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Increased accumulation of anthocyanins in Fragaria chiloensis fruits

by transient suppression of FcMYB1 gene

Ariel Salvatierra
, Paula Pimentel
, Mara Alejandra Moya-Len
, Ral Herrera
Instituto de Biologa Vegetal y Biotecnologa, Universidad de Talca, Casilla 747, Talca, Chile
Centro de Estudios Avanzados en Fruticultura (CEAF), Av. Salamanca s/n, Los Choapinos, Rengo, Chile
a r t i c l e i n f o
Article history:
Received 13 March 2012
Received in revised form 30 November 2012
Accepted 19 February 2013
Available online 20 March 2013
White strawberry
Fragaria chiloensis ssp. chiloensis
Fruit color
RNAi silencing
Flavan 3-ol
a b s t r a c t
Anthocyanins and proanthocyanidins (PAs), avonoid-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 owers suppressed avonoid-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 avan 3-ols. Altogether, these results indicate a role for FcMYB1 in
regulation of the branching-point of the anthocyanin/PA biosynthesis determining the discolored pheno-
type of the white Chilean strawberry fruit.
2013 Elsevier Ltd. All rights reserved.
1. Introduction
The native Chilean strawberry (Fragaria chiloensis (L.) Mill. ssp.
chiloensis Staudt) is an octoploid species (2n = 8x = 56) of the Ros-
aceae family. Two botanical forms are recognizable in wild lands.
F. chiloensis ssp. chiloensis f. patagonica is a plant with small
fruits, red receptacle, and yellow or red achenes. On the other
hand, F. chiloensis ssp. chiloensis f. chiloensis is a robust plant cul-
tivated on a small scale that bears larger fruits, which are com-
posed of a pinkish-white receptacle and red achenes when fully
ripe. This strawberry is characterized as having a high and partic-
ular aroma (Gonzlez et al., 2009a), large fruit size (compared
with all other wild species) and a remarkable tolerance to Botrytis
infection (Gonzlez et al., 2009b). However, the fruit depigmenta-
tion of the white native strawberry constitutes its most character-
istic trait.
In the Fragaria genus, fruit color is determined by accumulation
of anthocyanins, the most abundant avonoid-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
avonoid 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 avonoid 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 identied
0031-9422/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
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,
dihydroavonol reductase; daa, days after anthesis; FLS, avonol synthase; F3H,
avanone 3-hydroxylase; GSP, gene-specic primer; HPLC-DAD, high performance
liquid cromatography-diode array detector; LAR, leucoanthocyanidin reductase;
LSD, Least Signicant Differences; PA, proanthocyanidin; qRT-PCR, quantitative
real-time PCR; RACE, Rapid Amplication cDNA Extremes; RNAi, RNA interference;
TF, transcription factor; UFGT, UDP glucose:avonoid 3-O-glucosyl transferase.

Corresponding author. Tel.: +56 (71) 200277; fax: +56 (71) 200276.
E-mail address: (R. Herrera).
Both authors contributed equally to this work.
Present address: Centro de Estudios Avanzados en Fruticultura (CEAF), Av.
Salamanca s/n, Los Choapinos, Rengo, Chile.
Phytochemistry 90 (2013) 2536
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in the C-terminal, and it has been suggested that members of the
same subgroup have similar functions (Dubos et al., 2010; Stracke
et al., 2001). Initially, action of the MYB family in this branch of
secondary metabolism has been reported as related to the biosyn-
thesis of anthocyanins in grain (Paz-Ares et al., 1987). In A. thaliana,
O. sativa and V. vinifera, several MYBs belonging to subfamily R2R3
have been identied 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 identied in plants (Kagale and
Rozwadowski, 2011). AtMYB4, ortholog of AmMYB308, was the rst
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 avonoid compounds in tobacco owers
(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 nal steps of avonoid 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 avonoid levels and
increased carotenoid levels in tomato (Mahjoub et al., 2009), while
in tobacco the avonoid 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 difculties 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 avan 3-ols
(Griesser et al., 2008). Currently, RNAi is widely used in plant bio-
technology because it is a fast, simple, and a sequence-specic 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 modied 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 avonoid 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 avan 3-ol monomers, (+)-catechin (3)
and ()-epicatechin (4; Fig. 1), were analyzed by HPLC-DAD.
