Comparison of transcriptional proles of avonoid genes and anthocyanin

contents during fruit development of two botanical forms

of Fragaria chiloensis ssp. chiloensis
Ariel Salvatierra, Paula Pimentel, Maria Alejandra Moya-Leon, Peter D.S. Caligari, Raul Herrera

Instituto de Biologa Vegetal y Biotecnologa, Universidad de Talca, Casilla 747, Talca, Chile
a r t i c l e i n f o
Article history:
Received 14 December 2009
Received in revised form 27 July 2010
Available online 26 August 2010
Keywords:
Fragaria chiloensis ssp. chiloensis
Rosaceae
Fruit color
Developmental gene expression
Anthocyanins
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-glucoside
as the major one in f. chiloensis fruit. Partial gene sequences of the phenylpropanoid and avonoid bio-
synthesis pathways were isolated from the native Chilean strawberry fruits, and used to design gene-spe-
cic 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 was
concomitant with the particular progress of the transcriptional activity of genes involved in the biosyn-
thesis of avonoid 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.
2010 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. This native strawberry has two botanical forms
which are readily recognizable. F. chiloensis ssp. chiloensis f. patago-
nica is a wild plant with small fruits, red receptacle, and yellow or
red achenes. On the other hand, F. chiloensis ssp. chiloensis f. chilo-
ensis is a robust plant cultivated on a small scale that bears larger
fruits, which are composed of a pinkish-white receptacle and red
achenes when fully ripened. The latter is the maternal progenitor
of the widely cultivated strawberry, Fragaria ananassa Duch.
(Hancock et al., 1999). F. chiloensis is characterized as having a high
and particular aroma (Gonzlez et al., 2009a), large fruit size (com-
pared with all other wild species), and remarkable tolerance to
infection by Botrytis (Gonzlez et al., 2009b). These advantages,
along with other characteristics, make it an important germplasm
source both for its own development as a new exotic fruit crop, as
well as for further development of new cultivars of the commercial
strawberry (F. ananassa).
These thus offer an interesting model to study fruit pigmenta-
tion by anthocyanins, considering that the color of the white and
red botanical forms reect the presence of such phytochemicals.
Anthocyanins are natural colorants belonging to the avonoid fam-
ily of compounds, a secondary class of metabolites that are respon-
sible for the red, violet and blue colors observed in owers and
fruits in a large number of plants. Due to the broad distribution
of 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 information
concerning 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 avonoids in strawberry
0031-9422/$ - see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2010.08.005
Abbreviations: ANR, anthocyanidin reductase; ANS, anthocyanidin synthase;
CTAB, cetyltrimethylammonium bromide; CHI, chalcone isomerase; CHS, chalcone
synthase; C4H, cinnamate 4-hydroxylase; DAA, days after anthesis; DFR, dihydro-
avonol reductase; FLS, avonol synthase; F3H, avanone 3-hydroxylase; GSP, gene
specic primer; HPLC-DAD, high performance liquid chromatography-diode array
detector; LAR, leucoanthocyanidin reductase; PA, proanthocyanidin; PAL, phenyl-
alanine ammonia-lyase; qRT-PCR, quantitative reverse transcription-polymerase
chain reaction; TF, transcription factor; UFGT, UDP glucose:avonoid 3-O-glucosyl
transferase; 4CL, 4-coumarate:CoA ligase; f. chiloensis, Fragaria chiloensis ssp.
chiloensis f. chiloensis; f. patagonica, Fragaria chiloensis ssp. chiloensis f. patagonica.

Corresponding author. Tel.: +56 71 200277; fax: +56 71 200276.

E-mail address: raherre@utalca.cl (R. Herrera).
