Phytochemistry 71 (2010) 1839–1847

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Phytochemistry

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Comparison of transcriptional profiles of flavonoid genes and anthocyanincontents during fruit development of two botanical formsof Fragaria chiloensis ssp. chiloensis

Ariel Salvatierra, Paula Pimentel, Maria Alejandra Moya-Leon, Peter D.S. Caligari, Raul Herrera ⇑Instituto de Biología Vegetal y Biotecnología, Universidad de Talca, Casilla 747, Talca, Chile

a r t i c l e i n f o

Article history:Received 14 December 2009Received in revised form 27 July 2010Available online 26 August 2010

Keywords:Fragaria chiloensis ssp. chiloensisRosaceaeFruit colorDevelopmental gene expressionAnthocyanins

0031-9422/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.phytochem.2010.08.005

Abbreviations: ANR, anthocyanidin reductase; ACTAB, cetyltrimethylammonium bromide; CHI, chalcosynthase; C4H, cinnamate 4-hydroxylase; DAA, days aavonol reductase; FLS, flavonol synthase; F3H, flavanospecific primer; HPLC-DAD, high performance liquiddetector; LAR, leucoanthocyanidin reductase; PA, proalanine ammonia-lyase; qRT-PCR, quantitative revechain reaction; TF, transcription factor; UFGT, UDP gltransferase; 4CL, 4-coumarate:CoA ligase; f. chiloechiloensis f. chiloensis; f. patagonica, Fragaria chiloensi⇑ Corresponding author. Tel.: +56 71 200277; fax:

E-mail address: raherre@utalca.cl (R. Herrera).

a b s t r a c t

Difference in fruit pigmentation observed between two botanical forms of Fragaria chiloensis ssp. chiloen-sis (form chiloensis and form patagonica) was studied through transcriptional and chemical approaches.The proportion of different anthocyanins was demonstrated to be characteristic of each botanical form,with pelargonidin 3-glucoside being the most abundant in f. patagonica fruit and cyaniding 3-glucosideas the major one in f. chiloensis fruit. Partial gene sequences of the phenylpropanoid and flavonoid bio-synthesis pathways were isolated from the native Chilean strawberry fruits, and used to design gene-spe-cific primers in order to perform transcriptional analyses by qRT-PCR. These genes showed spatial,developmental, and genotypic associated patterns. The red fruit of f. patagonica exhibited higher tran-script levels of anthocyanin-related genes and higher levels of anthocyanins compared to the barely pig-mented fruit of f. chiloensis. The anthocyanin accumulation in F. chiloensis ssp. chiloensis fruits wasconcomitant with the particular progress of the transcriptional activity of genes involved in the biosyn-thesis of flavonoid pigments. The differences in anthocyanin contents, both in terms of type and quantity,between the two botanical forms of F. chiloensis ssp. chiloensis were coincident with the differential tran-scriptional patterns found in the anthocyanin-related genes.

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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. This native strawberry has two botanical formswhich are readily recognizable. F. chiloensis ssp. chiloensis f. patago-nica is a wild plant with small fruits, red receptacle, and yellow orred achenes. On the other hand, F. chiloensis ssp. chiloensis f. chilo-ensis is a robust plant cultivated on a small scale that bears largerfruits, which are composed of a pinkish-white receptacle and redachenes when fully ripened. The latter is the maternal progenitor

ll rights reserved.

NS, anthocyanidin synthase;ne isomerase; CHS, chalconefter anthesis; DFR, dihydrofl-ne 3-hydroxylase; GSP, genechromatography-diode arrayanthocyanidin; PAL, phenyl-

rse transcription-polymeraseucose:flavonoid 3-O-glucosylnsis, Fragaria chiloensis ssp.s ssp. chiloensis f. patagonica.+56 71 200276.

of the widely cultivated strawberry, Fragaria � ananassa Duch.(Hancock et al., 1999). F. chiloensis is characterized as having a highand particular aroma (González et al., 2009a), large fruit size (com-pared with all other wild species), and remarkable tolerance toinfection by Botrytis (González et al., 2009b). These advantages,along with other characteristics, make it an important germplasmsource both for its own development as a new exotic fruit crop, aswell as for further development of new cultivars of the commercialstrawberry (F. � ananassa).

These thus offer an interesting model to study fruit pigmenta-tion by anthocyanins, considering that the color of the white andred botanical forms reflect the presence of such phytochemicals.Anthocyanins are natural colorants belonging to the flavonoid fam-ily of compounds, a secondary class of metabolites that are respon-sible for the red, violet and blue colors observed in flowers andfruits in a large number of plants. Due to the broad distributionof anthocyanins in the plant kingdom, their chemistry, distribu-tion, biosynthesis and regulation have been extensively studied.This has resulted in the collection of a large amount of informationconcerning the production of plant pigments (Dooner et al., 1991;Holton and Cornish, 1995; Grotewold, 2006; Ferrer et al., 2008).