2. Results and discussion
2.1. Isolation and characterization of full-length FcMYB1 cDNA
A partial gene fragment of 570 bp was amplied 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 specic 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
untranslated region (UTR) of both genes
was identical, at least to the extent available for FaMYB1. Simi-
larly, FcMYB1 3
-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 identied in several proteins such
as AUX-IAA, ERF and TFIIIA zinc nger type. The FcMYB1 protein se-
quence matches the motif dened 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 rst Arabidopsis MYB
described as transcriptional repressor (Jin et al., 2000). Alignment
Fig. 1. Structures of compounds analyzed in this study. (1) Cyanidin 3-glucoside;
(2) pelargonidin 3-glucoside; (3) (+)-catechin; (4) ()-epicatechin.
26 A. Salvatierra et al. / Phytochemistry 90 (2013) 2536
of amino acid sequences grouped FcMYB1in the clade of R2R3-MYB
repressors. These proteins shares the LXLXL domain dened 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 FcMYB1and 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 avonoid biosynthesis.
2.2. FcMYB1 temporal and organ-specic expression analysis
The expression prole 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-specic and temporal expression patterns were analyzed
by qRT-PCR. RNA was isolated from fruit at different developmen-
tal stages and other tissues (ower, leaf, runner and root). Compar-
ative analysis of temporal FcMYB1gene expression in both white
and commercial strawberry fruits showed differences, in transcript
level and in expression prole trend (Fig. 3A). A sustained increase
in mRNA levels was observed for this TF concomitant with the pro-
gress of fruit development and ripening of white strawberry fruit.
On the other hand, levels of FcMYB1 transcripts decreased in
F. ananassa cv. Camarosa, while exhibiting a slight rise at stage
4 (Fig. 3A). A similar expression prole 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. 3A). Since Aharoni et al. (2001) demonstrated that over-
expression of FaMYB1 in tobacco plants suppressed ower pigmen-
tation, the high level of transcripts of this TF detected in white
Chilean strawberry may contribute to the repression described in
Fig. 2. Phylogenic relationships of selected MYB proteins from different plant species. The scale represents the number of substitutions per site and the numbers
next to the nodes correspond to bootstrap values of 1000 replicates. The tree was rooted using the human c-MYB protein as outgroup. TFs have been reported
with functions in development of stamen or trichomes (sky blue circles), biosynthesis of phenylpropanoids/lignin (green circles), anthocyanins (red circles),
proanthocyanidins (orange circles) and avonols (purple circles). Accession numbers are shown in parentheses: AtMYB4 (BAA21619), AmMYB308 (JQ0960),
VvMYB4b (ACN94269), ZmMYB42 (Q2A700), AtMYB3 (BAA21618), AtMYB6 (Q38851), AmMYB330 (JQ0957), AtMYB32 (O49608), AtMYB7 (NP_179263), FcMYB1
(GQ867222), FaMYB1 (AAK84064), VvMYBPA1 (CAJ90831), AtMYB111 (NP_199744), AtMYB12 (NP_182268), AtMYB11 (NP_191820), AtMYB5 (NP_187963),
AtMYB123 (Q9FJA2), AtMYB82 (Q9LTF7), MdMYB8 (ABB84756), MdMYBA (BAF80582), MdMYB10 (ABB84753), PaMYB10 (ACA04854), GhMYB10 (AAK19615),
PhAn2 (AAF66727), NtAn2 (ACO52470), LeANT1 (AAQ55181), VvMYBA (ABB87013), AtMYB113 (Q9FNV9), AtMYB75 (Q9FE25), AtMYB90 (Q9ZTC3), AtMYB114
(Q9FNV8) and c-MYB (X52125).
A. Salvatierra et al. / Phytochemistry 90 (2013) 2536 27
avonoid biosynthesis genes and anthocyanin accumulation, and
could explain the origin of pigment-decient phenotype character-
istic of its fruit.
