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Phytochemistry 90 (2013) 25–36 Contents lists available at SciVerse ScienceDirect Phytochemistry journal homepage:

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Phytochemistry

journal homepage: www.elsevi er.com/locate/phytochem Increased accumulation of anthocyanins in Fragaria

Increased accumulation of anthocyanins in Fragaria chiloensis fruits by transient suppression of FcMYB1 gene

Ariel Salvatierra a , 1 , 2 , Paula Pimentel b , 1 , María Alejandra Moya-León a , Raúl Herrera a ,

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

article info

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 chiloensis ssp. chiloensis Rosaceae Fruit color RNAi silencing Anthocyanin Flavan 3-ol

abstract

Anthocyanins and proanthocyanidins (PAs), flavonoid-derived metabolites with different physiological 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 of FaMYB1 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 in F . chiloensis , FcMYB1 was transiently down-regulated in planta using an RNAi-based approach. Quantitative real-time PCR on FcMYB1 down-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 (2 n = 8 x = 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-

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: raherre@utalca.cl (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.

0031-9422/$ - see front matter 2013 Elsevier Ltd. All rights reserved.

posed of a pinkish-white receptacle and red achenes when fully ripe. This strawberry is characterized as having a high and partic- ular aroma ( González et al., 2009a ), large fruit size (compared with all other wild species) and a remarkable tolerance to Botrytis infection ( González et al., 2009b ). However, the fruit depigmenta- tion of the white native strawberry constitutes its most character- istic trait. In the Fragaria genus, 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

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A. Salvatierra et al. / Phytochemistry 90 (2013) 25–36

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 ). In A. thaliana, O. sativa and V. vinifera , several MYBs belonging 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 ). In A. 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 of AmMYB308 , 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 of Lotus corniculatus ( Paolocci et al., 2011). Thus, both studies demonstrated over-expression of FaMYB1 in 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 of CHS gene 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 of this 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.

)-epicatechin ( 4 ; Fig. 1 ), were analyzed by HPLC-DAD. Fig. 1. Structures of compounds

Fig. 1. Structures of compounds analyzed in this study. ( 1 ) Cyanidin 3-glucoside; ( 2 ) pelargonidin 3-glucoside; ( 3 ) (+)-catechin; ( 4 ) ( )-epicatechin.

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 were sequenced and a cDNA sequence of 1014 bp named FcMYB1 (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 5 0 untranslated region (UTR) of both genes was identical, at least to the extent available for FaMYB1 . Simi- larly, FcMYB1 3 0 -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 of FaMYB1 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 of A. 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 TFIIIA zinc finger type. The FcMYB1 protein se- quence matches the motif defined for R2R3-MYB proteins belong- ing to subgroup 4, as well as AtMYB4 ( Jin et al., 2000 ). Interestingly, AtMYB4 has been proven to be a negative regulator of cinnamate 4-hydroxylase (C4H) gene expression, being the first Arabidopsis MYB described as transcriptional repressor ( Jin et al., 2000 ). Alignment

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A. Salvatierra et al. / Phytochemistry 90 (2013) 25–36 27 Fig. 2. Phylogenic relationships of selected

Fig. 2. Phylogenic relationships of selected MYB proteins from different plant species. The scale represents the number of substitutions per site and the nu mbers 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 report ed 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 (JQ09 60), 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).

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 between FcMYB1 and its ortholog FaMYB1 . Therefore, it was assumed that FcMYB1 retains 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 of FaMYB1 increased during, ripening of the F . 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 of FcMYB1 in white Chilean strawberry, organ-specific and temporal expression patterns were analyzed by 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 FcMYB1 gene expression in both white and commercial strawberry fruits showed differences, in transcript level and in expression profile trend ( Fig. 3 A). 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. 3 A). A similar expression profile was described in a previous transcriptional analysis which included three developmental stages of white Chilean strawberry and commercial strawberry cv. Chandler ( Saud et al., 2009 ). In our study, FcMYB1 showed high- er transcript levels along development and ripening of the white Chilean strawberry fruit compared to F . ananassa cv. Camarosa ( Fig. 3 A). Since Aharoni et al. (2001) demonstrated that over- expression of FaMYB1 in tobacco plants suppressed flower pigmen- tation, the high level of transcripts of this TF detected in white Chilean strawberry may contribute to the repression described in

