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Characterization of the trans-resveratrol production by different inducing factors in Monastrell grapevine cell
cultures
Journal: Biotechnology and Bioengineering
Manuscript ID: Draft
Wiley - Manuscript type: Article
Date Submitted by the Author:
Complete List of Authors: Belchi-Navarro, Sarai; University of Murcia, Plant Biology Almagro, Lorena; University of Murcia, Plant Biology Lijavetzky, Diego; Universidad Nacional de Cuyo, Instituto de Biología Agrícola de Mendoza, Consejo Nacional de Investigaciones Científicas y Técnicas Bru, Roque; University of Alicante, 1Departamento de Agroquímica y Bioquímica Pedreño, Maria; University of Murcia, Plant Biology
Key Words: cell culture , cyclodextrin , elicitation, methyljasmonate, resveratrol, Vitis vinifera
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Characterization of the trans-resveratrol production by different inducing
factors in Monastrell grapevine cell cultures
Sarai Belchí-Navarro, Lorena Almagro, Diego Lijavetzky2*
, Roque Bru1 and Maria A.
Pedreño
Departamento de Biología Vegetal, Facultad de Biología, Universidad de Murcia, Campus de
Espinardo, 30100 Murcia, Spain; 1Departamento de Agroquímica y Bioquímica, Facultad de
Ciencias, Universidad de Alicante, Apartado 99, 03080 Alicante, Spain. 2Departamento de
Genética Molecular de Plantas, Centro Nacional de Biotecnología, Consejo Superior de
Investigaciones Científicas (CSIC), Cantoblanco, 28049 Madrid, Spain;
*Permanent address: Instituto de Biología Agrícola de Mendoza, Consejo Nacional de
Investigaciones Científicas y Técnicas, Facultad de Ciencias Agrarias, Universidad Nacional
de Cuyo (IBAM-FCA-UNCU), Chacras de Coria (5505), Mendoza, Argentina.
Corresponding author: Maria A. Pedreño ([email protected] )
Department of Plant Biology, Faculty of Biology, University of Murcia, Campus de
Espinardo, 30100 Murcia, Spain
Telephone: +34868887000; Fax: +34868883963
Short running title: resveratrol production by cell cultures
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Abstract
trans-Resveratrol is one of the most thoroughly studied bioactive molecules due to its
potential benefits for human health. For this reason too, new strategies based on Vitis vinifera
cell cultures have been used to increase trans-resveratrol production. These strategies include
the selection of highly productive Vitis cell lines, elicitor treatments, the addition of
biosynthetic precursors and stilbene metabolic engineering. V. vinifera cell cultures have been
used to investigate the factors involved in the induction and regulation of stilbene
biosynthesis and its metabolism. One common goal of such studies has been optimisation of
in vitro stilbene production through the use of biotic or abiotic elicitors, in an attempt to
attach an economically viable technology for commercial application.
In this work, the effect of different inducing factors on trans-resveratrol production in
Monastrell grapevine cell cultures is evaluated. A detailed analysis provides the optimal
concentrations of cyclodextrins and methyljasmonate and dosage UV irradiation, optimal cell
density, elicitation time and sucrose content in the culture media. The results indicate that
trans-resveratrol productivity decreases as the initial cell density increases in grapevine cells
treated with cyclodextrins individually or in combination with methyljasmonate, the decrease
observed with cyclodextrins alone being far more drastic than the combined treatment.
The synergistic effect observed on extracellular trans-resveratrol accumulation provoked by
the joint use of cyclodextrins and methyljasmonate is lower when these chemical compounds
are combined with UV exposure. Likewise, trans-resveratrol production is dependent on
levels of sucrose in the elicitation medium since, when its concentration in the culture
medium is low, trans-resveratrol biosynthesis is restricted. In contrast, when the supply of
carbon source is not limiting, the highest levels of trans-resveratrol are obtained by the joint
action of cyclodextrins and methyljasmonate.
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Much of the success of this strategy is due to the use of cyclodextrins, which act not only as
inducers of trans-resveratrol biosynthesis but also as promoters of adducts that remove trans-
resveratrol from the medium, reducing feedback inhibition and trans-resveratrol degradation
and allowing its accumulation in high concentrations.