Phytochemistry 71 (2010) 18391847
Contents lists available at ScienceDirect
Phytochemistry
j our nal homepage: www. el sevi er . com/ l ocat e/ phyt ochem
fruits (Hannum, 2004). Pelargonidin 3-glucoside (1) is the predom-
inant anthocyanin in several varieties of red strawberries, usually
followed by pelargonidin 3-rutinoside and cyanidin 3-glucoside
(2) (Gil et al., 1997; Kosar et al., 2004; Tulipani et al., 2008). These
three 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 (Kosar
et al., 2004), when fully ripe. However, at the same developmental
stage, f. chiloensis had lower levels of anthocyanins, with cyanidin
3-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 botanical
forms of the native Chilean species of F. chiloensis ssp. chiloensis.
Flavonoid genes involved in anthocyanin biosynthesis exhibit
up-regulation during ripening leading to fruit pigmentation in
F. ananassa thereby establishing a positive correlation between
transcript levels of avonoid genes and anthocyanin accumulation
(Manning, 1998; Almeida et al., 2007; Carbone et al., 2009).
In the present work, cDNA fragments of genes fromphenylprop-
anoid (PAL, C4H and 4CL) and avonoid biosynthetic pathway (CHS,
CHI, F3H, DFR, ANS, UFGT, LAR and ANR) were isolated from the na-
tive Chilean strawberry. The differential transcriptional proles of
these genes were analyzed in f. chiloensis and f. patagonica
through four different fruit developmental stages and tissues by
qRT-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 quantied by HPLC-DAD.
The relationship between transcriptional proles and anthocyanin
contents present in both botanical forms is discussed in terms of
the modulation of avonoid gene expression in the determination
of the different pigmentation patterns observed in the white- and
red-fruited Chilean native strawberries.
2. Results and discussion
2.1. Transcriptional proles of genes involved in biosynthesis of
phenolic compounds in fruits at different developmental stages
The phenylpropanoid biosynthesis pathway is part of the sec-
ondary metabolismof plants, and its branches transformthe amino
acid, phenylalanine, into a variety of important phytochemicals,
including lignins, stilbenes, coumarins, salicylates, sinapate esters
and avonoids. The structural diversity of compounds derived
from phenylalanine is due to the action of enzymes and enzyme
complexes that bring about regio-specic condensation, cycliza-
tion, aromatization, hydroxylation, glycosylation, acylation, pre-
nylation, sulfation and methylation reactions (Noel et al., 2005).
In order to study the transcriptional proles of avonoid genes
in the Chilean native strawberry of red and white fruit, fragments
of genes involved in this biosynthetic pathway were isolated. These
gene fragments showed a high nucleotide homology with phenyl-
propanoid and avonoid genes from other plant species (Supple-
mentary Table 2). Suitable primers for transcriptional analysis by
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. Four
different developmental stages for F. chiloensis ssp. chiloensis fruits: (A) red fruited
botanical form patagonica; (B) whitepinkish fruited botanical form chiloensis. Bars
are equivalent to 1 cm.
Table 1
Primer sequences of the phenylpropanoid and avonoid genes and housekeeping
gene (GAPDH) used for qRT-PCR. All primers were designed from partial sequences
isolated in this work, except for FLS primer, which was designed directly from a
F. ananassa sequence found in public database (GenBank number accession
DQ087252).
