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Increased accumulation of anthocyanins in Fragaria chiloensisfruitsby transient suppression ofFcMYB1gene

Ariel Salvatierra a,1,2, Paula Pimentel b,1, Mara Alejandra Moya-Len a, Ral Herrera a,

a Instituto de Biologa Vegetal y Biotecnologa, Universidad de Talca, Casilla 747, Talca, Chileb Centro de Estudios Avanzados en Fruticultura (CEAF), Av. Salamanca s/n, Los Choapinos, Rengo, Chile

a r t i c l e i n f o

Article history:

Received 13 March 2012

Received in revised form 30 November 2012

Accepted 19 February 2013

Available online 20 March 2013

Keywords:

White strawberry

Fragaria chiloensisssp.chiloensis

Rosaceae

Fruit color

RNAi silencing

Anthocyanin

Flavan 3-ol

a b s t r a c t

Anthocyanins and proanthocyanidins (PAs), flavonoid-derived metabolites with different physiologica

roles, are produced by plants in a coordinated manner during fruit development by the action of tran

scription factors (TFs). These regulatory proteins have either an activating or repressing effect over struc

tural genes from the biosynthetic pathway under their control. FaMYB1, a TF belonging to the R2R3-MYB

family and isolated from commercial strawberry fruit (Fragaria ananassa), was reported as a transcrip-

tional repressor and its heterologous over-expression in tobacco flowers suppressed flavonoid-derived

compound accumulation. FcMYB1, an ortholog ofFaMYB1 isolated from the white Chilean strawberry

(Fragaria chiloensis ssp. chiloensis f. chiloensis), showed higher transcript levels in white (F. chiloensis

than in red (F. ananassa cv. Camarosa) fruits. In order to assess its contribution to the discolored phe-

notype inF.chiloensis,FcMYB1was transiently down-regulatedin planta using an RNAi-based approach

Quantitative real-time PCR onFcMYB1down-regulated fruits resulted an up-regulation of anthocyanidin

synthase (ANS) and a strong repression of anthocyanidin reductase (ANR) and leucoanthocyanidin reduc

tase (LAR) transcript accumulation. In addition, these fruits showed increased concentrations of anthocy-

anins and undetectable levels of flavan 3-ols. Altogether, these results indicate a role for FcMYB1 in

regulation of the branching-point of the anthocyanin/PA biosynthesis determining the discolored pheno

type of the white Chilean strawberry fruit.2013 Elsevier Ltd. All rights reserved

1. Introduction

The native Chilean strawberry (Fragaria chiloensis (L.) Mill. ssp.

chiloensis Staudt) is an octoploid species (2n= 8x= 56) of the Ros-

aceae family. Two botanical forms are recognizable in wild lands.

F. chiloensis ssp. chiloensis f. patagonica is a plant with small

fruits, red receptacle, and yellow or red achenes. On the other

hand,F. chiloensis ssp. chiloensis f. chiloensis is a robust plant cul-

tivated on a small scale that bears larger fruits, which are com-

posed of a pinkish-white receptacle and red achenes when fully

ripe. This strawberry is characterized as having a high and partic-

ular aroma (Gonzlez et al., 2009a), large fruit size (compared

with all other wild species) and a remarkable tolerance to Botrytis

infection (Gonzlez et al., 2009b). However, the fruit depigmenta-

tion of the white native strawberry constitutes its most character-

istic trait.

In theFragariagenus, fruit color is determined by accumulation

of anthocyanins, the most abundant flavonoid-derived constitu

ents in strawberry fruits (Hannum, 2004). Fruit pigmentation in

white native strawberry thus appears to be determined by the

expression of a R2R3 MYB and the down-regulation of genes from

flavonoid pathway (Saud et al., 2009; Salvatierra et al., 2010).

Production of anthocyanin is given by the action of a set of en-

zymes belonging to the flavonoid biosynthesis pathway, whose

gene transcription is coordinated by regulatory proteins called

transcription factors (Broun, 2004). Several TF families such as

MYB, bHLH, MADS, WRKY and WIP have been reported to be in-

volved in the molecular regulation of different phytochemicals in

different plant species (Quattrocchio et al., 2006 and references

therein). In Arabidopsis thaliana, MYBs have been divided into 22

subgroups according to the conserved amino acid motifs identified

0031-9422/$ - see front matter 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.phytochem.2013.02.016

Abbreviations: ANOVA, analysis of variance; ANR, anthocyanidin reductase; ANS,

anthocyanidin synthase; C4H, cinnamate 4-hydroxylase; CTAB, cetyltrimethylam-

monium bromide; CHI, chalcone isomerase; CHS, chalcone synthase; DFR,

dihydroflavonol reductase; daa, days after anthesis; FLS, flavonol synthase; F3H,

flavanone 3-hydroxylase; GSP, gene-specific primer; HPLC-DAD, high performance

liquid cromatography-diode array detector; LAR, leucoanthocyanidin reductase;

LSD, Least Significant Differences; PA, proanthocyanidin; qRT-PCR, quantitative

real-time PCR; RACE, Rapid Amplification cDNA Extremes; RNAi, RNA interference;

TF, transcription factor; UFGT, UDP glucose:flavonoid 3-O-glucosyl transferase. Corresponding author. Tel.: +56 (71) 200277; fax: +56 (71) 200276.

E-mail address: [email protected](R. Herrera).1 Both authors contributed equally to this work.2 Present address: Centro de Estudios Avanzados en Fruticultura (CEAF), Av.

Salamanca s/n, Los Choapinos, Rengo, Chile.