In the Fragaria genus, fruit color is determined by the accumu-lation of anthocyanins, the most abundant flavonoids in strawberry

Fig. 1. Chemical structures of anthocyanins measured in Fragaria chiloensis ssp.chiloensis f. chiloensis and f. patagonica fruits. (1), Pelargonidin 3-glucoside; (2),cyanidin 3-glucoside.

Fig. 2. Developmental and ripening stages of native Chilean strawberry fruit. Fourdifferent developmental stages for F. chiloensis ssp. chiloensis fruits: (A) red fruitedbotanical form patagonica; (B) white–pinkish fruited botanical form chiloensis. Barsare equivalent to 1 cm.

Table 1Primer sequences of the phenylpropanoid and flavonoid genes and housekeepinggene (GAPDH) used for qRT-PCR. All primers were designed from partial sequencesisolated in this work, except for FLS primer, which was designed directly from aF. � ananassa sequence found in public database (GenBank number accessionDQ087252).

Targetgene

Primers (forward/reverse) Ampliconsize (bp)

Efficiency(%)

PAL 50-CAAGGGCGGCGATGCTAGTAAG-30 153 96.550-CCAAGTCACCCGACGACGAGAT-30

C4H 50-CTGTAAGGAGGTGAAGGAGAAGAGG-30

139 97.1

50-CTGTTGAGCGTCCAGGATGTG-30

4CL 50-ACTTGGTCAGGGATATGGGATG-30 150 95.450-GCACCAGTTTCAGGGTCTACG-30

CHS 50-CCGACTACTACTTTCGTATCACCA-30

190 94.3

50-ACTACCACCATGTCTTGTCTTGC-30

CHI 50-TTTTCAATGGCTTTCGCTTCTG-30 119 94.750-GTGACAATGATACTACCGCTGACG-30

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fruits (Hannum, 2004). Pelargonidin 3-glucoside (1) is the predom-inant anthocyanin in several varieties of red strawberries, usuallyfollowed by pelargonidin 3-rutinoside and cyanidin 3-glucoside(2) (Gil et al., 1997; Kosar et al., 2004; Tulipani et al., 2008). Thesethree compounds represent more than 95% of the total anthocya-nins in the cultivated strawberry (Lopes da Silva et al., 2007). F.chiloensis f. patagonica fruit also showed similar levels of pelargon-idin 3-glucoside (1) (Simirgiotis et al., 2009) to F. � ananassa (Kosaret al., 2004), when fully ripe. However, at the same developmentalstage, f. chiloensis had lower levels of anthocyanins, with cyanidin3-glucoside (2) being the major anthocyanin followed by pelargon-idin 3-glucoside (1) (Simirgiotis et al., 2009). This indicates differ-ences in anthocyanin compositions between the two botanicalforms of the native Chilean species of F. chiloensis ssp. chiloensis.

Flavonoid genes involved in anthocyanin biosynthesis exhibitup-regulation during ripening leading to fruit pigmentation inF. � ananassa thereby establishing a positive correlation betweentranscript levels of flavonoid genes and anthocyanin accumulation(Manning, 1998; Almeida et al., 2007; Carbone et al., 2009).

In the present work, cDNA fragments of genes from phenylprop-anoid (PAL, C4H and 4CL) and flavonoid biosynthetic pathway (CHS,CHI, F3H, DFR, ANS, UFGT, LAR and ANR) were isolated from the na-tive Chilean strawberry. The differential transcriptional profiles ofthese genes were analyzed in f. chiloensis and f. patagonicathrough four different fruit developmental stages and tissues byqRT-PCR. In parallel, the accumulation of the two major anthocya-nins, pelargonidin 3-glucoside (1) and cyaniding 3-glucoside (2;Fig. 1), in these native strawberries was quantified by HPLC-DAD.The relationship between transcriptional profiles and anthocyanincontents present in both botanical forms is discussed in terms ofthe modulation of flavonoid gene expression in the determinationof the different pigmentation patterns observed in the white- andred-fruited Chilean native strawberries.