Target
gene
Primers (forward/reverse) Amplicon
size (bp)
Efciency
(%)
PAL 5
0
-CAAGGGCGGCGATGCTAGTAAG-3
0
153 96.5
5
0
-CCAAGTCACCCGACGACGAGAT-3
0
C4H 5
0
-
CTGTAAGGAGGTGAAGGAGAAGAGG-
3
0
139 97.1
5
0
-CTGTTGAGCGTCCAGGATGTG-3
0
4CL 5
0
-ACTTGGTCAGGGATATGGGATG-3
0
150 95.4
5
0
-GCACCAGTTTCAGGGTCTACG-3
0
CHS 5
0
-CCGACTACTACTTTCGTATCACCA-
3
0
190 94.3
5
0
-ACTACCACCATGTCTTGTCTTGC-3
0
CHI 5
0
-TTTTCAATGGCTTTCGCTTCTG-3
0
119 94.7
5
0
-GTGACAATGATACTACCGCTGACG-
3
0
F3H 5
0
-GTGCGCCACCGTGACTACTC-3
0
157 95.4
5
0
-ATGCCTTTGTCAATGCCTCC-3
0
DFR 5
0
-GGGTGGTGTTTACATCTTCGG-3
0
156 96.5
5
0
-CTGCTTGCTCGGCTAGAGTTT-3
0
ANS 5
0
-ATCGTCATGCACATAGGCGACACC-
3
0
130 97.1
5
0
-CCTTGGGCGGCTCACAGAAAA-3
0
UFGT 5
0
-ATCGTGGCTTGACAAACAGAA-3
0
133 94.2
5
0
-TGACCACAAGAATGGAACCCTA-3
0
ANR 5
0
-CATCCAAGGCGAAGACCAT-3
0
167 96.5
5
0
-
TCATACTTAAACAACTGAGACCACC-3
0
LAR 5
0
-GGTGATGGCACGGTTAAAGC-3
0
156 100.4
5
0
-CTCCCACAGTGAAGCAAGTCC-3
0
FLS 5
0
-TTATCTTTGGGGTTAGGGCTTGAA-
3
0
162 98.1
5
0
-GAGAATGGTGAGGGCGGACA-3
0
GAPDH 5
0
-
TCCATCACTGCCACCCAGAAGACTG-3
0
132 93.0
5
0
-AGCAGGCAGAACCTTTCCGACAG-
3
0
1840 A. Salvatierra et al. / Phytochemistry 71 (2010) 18391847
qRT-PCR were thus then designed from the isolated sequences
(Table 1).
Along the fruit development process, PAL, the rst gene in-
volved in this metabolic pathway, showed similar expression level
in both botanical forms with a continuous increment in transcripts
during development (Fig. 3A). This step represents the connection
between 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.
Fig. 3. Transcriptional analysis of genes involved in avonoid biosynthesis during development of F. chiloensis ssp. chiloensis f. chiloensis (white circles) and f. patagonica
(black circles) fruits by qRT-PCR. The fruits were classied into four developmental and ripening stages as described in Section 4: S1 corresponding to C1 and P1; S2 to C2 and
P2; S3 to C3 and P3 and S4 to C4 and P4 of F. chiloensis ssp. chiloensis f. patagonica and f. chiloensis, respectively. Relative gene expression levels were normalized against
GAPDH transcript values. Values represent the average SD of three biological replicates with two technical replicates of each developmental stage.
A. Salvatierra et al. / Phytochemistry 71 (2010) 18391847 1841
The following genes in the phenylpropanoid biosynthesis pathway,
C4H and 4CL, displayed at the rst developmental stage high tran-
script levels which dropped in the following stages to basal levels
in both botanical forms (Fig. 3B and C). Similar transcript patterns
were observed in both fruits, although higher transcript levels
were recorded for F. chiloensis ssp. chiloensis f. patagonica at stage
1. Since this biosynthetic pathway is general for a large variety of
plant compounds, it does not seem to have a determining role in
the differences of fruit pigmentation recorded between the patago-
nica and chiloensis forms.
The rst committed step of the avonoid biosynthesis pathway
is mediated by CHS. All avonoids are derived from the chalcone
scaffold synthesized by this rst enzyme, which catalyzes the iter-
ative condensation and subsequent intramolecular cyclization of
three acetate units onto the p-coumaroyl-CoA end product of the
general phenylpropanoid pathway. At the initial developmental
stages, CHS transcripts exhibit a basal levels and only at the last
stage there was a signicant transcript increment in F. chiloensis
ssp. chiloensis f. chiloensis fruit (Fig. 3D). On the other hand, in F.
