LDOX, leucoanthocyanidindioxygenase; MT, methyltransferases (altered from Boss and Davies, 2009).

Plant Physiol. Vol. 169, 2015 1899

VvMYBA Regulates an Anthocyanin Acyltransferase


http://www.plantphysiol.org/cgi/content/full/pp.15.01255/DC1


The cv Chardonnay berries constitutively expressingVvMYBA1 (35S:VvMYBA) had purple/black pigmen-tation in their skin (Fig. 2C) and purple flesh, which waspresent throughout development and ripening, andmost other tissues of the plant were also pigmented. Thecv Shiraz (Fig. 2D) was used to create transgenic vinescontaining a VvMYBA-silencing construct (VvMYBAsi)that silenced both the VvMYBA1 and VvMYBA2 genes.Transgenic cv Shiraz berries possessed either rose-colored skin pigmentation (rose cv Shiraz; Fig. 2E) ornonpigmented skin (white cv Shiraz; Fig. 2F).

Conserved Transcriptomic Changes in TransgenicGrapevines with Altered VvMYBA Gene Expression

A comparison of the transcriptomes of transgenicwhole berries, close to ripeness, with altered VvMYBAgene expression and their controls was carried out byhybridizing RNA to a NimbleGen microarray con-taining probes that represent the 98.6% of the genespredicted in the 123 grapevine V1 genome (Micro-array Gene Expression Omnibus database accession no.GSE56915; http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=cnmxwmmsfjctvgt&acc=GSE56915). Therewere 188 and 160 genes significantly up- and down-regulated, respectively, using a false discovery rate of2.5% and a fold change (FC) of 2 or greater in theVvMYBA1-overexpressing red cv Chardonnay berriescomparedwith nonpigmented controls (SupplementalTable S1). Using the same parameters, there were 48

and 319 genes significantly up- and down-regulated,respectively, in transgenic white cv Shiraz berriescompared with nontransgenic red-berried controls(Supplemental Table S2). All the significantly modu-lated genes were automatically annotated against theV1 gene prediction version of the grapevine genome(http://genomes.cribi.unipd.it/grape/index.php) andmanually improved where possible.

To gain information about the genes probably con-trolled by VvMYBA, these microarray data were an-alyzed for genes whose expression was consistentlyup- or down-regulated in response to the presence orabsence of VvMYBA gene expression in both cv Char-donnay and cv Shiraz and their respective transgeniclines. Only 26 and one genes were up- and down-regulated, respectively (FC $ 2), in a consistent man-ner between the two sets of plants (Table I). Those withthe greatest expression changes were characterizedor putative flavonoid-related genes that were up-regulated in red berries compared with white. Theseincluded the anthocyanin transporter ANTHOCYANINMULTIDRUG AND TOXIC EFFLUX TRANSPORTER2(VvanthoMATE2; Gomez et al., 2011) andGLUTATHIONE-S-TRANSFERASE4 (VvGST4; Conn et al., 2008), which isinvolved in the transport of flavonoids either directly ordue to their modification before their transport, andseveral anthocyanin biosynthesis genes, including ninemembers of the FLAVONOID 39,59-HYDROXYLASE(F3959H; Falginella et al., 2010) gene family, threeputative CHALCONE SYNTHASE (CHS) genes, andVvUFGT. In addition, the list included one transcript

Figure 2. Photographs of berries from transgenicplants with alteredVvMYBA gene expression takenwhen harvested (approximately 22 degrees Brix).A, Control cv Chardonnay. B and C, Transgeniccv Chardonnay expressing VvMYBA1 under thecontrol of its own promoter (B) or the 35S con-stitutive promoter from the Cauliflower mosaicvirus (C). D, Control cv Shiraz. E and F, Trans-genic cv Shiraz expressing a VvMYBA-silencingconstruct with rose (E) and white (F) berry colorphenotypes.


Rinaldo et al.


http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=cnmxwmmsfjctvgt&acc=GSE56915





http://genomes.cribi.unipd.it/grape/index.php


corresponding toFLAVONOLANDANTHOCYANIDIN-GLUCOSIDE 39,59-O-METHYLTRANSFERASE (VvFAOMT;Hugueney et al., 2009; Lücker et al., 2010), involved inflavonol and anthocyanin methylation, another puta-tive anthocyanin methyltransferase, and a transcriptbelonging to the BAHD acyltransferase family, possiblyinvolved in the acylation of anthocyanin. Only onegene,PHENYLALANINEAMMONIALYASE2 (VvPAL2;Guillaumie et al., 2011), was related to general phenyl-propanoid biosynthesis. There were no flavonoid bio-synthetic genes thatwere consistently down-regulated inred berries in both grapevine cultivars. There were,however, some phenylpropanoid/flavonoid pathwaygenes that had modulated expression in only one of thecultivars (i.e. either cv Chardonnay or cv Shiraz trans-genic berries), and these are discussed in the “Supple-mental Data.”To validate the microarray data, the expression of a

subset of flavonoid biosynthesis genes was analyzed inthe transgenic berries using quantitative PCR (qPCR;Supplemental Figs. S2 and S3).Overall, the transcriptomic data confirm the specific

role of VvMYBA in the control of anthocyanin-relatedgenes. The core set of genes most up-regulated byoverexpression of VvMYBA and most down-regulated

by the silencing of this gene includes known targets ofthe TF as well as new putative players in the anthocy-anin biosynthetic pathway. Among these, the unchar-acterized gene annotated as belonging to the BAHDprotein superfamily (VIT_03s0017g00870) was hy-pothesized to function as an anthocyanin acyltransfer-ase and chosen for further characterization.

Vv3AT Belongs to the BAHD Superfamily ofAcyltransferases within a Clade Distinct from Most OtherAnthocyanin Acyltransferases

A full-length complementary DNA (cDNA) clone ofthe BAHD gene, identified from the microarrays, wasisolated from cv Cabernet Sauvignon (Vv3AT-CS).The cv Cabernet Sauvignon gene consists of a singleexon 1,284 bp in length. Two alleles were present inthis cultivar that differ by two nucleotides: an A/Gpolymorphism at position 238 of the putative codingregion does not alter the predicted amino acid se-quence, and a C/T polymorphism at 784 bp results inthe conversion of Arg-262 to Cys in the 428-aminoacid protein sequence. Allele 1 encodes the samepredicted protein as VIT_03s0017g00870, from the

Table I. Genes with altered transcription levels (FC $ 2) in response to VvMYBA gene expression in a consistent manner in both transgenic cvChardonnay and cv Shiraz berries

Transcript levels were determined using microarrays.

Gene Identifier Description Red Chardonnaya/Control FC White Shirazb/Control FC

VIT_04s0079g00690 Glutathione S-transferase (VvGST4) 807.18 220.84VIT_06s0009g02840 Flavonoid 39,59-hydroxylase 390.88 266.78VIT_06s0009g02810 Flavonoid 39,59-hydroxylase 289.81 244.81VIT_06s0009g02880 Flavonoid 39,59-hydroxylase 283.94 235.86VIT_06s0009g03040 Flavonoid 39,59-hydroxylase 249.34 223.12VIT_06s0009g02970 Flavonoid 39,59-hydroxylase 234.77 248.42VIT_06s0009g03110 Flavonoid 39,59-hydroxylase 210.96 216.17VIT_06s0009g02860 Flavonoid 39,59-hydroxylase 206.03 223.98VIT_06s0009g02920 Flavonoid 39,59-hydroxylase 150.77 262.91VIT_06s0009g03050 Flavonoid 39,59-hydroxylase 132.16 230.06VIT_01s0010g03510 Anthocyanin O-methyltransferase (VvFAOMT) 112.48 212.58VIT_01s0010g03490 Anthocyanin O-methyltransferase 111.20 231.71VIT_03s0017g00870 BAHD family acyltransferase 65.66 226.44VIT_16s0039g02230 UDP-Glc:flavonoid 3-O-glucosyltransferase (VvUFGT) 61.71 223.38VIT_16s0022g01020 Chalcone synthase 34.57 230.69VIT_16s0022g01140 Chalcone synthase 31.77 241.93VIT_16s0022g01190 Chalcone synthase 30.20 223.07VIT_00s0357g00130 Annexin ANN6 8.80 23.08VIT_16s0050g00910 AnthoMATE2 transport protein (VvanthoMATE2) 5.94 22.76VIT_14s0128g00160 Protein kinase constitutive differential growth1 3.38 23.00VIT_16s0022g00870 Invertase/pectin methylesterase inhibitor 3.30 24.93VIT_13s0067g01770 Steroid 5a-reductase 3.26 22.70VIT_13s0019g04460 Phe ammonia lyase2 (VvPAL2) 2.54 22.02VIT_08s0007g05430 Pyruvate kinase 2.48 22.70VIT_12s0028g01570 Zinc finger (C3HC4-type RING finger) 2.42 22.09VIT_14s0060g02390 Soybean gene regulated by cold2 2.32 22.09VIT_18s0001g14800 Lipase3 family II extracellular 22.25 4.18

aRed cv Chardonnay contained a 35S:VvMYBA1 construct and expressed the VvMYBA1 gene. Nontransgenic unpigmented cv Chardonnay berrieswere the control for these experiments. bWhite cv Shiraz contained a VvMYBAsi construct that completely silenced the expression of VvMYBA1/2genes. Nontransgenic red/black cv Shiraz berries were used as the control for these experiments.






PN40024 grapevine genome sequencing project(Jaillon et al., 2007).

A comprehensive protein alignment and a phyloge-netic treewere created using 72 BAHDprotein sequencesfrom 38 different plant species, including the predictedVv3AT protein sequence. These proteins included the listof genetically or biochemically characterized BAHDproteins reported previously by D’Auria (2006) plusadditional published BAHD proteins characterized since2006 (Supplemental Table S3; Supplemental ReferencesS1). The tree separated the proteins into five major clades(Fig. 3), with Vv3AT falling into clade IIIa (Tuominenet al., 2011), most similar to an anthocyanin acyl-transferase, ANTHOCYANIN5-O-GLUCOSIDE-4‴-O-MALONYLTRANSFERASE (Ss5MaT2), from Salviasplendens (Suzuki et al., 2004b). A nucleotide BLASTin the grapevine nucleotide database of the NationalCenter for Biotechnology Information server revealed 10related sequences ranging from 66% to 95% identity over68% to 100% coverage of the Vv3AT coding sequence. Atranslated nucleotide BLAST search in the grapevineprotein database of the National Center for Biotechnol-ogy Information found 45 hypothetical proteins with23% or greater identity to 54% or greater coverage of theVv3AT sequence. None of these genes or proteins hadbeen functionally characterized, so they were not in-cluded in this phylogenetic analysis.

Protein Sequence Changes and Gene Expression Patternsof Vv3AT Correlate with the Absence of AcylatedAnthocyanins in cv Pinot Noir and Pale-Fruited cvCabernet Sauvignon Mutants

The cv Pinot Noir, a red-berried grapevine cultivar,does not synthesize acylated anthocyanins (Van Burenet al., 1968) and, therefore, represents a mutant that canbe compared with most other cultivars that do containthese compounds, such as cv Cabernet Sauvignon andcv Shiraz. Sequence analysis of cDNA of Vv3AT-PNfrom cv Pinot Noir showed two single-nucleotidepolymorphisms compared with the cv Cabernet Sau-vignon cDNA sequences. The first of these is a C/Tpolymorphism at position 349 bp of the codingregion, which introduces a premature stop codon re-sulting in a truncated protein of 116 amino acids. Analignment of Vv3AT-CS and Vv3AT-PN against pre-viously characterized BAHD proteins showed that thepredicted Vv3AT-PN protein did not contain either ofthe functional motifs, HXXXDG and DFGWG, found inalmost all members of the BAHD family (St-Pierre andLuca, 2000), most likely rendering it inactive (Fig. 4).

