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A SQUAMOSA MADS-box gene regulates
anthocyanin accumulation in bilberry fruits
ARTICLE in PLANT PHYSIOLOGY · AUGUST 2010
Impact Factor: 6.84 · DOI: 10.1104/pp.110.158279 · Source: PubMed
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A SQUAMOSA MADS Box Gene Involved in the
Regulation of Anthocyanin Accumulation in
Bilberry Fruits1[W][OA]
Laura Jaakola, Mervin Poole2, Matthew O. Jones3, Terttu Kämäräinen-Karppinen, Janne J. Koskimäki,
Anja Hohtola, Hely Häggman, Paul D. Fraser, Kenneth Manning, Graham J. King,
Helen Thomson, and Graham B. Seymour*
Department of Biology University of Oulu, FIN–90014 Oulu, Finland (L.J., T.K.-K., J.J.K., A.H., H.H.); Division
of Plant and Crop Sciences, University of Nottingham, Sutton Bonington, Loughborough, Leicstershire LE12
5RD, United Kingdom (M.P., G.B.S.); School of Biological Sciences, Royal Holloway, University of London,
Egham, Surrey TW20 OEX, United Kingdom (M.O.J., P.D.F.); Warwick-HRI, University of Warwick,
Wellesbourne, Warwick CV35 9EF, United Kingdom (K.M.); Rothamsted Research, Harpenden, Hertfordshire
AL5 2JQ, United Kingdom (G.J.K.); and Unilever Research and Development, Colworth Laboratory,
Sharnbrook, Bedford, MK44 1LQ, United Kingdom (H.T.)
Anthocyanins are important health-promoting phytochemicals that are abundant in many fleshy fruits. Bilberry (Vaccinium
myrtillus) is one of the best sources of these compounds. Here, we report on the expression pattern and functional analysis of a
SQUAMOSA-class MADS box transcription factor, VmTDR4, associated with anthocyanin biosynthesis in bilberry. Levels of
VmTDR4 expression were spatially and temporally linked with color development and anthocyanin-related gene expression.
Virus-induced gene silencing was used to suppress VmTDR4 expression in bilberry, resulting in substantial reduction in
anthocyanin levels in fully ripe fruits. Chalcone synthase was used as a positive control in the virus-induced gene silencing
experiments. Additionally, in sectors of fruit tissue in which the expression of the VmTDR4 gene was silenced, the expression
of R2R3 MYB family transcription factors related to the biosynthesis of flavonoids was also altered. We conclude that VmTDR4
plays an important role in the accumulation of anthocyanins during normal ripening in bilberry, probably through direct or
indirect control of transcription factors belonging to the R2R3 MYB family.
Naturally occurring polyphenols in plants are
likely to make a major contribution to human health
and therefore life expectancy (Renaud and Delorgeril,
1992). Fruits are some of the most accessible dietary
sources of polyphenols, of which anthocyanins are
recognized for their high antioxidant capacity and
1
This work was supported by the Biotechnology and Biological
Sciences Research Council in the United Kingdom and Unilever
Research and Development (award no. D20300 to G.B.S.), the Academy of Finland (grant no. 09141 to L.J.), the Ella and Georg
Ehrnrooth Foundation and the Kone Foundation, and the EU-FP6
DEVELONUTRI project (to P.D.F.).
2
Present address: Campden BRI, Station Road, Chipping Campden, Gloucestershire GL55 6LD, UK.
3
Present address: School of Biological Sciences, Royal Holloway,
University of London, Egham, Surrey TW20 OEX, UK.
* Corresponding author; e-mail graham.seymour@nottingham.
ac.uk.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Graham B. Seymour (graham.seymour@nottingham.ac.uk).
[W]
The online version of this article contains Web-only data.
[OA]
Open Access articles can be viewed online without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.110.158279
reported positive effects on blood vessels (Kähkonen
et al., 2003; Vinson et al., 2005; Dragsted et al., 2006).
Furthermore, these secondary metabolites are thought
to have positive anticancer effects (Butelli et al., 2008)
and to promote brain function (Williams et al., 2008).
Anthocyanins generate the characteristic red, blue,
and purple pigments in many flowers and in fruits
such as bilberry (Vaccinium myrtillus), black currant
(Ribes nigrum), blackberry (Rubus fruticosus), and strawberry (Fragaria spp.).
Bilberry is one of the richest natural sources of
anthocyanins, the ripe fruit typically containing 29 mg
g21 dry weight (Lätti et al., 2008). In many fruits, these
colored compounds accumulate only in the skin, while
in bilberry they occur throughout the fruit flesh. At
least 15 different anthocyanidin glycosides contribute
to the anthocyanin profile of the fruit, which also
contains high levels of the flavonols quercetin and
myricetin in addition to hydroxycinnamic acids (Jaakola
et al., 2002, 2004; Riihinen et al., 2008).
The biosynthetic pathway in plants leading to anthocyanins is well established, and transcription factors
that directly regulate multiple steps of the expression of
genes in this metabolic pathway are known (Grotewold,
2006). This information has recently been used to
generate tomatoes (Solanum lycopersicum) containing
Plant PhysiologyÒ, August 2010, Vol. 153, pp. 1619–1629, www.plantphysiol.org Ó 2010 American Society of Plant Biologists
1619
Jaakola et al.
high concentrations of anthocyanins (Butelli et al.,
2008). In bilberry fruit, ripening-related gene expression has been studied in relation to flavonoid biosynthesis (Jaakola et al., 2002). Many berry species
accumulate anthocyanins in their fruits only during the
ripening phase, but while anthocyanins are markers of
ripening, the molecular circuits connecting their accumulation to the ripening process are poorly understood.
A major goal is to identify and understand how those
molecular circuits involved with anthocyanin biosynthesis are interlinked with the control of ripening and
then to harness this information to breed novel fruit
varieties with enhanced nutritional and health benefits.
