Genome-wide analysis of the grapevine stilbene synthase multigenic family: genomic organization and expression profiles upon biotic and abiotic stresses
BMC Plant Biology
Genome-wide analysis of the grapevine stilbene synthase multigenic family: genomic organization and expression profiles upon biotic and abiotic stresses
Alessandro Vannozzi 0 1 3 4
Ian B Dry 2
Marianna Fasoli 5
Sara Zenoni 5
Margherita Lucchin 0 1 3 4
0 Centro Interdipartimentale per la Ricerca in Viticoltura ed Enologia, Universita di Padova , Agripolis, viale dell'Universita 16, 35020, Legnaro, Padova , Italy
1 Department of Agronomy , Food, Natural resources, Animals and Environment , University of Padova , Agripolis, viale dell'Universita 16, 35020, Legnaro, Padova , Italy
2 CSIRO Plant Industry , PO Box 350, Glen Osmond, SA 5064 , Australia
3 Centro Interdipartimentale per la Ricerca in Viticoltura ed Enologia, Universita di Padova , Agripolis, viale dell'Universita 16, 35020, Legnaro, Padova , Italy
4 Department of Agronomy , Food, Natural resources, Animals and Environment , University of Padova , Agripolis, viale dell'Universita 16, 35020, Legnaro, Padova , Italy
5 Dipartimento di Biotecnologie, Universita degli Studi di Verona , Strada Le Grazie 15, 37134, Verona , Italy
Background: Plant stilbenes are a small group of phenylpropanoids, which have been detected in at least 72 unrelated plant species and accumulate in response to biotic and abiotic stresses such as infection, wounding, UV-C exposure and treatment with chemicals. Stilbenes are formed via the phenylalanine/polymalonate-route, the last step of which is catalyzed by the enzyme stilbene synthase (STS), a type III polyketide synthase (PKS). Stilbene synthases are closely related to chalcone synthases (CHS), the key enzymes of the flavonoid pathway, as illustrated by the fact that both enzymes share the same substrates. To date, STSs have been cloned from peanut, pine, sorghum and grapevine, the only stilbene-producing fruiting-plant for which the entire genome has been sequenced. Apart from sorghum, STS genes appear to exist as a family of closely related genes in these other plant species. Results: In this study a complete characterization of the STS multigenic family in grapevine has been performed, commencing with the identification, annotation and phylogenetic analysis of all members and integration of this information with a comprehensive set of gene expression analyses including healthy tissues at differential developmental stages and in leaves exposed to both biotic (downy mildew infection) and abiotic (wounding and UV-C exposure) stresses. At least thirty-three full length sequences encoding VvSTS genes were identified, which, based on predicted amino acid sequences, cluster in 3 principal groups designated A, B and C. The majority of VvSTS genes cluster in groups B and C and are located on chr16 whereas the few gene family members in group A are found on chr10. Microarray and mRNA-seq expression analyses revealed different patterns of transcript accumulation between the different groups of VvSTS family members and between VvSTSs and VvCHSs. Indeed, under certain conditions the transcriptional response of VvSTS and VvCHS genes appears to be diametrically opposed suggesting that flow of carbon between these two competing metabolic pathways is tightly regulated at the transcriptional level.
Conclusions: This study represents an overview of the expression pattern of each member of the STS gene family
in grapevine under both constitutive and stress-induced conditions. The results strongly indicate the existence of a
transcriptional subfunctionalization amongst VvSTSs and provide the foundation for further functional investigations
about the role and evolution of this large gene family. Moreover, it represents the first study to clearly show the
differential regulation of VvCHS and VvSTS genes, suggesting the involvement of transcription factors (TFs) in both
the activation and repression of these genes.
Approximately 450 million years ago, several pioneering
green algal ancestors, probably related to Charales ,
spread out from water to occupy a new bio-geographical
niche: dry land. This colonisation of dry land was
accompanied by the need to deal with important stresses
including desiccation, UV radiation, as well as attack by
already diversified microbial soil communities. This led
to a number of physiological adaptations, including the
evolutionary emergence of entirely new specialized
secondary metabolic pathways . One in particular was
crucial: the phenylpropanoid pathway, which represents
a ubiquitous and specific trait of land plants providing
vital compounds such as lignin and flavonoids .
Lignin is a structural polymer important for the structural
integrity necessary for the emergence of self-supporting
structures. Flavonoids, which often impart a
speciesspecific chemical signature upon an organism, serve
vital roles in the protection of plants against biotic and
abiotic stresses, reproduction and internal regulation of
cell physiology and signalling .
The role of phenylpropanoid compounds in defence
appears to be restricted to a minor class of compounds
that are often referred to as phytoalexins. The term
phytoalexins probably derives from the Greek language
and means warding off agents in plants and refers to
low mass, lipophilic, antimicrobial compounds that not
only accumulate rapidly at the site of interaction with
incompatible pathogens [5,6] but also accumulate in
response to abiotic stresses such as exposure to UV light,
wounding or treatment with chemicals such as salts and
heavy metals, respiratory inhibitors and surfactants .
Because of the agricultural and economic importance of
grapevine as a crop plant, the strategies it uses to defend
against phyto-pathogenic organisms, as well as deal with
abiotic stresses, has attracted considerable interest in
recent times. Amongst the arsenal of defence mechanisms
available to grapevine cells is the production of
phytoalexins. Phytoalexins from the Vitaceae family have been
the subject of numerous studies over the past decade,
not only because of their biological activities in planta,
but also because of their possible pharmacological
Although phytoalexins display an enormous chemical
diversity throughout the plant kingdom, in grapevine
they constitute a rather restricted group of molecules
belonging to the stilbene family . Plant stilbenes,
together with flavonoids, belong to the class of compounds
called polyketides, which represents a major group of
phenylpropanoids derived from the extension of the
activated form of coumaric acid with three acetyl moieties.
Apart from the Vitaceae, stilbenes have been detected in
at least 72 unrelated plant species distributed among 31
genera and 12 families including Fagaceae, Liliaceae,
Moraceae, Myrtaceae, Papilionaceae, Pinaceae, and
Poaceae [8-10]. Despite the multiplicity of forms detected in
these different plants, most plant stilbenes, including
those ones detected in grapevine, are derivatives of the
basic unit trans-resveratrol
(3,5,4-trihydroxy-transstilbene). In addition to resveratrol, more complex
compounds derived from its modification have also been
detected in grapevine such as cis- and trans- piceid
, viniferins, which represent oligomers arising from
the oxidative coupling of resveratrol, pterostilbene
[15,16] and piceatannol .
Several plant species, such as Polygonum cuspidatum
and Pinus spp. constitutively accumulate large amount
of stilbenes [18-23]. However, the majority of studies
conducted on cells and leaves of peanut, grapevine and
pine seedlings have shown that stilbenes are present at
only very low levels under normal conditions, but
strongly accumulate in response to a wide range of
biotic and abiotic stresses as a result of an increased
transcription of their biosynthetic genes and the
coordinated activation of upstream genes belonging to the
general phenylpropanoid pathway, such as PAL and
C4H. These abiotic stress treatments include mechanical
damage [24,25], UV-C light irradiation [26,27],
treatments with chemicals such as aluminium ions,
cyclodextrins and ozone [28-30] and the application of plant
hormones like ethylene and jasmonates [31-33]. In terms
of biotic stresses, the biosynthesis of stilbenes in
grapevine tissues is also particularly well documented, with
the accumulation of stilbenic compounds reported
following infection with a range of different pathogens,
including powdery mildew (Erysiphe necator) [34,35],
downy mildew (Plasmopara viticola) , gray mold
(Botrytis cinerea) [16,37,38] and Aspergillus carbonarius
Stilbene synthase (STS) is the key enzyme leading to
the biosynthesis of resveratrol and stilbenes and was
firstly extracted and purified from stressed cell
suspension cultures of peanut (Arachis hypogaea) . It
belongs to the type III polyketide synthase super family,
of which chalcone synthase (CHS) represents the
archetypal enzyme. The enzyme is a dimer of estimated
molecular weight 90 kDa with an iso-electric point (pI) of
4.8. A conserved cysteine residue, located in the central
section of these proteins has been shown to be essential
for the catalytic activity of both STS and CHS enzymes
and represents the binding site for the
p-coumaroylCoA starting substrate . The region around this
active site is well conserved and can be used as a signature
pattern for CHS and STS. The two proteins show a high
degree of similarity based on sequence homology (which
reaches approximately 75-90% amino acid sequence
identity depending on the species), and on the
comparison of their crystallographic structures , suggesting
that STS independently evolved from CHS several times
in the course of evolution . STSs, which, in contrast
with the ubiquitous CHSs, are only present in
stilbeneproducing plants, catalyse the formation, in a single
enzymatic reaction, of exactly the same linear tetraketide
intermediate (from p-coumaroyl-CoA and three
malonyl-CoA) produced by CHS in the flavonoid pathway,
but with a different cyclization that leads to the
production of stilbenes rather than chalcones (Figure 1).
To date, STS genes have been cloned from peanut
(A. hipogaea), Scots pine (P. sylvestris), Eastern white
pine (P. strobus), Japanese red pine (P. densiflora),
grapevine (V. vinifera L.) and sorghum (Sorghum bicolor). In
many of these plant species STSs exist as a family of
closely related genes. For example, two STS genes have
been found in peanut and Eastern white pine [45,46],
Scots pine has a small multigene family of at least five
pynosylvin synthase genes (PST1, PST2, PST3, PST4 and
PST5)  and Japanese red pine possesses three
members (PdSTS1, PdSTS2 and PdSTS3) . Apart from
Sorghum, for which only one STS member has been
identified [10,49], grapevine represents the only stilbene
producing plant species for which the entire genome has
been sequenced [50,51]. Forty-three VvSTS members
were predicted with GAZE and JIGSAW prediction tools
in the 8.4 X coverage genome draft of the PN40024
genotype (French-Italian consortium)  while only
twenty-one members were predicted from the genome
sequence of the PN ENTAV 115 genotype (IASMA)
. Sparvoli et al.  performing a molecular
characterization of structural genes involved in
anthocyanins and stilbene biosynthesis in V. vinifera has
previously hypothesized that these gene families
probably arose from the same ancestral gene and that
subsequent gene duplications and molecular divergence may
have contributed to the establishment of functionally
This aim of this study was to clarify the genome
organization of the entire STS family in grapevine and
investigate the transcriptional response of each VvSTS
member in different grapevine tissues, at different
developmental stages and under different stress conditions, in
order to determine if this gene family evolved into
different sub-groups characterized by specific role in the
response to different stresses or in the plant development.
Identification, annotation and chromosomal distribution
of grapevine STS genes
The genome sequence of the near-homozygous
PN40024 genotype of the V. vinifera cv. Pinot noir was
searched for predicted STS gene sequences. These were
predicted on the genome draft by combining ab initio
models together with V. vinifera complementary DNA
sequences, such as EST databases and alignment of
gene/protein models from other species . The
Hidden Markov Model (HMM) for the CHS/STS active site
[PS00441] was obtained from PROSITE and used in a
BLASTP search against the 8.4X, 12X V0 and 12X V1
proteome databases. In order to extend the search to
identify putative gene family members not predicted by
the GAZE and JIGSAW software programs, a tBLASTx
search of the HMM and of the entire amino acid
sequence of previously identified VvSTS was also
performed against the genome sequence. Fifty-one hits
were obtained. Three predictions carrying the CHS/STS
HMM were found to encode for chalcone synthase
genes and were excluded leaving a total of forty-eight
putative VvSTS gene sequences. These sequences were
designated as VvSTS1 to VvSTS48 based on their
chromosomal position (Table 1). VvSTS1-6 are located
in a region of approximately 90 Kb on chr10, whereas
VvSTS7-48 reside on chr16, within a 500 Kb region. Five
sequences corresponding to genes designated as
VvSTS11, VvSTS14, VvSTS34, VvSTS40 and VvSTS44 fall
in genome regions which are not predicted to contain
any gene based on GAZE and JIGSAW prediction tools.
