The Phytoalexin Resveratrol Regulates the Initiation of
Hypersensitive Cell Death in
Vitis
Cell
Xiaoli Chang
1
*, Ernst Heene
1
, Fei Qiao
2
, Peter Nick
1
1 Department of Molecular Cell Biology, Botanical Institute 1, Karlsruhe Institute of Technology, Karlsruhe, Germany, 2 Institute of Tropical Crops Genetic Resources,
Chinese Academy of Tropical Agricultural Sciences, Danzhou, China
Abstract
Resveratrol is a major phytoalexin produced by plants in response to various stresses and promotes disease resistance. The
resistance of North American grapevine Vitis rupestris is correlated with a hypersensitive reaction (HR), while susceptible
European Vitis vinifera cv. ‘Pinot Noir’ does not exhibit HR, but expresses basal defence. We have shown previously that in
cell lines derived from the two Vitis species, the bacterial effector Harpin induced a rapid and sensitive accumulation of
stilbene synthase (StSy) transcripts, followed by massive cell death in V. rupestris. In the present work, we analysed the
function of the phytoalexin resveratrol, the product of StSy. We found that cv. ‘Pinot Noir’ accumulated low resveratrol and
its glycoside trans-piceid, whereas V. rupestris produced massive trans-resveratrol and the toxic oxidative d-viniferin,
indicating that the preferred metabolitism of resveratrol plays role in Vitis resistance. Cellular responses to resveratrol
included rapid alkalinisation, accumulation of pathogenesis-related protein 5(PR5) transcripts, oxidative burst, actin
bundling, and cell death. Microtubule disruption and induction of StSy were triggered by Harpin, but not by resveratrol.
Whereas most responses proceeded with different amplitude for the two cell lines, the accumulation of resveratrol, and the
competence for resveratrol-induced oxidative burst differed in quality. The data lead to a model, where resveratrol, in
addition to its classical role as antimicrobial phytoalexin, represents an important regulator for initiation of HR-related cell
death.
Citation: Chang X, Heene E, Qiao F, Nick P (2011) The Phytoalexin Resveratrol Regulates the Initiation of Hypersensitive Cell Death in Vitis Cell. PLoS ONE 6(10):
e26405. doi:10.1371/journal.pone.0026405
Editor: Mikhail V. Blagosklonny, Roswell Park Cancer Institute, United States of America
Received August 12, 2011; Accepted September 26, 2011; Published October 28, 2011
Copyright: ß 2011 Chang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by fellowships of the Chinese Scholarship Council to Xiaoli Chang. The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: chang.xiaoli@bio.uni-karlsruhe.de
Introduction
Grapevine, an economically and culturally important crop, is
affected by a wide range of pathogens, causing yield losses and
impairing wine quality. During a long history of coevolution
between host and pathogens such as Downy and Powdery Mildew,
North American Vitis species have developed defence mechanisms
based on specific host receptors capable to activate defence after
recognition of pathogen effectors (effector-triggered immunity, ETI)
[1], often culminating in a hypersensitive reaction (HR). HR is a
plant-specific form of programmed cell death (PCD) associated with
plant resistance to pathogen infection and characterized by a rapid
and localized death of tissues at the site of infection to limit further
pathogen multiplication and spread [2,3]. Since Downy and
Powdery Mildew arrived in Europe only in 1869, ETI against
these pathogens is absent in the cultivated grapevine Vitis vinifera.
However, so called basal defence, effective against a broad variety of
pathogen strains, but not culminating in HR, is active in grapevine.
Although triggered by different receptors, both responses share
molecular components and cellular mechanisms, and differ on the
quantitative rather than on the qualitative level [4].
Phytoalexins, a functionally defined class of secondary metab-
olites, are generated de novo in response to stress factors such as
pathogen attack. In grapevine, stilbenes, in general, and
resveratrol (trans-3, 49, 5-trihydroxystilbene) in particular, are
well-known phytoalexins active against Downy and Powdery
Mildew. Transcription of stilbene synthases (StSy), key enzymes for
resveratrol synthesis, is induced by pathogens, activation of the
jasmonate pathway [5], or abiotic stresses [6]. Engineering the StSy
into plants of interest results in resveratrol accumulation and
elevates pathogen resistance in some crops such as rice [7], tomato
[8], or barley [9]. Stilbene synthases are typically organised in
gene families with high sequence homology of individual
members, but different regulatory features in their promotors
[10]. For the sake of simplicity, in this study, the term stilbene
synthase is used to designate this family of enzymes. Resveratrol
acts as a precursor for stilbene compounds of higher fungitoxicity
that accumulate in grapevine as a result of infection or stress [11].
Among those, especially d-viniferin inhibits zoospores mobility of
Downy Mildew (Plasmopara viticola), whereas the glycoside piceid is
not active [12,13].
In addition to its antimicrobial activity, resveratrol has also
attracted attention based on its health benefits to human including
prevention of cardiovascular disease, protection against cancers,
obesity, diabetes and neurodegenerative diseases, and extension of
lifespan by mimicking the caloric restriction effect [14,15,16,17]. A
famous phenomenon is termed as ‘‘French Paradox’’ which
describes that mild consumption of red wine can reduce the risk of
heart disease due to resveratrol content in the red wine [18].
North American Vitis rupestris has evolved sympatrically with
several of the major grapevine diseases, and can counteract
pathogen attack not only by induction of phytoalexins, but, in
PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue 10 | e26405
addition, initiation of HR [2]. HR is not only triggered by pathogens,
but also by the Harpin elicitor, a type-III bacterial effector derived
from Erwinia amylovora, the causative agent of fire blight in Rosaceae
[19]. In non-host plants, using Harpin, defence responses in two
grapevine cell lines derived from the pathogen-sensitive cultivar
‘Pinot Noir’ and the pathogen-resistant V. rupestris were compared
[20]. V. rupestris readily responded to Harpin with a massive HR-type
of cell death occurring within 48 h [21], and showed a strong, rapid,
and transient accumulation of StSy transcripts. This response was
accompanied by disruption of cortical microtubules, and massive
bundling of actin filaments. Pharmacological manipulation of
microtubules enhanced accumulation of StSy transcripts in the
absence of elicitor [20]. Conversely, the responses in cv. ‘Pinot Noir’
were weaker. This leads to the question – at what point these
quantitative differences are transformed into a qualitatively different
output (basal defence versus HR-mediated cell death)? The cellular
effects of resveratrol on fungi have been investigated in some studies
[22,23]. However, to our knowledge, the resveratrol responses of the
phytoalexin-producing plant cells themselves have not been ad-
dressed previously.