MYBs are involved in coordinating structural genes of the avo-
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 avonoid structural genes in determining
pigment-decient 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-specic
expression analysis established low levels of FcMYB1 mRNA in
ower, leaf, runner and roots of F. chiloensis compared to fruit
(Fig. 3B).
2.3. FcMYB1 transient gene silencing in F. chiloensis fruits
FcMYB1 was suppressed by RNA interference and the
expression of genes from the avonoid 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. 4A and B).
FcMYB1 transcript levels were assessed by qRT-PCR in agroinl-
trated fruits. Considering that the RNAi mechanism action and the
agroinltration technique involve a signicant 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 agroinltration events were made with pH12-FcMYB1
interference construction and four lines showed a signicant
reduction in FcMYB1 transcript levels compared to the expression
average in control fruit (Fig. 5).
The expression of genes involved in the avonoid biosynthe-
sis pathway was analyzed in four FcMYB1 suppressed lines
(pH12-FcMYB1.4 to .7) (Fig. 6). The expression patterns so ob-
tained dene three different groups: unaffected genes (CHS), re-
pressed genes (CHI, F3H, DFR, LAR and ANR) and over-expressed
genes (ANS and UFGT). The reduced transcript level of CHS has
been reported to cause loss of pigmentation in affected plant tis-
sue (Fukusaki et al., 2004; Koseki et al., 2005; Hoffmann et al.,
2006; Lunkenbein et al., 2006). CHS was the only gene whose
expression remained almost unaffected in fruits of FcMYB1 sup-
pressed lines (Fig. 6A). Similarly, expression of this gene was
not altered in transgenic tobacco lines over expressing
FaMYB1, despite the change in the color in owers (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 avonoid-derived compounds remains unaltered.
CHI, F3H and DFR genes exhibited a moderate reduction in
their transcript levels in FcMYB1 suppressed lines (Fig. 6BD).
Apparently, the expression levels of the intermediate genes of
the avonoid pathway seem not to be crucial for appearance
of pigmentation. LAR and ANR are enzymes responsible for the
synthesis of avan-3-ols from leucoanthocyanidins and anthocy-
anidins, respectively, and constitute a dichotomous point at the
end of the avonoid 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-
specic manner (Bogs et al., 2005). However, a decrease in the
transcript levels of these PA-related genes, as well as PAs and
avan 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. 6G and H). This level
of suppression on PA-related genes may contribute to the in-
creased pigmentation of these fruits. Similarly, owers of tobacco
lines over-expressing different apple ANR isoforms (i.e. MdANR1,
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-specic analysis of the expression in different tissues of F. chiloensis.
Vegetative tissues were ower (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).
28 A. Salvatierra et al. / Phytochemistry 90 (2013) 2536
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 ower 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 owers 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 ndings suggest a blockage at the transcriptional level in the
PA biosynthesis pathway, which may redirect precursors of
avonoids to the biosynthesis of anthocyanin pigments in white
Chilean strawberry fruits in FcMYB1 suppressed lines.
The nal steps of anthocyanin pigment biosynthesis are given
by the enzymatic activities of ANS and UFGT, which synthesize
anthocyanidins (from avan 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
Fig. 4. Phenotype of agroinltrated fruits. (A) Fruit injected with the control construction. (B) Fruit injected with the interference construction pH12-FcMYB1. Fruits were
harvested at the mature stage on day 14 post-injection (28 days after anthesis).
Fig. 5. FcMYB1 gene expression level of agroinltrated 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
agroinltrated lines with FcMYB1 expression levels signicantly 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.
A. Salvatierra et al. / Phytochemistry 90 (2013) 2536 29
Fig. 6. FcMYB1 suppression effect in gene expression of avonoid biosynthesis pathway. The analysis of expression was determined by qRT-PCR and calibrated against the
average of expression value of each gene in three control events (black line). The values and error bars represent the mean and standard deviation of three technical replicates
of each agroinltration event, respectively.