Phytochemistry 90 (2013) 2536

Contents lists available atSciVerse ScienceDirect

Phytochemistry

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / p h y t o c h e m
http://dx.doi.org/10.1016/j.phytochem.2013.02.016mailto:[email protected]://dx.doi.org/10.1016/j.phytochem.2013.02.016http://www.sciencedirect.com/science/journal/00319422http://www.elsevier.com/locate/phytochemhttp://www.elsevier.com/locate/phytochemhttp://www.sciencedirect.com/science/journal/00319422http://dx.doi.org/10.1016/j.phytochem.2013.02.016mailto:[email protected]://dx.doi.org/10.1016/j.phytochem.2013.02.016


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in the C-terminal, and it has been suggested that members of the

same subgroup have similar functions (Dubos et al., 2010; Stracke

et al., 2001). Initially, action of the MYB family in this branch of

secondary metabolism has been reported as related to the biosyn-

thesis of anthocyanins in grain (Paz-Ares et al., 1987).InA. thaliana,O. sativaandV. vinifera, severalMYBsbelonging to subfamily R2R3

have been identified and reported as activators or repressors of

transcription (Jin et al., 2000; Stracke et al., 2001; Yanhui et al.,2006; Matus et al., 2008). InA. thaliana, MYBs from subgroup 4 in-

clude transcriptional repressors (Dubos et al., 2010). They share

the consensus sequence LXLXL, which is the most important tran-

scriptional repression motif identified in plants (Kagale and

Rozwadowski, 2011).AtMYB4, ortholog ofAmMYB308, was the first

MYB functionally reported as transcriptional repressor in A. thali-

ana (Jin et al., 2000). Then, a MYB isolated from Fragaria ananassa

(FaMYB1) was reported as physiologically suppressive in the accu-

mulation of certain flavonoid compounds in tobacco flowers

(Aharoni et al., 2001). In addition, the ectopic expression of this

TF repressed the expression of PA related genes, reducing the accu-

mulation of PA polymers in leaves ofLotus corniculatus (Paolocci

et al., 2011). Thus, both studies demonstrated over-expression of

FaMYB1in its regulatory role on the final steps of flavonoid biosyn-

thesis pathway in two heterologous systems (i.e., Nicotiana tabacum

and L. corniculatus). However, the evidence obtained from the

analysis of this type of system is limited, because they may not

adjust entirely to what occurs in the plant of interest. For

example, over-expression of VvMYB5b, a grape TF involved in

anthocyanin and PA biosynthesis, decreased flavonoid levels and

increased carotenoid levels in tomato (Mahjoub et al., 2009), while

in tobacco the flavonoid levels increased but did not change the

carotenoid contents (Deluc et al., 2008). These results emphasize

the importance of the genetic background used in expression

experiments, and the need for caution in the interpretation of the

results. However, studies on gene function in plant species with

either long life cycles or difficulties in transformation and regener-

ation complicate to the research process. Transient genetic trans-

formation in crop plants has however, overcome this obstacle.

F. ananassa fruit has been shown to be susceptible to transient

expression of GUS reporter gene through the injection of an Agro-

bacterium suspension (Spolaore et al., 2001). In strawberry, tran-

sient transformation has been used for the silencing ofCHSgene

expression induced by RNAi (RNA interference), resulting in fruit

with either low or almost non-existent pigmentation at the ripe

stage (Hoffmann et al., 2006). Using the same approach, FaGT1 (gly-

cosyltransferase 1 of F. ananassa) gene silencing resulted in a

moderate reduction of anthocyanin pigment concentration in ripe

strawberry fruit and a redirection in biosynthesis to flavan 3-ols

(Griesser et al., 2008). Currently, RNAi is widely used in plant bio-

technology because it is a fast, simple, and a sequence-specific way

to decrease gene expression and has been used primarily for gene

discovery and validation of function (Small, 2007). Optimization ofthis methodology thus constitutes a great advantage, in terms of

the time especially in the analysis of fruit development-related

genes. This is because it is able to obviate the period of regenera-

tion and fruiting of genetically modified plant.

The aim of this study was thus to evaluate the suppression ef-

fect of a repressor TF of anthocyanin pigment accumulation over-

expressed in the native Chilean strawberry white fruited. For this

purpose, the FcMYB1 full length cDNA sequence was isolated and

a gene fragment was cloned to obtain a RNAi construct. The

FcMYB1 suppression degree and its impact on gene transcriptional

activity in the flavonoid pathway were examined using quantita-

tive real-time PCR (qRT-PCR). In parallel, changes in the content

of two major anthocyanins, cyanidin 3-glucoside (1) and pelargon-

idin 3-glucoside (2), and flavan 3-ol monomers, (+)-catechin (3)and ()-epicatechin (4;Fig. 1), were analyzed by HPLC-DAD.

2. Results and discussion

2.1. Isolation and characterization of full-length FcMYB1 cDNA

sequence

A partial gene fragment of 570 bp was amplified by PCR from a

pool of cDNA from F. chiloensis ssp. chiloensis f. chiloensis fruits

collected at different stages of development. The sequence of this

fragment allowed construction of specific primers for isolation of

a MYB full-length cDNA sequence by RACE. Thus, 20 clones weresequenced and a cDNA sequence of 1014 bp namedFcMYB1 (Gen-

Bank accession number GQ867222), ortholog to FaMYB1, was ob-

tained. These genes share 99% identity (558 identical bases out of

561) within the open reading frame. The sequence showed three

nucleotide variations given by the substitution of thymine by

cytosine at positions 141 and 414 and adenine instead of guanine

at position 201. The 50 untranslated region (UTR) of both genes

was identical, at least to the extent available for FaMYB1. Simi-

larly, FcMYB1 30-UTR shares a high identity to FaMYB1, although

it is shorter than that its ortholog (Suppl. Fig. 1). Comparison of

the deduced amino acid sequences ofFaMYB1 and FcMYB1 estab-

lished there were no differences at the amino acid level (Suppl.

Fig. 2).

Phylogenic analysis of the 31 MYB proteins that have been iso-lated from different plant species grouped FcMYB1 in a clade re-

lated to members of subgroup 4 of the R2R3-MYB gene

subfamily ofA. thaliana (AtMYB4, 32, 7 and 3) and other related

MYBs with repressor motifs (Fig. 2). TFs belonging to subgroup 4

share the amino acid motif LNL[E/D]L (Stracke et al., 2001; Dubos

et al., 2010). The consensus LXLXL sequence (where X is any amino

acid) is present in the EAR motif (ERF-associated amphiphilic

repression) described as a repressor domain (Ohta et al., 2001),

and similar motifs have been identified in several proteins such

as AUX-IAA, ERF and TFIIIAzinc fingertype. The FcMYB1 protein se-

quence matches the motif defined for R2R3-MYB proteins belong-

ing to subgroup 4, as well asAtMYB4(Jin et al., 2000). Interestingly,

AtMYB4 has been proven to be a negative regulator ofcinnamate

4-hydroxylase (C4H) gene expression, being the firstArabidopsisMYBdescribed as transcriptional repressor (Jin et al., 2000). Alignment

Fig. 1. Structures of compounds analyzed in this study. (1) Cyanidin 3-glucoside;

(2) pelargonidin 3-glucoside; (3) (+)-catechin; (4) ()-epicatechin.