In this study, we show that, in response to Harpin, the pathogen
sensitive cv. ‘Pinot Noir’ produces low resveratrol and its glycoside
piceid, whereas the resistant V. rupestris trends to accumulate
abundant resveratrol and the potent oxidised dimmer d-viniferin.
Exogenous resveratrol inhibits cell growth in a dose-dependent
manner and activates defence-related responses such as rapid
alkalinisation, and accumulation of transcripts for the pathogen-
related proteins 5 and 10 (PR5 and PR10), a mild elevation of ROS-
formation, actin bundling, and cell death. Unlike Harpin,
resveratrol does not induce transcripts for RS and StSy, nor does
it affect microtubule structure. In V. rupestris, Harpin induces rapid
and massive formation of ROS, and suppression of production
and/or scavenging of apoplastic ROS impairs the elicitor-induced
accumulation of StSy transcripts. The data are interpreted by a
model, where resveratrol, in addition to its classical function as
antimicrobial phytoalexin, acts as a signaling molecule in the
regulation of the initiation of HR-related cell death.
Results
Resveratrol production is triggered by the Harpin elicitor
The Harpin elicitor induced a transient accumulation of stilbene
synthase (StSy) transcripts, which was strong in V. rupestris as
compared to V. vinifera cv. ‘Pinot noir’ [20]. To investigate,
whether the product of StSy, i.e. the stilbenic resveratrol, or its
derivatives (Figure 1A), also accumulates in response to the elicitor.
The abundance of resveratrol was quantified by HPLC in both cell
lines after Harpin treatment.
Trans-resveratrol was detected from 2 h in both cell lines.
However, in V. rupestris, the amount of trans-resveratrol increased
sharply from 6 h after elicitation, reaching a maximum of more
than 21.1
mgg
21
f.w. at 10 h (corresponding to .90 mM),
followed by a sharp decline. In cv. ‘Pinot noir’, the response was
weaker and later with a maximal induction of 3.8
mgg
21
f.w.
(around 15
mM) at 24 h after elicitation (Figure 1B).
In addition to trans-resveratrol, its metabolic products, the
glycoside trans-piceid and the oxidised dimer d-viniferin, were
followed over time. Trans-piceid increased dramatically from 6 h,
and reached 23.1
mgg
21
f.w. at 48 h (corresponding to .60 mM) in
cv. ‘Pinot noir’ (Figure 1C), even during the later stages, when trans-
resveratrol decreased (compare Figures 1B, C), indicating rapid
glycosylation of the trans-resveratrol might produce in response to
Harpin. In V. rupestris, trans-piceid increased more slowly and to a
much lower level (only 1/10 of that reached in cv. ‘Pinot Noir’).
In contrast to piceid, Harpin strongly induced d-viniferin in V.
rupestris (Figure 1D). The increase of d-viniferin was first slow, but
proceeded steadily. From 10 h, the accumulation was accelerated
reaching 236.2
mgg
21
f.w. (corresponding to 450 mM) at 48 h.
Thus, the bulk of d-viniferin accumulation coincides with the
decline of its precursor resveratrol. In cv. ‘Pinot Noir’, d -viniferin
accumulated only to 23.5
mgg
21
f.w.
Resveratrol responses of cell growth and rapid
alkalinisation
To understand the biological function of the accumulation of
resveratrol, we further investigated the cellular responses to
exogenous resveratrol. Packed cell volume (PCV) at different
concentrations of resveratrol was measured at the stationary phase
after 7 days of growth (Figure 2A), and was found to decline from
10
mM resveratrol approached zero levels for 500 mM in both cell
lines with a significantly stronger influence on V. rupestris as
compared to cv. ‘Pinot Noir’. The time course of growth inhibition
(Figure 2B) showed a lag of 48 h and reached 80% (as compared
to the solvent control) at 96 hours in both cell lines. However, the
inhibition was much more pronounced at 96 h compared to the
stationary phase three days later (see the 50
mM values in
Figure 2A). Thus, the inhibition was compensated with time,
apparently more rapidly in cv. ‘Pinot Noir’ than in V. rupestris.
In the next step, extracellular alkalinisation, as a fast and
convenient cellular indicator of plant defence [24], was analysed in
response to 50
mM resveratrol (corresponding to the half-maximal
concentration in response to the elicitor). In both cell lines,
alkalinisation became detectable from 30 min, but developed more
rapidly in V. rupestris (Figure 2C). The dose-response of steady-state
pH (Figure 2D) showed an increase with rising concentrations of
resveratrol reaching a maximal value of 1.25 units (similar to the
maximal response achieved by Harpin elicitation as reported in
Qiao et al. [20]. Although this increase presented as well, it was not
as pronounced in cv. ‘Pinot Noir’. However, a reliable 500
mM
point could not be measured for this cell line, because most cells
collapsed leading to uncontrolled fluctuations of pH in consequence
of vacuolar breakdown and cell death.
Resveratrol-mediated defence-gene expression
To investigate whether exogenous resveratrol is able to activate
defence genes, we followed the expression of defence genes encoding
reveratrol synthase (RS), stilbene synthase (StSy), osmotin-type
pathogenesis-related protein 5 (PR5), a member of the pathogenesis
protein 10 (PR10) family, and polygalacturonase inhibiting protein
(PGIP), which have been used previously for the Harpin response in
Vitis cells [20]. For both cell lines, only minor fluctuations were
observed for PGIP expression (Figure 2E–H). In contrast, transcripts
for PR10 and, especially, PR5 increased rapidly and significantly from
30 min after addition of resveratrol. In V. rupestris (Figure 2E, F), the
accumulation was much faster and to higher levels as compared to cv.