chiloensis ssp. chiloensis f. patagonica fruit, a particularly distinct
transcriptional prole was observed. In this case, maximum tran-
script levels were reached at the turning fruit stage (S3) when
the receptacle begins its gradual pigmentation. Then, a slight de-
crease in CHS messenger is observable at S4. Similar transcription
patterns have been reported for CHS genes in other developmental
studies carried out in several F. ananassa genotypes (Manning,
1998; Almeida et al., 2007; Carbone et al., 2009; Saud et al.,
2009). CHI stereospecically directs and greatly accelerates the
spontaneous additional cyclization of chalcones to form the avo-
noid core. Since the activity of this enzyme is tightly related to CHS
activity, it was not surprising to observe the same trend in their
messenger proles (Fig. 3E). F3H, ANS and UFGT transcript levels
share a similar pattern with CHS and CHI, but a drop in S4 stage
in F. chiloensis ssp. chiloensis f. patagonica fruit was not noted. In
the particular case of UFGT, no increment is observed inF. chiloensis
ssp. chiloensis f. chiloensis fruit at S4 stage, which is unique behav-
ior compared with that of CHS, CHI, F3H, DFR and ANS transcript
proles (Fig. 3F, H and I). UFGTs catalyze transfer of glucose from
UDP-activated sugar donor molecule to the hydroxyl group at C3
of avonols and anthocyanidins. Glycosylation increases the water
solubility of polar avonoid compounds and improves their stabil-
ity. This is an essential nal step required to stabilize anthocyani-
dins so that they can accumulate as water soluble pigments in the
vacuoles, a subcellular compartment with an acidic environment
where anthocyanins exist in their colored form. Thus UFGT is re-
garded as indispensable in anthocyanin biosynthesis rather than
simply a modifying enzyme. In Vitis vinifera cv. Shiraz, the molec-
ular analyses of structural genes involved in anthocyanin synthesis
showed that all structural genes tested, except UFGT, were ex-
pressed in most berry tissues, whereas expression of UFGT was
only detected in the berry skin and was always associated with
anthocyanin accumulation (Boss et al., 1996a; Kobayashi et al.,
2001). This step was identied as a key point in the control of berry
color, although no differences were observed in either coding or
promoter sequences of this gene between colored and white culti-
vars (Kobayashi et al., 2002). In strawberry, RNAi down-regulation
of FaGT1, a UFGT gene phylogenetically related to VvGT1, showed
signicant reduced levels of the strawberry pigments pelargonidin
3-glucoside malonate and pelargonidin 3-glucoside (1) compared
to control fruit (Griesser et al., 2008). In the light of all of above,
the dramatic difference observed in UFGT mRNA levels seems to
be playing a pivotal role in the difference of pigmentation between
both botanical forms of native Chilean strawberry.
DFR catalyzes the stereo-specic reduction of dihydroavonols
to leucoanthocyanidins, using NADPH as a cofactor (Kristiansen
and Rohde, 1991). Leucoanthocyanidins are intermediate precur-
sors for synthesis of anthocyanins and proanthocyanidins (PAs).
As in the previously discussed avonoid genes, DFR transcripts
had an increasing trend through berry development (Fig. 3G). In
ripe fruits, F. chiloensis ssp. chiloensis f. chiloensis showed lowlevels
of anthocyanins compared to F. chiloensis ssp. chiloensis f. patago-
nica and F. ananassa cv. Chandler, but catechin and higher levels
of ellagic acid were detected in EtOAc soluble fractions (Simirgiotis
et al., 2009). Despite the scarcity of colored anthocyanins inF. chilo-
ensis ssp. chiloensis f. chiloensis fruit, the presence of tannin com-
pounds in ripe fruit could explain the pattern depicted by DFR
which showed equivalent levels in S4 stage in both botanical
forms.