26 A. Salvatierra et al. / Phytochemistry 90 (2013) 2536
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of amino acid sequences grouped FcMYB1in the clade of R2R3-MYB

repressors. These proteins shares the LXLXL domain defined for the

subgroup 4, suggesting a role as active transcriptional repressor(Suppl. Fig. 3). Bioinformatic structural analysis however did not

show amino acid polymorphisms betweenFcMYB1and its orthologFaMYB1. Therefore, it was assumed thatFcMYB1retains the biolog-

ical function described for its ortholog and is able to suppress

anthocyanin accumulation by regulating the expression of struc-

tural genes involved in flavonoid biosynthesis.

2.2. FcMYB1 temporal and organ-specific expression analysis

The expression profile ofFaMYB1 increased during, ripening of

theF. ananassa cv. Elsanta fruit, with a maximum of transcripts

detected at the red fruit stage (Aharoni et al., 2001). Aiming to as-

sess the transcript levels ofFcMYB1 in white Chilean strawberry,

organ-specific and temporal expression patterns were analyzedby qRT-PCR. RNA was isolated from fruit at different developmen-

tal stages and other tissues (flower, leaf, runner and root). Compar-

ative analysis of temporal FcMYB1gene expression in both white

and commercial strawberry fruits showed differences, in transcriplevel and in expression profile trend (Fig. 3A). A sustained increase

in mRNA levels was observed for this TF concomitant with the pro

gress of fruit development and ripening of white strawberry fruit

On the other hand, levels of FcMYB1 transcripts decreased in

F. ananassa cv. Camarosa, while exhibiting a slight rise at stage

4 (Fig. 3A). A similar expression profile was described in a previous

transcriptional analysis which included three developmenta

stages of white Chilean strawberry and commercial strawberry

cv. Chandler (Saud et al., 2009). In our study, FcMYB1showed high

er transcript levels along development and ripening of the white

Chilean strawberry fruit compared to F. ananassa cv. Camarosa

(Fig. 3A). Since Aharoni et al. (2001) demonstrated that over

expression ofFaMYB1 in tobacco plants suppressed flower pigmen

tation, the high level of transcripts of this TF detected in whiteChilean strawberry may contribute to the repression described in

Fig. 2. Phylogenic relationships of selected MYB proteins from different plant species. The scale represents the number of substitutions per site and the number

next to the nodes correspond to bootstrap values of 1000 replicates. The tree was rooted using the human c-MYB protein as outgroup. TFs have been reported

with functions in development of stamen or trichomes (sky blue circles), biosynthesis of phenylpropanoids/lignin (green circles), anthocyanins (red circles)

proanthocyanidins (orange circles) and flavonols (purple circles). Accession numbers are shown in parentheses: AtMYB4 (BAA21619), AmMYB308 (JQ0960)

VvMYB4b (ACN94269), ZmMYB42 (Q2A700), AtMYB3 (BAA21618), AtMYB6 (Q38851), AmMYB330 (JQ0957), AtMYB32 (O49608), AtMYB7 (NP_179263), FcMYB1

(GQ867222), FaMYB1 (AAK84064), VvMYBPA1 (CAJ90831), AtMYB111 (NP_199744), AtMYB12 (NP_182268), AtMYB11 (NP_191820), AtMYB5 (NP_187963)

AtMYB123 (Q9FJA2), AtMYB82 (Q9LTF7), MdMYB8 (ABB84756), MdMYBA (BAF80582), MdMYB10 (ABB84753), PaMYB10 (ACA04854), GhMYB10 (AAK19615

PhAn2 (AAF66727), NtAn2 (ACO52470), LeANT1 (AAQ55181), VvMYBA (ABB87013), AtMYB113 (Q9FNV9), AtMYB75 (Q9FE25), AtMYB90 (Q9ZTC3), AtMYB114

(Q9FNV8) and c-MYB (X52125).

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flavonoid biosynthesis genes and anthocyanin accumulation, and

could explain the origin of pigment-deficient phenotype character-

istic of its fruit.

MYBs are involved in coordinating structural genes of the flavo-

noid biosynthesis pathway in fruits such as grape (Kobayashi et al.,

2002; Walker et al., 2007), apple (Takos et al., 2006a; Ban et al.,

2007; Espley et al., 2007), pear (Feng et al., 2010; Pierantoni

et al., 2010), mangosteen (Palapol et al., 2009), Chinese bayberry

(Niu et al., 2010) and strawberry (Aharoni et al., 2001; Lin-Wanget al., 2010; Schaart et al., 2012), respectively.

Comparative expression analysis, carried out between two

botanical forms of Chilean native strawberry contrasting in fruit

pigmentation, indicated a coordinated down-regulation of antho-

cyanin related genes in white Chilean strawberry fruits (Salvatierra

et al., 2010). Genes of this pathway showed a high nucleotide iden-

tity, on comparing the partial sequences isolated from red

(F. ananassa) and white strawberry fruit, thereby discarding a

major role of flavonoid structural genes in determining

pigment-deficient phenotype. Most likely, it could be argued that

participation of one or more TFs are involved in regulation of fruit

pigmentation in the native Chilean strawberry. Organ-specific

expression analysis established low levels of FcMYB1 mRNA in

flower, leaf, runner and roots of F. chiloensis compared to fruit(Fig. 3B).

2.3. FcMYB1 transient gene silencing in F. chiloensis fruits

FcMYB1 was suppressed by RNA interference and the

expression of genes from the flavonoid biosynthetic pathway

was analyzed. Fruits injected with the pHELLSGATE12-FcMYB1i

(pH12-FcMYB1) construct and collected 28 days after anthesis

(daa) showed a phenotype with a more intense and homoge-

neous pigmentation on their surface than fruits injected withcontrol construction, corresponding to the empty vector

(Fig. 4A and B).

FcMYB1 transcript levels were assessed by qRT-PCR in agroinfil-

trated fruits. Considering that the RNAi mechanism action and the

agroinfiltration technique involve a significant degree of variability

in their biological effect, the average of FcMYB1 transcript levels

detected in three control lines was set as 100% of expression of this

gene. Ten agroinfiltration events were made with pH12-FcMYB1

interference construction and four lines showed a significant

reduction inFcMYB1 transcript levels compared to the expression

average in control fruit (Fig. 5).