‘Pinot Noir’ (Figure 2G, H). It should be noted that RS and StSy
transcripts that accumulated rapidly in response to Harpin [20], did
not show a significant response to resveratrol.
Resveratrol and Harpin induce reactive oxygen species
differentially
Generation of reactive oxygen species (ROS) is associated with
hypersensitive cell death [25]. To test, whether the elevated
growth inhibition of the V. rupestris cell line (Figure 2B) is related to
a facilitated induction of hypersensitive cell death, we used the
fluorescent dye dihydrorhodamine 123 (DHR 123) to follow ROS
production in response to either Harpin (9
mgl
21
), or resveratrol
Resveratrol Regulates HR Cell Death in Vitis Cell
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(50 mM) compared to a solvent control. No significant changes
were observed for the solvent control in two cell lines (Figure 3A,
B). This basal fluorescence was elevated at 15 minutes after
Harpin treatment in both cell lines. In V. rupestris, a further
increase (about 1.5 folds) was observed from about 30–35 min
after elicitation, but not obvious in cv. ‘Pinot Noir’. Application of
50
mM resveratrol nearly did not induce any increase of
fluorescence in cv. ‘Pinot Noir’. In V. rupestris, the signal did
increase, however, only distinct at around 40 min, i.e. later than in
response to the Harpin elicitor.
Are reactive oxygen species necessary for elicitor-
triggered induction of stilbene synthase?
To test, whether the ROS triggered by the Harpin elicitor are
necessary for the induction of StSy, we used gain- or loss-of-
function experiments employing H
2
O
2
as ROS-donor, whereas
the NADPH oxidase inhibitor DPI, or the ROS-scavenger
catalase were used to quell the increase of ROS abundance
following challenge with Harpin. Analysis by semiquantitative RT-
PCR showed that exogenous H
2
O
2
failed to induce accumulation
of StSy transcripts in absence of elicitor, nor could it amplify the
response to Harpin (Figure 3C–F). However, application of DPI
significantly suppressed the transcripts of StSy in both cell lines, but
this inhibition was much more pronounced in V. rupestris than that
in cv. ‘Pinot Noir’ (Figure 3E, F). Catalase inhibited StSy
transcripts as well. However, in cv. ‘Pinot Noir’, inhibition by
catalase was more efficient than by DPI, whereas this relation was
reversed in V. rupestris. As to be expected, neither DPI nor catalase
or H
2
O
2
did induce any accumulation of StSy transcripts in
absence of the elicitor. These results suggest that ROS are
Figure 1. Accumulation of stilbenes in response to Harpin in cv. ‘Pinot noir’ and
V. rupestris
.AAlternative pathways for the stilbenic
resveratrol by glycosylation leading to trans-piceid or oxidation leading to d-viniferin. Time courses for the accumulation of trans-resveratrol (B),
trans-piceid (C), and d-viniferin (D) after treatment with 9
mgl
21
Harpin are plotted as mean values and standard errors from at least five
independent experimental series.
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necessary for the induction of StSy transcripts in response to the
Harpin elicitor. However, they are not sufficient to trigger StSy
transcripts in the absence of the elicitor.
Resveratol and Harpin induce cytoskeletal response
differentially
A second aspect of hypersensitive-related programmed cell
death is the reorganisation of actin [25,26]. We have found in a
previous study [20] that the cytoskeleton was reorganised in Vitis
cells in response to the Harpin elicitor. We therefore compared the
elicitor responses of microtubules and actin filaments to the
responses to resveratrol.
When microtubules were visualised 30 min after treatment with
either the solvent (Figure 4A, B), or with 50
mM resveratrol
(Figure 4C, D), microtubules were not found to be different from
untreated cells. However, treatment with Harpin (9
mg.l
21
) led to
disintegration of microtubules in V. rupestris but not in cv. ‘Pinot
Noir’ (Figure 4E, F).
In contrast to microtubules, the response of actin filaments to
resveratrol was pronounced in V. rupestris (Figure 5A). As
Figure 2. Responses of cv. ‘Pinot noir’ and
V. rupestris
to resveratrol. A Dose-response relation for growth (measured as increment in packed
cell volume) over resveratrol concentration. Data show means from four independent experimental series. B Time course of growth inhibition in
response to 50
mM resveratrol as compared to the solvent control (equal concentration of ethanol), values show means from four independent
experimental series, bars standard errors. C Representative time course of extracellular alkalinisation induced by 50
mM resveratrol (+res) versus the
solvent control (2res). D Dose-response relation for the steady-state response of pH over resveratrol concentration (assessed two hours after
addition of resveratrol) E–H Response of defence-related genes to 50
mM resveratrol, including encoding reveratrol synthase (RS), stilbene synthase
(StSy), pathogenesis-related protein genes 5 and 10 (PR5, PR10), and polygalacturonase inhibiting protein gene (PGIP) detected by semiquantitative
RT-PCR. E,G shows a representative gel, F,H shows mean values and standard errors at con (control, white bars), 0.5 h (cross-hatched bars), 1 h
(horizontally striped bars), and 3 h (boldly striped bars) after addition of 50
mM resveratrol from at least three independent experimental series,
relative to the respective control value using elongation factor 1-a (EF1a) as internal standard.
doi:10.1371/journal.pone.0026405.g002
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compared to untreated controls, where fine strands of actin were
observed in the periphery of the cells, actin filaments were strongly
bundled and had contracted towards the nucleus 30 min after
treatment with resveratrol. Since transgenic grapevine marker
lines expressing GFP fusions of cytoskeletal markers are not
available for in vivo studies, we tested, whether resveratrol was able
to induce actin response in the transgenic tobacco BY-2 line GFP-
11 [27] expressing the fluorescently tagged FABD-actin marker.