Although the Genoscope integrated method for
deducing proteins is very exhaustive, some gene models were
found to be incorrect based on available EST sequences
and when compared with cloned VvSTS CDS sequences
already deposited on the GenBank database. With
particular reference to the 12X V1 coverage assembly,
the predictions designated as Vv10s0042g00840,
Vv10s0042g00850 and Vv10s0042g00860 are listed as
three different genes in the proteome database, but our
Figure 1 General phenylpropanoid pathway and flavonoid and stilbene branching pathways. The enzymes shown in these pathways are
as follows: PAL, phenylalanine ammonia-lyase; C4H, cinnamate-4-hydroxylase; 4CL, 4-cumaroyl: CoA-lyase; CHS, chalcone synthase; STS, stilbene
analysis indicates they represent one single unique VvSTS
gene, designated as VvSTS1 (Table 1). A similar
observation was made for the predictions Vv10s0042g00880 and
Vv10s0042g00890, which also represent a single gene
designated as VvSTS3. The opposite situation was
observed for genes designated as VvSTS37 and
VvSTS38, which are represented by the same prediction
Vv16s100g01110. Genomic sequences of five genes,
VvSTS12, VvSTS13, VvSTS14, VvSTS25 and VvSTS26
were obtained by corresponding sequences from the PN
ENTAV 115 genome sequence because of gaps in the
Based on the deduced amino acid sequences obtained
from Genoscope predictions and from manual analysis
using Vector NTI software, several VvSTS proteins were
found to be truncated because of SNP/mutations leading
to premature stop codons or in/del mutations causing
frame-shifts and changes in the protein primary
structure. In order to investigate whether these observations
were limited to the PN40024 genotype used by the
French-Italian Consortium or were also detectable in
other genotypes, the closest sequences in the PN
ENTAV 115 genotype and specifically matching
pairedend reads obtained from the whole-transcriptome
sequencing of Pinot noir clone 115, were screened for
these mutations (Additional file 1). Based on available
sequence information it was not possible to determine
with absolute certainty whether VvSTS1, VvSTS3,
VvSTS4, VvSTS8, VvSTS12, VvSTS13 or VvSTS25 encode
a complete ORF. VvSTS2, VvSTS33, VvSTS40 and
VvSTS44 were predicted to have premature stop codons
in all three genotypes screened or in both the PN40024
and PN ENTAV 115 where no specific paired-end reads
were available. VvSTS11 and VvSTS34 represent gene
fragments of 233 nt and 453 nt respectively with the
upstream and downstream sequences not coding for STS.
Finally VvSTS18 was predicted to be a coding gene
based on the fact that at least one allele at this locus was
predicted to encode a complete ORF based on the three
genotypes sequences screened.
The genomic sequences of the VvSTS genes detected in
the PN40024 genome ranged in size from a minimum
length of 1315 nt (VvSTS16) to a maximum of 1566 nt
(VvSTS1) depending on the length of the single introns
present in all members within the triplet coding for Cys-60.
Deduced protein length for all 36 full-length coding genes
was 392 aa (Additional file 2), whilst of those pseudogenes
that possessed the CHS/STS active site
VK]-x-[LIVMF]-[RAL]) VvSTS1 encodes for a 234 aa
Table 1 Grapevine STS members identified based on the PN40024 12X V1 coverage (Continued)
Genes have been named from VvSTS1 to VvSTS48 based on the chromosomal location. The chromosomal location and corresponding identifier on the 12X V1
coverage are also provided. The predicted open reading frame (ORF) prediction was assigned as follows: Yes: genes encoding a complete ORF; Unsure: genes for
which it was not possible to determine with certainty whether they encode a full length or truncated ORF based on available sequence information from the
PN40024 and PN ENTAV 115 genotypes and specifically matching paired-end reads obtained from the whole-transcriptome sequencing of Pinot noir clone 115;
No: genes encoding for a truncated ORF.
product, VvSTS2 for a 206 aa product (181 without
considering the first 46 nt which are probably wrongly predicted),
VvSTS4 for a 267 aa product and VvSTS18 for a 185 aa
product. All other genes give products lacking the active
site and were considered non functional.
Phylogenetic analyses of the deduced VvSTS proteins
In order to examine the phylogenetic relationship between
the predicted VvSTS proteins a phylogenetic tree was
constructed using the E-INSI tool of the MAFFT 6.0 software
as described in the tutorial for the Grapevine Genome
(http://www.vitaceae.org/index.php/Annotation_tutorial) provided by the International grape Genome
Program (IGGP) steering committee. Gene members
encoding a truncated ORF were not included in the
alignment, but for VvSTS genes where both coding and
noncoding alleles had been identified in different genotypes, the
coding ORFs were also included in the analyses. The three
VvCHS proteins corresponding to VvCHS1 [Genbank:
AB015872], VvCHS2 [Genbank: AB066275] and VvCHS3
[Genbank: AB066274], respectively Vv14s0068g00930,
Vv14s006800920 and Vv05s0136g00260 in the 12X V1
assembly of the PN40024 genotype, were also included in the
analysis to ascertain the evolutionary relationships between
VvSTS and VvCHS proteins. Figure 2 shows that VvSTS
proteins cluster in three main sub-families, which have
been designated as groups A, B and C. Group A is
composed entirely of those members located on the chr10
(i.e. VvSTS1-6), while groups B and C are composed of 22
and 13 members respectively all positioned on chr16. The
three VvCHS proteins were found to cluster outside the
tree as outgroups.
Microarray analysis of VvSTS and VvCHS expression
during grapevine development and post-harvest berry
The expression pattern of VvSTS genes encoding a
complete ORF were analysed in a global V. vinifera cv.
Corvina gene expression atlas of different organs at
various developmental stages (Fasoli et al., in preparation).
Although VvSTS37 and VvSTS38 were predicted to
encode a full-length protein, these genes were excluded from
the expression analyses as they are represented by the
same prediction in the reference genome, which could
lead to incorrect estimations of the expression values for
these genes. The expression atlas was generated using a
microarray technology based on gene predictions obtained
from the 12X V1 coverage assembly of the PN40024
genotype. Figure 3 shows a graphical representation of the
expression pattern of each VvSTS, together with the three
VvCHS genes identified in grapevine, and was generated
using MeV software. Raw VvSTS and VvCHS expression
values are reported in Additional file 3.
The first thing to note regarding the results shown in
Figure 3 is that the majority of VvSTS gene family
members show little or no constitutive expression in most
grapevine tissues including young leaves, stems, buds,
flowers and developing grape berries. The exceptions to
this appear to be roots and all stages of rachis
development in which members of all three VvSTS groups show
elevated levels of constitutive expression. As a group,
members of VvSTS group A also appear to have a higher
level of constitutive expression in young leaf (Y) tissues
than the majority of members of subgroups B and C.
Another important observation regarding VvSTS
expression in developing grape tissues is that members of
all groups are strongly induced during aging or
senescence. This was observed in both senescing leaves and in
berries undergoing the process of berry withering. Berry
withering is a post-harvest drying process used
specifically with Corvina berries for the production of dessert
and fortified wines. The drying process leads to
alterations in most quality characteristics and an increase in
the concentration of simple sugars. Berries were sampled
for expression analysis after the first, second and third
month of the withering phase. The results clearly show a
very strong induction of nearly all VvSTS family
members in the berry pericarp in response to the withering
Figure 2 Phylogenetic tree of predicted STS proteins in grapevine. Consensus phylogenetic tree generated after sequence alignment with
MAFFT 6.0 using the neighbour-joining method. VvSTS gene members predicted to encode for a truncated ORF were not considered. Deduced
protein for VvCHS1, VvCHS2 and VvCHS3 were also included in the analysis. Reliability of the predicted tree was tested using bootstrapping with
1000 replicates. Numbers at the forks indicate how often the group to the right appeared among bootstrap replicates. Different coloured bars
indicate three main sub-groups designated as A, B and C.
process. This organ appears to accumulate VvSTS
transcripts within the exocarp tissue, whereas the expression
is much lower in berry flesh (Additional file 4).
Figure 3 also shows a comparison between the
constitutive expression patterns of the VvSTS family
members with the three VvCHS genes in grapevine. What
is clear from this comparison is that the expression of
the three VvCHS genes appears to show an opposite
pattern to that of the VvSTS genes across a number of
different tissues and developmental stages. For
example, expression of at least one member of the
VvCHS gene family is found to be high in young
leaves, stems, buds, the rachis at fruit set and in
developing berries in which VvSTS expression is generally
very low. The converse is also true: in tissues where
VvSTS expression is strongly induced e.g. senescing
leaves, in vitro roots, the rachis from ripe berries and
withering berries, there is little or no VvCHS
mRNA-seq analysis of VvSTS and VvCHS expression in
grape leaves in response to stress
The same VvSTS and VvCHS gene sequences predicted
in the PN40024 genome sequence and analysed in the
grapevine expression atlas (Figure 3) were also studied
for their expression under biotic and abiotic stress
conditions. In order to overcome the difficulty posed by the
high sequence conservation between these genes, which
makes it difficult to clearly discriminate between
individual members using PCR-based expression analyses, a
whole transcriptome (mRNA-seq) approach was
performed using the Illumina Next Generation Sequencing
(NGS) technology. V. vinifera cv Pinot noir leaf discs
were collected at 0, 24 and 48 h after wounding, UV-C
exposure and infection with P. viticola. Seven pools of
RNA samples, representing each treatment and the
control sample, which was common for all three treatments,
were used to build libraries for high-throughput parallel
sequencing using an Illumina Genome Analyser II
Figure 3 Expression image of the complete VvSTS family in the
V. vinifera cv Corvina atlas. Expression data was normalised based
on the mean expression value of each gene in all tissues/organs
analysed. Different organs/tissues are displayed vertically above each
column. VvSTS gene names are displayed to the right of each row
and are clustered in different groups A, B, C according to protein
homology as shown in Figure 2. Expression data for VvCHS genes
are included for comparison. The colour scheme used to represent
expression level is orange/white: black boxes indicate a low variation
in expression, white boxes indicate a decrease and orange boxes
indicate a increase respect to the mean value of a given gene. Y,
young leaf; FS, fruit-set; S, senescence; G, green stem; AB, bud-burst;
PFS, post fruit-set; V, vraison; MR, mid-ripe; R, ripe; F, flowering (50%
cap-fall); PHWI, post-harvest withering I (1st month); PHWII,
postharvest withering II (2nd month); PHWIII, post-harvest withering III
(GAIIx). Each treatment was represented at least by 32
million reads, a tag density sufficient for quantitative
analysis of gene expression .
All three stress treatments resulted in a significant
induction of expression of at least some members
of the VvSTS gene family (Figure 4, Additional file 5).
Of the three stress treatments employed, UV-C
exposure led to the highest induction of the majority of
VvSTS members, followed by downy mildew infection
and wounding. The wounding and UV-C responses
appeared to peak within 24 h of treatment whereas
the downy mildew-treated discs continued to show an
increase in VvSTS transcription after 48 h, presumably
reflecting the establishment of the downy pathogen
within the leaf tissue. Interestingly, there appeared to
be only minor differences in VvSTS transcription
between wounded and downy mildew-inoculated discs
after 24 hours indicating that VvSTS genes are not
induced in the early stages of downy mildew infection
prior to haustorial formation.
Figure 4 Expression image of the complete VvSTS gene family
in V. vinifera cv. Pinot noir stressed leaves. The treatments
(wounding, exposure to UV-C and downy mildew infection) are
displayed vertically above each column. Genes are displayed to the
right of each row and clustered in different groups A, B, C and CHS
as evidenced by different colours. Relative levels of expression are
indicated by a colour gradient from low (white) to high (orange).
Expression data are expressed as the number of mapped reads per
Kb of exon per million mapped reads (RPKM).
In agreement with the microarray data shown in
Figure 3, VvSTS genes in group A, unlike those in
groups B and C, are characterised by significant levels of
constitutive expression in young leaves. Furthermore,
group A genes are not induced in response to wounding
and show only a minor increase in transcription in
response to UV-C treatment compared to control discs. In
contrast, VvSTS genes in group B are highly responsive
to abiotic stress treatments with wounding resulting in
increases in transcription ranging from 7 to 186 fold
after 24 h. When these discs are also exposed to UV-C
light there is a further increase in transcription ranging
from 11.3 to 27 fold. VvSTS genes in group C appear to
show transcriptional responses which are intermediate
between those of genes in groups A and B.