The regulation of ripening in fleshy fruits has been
most extensively studied and is best understood in
tomato (Giovannoni, 2007; Seymour et al., 2008). Tomato and bilberry differ in their ripening behavior.
Tomatoes are climacteric fruits where ripening is under the control of ethylene, while bilberry is a nonclimacteric fruit where ethylene appears to have little
influence on ripening. Both these fruits, however, are
classified as berries, and there is increasing evidence
that fleshy fruit species may share common mechanisms of ripening control related to developmental
cues that act above and beyond the influence of
ethylene (Giovannoni, 2004). Information on the identity of these ripening-control genes has now become
apparent through the study of nonripening mutants of
tomato. A small number of single gene mutants have
been identified that almost completely abolish ripening, including ripening inhibitor (rin) and Colorless
non-ripening (Cnr; Giovannoni, 2007). The gene at the
tomato rin locus is a member of the MADS box
SEPALLATA (SEP) subfamily, LeMADS-RIN (Vrebalov
et al., 2002). MADS box genes are normally associated
with floral development, but LeMADS-RIN is necessary for ripening. The gene at the Cnr locus encodes a
SQUAMOSA promoter-binding protein (SBP box)
transcription factor (Manning et al., 2006) that is likely
to interact with the promoters of the SQUAMOSA
(SQUA) subfamily of MADS box genes (Lännenpää
et al., 2004). SQUA MADS box genes are commonly
associated with floral development and especially the
regulation of floral meristem identity (Simpson et al.,
1999; Theissen, 2001; Scott et al., 2002). This class of
transcription factors has also been shown, however, to
have other functions. They have been implicated in the
regulation of tuber formation in vegetative meristems
of potato (Solanum tuberosum; Rosin et al., 2003b), and
the FRUITFULL gene (FUL) in Arabidopsis (Arabidopsis thaliana) mediates cell differentiation during fruit
development (Dinneny et al., 2005) as well as floral
meristem identity (Mandel and Yanofsky, 1995). In
tomato, TDR4, which is a likely FUL ortholog, shows
enhanced expression during fruit ripening (Eriksson
et al., 2004), although it has yet to be assigned a
function.
Elevated expression of SEP- and SQUA-class MADS
box genes during ripening has now been observed in a
wide range of climacteric and nonclimacteric fruits,
1620
including strawberry, banana (Musa spp.), and grape
(Vitis vinifera; Vrebalov et al., 2002; Rosin et al., 2003a;
Liu et al., 2009). LeMADS-RIN and LeSBP-CNR are
necessary for normal ripening in tomato, with the
latter likely controlling the expression of the SQUA
gene TDR4. Therefore, these genes and other related
transcription factors can be seen as part of the highlevel regulatory network controlling the ripening process. Their links to downstream effectors that bring
about changes in color, texture, and flavor, however,
are poorly understood.
The aim of this work was to investigate the regulatory mechanisms controlling anthocyanin accumulation in bilberry, and especially the higher level
regulatory network linking anthocyanin biosynthesis
to ripening. We have cloned a bilberry homolog of the
tomato TDR4 gene, which we name VmTDR4. Transcripts of this gene were found to have a strong
ripening-related pattern of expression. In this article,
we report on VmTDR4 expression in ripening bilberry
and how inhibition of VmTDR4 expression substantially reduces the accumulation of anthocyanins in the
fruit. Our data also suggest that the VmTDR4 gene
may act directly or indirectly on transcription factors
belonging to the R2R3 MYB family, the known regulators of flavonoid biosynthesis.
RESULTS
VmTDR4 Is a Bilberry Homolog of the Tomato TDR4 and
Arabidopsis FUL Genes
The aim of this work was to better understand the
molecular events controlling ripening in bilberry, a
fruit that accumulates high levels of health-promoting
anthocyanins. In preliminary experiments, we attempted
to isolate bilberry orthologs of the tomato ripening
regulatory genes CNR and TDR4. We were able to clone
a TDR4-like gene, VmTDR4 (accession no. FJ418852).
The full-length VmTDR4 cDNA is related by sequence
homology to FUL from Arabidopsis and TDR4/FULlike genes from tomato; VmTDR4 showed 72% identity
to tomato TDR4 at the nucleotide level and 70% identity at the amino acid level. A phylogenetic analysis
(Fig. 1) indicated a close relationship between protein
sequences from Arabidopsis, tomato, and bilberry. In
Arabidopsis, the FUL gene has pleiotrophic effects,
being involved in regulating the formation of the lignified dehiscence zone in the fruit as well as floral meristem identity (Dinneny et al., 2005). The role of TDR4
in tomato fruits is unknown.
VmTDR4 Is Expressed in Bilberry Fruit Tissues, and the
Expression Pattern Is Temporally and Spatially Related
to Anthocyanin Biosynthesis
In situ hybridization experiments revealed that
VmTDR4 transcripts are present in various tissues of
the unripe fruit (Fig. 2, A–G). These included pericarp
cells, the skin and developing embryo tissues, and
Plant Physiol. Vol. 153, 2010
A Bilberry MADS Box Gene Involved in Anthocyanin Biosynthesis
Figure 1. Phylogenetic analysis of the relationship between SQUAclass MADS box genes. Full-length protein sequences were aligned
using MAFFT version 5 (Katoh et al., 2005), and an average distance
tree was constructed based on percentage identity between sequences
in Jalview 2.4 (Clamp et al., 2004) of Arabidopsis (AGAMOUS, NP_
567569; AP1, NP_177074; CAULIFLOWER, Q39081; FUL, NP_568929),
tomato (MADS-MC, AAM15774; TDR4, AAM33098; LeFUL2, AY306156),
Antirrhinum (SQUA, CAA45228; DEFH28, AAK72467), and bilberry
(VmTDR4, FJ418852).
especially the vascular regions of the placenta. Ripe
fruits were unsuitable for in situ experiments due
to problems with effective fixation of the material.