The expression of genes involved in the flavonoid biosynthe-

sis pathway was analyzed in four FcMYB1 suppressed lines

(pH12-FcMYB1.4 to .7) (Fig. 6). The expression patterns so ob-

tained define three different groups: unaffected genes (CHS), re-

pressed genes (CHI, F3H, DFR, LAR and ANR) and over-expressed

genes (ANS and UFGT). The reduced transcript level of CHS has

been reported to cause loss of pigmentation in affected plant tis-

sue (Fukusaki et al., 2004; Koseki et al., 2005; Hoffmann et al.,

2006; Lunkenbein et al., 2006). CHS was the only gene whose

expression remained almost unaffected in fruits of FcMYB1 sup-

pressed lines (Fig. 6A). Similarly, expression of this gene was

not altered in transgenic tobacco lines over expressing

FaMYB1, despite the change in the color in flowers (Aharoni

et al., 2001).

A more intense pigmentation was observed in fruits ofFcMYB1

suppressed lines. It seems that this TF does not affect CHSexpres-

sion, suggesting that production of precursors for the biosynthesis

of flavonoid-derived compounds remains unaltered.

CHI, F3H and DFR genes exhibited a moderate reduction in

their transcript levels in FcMYB1 suppressed lines (Fig. 6BD).

Apparently, the expression levels of the intermediate genes of

the flavonoid pathway seem not to be crucial for appearance

of pigmentation. LAR and ANR are enzymes responsible for the

synthesis of flavan-3-ols from leucoanthocyanidins and anthocy-

anidins, respectively, and constitute a dichotomous point at the

end of the flavonoid pathway leading to the biosynthesis of

PAs instead of anthocyanins. Correlation between gene expres-

sion for these enzymes and PA content has been documented

in fruits. In pollination-constant and non-astringent (PCNA) per-

simmon, both ANR expression and soluble tannin content were

diminished during fruit development (Ikegami et al., 2005).

Moreover, a comparative analysis between PCNA and non-PCNApersimmon fruits established a lower content of soluble tannin

and ANR expression levels during fruit development (Akagi

et al., 2009). During grape berry development, expression pat-

terns ofANR and isoforms ofLAR (i.e. LAR1 andLAR2) determine

PA accumulation and composition in a tissue and temporal-

specific manner (Bogs et al., 2005). However, a decrease in the

transcript levels of these PA-related genes, as well as PAs and

flavan 3-ol contents during fruit development, have been

detected in other crops such as commercial strawberry (Carbone

et al., 2009) and apple (Takos et al., 2006b).

LAR andANR genes evidenced severe reduction in their expres-

sion levels inFcMYB1 suppressed fruits (Fig. 6G and H). This level

of suppression on PA-related genes may contribute to the in-

creased pigmentation of these fruits. Similarly, flowers of tobaccolines over-expressing different apple ANR isoforms (i.e. MdANR1,

Fig. 3. FcMYB1gene relative expression levels. (A) Comparative analysis ofFcMYB1

temporal expression in fruits of F. chiloensis ssp. chiloensis f. chiloensis (whitecircles) andF. ananassacv. Camarosa (black circles) at four developmental stages.

(B) Organ-specific analysis of the expression in different tissues of F. chiloensis.

Vegetative tissues were flower(F), leaf (L), runner(R) androot(Rt).Transcript levels

were analyzed by qRT-PCR and calibrated against gene expression in stage 1 ofF.

chiloensis (C1).



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MdANR2a and MdANR2b) showed petals with reduced pigmenta-

tion along with high levels of (+)-catechin (3) and ()-epicatechin

(4) and low levels of cyanidin (Han et al., 2012). On the other hand,

expression of ANR in tobacco flower petals and in Arabidopsis

leaves resulted in loss of anthocyanins and accumulation of PAs

(Xie et al., 2003; Routaboul et al., 2006). Conversely, down-regula-

tion ofANR gene depicts the opposite scenario. Low transcript lev-

els of this gene detected in soybean grains and clover flowers of

ANR suppressed lines implied the presence of plant tissues abnor-

mally pigmented owing to a high accumulation of anthocyanin

joined to a marked decrease in the content of PAs (Kovinich

et al., 2011; Abeynayake et al., 2012). In concordance with this,

our findings suggest a blockage at the transcriptional level in thePA biosynthesis pathway, which may redirect precursors of

flavonoids to the biosynthesis of anthocyanin pigments in white

Chilean strawberry fruits inFcMYB1 suppressed lines.

The final steps of anthocyanin pigment biosynthesis are given

by the enzymatic activities of ANS and UFGT, which synthesize

anthocyanidins (from flavan 3,4-diols) and anthocyanins (glycosyl

ated anthocyanidins), respectively. In fruits, expression of these

structural genes have been associated with color development

and/or accumulation of anthocyanins (Boss et al., 1996a; Manning

1998; Kobayashi et al., 2001; Jaakola et al., 2002; Bogs et al., 2005

Takos et al., 2006a; Almeida et al., 2007; Espley et al., 2007; Palapo

et al., 2009; Saud et al., 2009; Pierantoni et al., 2010; Salvatierra

et al., 2010). A genetic and transcriptional study carried out in

red and white fruited Duchesnea indica suggests that the lack ocolor in white fruit may be attributed to the low expression o

Fig. 4. Phenotype of agroinfiltrated fruits. (A) Fruit injected with the control construction. (B) Fruit injected with the interference construction pH12-FcMYB1. Fruits were

harvested at the mature stage on day 14 post-injection (28 days after anthesis).

Fig. 5. FcMYB1 gene expression level of agroinfiltrated fruits. Black bars correspond to the level ofFcMYB1 expression in fruits injected with the control construction. The

average value of gene expression in control lines was established as the threshold representing 100% of FcMYB1 relative expression (black line). Gray bars represen

agroinfiltrated lines withFcMYB1expression levels significantly reduced from the average of control events with ap< 0.05 according to LSD multiple comparison Fisher test

The values and error bars represent the mean and standard deviation of three technical replicates of each line, respectively.



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Fig. 6. FcMYB1 suppression effect in gene expression of flavonoid biosynthesis pathway. The analysis of expression was determined by qRT-PCR and calibrated against the

average of expression value of each gene in three control events (blackline). The valuesand error bars representthe mean andstandard deviationof three technical replicates

of each agroinfiltration event, respectively.