Here we could observe that after addition of resveratrol (50
mM)
actin filaments were progressively depleted from cell periphery,
whereas simultaneously perinuclear bundles of actin appeared
within the first 30 min. This actin reorganisation developed
progressively over the following time period (Figure 5B).
To functionally verify this resveratrol-induced response of actin
bundling, we assessed actin-dependent cellular events. Since
alterations of actin organisation interfere with the dynamic
localisation of the auxin-efflux component PIN1 [28], we tested
the resveratrol response in a transgenic tobacco BY-2 line
expressing AtPIN1 in fusion with RFP under control of its own
promoter [29]. When actin filaments were eliminated by
Latrunculin B, the reintegration of PIN1-RFP into the plasma
membrane was affected resulting in intracellular agglomerations
(Figure 5C, upper row). Likewise, 50
mM resveratrol were able to
induce a similar agglomeration, but with a delay of about 15 min
as compared to treatment with Latrunculin B (Figure 5C, lower
row). This rapid cellular response to resveratrol was followed, a
Figure 3. Production of reactive oxygen species (ROS) and effects on
stilbene synthase
(
StSy
) transcripts. A, B Time course accumulation
of ROS using dihydrorhodamine 123 in response to the solvent control, Harpin (9
mgl
21
), or resveratrol (50 mM). ROS relative fluorenscence was
quantified relative to the respective base fluorescence by Image J software. C–F Effect of ROS on StSy expression to Harpin as assessed by RT-PCR for
cv. ‘Pinot Noir’ (C, E) and V. rupestris (D, F). C, D Representative Gels for StSy transcripts 2 h after addition of Harpin (9
mgl
21
), H
2
O
2
(10 mM), Harpin
with H
2
O
2
, NADPH oxidase inhibitor DPI (10 mM), Harpin with DPI, catalase (100 U) or Harpin with catalase. Water was added and used as control. E, F
Mean values and standard errors from at least three independent experimental series, relative to the respective control value using elongation factor
1-a (EF1a) as internal standard.
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day later, by a decrease of mitotic index, and a stimulation of cell
death (Figure S1, Text S1). These responses were dependent on
the concentration of resveratrol and were affected already at the
lowest tested concentration of 1
mM resveratrol. In addition, the
synchrony of cell division, a diagnostic marker for the activity of
actin-dependent auxin transport [28], was progressively disrupted
resulting in a progressive decrease of a diagnostic frequency peak
of 6-celled over 5-celled files when the concentration of resveratrol
reached 10
mM.
These findings show that actin reorganisation, a cellular marker
for hypersensitive-reactive cell death, could be triggered by
resveratrol as reported earlier for the Harpin elicitor [30]. In
contrast, microtubules that are eliminated by the elicitor in V.
rupestris, do not respond markedly to resveratrol treatment. The
actin response to resveratrol is stronger in V. rupestris, weaker in cv.
‘Pinot Noir’. Similar to the situation in the Vitis cell lines,
treatment of BY-2 cells with resveratrol is followed by cell death
(Figure S1).
Discussion
The resistance of wild American Vitis species such as V. rupestris
is correlated with a readily expressed hypersensitive reaction [2].
European cultivated grapevines such as cv. ‘Pinot Noir’ express
only basal defence, which becomes manifest in the accumulation
of defence-related transcripts [20]. The comparison of the two cell
lines should therefore provide insight into differences and overlaps
between basal defence and HR. Here, we have shown that the
product of StSy, low trans -resveratrol and its glycoside trans-piceid
accumulated in cv. ‘Pinot Noir’, but formed abundant trans-
resveratrol and the oxidised dimer d-viniferin in V. rupestris.
Exogenous resveratrol inhibited cell growth, and activated
defence-related responses such as rapid alkalinisation, induction
of PR5 and PR10, actin bundling, and cell death. All of these
responses were manifest in both cell lines, but significantly stronger
in V. rupestris. Both cell lines induced formation of ROS by Harpin
elicitor, but resveratrol could trigger ROS-formation only in V.
rupestris. These observations indicate that resveratrol, in addition to
its classical role as phytoalexin, exerts additional roles that seem to
be linked with the execution of hypersensitive cell death.
Although the cellular responses to the Harpin elicitor are
complex, the results from the present work and our previous study
[20] allow to sketch down a first working model (Figure 6). The
initial step involves perception of Harpin, either by a receptor
(Figure 6A), or by membrane-permeabilisation in an ionophore-
like manner [30]. Perception initiates signaling including calcium
influx [20], probably through a mechanosensitive ion channel
[31,32], and apoplastic oxidative burst. The ROS, are activated
within minutes by Harpin in both lines, and within about 30 min
by resveratrol (only in V. rupestris) are necessary but not sufficient to
mediate the response of StSy to Harpin (Figure 3). DPI and
catalase inhibit Harpin-induced StSy induction differently, indi-
cating that different ROS species might trigger different signaling
pathways.
Signal perception (Figure 6A) generates a primary signal
(Figure 6B) connected with a disruption of microtubules. Besides
their classical functions, microtubules participate in the sensing of
stimuli [33]. The two cell lines differ in microtubular dynamics
manifest as altered abundance of detyrosinated a-tubulin accu-
mulating in stable microtubules [20]. In cv. ‘Pinot Noir’,
microtubules were more dynamic and remained intact, in V.
rupestris, they were more stable and disrupted in response to
Harpin. The exact timing of this microtubule response is difficult
to determine, since inspection of a cell population stained by
immunofluorescence, will detect only advanced stages of disrup-
tion obvious at 30 min. To detect earlier stages, it is necessary to
follow the response in individual cells over time in vivo. We have
therefore launched the generation of transgenic Vitis expressing
fluorescently tagged cytoskeletal markers. Irrespective of the exact
timing of the microtubule response, experiments where microtu-
bular disruption in V. rupestris was sufficient to trigger accumula-
tion of StSy, indicate a stable, detyrosinated, population of
microtubules participating in primary defence signaling. A
straightforward working hypothesis would propose a microtu-
bule-lever structure that participates in the gating of mechan-
osensitive calcium channels [31,34] driving defence signaling. In
contrast to Harpin, exogenous resveratrol does not trigger a
microtubular response (Figure 4). Parallel to microtubules, actin
filaments respond by progressive bundling and contraction
towards the nucleus, a response that is very pronounced in V.
rupestris, but barely detectable in cv. ‘Pinot Noir’ [20]. The function
of this actin response will be discussed below.