VmTDR4 expression was investigated in ripe fruit and
other organs by quantitative reverse transcription
(QRT)-PCR (Fig. 2, H and I). Transcripts were detected
mainly in the fruit and floral tissues, with the highest
levels in the ripe flesh of the berry (Fig. 2H). Limited
expression was detected in leaves, and none was
detected in the rhizome. The expression pattern of
VmTDR4 during berry development and ripening is
shown in Figure 2I. VmTDR4 transcripts continued to
accumulate until just before the fruit were fully ripe
and just prior to anthocyanins reaching their highest
levels (Jaakola et al., 2002). The expression patterns of
several genes known to be involved in anthocyanin
biosynthesis (Jaakola et al., 2002) are shown in Figure
3, in addition to novel MYB transcription factors
VmMYB1 and VmMYB2. Chalcone synthase (CHS;
accession no. AY123765), dihydroflavonol 4 reductase
(DFR; accession no. AY123767), and anthocyanin synthase (ANS; accession no. AY123768) all showed a
ripening-related pattern with similarities to that of
VmTDR4 (Fig. 3, A–D). The levels of anthocyanin reductase (ANR; accession no. FJ666338) are low during
the ripening phase, reflecting the flux toward colored
anthocyanin pigments and away from proanthocyanidins (Fig. 3D; see also pathway in Fig. 4). We have
recently cloned R2R3-MYB family transcription factors from ripening bilberry fruit, including VmMYB1
(accession no. GU904211) and VmMYB2 (accession no.
GU227356). The expression profile of the VmMYB2 gene
was very similar to VmTDR4, being strongly ripening
related and reaching its highest level before the fully
Plant Physiol. Vol. 153, 2010
ripe stage (Fig. 3E). In contrast, VmMYB1 expression
was at its highest level during early fruit development
(Fig. 3F).
A natural bilberry fruit color mutant, white bilberry,
which ripens with a white fleshy pericarp, has been
described (Jaakola et al., 2002). The mutant fruits have
greatly reduced levels of anthocyanins and decreased
expression of anthocyanin biosynthetic genes compared with the wild type (Jaakola et al., 2002). QRTPCR analyses of VmTDR4, VmMYB2, and CHS in
white bilberry showed that at the same stage of ripening (ripe fruits) genes are down-regulated in the
fruits of the white mutant compared with wild-type
controls (Fig. 5). Collectively, the expression data from
wild-type and mutant bilberry fruits are suggestive of
a relationship between VmTDR4 expression and anthocyanin biosynthesis, but they do not, of course,
provide a functional link.
Functional Analysis of VmTDR4 in Bilberry Using
Virus-Induced Gene Silencing
To further investigate the function of VmTDR4 in
relation to flavonoid biosynthesis in bilberry, we used
virus-induced gene silencing (VIGS) with a CHS construct as a positive control, following the lead of other
studies involving fruits where anthocyanins are the
principal pigments affecting fruit color (Hoffmann
et al., 2006). This is the first report, to our knowledge,
of VIGS in this species. Fragments of the VmTDR4 and
CHS genes were amplified by PCR and cloned into the
VIGS vectors (see “Materials and Methods”). Inoculations with the VmTDR4 sequence resulted in fruits
with altered pigmentation, which included green sectors (Fig. 6). Fruits were photographed during the
ripening process at 10 d (Fig. 6A) and 32 d (Fig. 6, B
and C) after injections. A similar experiment was
undertaken in a second year, whereby fruits were
collected 42 d after the injections when they were fully
ripe (Fig. 6D). Each gene construct was tested on 100
individual berries. In the VmTDR4 and CHS treatments, between 16% and 20% of berries showed sectors with abnormal pericarp color development (Fig. 6,
A–C). Cross sections of fruits at the fully ripe stage
revealed a substantial reduction in red pigmentation
in the pericarp in fruit from both the VmTDR4 and
CHS treatments (Fig. 6D). All untreated fruits or those
injected with the empty vector (negative control) ripened normally, developing a dark red/black pigmentation in the pericarp and skin. Green sectors were
completely absent.
Expression levels of VmTDR4, CHS, DFR, ANS, ANR,
VmMYB1, and VmMYB2 were examined in VmTDR4
and CHS VIGS-treated fruits by QRT-PCR (Fig. 7). RNA
was isolated just prior to the fully ripe stage at 32 d post
injection (Fig. 6, B and C), as this is the point at which
anthocyanin-related gene expression reaches a peak. In
VIGS experiments, the effects are often localized to
small tissue sectors; therefore, gene expression was
compared between the “green” and “red” sectors. It is
1621
Jaakola et al.
Figure 2. In situ hybridization and expression profiles for VmTDR4 in bilberry. A to G, In situ hybridization. Bluish color indicates the expression of
VmTDR4 in samples hybridized with the antisense
probe. A, Cross section of whole bilberry fruit. B,
Placenta and developing seed, antisense probe. C,
Closeup of developing seed, antisense probe. D,
Vascular tissue, antisense probe. E, Vascular tissue,
sense probe. F, Epidermis and pericarp, antisense
probe. G, Epidermis and pericarp, sense probe. Bars =
1 mm in A, 10 mm in B and C, and 1 mm in D to G. H
and I, Determination of VmTDR4 transcript abundance by QRT-PCR. H, VmTDR4 transcript levels in
bilberry leaf (L), flower (F), ripe berry skin (BS), ripe
berry flesh (BF), mature seed (S), and rhizome (R). I,
VmTDR4 transcript levels during bilberry fruit development and ripening in flowers (F), immature green
(IG), mature green (MG), turning (T), ripe (R), and
fully ripe (FR) fruits. Data are means 6 SE (n = 3).
difficult to compare absolute values with empty vectortreated material directly because individual fruits on
the same plant can ripen at slightly different rates.
However, the expression of genes related to flavonoid
biosynthesis in both untreated and empty vector treatments was consistent with transcript levels observed
during normal ripening in bilberry.