30 A. Salvatierra et al. / Phytochemistry 90 (2013) 2536
ANS (Debes et al., 2011). Additionally, the role of ANS in the avo-
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 avonoid 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. 6E). This result could be
related to the decrease in transcript levels of PA genes, since a tight
dependent relationship between expression of ANS and ANR has
been described in this branch point. Thus, an increase in expression
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 avonoid pre-
cursors towards accumulation of anthocyanin pigments at the ex-
pense of avan 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 avonoid biosynthesis pathway are expressed
during fruit development, but only UFGT mRNAs are detected in
the skin of red cultivars after vraison, demonstrating this to be
critical for the biosynthesis of anthocyanins (Boss et al., 1996a,b).
A white mutant of Malay apple (Syzygium malaccense) has no
detectable levels of anthocyanin, UFGT activity and expression in
its skin, contrary to the considerable expression level of several
other genes in the same biosynthetic pathways (Kotepong et al.,
2011), suggesting that the white mutation may be correlated with
a failure of the last step in anthocyanin synthesis. Moreover, down-
regulation of FaGT1 generated strawberry fruits with signicantly
reduced levels of pelargonidin and increased concentration of a-
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. 6F). This fruit
exhibited evident damage attributable to the agroinltration tech-
nique, which could explain its unexpected behavior in transcript
accumulation. By contrast, highest expression of UFGT was de-
tected in pH12-FcMYB1i.5, which is coincident with the lowest
expression level of PA related genes (Fig. 6G and H) and higher
ANS expression (Fig. 6E). The interrelationship between these
genes (i.e. LAR/ANR and ANS/UFGT) and the fate of precursors of a-
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 owers 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 FaMYB1repressed 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
conrms and validates the biological role of FcMYB1 in pigmenta-
tion of fruit tissue where both branches of the avonoid 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 avonoid 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. Quantication of anthocyanins and avan 3-ols contents
Contents of anthocyanins and avan 3-ols in agroinltrated
fruits were assessed by analyzing the suppression effect of FcMYB1
transcript accumulation on avonoid compound accumulation.
Along development and ripening of white strawberry fruits, there
is an opposite accumulation trend of nal avonoid metabolites
(Fig. 7). While the amounts of avan 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 signicant variation in the content
of cyanidin 3-glucoside (1) as compared to control fruit (Fig. 8A).
Analyses of metabolites showed a greater accumulation of pelarg-
onidin 3-glucoside (2) concomitant with a decrease in FcMYB1
transcript accumulation (Fig. 8B). This fact establishes the exis-
tence of a direct relationship between the 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 avan 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 avan 3-ols were not detected (Fig. 8C),
which is related to the severe reduction in the transcript levels of
genes involved in biosynthesis of PA. Conversely, silencing
glycosyltransferase FaGT1 reduced anthocyanins levels and
A. Salvatierra et al. / Phytochemistry 90 (2013) 2536 31
Fig. 7. Accumulation patterns of nal avonoid compounds during development of Fragaria chiloensis ssp. chiloensis f. chiloensis fruits. Anthocyanins (cyanidin 3-glucoside
(1) and pelargonidin 3-glucoside (2)) and avan 3-ols ((+)-catechin (3) and ()-epicatechin (4)) were determined by HPLC-DAD from a pool of fruits of each developmental
stage. Values are the mean standard deviation of three independent biological replicates and two technical replicates.
Fig. 8. Flavonoid metabolite levels (lg g
fresh weight) in agroinltrated fruits collected at the ripe stage. (A) Cyanidin 3-glucoside (1) content. (B) Pelargonidin 3-glucoside
(2) content. Values and error bars represent the mean and standard deviation of three technical replicates of a single anthocyanin extraction. Different letters represent
signicant 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 avan 3-ols. Different letters in the same column represent signicantly different values with p < 0.05 according to LSD multiple comparison Fisher test. nd, Not
32 A. Salvatierra et al. / Phytochemistry 90 (2013) 2536
increased avan 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
esh 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) identied 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 eshed (e.g. Red Field) and red skinned (e.g. Pacic
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 esh and skin.