In parallel to the microtubule response, Harpin induces drastic
changes of extracellular alkalinisation [24], inhibited by very low
concentrations of Gd
3+
, an inhibitor of mechanosensitive Ca
2+
channels [20]. Exogenous resveratrol, although inducing alkalin-
isation, failed to induce StSy transcripts (Figure 3), demonstrating
that alkalinisation (in contrast to microtubule disruption) was not
sufficient to trigger a defence response in grapevine cell system.
Figure 4. Responses of microtubules to Harpin and resveratrol.
Cells of cv. ‘Pinot Noir’ (A, C, E) and V. rupestris (B, D, F) were treated
with either ethanol as solvent control, with resveratrol (50
mM), or with
Harpin (9
mgl
21
), and microtubules were stained by means of
immunofluorescence. Representative geometrical projections of confo-
cal z-stacks are shown. Size bars = 20
mm.
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The primary signal is followed by the accumulation of StSy
transcripts at 30 min [20], resveratrol (Figure 6C) from about 2 h
after addition of Harpin (Figure 1). As pointed out above, the
induction of StSy requires ROS. However, H
2
O
2
was not sufficient
to trigger expression of StSy transcripts indicating concerted action
of parallel, non-redundant pathways, for instance, the mitogen-
associated protein kinases (MAPK) mediating calcium-indepen-
dent Harpin-induced expression of defence genes [30].
Due to its toxicity for the producing cell itself, resveratrol must
be either sequestered or secreted. In fact, both mechanisms seem
to be at work. In ripening berries that accumulate resveratrol
without pathogen challenge, StSy was found in vesicles near the
plasma membrane, suggesting secretion into the apoplast [35].
When resveratrol was induced by methyl jasmonate in grapevine
cells high amounts accumulated in the vacuole [36].
Resveratrol shows antifungal activity against grapevine patho-
gens and thus meets the criteria for a phytoalexin, but resveratrol
seems to be more than a final product. (Figure 6D). Resveratrol
can be further metabolised into oxidative d-viniferin that is even
more effective against P. viticola zoospores or into the ineffective
glycoside piceid [12,37]. However, all stilbenes show not same
toxicity against pathogen. Resveratrol had real inhibitory effects
on conidial germination of B. cinerea liquid cultures when used at
concentration ranging from 60
mg/ml (25% inhibition) to 160 mg/
ml (100% inhibition), i.e., from 2.6 to 7610
24
M [22]. Piceid has
never shown any toxic activity against P.viticola zoospores, even at
Figure 5. Response of actin filaments to resveratrol. A Actin organization in V. rupestris in a control cell and after 30 min treatment with 50 mM
resveratrol visualized by fluorescent phalloidin. B Actin response to 50
mM resveratrol in vivo using the actin marker tobacco GFP-11. Size
bars = 20
mm. C Relocation of the auxin-efflux regulator PIN1-RFP after treatment with the actin inhibitor LatB (2 mM) or with resveratrol (50 mM).
Arrows indicate relocalisation of the PIN1-RFP marker. Size bars = 20
mm. All images were captured using an AxioImager Z.1 microscope (Zeiss)
equipped with an ApoTome microscope slider through the filter sets 38 HE for FITC or GFP (excitation at 470 nm, beamspliter at 495 nm, and
emission at 525 nm) or 43 HE for PIN1-RFP (excitation at 550 nm, beamsplitter at 570 nm, and emission at 605 nm) respectively.
doi:10.1371/journal.pone.0026405.g005
Resveratrol Regulates HR Cell Death in Vitis Cell
PLoS ONE | www.plosone.org 7 October 2011 | Volume 6 | Issue 10 | e26405
concentration .100 mM [12], whereas viniferin has an antifungal
activity upon germination of B.cinerea conidia (ED50 = 36
mg/ml,
7.9610
25
M) similar to that of pterostilbene, which is the most
toxic stilbene [37]. The differential conversion of resveratrol in V.
rupestris versus cv. ‘Pinot Noir’ may be a branching point between
basal immunity and HR. Our results are supported by
circumstantial evidence from molecular farming of the therapeu-
tically interesting resveratrol. Here, induction of stilbene synthesis
by methyl jasmonate accumulated mostly piceid in V. vinifera [38],
but large quantities of resveratrol and viniferin in a hybrid with the
HR-competent American V. berlandieri [36].
These observations shift resveratrol-metabolising enzymes into
the focus. Glycosylation into piceid might be triggered. In contrast,
resveratrol-oxidising basic peroxidase isoenzymes [39,40] might be
of particular interest as regulatory targets, because they are
differentially localised either in the apoplast (isoenzyme A1, B3) or
the vacuole (isoenzyme B5), and have been associated with
constitutive defence of grapevine against fungi [41]. A key role of
Figure 6. Model for the resveratrol as a secondary signal of elicitor-triggered hypersensitive response in
Vitis
cell. A Perception by
binding of the elicitor (el) to a receptor (elr) interactimg with a mechanosensitive ion channel (msc) and submembraneous microtubules (mt) and
actin filaments (af). Binding activates the NADPH-oxidase Rboh leading to apoplastic reactive oxygen species (ROS), which can permeate into the
cytoplasm. B A primary signal generated by microtubule disruption activates defence-genes, especially stilbene synthases (StSy). In parallel, ROS
activate proton influx. C Synthesis of resveratrol by StSy is accompanied by progressive bundling of actin filaments (heralding commitment for
programmed cell death) and partial translocation of resveratrol into the vacuole, where it can be glycosylated into inactive piceid (in cv. ‘Pinot Noir’)
or accumulate as aglycon (in V. rupestris). D Resveratrol as secondary signal initiates accelerates hypersensitive cell death by a second oxidative burst,
and induction of PR5. In parallel, actin contraction is accentuated. E Execution of hypersensitive cell death results in vacuolar breakdown releasing
PR5 and resveratrol. Contact of resveratrol with ROS forms the highly cytotoxic d-viniferin.
doi:10.1371/journal.pone.0026405.g006
Resveratrol Regulates HR Cell Death in Vitis Cell
PLoS ONE | www.plosone.org 8 October 2011 | Volume 6 | Issue 10 | e26405
resveratrol metabolisation is also supported by the fact that
resveratrol could be identified as a target of fungal effectors.