In pTV00-VmTDR4-treated fruit, the levels of
VmTDR4 expression were approximately 25% lower
in the green than in the red sectors (Fig. 7), and this
difference was significant (P # 0.005). This initially
suggested an uncoupling between VmTDR4 expression and reduced pigment accumulation in the green
sectors, where a more dramatic reduction in VmTDR4
levels might have been expected. However, beneath
the skin in the green sectors of the fruits, partial color
development occurred in the fleshy pericarp tissue
(Fig. 6C). While VIGS may lead to “patchy” ripening
effects and make data interpretation more challenging,
it is an effective method for functional analysis in a
1622
species where analysis of stable transformants could
take several years. The small amount of material in the
sectors was insufficient for both RNA and metabolomic analysis, so the latter was performed on whole
fully ripe berries (Fig. 6D; see below), where the most
dramatic effects of the VIGS were seen. In contrast
with the VmTDR4-injected fruits, VmTDR4 expression
was not suppressed in the pTV00-CHS-treated material, although these fruits also developed green sectors. This is consistent with VmTDR4 being upstream
of CHS in a regulatory network. In pTV00-CHS-treated
fruits, CHS expression was markedly down-regulated
in the green sectors, as might be predicted, which
indicates effective silencing of this gene with the CHS
construct. However, CHS expression was also lower
in green compared with red sectors from pTV00VmTDR4-infected fruits. CHS expression in both treatments is relatively low, and this may reflect some
silencing of the target genes throughout the fruit by the
VIGS treatment. Alternatively, as the VIGS method
Plant Physiol. Vol. 153, 2010
A Bilberry MADS Box Gene Involved in Anthocyanin Biosynthesis
Figure 3. Determination of transcript abundance of
genes at different stages of bilberry fruit development
and ripening. A, CHS. B, DFR. C, ANS. D, ANR. E,
VmMYB2. F, VmMYB1. Stages are flower (F), immature green (IG), mature green (MG), turning (T), ripe
(R), and fully ripe (FR) fruit. Data are means 6 SE
(n = 3).
causes transient silencing, it is possible that at the
time samples were collected (32 d after injections), the
silencing effect was already weaker with respect to its
effect on VmTDR4, but silencing of VmTDR4 had
longer lasting effects on the suppression of CHS
mRNA levels.
We also examined the expression of novel bilberry
MYBs and several genes known to be involved in the
anthocyanin pathway (Fig. 7). VmMYB2 expression
was significantly (P # 0.005) lower in the pTV00VmTDR4 green sectors as compared with red sectors,
indicating a link between VmTDR4 and VmMYB2
expression and development of the red anthocyanin
pigmentation. There was, however, no significant difference in VmMYB2 expression in the red or green
sectors of the CHS VIGS fruit, indicating that the
effects of CHS on pigment accumulation are downstream of these two genes. Interestingly, the expression
of VmMYB1 was significantly higher (P # 0.005) in the
pTV00-VmTDR4 green sectors as compared with the
red sectors. VmMYB1 shares the closest sequence-level
Plant Physiol. Vol. 153, 2010
homology with FaMYB1, which is a known suppressor of anthocyanin biosynthesis in strawberry fruit
(Aharoni et al., 2001). The expression of DFR, ANS,
and ANR was also examined. DFR and ANS, which
are below CHS in the pathway leading to anthocyanins (Fig. 4), showed significant (P # 0.05) reductions
in their levels of expression in green sectors in both
pTV00-VmTDR4 and pTV00-CHS VIGS fruits. In
contrast, ANR expression was higher in the green
sectors from both VIGS treatments. ANR catalyzes
the synthesis of flavan-3-ols such as (2)-epicatechins,
a step in the pathway prior to proanthocyanidins,
which are abundant in bilberry fruit tissues prior to
the accumulation of anthocyanins.
We wanted to obtain metabolite data along with
information from the transcriptomics, but this proved
difficult with the small sectors from the VIGS fruits at
32 d after injection. However, we were able to repeat
the VIGS experiments in a second season with similar
results, and this time fully ripe bilberry samples were
collected 42 d after injection, when distinct effects on
1623
Jaakola et al.
DISCUSSION
Some of the key genes involved in the molecular
regulation of fruit ripening have been revealed through
the study of nonripening mutants in tomato, such as
rin, Cnr, Nr, and nor (Giovannoni, 2007; Seymour
et al., 2008). However, the links between these regulators and their downstream effectors that bring about
the changes in color, texture, and flavor are poorly
understood.
In many fleshy fruit species, flavonoids including
anthocyanins are the major colored pigments syn-
Figure 4. A schematic presentation of the flavonoid biosynthetic
pathway. Enzyme abbreviations not defined in the text are as follows: PAL, Phe ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL,
4-coumaroyl:CoA ligase; CHI, chalcone isomerase; F3H, flavanone
3-hydroxylase; UFGT, UDP Glc-flavonoid 3-O-glucosyl transferase; RT,
rhamnosyl transferase.
pigment accumulation could be seen throughout the
fruit (Fig. 6D). This provided sufficient tissue for
metabolite analysis. Furthermore, as the fruit were fully
ripe and the effect was apparent throughout the fruits,
they could also then be compared with whole fully ripe
empty vector or untreated fruits. The metabolite data
(Table I) demonstrated that the principal effect in
VmTDR4- and CHS-silenced fruits was a 2- to 3-fold
reduction in levels of anthocyanins compared with the
empty vector or nontreated fruits. Levels of quercetin
derivatives were low in all samples, and despite levels
in the VmTDR4- and CHS-silenced fruits being elevated, these values are within the normal variation of
these compounds in fully ripe bilberry fruits, unlike the
anthocyanin fraction. The biosynthesis of quercetin is
highest in the early stages of fruit development, and the
quercetin contents are 3- to 4-fold lower in the ripe
bilberries compared with the unripe fruits (Jaakola
et al., 2002). The VIGS treatments were made during
berry ripening and after the peak in quercetin content. It
is possible that quercetin derivatives seen in the VIGS
fruit accumulated prior to this stage. The empty vector
fruits showed evidence of an increase in the early parts
of the phenylpropanoid pathway (e.g. p-coumaric acid)
and in cyanidin that was not apparent in the VmTDR4and CHS-treated fruits. This may be the result of
defense-related responses by the fruits, arising from
the presence of the viral vector in the absence of
constructs that modulate the expression of genes in
the flavonoid pathway. Collectively, these data provide
strong evidence for a role for VmTDR4 in the regulation
of flavonoid biosynthesis in bilberry fruits.