The relationship between VvSTS and VvCHS
transcription in young leaf tissues subjected to abiotic stress
treatments (Figure 4) appears to be somewhat more
complicated than was observed for constitutive expression
patterns in different grapevine tissues (Figure 3). As
observed with VvSTS genes in groups B and C, wounding
led to an increase in transcription of all three VvCHS
genes ranging from 3.2-8.7 fold after 24 h. However, in
accordance with the inverse transcriptional responses of
VvSTS and VvCHS genes observed in a range of different
grapevine tissues in Figure 3, the further increase in
Figure 5 Expression of grapevine selected VvSTS6, VvSTS16 and VvSTS48 genes upon abiotic and biotic stresses. Selected members
representative for A- (VvSTS6), B- (VvSTS48) and C- (VvSTS16) subgroups were screened by quantitative RT-PCR in wounded, UV-C exposed and
downy mildew infected samples. Transcript are normalised to the expression of elongation factor (EF1) and plotted as actual transcript copy
number. Bars indicate standard error (SE) in three technical replicates. Empty bars represent wounded samples, which also represent the control
for the other treatments. Dark gray bars represent UV-C treated samples. Light gray bars represent downy mildew infected samples.
VvSTS transcription in response to UV-C treatment was
accompanied by an 820 fold reduction in expression of
the VvCHS genes (Figure 4) to levels below that found in
Quantitative RT-PCR analyses of selected VvSTS members
of groups A, B and C and VvCHSs in stressed leaves
The analysis of mRNA-seq data from leaf samples
treated by wounding, UV-C exposure and P. viticola
infection, together with analysis of gene expression atlas in
V. vinifera cv. Corvina indicated there are differential
expression patterns across different VvSTS groups and
between members of the VvSTS and VvCHS polyketide
synthase families. To confirm and investigate these
observations in more detail, the expression patterns of
selected members of the VvSTS groups A, B and C i.e.
VvSTS6, VvSTS48 and VvSTS16 and the three grapevine
VvCHS genes was monitored using quantitative RT-PCR
across a time course series following wounding, UV-C
irradiation, and P. viticola inoculation of Shiraz leaf
tissue (Figures 5 & 6).
Elongation factor EF1 was selected as the reference
gene as it was found to be more stable than 18 S and
actin in the wounded and UV-C irradiation treatments
(data not shown). Figure 5 illustrates changes in
selected VvSTSs mRNA transcript levels over time in
response to the three applied stresses. The qPCR
results confirmed the results of the mRNA-seq
experiment with the group B gene VvSTS48 showing much
higher levels of transcript accumulation than VvSTS
genes in groups A (VvSTS6) and C and (VvSTS16)
respectively under all stress treatments. However, it is
clear from this more detailed expression analysis that
VvSTS6 and VvSTS16 display a similar pattern of
induction in response to these three stress treatments
and that the pattern of induction of these group A
and C genes is clearly different to that observed for
the group B gene, VvSTS48. For example, both VvSTS6
and VvSTS16 show a peak of transcription at 8 h post
UV-C treatment. In contrast, VvSTS48 shows a
continual increase in transcript levels over the whole 48-h
period following UV-C treatment. This is well
illustrated in Figure 7A, which shows a comparison of
the fold-change in gene expression in response to
UVC treatment for these three VvSTS genes. Based on
this analysis it would appear that VvSTS6 and
VvSTS16 actually respond earlier than VvSTS48 to
UV-C treatment. However, it should be noted that the
Figure 6 Expression of grapevine CHS genes upon abiotic and biotic stresses. Grapevine CHS1, CHS2 and CHS3 genes were screened by
quantitative RT-PCR in wounded, UV-C exposed and downy mildew infected samples. Transcript are normalised to the expression of elongation
factor (EF1) and plotted as actual transcript copy number. Bars indicate standard error (SE) in three technical replicates. Empty bars represent
wounded samples, which also represent the control for the other treatments. Dark gray bars represent UV-C treated samples. Light gray bars
represent downy mildew infected samples.
transcriptional activity of VvSTS48 is such that the
level of transcription of this gene in response to
wounding alone, at 4 h, is still greater than that
observed for VvSTS6 and VvSTST16 at 4 h following
wounding plus UV-C treatment (Figure 5). Indeed, 8 h
after UV-C treatment the level of expression of this
group B gene is approximately 60 and 15 fold higher
than is observed for the subgroup A and C VvSTS
Differences in the timing of response of these three
VvSTS group representatives are also evident from the
downy mildew inoculation experiment. In agreement
with the mRNA-seq data (Figure 4) there appears little
transcriptional response from three VvSTS genes to
downy mildew infection within the first 24 h. However,
from 48 hpi both VvSTS6 and VvSTS16 show a
marked increase in transcription peaking at 72 hpi
(Figures 5 & 7B). In contrast, a significant increase in
downy-mildew induced transcription is not observed
for VvSTS48 until 72 hpi and continues to increase up
to 96 hpi. Even so, the copy number of this group B
gene transcript at 72 hpi is still significantly higher
than that observed for the group A and C genes
Figure 6 shows the patterns of expression observed
for the three VvCHSs genes in response to the same
treatments. As observed in both the Corvina gene
expression atlas (Figure 3) and the Pinot noir
mRNAseq analysis (Figure 4), constitutive levels of
expression of all three VvCHS genes are much higher than
VvSTS genes in young Shiraz leaves (cf. Figures 5 &
6). Both VvCHS1 and VvCHS2 show only a minor
increase in expression upon wounding, although a slight
decrease in expression was detected at 96 h after
treatment. In contrast, VvCHS3 showed a 5-fold
increase at 8 h after wound treatment, followed by a
decrease in expression as observed for the other two
Of greater significance is the observation that the
application of both the UV-C and downy treatments
led to a significant reduction in transcript
accumulation of all three VvCHS genes compared to control
(wounded-only) discs (Figure 6). This is most clearly
displayed in Figure 7, which show that as the
combined level of VvSTS transcription increased in
response to these biotic and abiotic stress treatments, so
the level of transcription of all three VvCHS genes was
suppressed by as much as 1841 fold at 48 h post
Figure 7 Relative changes in expression of VvSTS and VvCHS genes in response to UV-C irradiation and downy mildew infection. This
figure summarizes the fold changes of selected VvSTS6, VvSTS16 and VvSTS48 and VvCHSs genes in UV-C exposed (A) and downy mildew infected
leaf discs (B). Fold change was obtained by calculating the ratio between treated (UV-C or downy infected) and untreated (i.e. wounded discs)
samples at the same time point.
UV-C treatment (Figure 7A) and 1030 fold within
72 h of downy mildew inoculation (Figure 7B).
Expansion of the STS family in grapevine
To date, STS genes have been cloned from several plant
species including peanut, sorghum, pine and grapevine
. In peanut and pine STS genes are organised in
multigenic families composed of 25 members, although
in the absence of a whole genome sequence for these
species an accurate estimate of the number of family
members is difficult. Grapevine and sorghum represent
the only two species which possess stilbene biosynthetic
genes for which the genomes have been completely
sequenced. Screening of the sorghum genome sequence
revealed the presence of a single, unique STS gene [10;
49]. In this study, a search for STS genes in the most
update version of the genome assembly of the grape
PN40024 genotype referred to as 12X V1, led to the
identification of 48 members, designated VvSTS1 to
VvSTS48 and included at least 33 full-length coding
genes, 8 pseudogenes and 7 sequences that remain to be
resolved (Table 1).
The striking size of the grapevine STS gene family,
compared to other stilbene-producing plant species, is
not surprising given that analysis of the grape genome
sequence has already indicated an expansion in the size
of other gene families related to secondary metabolism
in grapevine [50,51]. For example, it is estimated that
there may be up to 35 terpene synthase (TPS) genes in
grapevine based on the genome assembly of the PN
ENTAV 115 genotype . The phenylalanine
ammonia-lyase (PAL) gene, which encodes for the key
enzyme of the phenylpropanoid pathway, has 13
members in grapevine, whereas only 48 genes are present in
Arabidopsis, rice, and poplar . More recently,
Falginella et al.  reported on the expansion and
subfunctionalization of the grapevine flavonoid 3,5-hydroxylase
(F35H) gene family, responsible for the biosynthesis of
precursors of blue anthocyanins. Large-scale (segmental
or whole) genome duplication has been recurring during
angiosperm evolution and is one of the driving forces in
the evolution of genomes and genetic systems [56,57].
Subsequent gene loss and gene rearrangements further
affect gene copy number and fractionate ancestral gene
lineages across multiple chromosomes. The expansion of
the F35H family, which is composed of 16 members,
appears to be the result of multiple events of segmental
and tandem duplications that occurred in the Vitaceae
lineage, after the separation from other dicots . Of
the 16 copies of F3'5'Hs present in the PN40024
genome, 15 reside in a tandem array within a 650 Kb region
on chr 6 with an isolated copy on chr 8. Although a
detailed study of the VvSTS evolution was not the major
aim of this study, the model proposed for the F35H
family could also be applied to the VvSTS gene family.
The majority of VvSTS members (VvSTS7-VvSTS48) are
located in a 500 Kb region on chr16, which shows
numerous paralogous zones, not only at the level of coding
regions, but also in non-coding regions (data not shown)
suggesting multiple events of tandem and segmental
duplication. Something similar could have happened for
members VvSTS1-VvSTS6 located within an 80 Kb
region of chr 10. A recent analysis of the genome
architecture of the PN40024 line and its high-identity
duplication content by , identified that 85 Mb out of
the 487 Mb comprising the grapevine genome is
duplicated. Furthermore, they found that chr 16, which
contains the majority of VvSTS family members, has the
highest percentage (25.08%) of segmental duplication
among the assembled non-random chromosomes.
It is noteworthy that duplicate genes involved in
secondary metabolism or involved in the response to
exogenous stimuli, appear to be more frequently
maintained than duplicate genes belonging to other
categories [59-61]. Moreover its generally assumed that the
maintenance of duplicate genes provides a foundation
for consolidation and refinement of established
functions, particularly in secondary metabolism, by
preserving extra copies that guarantee a gene reservoir for
adaptive evolution [62-64]. What is particularly
interesting in the case of the STS gene family is that the
majority of plants dont even possess a single STS gene, whilst
grapevine has evolved such a large STS gene-reservoir.
The fact that a single STS gene is present in the
monocot Sorghum [10,49] suggests that the evolution of STS
from CHS, the common ancestor of PKSs, occurred
before the monocot-dicot separation. Nevertheless, its
difficult to explain the lack of stilbene-producing genes in
the majority of plant species and the conservation and
retention of many duplicated STS genes in a restricted
group of unrelated species. It could be argued that the
production of stilbenes did not confer an evolutionary
advantage in those species that lost their biosynthetic
genes or, on the other hand, that the majority of species
were not able to cope with the production of
compounds such as resveratrol, that, although related to
benefits at low range of concentrations, are phytotoxic
to plant cells at higher concentrations .
Structure/Function of VvSTS proteins
All full-length VvSTS coding genes were found to
encode proteins of 392 amino acids in length and contain
the conserved CHS/STS active site (Additional file 2). In
some cases (Table 1), it was not possible, based on
currently available sequence information, to determine with
certainty whether the genes encode for a complete or
truncated ORF. This includes the genes VvSTS1 and
VvSTS4, which are particularly interesting as they
possess the conserved CHS/STS active site within the
truncated allele (Additional file 6).
In a previous study, which compared the
enzymological properties of three STS proteins (PdSTS1,
PdSTS2 and PdSTS3) and one CHS protein (PdCHSX)
from Japanese red pine, it was observed that PdSTS3,
which has a frame-shift mutation leading to a premature
stop codon, presents a functional divergence compared
to the other full-length STS/CHS proteins . In
particular, the PdSTS3 protein showed poor solubility
compared to PdSTS2, but despite being truncated, still
demonstrated a high potential for pinosylvin production.