Transcript levels of VvMYBA1 and VvMYBA2 genesare difficult to determine in grapevine due to theirsimilarity to, and the expression of, nonfunctionalVvMYBA genes and alleles within the grapevine ge-nome. VvUFGT gene expression has been correlatedpreviously to anthocyanin accumulation (Boss et al.,1996a) and is known to be transcriptionally activated byVvMYBA1 and VvMYBA2 TFs; hence, it is a useful

marker for the expression of these TFs (Kobayashi et al.,2002; Walker et al., 2007). The transcript level of Vv3ATand VvUFGT (as a marker for VvMYBA expression)was analyzed using qPCR over the development ofwhole berries from cv Cabernet Sauvignon and cv PinotNoir starting from 2 weeks post flowering (wpf)through to harvest at 14 wpf (Fig. 5). VvUFGT tran-scripts began accumulating postveraison in both cvCabernet Sauvignon and cv Pinot Noir, as shown pre-viously (Boss et al., 1996b). Vv3AT transcript levelsfollowed a very similar pattern to those of VvUFGT incv Cabernet Sauvignon, suggesting a link between thefunction and regulation of these two genes. In com-parison, Vv3AT transcript levels in cv Pinot Noir werelow throughout development, with only a slight in-crease postveraison.

Two color sports of cv Cabernet Sauvignon (cvMalianand cv Shalistin; Supplemental Fig. S4) have arisen dueto a deletion of the berry color locus carrying theVvMYBA genes (Walker et al., 2006). In cv Malian, thisdeletion occurred in the L2 cell layer, resulting inbronze/rose-colored berries, while in cv Shalistin, thedeletion is also present in the L1 cell layer, resulting inwhite berries. To further investigate the link betweenVv3AT gene expression and VvMYBA TFs, transcriptlevels of Vv3AT and VvUFGT were analyzed overearly (whole berries) and late (skins only) develop-ment of cv Cabernet Sauvignon, cv Malian, and cvShalistin berries (Fig. 6). The changes in transcriptlevels of Vv3AT were very similar to that of VvUFGT.In both cases, transcripts began accumulating afterveraison in cv Cabernet Sauvignon and cv Malian, butlevels were lower in cv Malian. No transcripts of eitherVv3AT orVvUFGTwere detected in cv Shalistin berries.It has been shown previously that VvUFGT transcrip-tion is activated by the VvMYBA1 and VvMYBA2 TFs(Walker et al., 2007). This would explain the expressionpattern of VvUFGT seen here, as VvMYBA genes areonly expressed in the L1 cell layer of the berry skin in cvMalian, while cv Shalistin does not contain functionalVvMYBA genes at all (Walker et al., 2006). These resultsdemonstrate a strong link between the presence of theVvMYBA TFs and Vv3AT gene expression.

The Grapevine Color Regulator VvMYBA1 Activates theExpression of Vv3AT in cv Chardonnay Suspension Cells

Transient promoter-binding assays using a lucifer-ase reporter system were performed to determine ifVvMYBA1 can activate expression from the Vv3ATpromoter. Genomic DNA 711 bp upstream of the pu-tative initiation codon of the Vv3AT gene was isolatedfrom cv Cabernet Sauvignon and cv Pinot Noir. Thesepromoter regions differed by six point mutations,which were all located more than 400 bp upstream ofthe predicted start codon. VvUFGT, Vv3AT-CS, andVv3AT-PN promoters were ligated upstream to a lu-ciferase reporter gene in a pLUC vector (Horstmannet al., 2004). These constructs, alongwith two constructs


Rinaldo et al.







Figure 3. Phylogenetic tree of characterized plant acyltransferases belonging to the BAHD protein family, including the putative anthocyanin acyl-transferase sequence from grapevine (Vv3AT; boxed). Protein sequences were aligned using ClustalW and manually edited to remove nonconservedregions. A phylogenetic tree and the posterior probability of the nodeswere generated using Bayesian inferencewithin BEAST version 1.7.5. The cladeshave been named based on the groupings discussed by D’Auria (2006). The scale bar represents the number of amino acid substitutions per site.Species, accession numbers, and references for each protein can be found in Supplemental Table S3.






expressing VvMYBA1 and a bHLH TF (ENHANCEROF GLABRA3 [AtEGL3], previously shown to be re-quired for TF complex formation in similar assays [Bogset al., 2007], and VvMYC1 [Hichri et al., 2010]; Fig. 7),were introduced into grapevine cell suspension cul-tures using biolistic-mediated transfection. Luciferaseactivity in cells bombarded with the VvUFGTpr:LUC,VvMYBA1, and either AtEGL3 or VvMYC1 genes wasover 60-fold higher than the activity in control cells(where an empty vector construct replaced that ofVvMYBA1). The luciferase activity in the grape cellswhen the promoters of Vv3AT-CS and Vv3AT-PNwereused was 25- to 40-fold higher than background con-trols, suggesting that VvMYBA1 can activate eitherpromoter of Vv3AT in planta. When VvMYC1 wasused as the bHLH TF, both Vv3AT-CS and Vv3AT-PNpromoters were activated to similar levels (Fig. 7;Supplemental Table S4).

Constitutive Expression of Vv3AT in Tobacco Results inthe Production of Acylated Anthocyanins

Vv3AT was constitutively expressed under the con-trol of the cauliflower mosaic virus 35S promoter intobacco (Nicotiana tabacum var Samsun) through Agro-bacterium tumefaciens-mediated stable transformation.Six transgenic lines from independent transformation

events were created that expressed Vv3AT in theirflowers at varying levels (Supplemental Fig. S5). Someflowers from one transgenic line (Vv3AT-1; Fig. 8)expressing Vv3AT visually displayed a slightly moreblue/purple hue compared with wild-type untrans-formed controls (Fig. 8A), while the others had similarphenotypes to the wild type. Anthocyanins wereextracted from flowers of all six lines and separatedusing HPLC, and peaks were identified using liquidchromatography-tandem mass spectrometry (LC-MS/MS). Two anthocyanin species could be detectedin wild-type tobacco flowers. The most abundant spe-cies was cyanidin-3-O-rutinoside (peak 1), with lesseramounts of pelargonidin-3-O-rutinoside also present(peak 2; Fig. 8B; Supplemental Table S5). Extractsfrom tobacco flowers constitutively expressing Vv3ATcontained six extra peaks not present in the wild-typesamples. These peaks were identified as cyanidin-3-O-acetylglucoside (peak 3), pelargonidin-3-O-acetylglucoside(peak 4), cyanidin-3-O-caffeoylglucoside (peak 5),pelargonidin-3-O-caffeoylglucoside (peak 6), cyanidin-3-O-coumaroylglucoside (peak 7), and pelargonidin-3-O-coumaroylglucoside (peak 8; Fig. 8B; Supplemental TableS5). Of the acylated anthocyanins, all of the transgenictobacco lines expressing Vv3AT contained a higher con-centration of coumaroylated anthocyanins than acety-lated anthocyanins andhad lowest amounts of caffeoylatedanthocyanins (Fig. 8C).

Figure 4. Amino acid sequence alignment of Vv3AT from cv Cabernet Sauvignon with previously characterized BAHD proteins.In cv Pinot Noir, Vv3AT contains a nonsense mutation resulting in a truncated protein. The black arrow indicates where thistruncated version of the protein ends, which does not contain either of the BAHD family functional motifs HXXXD (boxed in red)and DFGWG (boxed in blue). Vv3AT contains the clade III-specific motif KPSSTP (boxed in green) and two motifs found in cladeIIIa, the YPLAGR motif (boxed in pink) and QVTX(F/L)XCGG (boxed in light blue). The YFGNC motif common to most antho-cyanin acyltransferases in clade I is boxed in orange. The Ala residue corresponding to His-178 in Dm3MaT3 and predicted toaffect substrate preference is indicated by the red arrow. Other genes, species, and accession numbers are as follows: ArabidopsisAt3AT1, NP_171890; Clarkia breweri BENZOYL COENZYME A:BENZYL ALCOHOL BENZOYL TRANSFERASE (CbBEBT),AAN09796; S. splendens Ss5MaT1, AAL50566; S. splendens Ss5MaT2, AAR26385; D. morifolium MALONYL COENZYME A:ANTHOCYANIN 3-O-GLUCOSIDE-699-O-MALONYLTRANSFERASE3 (Dm3MaT3), AB290338; and C. breweri ACETYLCOENZYME A:BENZYLALCOHOL ACETYLTRANSFERASE (CbBEAT), AAC18062.1.


Rinaldo et al.








Recombinant Vv3AT Can Acylate Anthocyanins in Vitro

In order to study the function of Vv3AT and itssubstrate preferences in vitro, recombinant His-taggedVv3AT protein was generated and purified using af-finity chromatography (Supplemental Figs. S6 and S7).The kinetic properties of this enzymewere determinedusing various acyl donor and acceptor substrates(Table II; Supplemental Figs. S8 and S9). Due to theinability to purify the recombinant protein to homoge-neity, the concentration of the enzyme was estimatedbased on a His-tag ELISA and visualization on westernblots. Hence, the kinetic parameters described are esti-mates only but are comparable between the varioussubstrates tested. First, three acyl donors were assayedusing malvidin-3-O-glucoside as the anthocyanin acylacceptor, as this is the predominant anthocyanin foundin the berry skins of grapevine (Mazza, 1995). As acetyl-,

coumaroyl-, and caffeoyl-conjugated anthocyanins arepresent in grapevine, Vv3AT activity was tested usingacetyl-CoA, coumaroyl-CoA, and caffeoyl-CoA donors.It was found that Vv3AT was capable of using all threeof these acyl donors in our assay conditions. The lowestapparent Km was observed when coumaroyl-CoA wasthe acyl donor (1.256 0.16 mM), while the enzyme hadthe lowest affinity for acetyl-CoA, with an apparentKm of 16.86 6 6.43 mM. When acetyl-CoA was the acyldonor, an apparent catalytic rate constant (kcat) of 1.1160.61 s21 was observed, which was 8.5- and 3.8-foldhigher than the apparent kcat data when coumaroyl- andcaffeoyl-CoA were the acyl donors, respectively (0.13 60.04 and 0.29 6 0.09 s21). The apparent specificity con-stant, taking into account both the Km and kcat of thesubstrate, was similar for all three CoA donors: 0.08 60.07mM

21 s21 with acetyl-CoA, 0.106 0.03mM21 s21 with

coumaroyl-CoA, and 0.076 0.02 mM21 s21 with caffeoyl-

CoA. Malonyl-CoA was also tested to see if it could actas substrate for Vv3AT, as many other BAHD anthocy-anin acyltransferases use this compound as an acyl do-nor, including Ss5MAT2 (D’Auria, 2006), the closestcharacterized homolog of Vv3AT. While Vv3AT couldutilize malonyl-CoA as a substrate, the apparent Kmwas calculated at 588.77 6 74.48 mM, which was muchhigher than the other acyl donors tested, and the spec-ificity constant was approximately 100-fold lower at0.0016 6 0.0002 mM

21 s21. Therefore, it was concludedthat Vv3AT can catalyze the acylation of malvidin-3-O-glucoside with a range of CoA-conjugated acyl donors.

When coumaroyl-CoA and caffeoyl-CoA were usedas acyl donors in concentrations above 100 mM, it wasfound that the production of the expected acylatedanthocyanin was inhibited. No substrate inhibition wasobserved when using acetyl-CoA in concentrations upto 1 mM (Supplemental Fig. S10). For this reason,Vv3AT enzyme kinetics using various anthocyanin acylacceptors was conducted using acetyl-CoA as the acyldonor.