Ultimately, the anthocyanin-related genes of the native Chilean
strawberry, as reported in developmental studies in other fruits
such 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), showed
transcriptional proles that correlate positively with anthocyanin
accumulation throughout fruit development. In addition, compar-
ative transcriptional analysis carried out between fruits of both
botanical forms of F. chiloensis established higher transcript levels
of avonoid genes leading to anthocyanins (except by DFR) in the
red-fruited F. chiloensis ssp. chiloensis f. patagonica than in the
white-fruited F. chiloensis ssp. chiloensis f. chiloensis at ripe stage,
agreeing with similar comparative studies in species with fruits
contrasting 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 and
ANR, are competitive branches for anthocyanin biosynthesis in the
avonoid pathway. FLS, LAR and ANR showed a decreasing tran-
scriptional prole as fruit ripening proceeds (Fig. 3JL). Transgenic
anti-sense FLS owers 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 competition
among DFR (for anthocyanin production) and FLS (for avonol pro-
duction). In fruits of both native strawberries, FLS transcripts are
present at high levels in the S1 stage and then they decay steadily
to very low levels in S4 stage (Fig. 3J), showing an opposite trend to
what was observed with the anthocyanin-related genes, conrm-
ing the competition between these two pathways in this fruit.
High levels of avan 3-ol accumulated at early stages, which
could protect immature berries both from feeding by animals
and pest insects, and from pathogen attack (Gould and Lister,
2006). During ripening of commercial strawberry fruit, the concen-
tration of avan 3-ols, constituents of PAs, decreased continuously
while anthocyanin content increased, giving the fruit an attractive
appearance. Thus, there is an obvious redirection of avonoid bio-
synthesis from avan 3-ol to anthocyanin formation during the
complex developmental process of fruit ripening (Halbwirth
et 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 proles decrease from S1 to S4 stages as in commercial
strawberry, but a notable point is that F. chiloensis ssp. chiloensis
f. chiloensis showed higher levels than F. chiloensis ssp. chiloensis
f. patagonica of ANR transcripts (Fig. 3K and L). Since quantication
of these metabolites has not been reported for native Chilean
strawberry, the real impact of this particular behavior of ANR is still
unclear.
The temporal transcriptional analysis performed in this study
evidenced a coordinated up-regulation of genes associated to
anthocyanin biosynthesis, but at different scale, in fruits from both
botanical forms. These results suggest that the expression of struc-
1842 A. Salvatierra et al. / Phytochemistry 71 (2010) 18391847
tural genes was synchronously regulated in a form-specic man-
ner. Similar patterns have been reported in other species and the
regulation of structural genes by transcription factors has been
widely discussed (Palapol et al., 2009; Yuan et al., 2009). In red-
eshed apple, the transcript levels of anthocyanin-related genes
were higher throughout development than those found in white-
eshed 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
transcriptional proles associated to developmental stages, where
MYB proteins controlling the expression of PA related genes (e.g.,
VvMYBPA1, VvMYB5a and VvMYB5b) were up-regulated in unripe
stages 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 the
need of a complex network of regulatory genes in order to modu-
late the avonoid structural genes through fruit ripening. Cur-
rently, the regulation of expression of structural genes on
Fig. 4. Spatial transcriptional analysis of genes involved in avonoid biosynthesis by qRT-PCR. The gene expression in ower, leaf, runner and root from F. chiloensis ssp.
chiloensis f. chiloensis (white bars) and f. patagonica (black bars) was assessed in comparison with that of F. chiloensis ssp. chiloensis f. chiloensis fruit on stage 1 (C1). Relative
gene expression levels were normalized against GAPDH transcript values. Values represent the average SD of three biological replicates with two technical replicates of each
tissue.
A. Salvatierra et al. / Phytochemistry 71 (2010) 18391847 1843
avonoid pathway by transcription factors is a matter of analysis in
our ongoing work.
2.2. Transcriptional proles of genes involved in biosynthesis of
phenolic compounds in tissues
The spatial transcriptional analysis detected expression of genes
involved in phenylpropanoid and avonoid biosynthesis pathways
in all tissues assessed. A greater difference in transcript levels for
all genes from the phenylpropanoid biosynthesis pathway was ob-
served (Fig. 4). For PAL, the higher differences in transcript level
was observed for ower, leaf and roots (Fig. 4A). Roots and runners
exhibited higher mRNA levels of C4H and 4CL than the other tis-
sues, since their developmental dynamics involve continuous
growth of these woody structures and the concomitant synthesis
of lignin monomers (Fig. 4B and C).