F3H 50-GTGCGCCACCGTGACTACTC-30 157 95.450-ATGCCTTTGTCAATGCCTCC-30

DFR 50-GGGTGGTGTTTACATCTTCGG-30 156 96.550-CTGCTTGCTCGGCTAGAGTTT-30

ANS 50-ATCGTCATGCACATAGGCGACACC-30

130 97.1

50-CCTTGGGCGGCTCACAGAAAA-30

UFGT 50-ATCGTGGCTTGACAAACAGAA-30 133 94.250-TGACCACAAGAATGGAACCCTA-30

ANR 50-CATCCAAGGCGAAGACCAT-30 167 96.550-TCATACTTAAACAACTGAGACCACC-30

LAR 50-GGTGATGGCACGGTTAAAGC-30 156 100.450-CTCCCACAGTGAAGCAAGTCC-30

FLS 50-TTATCTTTGGGGTTAGGGCTTGAA-30

162 98.1

50-GAGAATGGTGAGGGCGGACA-30

GAPDH 50-TCCATCACTGCCACCCAGAAGACTG-30

132 93.0

50-AGCAGGCAGAACCTTTCCGACAG-30

2. Results and discussion

2.1. Transcriptional profiles of genes involved in biosynthesis ofphenolic compounds in fruits at different developmental stages

The phenylpropanoid biosynthesis pathway is part of the sec-ondary metabolism of plants, and its branches transform the aminoacid, phenylalanine, into a variety of important phytochemicals,including lignins, stilbenes, coumarins, salicylates, sinapate estersand flavonoids. The structural diversity of compounds derivedfrom phenylalanine is due to the action of enzymes and enzymecomplexes that bring about regio-specific condensation, cycliza-tion, aromatization, hydroxylation, glycosylation, acylation, pre-nylation, sulfation and methylation reactions (Noel et al., 2005).

In order to study the transcriptional profiles of flavonoid genesin the Chilean native strawberry of red and white fruit, fragments

of genes involved in this biosynthetic pathway were isolated. Thesegene fragments showed a high nucleotide homology with phenyl-propanoid and flavonoid genes from other plant species (Supple-mentary Table 2). Suitable primers for transcriptional analysis by

A. Salvatierra et al. / Phytochemistry 71 (2010) 1839–1847 1841

qRT-PCR were thus then designed from the isolated sequences(Table 1).

Along the fruit development process, PAL, the first gene in-volved in this metabolic pathway, showed similar expression levelin both botanical forms with a continuous increment in transcripts

Fig. 3. Transcriptional analysis of genes involved in flavonoid biosynthesis during deve(black circles) fruits by qRT-PCR. The fruits were classified into four developmental and riP2; S3 to C3 and P3 and S4 to C4 and P4 of F. chiloensis ssp. chiloensis f. patagonica andGAPDH transcript values. Values represent the average ± SD of three biological replicate

during development (Fig. 3A). This step represents the connectionbetween primary and secondary metabolism since the enzyme en-coded by this gene is responsible for catalyzing the trans-elimina-tion of ammonia from phenylalanine and the generation of trans-cinnamic acid, the substrate for the next step mediated by C4H.

lopment of F. chiloensis ssp. chiloensis f. chiloensis (white circles) and f. patagonicapening stages as described in Section 4: S1 corresponding to C1 and P1; S2 to C2 andf. chiloensis, respectively. Relative gene expression levels were normalized against

s with two technical replicates of each developmental stage.

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The following genes in the phenylpropanoid biosynthesis pathway,C4H and 4CL, displayed at the first developmental stage high tran-script levels which dropped in the following stages to basal levelsin both botanical forms (Fig. 3B and C). Similar transcript patternswere observed in both fruits, although higher transcript levelswere recorded for F. chiloensis ssp. chiloensis f. patagonica at stage1. Since this biosynthetic pathway is general for a large variety ofplant compounds, it does not seem to have a determining role inthe differences of fruit pigmentation recorded between the patago-nica and chiloensis forms.