The 3-O-glucosides of malvidin, cyanidin, delphi-nidin, peonidin, and petunidin are the five major an-thocyanin species found in grapes (He et al., 2010).The activity of Vv3AT was assayed using all of theseanthocyanins as acyl acceptors except petunidin-3-O-glucoside (not commercially available). The kineticparameters of Vv3AT when malvidin- and peonidin-3-O-glucosides were used as substrates were similar,with apparent Km values of 98.666 19.59 and 79.52 68.37 mM, apparent kcat values of 13.92 6 0.71 and14.2 6 1.97 s21, and apparent specificity constants of0.15 6 0.03 and 0.18 6 0.03 mM

21 s21, respectively.Apparent Km values calculated with cyanidin-3-O-glucoside and delphinidin-3-O-glucoside acyl accep-tors were much higher at 506.22 6 8.55 and 765.13 651.97 mM, respectively, but the apparent kcat valueswere also higher at 77 6 5.92 and 67.89 6 13.01 s21,indicating that the specificity constants of 0.15 6 0.01and 0.09 6 0.02 mM

21 s21, respectively, were not thatdissimilar to those of malvidin- and peonidin-3-O-glucosides. To determine if Vv3AT would also acylate

Figure 5. Relative gene expression of Vv3AT (A) and VvUFGT (B)throughout grape berry development in cv Cabernet Sauvignon (CabSauv) and cv Pinot Noir. Veraison occurred at 8 wpf (dashed lines).Gene expression was determined by qPCR and is shown relative to theaverage expression levels of three housekeeping genes, UBIQUITIN,ACTIN2, and ELONGATION FACTOR1a-2 (VvEF1a-2). All data arepresented as means of three technical replicates 6 SD.








anthocyanins with other glycosylation patterns, ac-tivity using cyanidin-3,5-O-diglucoside and cyanidin-3-O-rutinoside as acyl acceptors was also tested.Vv3AT was capable of acylating cyanidin-3,5-O-diglucoside, with an apparent Km of 64.17 6 3.87 mM, akcat of 0.18 6 0.02 s21, and an apparent specificity con-stant of 0.003 6 0.0004 mM

21 s21, the latter two beingmuch lower than those calculated when the mono-glucoside anthocyanins were used as substrates.Recombinant Vv3AT showed no activity with cyanidin-3-O-rutinoside.

DISCUSSION

VvMYBA Regulates the Later Stages of AnthocyaninSynthesis, Modification, and Transport

The production of transgenic grapevines with alteredexpression of VvMYBA genes has enabled what is, to ourknowledge, the first study of the effect of these TFs inberries. Expression or silencing of these genes resulted inaltered berry pigment phenotypes due to the role ofVvMYBA TFs in activating anthocyanin biosynthesis(Kobayashi et al., 2002;Walker et al., 2007; Cutanda-Perezet al., 2009). A comparison of the transcriptomic changesdetected in the transgenic cv Shiraz and cv Chardonnayberries revealed altered expression of 27 genes with FC.2 in both cultivars (Table I). Seventeen genes with FCsgreater than 10were up-regulated in red berries and havecharacterized roles in anthocyanin biosynthesis, while theonly gene that was down-regulated does not have thisfunction and the FCsweremuch lower. This suggests thatVvMYBA is a positive regulator of anthocyanin biosyn-thesis. Transcriptome studies in other plants over-expressing regulators of anthocyanin synthesis, such as

ANT1 (named for the activation-tagged insertion lineant1) in tomato and AtPAP1 in Arabidopsis, haveshown similar results (Mathews et al., 2003; Tohgeet al., 2005). The list of modulated genes within theindividual cultivars with altered VvMYBA expressionwas much larger than the 27 common genes affected inboth cultivars (Supplemental Tables S1 and S2). Whilethe VvMYBA1 transgene was clearly expressed in thetransgenic red cv Chardonnay berries, the combined

Figure 6. Relative gene expression of Vv3AT (Aand C) andVvUFGT (B andD) genes throughoutthe development of cv Cabernet Sauvignon(Cab Sauv), cv Malian, and cv Shalistin berries.Whole berries were used between 2 and 10 wpf(A and B), and skin sampleswere assayed from 9to 18wpf (C andD). Veraison occurred between9 and 10 wpf (dashed lines). Gene expressionwas determined by qPCR and is shown relativeto the average expression levels of threehousekeeping genes, UBIQUITIN, ACTIN2,and VvEF1a-2. All data are presented as meansof three technical replicates 6 SD.

Figure 7. Transcriptional activationofVvUFGT,Vv3AT-CS, andVv3AT-PNgene promoters by VvMYBA1. Luciferase activity was measured 48 h afterbombardment of grape suspension culture cells with promoter:pLUC con-structs along with 35S:VvMYBA1 in pART7, pRluc (Renilla spp. luciferaseplasmid for normalization), and a bHLH gene (i.e. either 35S:AtEGL3 inpFF19orVvMYC1 in pART7). Activitywas dividedbybackground luciferaseactivity of negative controls lacking the 35S:VvMYBA1 construct and isreported relative to Renilla spp. activity. Letters a, b, and c indicate where adifference is statistically significant as determinedbyone-wayANOVA. Errorbars represent SD of four technical replicates. This experimentwas carried outtwice with very similar results.


Rinaldo et al.




transcript levels of all three endogenous VvMYBA genes(nonfunctional) were similar in the mature transgenicberries compared with the cv Chardonnay control(Supplemental Figs. S2 and S3). This is consistent withthemicroarray results, where nonfunctionalMYBA geneexpression was not altered significantly.In grapevine, VvUFGT catalyzes the final step in

producing stable anthocyanins by adding a Glc mole-cule to anthocyanidins (Ford et al., 1998). Anthocyaninscan then be further modified bymethyltransferases and

acyltransferases before they are transported into thevacuole (Mazza, 1995; Fournier-Level et al., 2011).VvUFGT and genes involved in anthocyanin methyla-tion (VvFAOMT; Hugueney et al., 2009; Lücker et al.,2010) and modification for transport and vacuolartransport (VvGST4 and VvanthoMATE2; Conn et al.,2008; Gomez et al., 2011), plus Vv3AT encoding theBAHD anthocyanin acyltransferase characterized inthis study, were all strongly up-regulated in red berries.These results show that VvMYBA up-regulates the final

Figure 8. A, Wild type (WT) andtransgenic tobacco var Samsunflowers expressing theVv3AT geneunder the control of the 35S pro-moter (Vv3AT) representing therange of colors found on thoseplants. B, HPLC separation ofanthocyanins in wild-type andtransgenic Vv3AT tobacco flow-ers. Peak identities were deter-mined using LC-MS/MS. mAU,Milliabsorbance units. C, Totalanthocyanin content (ng g21 freshweight [FW]) of wild-type andtransgenic Vv3AT tobacco lineswith the relative proportion ofeach type of anthocyanin in thatsample. Anthocyanin content wasdetermined from three technicalreplicates, and error bars repre-sent SD.






steps of anthocyanin coloring of the berry and, hence, isan essential MYB TF required for the activation of theselater stages of anthocyanin synthesis and transport.While this was shown previously by Cutanda-Perezet al. (2009) in an analysis of transgenic grapevinehairy root cultures, the up-regulation ofVv3ATwas notdetected. Tohge et al. (2005), Luo et al. (2007), andButelli et al. (2008) also identified anthocyanin acyltrans-ferase genes that were up-regulated by MYB TFs in Arabidopsis and tomato, suggesting that this could be a wide-spread pattern. However the direct regulation of acyl-transferase genes was not demonstrated in their studies.

Many members of the VvF3959H family were up-regulated in red berries to similar levels as VvUFGTand Vv3AT. In grapes, anthocyanins can be eitherdihydroxylated or trihydroxylated at the 3949 (cyanidinand peonidin) or the 394959 (malvidin, delphinidin, andpetunidin) positions of the B ring, with VvF3959H en-zymes catalyzing the later reactions (Bogs et al., 2006),suggesting that VvMYBApreferentially up-regulates thesynthesis of trihydroxylated anthocyanins in berries.Many other early flavonoid pathway genes were up-regulated in red berries expressing VvMYBA (seeSupplemental Methods S1; Supplemental Figs. S2 andS3), albeit with much lower FCs compared with theaforementioned genes. The transcriptomic data showevidence that VvMYBA may also exert antagonistic ef-fects on genes related to the general phenylpropanoidmetabolism, such as several PAL genes, proanthocya-nidin biosynthesis, such as VvMYBPA1 (Bogs et al.,2007), and some PA-related structural genes. These ef-fects may be the result of ametabolic feedback and/or oftranscriptional interplays between regulators.

Evidence of Convergent Evolution of Vv3AT to otherBAHD Acyltransferases

The putative BAHD anthocyanin acyltransferasegene, Vv3AT, was identified as being up-regulated in

red berries from the microarray experiments. Thispreviously uncharacterized gene was not identified as acandidate involved in anthocyanin acylation variationin the QTL analysis recently published by Costantiniet al. (2015). A comprehensive phylogenetic analysis ofcharacterized BAHD plant acyltransferases, includingVv3AT, found that the proteins are grouped into fivemajor clades that could be classified predominantly bythe substrate specificities of the enzymes (Fig. 3;Supplemental Table S3), in agreement with previouslypublished phylogenetic trees (D’Auria, 2006; Yu et al.,2009; Tuominen et al., 2011). Most of the anthocyaninand flavonoid acyltransferases clustered together inclade I, but Vv3AT was placed in clade IIIa. The ma-jority of enzymes in clade I utilize malonyl-CoA as theirmajor acyl donor, while those in clade III mostly utilizeacetyl-CoA. Malonylated anthocyanins have not beendetected in grapevine, while acetylated anthocyaninsare common (for review, see He et al., 2010). However,another anthocyanin acyltransferase in this clade, fromS. splendens (Ss5MAT2), has been shown to utilizemalonyl-CoA to acylate anthocyanin-5-O-glucosides(Suzuki et al., 2004b). This enzyme and Vv3AT bothlack the characteristic YFGNC motif common to theanthocyanin transferases present in clade I (Fig. 3). Thissuggests that Vv3AT, like Ss5MAT2, has evolved froma different branch of the BAHD family than the otheranthocyanin acyltransferases designated as membersof clade I.

Due to the absence of acylated anthocyanins in thegrapevine cv Pinot Noir (Van Buren et al., 1968), acomparison of Vv3AT gene sequences and transcriptlevels in this cultivar with those of cv Cabernet Sau-vignon, which does produce acylated anthocyanins,was carried out. Very low or undetectable levels of theVv3AT transcript were present in cv Pinot Noir berriescompared with fruit from cv Cabernet Sauvignon,which accumulated the transcript postveraison, peakingat 10 wpf (2 weeks after veraison; Fig. 5). Furthermore,

Table II. Kinetics of recombinant Vv3AT enzyme with various acyl donor and acceptor substrates

Values were calculated from three independent biological replicates, from which the SD is reported.

Sample Km kcat Specificity Constant

mM s21 mM21 s21

Acyl donora

Acetyl-CoA 16.86 6 6.43 1.11 6 0.61 0.08 6 0.07Caffeoyl-CoA 4.10 6 0.37 0.29 6 0.09 0.07 6 0.02Coumaroyl-CoA 1.25 6 0.16 0.13 6 0.04 0.10 6 0.03Malonyl-CoA 588.77 6 74.48 0.92 6 0.03 0.0016 6 0.0002

Acyl acceptorb

Malvidin 3-glucoside 98.66 6 19.59 13.92 6 0.71 0.15 6 0.03Cyanidin 3-glucoside 506.22 6 8.55 77.00 6 5.92 0.15 6 0.01Delphinidin 3-glucoside 765.13 6 51.97 67.89 6 13.01 0.09 6 0.02Peonidin 3-glucoside 79.52 6 8.37 14.20 6 1.97 0.18 6 0.03Cyanidin 3,5-diglucoside 64.17 6 3.87 0.18 6 0.02 0.003 6 0.0004Cyanidin 3-rutinoside NAc NA NA

aThese reactions were carried out using malvidin 3-glucoside as an acyl acceptor. bThese reactionswere carried out using acetyl-CoA as an acyl donor. cNA, No activity.