Furthermore, neither pinosylvin nor pinocembrin
inhibited the PdSTS3 activity in vitro, whereas these
metabolites effectively inhibited the activity of both PdSTS2 and
PdCHSX. Thus, although the truncated ORFs of VvSTS1
and VvSTS4 are shorter than that observed for PdSTS3
(Additional file 6) we cannot rule out the possibility that
these truncated alleles may still contribute to stilbene
synthesis biosynthesis in grape cells.
Together with the CHSs, STSs represent the most
studied enzymes of the plant type III PKS proteins and
for this reason this group is often referred as the CHS/
STS type III PKS family. The two enzymes compete for
the same substrates, share very close amino acid
sequences, and possess very similar crystallographic
structures . Previous phylogenetic analyses of the
STS and CHS families indicated that STSs of Scots pine,
peanut and grapevine do not form a separate cluster, but
instead cluster with the CHSs proteins from the same or
related plants . This observation, reinforced by the
observation that only three amino acids exchanges were
required within the N-terminal 107 aa of CHS to shift
the activity to a STS-type function, suggests that STS
may have evolved from CHS several times during the
course of evolution . In this study, the three CHS
genes identified in the PN40024 genotype, based on
clones previously isolated in Cabernet Sauvignon ,
were included in the phylogenetic analysis performed on
the STS family to investigate whether any of the
predicted VvSTS proteins cluster more closely to the
VvCHS clade. Sequence alignment and phylogenetic tree
analyses revealed the existence of 3 VvSTS clades or
groups, designated as A, B and C (Figure 2). Group A is
composed of genes located on chr10, whereas groups B
and C are composed of members located on chr16.
However, neighbour-joining analysis indicated that all
predicted VvSTS proteins cluster separately from the
three VvCHSs, suggesting a conservation of function
amongst all VvSTS members. This observation is in
agreement with a recent functional study in which 10
different VvSTS genes (including members of each
group) were transiently expressed in tobacco and all led
to an accumulation of resveratrol and stilbenes, with no
evidence for the production of any other products
(Parage et al, in preparation).
Temporal and spatial patterns of STS gene expression in
Using an expression atlas of V. vinifera cv. Corvina (Fasoli
et al., in preparation), it was possible to investigate
patterns of expression of all of the predicted coding members
of the VvSTS and VvCHS gene families in different
grapevine tissues and at different developmental stages
Expression of the majority of VvSTS genes was found
to be very low in most non-stressed grapevine tissues
analysed. The two exceptions to this were in vitro roots
and the berry rachis. The high level of VvSTS expression
in in vitro roots is in agreement with the detection of
high levels of oligostilbenes in this organ . Moreover,
the propagation of this organ in vitro is an artificial
procedure that could represent a stress for the plant, leading
to the stress-induced transcription of VvSTS genes as
observed in Figure 4. The elevated levels of VvSTSs
expression in the berry rachis, however, are more
surprising. What is particularly striking is the dramatic increase
in transcription of group B and C VvSTS genes in the
rachis during maturation of the Corvina berries while
there is no detectable induction of VvSTS genes in the
berries themselves (Figure 3). As discussed in more
detail below, VvSTS expression in grape tissues such as
leaves and berries appears to be strongly associated with
senescence. Thus, the results shown in Figure 3 may
reflect the fact that the rachis on Corvina berries
undergoes maturation and senescence during berry
ripening. This is also supported by the fact that rachis
are generally brown, dehydrated and lignified by the
time berries reach full maturity.
Interestingly, the microarray results did not show any
significant increase in VvSTS expression in Corvina
berries during both vraison and ripening. This is in
contrast with previously reported studies, which indicate
that healthy grape berries synthesise stilbene compounds
under natural environmental conditions [14,68-70].
However, stilbene production during berry ripening has
been shown to be genotype dependent with high
producers such as Pinot noir producing up to 20 ug
resveratrol per g berry fresh wt at maturity  compared to a
low producer like Corvina which was found to
synthesize only 1.5 g g-1 at harvest . It would
appear, therefore, that the microarray technique was not
sufficiently sensitive to detect the low level changes in
VvSTS expression during ripening of the Corvina
In general, VvSTS expression was low in young grape
leaves except for two VvSTS gene members of group A.
However, as observed for the rachis, grapevine leaves
also show a dramatic increase in VvSTS transcription as
they reach maturity and begin to senesce (Figure 3). This
was true of gene members of each VvSTS group with
individual genes increasing by as much as 2 (VvSTS5-6) to
130 fold (VvSTS9) in senescing leaves compared to
young leaves. Leaf senescence is an active and highly
regulated process that involves an integrated response of
leaf cells to age information and other internal and
environmental signals . It is accompanied by a
decreased expression of genes related to photosynthesis
and protein synthesis and an increase in the expression
of hundreds of senescence-associated genes . Many
of these genes are associated with the remobilization of
nutrients to other developing organs . However, it is
not immediately clear as to what role stilbene
biosynthesis would play in such a process. The observation that
a number of pathogenesis-related (PR) genes are induced
during leaf senescence has lead to the suggestion that
the senescence program might have incorporated
features of the pathogen-defense response to protect the
senescing leaf against opportunistic pathogens .
Alternatively, the induction of STS genes in senescing
leaves may simply be a consequence of changes in the
levels of various phytohormones including abscissic acid
(ABA), salicylic acid (SA), jasmonates (JA) and ethylene
which are known to play an important role in regulating
leaf senescence and which have also been shown to be
involved in the induction of stilbene biosynthesis. For
example, treatment of Cabernet Sauvignon cuttings with
Ethephon, an ethylene-releasing compound, resulted in
an enhancement of both PAL and STS gene induction
leading to an increase in phytoalexins biosynthesis by
. Similarly, JA, another key hormone in the
senescence response, has been shown to induce high levels of
STS transcription in cell cultures of V. vinifera cv.
Cabernet Sauvignon . Therefore, it is likely that the
increased expression of STS genes during leaf
senescence is related to an accumulation of hormones such as
ethylene and jasmonates, which are well known to be
involved in these particular plants developmental stages.
Stress-induced VvSTS gene expression in grapevine
The majority of previous studies on the accumulation of
stilbene compounds and their biosynthetic genes
performed on peanut and grapevine tissues, indicated that
these genes are highly inducible in response to a number
of biotic and abiotic stresses including mechanical
damage [24,25], UV-C light irradiation [26,27], treatments
with chemicals such as aluminium ions, cyclodextrins
and ozone [28-30] and infection, including powdery
mildew, downy mildew and gray mold [35-40]. Although
these studies have made important contributions to our
general understanding of the behaviour of stilbene
biosynthetic genes, in light of the information we now have
regarding the size of the VvSTS gene family and the
strong sequence conservation amongst its members, the
interpretation of some of this data needs to be
reconsidered. To this end, we investigated the transcriptional
response of all of the predicted coding members of the
VvSTS and VvCHS gene families to three abiotic stress
treatments (post-harvest drying, wounding and exposure
to UV-C radiation) and one biotic treatment (downy
mildew infection) using either grape berries or grape
The process of post-harvest berry drying (berry
withering) involves harvesting of ripe grapes and allowing
them to dry over a period of three months in a naturally
ventilated room. Its primary purpose is to alter berry
quality characteristics and increase the concentration of
simple sugars in the production of dessert and fortified
wines typical of the Valpolicella region in Italy. However,
the drying of harvested grapes in this way results in a
loss of over 30% of their weight through evaporation
during this post-harvest treatment  and, as such,
imposes a significant water stress on the berries. It also
results in a dramatic induction of the majority of VvSTS
genes (Figure 3) demonstrating that drying berries are
still capable of undergoing a significant stress response.
Versari et al.  previously observed an increase in the
resveratrol content of skins sampled from Corvina
berries which had undergone an artificial berry withering
treatment. A later study by Zamboni et al.  showed
that berry withering was associated with an increase in
the transcription of a range of genes involved in hexose
metabolism and transport, cell wall composition, and
secondary metabolism including a number of VvSTS
genes. Our data extends these original observations to
show that nearly all of the VvSTS gene members are
markedly induced by the dehydration stress.
Furthermore the increase in VvSTS expression was detected
predominately within skin of the drying grape berry
(Additional files 3 and 4). This is in agreement with the
immuno-detection of STS proteins performed on berry
extracts by Fornara et al.  who showed that STS
protein is located mainly in berry exocarp during the
vraison phase and is detected only occasionally within the
In order to obtain more control over the stress
treatments imposed, the second set of experiments employed
young rapidly expanding leaves harvested from
glasshouse-grown V. vinifera cv. Pinot noir plants and
utilised whole transcriptome mRNA-seq analysis to
investigate the expression patterns of all of the predicted
coding members of the VvSTS and VvCHS gene families
in response to mechanical wounding, UV-C exposure
and downy mildew (P. viticola) infection. In agreement
with data obtained from the Corvina expression atlas
(Figure 3), there appears to be a much higher level of
constitutive expression of the group A VvSTS gene
family members (VvSTS5 and VvSTS6) than VvSTS gene
members belonging to groups B and C raising the
question as to the role of group A VvSTS proteins in young
leaves. In terms of stress-induced expression, the results
indicate that among the three stress treatments
examined, UV-C exposure resulted in the highest VvSTS
induction, followed by downy mildew infection and
wounding (Figure 4), confirming previous observations
. The much larger increase in VvSTS induction in
response to UV-C exposure may reflect the much larger
number of cells within the leaf disc that are subjected to
UV-C exposure compared to the wounding and downy
mildew treatments which are only affecting a subset of
cells. The data also indicates that members within the
same VvSTS groups are not only related through protein
homology (Figure 2) but also appear to show similar
transcriptional responses (Figure 4). Thus, members of
group B showed the highest response to all stress
treatments, whereas group C members showed a reduced
response, while the two group A genes showed little or no
transcriptional response to the three stress treatments
In an attempt to validate the different stress-induced
transcriptional responses within the VvSTS gene family,
a more detailed analysis of individual members of group
A (VvSTS6), group B (VvSTS48) and group C (VvSTS16)
was undertaken using qPCR (Figure 5). The qPCR
analysis confirmed the significant differences in the
quantitative response of these different group members to the
different abiotic and biotic stress treatments observed
using the mRNA-seq analysis (Figure 4). At the peak of
induction, the transcript copy number of VvSTS48 was
found to be 1550 fold higher than the levels of
VvSTS16 and VvSTS6. If one assumes there are no major
differences in translational efficiency between these
different transcripts, this means that the bulk of the
observed increase in the biosynthetic capacity of the
stilbene pathway under stress conditions would appear to
be contributed by the group B VvSTS family members.
Not only did qPCR analysis of stress-induced VvSTS
induction in grape leaves confirm the quantitative
differences in the transcriptional response of the different
group members, it also demonstrated clear differences in
the pattern and timing of the response to the different
abiotic and biotic stress treatments. The transcriptional
response of VvSTS6 and VvSTS16 to both UV-C
treatment and downy mildew infection appears to be similar
and more rapid than the response of VvSTS48 (Figures 5
& 7) leading one to speculate that the genes within the
VvSTS groups A and C may be responding to different
transcriptional signals to those in group B. The
differential timing in the stress-response of VvSTS genes from
the different groups provides an explanation for previous
observations that total STS transcription in grape cells,
as detected with Northern blot assays or PCR using
generic primers, following stress or elicitor treatment, is
often observed to be biphasic [27,76,77]. Indeed, Wiese
et al.  previously suggested that the biphasic nature
of the VvSTS response indicated that the VvSTS gene
family may be divided into two groups: some expressed
early with rapid degradation of the mRNA and others
which are expressed later, providing more stable mRNA.