The first committed step of the flavonoid biosynthesis pathwayis mediated by CHS. All flavonoids are derived from the chalconescaffold synthesized by this first enzyme, which catalyzes the iter-ative condensation and subsequent intramolecular cyclization ofthree acetate units onto the p-coumaroyl-CoA end product of thegeneral phenylpropanoid pathway. At the initial developmentalstages, CHS transcripts exhibit a basal levels and only at the laststage there was a significant transcript increment in F. chiloensisssp. chiloensis f. chiloensis fruit (Fig. 3D). On the other hand, in F.chiloensis ssp. chiloensis f. patagonica fruit, a particularly distincttranscriptional profile was observed. In this case, maximum tran-script levels were reached at the turning fruit stage (S3) whenthe receptacle begins its gradual pigmentation. Then, a slight de-crease in CHS messenger is observable at S4. Similar transcriptionpatterns have been reported for CHS genes in other developmentalstudies carried out in several F. � ananassa genotypes (Manning,1998; Almeida et al., 2007; Carbone et al., 2009; Saud et al.,2009). CHI stereospecifically directs and greatly accelerates thespontaneous additional cyclization of chalcones to form the flavo-noid core. Since the activity of this enzyme is tightly related to CHSactivity, it was not surprising to observe the same trend in theirmessenger profiles (Fig. 3E). F3H, ANS and UFGT transcript levelsshare a similar pattern with CHS and CHI, but a drop in S4 stagein F. chiloensis ssp. chiloensis f. patagonica fruit was not noted. Inthe particular case of UFGT, no increment is observed in F. chiloensisssp. chiloensis f. chiloensis fruit at S4 stage, which is unique behav-ior compared with that of CHS, CHI, F3H, DFR and ANS transcriptprofiles (Fig. 3F, H and I). UFGTs catalyze transfer of glucose fromUDP-activated sugar donor molecule to the hydroxyl group at C3of flavonols and anthocyanidins. Glycosylation increases the watersolubility of polar flavonoid compounds and improves their stabil-ity. This is an essential final step required to stabilize anthocyani-dins so that they can accumulate as water soluble pigments in thevacuoles, a subcellular compartment with an acidic environmentwhere anthocyanins exist in their colored form. Thus UFGT is re-garded as indispensable in anthocyanin biosynthesis rather thansimply a modifying enzyme. In Vitis vinifera cv. Shiraz, the molec-ular analyses of structural genes involved in anthocyanin synthesisshowed that all structural genes tested, except UFGT, were ex-pressed in most berry tissues, whereas expression of UFGT wasonly detected in the berry skin and was always associated withanthocyanin accumulation (Boss et al., 1996a; Kobayashi et al.,2001). This step was identified as a key point in the control of berrycolor, although no differences were observed in either coding orpromoter sequences of this gene between colored and white culti-vars (Kobayashi et al., 2002). In strawberry, RNAi down-regulationof FaGT1, a UFGT gene phylogenetically related to VvGT1, showedsignificant reduced levels of the strawberry pigments pelargonidin3-glucoside malonate and pelargonidin 3-glucoside (1) comparedto control fruit (Griesser et al., 2008). In the light of all of above,the dramatic difference observed in UFGT mRNA levels seems tobe playing a pivotal role in the difference of pigmentation betweenboth botanical forms of native Chilean strawberry.

DFR catalyzes the stereo-specific reduction of dihydroflavonolsto leucoanthocyanidins, using NADPH as a cofactor (Kristiansenand Rohde, 1991). Leucoanthocyanidins are intermediate precur-

sors for synthesis of anthocyanins and proanthocyanidins (PAs).As in the previously discussed flavonoid genes, DFR transcriptshad an increasing trend through berry development (Fig. 3G). Inripe fruits, F. chiloensis ssp. chiloensis f. chiloensis showed low levelsof anthocyanins compared to F. chiloensis ssp. chiloensis f. patago-nica and F. � ananassa cv. Chandler, but catechin and higher levelsof ellagic acid were detected in EtOAc soluble fractions (Simirgiotiset al., 2009). Despite the scarcity of colored anthocyanins in F. chilo-ensis ssp. chiloensis f. chiloensis fruit, the presence of tannin com-pounds in ripe fruit could explain the pattern depicted by DFRwhich showed equivalent levels in S4 stage in both botanicalforms.

Ultimately, the anthocyanin-related genes of the native Chileanstrawberry, as reported in developmental studies in other fruitssuch as grapevine (Boss et al., 1996b), apple (Kondo et al., 2002),bilberry (Jaakola et al., 2002), strawberry (Almeida et al., 2007;Carbone et al., 2009) and mangosteen (Palapol et al., 2009), showedtranscriptional profiles that correlate positively with anthocyaninaccumulation throughout fruit development. In addition, compar-ative transcriptional analysis carried out between fruits of bothbotanical forms of F. chiloensis established higher transcript levelsof flavonoid genes leading to anthocyanins (except by DFR) in thered-fruited F. chiloensis ssp. chiloensis f. patagonica than in thewhite-fruited F. chiloensis ssp. chiloensis f. chiloensis at ripe stage,agreeing with similar comparative studies in species with fruitscontrasting in pigmentation, such as grapevine (Boss et al.,1996a,c), apple (Honda et al., 2002) and bilberry (Jaakola et al.,2002).

Flavonols, synthesized by FLS, and PAs, synthesized by LAR andANR, are competitive branches for anthocyanin biosynthesis in theflavonoid pathway. FLS, LAR and ANR showed a decreasing tran-scriptional profile as fruit ripening proceeds (Fig. 3J–L). Transgenicanti-sense FLS flowers of Petunia and Nicotiana accumulate in-creased levels of anthocyanins (Holton et al., 1993; Nielsen et al.,2002), which supports the existence of a substrate competitionamong DFR (for anthocyanin production) and FLS (for flavonol pro-duction). In fruits of both native strawberries, FLS transcripts arepresent at high levels in the S1 stage and then they decay steadilyto very low levels in S4 stage (Fig. 3J), showing an opposite trend towhat was observed with the anthocyanin-related genes, confirm-ing the competition between these two pathways in this fruit.