Keywords: cell culture; cyclodextrin; elicitation; methyljasmonate, resveratrol, Vitis vinifera.
Introduction
Vitaceae phytoalexins constitute a group of molecules belonging to the stilbene family
(Langcake, and Pryce, 1977 a, b; Jeandet et al. 2002) which are derivatives of the trans-
resveratrol structure (t-R, 3,5,4´-trihydroxystilbene). These compounds are known as
viniferins and have been found in both grapevine tissue and cell cultures as the result of
infection or stress, displaying in some cases, strong antifungal activity (Pezet et al. 2004). Due
to their potential benefits for human health, t-R especially has been thoroughly studied. Thus,
multiple lines of compelling evidence indicate its beneficial effects on neurological (Okawara
et al. 2007) and cardiovascular systems (Bradamante et al. 2004). One of the most striking
biological activities of t-R investigated during recent years has been its anticancer activity and
it has been seen to prevent carcinogenesis in the stages of tumour initiation, promotion and
progression (Pervaiz, 2003). More recent data provide interesting insights into the effect of
this compound on the lifespan of yeast, worms and flies, suggesting that t-R could be
regarded as a potential antiaging agent in treating age-related human diseases (De la Lastra
and Villegas, 2005). In addition, effects described in mice subjected to a high-calorie diet
(Baur et al. 2006) point to new approaches for treating not only age-related diseases but also
obesity-related disorders (Kaeberlein and Rabinovitch, 2006). That is why new strategies
based on the use of Vitis vinifera cell cultures have been used to increase the level of t-R
production. These strategies include the selection of highly producing Vitis cell lines, elicitor
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treatments, the addition of biosynthetic precursors and stilbene metabolic engineering. V.
vinifera cell cultures have been used in several studies to investigate the factors involved in
the induction and regulation of stilbene biosynthesis and its metabolism (Waffo Teguo et al.
1998; Krisa et al. 1999; Decendit et al. 2002; Commun et al. 2003). One common goal of
such studies has been the optimisation of the in vitro stilbene production through the use of
biotic or abiotic elicitors such as UV light irradiation (Langcake, and Pryce, 1977b; Adrian et
al. 2000), aluminium ions (Dercks, and Creasy, 1989; López-Serrano et al. 1997), glucans
(Aziz et al. 2003), fungal products (Calderon et al. 1993) or oligosaccharides (Darvill et al.
2003). However, differences found in the stilbene production depend on methodology, Vitis
species and cultivars used. Thus, in response to UV-C irradiation, American Vitis species
showed a higher capacity for t-R biosynthesis than susceptible species. This response was
particularly high for V. rupestris which provided up to 750 µg t-R /g fresh weight (FW) within
48 h of irradiation. The production of t-R was dependent on the both time dose of UV light
and the time elapsing after treatment (Douillet-Breuil et al. 1999).
Jasmonic acid and its more active derivative methyljasmonate (MJ) are signal
molecules that act as key compounds of the signal transduction pathway involved in the
induction of the biosynthesis of secondary metabolites which take part in plant defence
reactions (Gundlach et al. 1992; Creelman, and Mullet, 1997; Staswick 1998, Chung et al.
2003). Thus, the production of secondary metabolites increases when plant cell cultures are
elicited with jasmonates (Gundlach et al. 1992; Blechert et al. 1995; Zhao et al. 2005;
Vasconsuelo and Boland, 2007). In V. vinifera, Krisa et al. (1999) showed that Cabernet
Sauvignon cell cultures respond to MJ with an increase in the total piceids (resveratrol
glycosylated forms) content when MJ is added at the beginning of exponential growth phase,
being the maximum level reached around 30 µmol/g dried weight (DW). However, the
amount of stilbenes secreted to the medium is negligible. Similar experiments were carried
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out in V. vinifera cv Barbera cell cultures responding to MJ (Tassoni et al. 2005). In this case,
the endogenous t-R accumulation was around 100 nmol/g DW and the t-R secreted to the
medium was over 30 nmol/g DW. Kiselev et al. (2007) were able to produce up to 138 µmol
/g DW of endogenous t-R using transgenic rolB V. amurensis callus cultures.