Our results show that the release of the transcriptional repres-
sion exerted by FcMYB1 over ANS channeled the ux of intermedi-
ate metabolites of the avonoid biosynthesis pathway into
anthocyanin pigments associated with the receptacle (i.e., pelarg-
onidin 3-glucoside (2)). Apparently, this redirection in metabolic
ux drained precursors (avan 3,4-diols and anthocyanidins) for
PA production resulting in undetectable levels of PA monomers
in fruits of FcMYB1 suppressed lines (Fig. 8C). The lack of avan
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 avonoid
precursors were redirected to the anthocyanin biosynthesis at ex-
pense of PA production in white Chilean strawberries owing to the
suppression of this transcriptional repressor on accumulation of
anthocyanins. More recently, a group of three different TFs have
been reported to be involved controlling PA synthesis in F. anan-
assa and the interaction of MYB1 to bHLH3D has been suggested
(Schaart et al., 2012).
3. Conclusions
In this study, the FcMYB1 gene was isolated from the native
white Chilean strawberry fruit (F. chiloensis ssp. chiloensis f. chilo-
ensis) and its biological role was assessed in fruits attached to
the plant. An aim was to obtain a reduction in the transcript level
of FcMYB1 in white strawberry fruit using the agroinltration 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 avonoid 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 avan 3-ol content. The results showed that FcMYB1 exerts a
regulatory effect at transcriptional level in a biosynthesis avo-
noid-derived compounds aimed at promoting anthocyanin produc-
tion at the expenses of avan 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 avonoid 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 avan 3-ols, it can be hypothesized
that severe down-regulation of LAR and ANR was a side-effect
driven by redirection of ux of avonoid precursors towards syn-
thesis of anthocyanins in white Chilean strawberry fruits. This re-
sult highlights the possibility of using this TF to direct the ux of
avonoid 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 3804
S; longitude
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 eld from Chanco, Maule Re-
gion, Chile (latitude 3541
S; longitude 7232
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 agroinltration
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, agroinltrated fruits,
and from ower, leaf, runner and root tissue using the CTAB method
with minor modications (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
Specic 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
MYBorf rev 5
). 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 specic primers were
designed (MYB1race fwd 5
; MYB1race rev 5
) for RACE reaction to obtain full length FcMYB1 sequence. Total
RNA (5 lg) from a mix of F. chiloensis fruit was used for RACE-
Ready cDNAs (BD Biosciences, Clontech) following the users man-
ual. The 5
and 3
specic 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 Amplication 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
A. Salvatierra et al. / Phytochemistry 90 (2013) 2536 33
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 amplied by PCR using the primers MYBdir fwd
, and MYBdir 5
, 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 nal 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-specic primers (fwd MYBdir) and M13
(M13 rev 5
). For suppressing FcMYB1
expression, pHELLSGATE12 (Helliwell and Waterhouse, 2003)
was used as the destination vector. Recombination was performed
using Gateway

LR Clonase II Enzyme Mix kit (Invitrogen)

according to the manufacturers instructions and using equivalent
amounts of entry vector (160 ng/ul) and destination vector
(160 ng/ul). Transformed bacteria (One Shot

TOP10 chemically
Competent E. coli (Invitrogen)) were plated on LB agar supple-
mented with Spectinomycin (PhytoTechnology Laboratories) at a
nal concentration of 100 lg/ml. Resistant colonies were analyzed
using restriction enzymes XhoI (Promega) and XbaI (Promega) to
assess the presence of the gene fragment in the construction. As
empty vector control, pHELLSGATE12-GUSi construction was car-
ried out using a GUS gene fragment.
4.5. White strawberry fruit transfection by agroinltration
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 nal con-
centration of 100 lg/ml of each antibiotic. Resistant colonies were
analyzed by PCR for the presence of FcMYB1 gene fragment using
FcMYB1 specic primers. A positive colony was cultured in selec-
tive YM (100 ml) and incubated at 30 C until an O.D.
0.6 and 0.8. Then, the culture was centrifuged for 3 min at 1620g
in a swinging bucket centrifuge (5804 R centrifuge, Eppendorf).
The pellet obtained was resuspended in one third of its original
culture volume, with MMA agroinltration medium (MS salt
4.32 mg/l; 10 mM MES; 20 g/l sucrose; 200 mM acetosyringone).