High levels of flavan 3-ol accumulated at early stages, whichcould protect immature berries both from feeding by animalsand pest insects, and from pathogen attack (Gould and Lister,2006). During ripening of commercial strawberry fruit, the concen-tration of flavan 3-ols, constituents of PAs, decreased continuouslywhile anthocyanin content increased, giving the fruit an attractiveappearance. Thus, there is an obvious redirection of flavonoid bio-synthesis from flavan 3-ol to anthocyanin formation during thecomplex developmental process of fruit ripening (Halbwirthet al., 2006).

In PA biosynthesis, LAR catalyzes the synthesis of catechin/afz-elechin and ANR the synthesis of epicatechin/epiafzelechin,depending on the degree of hydroxylation of their respective sub-strates. In the native Chilean strawberry, LAR and ANR transcrip-tional profiles decrease from S1 to S4 stages as in commercialstrawberry, but a notable point is that F. chiloensis ssp. chiloensisf. chiloensis showed higher levels than F. chiloensis ssp. chiloensisf. patagonica of ANR transcripts (Fig. 3K and L). Since quantificationof these metabolites has not been reported for native Chileanstrawberry, the real impact of this particular behavior of ANR is stillunclear.

The temporal transcriptional analysis performed in this studyevidenced a coordinated up-regulation of genes associated toanthocyanin biosynthesis, but at different scale, in fruits from bothbotanical forms. These results suggest that the expression of struc-

A. Salvatierra et al. / Phytochemistry 71 (2010) 1839–1847 1843

tural genes was synchronously regulated in a form-specific man-ner. Similar patterns have been reported in other species and theregulation of structural genes by transcription factors has beenwidely discussed (Palapol et al., 2009; Yuan et al., 2009). In red-fleshed apple, the transcript levels of anthocyanin-related geneswere higher throughout development than those found in white-fleshed apple and strongly correlated to MdMYB10 expression (Esp-ley et al., 2007). In berry skin of V. vinifera cv Shiraz, qRT-PCR anal-yses of a number of transcription factors have reported

Fig. 4. Spatial transcriptional analysis of genes involved in flavonoid biosynthesis by qchiloensis f. chiloensis (white bars) and f. patagonica (black bars) was assessed in comparigene expression levels were normalized against GAPDH transcript values. Values representissue.

transcriptional profiles associated to developmental stages, whereMYB proteins controlling the expression of PA related genes (e.g.,VvMYBPA1, VvMYB5a and VvMYB5b) were up-regulated in unripestages and those controlling the anthocyanin-related genes (e.g.,VvMYB5b, VvMYBA1 and VvMYBA2) showed higher transcript lev-els at ripe stages (Deluc et al., 2008). This fact makes evident theneed of a complex network of regulatory genes in order to modu-late the flavonoid structural genes through fruit ripening. Cur-rently, the regulation of expression of structural genes on

RT-PCR. The gene expression in flower, leaf, runner and root from F. chiloensis ssp.son with that of F. chiloensis ssp. chiloensis f. chiloensis fruit on stage 1 (C1). Relativet the average ± SD of three biological replicates with two technical replicates of each

Table 2Changes in main anthocyanin content (lg g�1 fresh weight) during development and ripening of both botanical forms of F. chiloensis ssp. chiloensis.

F. chiloensis ssp. chiloensis Developmental stage Pelargonidin 3-glucoside (1) Cyanidin 3-glucoside (2)

f. chiloensis S1 nd 3.03 ± 0.08aS2 nd 4.98 ± 0.50aS3 nd 7.80 ± 0.17aS4 3.95 ± 0.13a 5.86 ± 1.18a

f. patagonica S1 nd 4.66 ± 0.66bS2 nd 6.55 ± 0.64bS3 6.42 ± 0.73 11.84 ± 2.20bS4 12.47 ± 1.15b 6.63 ± 0.57a

Values represent the average ± SD of three biological replicates. For each anthocyanin, different letters at the same developmental stage indicate significant differencesbetween botanical forms at p < 0.05.S1: developmental stage 1 (C1/P1); S2: developmental stage 2 (C2/P2); S3: developmental stage 3 (C3/P3); S4: developmental stage 4 (C4/P4); nd: compound not detected.

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flavonoid pathway by transcription factors is a matter of analysis inour ongoing work.

2.2. Transcriptional profiles of genes involved in biosynthesis ofphenolic compounds in tissues

The spatial transcriptional analysis detected expression of genesinvolved in phenylpropanoid and flavonoid biosynthesis pathwaysin all tissues assessed. A greater difference in transcript levels forall genes from the phenylpropanoid biosynthesis pathway was ob-served (Fig. 4). For PAL, the higher differences in transcript levelwas observed for flower, leaf and roots (Fig. 4A). Roots and runnersexhibited higher mRNA levels of C4H and 4CL than the other tis-sues, since their developmental dynamics involve continuousgrowth of these woody structures and the concomitant synthesisof lignin monomers (Fig. 4B and C).