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28 A. Salvatierra et al. / Phytochemistry 90 (2013) 25–36 Fig. 3. FcMYB1 gene relative expression

Fig. 3. FcMYB1 gene relative expression levels. (A) Comparative analysis of FcMYB1 temporal expression in fruits of F . chiloensis ssp. chiloensis f. chiloensis (white circles) and F . ananassa cv. 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) and root (Rt). Transcript levels were analyzed by qRT-PCR and calibrated against gene expression in stage 1 of F . chiloensis (C1).

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-Wang et 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. 3 B).

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 with control construction, corresponding to the empty vector ( Fig. 4 A 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 in FcMYB1 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. 6 A). 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 of FcMYB1 suppressed lines. It seems that this TF does not affect CHS expres- 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. 6 B–D). 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-PCNA persimmon 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 of ANR and isoforms of LAR (i.e. LAR1 and LAR2 ) 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 and ANR genes evidenced severe reduction in their expres- sion levels in FcMYB1 suppressed fruits ( Fig. 6 G and H). This level of suppression on PA-related genes may contribute to the in- creased pigmentation of these fruits. Similarly, flowers of tobacco lines over-expressing different apple ANR isoforms (i.e. MdANR1,

A. Salvatierra et al. / Phytochemistry 90 (2013) 25–36

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A. Salvatierra et al. / Phytochemistry 90 (2013) 25–36 29 Fig. 4. Phenotype of agroinfiltrated fruits.

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).

stage on day 14 post-injection (28 days after anthesis). Fig. 5. FcMYB1 gene expression level of

Fig. 5. FcMYB1 gene expression level of agroinfiltrated fruits. Black bars correspond to the level of FcMYB1 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 represent agroinfiltrated lines with FcMYB1 expression levels significantly reduced from the average of control events with a p < 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.

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 of ANR 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 the PA biosynthesis pathway, which may redirect precursors of

flavonoids to the biosynthesis of anthocyanin pigments in white Chilean strawberry fruits in FcMYB1 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; Palapol 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 of color in white fruit may be attributed to the low expression of

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30 A. Salvatierra et al. / Phytochemistry 90 (2013) 25–36 Fig. 6. FcMYB1 suppression effect in

Fig. 6. FcMYB1 suppression effect in gene expression of flavonoid biosynthesis pathway. The analysis of expression was determined by qRT-PCR and calibrated agains t the average of expression value of each gene in three control events (black line). The values and error bars represent the mean and standard deviation of th ree technical replicates of each agroinfiltration event, respectively.

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ANS ( Debes et al., 2011 ). Additionally, the role of ANS in 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 ANS in an indica rice mutant channeled PA precursors to the syn- thesis of anthocyanins in pericarp tissue ( Reddy et al., 2007 ). More- over, silencing ANS in a red-leaved apple cultivar showed an almost complete loss of cyanidin 3-galactoside and higher concentrations

of other flavonoid compounds, such as (+)-catechin ( 3 ) and oligo-

mers ( Szankowski et al., 2009 ). In FcMYB1 suppressed fruits, ANS was the only one gene showing higher levels of transcripts in all si- lenced lines compared to the control ( Fig. 6 E). 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