1624
Figure 5. Gene expression in wild-type and white mutant bilberry.
Determination of VmTDR4 and CHS transcript abundance by quantitative PCR in wild-type (black bars) and white mutant (white bars)
bilberry fruits. Data are means 6 SE (n = 3).
Plant Physiol. Vol. 153, 2010
A Bilberry MADS Box Gene Involved in Anthocyanin Biosynthesis
VmTDR4 Is a SQUA MADS Box Gene Involved in
Anthocyanin Biosynthesis in Ripening Bilberry
Figure 6. Images of bilberry fruits during development and ripening
after injection of VmTDR4- and CHS-silencing constructs. Bilberry fruit
are shown at 10 d (A), 32 d (B and C), and 42 d (D) following injections
with VmTDR4 and CHS VIGS vectors in comparison with the nontreated control (NTC) and empty vector control (EV) fruits. Fruit in D are
fully ripe.
thesized during ripening. Regulation of flavonoid
biosynthesis occurs via coordinated transcriptional
control of enzymes in the biosynthetic pathway by
the interaction of DNA-binding R2R3 MYB transcription factors and MYC-like basic helix-loop-helix and
WD40-repeat proteins (Stracke et al., 2001; Broun,
2005; Koes et al., 2005; Ramsay and Glover, 2005;
Grotewold, 2006). It is also known that other factors,
such as light, temperature, and nutrients, play a role in
flavonoid accumulation (Peng et al., 2008). A major
missing part of the network is the association between
the regulatory genes controlling fruit ripening and the
downstream pathways that lead to the synthesis of
important secondary products such as anthocyanins.
In this work, we report a functional link between a
SQUA MADS box transcription factor and the control
of flavonoid biosynthesis in bilberry fruit.
Plant Physiol. Vol. 153, 2010
A role for MADS box transcription factors in regulating anthocyanin biosynthesis in a range of plant
tissues has been reported previously. A sweet potato
(Ipomoea batatas) SQUA transcription factor, IbMADS10,
showed a pattern of expression that was tightly correlated with anthocyanin levels in pigmented tissues,
especially in the red roots. Also, when ectopically
expressed in sweet potato callus, it induced anthocyanin accumulation (Lalusin et al., 2006). IbMADS10
shares significant sequence homology with DEFH28
from Antirrhinum majus (Müller et al., 2001), a likely
ortholog of VmTDR4 from bilberry, TDR4 from tomato,
and FUL from Arabidopsis (Fig. 1). Furthermore, a link
between the expression of a MADS box gene and the
accumulation of flavonoids has also been reported
in Arabidopsis for seed coat pigmentation. The TT16/
ABS MADS domain protein is required for normal
development and pigmentation of the seed coat and is
postulated to modulate anthocyanin biosynthesis by
interacting directly with an R2R3-MYB DNA-binding
domain protein (Nesi et al., 2001; Debeaujon et al.,
2003).
In bilberry fruits, high levels of anthocyanins accumulate in the skin and flesh during ripening, and these
changes are correlated with the expression of the
SQUA MADS box gene, VmTDR4. The in situ hybridization experiments and QRT-PCR indicated that
VmTDR4 expression was associated with numerous
different tissues in the unripe fruit, including the
vascular tissues, and with anthocyanin biosynthesis
in ripe fruits. Interestingly, VmTDR4 expression is low
in a white mutant of bilberry that ripens normally.
These observations are suggestive of a link between
VmTDR4 and flavonoid biosynthesis but do not establish any functional relationship.
Functional analysis of the VmTDR4 gene in bilberry was undertaken by VIGS. Down-regulation of
VmTDR4 in the fruit suppressed the expression of
several flavonoid biosynthesis genes and inhibited
anthocyanin biosynthesis. VmTDR4 and VmMYB2
were not down-regulated in the CHS VIGS fruits,
indicating that their effects are upstream of this gene.
The down-regulation of VmTDR4 also suppressed
the expression of VmMYB2, a bilberry R2R3-MYB
family member transcription factor, the expression of
which is closely associated with accumulation of
anthocyanins during the fruit development. The
partial sequence we obtained for VmMYB2 shares
62% to 63% amino acid identity with the R2R3 region
of Arabidopsis AtPAP1 and AtPAP2 and 61.5% identity with VvMYBA1, an ortholog of AtPAP genes of
grapevine berry (Supplemental Fig. S1). However,
VmMYB2 shares closest identity with VvMYBPA1
(86%) and VvMYB5b (73%; Bogs et al., 2007; Deluc
et al., 2008). Moreover, another R2R3-MYB family
member, VmMYB1, was isolated from bilberry fruits
recently. VmMYB1 shares 78% identity with FaMYB1,
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Jaakola et al.
Figure 7. Effects of VmTDR4- and CHSsilencing constructs on bilberry gene expression during ripening. Relative expression is
shown for VmTDR4, VmMYB1, VmMYB2,
and flavonoid biosynthetic genes CHS, DFR,
ANS, and ANR in green and red sectors of
bilberry fruits 32 d after injection of VIGS
vectors. VmTDR4-silenced fruit green sector
(TG), VmTDR4-silenced fruit red sector (TR),
CHS-silenced fruit green sector (CG), CHSsilenced fruit red sector (CR), and empty
vector-injected fruits are indicated. Data are
means 6 SE (n = 3).
a negative regulator and suppressor of anthocyanin
biosynthesis from strawberry (Aharoni et al., 2001).