Runners of F. chiloensis ssp. chiloensis f. patagonica present a redpigmentation. In this case, flavonoid biosynthesis pathway genesleading to anthocyanins are up-regulated, except by ANS, com-pared to what happens in green runners of F. chiloensis ssp. chiloen-sis f. chiloensis. The low level of ANS transcripts found in the redrunners of F. chiloensis ssp. chiloensis f. patagonica could be a resultof the incapacity to detect a putative tissue-specific ANS paralogousgene. The most prominent difference was noted at the level of theUFGT messengers, where quantities of mRNAs of this gene werethree times higher in red runners relative to green ones. Again, inthese contrasting colored tissues, UFGT seems to be the determin-ing factor in the anthocyanin-related pigmentation of plant struc-tures in the native Chilean strawberries (Fig. 4D–I).

Roots of F. chiloensis ssp. chiloensis f. chiloensis have a reddishhue and the situation described above occurs in an opposite way,since F. chiloensis ssp. chiloensis f. patagonica roots are green, buton a minor scale (Fig. 4D–I). In addition, DFR, LAR and ANR tran-scripts showed higher levels in F. chiloensis ssp. chiloensis f. chiloen-sis which could suggest a redirection in the flavonoid pathway tothe synthesis of PAs in this tissue (Fig. 4K and L).

Flowers of both botanical forms gave highest expression of FLSamong the native Chilean strawberry tissues analyzed (Fig. 4J).This behavior has already been reported for the commercial straw-berry (Almeida et al., 2007) and correlates very well with the phys-iological role of flavonols in planta since their biosynthesis isproposed as key point for biological events that happen in theflower organ, such as pollinator attraction (Tanaka et al., 2008)and pollen germination (van der Meer et al., 1992; Napoli et al.,1999).

Leaves of F. chiloensis ssp. chiloensis f. chiloensis exhibited hightranscript levels of PA genes (Fig. 4K and L); this fact could explainthe enhanced tolerance to Botrytis infection (González et al.,2009b) as reported for other plant species (Ardi et al., 1998; Miran-da et al., 2007).

The existence of a spatial regulation of the flavonoid geneexpression is evident which would lead to the biosynthesis of di-verse phenolic compounds. These metabolites have multiple bio-logical functions in each tissue as a means for sustainingsuccessful adaptability, growth and reproduction of the plant.

2.3. Accumulation of anthocyanins during fruit development

Accumulation of anthocyanins during ripening has been re-ported for several fruits such as grape (Boss et al., 1996c; Ryanand Revilla, 2003; Vian et al., 2006), mangosteen (Palapol et al.,2009), bilberry (Jaakola et al., 2002), apple (Kondo et al., 2002)and strawberry (Kosar et al., 2004; Halbwirth et al., 2006; Carboneet al., 2009; Saud et al., 2009). The most commonly occurringanthocyanins in strawberry are cyanidin and pelargonidin deriva-tives. The main pigment in cultivated strawberries has been iden-tified as pelargonidin 3-glucoside (1) (Gil et al., 1997; Määttää-Riihinen et al., 2004) and the presence of cyanidin 3-glucoside(2) is widely documented (Nyman and Kumpulainen, 2001; Wanget al., 2003; Kosar et al., 2004; Tulipani et al., 2008).

The composition and contents of anthocyanins were deter-mined in all developmental and ripening stages described for F.chiloensis ssp. chiloensis f. chiloensis and f. patagonica fruits. Cyani-din 3-glucoside (2) was present at all developmental stages infruits of both botanical forms, with a maximum level at S3 stage;nevertheless, a higher concentration of this pigment was observedin fruits from F. chiloensis ssp. chiloensis f. patagonica. The presenceof cyanidin 3-glucoside (2) in the unripe stages could be explainedby the early pigmentation of achenes seen in fruits of both botan-ical forms. Pelargonidin 3-glucoside (1), the typical receptacleanthocyanin, was detected in F. chiloensis ssp. chiloensis f. patago-nica at S3 stage, with a maximum level at S4 (12.5 lg g�1), whilethis anthocyanin was only detected at a low level in the S4 stagein F. chiloensis ssp. chiloensis f. chiloensis (4.0 lg g�1), representingonly one third of pelargonidin 3-glucoside (1) found in ripe fruits off. patagonica (Table 2).