Agroinltration 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). Agroinltrated 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, 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 template in a 20 ll reaction mix-
ture. Each reaction mixture contained template RNA (2 lg), 2x
cDNA Synthesis Master Mix (10 ll), Oligo (dT) Primer (3 ll) and
AfnityScript RT/TNasa Block Enzyme Mixture (1 ll). cDNA was di-
luted 1:4, and 2 ll of the dilution was used in a SYBR Green RT-
PCR. cDNA (50 ng) was used for qRT-PCR assays, carried out with
gene-specic primers: qPCR-MYB1 fwd 5
; qPCR-MYB1 rev 5
; and
for structural genes of avonoid pathway see Salvatierra et al.
(2010), using a DNA Engine Opticon2 thermocycler (MJ Research)
and Brilliant II SYBR Green QPCR master mix kit (Stratagene) fol-
lowing the manufacturers instructions. Biological replicates were
analyzed in duplicate. Specicity of amplication products was
conrmed 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
efciency (E) for each specic 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 (Pfaf, 2001).
4.7. Chemical analysis
Quantication of anthocyanins (cyanidin 3-glucoside (1) and
pelargonidin 3-glucoside (2)) and avan 3-ols ((+)-catechin (3)
and ()-epicatechin (4)) was performed after construction of
standard calibration curves. Pelargonidin 3-glucoside (2) was
purchased as calistephin chloride (Extrasynthse) and cyanidin
3-glucoside (1) as kuromanin chloride (SigmaAldrich). (+)-Catechin
(3) and ()-epicatechin (4) were purchased from Extrasynthse.
Anthocyanin contents in fruits were processed from fruit (2.5 g)
as described in Salvatierra et al. (2010). To determine avan 3-ol
contents in fruits, fruit (2.5 g) was blended with an Ultra-Turrax
T25 digital (IKA) in ultrapure H
O (50 ml). The homogenate was
extracted with Et
O (20 ml 3) and EtOAc (20 ml 3). The six ex-
tracts were combined, dried (0.5 mg Na
anhydrous), and the
organic extract was concentrated to dryness, reconstituted in
O (2 ml) (1:1, v/v) and ltered before analysis. The ow
rate of elution was 600 ll/min of solvent A (AcOH 2%, in ultrapure
O) at initial (0.012.0 min) and nal phase of the run (23.5
25.0 min). Intermediate steps consisted in a gradient elution with a
ow rate of 800 ll/min with a mixture of 30% A and 70% B (CH
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 (MeOHH
(95:5, V/V)) up to 22.5 min. Separation and quantication of antho-
cyanins and avan 3-ols was performed using a 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 manager
software (HewlettPackard). A reversed phase column Kromasil
C18 100-3.5 (Akzo Nobel) 150 4.6 mm, equipped with a precol-
umn C18 Kromasil (Akzo Nobel) was used for the separation. Diode
array detector was programmed to perform chromatographic read-
ings in a range of wavelengths from 280 to 600 nm in steps of 2 nm
and detection at 280 nm (detection of (PAs), avan 3-ols, gallic acid
and galloil esters), 320 nm (hydroxycinnamic acid test), 360 nm
(avonols), 510 nm and 520 nm (anthocyanins) (Kosar et al.,
2004; Mtt-Riihinen et al., 2004; Oszmianski et al., 2009; Vasco
et al., 2009). Means of two technical replicates of three indepen-
dent quantications were subjected to one-way ANOVA and LSD
pairwise comparisons using Statistica 4.0 software (Statsoft Inc).
34 A. Salvatierra et al. / Phytochemistry 90 (2013) 2536
We thank anonymous reviewers for helpful comments on this
manuscript. This work has been funded by grant PBCT Anillo Cien-
cia y Tecnologa (ACT-41). A.S. thanks University of Talca, MeceSup
and Anillo ACT-41 for Ph.D. fellowships. P.P. thanks Conicyt for a
Ph.D. fellowship. We are grateful to CSIRO (Australias Common-
wealth Scientic and Industrial Research Organization) for provid-
ing the pHELLSGATE12 vector.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
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