Rinaldo et al.







any transcript that was expressed in cv Pinot Noirwould contain a premature stop codon, due to a non-sense mutation resulting in a truncated protein lackingtwo biochemically important motifs, HXXXDG andDFGWG (St-Pierre and Luca, 2000; Fig. 4). Hence, cvPinot Noir does not possess a functional Vv3AT pro-tein, despite the presence of an annotated sequence offull length in the backcrossed PN40024 genome. Thisgene sequence, VIT_03s0017g00870, could have beencontributed by another probable parent, Frankenthal,rather than cv Pinot Noir (Jaillon et al., 2007). TheVvMYBA1/2 genes were analyzed by Walker et al.(2007) and shown to be similar to those of cv Shiraz andcv Cabernet Sauvignon. It is unlikely that MYBA playsa direct role in the lack of acylated anthocyanins in thiscultivar, as anthocyanin biosynthesis is still prevalent.Taken together, the results from the protein assays andanalysis of the cv Pinot Noir Vv3AT gene strongly in-dicate that Vv3AT is the gene responsible for the acyl-ation of anthocyanins in grapevine.

Transcriptional Regulation of Vv3AT

Transient promoter-binding luciferase gene expres-sion assays using grapevine suspension cells showedthat VvMYBA can activate the expression of Vv3AT(Fig. 7). This result was supported by the pattern ofVv3AT transcription compared in developing berries ofcv Cabernet Sauvignon, cvMalian, and cv Shalistin thatdiffer in VvMYBA gene expression (Fig. 6). VvMYBAhas been shown to activateVvUFGT transcription (Walkeret al., 2007), and the expression of this gene followedalmost identical patterns to that of Vv3AT in the threecultivars over berry development and ripening. Therehas been relatively little work done on the regulation ofBAHDgene transcription, as studies havemostly focusedon their enzymatic functions. A limited number of studieshave used promoter fusion constructs to investigate thespatial and developmental expression of the BAHD ac-yltransferase genes: SUBERIN FERULOYL TRANSFER-ASE, tomatoACYLTRANSFERASE2, FATTY ALCOHOL:CAFFEOYL-COENZYME A CAFFEOYLTRANSFERASE,ALIPHATIC SUBERIN FERULOYL TRANSFERASE,DWARF AND ROUND LEAF1, DEFICIENT IN CUTINFERULATE, and SPERMIDINEHYDROXYCINNAMOYLTRANSFERASE (Grienenberger et al., 2009; Molina et al.,2009; Kosma et al., 2012; Rautengarten et al., 2012;Schilmiller et al., 2012; Boher et al., 2013; Zhu et al.,2013). Several studies have also shown links betweenthe expression of MYB TFs and BAHD acyltransferasegenes. For example, Onkokesung et al. (2012) identifiedthe putrescine and spermidine acyltransferases, NaAT1and NaDH29, through microarray studies utilizingN. attenuata plants, where the NaMYB8 gene, encodinga TF known to regulate phenolamide biosynthesis, wassilenced. Strong links between the expression ofNaMYB8, NaAT1, and NaDH29 were demonstrated,suggesting that their expression was controlled by thisTF. In another study, Luo et al. (2008) expressed the

AtMYB12 gene in tomato, which activates the biosyn-thesis of chlorogenic acids in Arabidopsis, compoundsderived from the phenylpropanoid pathway. The ex-pression of a number of tomato genes involved inchlorogenic acid biosynthesis was increased inAtMYB12-expressing lines, including HYDROXYCINNAMOYL-COENZYME A QUINATE TRANSFERASE andHYDROXYCINNAMOYL-COENZYME A SHIKIMATE/QUINATE HYDROXYCINNAMOYL TRANSFERASE,two characterized BAHD acyltransferases. The researchpresented in this article provides further insight intoBAHD gene regulation and presents additional evi-dence that MYB TFs may directly activate their tran-scription (Fig. 7).

The cv Cabernet Sauvignon and cv Pinot NoirVv3ATgene promoters, which differed by only six point mu-tations, were activated to similar levels by VvMYBA intransient assays (Fig. 7), even though very low geneexpression was detected in cv Pinot Noir berries post-veraison compared with cv Cabernet Sauvignon (Fig.5). Premature stop codons in mRNA transcripts canlead to the activation of the nonsense-mediated mRNAdecay pathway resulting in the rapid degradation ofnonsense transcripts (for review, see van Hoof andGreen, 2006). There is evidence that nonsense-mediatedmRNA decay acts on many plant gene mutants, in-cluding the flavonoid biosynthetic pathway gene CHSin petunia (Que et al., 1997). It is possible that the pre-mature stop codon in Vv3AT-PN is detected by thispathway, resulting in rapid degradation of the tran-script and, hence, the low levels of Vv3AT transcriptsdetected in cv Pinot Noir berries.

Vv3AT Functions as a BAHD Acyltransferase That CanUse Both Aliphatic and Aromatic Acyl Donors

The in planta function of Vv3AT was tested by con-stitutive expression of this gene in tobacco. One trans-genic line displayed a slightly more blue/purple hue tothe flowers compared with wild-type controls (Fig. 7),but this was not consistent in all lines, most of whichdisplayed no significant difference from the controls.Biochemical analyses showed that wild-type flowerscontained cyanidin- and pelargonidin-3-O-rutinosides,while the flowers expressing Vv3AT also contained cya-nidin- and pelargonidin-3-O coumaroyl-, caffeoyl-, andacetyl-monoglucosides (Fig. 8). No acylated rutinosideanthocyanin conjugates were detected in the transgenictobacco flowers, suggesting that Vv3AT is not able toacylate such anthocyanins. Rutinose is a disaccharidemade up of Glc with Rha attached at the 699 position,the same position where acylation occurs in grapevine.Luo et al. (2007), who expressed the ArabidopsisCOUMAROYL-COENZYME A:ANTHOCYANIDIN 3-O-GLUCOSIDE-699-O-COUMAROYLTRANSFERASE1/2 (At3AT1 and At3AT2) genes in tobacco, also reportedno visible significant difference in transgenic flower colorcompared with control flowers, despite the accumula-tion of coumaroylated anthocyanins.





Substrate preferences and enzyme kinetics of Vv3ATwere studied using in vitro assays with His-tagged re-combinant Vv3AT protein. The activity of this enzymewas tested with four anthocyanin acyl acceptor mole-cules found in grapevine and the three most commonacyl donors (acetyl-CoA, coumaroyl-CoA, and caffeoyl-CoA). Vv3AT catalyzed acylation using all of thesesubstrates. A recent study showed that berries of 34grapevine cultivars contained varying proportions ofacylated anthocyanins, but the majority containedmuch less caffeoylated anthocyanins compared withtheir acetylated and coumaroylated counterparts(Ferrandino et al., 2012). Considering the similar kineticproperties of Vv3AT with all three acyl donors (TableII), other factors must impact anthocyanin acylation,the most likely being the availability of the CoA sub-strates and the involvement of other enzymes in an-thocyanin acylation, such as the candidate genesidentified by Costantini et al. (2015). The aromatic acyldonors, caffeoyl- and coumaroyl-CoA, were shown toinhibit the production of the corresponding acylatedanthocyanin by recombinant Vv3AT at concentrationsgreater than 100 mM (Supplemental Fig. S10). This wasnot observed for the aliphatic acyl donor acetyl-CoA,suggesting that the aromatic ring of caffeoyl- andcoumaroyl-CoA may interfere with Vv3AT enzymefunction when present in high concentrations. Thissuggests that in planta substrate availabilitymay have a2-fold effect on the types of acylated anthocyaninsfound in a particular cultivar, as aromatic acyl donorconcentrations, either too low or too high, will have anegative effect on the occurrence of that type of acyla-tion event. An intriguing observation, reported also forthe kinetic parameters of Arabidopsis BAHD familyacyltransferase (D’Auria et al., 2007), was the approxi-mately 10-fold discrepancy in kcat values of Vv3AT de-termined for the donor or acceptor substrate, withthe other substrate at a fixed concentration above itsKm. The Arabidopsis BAHD family acyltransferaseAt5MAT showed comparable kcat values (0.12 and0.2 s21, respectively) for the acceptor substrate cyanidin3-O-[299-O-(xylosyl)-699-O-(p-O-(glucosyl)-p-coumaroyl)glucoside]5-O-glucoside and the donor substratemalonyl-CoA but an approximately 10-fold difference between thekcat values for a second acceptor substrate, cyanidin 3-O-[299-O-(2‴-O-(sinapoyl) xylosyl)699-O-(p-O-(glucosyl)p-coumaroyl) glucoside]5-O-glucoside, and the donorsubstrate malonyl-CoA (0.00012 and 0.0013 s21, re-spectively). Vv3AT kcat valueswere 13.93 and 1.11 s21 formalvidin-3-glucoside and acetyl-CoA, respectively.

Vv3AT activity was also tested with donor substratesused by other BAHD protein family members (Fig. 3;Supplemental Table S3). Most characterized anthocya-nin acyltransferases utilize malonyl-CoA as the majoracyl donor. Vv3AT was able to utilize malonyl-CoA,but it had much lower affinity for this substrate com-pared with acetyl-, coumaroyl-, and caffeoyl-CoA(Table II). This may explain the lack of malonylatedanthocyanins in grapevine, although the availability ofthis acyl donor may also play a role. Vv3AT could also

acylate cyanidin-3,5-O-diglucoside, but at lower effi-ciency compared with the monoglucosides tested (Ta-ble II). This preference for a particular glycosylationpattern has been reported before. For example, theArabidopsis anthocyanin acyltransferases, At3AT1 andAt3AT2, had over 100-fold lower kcat values whendiglucosides were used as substrates compared withmonoglucosides (Luo et al., 2007), while antho-cyanin malonyltransferases from chrysanthemum(Dendranthema 3 morifolium; Suzuki et al., 2004a) andDahlia variabilis (Suzuki et al., 2002) could not acylatepelargonidin-3,5-O-diglucoside. Recombinant Vv3ATcould not acylate cyanidin-3-O-rutinoside in vitro, inagreement with the observations of transgenic tobaccoflowers constitutively expressing Vv3AT (Fig. 8).Vv3AT uses 3-O-glucoside substrates and acylates thesugarmolecule at the 699 position. As such, it has similaractivity to a number of previously characterized BAHDenzymes with anthocyanin-3-O-glucoside-699-O-acyl-transferase activity. These include the aromatic acyl-transferases Perilla frutescensHYDROXYCINNAMOYLCOENZYME A:ANTHOCYANIN 3-O-GLUCOSIDE-699-O-ACYLTRANSFERASE as well as At3AT1 andAt3AT2 and the aliphatic acyltransferases D. variabilisMALONYL COENZYME A:ANTHOCYANIN 3-O-GLUCOSIDE-699-O-MALONYLTRANSFERASE, Sc3MaT(from Senecio cruentus), and MALONYL COENZYMEA:ANTHOCYANIN 3-O-GLUCOSIDE-699-O-MALO-NYLTRANSFERASE1 (from D. morifolium; Yonekura-Sakakibara et al., 2000; Suzuki et al., 2002, 2003, 2004a;Luo et al., 2007). Yet, Vv3AT is set apart from theseenzymes by its ability to utilize both aliphatic and ar-omatic acyl donor substrates with similar specificities, anovel activity for this enzyme family.