The different patterns of transcriptional response
between the VvSTS groups further suggest that these genes
may be responding to different signalling pathways. Both
the JA and ethylene signalling pathways have previously
been shown to have a role in STS transcription
[3133,74,78,79]. Faurie et al.  were able to show that
cotreatment of Cabernet sauvignon suspension cells with
methyl-jasmonate (MeJ) + Ethephon (ethylene) not only
led to both a higher level of total stilbenes and VvSTS
transcription compared to treatment with either elicitor
alone, but also resulted in a biphasic pattern of
transcription which was not observed in cells treated with
MeJ or Ethephon only. These observations lend support
to the hypothesis that VvSTS genes within the different
groups respond to different stress/defense signalling
Transcriptional subfunctionalization has also been
reported between the 15 members of the F35H family
, where the development of structural variation in
the promoter regions of recently duplicated gene copies
has led to differences in member-specific patterns of
accumulation across organs, developmental stages and
cultivars. Indeed, in the absence of transcriptional
subfunctionalization, it would be hard to explain the
retention of so many functionally identical VvSTS gene family
One question yet to be resolved is the identity of the
transcription factor(s) which regulate VvSTS
transcription. The expression of phenylpropanoid pathway genes
is regulated by the binding of R3R3-type MYB
transcription factors (TFs) to highly conserved cis-elements in
their promoters [81,82]. Over the last few years a
number of R2R3-type MYB TFs have been identified which
regulate flavonol pathway genes in grapevine [83-87],
however, to date, no transcription factor responsible for
the regulation of VvSTS transcription has been reported.
We have undertaken a PTM (Pavlidis Template
matching) analysis of the whole mRNA-seq dataset for all
26,346 genes annotated in the 12X V1 PN40024
assembly to identify TF genes that show co-expression with
VvSTS under the different stress conditions applied.
This has resulted in the identification of two R3R3-MYB
candidates which we believe have a role in the
transcriptional regulation of the stilbene biosynthetic pathway
(Vannozzi et al., in preparation).
Differential regulation of VvSTS and VvCHS genes in
grapevine during development and in response to stress
Although there appears to have been little divergence in
sequence since the evolution of STS from CHS, there
has been sufficient mutation to lead to changes in the
products synthesised. These products clearly fulfill very
different roles in plant growth and development.
Chalcone synthase catalyses the first committed step of the
flavonoid biosynthetic pathway, which leads to the
synthesis of anthocyanins, tannins and flavonols. Stilbene
synthase, on the other hand, appears to function
primarily as a stress-response protein, and has been implicated
to have a role in defence against pathogens including
powdery mildew, downy mildew and Botrytis cinerea
[88,89]. As these two proteins represent branch points
in the same pathway, the diversion of carbon skeletons
into either secondary metabolism via CHS or stilbenic
defence compounds via STS would be expected to be
under tight control.
Evidence for the existence of crosstalk between these
two pathways in grapevine cells is clearly evident from
the analysis of gene expression data in Corvina tissues at
various developmental stages (Figure 3). Tissues in
which VvSTS expression levels are generally low i.e.
stem, bud, young leaves, rachis at fruit set and
developing berries are characterised by high constitutive
expression of at least one of the three different VvCHS genes
(Figure 3). Conversely, expression of all three VvCHS
genes is suppressed in tissues in which VvSTS
transcription is strongly induced i.e. roots, senescing
leaves, maturing rachi and berries undergoing withering
treatment. A similar pattern of inverse expression
patterns between the members of the VvSTS and VvCHS
gene families is also evident in grape leaves exposed to
UV-C or inoculated with downy mildew (Figure 7).
While both stress treatments resulted in dramatic
increase in VvSTS transcription, the expression of all three
VvCHS genes was strongly suppressed relative to the
untreated leaf discs.
While a number of previous studies have shown that
the expression of CHS can be induced by UV-A and
UV-B light and pathogen infection (reviewed in ),
this is the first study, to our knowledge, that has
investigated the effect of UV-C light on CHS transcription. The
other major difference between our study and previous
investigations is that our research has been carried
out on grapevine which has a highly evolved stilbene
biosynthetic pathway which is strongly induced by both
UV-C and downy mildew infection. As such, one might
expect there to be an enhanced level of cross-talk
between the flavonoid and stilbene biosynthetic
pathways in grapevine.
It has been well documented that the triggering of
defence pathways in plants causes a suppression of genes
associated with photosynthesis and basic metabolism
leading to the suggestion that there is a diversion of
metabolic resources from general metabolism to
defense-related metabolism, during pathogen attack.
This is particularly true for the flavonoid pathway, which
has been shown to be suppressed in a number of
different plant species following exposure to fungal pathogens
or fungal elicitors [91-94]. Recently Schenke et al. 
demonstrated that the induction of biosynthetic
pathways, in Arabidopsis, responsible for the synthesis of
lignin and the phytoalexin scopoletin, by the bacterial
elicitor flg22, was associated with a strong suppression
of flavonol biosynthesis genes including CHS. They
concluded that as flavonols, lignin and scopoletin are all
derived from phenylalanine, that under stress conditions,
the plant appears to refocuses its metabolism on the
production of scopoletin and lignin, at the expense of
flavonol. We propose that a similar antagonistic
relationship exists between flavonol biosynthesis and stilbene
biosynthesis in grapevine and that during periods of
abiotic or biotic stress, stilbene biosynthesis takes
precedence over flavonol biosynthesis.
How might this antagonistic relationship be regulated?
In Arabidopsis, it appears that the antagonistic
relationship between the flavonol and stress/defense
biosynthetic pathways involves the action of at least two
opposing MYB TFs: MYB12 (positive regulator) and
MYB4 (negative regulator), which compete for binding
to MYB-recognition elements within the promoters of
the flavonol biosynthetic pathway genes. We are
currently investigating whether R2R3-MYB candidates in
grapevine might also repress the transcription of the
VvCHS genes during the induction of the stilbene
The sequencing of the grapevine genome, together with
the vertiginous development of next generation
sequencing technologies constitute a powerful tool for gene
search and studies concerning their evolution,
expression and function. This study embodies a particularly
significant example of the advantages provided by these
new tools, providing a detailed description of the
expression patterns of each VvSTS genes in an extremely
conserved gene family such as the one here described. This
is the first study to our knowledge that describes the
behaviour of the VvSTS gene family focusing on each
single member and taking into account the strong
sequence conservation that characterizes it. Using this
approach we have demonstrated transcriptional
subfunctionalization amongst different members of the VvSTS
gene family. Furthermore we provide evidence for the
co-ordinated transcriptional regulation of the VvSTS and
VvCHS gene families which may serve to regulate the
flow of carbon via these two competing metabolic
For mRNA-sequencing analysis leaves were obtained
from field grown vines at the Lucio Toniolo
experimental farm of the University of Padova (Legnaro, PD,
Italy). V. vinifera cv. Pinot noir plants (clone 115 on
K5BB rootstock) were obtained from a certified nursery
(Vitis Rauscedo, Pordenone, Italy). For quantitative
RTPCR analyses leaves of V. vinifera cv Shiraz were
obtained and samples from potted glasshouse vines at
the Waite Campus (Adelaide, South Australia, latitude
3456 south, longitude 13836 east). Grapevines were
propagated from dormant cuttings obtained from the
Riverland Vine Improvement Committee (Monash,
Database search, gene structure determination and
chromosomal locations of grapevine STS genes
Protein sequences encoded by STS genes in grapevine
were identified using BLAST  at the Genoscope
BLAST server  providing the 8.4X and 12X V0
assembly coverage of the PN40024 genotype , and at
the National Centre for Biotechnology Information
(NCBI) . The search was extended by consulting an
uploaded version of the PN40024 12X assembly
coverage, designated as V1, kindly provided by Prof. Giorgio
Valle (University of Padova, Italy) . A BLASTP
search of the proteome database of the Genoscope
Genome Project was carried out using the HMM (Hidden
Markov Model) for the CHS/STS active site (PS00441)
obtained from Prosite . An e-value of 1e-3 was set
to avoid false positives. To further increase the extent of
the database search results, a tBlastN search of the
genome sequence using one of the deduced protein
sequences obtained from the Genoscope protein
database was also performed in an attempt to capture VvSTS
members that might have been missed using the GAZE
and JIGSAW predictions and not included in the
grapevine proteome database. Sequences were edited and
analysed using Vector NTI v9 (Invitrogen) and gene
structure was deduced from Genoscope gene annotation
or from manual annotation based on the genomic
sequences provided by Genoscope and comparison with
the corresponding EST and deduced protein sequences
for paralogous VvSTS genes. The chromosomal location
of VvSTS genes was deduced using the BLAT server and
additional physical localization tools at the Genoscope
Genome Project website. Fragmentary predictions in the
12X PN40024 genomic sequence due to mistakes in the
V1 assembly were substituted by corresponding
sequences obtained from the parallel IASMA sequencing
project obtained from the PN ENTAV 115 genotype 
available at the NCBI database server.
Phylogeny reconstruction and bootstrap analysis
A multiple sequence alignment (MSA) of the VvSTS
deduced proteins, was performed using the E-INSI tool of
the MAFFT 6.0 software , which takes into account
the possibility of large gaps in the alignments. Three CHS
proteins corresponding to CHS1 (AB015872; Vv14s0068
g00930), CHS2 (AB066275; Vv14s0068g00920) and CHS3
(AB066274; Vv05s0136g00260)  were also included in
the analysis. An unrooted phylogenetic tree was generated
with the neighbour-joining method  using MEGA 5.0
software . The best protein substitution model was
chosen using the ProtTest suite . Reliability of tree
obtained was tested using bootstrapping with 1000
replicates. Resulting trees were edited and modified using
Treedyn software (http://www.treedyn.org).
Analysis of a gene expression atlas of V. vinifera cv.
The expression patterns of VvSTS genes predicted from
the analysis of the grapevine genome releases was
analysed in a global V. vinifera cv. Corvina (clone 48) gene
expression atlas of different organs at various
developmental stages. Microarray data were kindly provided
from Prof. Mario Pezzotti (University of Verona, Italy)
for the following tissues: in vitro roots, green stem, buds
after budburst (rosette of leaf tips visible), young leaves
(leaves collected from shoots with only 5 leaves),
senescing leaves (leaves at the beginning of leaf-fall), berry
rachis (from fruit-set to ripening), flowers (50% cap-fall)
and berry pericarp (from fruit set to ripe). In addition,
berries were also examined which had undergone
postharvesting withering for 13 months after harvest.
VvSTS genes encoding for an incomplete ORF were
excluded from the analysis. Genes not represented by a
12X V1 identifier were also excluded. Data were
analysed and expressed graphically by mean of MeV (Multi
Experiment Viewer) software .
mRNA-seq samples preparation and sequencing
For mRNA-seq analysis, leaf discs (15 mm diameter)
were punched from healthy leaves detached from V.
vinifera cv. Pinot noir glasshouse-grown vines. Discs
were randomly selected from the third/forth leaves
collected from different vines, subjected to abiotic and
biotic stresses as described below and incubated upside
down on moist 3MM filter paper in large Petri dishes.
Punching of discs was considered as a wounding
treatment per se, and as a control for other treatments. The
UV-C treatment was achieved by exposing the abaxial
surface of the discs to 30 W UV-C light for 10 mins at a
distance of 10 cm. Downy mildew (Plasmopara viticola)
infection was carried out spraying a solution containing
downy mildew sporangia at concentration of 105
sporangia ml-1. Pinot noir leaf discs were sampled at 0, 24 and
48 h after each treatment and total RNA extracted using
the Spectrum Plant total RNA Kit (Sigma) according to
manufacturers instructions. RNA samples obtained from
different plants were pooled, and 1g of total RNA was
retrotranscribed using the SuperScript III First Strand
Synthesis System for RT-PCR (Invitrogen) with the
oligo (dT)20 primer according to manufacturers
instructions. The first-strand cDNA was initially analysed
for the presence of VvSTS transcripts by PCR using the
degenerate oligonucleotides GGTGACTAAGTCCGAN
CAYATGAC and GACTTTGGCTGTCCCCAYTCYTT
designed using CODEHOP (Consensus-Degenerate
Hybrid Oligonucleotide Primers) software  to ensure
that the desired induction had been obtained. .
Subsequently, 5 g of the same RNA pools were used for
mRNA-seq library preparation and IlluminaW
sequencing at the Institute of Applied Genomic (IGA, Udine,
Italy). Each library had an insert size of 200 bp, and 36
to 39 bp paired ends reads sequenced on an Illumina
Genome Analyzer IIx (GAIIx).
Alignment and analysis of Illumina reads against the
V. vinifera genome
Paired end reads obtained by Illumina mRNA-seq
sequencing were aligned using both the 8.4X and 12X V1
coverage assembly of the PN40024 genotype sequence.