Fungal laccases of Botrytis cinerea cause an oxidative degradation of
resveratrol [42] allowing the fungus to escape from the action of
grapevine phytoalexins [43]. If resveratrol metabolism acts as a
switch between different types of immunity, selective pressure on
coevolving pathogens would be expected to favour effectors
targeted to this developmental switch.
When resveratrol and its derivatives act as secondary signals,
specific resveratrol responses must exist. We observed that
resveratrol stimulated oxidative burst, reorganisation of the actin
cytoskeleton, and induction of defence genes such as PR5. Despite
a certain overlap with Harpin-triggered responses, specific
differences exist:
i. Harpin caused disintegration of microtubules, resveratrol
failed to do so, even in the highly responsive V. rupestris
(Figure 4).
ii. Oxidative burst in response to Harpin was detected already at
5 min even in cv. ‘Pinot Noir’ (Figure 3), but not earlier than
30 min in response to resveratrol, even in V. rupestris.
Moreover, alkalinisation responded slowly to exogenous
resveratrol (Figure 2), but rapidly to Harpin [20]. The shift
in timing (about 30 min) would be consistent with a model,
where the proton channel was activated by the resveratrol-
triggered oxidative burst.
iii. The pattern of gene expression differed. Whereas Harpin
triggered a rapid, but transient response of StSy and RS
(30 min, peak at 2 h), these genes did not respond to
resveratrol. Instead, resveratrol triggered a slower, but
sustained response of PR10, and, prominently, of the
osmotin-type PR5 (Figure 2). PR10 was also among the genes
tested for their response to Harpin [20] and was found to
accumulate from about 2 h (but exclusively in V. rupestris, not
in cv ‘Pinot Noir’) – a temporal pattern consistent with a
mechanism, where the resveratrol generated by the Harpin-
induced StSy /RS triggers a second, delayed, but sustained
wave of gene expression.
The biological role of these secondary signals (second oxidative
burst, synthesis of osmotin-type PR5, progressive actin bundling)
seems to unfold during the execution of hypersensitive cell death
(Figure 6E): The oxidative burst generated by resveratrol could be
used by peroxidases in apoplast and vacuole [44] to convert
resveratrol into the highly potent oxidative oligomers, as shown for
a HR-like response triggered in grapevine by an elicitor from
Trichoderma viride [45]. Resveratrol would, thus, trigger a response
that drives its own conversion towards the more potent viniferins
representing the actual phytoalexins. Additionally, resveratrol-
triggered ROS might further activate downstream signal reactions
such as defence-related gene expression and HR [3]. Interestingly,
resveratrol failed to induce an oxidative burst in cv. ‘Pinot Noir’,
i.e. the two cell lines differ in their competence for resveratrol-
dependent oxidative burst, which means that the generation of
ROS is not a molecular property of resveratrol per se.
PR5 protein belongs to a widely distributed group of defence
proteins sharing sequence similarity with the intensely sweet
protein, thaumatin [46], which were proved to inhibit the
development of fungal pathogens, probably by binding fungal
1,3-b-D-glucans [47], but also can stimulate phytoalexin accumu-
lation in Arabidopsis thaliana [48]. In this study, the resveratrol-
responsive PR5 protein belongs to osmotin-like subset of these
proteins [49], contains an N-terminal signal peptide, but no ER-
retention signal suggesting secretion or transport into the vacuole.
Irrespective of its exact localisation, the induction of PR5 by
resveratrol proceeds to a very similar extent in both grapevine cell
lines, i.e. in contrast to the resveratrol-triggered oxidative burst
there seems to be no difference in competence for triggering PR5.
The progressive bundling of actin initiates earlier than the other
two responses and is observed in response to both Harpin [20] and
resveratrol (Figure 5). It can be followed in vivo in BY-2 cells using
GFP-tagged actin-marker lines (GFP-11) after Harpin and
resveratrol (Figure 5) treatment. Reorganisation of actin is
common in plant defence and has been traditionally interpreted
in the context of actin-dependent transport of secretory products
to the infection site and local activation of callose synthesis [49].
Exogenous resveratrol affected the polar localization of the auxin-
efflux component PIN1 (Figure 5) similarly to Latrunculin B,
consistent with actin-dependent recycling of PIN-proteins between
plasma membrane and endosomal compartments [50]. Resvera-
trol-triggered bundling of actin should therefore affect auxin
transport. In fact, cell division synchrony, a highly sensitive
reporter for auxin transport [51] was affected by resveratrol in
tobacco BY-2 wild type cell line (Figure S1, Text S1). However,
bundling of actin filaments also represents an evolutionary
conserved element of programmed cell death [26]. Electrical
detachment of submembraneous actin by nanosecond pulses can
trigger actin contraction, loss of membrane integrity, and
programmed cell death in the absence of pathogens or elicitors
[52,53]. The bundling of actin triggered by Harpin and resveratrol
has therefore to be seen in the context of a developmental program
that culminates in loss of membrane integrity and thus mediates in
the execution of cell death. It should be noted that actin bundling
initiates earlier than the significant quantity of resveratrol has been
synthetised and therefore must be controlled by a different
pathway – probably at the membrane-cytoskeleton interface
(Figure 6A). However, the response might be potentiated by
resveratrol.
As a result of these three mechanisms triggered by resveratrol,
highly toxic oxidative products (d-viniferin) are produced, proteins
that can attack fungal cell walls (PR5) accumulate, and the
(programmed) loss actin-dependent membrane integrity is poten-
tiated. This will culminate in the final blow: vacuolar breakdown
and release of toxic phytoalexins and PR5 contributing to the
efficient and active of HR-competent host cells to pathogenic
invaders.