Suzuki et al. (2007) showed the versatility of theseenzymes by engineering in vitro the aliphatic antho-cyanin acyltransferase Ss5MaT1 so that it could useboth aliphatic and aromatic acyl donors. They con-verted the Val-39-Arg-40-Arg-41 amino acids inSs5MaT1, the two Arg residues having positive charge,to the uncharged amino acidsMet-Leu-Gln to create themutant Ss5AT306. This mutant enzyme could usep-coumaroyl-CoA and caffeoyl-CoA acyl donors inaddition to malonyl-CoA, the major donor used bySs5MaT1. Sequence comparison of Ss5MaT1 withDm3MaT3, a malonyl-CoA:anthocyanin 3-O-glucoside-699-O-malonyltransferase for which the crystal structurewas solved by Unno et al. (2007), suggests that thesethree residues form part of the binding pocket for theacyl acceptor rather than the acyl donor. Suzuki et al.(2007) hypothesized that the amino acid substitutionsin Ss5AT306 were not directly involved in acyl donorbinding but rather resulted in a conformational changeof the active site, which in turn allowed for the bindingof aromatic acyl donors. The equivalent amino acids inVv3AT are also uncharged (i.e. Ala-38-Ser-39-Asn-40),which may contribute to the flexibility of this enzymetoward acyl donor substrates. Homology modelingof the grapevine enzyme and Ss5AT306 indicatedthat the corresponding regions in both proteins share


Rinaldo et al.





significant structural similarities and are in each casedistal from the likely binding site for malonyl-CoA.Additionally, His-151, which corresponds to thestrictly conserved His-170 in members of the BAHDfamily of enzymes, is located farther from malonyl-CoAin Vv3AT (Supplemental Methods, Results, andDiscussion; Supplemental Figs. S11 and S12). The ho-mology model shows also that the channel into whichthe acyl donor substrate is predicted to bind is slightlynarrower in Vv3AT than in the other enzymes for whichstructural data are available (Supplemental Fig. S13).Taken together, these data provide good preliminaryevidence to explain the substrate specificity of thegrapevine enzyme.Interestingly, the four amino acids in Vv3AT (Ile-Phe-

Tyr-Tyr) following the Ala-38-Ser-39-Asn-40 residuesdiscussed above are quite divergent from those seen inmany other flavonoid acyltransferases, which weredemonstrated to contain the conserved motif (Leu/Val)-X-Phe-Tyr by Suzuki et al. (2007; Fig. 4). Anotherkey amino acid hypothesized to be involved in gov-erning acyl donor preferences corresponds to the posi-tively charged Arg-178 in Dm3MaT3. Suzuki et al.(2007) showed that aromatic anthocyanin acyltransfer-ases share a Phe at this residue, which may dictate theircoumaroyl-CoA preference. The equivalent amino acidresidue in Vv3AT is an Ala, divergent from that in otheranthocyanin acyltransferases capable of utilizing aro-matic acyl donors.

CONCLUSION

In this study, transgenic grapevines with altered ex-pression of VvMYBA genes were used in microarrayanalyses to show that VvMYBA is a positive regulatorof the later stages of anthocyanin biosynthesis, includ-ing their glycosylation, methylation, acylation, andtransport into the vacuole. Some genes controlling en-zymatic steps upstream of VvUFGT were also affected.From this microarray analysis, we identified and sub-sequently characterized, to our knowledge, the firstanthocyanin acyltransferase (Vv3AT) from grapevineand demonstrated this enzyme activity in planta and invitro. It has a preference for anthocyanin monogluco-side molecules and can use both aromatic and aliphaticacyl-CoA thioesters as substrates, an activity that isnovel for previously characterized naturally occurringBAHD acyltransferases, which show a strong prefer-ence for one type of thioester. Vv3AT is capable ofsynthesizing the common acylated anthocyanins iden-tified in grapevine. This study also shows that tran-scription from the promoter of the Vv3AT gene isactivated by VvMYBA1. This provides an example ofdirect TF-driven regulation of a BAHD gene familymember and adds to the current understanding of theanthocyanin biosynthetic pathway and how it is regu-lated in plants. The cv Pinot Noir does not produceacylated anthocyanins, as it appears to contain a trun-cated version of Vv3AT. This research will assist

breeding programs aimed at producing varieties withgood potential for stable red wine color and providesvaluable information to those wishing to engineer antho-cyanin acyltransferases and/or anthocyanin structures.

MATERIALS AND METHODS

Plant Material

Transgenic grapevines (Vitis vinifera) plus controls were all glasshousegrown in potting mix (20 L of composted pine [Pinus spp.] bark, 10 L of riversand, 30 g of FeSO4, 60 g of pH amendment, and 140 g of long-life Osmocote) inambient light with a night break. Day and night temperatures were approxi-mately 27°C and 22°C, respectively. For microarray experiments, whole berrieswere sampled from three independent transgenic lines of cv Chardonnay andcv Shiraz. Bunches were harvested close to ripeness based on average totalsoluble solids, whichwere between 20 and 24 degrees Brix (Supplemental TableS6). A sample consisted of a single bunch except when there were fewer than100 berries, in which case bunches were pooled. Skin samples were first re-moved from fresh berries. All samples were frozen in liquid N2 and stored at280°C. Due to unsynchronized flowering of the glasshouse-grown vines,sampling occurred throughout the year.

Berry samples of cv Cabernet Sauvignon and cv Malian and cv Shalistin va-rieties were collected from grapevines grown in a commercial vineyard at Lang-horne Creek, South Australia (35°1793099 south, 139°293399 east), in the season of2010/11. Samples were collected at 2, 4, 6, 8, 9, 10, 12, 15, and 18 wpf. Four to sixbunches were randomly selected from the same 20 vines (per variety), and berriesfrom thesewere pooled and frozen in liquidN2. Veraison occurred at 9wpf (when50% of the cv Cabernet Sauvignon and cv Malian berries were colored).

Extraction of RNA from cv Cabernet Sauvignon and cv Pinot Noir berrysampleswas described byDunlevy et al. (2013). GenomicDNAextractionswereperformed previously by Walker et al. (2007).

Stable Transformation of Grapevines

A pART7 vector containing the VvMYBA1 cDNA sequence between acauliflower mosaic virus 35S promoter and an octopine synthase gene) tran-scriptional terminator (Gleave, 1992) had been constructed previously (Walkeret al., 2007). This expression cassette was excised from pART7 using a NotIrestriction enzyme and ligated to the plant expression vector pART27 (Gleave,1992) to create the 35S:VvMYBA1 construct.

Primers A13pfxho and A13prasp (containing XhoI and Asp718 restrictionsites, respectively; Supplemental Table S7) were used to amplify a 427-bp sensefragment from the 39 end of the VvMYBA1 gene, and primers A13pfxba andA13prcla (containing XbaI and ClaI restriction sites, respectively; SupplementalTable S7) amplified the same fragment in the antisense direction by PCR (REDTAQ; Invitrogen). PCR cycling conditions were as follows: 94°C for 3 min;followed by 35 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 2 min; then afinal extension at 72°C for 10 min. Fragments were ligated to pDrive (Qiagen),fromwhich the sense and antisense fragments were excised using XhoI/Asp718and XbaI/ClaI restriction enzymes, respectively. These fragments were se-quentially ligated to the pN6 vector (Wesley et al., 2001). The silencing cassettewas excised from pN6 using a NotI restriction enzyme, and this fragment wasligated into pART27 to create the VvMYBA-silencing (VvMYBAsi) construct.

The transformation vector with VvMYBA1 expressed under its own pro-moter was generated by PCR ligation to join the promoter amplified frombacterial artificial chromosome CS63P1 with the cDNA sequence of the gene,both from cv Cabernet Sauvignon (Walker et al., 2007). PCR was performedwith Pfu DNA polymerase (Stratagene) using primers A1prof1 and A1promr1and the bacterial artificial chromosome template, while the cDNA was ampli-fied with primers A1rxba and A1Ex2f with annealing temperature of 58°C.Products were extracted from a TAE agarose gel and used as the template in afurther 10 rounds of PCR. A 3-kb fragment was then ligated to Zero Blunt(Invitrogen), sequenced, and then transferred to pART7NAPX, a promoterlessderivative of pART7 (Goetz et al., 2006), usingAsp718 andXbaI restriction sites.The construct was transferred to pART27 utilizing NotI restriction sites.

VvMYBA expression or silencing pART27 constructs were inserted intoAgrobacterium tumefaciens strain EHA105, which was used to transform em-bryogenic callus utilizing the method of Iocco et al. (2001). Anthers of immatureflowers of cv Shiraz (BVRC12) and cv Chardonnay (I10V1) from Coombe












Vineyard, University of Adelaide, Waite Campus, were used to initiate calli onPIV and HT media, respectively. Embryogenic callus from both cultivars wasmaintained on C1 for 12 to 20 months prior to transformation. Transformedcallus was selected using 100 mg mL21 kanamycin. Germinated embryos werecut to remove the roots and the tips, comprising the cotyledons supported onapproximately 5 mm of hypocotyl, and transferred to shooting medium con-taining 10 mM benzylaminopurine (BAP) for shoot development. Rootedplantlets, whose leaves tested positive by PCR to the transgene, were deflaskedinto potting mix and hardened off over 3 weeks in the glasshouse.

RNA Extractions, cDNA Synthesis, Labeling, andMicroarray Experiments

Frozen whole berry samples were ground to a fine powder under liquidnitrogen using a chilled grindingmill (IKA) and amortar and pestle. Total RNAwas extracted using a modified perchlorate method described previously byBoss et al. (2001). Genomic DNA was removed using DNase (RNase-freeDNase; Qiagen) in conjunction with the RNeasy Mini kit (Qiagen). A spectro-photometer (NanoDrop 1000 V3.7.1; Thermo Fisher Scientific) and a bio-analyzer (Bioanalyser Chip RNA 7500 series II; Agilent) were used to determineRNA quantity and quality by ensuring that the absorbance ratios A260/A280 andA260/A230 were 1.8 or greater and the RNA integrity number was 1.7 or greater.

The cDNA synthesis, labeling, and chip hybridization were all carried outaccording to the NimbleGen Arrays User’s Guide: Gene Expression Analysisv3.2 protocols (Roche) using the NimbleGen microarray 090818 Vitis exp HX12(Roche). The microarray was scanned (ScanArray 4000XL; Perkin-Elmer) at 532nm (Cy-3 absorption peak) in conjunction with GenePix Pro-7 software (Mo-lecular Devices) to produce high-resolution images. Images were then analyzedusing NimbleScan version 2.5 software (Roche) which used a robust multichipaverage procedure to produce normalized expression data for each gene de-rived from the average of the signal intensities of the four probes on themicroarray for that gene.

Normalized expression values were converted to log2 values, and a Pearsoncorrelation analysis was carried out to evaluate the robustness of the biologicalreplicates in each sample. A gene was considered to be expressed if the nor-malized expression value for at least two of the three biological replicates washigher than the value obtained by averaging the fluorescence of negative con-trols present on the chip. A two-class unpaired significance analysis of micro-arrays was utilized using TMeV software (Saeed et al., 2003) to compare theexpression values between transgenic lines and the respective controls. The falsediscovery rate was set to 2.5% for the cv Chardonnay and cv Shiraz data sets.

To find gene expression that was altered due to VvMYBA in a consistentmanner in both cv Chardonnay and cv Shiraz berries, genes that were up-regulated in the red cv Chardonnay berries (positive red-white ratio) andconversely down-regulated inwhite cv Shiraz berries (negativewhite-red ratio),or vice versa,were of interest. Only geneswith FC$ 2 in both the cvChardonnayand cv Shiraz data sets were included.

Nucleic Acid Extractions and cDNA Synthesis for qPCR

RNA extraction and cDNA synthesis for cv Cabernet Sauvignon and cvPinot Noir berry developmental series were conducted by Dunlevy et al. (2010).