Alignment of reads against the 8.4X reference genome
assembly was carried out using CLC Genomic
Workbench software (http://www.clcbio.com) at the Institute
of Applied Genomics (IGA, Udine, Italy). Sequence
alignment against the 12X V1 coverage, was performed
using ELAND, an un-gapped alignment software
package, which is part of the Illumina pipeline version 1.32.
In both the alignments a maximum of two mismatches
per read was set and, for an accurate measurement of
gene expression, both unique reads and reads that occur
up to ten times were included, to avoid underestimating
the number of genes with closely related paralogues such
as VvSTS. VvSTS members wrongly predicted (VvSTS1,
VvSTS3, VvSTS33 and VvSTS34) or encoding for an
incomplete ORF were excluded from the analysis. Genes
not represented by a 12X V1 identifier were also
excluded. Data were analysed and expressed graphically
by mean of MeV (Multi Experiment Viewer) software
Differential gene expression analysis
The evaluation of gene expression was performed on the
mRNA-seq data obtained from the 8.4X and the 12X V1
coverage respectively with CLC Genomic workbench
and ERANGE 3.1 programs . In both cases, the
transcriptional activity of each gene was defined as the
number of mapped reads per kilobase of exon per
million mapped reads (RPKM):
total exon reads
RPKM mapped readsmillion exon lengthKb
Both programs compute the normalized gene locus
expression level by assigning reads to their site of origin
and counting them. In the case of reads that match
equally in multiple loci, they are distributed
proportionally to the weight of expression level given by specific
single-matching reads. This means that if there are 10
reads that match two different genes with equal exon
length, the two reads will be distributed according to the
number of unique matches for these two genes. The
gene that has the highest number of unique matches will
thus get a greater proportion of the 10 reads. If a read
has more hits than specified with this maximum number
of hits setting, it will be ignored. Expression values were
graphically represented using Multi Experiment Viewer
software (MeV; http://www.tm4.org/mev/; ).
Validation of mRNA-seq data by quantitative real-time
PCR expression analysis
Leaf discs (15 mm diameter) were punched from
healthy leaves detached from glasshouse-grown V.
vinifera cv. Shiraz vines. Discs were obtained from leaves
belonging to different plants and showing similar age
based on size and node positions in plants, treated
with the same different biotic and abiotic stresses
previously described and incubated upside down on 3MM
moist filter paper in large Petri dishes at 22C under
12 h light / 12 h dark conditions until harvest at
which point discs were immediately frozen in liquid
nitrogen and stored at 80C until RNA extraction.
Five discs were randomly chosen from different
treatments, at 0, 8, 16, 24, 48, 72 and 96 h after wound
treatment, 0, 4, 8, 24 and 48 h after UV-C treatment
and 0, 8, 24, 48 an 48 h after downy inoculation, dried
with absorbent paper and immediately frozen in liquid
nitrogen until extraction.
Selective primers were designed across dissimilar
exonic DNA stretches or using a 3-terminal SNP between
the perfect match of the target gene-copy and the
mismatched annealing site of paralogous sequences. Melt
curve analysis, agarose gel electrophoresis, and DNA
sequencing validated the absence of illegitimate
crossamplification of other paralogues. Expression analyses
were carried by quantitative real-time PCR using a Sybr
green method on a Rotor-Gene 3000 (Corbett Research,
Mortlake, Australia) thermal cycler. Each 15ul PCR
reaction contained 330 nM of each primer, 2ul of diluted
cDNA, 1X FastStart Sybr green (Roche) and sterile
water. The thermal cycling conditions used were 94C
for 10 min followed by 40 cycles of: 95C for 30 s, 60C
for 30 s, and 72C for 30 s, followed by a melt cycle with
1C increments from 55 to 96C. Real time PCR data
processing was performed using the standard curve
method. Standard curves were constructed using 10-fold
serial dilutions, using cDNA from samples and stages in
which the specific gene-copy was expressed or, if not
possible, genomic DNA. In order to compare the
expression level of different members belonging to the same
PKS family, the actual transcript copy number was
calculated based on the length of the product of
amplification and its concentration in standard dilutions used to
calculate the expression level. After testing the suitability
of 18 S, actin and elongation factor EF1 for use of
reference genes, elongation factor was selected for
normalization of all samples analysed. The expression of
each target gene was calculated relative to the expression
of elongation factor in each cDNA using Rotor-Gene 6
Software (Corbett Research, Mortlake, Australia) to
calculate CT values, observe melt profiles, extrapolate the
concentration and measure primer pairs efficiencies. The
primers used were: VvSTS6, VvSTS6F2
5GTTGTGCTGCATAGCGTTGC-3 and 5-GATTTAA
TTGGAAATTGTCCCCTTC-3; VvSTS16, VvSTS16F2
5CTTTTGACCCAATTGGAATCAAC-3 and VvSTS16R3
VvSTS48F 5-CTTGAAGGGGGAAAATGCT-3 and
VvSTS48R 5-TTACTGCATTGAAGGGTA AACC-3.
Additional file 1: Description of mutations/SNPs in predicted VvSTS
gene sequences from comparison of published grape genome
sequences (PN40024 & PN ENTAV 115) and reads obtained from
mRNA-seq analysis in this study.
Additional file 2: Alignment of VvSTS and VvCHS protein
sequences. This figure shows the alignment of three entire VvSTSs
deduced protein sequences representative of A- (VvSTS6), B- (VvSTS48)
and C- (VvSTS16) groups with the three VvCHS proteins. The alignment
was determined using MAFFT software and edited with GeneDoc
software. The conserved CHS/STS active site is highlighted in green and
differences in amino acid residues between VvSTS and VvCHS are
highlighted in red.
Additional file 3: Robust Multichip Average (RMA) normalised
expression data for selected VvSTS and VvCHS genes in the V.
vinifera cv Corvina atlas. Each hybridization was carried out on a
NibleGen microarray 090818 Vitis exp HX12 (Roche, NimbleGen Inv.,
Madison, WI), representing 29549 predicted genes on the basis of the
12X grapevine V1 gene prediction version (http://srs.ebi.ac.uk/). The chip
probe design is available at the following URL: http.//ddlab.sci.univr.it/
FunctionalGenomics/. Normalised expression data here reported are
limited to a subset of selected genes (VvSTS and VvCHS) and tissues from
the whole data set (Fasoli et al., in preparation) and represent the
averaged intensity of each gene in three biological replicates of each
sample. A Pearson Correlation was previously carried out to evaluate the
consistency of the biological replicates in each sample (R software). Y,
young leaf; FS, fruit-set; S, senescence; G, green stem; AB, bud-burst; PFS,
post fruit-set; V, vraison; MR, mid-ripe; R, ripe; F, flowering (50% cap-fall);
PHWI, post-harvest withering I (1st month); PHWII, post-harvest withering
II (2nd month); PHWIII, post-harvest withering III (3rd month).
Additional file 4: Expression image of the complete VvSTS family in
Corvina Berries undergoing withering process. This picture illustrate
more in detail the expression of VvSTS and VvCHS genes in berry tissues
(skin and flesh) during the last developmental phases and withering
process. Expression values are normalised based on the mean expression
value of each gene in all tissues/organs analysed. Different organs/tissues
are displayed vertically above each column. VvSTS gene names are
displayed to the right of each row and are clustered in different groups
A, B, C according to protein homology as shown in Figure 2.
Additional file 5: VvSTS and VvCHS genes RPKM expression data
obtained from Illumina Genome Analyser II (GAII). Here we report
the RPKM expression values of all VvSTS and VvCHS considered in this
study (Figure 4). Data shown were obtained by aligning paired end reads
on the 12X V1 coverage assembly of the PN40024 genome sequence
with ELAND, an ungapped alignment software package, which is part of
the Illumina pipeline version 1.32. Differential gene expression analyses
were performed by ERANGE 3.1 program. A maximum of two
mismatches was set and, for an accurate measurement of gene
expression, both unique reads and reads that occur up to ten times were
included, to avoid underestimating the number of genes with closely
related paralogues such as VvSTSs.
Additional file 6: Alignment of truncated STS protein sequences.
VvSTS1 and VvSTS4 deduced truncated proteins were aligned with a
grapevine full-length STS (VvSTS48) and the three STS genes from P.
densiflora (PdSTS1, PdSTS2 and PdSTS3). Alignment was obtained using
MAFFT software and edited with GeneDoc software. The CHS/STS active
site is highlighted in green. Stop codons are highlighted in red for those
sequences considered of interest because they still contain the active
Authors declare that in the past five years have not received
reimbursements, fees, funding, or salary from an organization that may in
any way gain or lose financially from the publication of this manuscript,
either now or in the future. Authors do not hold any stocks or shares in an
organization that may in any way gain or lose financially from the
publication of this manuscript, either now or in the future. Authors do not
hold or are currently applying for any patents relating to the content of the
manuscript. Authors did not receive reimbursements, fees, funding, or salary
from an organization that holds or has applied for patents relating to the
content of the manuscript. Authors do not have any other financial or
non-financial (political, personal, religious, ideological, academic, intellectual,
commercial or any other) competing interest to declare in relation to this
AV conceived the design of this study, planned and conducted most of the
lab experiments, performed the bioinformatic data analysis and wrote the
manuscript; IBD strongly contributed to the experimental planning, to the
interpretation of results and participated in drafting the manuscript; MF and
SZ analyzed and kindly provided data obtained from V. vinfera cv. Corvina
expression Atlas; ML devised and supervised the study, contributed in
interpretation of results and critically revised the manuscript. All authors have
read and approved the final manuscript. The mRNA-seq data were
submitted to Gene Expression Omnibus (NCBI) and are accessible through
GEO accession number GSE37743.
Authors would like to thank Dr Marzia Salmaso and Dr Sabrina Canova
(Department of Environmental Agronomy and Crop Protection of the
University of Padova, Legnaro, Italy) for their technical assistance, Prof.
Michele Morgante and Dr Federica Cattonaro (Istituto di Genomica
Applicata, Udine, Italy) for providing mRNA-seq data analysis on the 8X
coverage of the PN40024 genotype, Prof. Mario Pezzotti, who provided the
grapevine expression atlas for the expression analysis of STS genes in
grapevine tissues, Dr. Alberto Ferrarini (Department of biotechnology,
University of Verona, Italy) for his assistance with the alignments and analysis
of mRNA-seq output data on the grapevine 12X V1 coverage genome
assembly, and Prof. Annalisa Polverari who provided the P. viticola inoculum.
Thanks also to Dr. Mandy Walker for providing some VvCHS oligos and
Angelica Jermakow and Angela Feechan for their technical support at the
CSIRO Plant Industry (Urrbrae, SA). This work was partially supported by the
Veneto region Distretto del vino Progetto ValViVe and by the Italian
project AGER-SERRES, 20102105.
1. Kenrick P , Crane PR : The origin and early evolution of plants on land . Nature 1997 , 387 : 33 - 39 .
2. Waters E : Molecular adaptation and the origin of land plants . Mol Phylogenet Evol 2003 , 29 : 456 - 460 .
3. Emiliani G , Fondi M , Fani R , Gribaldo S : A horizontal gene transfer at the origin of phenylpropanoid metabolism: a key adaptation of plants to land . Biol Direct 2009 , 4 : 7 .
4. Noel JP , Austin MB , Bomati EK : Structure-function relationship in plant phenylpropanoid biosynthesis . Curr Opin Plant Biol 2005 , 8 : 249 - 253 .
5. Kuc J : Phytoalexins, stress metabolism, and disease resistance in plants . Annu Rev Phytopathol 1995 , 33 : 275 - 297 .
6. Purkayashta RP : Progress in phytoalexin research during the past 50 years . In Handbook of Phytoalexin Metabolism and Action . Edited by Daniel M, Purkayashta RP . New York : Marcel Dekker ; 1995 : 1 - 39 .
7. Harborne JB : The comparative biochemistry of phytoalexins induction in plants . Biochem Syst Ecol 1999 , 27 : 335 - 367 .
8. Counet C , Callemien D , Collin S : Chocolate and cocoa: new sources of trans- resveratrol and trans-piceid . Food Chem 2006 , 98 : 649 - 657 .