The QRT-PCR analysis of VmMYB1 shows that transcripts are more abundant at the early stages of bilberry fruit development and in the green sectors
of the VmTDR4 VIGS berries. These results indicate
that VmTDR4 functions via known regulators of
flavonoid biosynthesis, including R2R3-MYB family
members. The metabolic analyses of VIGS-treated fruits
revealed that the levels of anthocyanins were 2- to
3-fold lower in VmTDR4- and CHS-silenced fruits,
whereas the contents of hydroxycinnamic acids or flavonols were not affected. The lack of an effect on hydroxycinnamic acids may be because they are produced
in an earlier part of the phenylpropanoid pathway and
prior to the regulatory step affected by VmTDR4. Also,
flavonols such as quercetin are generated at the beginning of bilberry fruit ripening (Fig. 4), at a stage before
the VIGS injections were made. It is still unclear if
VmTDR4 only regulates anthocyanin biosynthesis or if
it also is involved in the regulation of other flavonoid
subgroups. From grapevine, it is known that different
R2R3-MYB family members can control separately the
biosynthesis of anthocyanins, flavonols, and proanthocyanins (Bogs et al., 2005, 2007; Azuma et al., 2008;
Czemmel et al., 2009).
In the VmTDR4-suppressed VIGS fruits, transcript
levels of ANR, leading to the synthesis of proanthocyanidins, were elevated. In many species, proanthocyanidins are produced at the early phases of fruit
development, and it has been suggested that they act
as defensive and astringent compounds to provide
protection against fungal pathogens and predation
of unripe fruits (Harborne, 1997; Jaakola et al., 2002;
Bogs et al., 2005). In the skin of red grape, the
flavonoid pathway switches to the production of anthocyanins instead of proanthocyanidins at the onset
of ripening, and this phenomenon is controlled by
MYB transcription factors (Bogs et al., 2007). In bilberry, the content of proanthocyanidins and the flavonol quercetin are higher at the early stages of fruit
development. At the onset of ripening, the level of
proanthocyanidins declines whereas quercetin remains at a constant but low level during the ripening
phase. The flavonol myricetin is synthesized in bilberry skin and flesh at the same time as the accumulation of anthocyanins (Jaakola et al., 2002). Therefore,
the higher levels of ANR transcripts in VmTDR4silenced bilberry fruit sections could suggest a delay
in ripening in the sectors. However, QRT-PCR analysis
of another ripening-related MADS box transcription factor, VmTAGL1, which is expressed at a higher
Table I. Phenolic-related metabolite changes in bilberry VIGS fruits
Determination of phenolics present in ripe bilberry fruit 42 d after injection of VIGS VmTDR4 and CHS
constructs. HPLC analysis was undertaken on pooled samples of three individual fruits in each treatment.
All values are provided in mg mg21 dry weight, according to the protocol described in “Materials and
Methods.” Data are means 6 SE. Values in the same column that are significantly different (P , 0.001)
from the empty vector treatment are shown in boldface.
Tissue Sample
Untreated fruits
Empty vector
VmTDR4 VIGS
CHS VIGS
1626
Chlorogenic
Acid
200
280
208
196
6
6
6
6
38.0
36.0
22.0
16.0
p-Coumaric
Acid
Quercetin
Derivatives
Delphinidin
Cyanidin
126 ± 10.0
276 6 12.0
172 ± 10.0
136 ± 0.06
0.23 6 0.02
0.2 6 0.1
0.3 ± 0.04
0.9 ± 0.20
78.7 6 4.5
95.0 6 10.6
33.8 ± 2.7
42.5 ± 1.8
55.0 ± 3.1
67.7 6 5.0
14.8 ± 0.6
33.8 ± 2.7
Plant Physiol. Vol. 153, 2010
A Bilberry MADS Box Gene Involved in Anthocyanin Biosynthesis
level in earlier stages of bilberry fruit development,
did not show differences between the green and red
sectors of the VmTDR4- or CHS-silenced fruits (data
not shown). An alternative metabolic explanation
could be that lack of anthocyanidin intermediates in
the VmTDR4-silenced sectors stimulates ANR transcription.
Role of TDR4-Like Genes in Other Plant Species
Elegant work in the model plant Arabidopsis has
demonstrated that a range of MADS box and other
transcription factors are involved in regulating the
development of their dry fruits. This includes the
expression of FUL, a SQUA gene that is primarily
responsible for proper valve development (Dinneny
et al., 2005). In the fleshy fruits of tomato, the TDR4
gene, which shares sequence homology with FUL,
shows a strong ripening-related pattern of expression
(Eriksson et al., 2004), but its function is not known.
Studies we have undertaken to reveal the role of TDR4
in tomato fruits were inconclusive, with no obvious
phenotypes where the gene was down-regulated in
RNA interference lines (G.B. Seymour and M. Poole,
unpublished data). This is probably due to the presence of other TDR4-like genes in the tomato genome
(Hileman et al., 2006). However, we observed that
tomato TDR4 induced anthocyanin biosynthesis when
it was expressed ectopically in Arabidopsis siliques
(Supplemental Fig. S2). These effects are consistent
with those observed for VmTDR4 in bilberry in that
they indicate a link between SQUA genes and the
regulation of the phenylpropanoid pathway. Furthermore, in Arabidopsis, the endogenous FUL gene,
which is strongly related to TDR4 and VmTDR4 by
sequence homology, is necessary for the expression of
the MYB transcription factor PAP2 under the condition
of nitrogen starvation (Supplemental Fig. S3). This
MYB is known to be involved in the regulation of
anthocyanin biosynthesis (Borevitz et al., 2000).