Cyanidin derived anthocyanins have been reported in commer-cial strawberries as being present mainly in achenes (Aaby et al.,2005). Cyanidin-3-O-b-D-glucopyranoside was the main anthocya-nin in F. chiloensis ssp. chiloensis f. chiloensis fruits and was foundonly in achenes (Cheel et al., 2005). The unusual quantity of cyani-din derived pigment detected in F. chiloensis ssp. chiloensis f. chilo-ensis fruits is given by the achene anthocyanin input, due to thefact that achenes are the most colored tissues in the whole fruitof this form.

In addition, a different anthocyanin ratio is evident between theripe fruits of this native strawberry, since F. chiloensis ssp. chiloensisf. patagonica showed a pelargonidin 3-glucoside (1) content 1.88times higher than cyanidin 3-glucoside (2) and in F. chiloensisssp. chiloensis f. chiloensis, cyanidin 3-glucoside (2) content was

A. Salvatierra et al. / Phytochemistry 71 (2010) 1839–1847 1845

1.48 times higher than pelargonidin 3-glucoside (1) according toour extraction procedures and HPLC-DAD measurements. This par-ticular trait could be useful for distinguishing the botanical formsby mean of a chemotaxonomic approach.

3. Conclusions

The current work establishes for the first time a clear relationshipbetween fruit pigmentation, transcriptional patterns of phenylprop-anoid and flavonoid biosynthesis pathways and accumulation ofrepresentative anthocyanins of both botanical forms of F. chiloensisssp. chiloensis during their fruit developmental process. The resultsindicate the existence of differential transcriptional patterns in theanthocyanin-related genes during fruit ripening among F. chiloensisssp. chiloensis f. chiloensis and f. patagonica with a clear down-regu-lation of these genes in F. chiloensis ssp. chiloensis f. chiloensis fruits,which could explain the scarce pigmentation in the white straw-berry where UFGT transcript levels seem to be critical for fruit antho-cyanin accumulation.

Moreover, the differences are not a reduction in the quantity ofanthocyanin but also in the quality of them. Further studies are re-quired for elucidating the role of transcription factors in the spatialand developmental regulation of structural genes responsible forphenolic compounds production in order to explain the differencesin their gene patterns observed among the two native Chileanstrawberries.

4. Experimental

4.1. Plant material

Fruit and plant tissues (flower, leaf, runner and root) of F. chilo-ensis ssp. chiloensis f. chiloensis were obtained from a commercialplantation in Contulmo, Bio-Bio Region, Chile (latitude 38� 040

8.600 S; longitude 73� 140 2.9600 W). Fruits were harvested in 2006and 2007. Fruit and plant tissues of F. chiloensis ssp. chiloensis f. pat-agonica were obtained from collections in its native habitat in Ter-mas de Chillán, Bío-Bío Region, Chile (S 36� 540 58.3500, W 71� 250

18.1900). Fruits were harvested during the 2006 and 2007 seasons(December–January).

Fruits of f. chiloensis were classified into four development andripening stages, according to Figueroa et al. (2008), based onweight and color of the receptacle and achene (Fig. 2): C1, smallfruit with green receptacle and green achenes (7 days after anthe-sis, daa); C2, large fruit with green receptacle and red achenes (14daa); C3, turning stage, white receptacle and red achenes (21 daa);and C4, ripe fruit with pink receptacle and red achenes (28 daa).Similarly, fruits of f. patagonica were classified for the first timeinto four development and ripening stages: P1, green receptacleand green achenes (7 daa); P2, green receptacle and red achenes(14 daa); P3, turning stage, pink receptacle and red achenes (21daa); and P4, ripe fruit with red receptacle and red achenes (28daa). After classification, samples were immediately frozen with li-quid N2 and stored at �80 �C until needed.

4.2. RNA extraction

Three independent total RNA samples were isolated from poolsof fruits prepared for each developmental stage, and also fromflower, leaf, runner and root tissue using the CTAB method withminor modifications (Chang et al., 1993). A DNAse treatment(Invitrogen) was carried out with the aim to remove contaminantgenomic DNA. Integrity of isolated RNAs was checked on agarosegels stained with ethydium bromide and their concentration mea-

sured in a ND-1000 UV spectrophotometer (NanodropTechnologies).

4.3. Cloning of partial sequences of flavonoid genes

Primers for amplifying partial sequences of genes involved inthe phenylpropanoid (PAL, C4H and 4CL) and flavonoid biosynthe-sis pathway (CHS, CHI, F3H, DFR, ANS, UFGT, LAR and ANR) (Supple-mentary Table 1) were designed from conserved nucleotideregions identified by multiple alignments of sequences from spe-cies phylogenetically related to Fragaria genus found in public dat-abases (Supplementary Fig. 1).