Many researchers have investigated the mechanism controlling the influence of sugar
on anthocyanin and stilbene accumulation (Larronde et al. 1998). Thus, after adding sucrose
to the culture medium of grapevine cell cultures, anthocyanin accumulation is increased,
while stilbene levels were only slightly affected (Larronde et al. 1998). Moreover, sugars are
essential to induce the expression of chalcone synthase gene, the level of which coincides
with the intracellular level of sugars, in particular sucrose and glucose (Tsukaya et al. 1991;
Takeuchi et al. 1994).
Amongst the different strategies known to increase the production of t-R using
grapevine cell cultures, we have chosen elicitation with β-cyclodextrins (CDs) since these
compounds chemically resemble to the alkyl-derived pectic oligosaccharides naturally
released from the cell walls during fungal attack (Bru et al. 2006). Recently, Zamboni et al.
(2009) reported that CDs trigger a signal transduction cascade which activates different
families of transcription factors in grapevine cells, inducing a halt in cell division,
reinforcement of the cell wall and the production of t-R and defence-related proteins. Thus,
the productivity of t-R in the spent medium of Monastrell cell cultures elicited with CDs was
around 300 µmol /g DW (Lijavetzky et al. 2008). This innovative method (Bru and Pedreño.
2003) differs from those that use other elicitors not only in the high levels of t-R produced but
also in the extraction process of this compound. Thus, in traditional elicitation methods, t-R
extraction from elicited cells gives low yields, whereas in this new process, t-R is secreted as
it is produced by cells and recovered directly from the spent media with no biomass
destruction. In addition, the high levels of t-R accumulated in the spent medium were seen to
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have no toxic effect on the cell lines, allowing successful subcultures. This innovative
elicitation process is mainly based on the characteristics of CDs.
Cyclodextrins are cyclic oligosaccharides derived from starch with 6, 7 or 8 glucose
residues linked by α (1 � 4) glycosidic bonds, namely α, β and γ cyclodextrins. They have a
hydrophilic external surface and hydrophobic central cavity that can trap apolar compounds,
including resveratrol (Morales et al. 1998). This hydrophobic cavity forms inclusion
complexes, which, in the case of CDs with t-R, is of the 1:1 type, altering its physicochemical
behaviour and making it a highly water-soluble compound.
In a previous work, we analysed the effects of MJ, CDs and a combination of both on
t-R production and the expression of stilbene biosynthetic genes in grapevine cell
suspensions. MJ and CDs significantly but transiently induced the expression of stilbene
biosynthetic genes when used independently to treat grapevine cells. Such expression
correlated with t-R production in CD-treated cells but not in MJ-treated cells. In the combined
treatment involving CDs and MJ, t-R production was almost one order of magnitude higher
(1400 µmol/g DW) and was correlated with the maximum expression values for stilbene
biosynthetic genes, demonstrating the synergistic effect of the combination of MJ with a true
and strong elicitor like CDs (Lijaveztky et al. 2008).
The final goal of our research is to choose the best operating conditions to achieve
high t-R levels and to increase t-R productivity evaluating the effect of different inducing
factors on Monastrell grapevine cell cultures. For this, a detailed analysis of these factors in t-
R production will provide the optimal concentrations of CDs and MJ and UV irradiation
dosage, optimal cell density, time elicitation and sucrose content in the culture media.
Materials and Methods
Establishment of callus and suspension cell cultures
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V. vinifera L. cv. Monastrell albino calli were established in our laboratory in 1990 as
described by Calderon et al. (1993). Since then, calli have been maintained at 25 ºC in
darkness in 250 ml flasks containing 100 ml of growth medium (Gamborg B5 medium
supplemented with Morel vitamins (Morel. 1970), 0.25 g/L casein hydrolysate, 20 g/L
sucrose, 0.2 mg/L kinetin, 0.1mg/L 1-naphthaleneacetic acid (NAA)), 8 g/L agar as
solidifying agent and pH adjusted at 6.0. Grapevine calli were subcultured on solid growth
medium every month.