9. Sotheeswaran S , Pasuphaty V : Distribution of resveratrol oligomers in plants . Phytochemistry 1993 , 32 : 1083 - 1092 .
10. Yu CK , Springob K , Schmidt J , Nicholson RL , Chu IK , Yip WK , Lo C : A stilbene synthase gene (SbSTS1) is involved in host and non-host defense responses in sorghum . Plant Physiol 2005 , 138 : 393 - 401 .
11. Waterhouse AL , Lamuela-Raventos RM : The occurrence of piceid, a stilbene glucoside , in grape berries. Phytochemistry 1994 , 37 : 571 - 573 .
12. Waffo-Teguo P , Descendit A , Deffieux G , Vercauteren J , Mrillon JM : Transresveratrol-3-O--glucoside (piceid) in cell-suspension cultures of Vitis vinifera . Phytochemistry 1996 , 42 : 1591 - 1593 .
13. Romero-Prez AI , Lamuela-Raventos RM , Andreas-Lacueva C , de la TorreBoronat MC : Method for the quantitative extraction of resveratrol and piceid isomers in grape berry skins. Effect of powdery mildew on the stilbene content . J Agric Food Chem 2001 , 49 : 210 - 215 .
14. Gatto P , Vrhovsek U , Muth J , Segala C , Romualdi C , Fontana P , Prueafer D , Stefanini M , Moser C , Mattivi F , Velasco R : Ripening and genotype control stilbene accumulation in healthy grapes . J Agric Food Chem 2008 , 56 : 11773 - 11785 .
15. Langcake P : Disease resistance of Vitis spp. and the production of stress metabolites resveratrol, e-viniferin, -viniferin and pterostilbene . Physiol Plant Pathol 1981 , 18 : 213 - 226 .
16. Langcake P , McCarty WV : The relationship of resveratrol production to infection of grapevine leaves by Botrytis cinerea . Vitis 1979 , 18 : 244 - 253 .
17. Bavaresco L , Fregoni M , Trevisan M , Mattivi F , Vrhovsek U , Falchetti R : The occurrence of the stilbene piceatannol in grapes . Vitis 2002 , 41 : 133 - 136 .
18. Hart JH : Role of phytostilbenes in decay and disease resistance . Ann Rev Phytopathol 1981 , 19 : 437 - 445 .
19. Jayatilake GS , Jayasuriya H , Lee ES , Koonchanok NM , Geahlen RL , Ashendel CL , McLaughlin JL , Chang CJ : Kinase inhibitors from Polygonum cuspidatum . J Nat Prod 1993 , 56 : 1805 - 1810 .
20. Benova B , Adam M , Onderkov K , Krlowsk J , Krajcek M : Analysis of selected stilbenes in Polygonum cuspidatum by HPLC coupled with CoulArray detection . J Sep Sci 2008 , 31 : 2404 - 2409 .
21. Barnes RA , Gerber NN : The antifungal agent from Osage orange wood . J Am Chem Soc 1955 , 77 : 3259 - 3262 .
22. Hathway DE : The use of hydroxystilbene compounds as taxonomic tracers in the genus Eucalyptus . Biochem 1962 , 83 : 80 - 84 .
23. Hillis WE : Properties of eucalypt woods of importance to the pulp and paper industries . Appita 1972 , 26 : 113 - 122 .
24. Chiron H , Drouet A , Lieutier F , Payer HD , Ernst D , Sandermann H : Gene induction of stilbene biosynthesis in Scots pine in response to ozone treatment, wounding and fungal infection . Plant Physiol 2000 , 124 : 865 - 872 .
25. Pezet R , Perret C , Jean-Denis JB , Tabacchi R , Gindro K , Viret O : -viniferin, a resveratrol dehydrodimer: one of the major stilbenes synthesized by grapevine leaves . J Agric Food Chem 2003 , 51 : 5488 - 5492 .
26. Adrian M , Jeandet P , Douillet-Breuil AC , Tesson L , Bessis R : Stilbene content of mature Vitis vinifera berries in response to UV-C elicitation . J Agric Food Chem 2000 , 48 : 6103 - 6105 .
27. Wang W , Tang K , Yang HR , Wen PF , Zhang P , Wang HL , Huang WD : Distribution of resveratrol and stilbene synthase in young grape plants (Vitis vinifera L. cv. Cabernet Sauvignon) and the effect of UV-C on its accumulation . Plant Physiol Biochem 2010 , 48 : 142 - 152 .
28. Rosemann D , Heller W , Sandermann H : Biochemical plant responses to ozone: II induction of stilbene biosynthesis in Scots pine ( Pinus sylvestris L.) seedlings. Plant Physiol 1991 , 97 : 1280 - 1286 .
29. Adrian M , Jeandet P , Bessis R , Joubert JM : Induction of phytoalexins (resveratrol) synthesis in grapevine leaves treated with aluminium chloride (AlCl3) . J Agric Food Chem 1996 , 44 : 1979 - 1981 .
30. Zamboni A , Minoia L , Ferrarini A , Tornielli GB , Zago E , Delledonne M , Pezzotti M : Molecular analysis of post-harvest withering in grape by AFLP transcriptional profiling . J Exp Bot 2008 , 59 : 4145 - 4159 .
31. Belhadj A , Telef N , Cluset S , Bouscaut J , Corio-Costet MF , Merillon JM : Ethephon elicits protection against Erisyphe necator in grapevine . J Agric Food Chem 2008 , 56 : 5781 - 5787 .
32. Belhadj A , Telef N , Saigne C , Cluzet S , Barrieu F , Hamdi S , Merillon JM : Effect of methyl jasmonate in combination with carbohydrates on gene expression of PR proteins, stilbene and anthocyanin accumulation in grapevine cell cultures . Plant Physiol Biochem 2008 , 46 : 493 - 499 .
33. Vezzulli S , Civardi S , Ferrari F , Bavaresco L : Methyl jasmonate treatment as a trigger of resveratrol synthesis in cultivated grapevine . Am J Enol Vitic 2007 , 58 : 530 - 533 .
34. Fung RWM , Gonzalo M , Fekete C , Kovacs LG , He Y , Marsh E , McIntyre LM , Schachtman DP , Qiu W : Powdery mildew induces defense-oriented reprogramming of the transcriptome in a susceptible but not in a resistant grapevine . Plant Physiol 2008 , 146 : 236 - 249 .
35. Schnee S , Viret O , Gindro K : Role of stilbenes in the resistance of grapevine to powdery mildew . Physiol Mol Plant Pathol 2008 , 72 : 128 - 133 .
36. Langcake P , Pryce RJ : The production of resveratrol by Vitis vinifera and other members of the Vitaceae as a response to infection or injury . Physiol Plant Pathol 1976 , 9 : 77 - 86 .
37. Adrian M , Jeandet P , Duillet-Breuil AC , Tesson L , Bessis R : Biological activity of resveratrol, a stilbenic compound from grapevines, against Botrytis cinerea, the causal agent for gray mold . J Chem Ecol 1997 , 23 : 1689 - 1702 .
38. Bzier A , Lambert B , Baillieul F : Study of defense-related gene expression in grapevine leaves and berries infected with Botrytis cinerea . Euro J Plant Pathol 2002 , 108 : 111 - 120 .
39. Bavarersco L , Vezzulli S , Battilani P , Giorni P , Pietri A , Bertuzzi T : Effect of ochratoxin A-producing Aspergilli on stilbenic phytoalexin synthesis in grapes . J Agric Food Chem 2003 , 51 : 6151 - 6157 .
40. Vezzulli S , Battilani P , Bavaresco L : Stilbene-synthase gene expression after Aspergillus carbonarius infection in grapes . Am J Enol Vitic 2007 , 58 : 132 - 134 .
41. Schppner A , Kindl H : Purification and properties of a stilbene synthase induced cell suspension of peanut . J Biol Chem 1984 , 259 : 6806 - 6811 .
42. Lanz T , Tropf S , Marner FJ , Schrder J , Schrder G : The role of cysteines in polyketide synthases. Site-directed mutagenesis of resveratrol and chalcone synthases, 2 key enzymes in different plant-specific pathways . J Biol Chem 1991 , 266 : 9971 - 9976 .
43. Ferrer JL , Austin MB , Stewart C , Noel JP : Structure and function of enzymes involved in the biosynthesis of phenylpropanoids . Plant Physiol Biochem 2008 , 46 : 356 - 370 .
44. Tropf S , Lanz T , Rensing SA , Schrder J , Schrder G : Evidence that stilbene synthases have developed from chalcone synthases several times in the course of evolution . J Mol Evol 1994 , 38 : 610 - 618 .
45. Schrder G , Brown JWS , Schrder J : Molecular analysis of resveratrol synthase cDNA, genomic clones and relationship with chalcone synthase . Euro J Biochem 1988 , 172 : 161 - 169 .
46. Raiber S , Schrder G , Schrder J : Molecular and enzymatic characterization of two stilbene synthases from Eastern white pine (Pinus strobus). A single Arg/His difference determines the activity and the pH dependence of the enzyme . FEBS Lett 1995 , 361 : 299 - 302 .
47. Preising-Mller R , Schwekendiek A , Brehm I , Reif HJ , Helmut K : Characterization of a pine multigenic family containing elicitor responsive stilbene synthase genes . Plant Mol Biol 1999 , 39 : 221 - 229 .
48. Kodan A , Kuroda H , Sakai F : A stilbene synthase from Japanese red pine (Pinus densiflora): implications for phytoalexins accumulation and downregulation of flavonoid biosynthesis . Proc Natl Acad Sci U S A 2002 , 99 : 3335 - 3339 .
49. Paterson AH , Bowers JE , Bruggmann R , Dubchak I , Grimwood J , Gundlach H , Haberer G , Hellsten U , Mitros T , Poliakov A , Schmutz J , Spannagl M , Tang H , Wang X , Wicker T , Bharti AK , Chapman J , Feltus FA , Gowik U , Grigoriev IV , Lyons E , Maher CA , Martis M , Narechania A , Otillar RP , Penning BW , Salamov AA , Wang Y , Zhang L , Carpita NC , et al: The Sorghum bicolor genome and the diversification of grasses . Nature 2009 , 457 : 551 - 556 .
50. Jaillon O , Aury JM , Noel B , Policriti A , Clepet C , Casagrande A , Choisne N , Aubourg S , Vitulo N , Jubin C , Vezzi A , Legeai F , Hugueney P , Dasilva C , Horner D , Mica E , Jublot D , Poulain J , Bruyere C , Billault A , Segurens B , Gouyvenoux M , Ugarte E , Cattonaro F , Anthouard V , Vico V , Del Fabbro C , Alaux M , Di Gaspero G , Dumas V , et al: The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla . Nature 2007 , 449 : 463 - 467 .
51. Velasco R , Zharkikh A , Troggio M , Cartwright DA , Cestaro A , Pruss D , Pindo M , Fitzgerald LM , Vezzulli S , Reid J , Malacarne G , Iliev D , Coppola G , Wardell B , Micheletti D , Macalma T , Facci M , Mitchell JT , Perazzolli M , Eldredge G , Gatto P , Oyzerski R , Moretto M , Gutin N , Stefanini M , Chen Y , Segala C , Davenport C , Dematte L , Mraz A , et al: A high quality draft consensus sequence of the genome of a heterozygous grapevine variety . PLoS One 2007 , 2 : e1326 .
52. Sparvoli F , Martin C , Scienza A , Gavazzi G , Tonelli C : Cloning and molecular analysis of structural genes involved in flavonoid and stilbene biosynthesis in grape ( Vitis vinifera L.). Plant Mol Biol 1994 , 24 : 743 - 755 .
53. Zenoni S , Ferrarini A , Giacomelli E , Xumerle L , Fasoli M , Malerba G , Bellin D , Pezzotti M , Delledonne M : Characterization of transcriptional complexity during berry development in Vitis vinifera using RNA-seq . Plant Physiol 2010 , 152 : 1787 - 1795 .
54. Morales M , Ros Barcelo A , Pedreno MA : Plant stilbenes: recent advances in their chemistry and biology . In, Advances in Plant Physiology, Volume 3 . Edited by Hemantaranjan A. Jodhpur, India: Scientific Publishers ; 2000 : 39 - 70 .