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ANS(Debes et al., 2011). Additionally, the role ofANSin the flavo-

noid pathways has been addressed through genetic manipulation,

demonstrating this to be an effective approach to modify both PA

and anthocyanin accumulation in plant tissues. So, over-expressing

ANSin an indica rice mutant channeled PA precursors to the syn-

thesis of anthocyanins in pericarptissue (Reddy et al., 2007). More-

over, silencingANSin a red-leaved apple cultivar showed an almost

complete loss of cyanidin 3-galactoside and higher concentrationsof other flavonoid compounds, such as (+)-catechin (3) and oligo-

mers (Szankowski et al., 2009). InFcMYB1 suppressed fruits, ANS

was the only one gene showing higher levels of transcripts in all si-

lenced lines compared to the control (Fig. 6E). This result could be

related to the decrease in transcript levels of PA genes, since a tight

dependent relationship between expression of ANS and ANR has

been described in this branch point. Thus, an increase in expression

ofANSmay be key for determination of the pigmented phenotype

on FcMYB1 suppressed fruits. Higher levels of ANS transcripts

might also imply a redirection of the metabolism of flavonoid pre-

cursors towards accumulation of anthocyanin pigments at the ex-

pense of flavan 3-ol and PA production. More recently, the

involvement of other TFs in the regulation of ANS has been shown,

and not surprisingly an interaction between FaMYB1 and bHLH TF

(Schaart et al., 2012).

UFGT catalyzes transfer of glucose from UDP-activated sugar

donor molecule to the hydroxyl group at C3 of anthocyanidins,

facilitating their transport to vacuoles where the acidic environ-

ment is essential for coloring (Pourcel et al., 2010). In grape berries,

almost all genes of flavonoid biosynthesis pathway are expressed

during fruit development, but only UFGT mRNAs are detected in

the skin of red cultivars after vraison, demonstrating this to be

critical for the biosynthesis of anthocyanins (Boss et al., 1996a,b).

A white mutant of Malay apple (Syzygium malaccense) has no

detectable levels of anthocyanin, UFGT activity and expression in

its skin, contrary to the considerable expression level of several

other genes in the same biosynthetic pathways (Kotepong et al.,

2011), suggesting that the white mutation may be correlated with

a failure of the last step in anthocyanin synthesis. Moreover, down-

regulation ofFaGT1 generated strawberry fruits with significantly

reduced levels of pelargonidin and increased concentration of fla-

van 3-ols (Griesser et al., 2008). InFcMYB1suppressed fruits,UFGT

presented either similar or slightly higher transcript levels than the

control, except in the fruit pH12-FcMYB1i.6 (Fig. 6F). This fruit

exhibited evident damage attributable to the agroinfiltration tech-

nique, which could explain its unexpected behavior in transcript

accumulation. By contrast, highest expression of UFGT was de-

tected in pH12-FcMYB1i.5, which is coincident with the lowest

expression level of PA related genes (Fig. 6G and H) and higher

ANS expression (Fig. 6E). The interrelationship between these

genes (i.e.LAR/ANRandANS/UFGT) and the fate of precursors of fla-

vonoid biosynthesis pathway is evident, since both down-regula-

tion of PA related genes and over-expression of anthocyaninrelated genes (mainly ANS) led to the pigmented phenotype of

the white Chilean strawberry fruit.

Interestingly, transgenic tobacco over-expressing FaMYB1

showed no pigmentation in flowers or reduced amounts of antho-

cyanin and low expression and activity ofANSand UFGT, respec-

tively (Aharoni et al., 2001). On the other hand, the ectopic

expression ofFaMYB1repressed the expression of PA related genes

and reduced the accumulation of PA polymers (Paolocci et al.,

2011). Both studies showed a reduction inANSmRNA levels. How-

ever, over-expression of FaMYB1 in L. corniculatus also reduces

markedly the transcript levels of ANR and LAR1. It was proposed

that FaMYB1 could compete with an endogenous PA activator

MYBs (e.g. LcTT2) for a common binding site in the ternary complex

MYB-bHLH-WDR, promoting a transcriptional repression of PA re-lated genes (Paolocci et al., 2011). However, L. corniculatus leaves

did not accumulate anthocyanin pigments. In this sense, our study

confirms and validates the biological role ofFcMYB1 in pigmenta-

tion of fruit tissue where both branches of the flavonoid pathway

coexist. LDOX (Leucoanthocyanidin dioxygenase, ANS) promoter is

directly controlled by different MYB/BHLH/WDR transcription

factor complexes containing the bHLH factors EGL3 and TT8

(Appelhagen et al., 2011). It is possible that the transcriptiona

repressor FcMYB1 may compete with other R2R3 MYBs for bindingbHLH factors as in the MYB/bHLH/WDR ternary complex that is in

volved in the regulation of anthocyanin production found in Ara-

bidopsis (Appelhagen et al., 2011; Schaart et al., 2012). In

addition, FcMYB1 has the consensus LXLXL sequence present in

the EAR motif that could act as an active transcriptional repressor

of regulatory partners (e.g., bHLHs and WDRs) or structural genes

of the flavonoid biosynthesis pathway. Thus, higher mRNA levels

of this transcriptional repressor could explain the low ANS tran-

script level reported in white Chilean strawberry and its concomi-

tant unpigmented phenotype (Salvatierra et al., 2010). Therefore

FcMYB1 suppression in white Chilean strawberry meant a higher

expression ofANSresulting in a higher accumulation of anthocya-

nins and more pigmented fruits.

2.4. Quantification of anthocyanins and flavan 3-ols contents

Contents of anthocyanins and flavan 3-ols in agroinfiltrated

fruits were assessed by analyzing the suppression effect ofFcMYB1

transcript accumulation on flavonoid compound accumulation

Along development and ripening of white strawberry fruits, there

is an opposite accumulation trend of final flavonoid metabolite

(Fig. 7). While the amounts of flavan 3-ols, ((+)-catechin (3) and

()-epicatechin (4)), had a maximum at immature stages (C1 and

C2), the amounts of anthocyanins (cyanidin 3-glucoside (1) and

pelargonidin 3-glucoside (2)) increased while fruit ripening pro-

gressed, reaching their maximum level at the ripe fruit stage. Sim

ilar trends have been described in studies in commercia

strawberry (Halbwirth et al., 2006; Carbone et al., 2009).