Grapevine cell suspensions were initiated by inoculating friable callus pieces in 250
mL Erlenmeyer flasks containing 100 mL of liquid growth medium adjusted to pH 6.0 and,
maintained in a rotary shaker (110 rpm) at 25 °C in the dark. The cell suspension was
routinely maintained by periodical subcultures every 14-16 days by diluting with one volume
of fresh growth medium and then distributing into two flasks.
Elicitor treatments and effect of sucrose content on t-R production in grapevine cell
cultures
Elicitation experiments were performed in triplicate using 12-14 day old grapevine
cell suspensions. For this, 20 g of washed cells (FW) were transferred to a 250 ml Erlenmeyer
flask and suspended in 100 ml of sterile fresh growth medium supplemented with either
methylated CDs (CAVASOL® W7M, Wacker) or MJ or a combination of both CDs and MJ,
followed by at least 96 h of incubation at 25 °C in darkness in a rotary shaker (110 rpm).
Control cultures without elicitors were always run in parallel. After elicitation, cells were
separated from the culture medium under a gentle vacuum, rapidly washed with cold distilled
water, weighed and frozen at -80 ºC until use. The spent medium was used for measuring the
t-R content.
Suspension cell cultures were also elicited under UV light at short and long time
exposition in the presence or in the absence of the above chemical elicitors. For this, flasks
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were opened in a laminar flow hood and exposed to UV-C light (254 nm) for either 15 or 120
minutes, at an irradiation distance of 15 cm. During this time and after irradiation, flasks were
kept in continuous agitation for at least 96 h.
The effect of sucrose content on grapevine cell cultures was assessed in two different
ways: either by transferring cells into sterile fresh Gamborg B5 medium with sucrose at 10 or
20 g/L at the moment the elicitation experiments were carried out, in the presence or in the
absence of the above chemical elicitors or by maintaining suspension cell cultures for at least
three subcultures in fresh growth media with sucrose at 10 or 20 g/L before carrying out the
elicitation experiments.
Determination of cell growth and viability
A time course of cell growth was followed by measuring the packed cell volume
(PCV, expressed in percentage). For this, 2 mL of cell suspension homogeneous aliquots were
periodically withdrawn and centrifuged at 100 x g for 5 min. The conductivity and the pH
were also determined in the spent medium using a Crison micro CM 2100 conductivity meter
and a Crison 2002 pHmeter respectively. Cell viability was assessed by incubating the cells
for 1-2 min in fresh Gamborg B5 medium containing 100 µg ml-1
fluorescein diacetate (DAF)
(Huang et al. 1986) and observing the fluorescence emission by living cells with a DMRB
Leica optical microscope (λexc = 490 nm, λemi = 520 nm) using a Leica filter I3.
Analysis of t-R in the spent medium
Aliquots of the spent medium were diluted with water and methanol to a final
concentration of 80% methanol (v/v). 20 µl of diluted and filtered (Anopore 0.2µm) samples
were analyzed in a HPLC (Waters 600E) with Diode Array Detector system (Waters 996).
Sample separation was performed using a Spherisorb ODS2 C-18 column (250 x 4.6 mm,
particle size 5µ) and they were eluted with a mobile phase of 1% acetic acid (A)-acetonitrile
(B) at a flow rate of 1 ml min−1
. The gradient consisted of 0 min, 85 % A; 5 min, 80 % A; 10-
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15 min, 65% A; 17-25 min, 10 % A; 25-30 min, 65% A, 30-35 min, 85% A. The t-R was
identified and quantified by comparison with authentic t-R of >99% purity (Sigma-Aldrich,
Madrid, Spain).