First-strand cDNAs from each developmental stage and planttissue were synthesized from total RNA (1 lg) using the Thermo-Script RT-PCR System kit (Invitrogen), according to manufacturer’sinstructions. Gene fragments were amplified from a fruit cDNApool including all developmental stages. Amplicons ranging from200 to 500 bp were cloned, sequenced and deduced amino acid se-quences were analyzed by BLASTp (Altschul et al., 1997) in order toassess their homologies. The amino acid sequences were deducedby means of Expasy Translate Tool available in Expasy website.

4.4. Transcriptional analysis

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 eachbiological replicate were used as a template in a 20 lL reactionmixture. Each reaction mixture contained template RNA (2 lg),2� cDNA Synthesis Master Mix (10 lL), Oligo (dT) Primer (3 lL)and AffinitySript RT/TNasa Block Enzyme Mixture (1 lL). cDNAwas diluted 1:4, and 2 lL of the dilution was used in a SYBR GreenRT-PCR. cDNA (50 ng) was used for qRT-PCR assays, carried outwith gene-specific primers (GSP) (Table 1) designed with PrimerPremier software 5.0 (Premier Biosoft International), using a DNAEngine Opticon2 thermocycler (MJ Research) and Brilliant II SYBRGreen QPCR master mix kit (Stratagene) following the manufac-turer’s instructions. Biological replicates were analyzed in dupli-cate. Specificity of amplification products was confirmed by theregistration of a single peak in melting curves of the PCR productsand the visualization of a single band on agarose gels.

Seven 10-fold dilutions of each gene fragment were used to cal-culate PCR efficiency (E) for each gene specific primer and house-keeping gene using the slope of a linear regression model:

E ¼ 10½�1=slope� ð1Þ

A GAPDH gene with constant expression levels through all fruitdevelopmental stages and tissues (Supplementary Fig. 2) was usedto normalize raw data and to calculate relative expression levels.S1 from f. chiloensis fruit (C1) was taken as the calibrator samplein this study.

Normalized Ct values were used for determining gene expres-sion variations in the samples analyzed according to the followingmodel (Pfaffl, 2001):

Relative expression ratio ðRÞ ¼ ðEtargetÞDCttarget ðcalibrator-sampleÞ

ðEhousekeepingÞDCthousekeeping ðcalibrator-sampleÞ

ð2Þ

Etarget was the PCR efficiency for a target gene; Ehousekeeping wasthe PCR efficiency for housekeeping gene GAPDH; DCttarget andDCthousekeeping corresponded to the subtraction of the calibratorCt value by the sample Ct value for each gene of interest and forthe normalizer gene, respectively.

1846 A. Salvatierra et al. / Phytochemistry 71 (2010) 1839–1847

4.5. Quantification of anthocyanins content

For anthocyanin extraction, frozen samples were powderedwith N2 in a mortar. Each biological replicate consisted of frozentissue (2.5 g), and three replicates were assessed separately. Eachsample was extracted with MeOH (12.5 mL) containing glacialHOAc (99:1, v/v) and homogenized by sonication during 10 min.Samples were then centrifuged at 16,000g for 20 min and thesupernatants were filtered through a 0.22 lm cellulose acetate fil-ter disc. The filtrate was concentrated five times in a Sep-Pak VacC18 cartridge. Concentrated extracts were stored until use at�80 �C.

The analysis of anthocyanins was carried out using an Agilent1100 series HPLC 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 man-ager software (Hewlett–Packard). Separations were performed ona reverse phase C18 analytical column (Kromasil 100, 25 cm �4.6 mm � 5 lm), equipped with a C18 precolumn (Kromasil) oper-ated at 35 �C with a flow rate of 700 lL/min. Quantifications ofanthocyanins were carried out between the wavelengths of 280and 600 nm, monitoring them at 520 nm. Elution was performedusing a gradient of solvents: 4% HCO2H in H2O (solvent A) andMeOH/H2O (95:5) (solvent B). The gradient used was 0–10 min,20% B; 10–15 min, 30% B; 15–20 min, 40% B; and 20–25 min, 100%B. Components were identified by comparison of their retentiontimes to those of authentic standards under the same analysis condi-tions. Calibration curves were prepared for pelargonidin 3-glucoside(1) and cyanidin 3-glucoside (2), and standards were purchasedfrom Sigma–Aldrich and Extrasynthèse, respectively. MeOH, glacialHOAc and HCO2H were purchased from J.T. Baker, Merck and Schar-lau, respectively. Means from two technical replicates of three inde-pendent quantifications were subjected to one-way ANOVA and LSDpairwise comparisons using Statistica 4.0 software (Statsoft Inc.).

Acknowledgments

This work has been funded by grant Proyecto PBCT Anillo Cien-cia y Tecnología (ACT-41) and the University of Talca project Frutil-la Chilena Integral. As thanks University of Talca, MeceSup andAnillo ACT-41 for Ph.D. fellowships. P.P. thanks Conicyt for aPh.D. fellowship.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.phytochem.2010.08.005.

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