Cyanidin 3-glucoside (1) is the major anthocyanin in white

strawberry (Cheel et al., 2005) and it was detected in all stages

of fruit development due to early achene pigmentation (Aaby

et al., 2005). Pelargonidin 3-glucoside (2) is the anthocyanin pig-

ment responsible for the red color in the genus Fragaria (Kosar

et al., 2004; Tulipani et al., 2008). However, in the white straw-

berry there is little accumulation of this pigment and it is almost

exclusively concentrated in stage 4 of development (ripe fruit)

when the receptacle has a slight pink color (Fig. 7). FcMYB1-

suppressed fruits showed no significant variation in the conten

of cyanidin 3-glucoside (1) as compared to control fruit (Fig. 8A)

Analyses of metabolites showed a greater accumulation of pelarg-

onidin 3-glucoside (2) concomitant with a decrease in FcMYB1

transcript accumulation (Fig. 8B). This fact establishes the exis

tence of a direct relationship between the FcMYB1transcript levelsand content of pelargonidin 3-glucoside (2), the major anthocyanin

detected in the receptacle of strawberry (Aaby et al., 2005; Fait

et al., 2008).

White strawberry fruit at the ripe stage showed the lowest lev-

els of flavan 3-ols and the highest levels of anthocyanin pigments

during the ripening process (Fig. 7). Despite this, it is noteworthy

that, in a comparative chemical analysis of phenolic composition

between the two botanical forms (red and white fruited) of native

Chilean strawberry andF. ananassacv. Camarosa, only F.chiloen-

sis ssp.chiloensis f. chiloensis (white fruited form) showed detect-

able levels of (+)-catechin (3) (Simirgiotis et al., 2009). However, in

FcMYB1 suppressed fruits flavan 3-ols were not detected (Fig. 8C)

which is related to the severe reduction in the transcript levels of

genes involved in biosynthesis of PA. Conversely, silencingglycosyltransferase FaGT1 reduced anthocyanins levels and



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Fig. 7. Accumulation patterns of final flavonoid compounds during development ofFragaria chiloensisssp.chiloensisf. chiloensis fruits. Anthocyanins (cyanidin 3-glucoside

(1) and pelargonidin 3-glucoside (2)) and flavan 3-ols ((+)-catechin (3) and ()-epicatechin (4)) were determined by HPLC-DAD from a pool of fruits of each developmental

stage. Values are the mean standard deviation of three independent biological replicates and two technical replicates.

Fig. 8. Flavonoid metabolite levels (lg g1 fresh weight) in agroinfiltrated fruits collected at the ripe stage. (A) Cyanidin 3-glucoside (1) content. (B) Pelargonidin 3-glucoside

(2) content. Values and error bars represent the mean and standard deviation of three technical replicates of a single anthocyanin extraction. Different letters represent

significant differences withp< 0.05 according to LSD multiple comparison Fisher test. (C) Flavan 3-ols content. Values are the meanand standard deviation of three technical

replicates of flavan 3-ols. Different letters in the same column represent significantly different values withp< 0.05 according to LSD multiple comparison Fisher test. nd, Not

detected.



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increased flavan 3-ols content in F. ananassa fruits (Griesser

et al., 2008).

FcMYB1suppressed fruits showed a more pigmented surface gi-

ven by higher pelargonidin 3-glucoside (2) levels. However, fruit

flesh remained unpigmented as in the control fruits. It seems that

the biological effect ofFcMYB1 is relegated to the skin like other

R2R3 MYB TFs (i.e. MdMYB1 and MdMYBA) identified in apple

(Takos et al., 2006a; Ban et al., 2007). Yet the expression of anotherTF of this family, namedMdMYB10 (ortholog of Arabidopsis PAP1/AtMYB075), has been correlated with transcriptional activation of

anthocyanidin related genes and accumulation of anthocyanin pig-

ments in red fleshed (e.g. Red Field) and red skinned (e.g. Pacific

Rose) cultivars (Espley et al., 2007). This TF could be an interesting

candidate for further studies in white Chilean strawberry fruit due

to its lack of anthocyanins in both flesh and skin.

Our results show that the release of the transcriptional repres-

sion exerted by FcMYB1 overANSchanneled the flux of intermedi-

ate metabolites of the flavonoid biosynthesis pathway into

anthocyanin pigments associated with the receptacle (i.e., pelarg-

onidin 3-glucoside (2)). Apparently, this redirection in metabolic

flux drained precursors (flavan 3,4-diols and anthocyanidins) for

PA production resulting in undetectable levels of PA monomers

in fruits of FcMYB1 suppressed lines (Fig. 8C). The lack of flavan

3-ols besides the severe drop in mRNA levels of PA related genes

seems to be a side-effect ofFcMYB1 suppression as a consequence

of the transcriptional activation ofANSand a higher biosynthesis of

anthocyanins in receptacle. Based on transcript and chemical anal-

yses ofFcMYB1 suppressed fruits, it can be argued that flavonoid

precursors were redirected to the anthocyanin biosynthesis at ex-

pense of PA production in white Chilean strawberries owing to the

suppression of this transcriptional repressor on accumulation of

anthocyanins. More recently, a group of three different TFs have

been reported to be involved controlling PA synthesis in F. anan-

assa and the interaction of MYB1 to bHLH3D has been suggested

(Schaart et al., 2012).

3. Conclusions

In this study, the FcMYB1 gene was isolated from the native

white Chilean strawberry fruit (F.chiloensis ssp.chiloensis f. chilo-

ensis) and its biological role was assessed in fruits attached to

the plant. An aim was to obtain a reduction in the transcript level

ofFcMYB1in white strawberry fruit using the agroinfiltration tech-

nique combined with an RNAi construction for silencing this gene.

A red pigmented fruit was obtained in FcMYB1 down-regulated

Chilean strawberry fruit. Transient suppression affected expression

of the flavonoid genes, increasing the transcript level of those

genes closely related to anthocyanin biosynthesis and decreasing

that of genes involved in PA production. Coincidentally, an increase

in pelargonidin 3-glucoside (2) content at the metabolite level wasobserved in the receptacle-associated anthocyanin, and a decrease

in flavan 3-ol content. The results showed that FcMYB1 exerts a

regulatory effect at transcriptional level in a biosynthesis flavo-

noid-derived compounds aimed at promoting anthocyanin produc-

tion at the expenses of flavan 3-ols in the Chilean white strawberry

fruits. These data provide experimental information that supports,

in F.chiloensisssp.chiloensisf. chiloensis fruit, the biological role of

FcMYB1 on the regulation of anthocyanin biosynthesis as previ-

ously reported heterologously for FaMYB1 in tobacco. In addition,

this extends evaluation of the FcMYB1 effect over transcription of

PA related genes in fruit tissue where the flavonoid branches of

anthocyanin and PA coexist. Since FcMYB1 suppression triggered

a transcriptional activation of ANS, higher levels of pelargonidin

3-glucoside (2) and the lack of flavan 3-ols, it can be hypothesizedthat severe down-regulation of LAR and ANR was a side-effect

driven by redirection of flux of flavonoid precursors towards syn-

thesis of anthocyanins in white Chilean strawberry fruits. This re-

sult highlights the possibility of using this TF to direct the flux o

flavonoid intermediary metabolites in the last steps of this path-

way through genetic manipulation.