Runners of F. chiloensis ssp. chiloensis f. patagonica present a red
pigmentation. In this case, avonoid biosynthesis pathway genes
leading 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 red
runners of F. chiloensis ssp. chiloensis f. patagonica could be a result
of the incapacity to detect a putative tissue-specic ANS paralogous
gene. The most prominent difference was noted at the level of the
UFGT messengers, where quantities of mRNAs of this gene were
three times higher in red runners relative to green ones. Again, in
these 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. 4DI).
Roots of F. chiloensis ssp. chiloensis f. chiloensis have a reddish
hue and the situation described above occurs in an opposite way,
since F. chiloensis ssp. chiloensis f. patagonica roots are green, but
on a minor scale (Fig. 4DI). In addition, DFR, LAR and ANR tran-
scripts showed higher levels inF. chiloensis ssp. chiloensis f. chiloen-
sis which could suggest a redirection in the avonoid pathway to
the synthesis of PAs in this tissue (Fig. 4K and L).
Flowers of both botanical forms gave highest expression of FLS
among 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 avonols in planta since their biosynthesis is
proposed as key point for biological events that happen in the
ower 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 high
transcript levels of PA genes (Fig. 4K and L); this fact could explain
the enhanced tolerance to Botrytis infection (Gonzlez 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 avonoid gene
expression 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 sustaining
successful 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; Ryan
and 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; Carbone
et al., 2009; Saud et al., 2009). The most commonly occurring
anthocyanins in strawberry are cyanidin and pelargonidin deriva-
tives. The main pigment in cultivated strawberries has been iden-
tied as pelargonidin 3-glucoside (1) (Gil et al., 1997; Mtt-
Riihinen et al., 2004) and the presence of cyanidin 3-glucoside
(2) is widely documented (Nyman and Kumpulainen, 2001; Wang
et 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 in
fruits of both botanical forms, with a maximum level at S3 stage;
nevertheless, a higher concentration of this pigment was observed
in fruits fromF. chiloensis ssp. chiloensis f. patagonica. The presence
of cyanidin 3-glucoside (2) in the unripe stages could be explained
by the early pigmentation of achenes seen in fruits of both botan-
ical forms. Pelargonidin 3-glucoside (1), the typical receptacle
anthocyanin, was detected in F. chiloensis ssp. chiloensis f. patago-
nica at S3 stage, with a maximum level at S4 (12.5 lg g
1
), while
this anthocyanin was only detected at a low level in the S4 stage
in F. chiloensis ssp. chiloensis f. chiloensis (4.0 lg g
1
), representing
only one third of pelargonidin 3-glucoside (1) found in ripe fruits of
f. 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 found
only 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 the
fact that achenes are the most colored tissues in the whole fruit
of this form.
In addition, a different anthocyanin ratio is evident between the
ripe fruits of this native strawberry, since F. chiloensis ssp. chiloensis
f. patagonica showed a pelargonidin 3-glucoside (1) content 1.88
times higher than cyanidin 3-glucoside (2) and in F. chiloensis
ssp. chiloensis f. chiloensis, cyanidin 3-glucoside (2) content was
Table 2
Changes 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 0.08a
S2 nd 4.98 0.50a
S3 nd 7.80 0.17a
S4 3.95 0.13a 5.86 1.18a
f. patagonica S1 nd 4.66 0.66b
S2 nd 6.55 0.64b
S3 6.42 0.73 11.84 2.20b
S4 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 signicant differences
between 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.
1844 A. Salvatierra et al. / Phytochemistry 71 (2010) 18391847
1.48 times higher than pelargonidin 3-glucoside (1) according to
our extraction procedures and HPLC-DAD measurements. This par-
ticular trait could be useful for distinguishing the botanical forms
by mean of a chemotaxonomic approach.