All other RNA extractions were performed using the Spectrum Plant TotalRNA Kit (Sigma-Aldrich) and On-column DNase I Digestion Kit (Sigma-Aldrich) for genomic DNA removal. cDNA was synthesized with a reversetranscriptase kit (Phusion RT-PCR Kit; Finnzymes).

DNAwas extracted fromyoung leaves of cvCabernet Sauvignon and cv PinotNoir vines previously byWalker et al. (2007) and from young transgenic tobacco(Nicotiana tabacum) leaves using the ISOLATE Plant DNA mini kit (Bioline).

Analysis of Gene Expression

Specific primers were designed to amplify 100- to 300-bp products fromVv3AT and reporter genes (Supplemental Table S7). The specificity of eachprimer pair was confirmed by PCR, sequencing, and detection of a single peakof fluorescence frommelt curves during qPCR. cDNAwas diluted 1:40 in sterileNanopure water (Thermo Fisher Scientific) before use. qPCR experiments wereconducted using a thermocycler (LightCycler 480 II instrument; Roche). Eachsample was assayed in triplicate in a reaction volume of 15 mL made up of 5 mLof diluted cDNA and 0.5 mM of each primer (Supplemental Table S7) in 13LightCycler 480 SYBR Green I Master Mix (Roche). Thermocycling conditionswere as follows: initial activation at 95°C for 5 min; followed by 45 cycles of

95°C for 20 s, 58°C for 20 s, and 72°C for 20 s; then a final extension at 72°C for5min. Reactionswere then heated to 95°C for 5min, cooled to 50°C for 45 s, andthen heated to 95°C at a 0.11°C s21 ramping rate to produce melt curves. Foreach gene, standard curves were produced from a linear dilution series of targetDNA fragments created by PCR, from which sample transcript concentrationwas determined. These concentrations were normalized against an averagevalue obtained from three housekeeping genes, UBIQUITIN, ACTIN2, andVvEF1a-2 (GenBank accession nos. CF406001, AF369525.1, and TC38276, re-spectively), and are reported as relative transcript levels.

Isolation of the Vv3AT Gene from Grapevine

Primers flanking the predicted start and stop codons of theVIT_03s0017g00870 gene (Supplemental Table S7) based on the 123 grapevinegenome (V.1 version; http://genomes.cribi.unipd.it/grape/) were used toamplify cv Cabernet Sauvignon and cv Pinot Noir genomic DNA and cDNAclones using PCR (Platinum Taq DNA Polymerase; Invitrogen). Cycling con-ditions were as follows: 95°C for 5 min; followed by 35 cycles of 95°C for 30 s,55°C for 30 s, and 72°C for 2.5 min; then a final extension at 72°C for 10 min.Fragments were ligated to pDrive (Qiagen) and sequenced.

Sequence Analysis and Phylogenetic Tree Construction

All sequencingwas carried out at theAustralianGenomeResearch Facility inAdelaide using their purified DNA sequencing service. Nucleotide and proteinsequence alignmentswere carried out usingAlignX (a component of VectorNTIAdvance 11.0; Invitrogen) except for the alignment used in the phylogeneticanalysis, for which a ClustalW alignment was used. In this case, the final se-quence alignment was generated by manually editing a ClustalW alignment(version 1.83) to select for conserved positions (Larkin et al., 2007). Phylogenieswere constructed using the BEAST (Bayesian Evolutionary Analysis by Sam-pling Trees) version 1.7.5 package (Ayres et al., 2012; Drummond et al., 2012).The WAG (Whelan and Goldman) substitution model with gamma + invariantsite heterogeneity was used with a strict molecular clock. A random startingtree was used with the Yule Process tree prior to determining speciation. AMarkov chain Monte Carlo maximum chain length of 100 million was set, andthe tree was run until completion. Tracer version 1.5 was used to ensure con-vergence by assessing the estimated sample size and the likelihood of the es-timated parameters. TreeAnnotator version 1.7.5 was used to generate themaximum clade credibility tree with a burn in of 50,000. The tree was thenannotated using FigTree (Rambaut, 2009). RAxML was used to confirm thestructure of the BEAST tree, and the ZmGlossy2 and AtCER2 sequences wereset as the outgroup (Stamatakis et al., 2008).

Production of Genetically Modified Tobacco Expressingthe Vv3AT Coding Sequence

Primers were designed to the Vv3AT gene to include an XhoI restriction siteimmediately 59 of the start codon and an Asp718 site immediately 39 of the stopcodon (Supplemental Table S7). The gene fragment was amplified by PCR, li-gated to pDrive (Qiagen), and sequenced. The gene was then inserted into themultiple cloning site of the pART7 vector (Gleave, 1992). This expression cas-sette was excised from pART7 using NotI restriction enzyme and ligated to theplant transformation vector pART27uGFP (derived from pART27 containingGFP driven by the Arabidopsis [Arabidopsis thaliana] ubiquitin promoter) tocreate the 35S:Vv3AT construct.

A. tumefaciens strain LBA4404 containing the 35S:Vv3AT construct was usedto transform tobacco var Samsun, based on the methods of Horsch et al. (1985).Bacteria were grown on Luria broth plates with appropriate antibiotics and200 mM acetosyringone at 28°C for 4 d, then suspended inMurashige and Skoog(MS) medium (13 MS salts, 13 Gamborg’s vitamins, and 30 g L21 Suc) to anoptical density at 600 nm of 1. Incisions were made on the abaxial side of tobaccoleaves parallel to the midrib, submerged in the MS medium/A. tumefaciensmixture for 10 min, blotted onto sterile filter paper, and transferred (abaxial sideup) to MS plates containing 1 mM each of a-naphthaleneacetic acid and BAP(Sigma) for 4 d at 20°C. Leaf pieceswerewashed inMSmedium containing 500mgmL21 cefotaxime, blotted on sterile filter paper, and transferred (abaxial sidedown) ontoMS plates containing 1mM each of a-naphthaleneacetic acid and BAP,500 mg mL21 cefotaxime, and 100 mg mL21 kanamycin. These were kept at 27°Cunder a 16/8-h photoperiod and transferred to fresh medium every 2 weeks.Shoots about 1 cm in length were transferred onto MS plates containing100 mg mL21 kanamycin. Six independent transformants, positive for the Vv3AT


Rinaldo et al.





http://genomes.cribi.unipd.it/grape/



construct by PCR analysis, were transferred to potting mix and hardened offin the glasshouse under the same conditions as the transgenic grapevines.Control tobacco plants were grown from cuttings in tissue culture on MSmedium and transferred to the glasshouse simultaneously. Vv3AT transcriptlevels in flowers of all six transformant lines were determined by qPCR asdescribed above.

Vv3AT Protein Expression and Purification

Vv3AT was amplified using primers VvBAHDNotI_F1 and VvBAHDX-hoI_R1 (Supplemental Table S7) and ligated to the pET30a(+) expression vector(Novagen, Merck), using the XhoI and NotI restriction sites, to generate anN-terminal His-Vv3AT fusion protein.

Escherichia coli [pBL21(DE3)] cells were cotransformed with pRIL (Stra-tagene) and either pET30a:His-Vv3AT or the empty pET30a. Cultures wereused to produce recombinant His-Vv3AT protein according to an auto-induction, high-density culturing method described by Studier (2005). Cellswere pelleted and lysed, and the lysate was clarified as described previously byDunlevy et al. (2010). His-tagged protein was purified, although not to homo-geneity, by His-tag affinity chromatography, and the final concentration ofrecombinant protein was estimated using a His-Tag Protein ELISA Kit (CellBiolabs) according to the manufacturer’s instructions (Supplemental Figs. S6and S7) and visualization of anti-His immunoreactive bands on western blots.

Enzyme Assays

Recombinant enzyme assayswere conductedusing recombinantHis-Vv3ATprotein from a single purification batch and repeated with two other batchesgrownandpurified independently.All reactionswere carriedout in triplicate forthe first batch and in duplicate thereafter; the same protein fraction from E. colicells containing an empty pET30a vector was used as a negative control. Suit-able assay parameters were determined by first testing various buffer, pH, andtemperature conditions (Supplemental Fig. S8). Reactions were conducted in avolume of 50 mL using 0.1 M sodium phosphate buffer, pH 6.5, and containing5mL of CoA-conjugated acyl donor dissolved in 0.1 M sodium phosphate buffer,1 mL of anthocyanin acyl acceptor dissolved in 100% methanol, and 0.5, 2, or0.67 mL of concentrated protein fraction from purification batches 1, 2, and 3,respectively. When determining Km, acyl donor and acceptors were maintainedat 200 mM, while the concentration of the other substrate was varied. Reactionswere carried out at 30°C for 20 min, which was within the linear range of thereaction based on preliminary tests (Supplemental Fig. S9). The reaction wasstopped by the addition of 50 mL of 100% methanol. The Km was calculatedby creating a Lineweaver-Burk plot and using the following equation: 1/V =(1/Vmax) + ((Km/Vmax) 3 (1/[S])).

Extraction and Detection of Anthocyanins

Anthocyanins were extracted from 100-mg aliquots of ground, frozen to-bacco flowers with 300 mL of 0.3% (v/v) formic acid in 70% (v/v) methanol,sonicated for 20 min in an ice bath, and centrifuged to pellet debris. A total of25 mL of the tobacco flower extraction supernatants and recombinant enzymeassay reactions was used for anthocyanin separation and quantification.

Anthocyanins were separated using a Hewlett Packard 1100 HPLC systemwith aWakosil C18 analytical column (3mm, 150mm3 4.6mm; SGE) protectedby a C18 guard column (SGE), following the method described by Downey andRochfort (2008). Anthocyanin concentrations in tobacco extracts were deter-mined by comparison of peak areas with a standard curve of cyanidin-3-O-rutinoside (0–37.8 ng mL21, r2 = 0.998). Acylated anthocyanin productconcentrations within the recombinant enzyme assays were determined bycomparison with a malvidin-3-O-glucoside standard curve (0–144.1 ng mL21,r2 = 0.998). Anthocyanin peaks were identified by their tandem mass spec-trometry parent and major daughter ions utilizing the HPLC method as de-scribed above coupled to a 6410 triple quadrupole mass spectrometer (Agilent)as described by Downey and Rochfort (2008). Supplemental Table S5 summa-rizes the ions detected for each compound, which were similar to reportedvalues (Luo et al., 2007; Downey and Rochfort, 2008).

Luciferase-Binding Assays

Promoter regions, 711 bp upstream of the putative start codon, were am-plified from cv Cabernet Sauvignon and cv Pinot Noir DNA using primers

VvBAHDPrF2_SacI and VvBAHDPrR1_BglII (Supplemental Table S7). Prod-ucts were ligated to the firefly (Photinus pyralis) luciferase (LUC) plasmid pLUC(Horstmann et al., 2004) using the SacI and BglII restriction sites. VvMYC1(NM001281253) was amplified using primers VvMyc1 F2 and R2(Supplemental Table S7) and ripe cv Shiraz berry skin cDNA, sequenced, andinserted in pART7 (Gleave, 1992). Transient transfection of cv Chardonnay sus-pension cultures and luciferase assays were carried out as described by Harriset al. (2013). All transfectionswere done in at least triplicate and repeated twice. Astatistical comparison of relative luciferase activity from the different promotersand bHLH TFs used in these experiments was conducted using a one-wayANOVA (P , 0.05) within the SPSS 16.0 statistical software package (SPSS).

Sequence data from this article can be found in the Microarray GeneExpression Database (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=cnmxwmmsfjctvgt&acc=GSE56915) under accession numbers Vv3AT-CS1, KM267559; Vv3AT-CS2, KM267560; and Vv3AT-PN, KM267561.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Total anthocyanin concentration in wild-typeand transgenic cv Chardonnay and cv Shiraz whole berries with alteredVvMYBA expression.