of ANS may 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 véraison, 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 of FaGT1 generated strawberry fruits with significantly reduced levels of pelargonidin and increased concentration of fla- van 3-ols ( Griesser et al., 2008 ). In FcMYB1 suppressed fruits, UFGT presented either similar or slightly higher transcript levels than the control, except in the fruit pH12 - FcMYB1i.6 ( Fig. 6 F). 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. 6 G and H) and higher ANS expression ( Fig. 6 E). The interrelationship between these genes (i.e. LAR / ANR and ANS / 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 anthocyanin related 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 of ANS and UFGT, respec- tively ( Aharoni et al., 2001 ). On the other hand, the ectopic expression of FaMYB1 repressed the expression of PA related genes and reduced the accumulation of PA polymers ( Paolocci et al., 2011 ). Both studies showed a reduction in ANS mRNA 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 of FcMYB1 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 transcriptional repressor FcMYB1 may compete with other R2R3 MYBs for binding bHLH 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 of ANS resulting 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 of FcMYB1 transcript accumulation on flavonoid compound accumulation. Along development and ripening of white strawberry fruits, there is an opposite accumulation trend of final flavonoid metabolites ( 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 commercial 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 content of cyanidin 3-glucoside ( 1 ) as compared to control fruit ( Fig. 8 A). Analyses of metabolites showed a greater accumulation of pelarg- onidin 3-glucoside ( 2 ) concomitant with a decrease in FcMYB1 transcript accumulation ( Fig. 8 B). This fact establishes the exis- tence of a direct relationship between the FcMYB1 transcript levels and 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 and F . ananassa cv. 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. 8 C), which is related to the severe reduction in the transcript levels of genes involved in biosynthesis of PA. Conversely, silencing glycosyltransferase FaGT1 reduced anthocyanins levels and

32

A. Salvatierra et al. / Phytochemistry 90 (2013) 25–36

32 A. Salvatierra et al. / Phytochemistry 90 (2013) 25–36 Fig. 7. Accumulation patterns of final

Fig. 7. Accumulation patterns of final flavonoid compounds during development of Fragaria chiloensis ssp. chiloensis f. 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.

biological replicates and two technical replicates. Fig. 8. Flavonoid metabolite levels ( l g g 1

Fig. 8. Flavonoid metabolite levels ( l g g 1 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. Differe nt letters represent significant differences with p < 0.05 according to LSD multiple comparison Fisher test. (C) Flavan 3-ols content. Values are the mean and standard deviation of three technical replicates of flavan 3-ols. Different letters in the same column represent significantly different values with p < 0.05 according to LSD multiple comparison Fisher test. nd, Not detected.

A. Salvatierra et al. / Phytochemistry 90 (2013) 25–36

33

increased flavan 3-ols content in F . ananassa fruits ( Griesser et al., 2008 ). FcMYB1 suppressed 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 of FcMYB1 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 another TF of this family, named MdMYB10 (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 over ANS channeled 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. 8 C). The lack of flavan

3-ols besides the severe drop in mRNA levels of PA related genes seems to be a side-effect of FcMYB1 suppression as a consequence

of the transcriptional activation of ANS and a higher biosynthesis of

anthocyanins in receptacle. Based on transcript and chemical anal- yses of FcMYB1 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 bHLH3 D 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

of FcMYB1 in 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 was

observed 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 . chiloensis ssp. chiloensis f. 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 hypothesized that 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 of flavonoid intermediary metabolites in the last steps of this path- way through genetic manipulation.

4. Experimental

4.1. Plant material

F. chiloensis ssp. chiloensis f. chiloensis (f. chiloensis) plants from Contulmo, Bio-Bio Region, Chile (latitude 38 04 0 8.6 00 S; longitude 73 14 0 2.96 00 W), were grown in pots and maintained at the Univer- sity of Talca (2009). Four development and ripening stages were considered as reported in Figueroa 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 35 41 0 49.21 00 S; longitude 72 32 0 34.60 00 W) at equivalent times to those described for white native strawberry. Fruits in plants of F . chiloensis ssp. chiloensis f. 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 of 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 treatment (Invitrogen) was carried out to remove contaminant genomic DNA. Integrity 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 partial sequence of this gene from a cDNA pool prepared from F . chiloensis ssp. chiloensis f. chiloensis fruit at different developmental and rip- ening stages (MYBorf fwd 5 0 -TGGAATTCGCTTCCTAAGGCTG-3 0 ; MYBorf rev 5 0 -TCCAATCCCAGAATC CAAGCAC-3 0 ). A 570 bp gene fragment obtained by PCR was cloned into the vector pSCA follow- ing manufacture instructions (StrataClone PCR Cloning Kit) and se- quenced at Macrogen (Seoul, Korea). Internal specific primers were designed (MYB1race fwd 5 0 -GGTCTCATCAGCCGATTCTGCTGC TGGC-3 0 ; MYB1race rev 5 0 -TATCTGTCCTTCCAGGCAGTCTTCCAGC- 3 0 ) for RACE reaction to obtain full length FcMYB1 sequence. Total RNA (5 l g) from a mix of F . chiloensis fruit was used for RACE- Ready cDNAs (BD Biosciences, Clontech) following the user’s man- ual. The 5 0 and 3 0 specific primers were designed based on partial 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 per 3 min; 1 cycle at 72 C per 15 min. Gene fragments obtained were cloned and sequenced as described above. Partial sequences of FcMYB1 were aligned to get the full-length cDNA