The data reported in this article provide strong
evidence that VmTDR4 plays an important role in
the control of anthocyanin biosynthesis in bilberry,
acting directly or indirectly through MYB transcription factors to control carbon flux through the phenylpropanoid pathway. Further work is required to
determine the precise molecular links between the
regulatory factors involved in this process. However,
this study provides, to our knowledge, the first evidence for a functional association between SQUA
MADS box genes and anthocyanin biosynthesis during ripening in a fleshy fruit species.
MATERIALS AND METHODS
Plant Material
The flowers and fruits of wild bilberry (Vaccinium myrtillus), growing in a
natural forest stand in Oulu (65°01# N, 25°28# E), Finland, were collected at six
different ripening stages, from flower to ripe fruit. Samples were immediately
Plant Physiol. Vol. 153, 2010
frozen in liquid nitrogen and stored at 270°C until analyzed. Arabidopsis
(Arabidopsis thaliana) Landsberg erecta and Columbia seeds were obtained
from the European Arabidopsis Stock Centre at the University of Nottingham.
ful mutant lines were donated by Prof. M. Yanofsky (University of California
San Diego).
Cloning of VmTDR4 and TDR4
Total RNA was isolated from bilberry fruit samples with the method
described by Jaakola et al. (2002). The quality of the isolated RNA was verified
by measuring the absorbance spectrum with NanoDrop N-1000 spectrophotometer (NanoDrop Technologies) and on a 1% (w/v) ethidium bromidestained agarose gel.
Degenerate primers VmTDR4f (5#-GTGATGCWGAGGTTGSTTTGA-3#)
and VmTDR4r (5#-AGCWGGTTRTTTTGCTCCTGC-3#) for VmTDR4 were
designed for isolating the VmTDR4 based on homology of the MADS box
genes from tomato (Solanum lycopersicum; TDR4) and Arabidopsis (FUL). The
full-length sequence of VmTDR4 was cloned using the SMART RACE cDNA
Amplification Kit (Clontech) with the gene-specific primers gspTDRf
(5#-TGGAACCTGGAATACCCGAAGCTCA-3#) and gspTDRr (5#-TGAAGCTCTCTCAGGGTCAAGGTGTCA-3#). The full-length cDNA sequence was
deposited in GenBank under accession number FJ418852.
Phylogenetic Estimation
Full-length protein sequences were aligned using MAFFT version 5 (Katoh
et al., 2005), and an average distance tree was constructed based on percentage
identity between sequences in Jalview 2.4 (Clamp et al., 2004).
Quantification of Transcript Abundance
Transcript accumulation of VmTDR4, VmMYB2, VmMYB1, and the flavonoid pathway genes (CHS, DFR, ANS, and ANR) was detected using the
DyNAmo Capillary SYBR Green qPCR Kit (Finnzymes). Glyceraldehyde-3phosphate dehydrogenase (AY123769) was used as a control gene for relative
quantification. QPCR analyses were performed with a LightCycler 2.0 instrument and software (Roche). The PCR conditions were 95°C for 10 min,
followed by 45 cycles of 95°C for 10 s, 60°C for 20 s, and 72°C for 10 s.
Concerning the transcript abundance of VmTDR4 and VmMYB2, a t test was
used to determine if the relative quantity was significantly lower (P # 0.05) in
green versus red parts of the VIGS-treated fruits based on data from three to
four technical replicates. We separated those visible sectors of VmTDR4- or
CHS-silenced fruits with a scalpel.
In Situ Hybridization
Unripe bilberry fruits were cut into cubes of 5 mm2 and fixed in 4% (w/v)
paraformaldehyde and 0.25% (v/v) glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.0) overnight at 4°C. The samples were rinsed in 0.1 M
sodium phosphate buffer (pH 6.8) and dehydrated in a graded series of
ethanol up to absolute. The ethanol was replaced by a series of xylene (25%,
50%, 75%, and 100%, v/v), after which the samples were gradually infiltrated
with paraffin (Merck). Paraffin-embedded samples were sectioned to a
thickness of 8 mm with a microtome (Microm HM 325). The sections were
spread on glass slides previously coated with 2% (v/v) 3-aminopropyltriethoxysilane (Sigma) in acetone and dried overnight at 40°C. Two 20-min
incubations in xylene were used for removing paraffin from the samples. For
in situ hybridizations, samples were rehydrated in a graded ethanol series up
to water.
Using primers 5#-CACCTTGACCCTGAGAGAGC-3# and 5#-GTCCACCTTGGTTTTGTTGC-3#, a 218-bp fragment from VmTDR4 was amplified
from bilberry fruit cDNA by PCR with DyNazyme II DNA polymerase
(Finnzymes) under standard PCR conditions. The PCR fragment was gel
purified using the Montage DNA Gel Extraction KIT (Millipore) and ligated
into pGEM-T Easy vector (Promega). Digoxigenin (DIG)-labeled sense and
antisense probes were prepared from the linearized plasmid by in vitro
transcription with SP6 or T7 RNA polymerase using the DIG RNA Labeling
Kit (Roche) according to the manufacturer’s instructions. Before hybridization, rehydrated tissue sections were treated with proteinase K (1 mg mL21 in
Tris-HCl and 50 mM EDTA, pH 7.5) for 30 min at 37°C followed by dehydration in a graded ethanol series up to absolute. For the hybridization, 100 mL of
1627
Jaakola et al.