3. Conclusions
The current workestablishes for the rst time a clear relationship
betweenfruit pigmentation, transcriptional patterns of phenylprop-
anoid and avonoid biosynthesis pathways and accumulation of
representative anthocyanins of both botanical forms of F. chiloensis
ssp. chiloensis during their fruit developmental process. The results
indicate the existence of differential transcriptional patterns in the
anthocyanin-related genes during fruit ripening among F. chiloensis
ssp. 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-
berrywhere UFGT transcript levels seemtobe critical for fruit antho-
cyanin accumulation.
Moreover, the differences are not a reduction in the quantity of
anthocyanin but also in the quality of them. Further studies are re-
quired for elucidating the role of transcription factors in the spatial
and developmental regulation of structural genes responsible for
phenolic compounds production in order to explain the differences
in their gene patterns observed among the two native Chilean
strawberries.
4. Experimental
4.1. Plant material
Fruit and plant tissues (ower, leaf, runner and root) of F. chilo-
ensis ssp. chiloensis f. chiloensis were obtained from a commercial
plantation in Contulmo, Bio-Bio Region, Chile (latitude 38 04
0
8.6
00
S; longitude 73 14
0
2.96
00
W). Fruits were harvested in 2006
and 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 Chilln, Bo-Bo Region, Chile (S 36 54
0
58.35
00
, W 71 25
0
18.19
00
). Fruits were harvested during the 2006 and 2007 seasons
(DecemberJanuary).
Fruits of f. chiloensis were classied into four development and
ripening stages, according to Figueroa et al. (2008), based on
weight and color of the receptacle and achene (Fig. 2): C1, small
fruit with green receptacle and green achenes (7 days after anthe-
sis, 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).
Similarly, fruits of f. patagonica were classied for the rst time
into four development and ripening stages: P1, green receptacle
and green achenes (7 daa); P2, green receptacle and red achenes
(14 daa); P3, turning stage, pink receptacle and red achenes (21
daa); and P4, ripe fruit with red receptacle and red achenes (28
daa). After classication, samples were immediately frozen with li-
quid N
2
and stored at 80 C until needed.
4.2. RNA extraction
Three independent total RNA samples were isolated from pools
of fruits prepared for each developmental stage, and also from
ower, leaf, runner and root tissue using the CTAB method with
minor modications (Chang et al., 1993). A DNAse treatment
(Invitrogen) was carried out with the aim to remove contaminant
genomic DNA. Integrity of isolated RNAs was checked on agarose
gels stained with ethydium bromide and their concentration mea-
sured in a ND-1000 UV spectrophotometer (Nanodrop
Technologies).
4.3. Cloning of partial sequences of avonoid genes
Primers for amplifying partial sequences of genes involved in
the phenylpropanoid (PAL, C4H and 4CL) and avonoid biosynthe-
sis pathway (CHS, CHI, F3H, DFR, ANS, UFGT, LAR and ANR) (Supple-
mentary Table 1) were designed from conserved nucleotide
regions identied 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 plant
tissue were synthesized from total RNA (1 lg) using the Thermo-
Script RT-PCR System kit (Invitrogen), according to manufacturers
instructions. Gene fragments were amplied from a fruit cDNA
pool including all developmental stages. Amplicons ranging from
200 to 500 bp were cloned, sequenced and deduced amino acid se-
quences were analyzed by BLASTp (Altschul et al., 1997) in order to
assess their homologies. The amino acid sequences were deduced
by means of Expasy Translate Tool available in Expasy website.