Supplemental Figure S2. Transcript abundance, determined by qPCR, ofVvMYBA transgene, VvMYBA, VvUFGT, and Vv3AT in wild-type andtransgenic cv Chardonnay and cv Shiraz whole berries with alteredVvMYBA expression using samples that were used in microarray exper-iments.

Supplemental Figure S3. Transcript abundance, determined by qPCR, ofVvCHS1, VvCHS2, VvCHS3, and DFR in wild-type and transgenic cvChardonnay and cv Shiraz whole berries with altered VvMYBA expres-sion using samples that were used in microarray experiments.

Supplemental Figure S4. Photographs of cv Cabernet Sauvignon, cv Malian,and cv Shalistin berries.

Supplemental Figure S5. Vv3AT transcript levels in flowers of transgenictobacco lines expressing Vv3AT.

Supplemental Figure S6. Protein gels from the His-tag affinity chromatog-raphy purification and quantification of Vv3AT.

Supplemental Figure S7. Western blots from the His-tag affinity chroma-tography purification and quantification of Vv3AT.

Supplemental Figure S8. Optimization of recombinant Vv3AT protein as-say buffer, pH, and temperature conditions.

Supplemental Figure S9. Determination of the linear range of recombinantVv3AT protein assay reactions for a single purification.

Supplemental Figure S10. Activity of Vv3AT protein with different acylacceptor molecules at varying concentrations.

Supplemental Figure S11. Structure superimposition of Ss5MaT1,Ss5AT306, and Vv3AT with Dm3MaT3.

Supplemental Figure S12. The interaction potentials of key residues withmalonyl-CoA ligand in Dm3MaT3, Ss5AT306, Ss5MaT1, and Vv3AT.

Supplemental Figure S13. The overall hydrophobicity profile of the acyldonor-binding pocket and modeled interaction with a malonyl-CoA lig-and for Dm3MaT3, Ss5AT306, Ss5MaT1, and Vv3AT.

Supplemental Table S1. Differentially expressed genes in red cv Chardon-nay 35S:VvMYBA1 berries by SAM analysis (provided as Excel file).

Supplemental Table S2. Differentially expressed genes in white cv ShirazVvMYBAsi berries by SAM analysis (provided as Excel file).

Supplemental Table S3. Characterized BAHD proteins used in phyloge-netic tree analysis.

Supplemental Table S4. Absolute chemiluminescence values from lucifer-ase reporter promoter activation assays in grapevine suspension cells.

Supplemental Table S5. Mass spectrometry parent ion and mass spec-trometry 2 major daughter ions detected by LC-MS/MS used to identify

































acylated anthocyanins in recombinant Vv3AT bioassays and transgenictobacco flowers.

Supplemental Table S6. Total soluble sugars in berries used in microarrayexperiments.

Supplemental Table S7. Details of primers.

Supplemental Methods S1. Additional methods, results, and discussion.

Supplemental References S1. Additional references.

ACKNOWLEDGMENTS

We thankMac Cleggett andAnneMcLennan (CleggettWines) for providingsamples of the cv Cabernet Sauvignon mutant grapes from the vineyard inLanghorne Creek; Christine Böttcher for expert advice on protein expressionand purification techniques as well as article preparation; and Elizabeth Lee,Karin Sefton, Lorraine Carruthers, Sue Maffei, Emily Nicholson, Adelle Craig,and Bronwyn Smithies for technical assistance.

Received August 13, 2015; accepted August 13, 2015; published September 22,2015.

LITERATURE CITED

Ayres DL, Darling A, Zwickl DJ, Beerli P, Holder MT, Lewis PO,Huelsenbeck JP, Ronquist F, Swofford DL, Cummings MP, et al(2012) BEAGLE: an application programming interface and high-performance computing library for statistical phylogenetics. Syst Biol61: 170–173

Bogs J, Ebadi A, McDavid D, Robinson SP (2006) Identification of theflavonoid hydroxylases from grapevine and their regulation during fruitdevelopment. Plant Physiol 140: 279–291

Bogs J, Jaffé FW, Takos AM, Walker AR, Robinson SP (2007) Thegrapevine transcription factor VvMYBPA1 regulates proanthocyanidinsynthesis during fruit development. Plant Physiol 143: 1347–1361

Boher P, Serra O, Soler M, Molinas M, Figueras M (2013) The potatosuberin feruloyl transferase FHT which accumulates in the phellogen isinduced by wounding and regulated by abscisic and salicylic acids. JExp Bot 64: 3225–3236

Boss PK, Davies C (2009) Molecular biology of anthocyanin accumulationin grape berries. In KA Roubelakis-Angelakis, ed, Molecular Biologyand Biotechnology of Grapevine. Springer, Berlin, pp 263–292

Boss PK, Davies C, Robinson SP (1996a) Analysis of the expression ofanthocyanin pathway genes in developing Vitis vinifera L. cv Shirazgrape berries and the implications for pathway regulation. Plant Physiol111: 1059–1066

Boss PK, Davies C, Robinson SP (1996b) Anthocyanin composition andanthocyanin pathway gene expression in grapevine sports differing inberry skin colour. Aust J Grape Wine Res 2: 163–170

Boss PK, Vivier M, Matsumoto S, Dry IB, Thomas MR (2001) A cDNAfrom grapevine (Vitis vinifera L.), which shows homology to AGAMOUSand SHATTERPROOF, is not only expressed in flowers but alsothroughout berry development. Plant Mol Biol 45: 541–553

Butelli E, Titta L, Giorgio M, Mock HP, Matros A, Peterek S, SchijlenEGWM, Hall RD, Bovy AG, Luo J, et al (2008) Enrichment of tomatofruit with health-promoting anthocyanins by expression of select tran-scription factors. Nat Biotechnol 26: 1301–1308

Conn S, Curtin C, Bézier A, Franco C, Zhang W (2008) Purifica-tion, molecular cloning, and characterization of glutathione S-transferases (GSTs) from pigmented Vitis vinifera L. cell suspension cul-tures as putative anthocyanin transport proteins. J Exp Bot 59:3621–3634

Costantini L, Malacarne G, Lorenzi S, Troggio M, Mattivi F, Moser C,Grando MS (2015) New candidate genes for the fine regulation of thecolour of grapes. J Exp Bot 66: 4427–4440

Cutanda-Perez MC, Ageorges A, Gomez C, Vialet S, Terrier N, Romieu C,Torregrosa L (2009) Ectopic expression of VlmybA1 in grapevine acti-vates a narrow set of genes involved in anthocyanin synthesis andtransport. Plant Mol Biol 69: 633–648

D’Auria JC (2006) Acyltransferases in plants: a good time to be BAHD.Curr Opin Plant Biol 9: 331–340

D’Auria JC, Reichelt M, Luck K, Svatos A, Gershenzon J (2007) Identifi-cation and characterization of the BAHD acyltransferase malonyl CoA:anthocyanidin 5-O-glucoside-699-O-malonyltransferase (At5MAT) inArabidopsis thaliana. FEBS Lett 581: 872–878

Dixon RA, Paiva NL (1995) Stress-induced phenylpropanoid metabolism.Plant Cell 7: 1085–1097

Downey MO, Rochfort S (2008) Simultaneous separation by reversed-phase high-performance liquid chromatography and mass spectralidentification of anthocyanins and flavonols in Shiraz grape skin. JChromatogr A 1201: 43–47

Drummond AJ, Suchard MA, Xie D, Rambaut A (2012) Bayesian phylo-genetics with BEAUti and the BEAST 1.7. Mol Biol Evol 29: 1969–1973

Dunlevy JD, Dennis EG, Soole KL, Perkins MV, Davies C, Boss PK (2013)A methyltransferase essential for the methoxypyrazine-derived flavourof wine. Plant J 75: 606–617

Dunlevy JD, Soole KL, Perkins MV, Dennis EG, Keyzers RA, Kalua CM,Boss PK (2010) Two O-methyltransferases involved in the biosynthesisof methoxypyrazines: grape-derived aroma compounds important towine flavour. Plant Mol Biol 74: 77–89

Falginella L, Castellarin SD, Testolin R, Gambetta GA, Morgante M, DiGaspero G (2010) Expansion and subfunctionalisation of flavonoid 39,59-hydroxylases in the grapevine lineage. BMC Genomics 11: 562–579

Ferrandino A, Carra A, Rolle L, Schneider A, Schubert A (2012) Profilingof hydroxycinnamoyl tartrates and acylated anthocyanins in the skin of34 Vitis vinifera genotypes. J Agric Food Chem 60: 4931–4945

Ford CM, Boss PK, Høj PB (1998) Cloning and characterization of Vitisvinifera UDP-glucose:flavonoid 3-O-glucosyltransferase, a homologue ofthe enzyme encoded by the maize Bronze-1 locus that may primarilyserve to glucosylate anthocyanidins in vivo. J Biol Chem 273: 9224–9233

Fournier-Level A, Hugueney P, Verriès C, This P, Ageorges A (2011)Genetic mechanisms underlying the methylation level of anthocyaninsin grape (Vitis vinifera L.). BMC Plant Biol 11: 179–192

Fujiwara H, Tanaka Y, Yonekura-Sakakibara K, Fukuchi-Mizutani M,Nakao M, Fukui Y, Yamaguchi M, Ashikari T, Kusumi T (1998) cDNAcloning, gene expression and subcellular localization of anthocyanin5-aromatic acyltransferase from Gentiana triflora. Plant J 16: 421–431

Gleave AP (1992) A versatile binary vector system with a T-DNA organ-isational structure conducive to efficient integration of cloned DNA intothe plant genome. Plant Mol Biol 20: 1203–1207

Goetz M, Vivian-Smith A, Johnson SD, Koltunow AM (2006) AUXINRESPONSE FACTOR8 is a negative regulator of fruit initiation in Arabi-dopsis. Plant Cell 18: 1873–1886

Gomez C, Conejero G, Torregrosa L, Cheynier V, Terrier N, Ageorges A(2011) In vivo grapevine anthocyanin transport involves vesicle-mediated trafficking and the contribution of anthoMATE transportersand GST. Plant J 67: 960–970

Grienenberger E, Besseau S, Geoffroy P, Debayle D, Heintz D, LapierreC, Pollet B, Heitz T, Legrand M (2009) A BAHD acyltransferase is ex-pressed in the tapetum of Arabidopsis anthers and is involved in thesynthesis of hydroxycinnamoyl spermidines. Plant J 58: 246–259

Guillaumie S, Fouquet R, Kappel C, Camps C, Terrier N, Moncomble D,Dunlevy JD, Davies C, Boss PK, Delrot S (2011) Transcriptional anal-ysis of late ripening stages of grapevine berry. BMC Plant Biol 11: 165–191

Harris NN, Luczo JM, Robinson SP, Walker AR (2013) Transcriptionalregulation of the three grapevine chalcone synthase genes and their role inflavonoid synthesis in Shiraz. Aust J Grape Wine Res 19: 221–229

He F, Mu L, Yan GL, Liang NN, Pan QH, Wang J, Reeves MJ, Duan CQ(2010) Biosynthesis of anthocyanins and their regulation in coloredgrapes. Molecules 15: 9057–9091

He J, Giusti MM (2010) Anthocyanins: natural colorants with health-promoting properties. Annu Rev Food Sci Technol 1: 163–187

Hichri I, Heppel SC, Pillet J, Léon C, Czemmel S, Delrot S, Lauvergeat V,Bogs J (2010) The basic helix-loop-helix transcription factor MYC1 isinvolved in the regulation of the flavonoid biosynthesis pathway ingrapevine. Mol Plant 3: 509–523

Horsch RB, Fry JE, Hoffmann NL, Wallroth M, Eichholtz D, Rogers SG,Fraley RT (1985) A simple and general method for transferring genesinto plants. Science 227: 1229–1231

Horstmann V, Huether CM, Jost W, Reski R, Decker EL (2004) Quantita-tive promoter analysis in Physcomitrella patens: a set of plant vectors ac-tivating gene expression within three orders of magnitude. BMCBiotechnol 4: 13


Rinaldo et al.