hybridization mixture (0.1 mg mL21 DIG-labeled antisense or sense probe, 50%
[v/v] deionized formamide [Sigma], 0.3 M NaCl, 10 mM Tris-HCl [pH 7.5],
1 mM EDTA, 13 Denhardt’s solution [Sigma], 150 mg mL21 tRNA [Roche], 500
mg mL21 polyadenylic acid [Sigma], 10% dextran sulfate, and 0.06 M dithiothreitol) was dispersed on the sections, and the hybridization was carried out
at 50°C overnight. After hybridization, slides were washed in 23 SSC (300 mM
NaCl, 30 mM sodium citrate, pH 7.0), in 13 SSC at 37°C and 0.53 SSC at 37°C,
for 10 min in each. Excess RNA probes were removed in RNase A treatment at
37°C for 60 min following by four 15-min washes in 10 mM Tris-HCl, 500 mM
NaCl, and 1 mM EDTA and one 30-min incubation in 23 SSC. For localization
of the hybridized transcripts, slides were washed in 100 mM Tris-HCl, 150 mM
NaCl, and 0.3% (v/v) Triton X-100 for 5 min and blocked with 2% (w/v)
blocking reagent (Roche) for 30 min, followed by a 2-h incubation at room
temperature with a 1:750 dilution of anti-(DIG-alkaline phosphatase) conjugate (Roche) and four 10-min washes in the same buffer. For color development, slides were washed for 5 min in 100 mM Tris-HCl, pH 9.5, 100 mM NaCl,
and 50 mM MgCl2 and immersed in 5-bromo-4-chloro-3-indolyl phosphate
and blue tetrazolium chloride (Roche) overnight in the same buffer. The next
day, slides were washed in water, dehydrated in a graded series of ethanol up
to absolute, and air dried before sealing with coverslips.
VIGS
The pBINTRA6 and pTV00 vectors were obtained from David Baulcombe
and PBL at the Sainsbury Laboratory. For pTV00-VmTDR4 construction, a
150-bp fragment of the VmTDR4 gene was PCR amplified from bilberry
fruit cDNA using primers (forward, 5#-CTCGGATCCGGTGGACAAAGTTCATCC-3#; reverse, 5#-GCTAAGCTTCGGCGGCATCAAAGTGTT-3#). The resulting PCR product was cloned into pTV00 to form pTV00-VmTDR4. For
pTV00-VmCHS construction, a 100-bp fragment of the VmCHS (AY123765)
gene was PCR amplified from bilberry fruit cDNA using primers (forward,
5#-CTCGGATCCAAGATCACCCACTCAGTCTTTTG-3#; reverse, 5#-GCTAAGCTTGCTTCACGGAGGGACGGAGCC-3#). The resulting PCR product was
cloned into pTV00 to form pTV00-VmCHS. The pTV00-VmTDR4 and pTV00VmCHS vectors were transformed into Agrobacterium tumefaciens strain
GV3101 for the inoculations.
Plant infiltration was performed as described previously (Ratcliff et al.,
2001). The Agrobacterium strains GV3101 containing pTV00-VmTDR4 or
pTV00-VmCHS and C58c1 containing pBINTRA6 were grown at 28°C in
liquid Luria-Bertani medium including antibiotics (50 mg mL21 kanamycin,
5 mg mL21 tetracycline, and 50 mg mL21 rifampicillin, pH 5.6). After 24 h, the
cells were harvested by centrifugation and resuspended in the infiltration
buffer (10 mM MgCl2 with 200 mM acetosyringone and 10 mM MES, pH 5.6) to a
final optical density at 600 nm of approximately 1 and shaken for 2 h (+28°C)
before mixing in a 1:1 ratio. The Agrobacterium mix containing either
pBINTRA6 + pTV00-VmTDR4 or VmCHS vectors was injected in bilberry
fruits that were in the middle of fruit development with a 1-mL syringe and
needle. As a control, only Agrobacterium with pBINTRA6 or infection buffer
was injected in the fruits.
Metabolite Analysis
Contents of anthocyanins, flavonols, and hydroxycinnamic acids from
fully ripe bilberry fruits 35 to 42 d following VIGS injections with VmTDR4,
CHS, or empty vector constructs were determined. Phenolics were extracted
from homogenized freeze-dried material (20–80 mg) with methanol (1 mL)
containing salicylic acid as an internal standard (10 mg). The mixture was
heated to 90°C for 60 min; after cooling, the suspension was centrifuged at
3,000g for 5 min; the supernatant was removed, and extracts were passed
through a 0.2-mm syringe-fitting filter before HPLC analysis. An Agilent 1100
HPLC solvent delivery system was used to separate phenolics on a reversephase C18 column (250 mm 3 4.6 mm, 5 mm; Hichrom). The mobile phase
consisted of 2% water in methanol acidified with 0.015% HCl by volume (A)
and acetonitrile (B). The initial gradient conditions used were 95% A, 5% B for
10 min, followed by a linear gradient to 50% B over 30 min. An online photo
diode array detector enabled detection and identification from characteristic
UV/visible light spectra. Authentic standards were used to confirm the
identity of the phenolics. Relative quantification was carried out by comparison of integrated peak areas with the internal standard at the lmax of
the phenolics detected. Previously, the identity of the phenolics had been
confirmed with liquid chromatography-electrospray ionization-mass spectrometry using identical conditions except that formic acid was used instead
of HCl.
1628
Sequence data from this article can be found in the GenBank/EMBL data
libraries under accession numbers FJ418852 (VmTDR4), AY123765 (CHS),
AY123767 (DFR), AY123768 (ANS), FJ666338 (ANR), GU904211 (VmMYB1),
and GU227356 (VmMYB2).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Alignment of R2 and R3 DNA-binding regions of
VmMYB2 with Arabidopsis PAP1, PAP2, ATMYB113, and ATMYB12,
grape VvMYBA1, VvMYBPA1, and VvMYB5b, and maize (Zea mays) C1
anthocyanin regulator.
Supplemental Figure S2. Effects of ectopic expression of tomato TDR4 in
Arabidopsis.
Supplemental Figure S3. Determination of AtPAP2 transcript abundance
by QPCR of cDNA in wild-type and ful mutant leaves in response to
growth with and without nitrogen (N; error bars indicate SE; n = 3).
Supplemental Materials and Methods S1.
ACKNOWLEDGMENTS
We thank Cathie Martin at the John Innes Centre and Jacquie De Silva at
Unilever for useful discussions during the preparation of this article. Also,
Chris Gerrish at Royal Holloway is thanked for assistance in the analysis
of the phenolics.
Received April 23, 2010; accepted June 18, 2010; published June 21, 2010.
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