Results and Discussion
Characterization of Monastrell cell culture growth
Prior to the elicitation treatments, Monastrell cell culture growth was characterized by
measuring PCV, pH and conductivity in Gamborg B5 medium supplemented with two
different sucrose concentrations (10 or 20 g/l, namely G10 and G20, respectively). As shown
in Figure 1A, these grapevine cell suspensions exhibited a typical sigmoid-shaped growth
curve with no significant differences in the lag or exponential phases. Differences existed in
the stationary phases of the cell lines grown in G20 and G10. Thus, the kinetic parameters of
cells growing in G20 pointed to a maximum biomass of 85% (PCVmax) and a biomass yield of
2.35 (∆PCV/g sucrose), 16 days being necessary to reach the stationary phase.
The corresponding values for cell suspensions grown in G10 were lower, and the
maximum PCV was around 78% (Figure 1A). As can be observed from Figures 1A and 1C,
the trend of the conductivity in the spent medium was contrary to that of PCV. The decrease
in conductivity was directly related with the depletion of mineral ions in the culture medium
since the cell culture uses them for growth.
The pH of the spent medium (Figure 1B), which was initially adjusted to 6.0,
underwent acidification during the first 48 hours and then increased before reaching a plateau
(from day 11) which was maintained until the end of the exponential growth phase. The pH of
the spent medium is conditioned by the cell metabolism and it is tightly related to the
consumption of the nitrogen source and the energetic state of cells (McDonald and Jackman,
1989).
Cell growth and t-R production is dependent on MJ dose and CDs
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The effect of the elicitors on cell growth was checked by determining cell FW
(initially 20 g in all treatments) after 96 hours of treatment. As shown in Figure 2, the FW of
MJ-treated cells remains constant at first or decreased slightly (16.7%) while in the combined
CDs + MJ treatment, cell growth was clearly reduced (27.1%). These results show that the
joint action of CDs and MJ leads to a reduction of cell growth, at least during the first 96 h of
treatment. The use of chemical compounds like jasmonates to induce the production of
stilbenes in grapevine cell cultures has been associated to modifications in the pattern of cell
growth (Krisa et al. 1999). Tassoni et al. (2005) described the undesirable effect of a
reduction of cell growth in MJ treated V. vinifera cv Barbera cell cultures. Kiselev et al.
(2007) also described the inhibition of cell growth in V. amurensis callus cultures as a result
of treatment with different elicitors. This effect of MJ on cell growth has been suggested to be
caused by the diversion of the metabolic flux (carbon allocation), favouring the activation of
the secondary metabolism over the primary metabolism (Larronde et al. 2003; Sivakumar and
Paek, 2005), although a direct effect of MJ on the repression of cell multiplication and/or
growth has recently been reported by Pauwels et al. (2008). Although cell growth was halted
in elicitor treated-cells, cell viability, as assessed by fluorescence microscopy of cells treated
with DAF (Figure 3), seems to be unaffected.
Figure 2 also shows the effect of different concentrations of MJ on t-R productivity by
Monastrell cell cultures elicited during 96 h, in the absence or in the presence of CDs (50
mM). No t-R was detected in cells treated with ethanol, the solvent in which MJ is delivered
to the culture (data not shown). As can be observed in Figure 2, the maximum level of t-R
produced and secreted by cells to the medium was reached when cell suspensions were
incubated in the joint presence of CDs and 100 µM MJ, this level being around 4.3-fold more
(1447.78 µmol t-R/g DW, Figure 2, solid line) than when cells were treated only with CDs
(338.60 µmol t-R/g DW). However, when cell cultures were elicited only with MJ, very low
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amounts of t-R were detected in the spent medium (16.36 µmol t-R/g DW with 100 µM MJ).
Krisa et al. (1999) described how the amount of total stilbenes secreted to the culture medium
was negligible in both MJ and control cultures of three V. vinifera cultivars. In addition, they
reported that stilbene accumulation was notably induced in grape cell CS4 (Cavernet
Sauvignon) cultivar when 25 µM MJ was added to the induction medium on day 6. In these
conditions and after 8 days of treatment, they obtained 771 µmol/L in 27.8 g DW /L, which
means 27.73 µmol t-R /g DW. In the presence of 25 µM MJ, we detected similar levels of t-R
in the spent medium (15.86 µmol t-R /g DW). However, t-R production levels increased 58.8
times (932.46 µmol t-R /g DW) when 25 µM MJ and 50 mM CDs were jointly used as
inducers.