4.4. Transcriptional analysis
For quantitative Real-Time reverse transcription PCR (qRT-PCR)
assays, rst-strand cDNA synthesis was performed using an Afn-
ityScript QPCR cDNA Synthesis kit (Stratagene, Agilent Technolo-
gies). For cDNA synthesis, total RNAs isolated from each
biological replicate were used as a template in a 20 lL reaction
mixture. Each reaction mixture contained template RNA (2 lg),
2 cDNA Synthesis Master Mix (10 lL), Oligo (dT) Primer (3 lL)
and AfnitySript RT/TNasa Block Enzyme Mixture (1 lL). cDNA
was diluted 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-specic primers (GSP) (Table 1) designed with Primer
Premier software 5.0 (Premier Biosoft International), using a DNA
Engine Opticon2 thermocycler (MJ Research) and Brilliant II SYBR
Green QPCR master mix kit (Stratagene) following the manufac-
turers instructions. Biological replicates were analyzed in dupli-
cate. Specicity of amplication products was conrmed by the
registration of a single peak in melting curves of the PCR products
and the visualization of a single band on agarose gels.
Seven 10-fold dilutions of each gene fragment were used to cal-
culate PCR efciency (E) for each gene specic primer and house-
keeping gene using the slope of a linear regression model:
E 10
1=slope
1
A GAPDH gene with constant expression levels through all fruit
developmental stages and tissues (Supplementary Fig. 2) was used
to normalize raw data and to calculate relative expression levels.
S1 from f. chiloensis fruit (C1) was taken as the calibrator sample
in this study.
Normalized Ct values were used for determining gene expres-
sion variations in the samples analyzed according to the following
model (Pfaf, 2001):
Relative expression ratio R
E
target

DCt
target calibrator-sample
E
housekeeping

DCt
housekeeping calibrator-sample
2
E
target
was the PCR efciency for a target gene; E
housekeeping
was
the PCR efciency for housekeeping gene GAPDH; DCt
target
and
DCt
housekeeping
corresponded to the subtraction of the calibrator
Ct value by the sample Ct value for each gene of interest and for
the normalizer gene, respectively.
A. Salvatierra et al. / Phytochemistry 71 (2010) 18391847 1845
4.5. Quantication of anthocyanins content
For anthocyanin extraction, frozen samples were powdered
with N
2
in a mortar. Each biological replicate consisted of frozen
tissue (2.5 g), and three replicates were assessed separately. Each
sample was extracted with MeOH (12.5 mL) containing glacial
HOAc (99:1, v/v) and homogenized by sonication during 10 min.
Samples were then centrifuged at 16,000g for 20 min and the
supernatants were ltered through a 0.22 lm cellulose acetate l-
ter disc. The ltrate was concentrated ve times in a Sep-Pak Vac
C18 cartridge. Concentrated extracts were stored until use at
80 C.
The analysis of anthocyanins was carried out using an Agilent
1100 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 (HewlettPackard). Separations were performed on
a 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 ow rate of 700 lL/min. Quantications of
anthocyanins were carried out between the wavelengths of 280
and 600 nm, monitoring them at 520 nm. Elution was performed
using a gradient of solvents: 4% HCO
2
H in H
2
O (solvent A) and
MeOH/H
2
O (95:5) (solvent B). The gradient used was 010 min,
20% B; 1015 min, 30% B; 1520 min, 40% B; and 2025 min, 100%
B. Components were identied by comparison of their retention
times tothose of authentic standards under the sameanalysis condi-
tions. Calibrationcurves were preparedfor pelargonidin3-glucoside
(1) and cyanidin 3-glucoside (2), and standards were purchased
from SigmaAldrich and Extrasynthse, respectively. MeOH, glacial
HOAc and HCO
2
Hwere purchased fromJ.T. Baker, Merck and Schar-
lau, respectively. Means fromtwo technical replicates of three inde-
pendent quantications were subjectedto one-way ANOVAand LSD
pairwise comparisons using Statistica 4.0 software (Statsoft Inc.).
Acknowledgments
This work has been funded by grant Proyecto PBCT Anillo Cien-
cia y Tecnologa (ACT-41) and the University of Talca project Frutil-
la Chilena Integral. As thanks University of Talca, MeceSup and
Anillo ACT-41 for Ph.D. fellowships. P.P. thanks Conicyt for a
Ph.D. fellowship.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.phytochem.2010.08.005.
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