55. Falginella L , Di Castellarin S , Testolin R , Gambetta GA , Morgante M , Di Gaspero G : Expansion and subfunctionalization of flavonoid 3',5'-hydroxylases in the grapevine lineage . BMC Genomics 2010 , 11 : 562 .
56. Moore RC , Purugganan MD : The early stages of duplicate gene evolution . Proc Natl Acad Sci U S A 2003 , 100 : 15682 - 15687 .
57. Tang H , Wang X , Bowers JE , Ming R , Alan M , Paterson AH : Unravelling ancient hexaploidy through multiply-aligned angiosperm gene maps . Genome Res 2008 , 18 : 1944 - 1954 .
58. Giannuzzi G , D'Addabbo P , Gasparro M , Martinelli M , Carelli FN , Antonacci D , Ventura M : Analysis of high-identity segmental duplications in the grapevine genome . BMC Genomics 2011 , 12 : 436 .
59. Casneuf T , De Bodt S , Raes J , Maere S , Van de Peer Y : Nonrandom divergence of gene expression following gene and genome duplications in the flowering plant Arabidopsis thaliana . Genome Biol 2006 , 7 : R13 .
60. Hanada K , Zou C , Lehti-Shiu MD , Shinozaki K , Shiu SH : Importance of lineage-specific expansion of plant tandem duplicates in the adaptive response to environmental stimuli . Plant Physiol 2008 , 148 : 993 - 1003 .
61. Keeling CI , Weisshaar S , Lin RPC , Bohlmann J : Functional plasticity of paralogous diterpene synthases involved in conifer defense . Proc Natl Acad Sci U S A 2008 , 105 : 1085 - 1090 .
62. Ober D : Seeing double: gene duplications and diversification in plant secondary metabolism . Trends Plant Sci 2005 , 10 : 444 - 449 .
63. Chapman BA , Bowers JE , Feltus FA , Paterson AH : Buffering of crucial functions by paleologus duplicated genes may contribute cyclicality to angiosperm genome duplication . Proc Natl Acad Sci U S A 2006 , 103 : 2730 - 2735 .
64. Ha M , Li WH , Chen J : External factors accelerate expression divergence between duplicate genes . Trends Genet 2007 , 23 : 162 - 166 .
65. Chang X , Heene E , Qiao F , Nick P : The phytoalexin resveratrol regulates the initiation of hypersensitive cell death in Vitis cell . PLoS One 2011 , 6 : e26405 .
66. Goto-Yamamoto N , Wan GH , Masaki K , Kobayashi S : Structure and transcription of three chalcone synthase genes of grapevine (Vitis vinifera) . Plant Sci 2002 , 162 : 867 - 872 .
67. Korhammer S , Reniero F , Mattivi F : An oligostilbene from Vitis roots . Phytochem 1995 , 38 : 1501 - 1504 .
68. Versari A , Parpinello GP , Tornielli GB , Ferrarini R , Giulivo C : Stilbene compounds and stilbene synthase expression during ripening, wilting and UV treatment in grape cv . Corvina. J Agric Food Chem 2001 , 92 : 729736 .
69. Burns J , Yokota T , Ashiara H , Lean MEJ , Crozier A : Plant food and herbal sources of resveratrol . J Agric Food Chem 2002 , 50 : 3337 - 3340 .
70. Hall D , De Luca V : Mesocarp localization of a bi-functional resveratrol/ hydroxicinnamic acid glucosyltransferase of Concord grape (Vitis lambrusca) . Plant J 2007 , 49 : 579 - 591 .
71. Espinoza C , Medina C , Somerville S , Arce-Johnson P : Senescence-associated genes induced during compatible viral interactions with grapevine and Arabidopsis . J Exp Bot 2007 , 58 ( 12 ): 3197 - 3212 .
72. Guiboileau A , Sormani R , Meyer C , Masclaux-Daubresse C : Senescence and death of plant organs: Nutrient recycling and developmental regulation . CR Biol 2010 , 333 : 382 - 391 .
73. Quirino BF , Noh YS , Himelblau E , Amasino RM : Molecular aspects of leaf senescence . Trends Plant Sci 2000 , 5 : 278 - 282 .
74. D'Onofrio C , Cox A , Davies C , Boss PK : Induction of secondary metabolism in grape cell cultures by jasmonates . Funct Plant Biol 2009 , 36 : 323 - 338 .
75. Fornara V , Onelli E , Sparvoli F , Rossoni M , Aina R , Marino G , Citterio S : Localization of stilbene synthase in Vitis vinifera L. during berry development . Protoplasma 2008 , 233 : 83 - 93 .
76. Borie B , Jeandet P , Parize A , Bessis R , Adrian M : Resveratrol and stilbene synthase mRNA production in grapevine leaves treated with biotic and abiotic phytoalexin elicitors . Amer J Enol Vitic 2004 , 55 : 60 - 64 .
77. Wiese W , Vornam B , Krause E , Kindl H : Structural organization and differential expression of three stilbene synthase genes located on a 13 kb grapevine DNA fragment . Plant Mol Biol 1994 , 26 : 667 - 677 .
78. Grimmig B , Gonzalez-Perez MN , Leubner-Metzger G , Vogeli-Lange R , Meins F , Hain R , Penuelas J , Heidenreich B , Langebar-Tels C , Ernst D , Sandermann H : Ozone-induced gene expression occurs via ethylene-depedent and -independent signalling . Plant Mol Biol 2003 , 51 : 599 - 607 .
79. Tassoni A , Fornale S , Franceschetti M , Musiani F , Michael AJ , Perry B , Bagni N : Jasmonates and Na-orthovanadate promote resveratrol production in Vitis vinifera cv . Barbera cell cultures. New Phytol 2005 , 166 : 895 - 905 .
80. Faurie B , Cluzet S , Corio-Costet MF , Mrillon JM : Methyl jasmonates/ Ethephon synergistically induces stilbene production in Vitis vinifera cell suspensions but fails to trigger resistance to Erysiphe necator . J Int Sci Vigne Vin 2009 , 43 : 99 - 110 .
81. Zhao J , Davis LC , Verpoorte R : Elicitor signal transduction leading to production of plant secondary metabolites . Biotechnol Adv 2005 , 23 : 283 - 333 .
82. Stracke R , Ishihara H , Huep G , Barsch A , Mehrtens F , Niehaus K , Weisshaar B : Differential regulation of closely related R2R3-MYB transcription factors controls flavonol accumulation in different parts of the Arabidopsis thaliana seedling . Plant J 2007 , 50 : 660 - 677 .
83. Deluc L , Barrieu F , Marchive C , Lauvergeat V , Decendit A , Richard T , Carde JP , Merillon JM , Hamdi S : Characterization of a grapevine R2R3-MYB transcription factor that regulates the phenylpropanoid pathway . Plant Physiol 2006 , 140 : 499 - 511 .
84. Bogs J , Jaff FW , Takos AM , Walker AR , Robinson SP : The grapevine transcription factor VvMybPA1 regulates proanthocyanidin synthesis during fruit development . Plant Physiol 2007 , 143 : 1347 - 1361 .
85. Deluc L , Bogs J , Walker AR , Ferrier T , Decendit A , Merillon JM , Robinson SP , Barrieu F : The transcription factor VvMYB5b contributes to the regulation of anthocyanin and proanthocyanidin biosynthesis in developing grape berries . Plant Physiol 2008 , 147 : 2041 - 2053 .
86. Czemmel S , Stracke R , Weisshaar B , Cordon N , Harris NN , Walker AR , Robinson SP , et al: The grapevine R2R3-MYB transcription factor VvMYBF1 regulates flavonol synthesis in developing grape berries . Plant Physiol 2009 , 151 : 1513 - 1530 .
87. Terrier N , Torregrosa L , Ageorges A , Vialet S , Verries C , Cheynier V , Romieu C : Ectopic expression of VvMybPA2 promotes proanthocyanidin biosynthesis in Vitis vinifera L. and suggests additional targets in the pathway . Plant Physiol 2009 , 149 : 1028 - 1041 .
88. Chong J , Poutaraud A , Hugueney P : Metabolism and roles of stilbenes in plants . Plant Sci 2009 , 177 : 143 - 155 .
89. Jeandet P , Delaunois B , Conreux A , Donnez D , Nuzzo V , Cordelier S , Clment C , Courot E : Biosynthesis, metabolism, molecular engineering, and biological functions of stilbene phytoalexins in plants . BioFactors (Oxford, England) 2010 , 36 : 331 - 341 .
90. Dao TTH , Linthorst HJM , Verpoorte R : Chalcone synthase and its functions in plant resistance . Phytochem Rev 2011 , 10 : 397 - 412 .
91. Lozoya E , Block A , Lois R , Hahlbrock K , Scheel D : Transcriptional repression of light-induced flavanoid synthesis by elicitor treatment of cultured parsley cells . Plant J 1991 , 1 : 227 - 234 .
92. Lo SC , Nicholson RL : Reduction of light-induced anthocyanin accumulation in inoculated sorghum mesocotyls. Implications for a compensatory role in the defense response . Plant Physiol 1998 , 116 : 979 - 989 .
93. McLusky SR , Bennett MH , Beale MH , Lewis MJ , Gaskin P , Mansfield JW : Cell wall alterations and localized accumulation of feruloyl-3- methoxytyramine in onion epidermis at sites of attempted penetration by Botrytis allii are associated with actin polarisation, peroxidase activity and suppression of flavonoid biosynthesis . Plant J 1999 , 17 : 523 - 534 .
94. Logemann E , Wu SC , Schrder JJ , Schmelzer E , Somssich IE , Hahlbrock K : Gene activation by UV light, fungal elicitor or fungal infection in Petroselinum crispum is correlated with repression of cell cycle-related genes . Plant J 1995 , 8 : 865 - 876 .
95. Schenke D , Bttcher C , Scheel D : Crosstalk between abiotic ultraviolet-B stress and biotic (flg22) stress signalling in Arabidopsis prevents flavonol accumulation in favour of pathogen defence compound production . Plant Cell Environ 2011 , 34 : 1849 - 1864 .
96. Altschul SF , Gish W , Miller W , Myers EW , Lipman DJ : Basic local alignment search tool . J Mol Biol 1990 , 215 : 403 - 410 .
97. The grape genome Browser . http://www.genoscope.cns.fr/externe/ GenomeBrowser/Vitis/
98. The National Center for Biotechnology information . http://www.ncbinlm. nih.gov/
99. Grape Genome 12X. http://gbrowse.cribi.unipd.it/private/gbrowse/ vitis_vinifera/
100. Sigrist CJA , Cerutti L , de Castro E , Lagendijk-Genevaux PS , Bulliard V , Bairoch A , Hulo N : PROSITE, a protein domain database for functional characterization and annotation . Nucleic Acids Res 2010 , 38 : 161 - 166 .
101. Katoh T : Parallelization of the MAFFT multiple sequence alignment program . Bioinformatics 2010 , 26 : 1899 - 1900 .
102. Saitou N , Nei M : The neighbour-joining method: a new method for reconstructing phylogenetic trees . Mol Biol Evol 1987 , 4 : 406 - 425 .
103. Tamura K , Peterson D , Peterson N , Stecher G , Nei M , Kumar S : MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods . Mol Biol Evol 2011 , 28 : 2731 - 2739 .
104. Abascal F , Zardoya R , Posada D : ProtTest: Selection of best-fit models of protein evolution . Bioinformatics 2005 , 21 : 2104 - 2105 .
105. Saeed A , Bhagabati NK , Braisted JC , Liang W , Sharov V , Howe EA , Li J , Thiagarajan M , Whaite JA , Quackenbush J : TM4 microarray software suite . Methods Enzymol 2006 , 411 : 134 - 193 .
106. Rose TM , Schultz ER , Henikoff JG , Pietrokovsky S , McCallum CM , Henikoff S : Consensus-degenerate hybrid oligonucleotide primers for amplification of distantly-related sequences . Nucleic Acids Res 1998 , 26 : 1628 - 1635 .
107. Wold Lab Caltech Biology. [http://woldlab.caltech.edu/RNA-seq]