4. Experimental

4.1. Plant material

F. chiloensisssp.chiloensisf. chiloensis (f. chiloensis) plants from

Contulmo, Bio-Bio Region, Chile (latitude 380408.600S; longitude

731402.9600W), were grown in pots and maintained at the Univer-

sity of Talca (2009). Four development and ripening stages were

considered as reported inFigueroa et al. (2008), based on weight

and color of the receptacle and achene: C1, small fruit with

green receptacle and green achenes (7 daa); C2, large fruit with

green receptacle and red achenes (14 daa); C3, turning stage, white

receptacle and red achenes (21 daa); and C4, ripe fruit with pink

receptacle and red achenes (28 daa). F. ananassa cv. Camarosa

fruits were collected in a commercial field from Chanco, Maule Re

gion, Chile (latitude 35410

49.2100

S; longitude 72320

34.6000

W) atequivalent times to those described for white native strawberry

Fruits in plants ofF.chiloensisssp.chiloensisf. chiloensis were used

to perform transient expression assays. Fruit agroinfiltration

started at C2 developmental stage (14 daa) and they were har-

vested at full ripe stage (28 daa). Fruits collected were immediately

frozen and stored at 80 C until analysis.

4.2. RNA extraction

Three independent total RNA samples were isolated from pools o

fruits prepared from each developmental stage, agroinfiltrated fruits

and from flower, leaf, runner and root tissue using the CTAB method

with minor modifications (Chang et al., 1993). DNAse I treatmen

(Invitrogen) was carried out to remove contaminant genomic DNAIntegrity of isolated RNAs was checked on agarose gels stained with

ethydium bromide and their concentration measured in a ND-1000

UV spectrophotometer (Nanodrop Technologies).

4.3. Isolation of full-length sequences of FcMYB1 gene

Specific primers were designed from FaMYB1 nucleotide se

quence (GenBank accession number AF401220) to isolate a partia

sequence of this gene from a cDNA pool prepared from F.chiloensis

ssp.chiloensisf. chiloensis fruit at different developmental and rip-

ening stages (MYBorf fwd 50-TGGAATTCGCTTCCTAAGGCTG-3 0

MYBorf rev 50-TCCAATCCCAGAATC CAAGCAC-30). A 570 bp gene

fragment obtained by PCR was cloned into the vector pSCA follow-

ing manufacture instructions (StrataClone PCR Cloning Kit) and sequenced at Macrogen (Seoul, Korea). Internal specific primers were

designed (MYB1race fwd 50-GGTCTCATCAGCCGATTCTGCTGC

TGGC-30; MYB1race rev 50-TATCTGTCCTTCCAGGCAGTCTTCCAGC-

30) for RACE reaction to obtain full length FcMYB1 sequence. Tota

RNA (5 lg) from a mix of F. chiloensis fruit was used for RACE-

Ready cDNAs (BD Biosciences, Clontech) following the users man-

ual. The 50 and 30 specific primers were designed based on partia

sequences using Primer Premier V5.0 software (Premier Biosoft

International) and synthesized by Alpha DNA (Montreal, Canada)

PCR-RACE reactions were performed using BD SMART RACE

cDNA Amplification kit (Clontech), with the following conditions

1 cycle at 94 C per 3 min; 25 cycles at 94 C per 1 min, 68 C per

1 min, 72 C per3 min; 1 cycleat 72 C per 15 min. Gene fragment

obtained were cloned and sequenced as described above. Partialsequences of FcMYB1 were aligned to get the full-length cDNA



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using BioEdit Sequence Alignment Editor software v 7.0 (Hall,

1999). Phylogenic and evolutionary analyses were developed using

the neighbor-joining method using MEGA program version 4.0

(Tamura et al., 2007).

4.4. Construction of FcMYB1 RNAi plasmid

A partial fragment of 555 bp from FcMYB1 full-length cDNAsequence was amplified by PCR using the primers MYBdir fwd

50-CACCATGAGGAAGCCCTGCTGC-3 0, and MYBdir 50-GGATCAGCCA

TTCCAGTACTA GTC-30, respectively. This fragment was cloned into

the vector pENTR/SD/D-TOPO included in the pENTR Direc-

tional TOPO Cloning kit (Invitrogen). The cloning product was

used to transform Escherichia coli (One Shot TOP10, Invitrogen),

which were cultured on LB agar plates supplemented with Kana-

mycin (PhytoTechnology Laboratories) at a final concentration of

25 mg/ml. Resistant colonies were analyzed by PCR to assess the

presence and cloning direction ofFcMYB1 gene fragment using a

combination of gene-specific primers (fwd MYBdir) and M13

(M13 rev 50-CAGGAAACAGCTATGAC-30). For suppressing FcMYB1

expression, pHELLSGATE12 (Helliwell and Waterhouse, 2003)

was used as the destination vector. Recombination was performed

using Gateway LR Clonase II Enzyme Mix kit (Invitrogen)

according to the manufacturers instructions and using equivalent

amounts of entry vector (160 ng/ul) and destination vector

(160 ng/ul). Transformed bacteria (One Shot TOP10 chemically

Competent E. coli (Invitrogen)) were plated on LB agar supple-

mented with Spectinomycin (PhytoTechnology Laboratories) at a

final concentration of 100 lg/ml. Resistant colonies were analyzed

using restriction enzymes XhoI (Promega) and XbaI (Promega) to

assess the presence of the gene fragment in the construction. As

empty vector control, pHELLSGATE12-GUSi construction was car-

ried out using aGUSgene fragment.

4.5. White strawberry fruit transfection by agroinfiltration

pHELLSGATE12-FcMYB1iconstruction was introduced into Agro-bacterium tumefaciens strain LBA4404 ElectroMAX (Invitrogen)

by electroporation in a Cell-Porator electroporator (Gibco BRL).

Transformed bacteria were plated on a selective medium YM agar

supplemented with Streptomycin (PhytoTechnology Laboratories)

and Spectinomycin (PhytoTechnology Laboratories) at a final con-

centration of 100 lg/ml of each antibiotic. Resistant colonies were

analyzed by PCR for the presence ofFcMYB1 gene fragment usingFcMYB1 specific primers. A positive colony was cultured in selec-

tive YM (100 ml) and incubated at 30 C until an O.D.600 between

0.6 and 0.8. Then, the culture was centrifuged for 3 min at 1620g

in a swinging bucket centrifuge (5804 R centrifuge, Eppendorf).