Hugueney P, Provenzano S, Verriès C, Ferrandino A, Meudec E, BatelliG, Merdinoglu D, Cheynier V, Schubert A, Ageorges A (2009) A novelcation-dependent O-methyltransferase involved in anthocyanin meth-ylation in grapevine. Plant Physiol 150: 2057–2070

Iocco P, Franks T, Thomas MR (2001) Genetic transformation of majorwine grape cultivars of Vitis vinifera L. Transgenic Res 10: 105–112

Jaillon O, Aury JM, Noel B, Policriti A, Clepet C, Casagrande A, ChoisneN, Aubourg S, Vitulo N, Jubin C, et al (2007) The grapevine genomesequence suggests ancestral hexaploidization in major angiospermphyla. Nature 449: 463–467

Kobayashi S, Goto-Yamamoto N, Hirochika H (2004) Retrotransposon-induced mutations in grape skin color. Science 304: 982

Kobayashi S, Ishimaru M, Hiraoka K, Honda C (2002) Myb-related genesof the Kyoho grape (Vitis labruscana) regulate anthocyanin biosynthesis.Planta 215: 924–933

Kosma DK, Molina I, Ohlrogge JB, Pollard M (2012) Identification of anArabidopsis fatty alcohol:caffeoyl-coenzyme A acyltransferase requiredfor the synthesis of alkyl hydroxycinnamates in root waxes. PlantPhysiol 160: 237–248

Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA,McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, et al (2007)Clustal W and Clustal X version 2.0. Bioinformatics 23: 2947–2948

Lücker J, Martens S, Lund ST (2010) Characterization of a Vitis vinifera cv.Cabernet Sauvignon 39,59-O-methyltransferase showing strong prefer-ence for anthocyanins and glycosylated flavonols. Phytochemistry 71:1474–1484

Luo J, Butelli E, Hill L, Parr A, Niggeweg R, Bailey P, Weisshaar B,Martin C (2008) AtMYB12 regulates caffeoyl quinic acid and flavonolsynthesis in tomato: expression in fruit results in very high levels of bothtypes of polyphenol. Plant J 56: 316–326

Luo J, Nishiyama Y, Fuell C, Taguchi G, Elliott K, Hill L, Tanaka Y,Kitayama M, Yamazaki M, Bailey P, et al (2007) Convergent evolutionin the BAHD family of acyl transferases: identification and characteri-zation of anthocyanin acyl transferases from Arabidopsis thaliana. Plant J50: 678–695

Markham KR (1988) Distribution of flavonoids in the lower plants and itsevolutionary significance. In JB Harborne, ed, The Flavonoids: Ad-vances in Research since 1986, Vol 4. CRC Press, Boca Raton, FL, pp427–468

Mathews H, Clendennen SK, Caldwell CG, Liu XL, Connors K, MatheisN, Schuster DK, Menasco DJ, Wagoner W, Lightner J, et al (2003)Activation tagging in tomato identifies a transcriptional regulator ofanthocyanin biosynthesis, modification, and transport. Plant Cell 15:1689–1703

Matus JT, Poupin MJ, Cañón P, Bordeu E, Alcalde JA, Arce-Johnson P(2010) Isolation of WDR and bHLH genes related to flavonoid synthesisin grapevine (Vitis vinifera L.). Plant Mol Biol 72: 607–620

Mazza G (1995) Anthocyanins in grapes and grape products. Crit Rev FoodSci Nutr 35: 341–371

Molina I, Li-Beisson Y, Beisson F, Ohlrogge JB, Pollard M (2009) Identi-fication of an Arabidopsis feruloyl-coenzyme A transferase required forsuberin synthesis. Plant Physiol 151: 1317–1328

Nakayama T, Suzuki H, Nishino T (2003) Anthocyanin acyltransferases:specificities, mechanism, phylogenetics, and applications. J Mol Catal BEnzym 23: 117–132

Onkokesung N, Gaquerel E, Kotkar H, Kaur H, Baldwin IT, Galis I (2012)MYB8 controls inducible phenolamide levels by activating three novelhydroxycinnamoyl-coenzyme A:polyamine transferases in Nicotiana at-tenuata. Plant Physiol 158: 389–407

Petroni K, Tonelli C (2011) Recent advances on the regulation of antho-cyanin synthesis in reproductive organs. Plant Sci 181: 219–229

Que Q, Wang HY, English JJ, Jorgensen RA (1997) The frequency anddegree of cosuppression by sense chalcone synthase transgenes are de-pendent on transgene promoter strength and are reduced by prematurenonsense codons in the transgene coding sequence. Plant Cell 9: 1357–1368

Rambaut A (2009) FigTree version 1.4.0. http://tree.bio.ed.ac.uk/software/figtree (December 12, 2014)

Rautengarten C, Ebert B, Ouellet M, Nafisi M, Baidoo EEK, Benke P,Stranne M, Mukhopadhyay A, Keasling JD, Sakuragi Y, et al (2012)Arabidopsis Deficient in Cutin Ferulate encodes a transferase required forferuloylation of v-hydroxy fatty acids in cutin polyester. Plant Physiol158: 654–665

Saeed AI, Sharov V, White J, Li J, Liang W, Bhagabati N, Braisted J, KlapaM, Currier T, Thiagarajan M, et al (2003) TM4: a free, open-sourcesystem for microarray data management and analysis. Biotechniques34: 374–378

Sainz MB, Grotewold E, Chandler VL (1997) Evidence for direct activa-tion of an anthocyanin promoter by the maize C1 protein and com-parison of DNA binding by related Myb domain proteins. Plant Cell 9:611–625

Schaefer HM, Schaefer V, Levey DJ (2004) How plant-animal interactionssignal new insights in communication. Trends Ecol Evol 19: 577–584

Schilmiller AL, Charbonneau AL, Last RL (2012) Identification of a BAHDacetyltransferase that produces protective acyl sugars in tomato tri-chomes. Proc Natl Acad Sci USA 109: 16377–16382

Smart R (1992) Pinot Noir: the ultimate viticultural challenge? AustGrapegrower Winemaker 340: 77–85

Stamatakis A, Hoover P, Rougemont J (2008) A rapid bootstrap algorithmfor the RAxML Web servers. Syst Biol 57: 758–771

St-Pierre B, Luca VD (2000) Evolution of acyltransferase genes: origin anddiversification of the BAHD superfamily of acyltransferases involved insecondary metabolism. Recent Adv Phytochem 34: 285–315

Studier FW (2005) Protein production by auto-induction in high densityshaking cultures. Protein Expr Purif 41: 207–234

Suzuki H, Nakayama T, Yamaguchi M, Nishino T (2004a) cDNA cloningand characterization of two Dendranthema3morifolium anthocyaninmalonyltransferases with different functional activities. Plant Sci 166:89–96

Suzuki H, Nakayama T, Yonekura-Sakakibara K, Fukui Y, Nakamura N,Yamaguchi MA, Tanaka Y, Kusumi T, Nishino T (2002) cDNA cloning,heterologous expressions, and functional characterization of malonyl-coenzyme A:anthocyanidin 3-O-glucoside-699-O-malonyltransferasefrom dahlia flowers. Plant Physiol 130: 2142–2151

Suzuki H, Nishino T, Nakayama T (2007) Anthocyanin acyltransferaseengineered for the synthesis of a novel polyacylated anthocyanin. PlantBiotechnol 24: 495–501

Suzuki H, Sawada S, Watanabe K, Nagae S, Yamaguchi MA, NakayamaT, Nishino T (2004b) Identification and characterization of a novel an-thocyanin malonyltransferase from scarlet sage (Salvia splendens) flow-ers: an enzyme that is phylogenetically separated from otheranthocyanin acyltransferases. Plant J 38: 994–1003

Suzuki H, Sawada S, Yonekura-Sakakibara K, Nakayama T, YamaguchiM, Nishino T (2003) Identification of a cDNA encoding malonyl-coenzyme A:anthocyanidin 3-O-glucoside 699-O-malonyltransferasefrom cineraria (Senecio cruentus) flowers. Plant Biotechnol 20: 229–234

Tohge T, Nishiyama Y, Hirai MY, Yano M, Nakajima J, Awazuhara M,Inoue E, Takahashi H, Goodenowe DB, Kitayama M, et al (2005)Functional genomics by integrated analysis of metabolome and tran-scriptome of Arabidopsis plants over-expressing an MYB transcriptionfactor. Plant J 42: 218–235

Tuominen LK, Johnson VE, Tsai CJ (2011) Differential phylogenetic ex-pansions in BAHD acyltransferases across five angiosperm taxa andevidence of divergent expression among Populus paralogues. BMC Ge-nomics 12: 236–252

Unno H, Ichimaida F, Suzuki H, Takahashi S, Tanaka Y, Saito A, NishinoT, Kusunoki M, Nakayama T (2007) Structural and mutational studiesof anthocyanin malonyltransferases establish the features of BAHD en-zyme catalysis. J Biol Chem 282: 15812–15822

Van Buren J, Bertino J, Robinson W (1968) The stability of wine antho-cyanins on exposure to heat and light. Am J Enol Vitic 19: 147–154

van Hoof A, Green PJ (2006) NMD in plants. In L Maquat, ed, Nonsense-Mediated mRNA Decay. Landes Bioscience, New York, pp 167–172

Walker AR, Lee E, Bogs J, McDavid DAJ, Thomas MR, Robinson SP(2007) White grapes arose through the mutation of two similar andadjacent regulatory genes. Plant J 49: 772–785

Walker AR, Lee E, Robinson SP (2006) Two new grape cultivars, budsports of Cabernet Sauvignon bearing pale-coloured berries, are theresult of deletion of two regulatory genes of the berry colour locus. PlantMol Biol 62: 623–635

Wesley SV, Helliwell CA, Smith NA, Wang MB, Rouse DT, Liu Q,Gooding PS, Singh SP, Abbott D, Stoutjesdijk PA, et al (2001) Con-struct design for efficient, effective and high-throughput gene silencingin plants. Plant J 27: 581–590




http://tree.bio.ed.ac.uk/software/figtree

http://tree.bio.ed.ac.uk/software/figtree


Yabuya T, Yamaguchi M, Fukui Y, Katoh K, Imayama T, Ino I (2001)Characterization of anthocyanin p-coumaroyltransferase in flowers ofIris ensata. Plant Sci 160: 499–503

Yonekura-Sakakibara K, Nakayama T, Yamazaki M, Saito K (2008)Modification and stabilization of anthocyanins. In K Gould, K Davies, CWinefield, eds, Anthocyanins: Biosynthesis, Functions, and Applica-tions. Springer, New York, pp 169–190

Yonekura-Sakakibara K, Tanaka Y, Fukuchi-Mizutani M, FujiwaraH, Fukui Y, Ashikari T, Murakami Y, Yamaguchi M, Kusumi T

(2000) Molecular and biochemical characterization of a novelhydroxycinnamoyl-CoA:anthocyanin 3-O-glucoside-699-O-acyltransferasefrom Perilla frutescens. Plant Cell Physiol 41: 495–502

Yu XH, Gou JY, Liu CJ (2009) BAHD superfamily of acyl-CoA dependentacyltransferases in Populus and Arabidopsis: bioinformatics and geneexpression. Plant Mol Biol 70: 421–442

Zhu W, Wang H, Fujioka S, Zhou T, Tian H, Tian W, Wang X (2013)Homeostasis of brassinosteroids regulated by DRL1, a putative acyl-transferase in Arabidopsis. Mol Plant 6: 546–558


Rinaldo et al.



a grapevine anthocyanin acyltransferase, transcriptionally … · in cv pinot noir, a red-berried...

Documents