Interestingly, in addition to its function as a signaling regulator
of HR cell death above, resveratrol is also well-known due to its
pharmacological effects on human health. Recently, autophagy, a
self-destructive mechanism, by which eukaryotic cells clear
damaged proteins and organelles, remobilise of cell contents and
maintain energetic requirements, has been considered conferring
longevity under nutrient limitation [54]. By activation of SIRT-1,
a NAD+-dependent deacetylase, resveratrol not only induces
autophagy and extends lifespan, but also suppresses colitis and
colon cancer [15,54,55,56]. Inhibition of S6 kinase by resveratrol
also suppresses the starvation-induced autophagy [57,58]. Two
alternative pathways may act in concert or in parallel to exert the
anti-aging effect of resveratrol. These findings raise a question:
Why can antimicrobial molecules produced by plants benefit to
human health? Obviously, the underlying mechanism is unlikely
explained by a fortuitious coincidence. One possibility is called
‘‘common origin hypothesis’’ that animals and plants share the
common ancestor in the biosynthetic pathway, or, since animals
have lost the ability to synthesize certain polyphenols, but they
have retained the ability to be activated by these molecules
[56,59]. Another popular explanation is, according to xenohorm-
esis hypothesis proposed by Howitz and Sinclair [17], organisms
Resveratrol Regulates HR Cell Death in Vitis Cell
PLoS ONE | www.plosone.org 9 October 2011 | Volume 6 | Issue 10 | e26405
have evolved the ability to sense stress-induced molecules i.e.
polyphenols from other species in their environment, and used the
cues to prepare in advance for loss of food supply and adversity
[60].
In conclusion, the comparison of the two grapevine cell lines has
uncovered that resveratrol functions as a secondary signaling
molecule, contrast with its normal phytoalexin role, to regulate the
key switch of the hypersensitive cell death in Vitis resistance. This
leads new questions: What are the direct targets of resveratrol in
plant defence signaling? Why can resveratrol trigger oxidative
burst in V. rupestris, but not in cv. ‘Pinot Noir’? Is actin-dependent
membrane stability involved in the signaling preparing a cell for
the ‘‘final call’’ to undergo programmed cell death in response to
resveratrol? Is PR5 simply a component of basal defence? Last, but
not least: what are the receptors that trigger basal defence and/or
HR?
Materials and Methods
Cell culture
Cell lines for Vitis rupestris and Vitis vinifera cv. ‘Pinot noir’
generated from leaves were cultivated as described in Qiao et al.
[20]. The transgenic tobacco (Nicotiana tabacum L. cv. Bright Yellow
2) cell line BY-2 GFP-11 [27] stably expressing the actin marker
Fimbrin Actin-Binding Domain 2 (FABD2) fused with GFP was
cultivated in presence of 30 mg l
21
hygromycin. The tobacco cell
line PIN1-RFP [29] expressed stably the auxin-efflux regulator
AtPIN1 in fusion with RFP under control of the AtPin1 promoter
was cultivated in addition with 100 mg l
21
kanamycin.
The Harpin elicitor (Messenger, EDEN Bioscience Corpora-
tion, Washington, USA; 3% active ingredient Harpin protein) was
dissolved in water to yield a stock solution of 300 mg.ml
21
.
Resveratrol (Sigma-Aldrich, Deisenhofen, Germany) was dissolved
in absolute ethanol to a stock solution of 100 mM. Diphenylene-
iodonium chloride (DPI) and Latrunculin B (Lat B) were
purchased from Sigma-Aldrich, Deisenhofen in Germany and
prepared in dimethylsulfoxide (DMSO) to stock solution of
10 mM and 1 mM respectively. Hydrogen peroxide [H
2
O
2
,
Sigma-Aldrich, 30% (w/w) in water] was diluted with water to a
stock solution of 10 mM. Catalase was dissolved in 50 mM Tris-
HCl, pH 7.0 to obtain a stock solution of 100 U.
ml
21
. All
treatments were accompanied by solvent controls, where the
maximal concentration of solvent used in the test samples was
administered.
Dose-response of cell growth and extracellular
alkalinisation over resveratrol
Cell growth was quantified by measuring packed cell volume
(PCV) at 7 days after subcultivation with or without the presence
of different concentrations of resveratrol and equal volumes of
solvent ethanol [61]. Time courses of growth inhibition were also
performed. In parallel, mortality was assessed using Evan’s Blue
[62]. The experiment was repeated four times. Extracellular
alkalinisation was evaluated according to Qiao et al. [20]. The
course of pH changes was plotted over time. Dose-response curves
were obtained by plotting the maximal change of pH over
resveratrol concentration.
Expression analysis
To evaluate the effect of exogenous resveratrol on the
transcription of defence-related genes, 1 ml cells (5 d) were treated
with 50
mM resveratrol or ethanol as a control for indicated time
points (0.5, 1 or 3 h). Transcripts of genes encoding resveratrol
synthase (RS), stilbene synthase (StSy), pathogenesis-related
proteins 5 and 10 (PR5, PR10), and polygalacturonase inhibiting
protein (PGIP) were quantified by semi-quantified reversible
transcription PCR (RT-PCR) using the primer combinations
defined in Kortekamp [63]. PCR products were quantified using
the Image J software (http://rsbweb.nih.gov/ij/) and standardized
relative to elongation factor 1-a as internal standard [64]. The
results were plotted as fold increase of transcript abundance as
compared with the untreated control. The data represent the
mean 6 standard errors from at least three independent
experimental series.
To determine the influence of reactive oxygen species (ROS) on
the expression of the marker gene StSy, 1 ml cells was induced for
2 h in the presence of different combinations of the elicitor Harpin
(9
mgl
21
), H
2
O
2
as ROS donor (10 mM), the NADPH oxidase
inhibitor DPI (10
mM) or the ROS scavenger catalase (100 U).
Experiments were performed three biologically repeats.