The pellet obtained was resuspended in one third of its original

culture volume, with MMA agroinfiltration medium (MS salt

4.32 mg/l; 10 mM MES; 20 g/l sucrose; 200 mM acetosyringone).Agroinfiltration suspension was injected into F. chiloensis ssp. chilo-

ensis f. chiloensis fruits in planta. In C2 fruits (14 daa), multiple

injections were performed each third day until ripe fruit stage

(28 daa). Agroinfiltrated ripe fruits were harvested, pulverized

and stored at 80 C for transcripts and chemical analyses.

4.6. Analysis of transcripts

For quantitative Real-Time reverse transcription PCR (qRT-PCR)

assays, first-strand cDNA synthesis was performed using an Affin-

ityScript QPCR cDNA Synthesis kit (Stratagene, Agilent Technolo-

gies). For cDNA synthesis, total RNAs isolated from each

biological replicate were used as template in a 20 ll reaction mix-

ture. Each reaction mixture contained template RNA (2 lg), 2xcDNA Synthesis Master Mix (10 ll), Oligo (dT) Primer (3 ll) and

AffinityScript RT/TNasa Block Enzyme Mixture (1ll). cDNA was di-

luted 1:4, and 2 ll of the dilution was used in a SYBR Green RT-

PCR. cDNA (50 ng) was used for qRT-PCR assays, carried out with

gene-specific primers: qPCR-MYB1 fwd 50-GGTGCCTGAGTT-

GAATCTC-30; qPCR-MYB1 rev 50-GCAACTTGAGGATCAGCC-3 0; and

for structural genes of flavonoid pathway see Salvatierra et al.

(2010), using a DNA Engine Opticon2 thermocycler (MJ Research)

and Brilliant II SYBR Green QPCR master mix kit (Stratagene) fol-lowing the manufacturers instructions. Biological replicates were

analyzed in duplicate. Specificity of amplification products was

confirmed by the registration of a single peak in PCR melting

curves and the visualization of a single band on agarose gels. Seven

10-fold dilutions of each gene fragment were used to calculate PCR

efficiency (E) for each specific and housekeeping gene using the

slope of a linear regression model. GAPDH gene with constant

expression levels through all fruit developmental stages and tis-

sues was used to normalize raw data and to calculate relative

expression levels as reported in Pimentel et al. (2010). Stage 1 from

F. chiloensis ssp. chiloensis f. chiloensis fruit (C1) was taken as the

calibrator sample. GAPDHgene was also used as normalizer gene

in transiently transformed strawberry fruits since it showed a con-

stant expression in this assay (Suppl. Fig. 4), and control fruits were

used as calibrator sample in this study. Normalized Ct values were

used for determining gene expression variations in the samples

analyzed according to the following model (Pfaffl, 2001).

4.7. Chemical analysis

Quantification of anthocyanins (cyanidin 3-glucoside (1) and

pelargonidin 3-glucoside (2)) and flavan 3-ols ((+)-catechin (3)

and ()-epicatechin (4)) was performed after construction of

standard calibration curves. Pelargonidin 3-glucoside (2) was

purchased as calistephin chloride (Extrasynthse) and cyanidin

3-glucoside (1) as kuromanin chloride (SigmaAldrich). (+)-Catechin

(3) and ()-epicatechin (4) were purchased from Extrasynthse.

Anthocyanin contents in fruits were processed from fruit (2.5 g)

as described inSalvatierra et al. (2010). To determine flavan 3-ol

contents in fruits, fruit (2.5 g) was blended with an Ultra-Turrax

T25 digital (IKA) in ultrapure H2O (50 ml). The homogenate was

extracted with Et2O (20 ml3) and EtOAc (20 ml3). The six ex-

tracts were combined, dried (0.5 mg Na2SO4 anhydrous), and the

organic extract was concentrated to dryness, reconstituted in

MeOH:H2O (2 ml) (1:1, v/v) and filtered before analysis. The flow

rate of elution was 600 ll/min of solvent A (AcOH 2%, in ultrapure

H2O) at initial (0.012.0 min) and final phase of the run (23.5

25.0 min). Intermediate steps consisted in a gradient elution with a

flow rate of 800ll/min with a mixture of 30% A and 70% B (CH3CN,

AcOH, H2O; 20:2:78, v/v) up to 12.0 min, 20% A and 80% B up to

14.5 min, 10% A and 90% B up to 16.0 min and 100% C (MeOHH2O

(95:5, V/V)) up to 22.5 min. Separation and quantification of antho-

cyanins and flavan 3-ols was performed using a Agilent 1100 seriesHPLC system provided by a photodiode array detector (DAD)

equipped with a manual injector (20 ll injection volume) and

interfaced to a PC running ChemStation chromatography manager

software (HewlettPackard). A reversed phase column Kromasil

C18 100-3.5 (Akzo Nobel) 150 4.6 mm, equipped with a precol-

umn C18 Kromasil (Akzo Nobel) was used for the separation. Diode

array detector was programmed to perform chromatographic read-

ings in a range of wavelengths from280 to 600 nmin steps of 2 nm

and detection at 280 nm (detection of (PAs), flavan 3-ols, gallic acid

and galloil esters), 320 nm (hydroxycinnamic acid test), 360 nm

(flavonols), 510 nm and 520 nm (anthocyanins) (Kosar et al.,

2004; Mtt-Riihinen et al., 2004; Oszmianski et al., 2009; Vasco

et al., 2009). Means of two technical replicates of three indepen-

dent quantifications were subjected to one-way ANOVA and LSDpairwise comparisons using Statistica 4.0 software (Statsoft Inc).

http://-/?-http://-/?-


11/12

Acknowledgments

We thank anonymous reviewers for helpful comments on this

manuscript. This work has been funded by grant PBCT Anillo Cien-

cia y Tecnologa (ACT-41). A.S. thanks University of Talca, MeceSup

and Anillo ACT-41 for Ph.D. fellowships. P.P. thanks Conicyt for a

Ph.D. fellowship. We are grateful to CSIRO (Australias Common-

wealth Scientific and Industrial Research Organization) for provid-ing the pHELLSGATE12 vector.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in

the online version, at http://dx.doi.org/10.1016/j.phytochem.2013.

02.016.

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