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A. Salvatierra et al. / Phytochemistry 90 (2013) 25–36

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 cDNA sequence was amplified by PCR using the primers MYB dir fwd 5 0 -CACC ATGAGGAAGCCCTGCTGC-3 0 , and MYB dir 5 0 -GGATCAGCCA TTCCAGTACTA GTC-3 0 , 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 of FcMYB1 gene fragment using a combination of gene-specific primers (fwd MYBdir) and M13 (M13 rev 5 0 -CAGGAAACAGCTATGAC-3 0 ). 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 manufacturer’s 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 l g/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 a GUS gene fragment.

4.5. White strawberry fruit transfection by agroinfiltration

pHELLSGATE12-FcMYB1i construction 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 l g/ml of each antibiotic. Resistant colonies were analyzed by PCR for the presence of FcMYB1 gene fragment using FcMYB1 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 1620 g 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 l l reaction mix- ture. Each reaction mixture contained template RNA (2 l g), 2x cDNA Synthesis Master Mix (10 l l), Oligo (dT) Primer (3 l l) and

AffinityScript RT/TNasa Block Enzyme Mixture (1 l l). cDNA was di- luted 1:4, and 2 l l 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 5 0 -GGTGCCTGAGTT- GAATCTC-3 0 ; qPCR-MYB1 rev 5 0 -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 manufacturer’s 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. GAPDH gene 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 (Extrasynthèse) and cyanidin 3-glucoside (1) as kuromanin chloride (Sigma–Aldrich). (+)-Catechin ( 3 ) and ( )-epicatechin ( 4 ) were purchased from Extrasynthèse. Anthocyanin contents in fruits were processed from fruit (2.5 g) as described in Salvatierra 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 H 2 O (50 ml). The homogenate was extracted with Et 2 O (20 ml 3) and EtOAc (20 ml 3). The six ex- tracts were combined, dried (0.5 mg Na 2 SO 4 anhydrous), and the organic extract was concentrated to dryness, reconstituted in MeOH:H 2 O (2 ml) (1:1, v/v) and filtered before analysis. The flow rate of elution was 600 l l/min of solvent A (AcOH 2%, in ultrapure H 2 O) at initial (0.0–12.0 min) and final phase of the run (23.5– 25.0 min). Intermediate steps consisted in a gradient elution with a flow rate of 800 ll/min with a mixture of 30% A and 70% B (CH 3 CN, AcOH, H 2 O; 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 (MeOH–H 2 O (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 series HPLC system provided by a photodiode array detector (DAD) equipped with a manual injector (20 l l injection volume) and interfaced to a PC running ChemStation chromatography manager software (Hewlett–Packard). 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 from 280 to 600 nm in 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; Määttää-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 LSD pairwise comparisons using Statistica 4.0 software (Statsoft Inc).

A. Salvatierra et al. / Phytochemistry 90 (2013) 25–36

35

Acknowledgments

We thank anonymous reviewers for helpful comments on this manuscript. This work has been funded by grant PBCT Anillo Cien- cia y Tecnología (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 (Australia’s 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.

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