Fluorescent detection of ROS
To examine production of ROS induced by Harpin or
resveratrol, 200
ml cells (at day 3 to 4 after subcultivation) were
suspended into 800
ml PBS, preequilibrated on a shaker for at 1 h
and then supplemented with dihydrorhodamine 123 (DHR 123,
final concentration 10
mM), a cell-permeable fluorogenic probe
reporting oxidative burst [65]. After 30 min incubation, cells were
washed 3 times using pre-warmed PBS at 37uC and resuspended in
1 ml PBS in combination with 50
mM resveratrol, or with 9 mgl
21
Harpin as positive control, or with the solvent ethanol as negative
control. Changes of the fluorescent signal were followed over time
under an AxioImager Z.1 microscope (Zeiss, Jena, Germany) using
a the filter set 38 HE (excitation at 470 nm, beamsplitter at 495 nm,
and emission at 525 nm), a 406 objective and a constant exposure
time of 300 ms. Production of ROS fluorescence was quantified by
Image J software (http://rsbweb.nih.gov/ij/).
Cytoskeletal visualisation in Vitis cells
The response of the cytoskeleton was visualized as described
previously [19] in fully expanded cells 10 days after subcultivation
and treatment with either the solvent control, or 50
mM
resveratrol, or 9
mgl
21
Harpin for 30 min, respectively. Cells
were observed under a confocal laser scanning microscope (TCS
SP1; Leica, Bensheim, Germany) using 663 oil immersion
objective with excitation by the 488 nm laser line of the ArKr
laser and a four-frame averaging protocol.
In vivo observation of resveratrol responses in transgenic
BY2 cell lines
For in vivo observation of cellular responses to resveratrol, 200 ml
aliquots of GFP-11 cells were collected at day 4 and diluted into
800
ml MS liquid medium supplemented with 50 mMresveratroland
then immediately examined. The localisation of PIN-RFP [28] was
followed after treatment with either Latrunculin B (final concentra-
tion 2
mM) or resveratrol (final concentration 50 mM) over time. All
time series were recorded under an AxioImager Z.1 microscope
(Zeiss) equipped with an ApoTome microscope slider through the
filter sets 38 HE (excitation at 470 nm, beamsplitter at 495 nm, and
emission at 525 nm) for GFP imaging, and 43 HE (excitation at
550 nm, beamsplitter at 570 nm, and emission at 605 nm) for RFP
imaging. All images were processed and analysed using the
AxioVision software (Rel. 4.5; Zeiss) as described earlier [66].
Quantification of stilbenes
After treatment with Harpin (9 mgl
21
), cells were collected at
indicated time points (0, 2, 4, 6, 8, 10, 24 or 48 h) by
Resveratrol Regulates HR Cell Death in Vitis Cell
PLoS ONE | www.plosone.org 10 October 2011 | Volume 6 | Issue 10 | e26405
centrifugation (5 000 rpm, 5 min). Stilbenes were extracted
according to Tassoni et al. [4] with minor modifications. 3 g fresh
weight of cells was mixed with 20 ml methanol and homogenised
by an ultrasonic processor (UP100H, Hielscher, Germany) for
3 min. The homogenate was incubated for 1 h in the dark at room
temperature on a rotatory shaker at 150 rpm and filtered through
filter paper (WhatmanH, Schleicher & Schu¨ll, Germany). The
filtrate was concentrated to a residual volume of 5 ml in a glass
tube at 40uC (Heating Bath B490, BU
¨
CHI, Germany) at 280 rpm
(Rotavapor R-205, BU
¨
CHI, Germany) and a pressure of 80 Pa
(Vacuubrand CVC2, Brand, Germany). Stilbenes were isolated
from the aqueous phase by adding 2 ml 5% (w/v) sodium
bicarbonate to buffer pH, and three aliquots of 5 ml ethyl acetate.
The pooled ethyl acetate phase was completely dried and the
residue suspended in 2 ml methanol prior to injection into the
HPLC.
Stilbenes were analysed using a high performance liquid
chromatograph, HPLC (Agilent, 1200 series, Waldbronn, Ger-
many) equipped with a Phenomenex Synergi hydro RP column
(15064.6 mm, particle size 4
mm, Phenomenex; Aschaffenburg,
Germany), a DAD detector, and a quaternary valve. The flow rate
was 0.8 ml min
21
, and the injection volume is 20 ml. The mobile
phases included acetonitrile (ACN), methanol and water in the
following gradient: 2 min ACN/water (10/90 v/v); 15 min ACN/
water (40/60 v/v); 30 min ACN/methanol (50/50 v/v); 32 min
ACN/methanol (5/95 v/v); 35 min ACN/methanol (5/95 v/v);
39 min ACN/water (10/90 v/v); 42 min ACN/water (10/90 v/
v).Trans-resveratrol, trans-piceid, and d-viniferin were quantified
and identified using an external standard on the basis of retention
time and UV-VIS spectra. The standards for trans-resveratrol,
trans-piceid (Phytolab, Vestenbergsgreuth, Germany) and d-
viniferin were dissolved in methanol at a concentration of
100 mg l
21
respectively. Calibration curves determined using
these standards were linear (r
2
.0.99) and used for quantification
of the samples. At least five independent experimental series were
conducted.
Supporting Information
Figure S1 Dose-dependent cellular responses of tobacco
BY-2 wild type cell to resveratrol treatment.
(DOC)
Text S1 Description of cell pattern experiments of
tobacco BY-2 wild type.
(DOC)
Acknowledgments
We would like to thank PD Dr. Hanns-Heinz Kassemeyer (State Institute
of Viticulture, Freiburg) for providing the standards d-viniferin used to
carry the HPLC analysis in this study.
Author Contributions
Conceived and designed the experiments: XC PN. Performed the
experiments: XC. Analyzed the data: XC EH FQ PN. Contributed
reagents/materials/analysis tools: XC EH FQ PN. Wrote the paper: XC
PN.
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PLoS ONE | www.plosone.org 12 October 2011 | Volume 6 | Issue 10 | e26405