On the other hand, Kiselev et al. (2007) reported high t-R production by V. amurensis
callus cultures transformed with the rolB gene of Agrobacterium rhizogenes. These callus
cultures accumulated up to 138.16 µmol t-R/g DW (3.15 % DW of t-R). This value, which is
two orders of magnitude higher that reported for other resveratrol-producing plant cell
cultures (Ku et al. 2005; Tassoni et al. 2005), is 10.4 times lower than that obtained in our
best conditions.
t-R production is dependent on elicitation time
The time course of t-R accumulation in Monastrell cell suspensions treated with CDs
and the optimal concentration of MJ (100 µM), separately or in combination, was followed by
periodically analyzing the t-R concentration in the spent medium (Table 1). Only a negligible
amount of t-R was detected in control and MJ treated-cells (data not shown). As can be
observed in Table 1, in both cases (using only CDs or a combination of CDs and MJ) a
continuous accumulation of t-R (measured as t-R production) was observed. The higher
accumulation rate in the combined treatment compared with the CD treatment (>4.5-fold) and
the larger accumulation with time are the driving forces behind the greater overall production
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of t-R. Since t-R production in the combined treatment was higher than the sum of the
individual treatments (due to the low production in MJ treated-cells), a synergistic effect
between both elicitors is assumed, as described Lijaveztky et al. (2008). Zamboni et al. (2006)
analysed variations in the response to CDs in four different grape genotypes. Thus, cell
cultures obtained from the crossing between V. riparia and V. berlandieri and V. amurensis
cell lines were capable of producing more t-R (911.25 and 225.22 mg/L, respectively) than
grapevine cell cultures of Pinot Noir (0.51 mg/L) or Merzling (4.31 mg/L) after 48 h of CD-
treatment. The results obtained by these authors using high-producer cell lines are in
accordance with those obtained from Monastrell cell cultures (Table 1). Differences in t-R
levels amongst Vitis genotypes could be related with their different levels of defence
response. This variability of the response of Vitis cultures to elicitation in presence of CDs
under the same conditions demonstrates that is very important to select genotypes which have
the ability to produce the highest levels of t-R.
t-R production is dependent on initial cell density
Once the optimal MJ concentration and time elicitation was fixed for the different
treatments, we analyzed the influence of initial cell density (expressed as % PCV) on t-R
production. As can be observed in Figure 4, the specific productivity of t-R decreased as the
initial cell density increased in grapevine cells treated with CDs (black bars) and CDs in
combination with MJ (white bars). As the response of cell cultures to elicitation depends on
cell density, and since the maximum levels of t-R are reached with a low cell density, it would
seem that this need to work at low cell density would make the scaling up process and the
transition to bioreactors would not be economically viable. Despite this fact, the levels of t-R
in all the combined treatments (CDs and MJ) are higher than those in the absence of MJ
(white bars vs black bars in Figure 4). In fact, normalization of the values in relation to the
specific productivity with a cell density of 25% PCV clearly shows that the decrease in t-R
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productivity is more dramatic for the treatment with CD alone (plaid bars) than for the
combined treatment with CDs and MJ (striped bars). For this reason, we decided to work at
intermediate cell density (50% PCV), using both CDs and MJ and to explore the possibility of
increasing t-R productivity through the addition of a third elicitor, in this case, UV light.
Effect of UV light exposure and both CDs and MJ on t-R production
UV light has been described as a physical elicitor of stilbene biosynthesis in grapevine
(Douillet-Breuil et al. 1999; González-Barrio et al. 2006). For this reason, the effect of the
exposure of grapevine cells to UV light in the presence of CDs, separately or in combination
with MJ, on t-R productivity was studied (Figure 5).
Cells treated with both white and UV-light, with and without MJ, produced a
negligible amount of t-R (data not shown). In addition, cell suspensions elicited only with
CDs and exposed for short times to UV light (15 min) showed a similar increase in t-R levels
as seen in UV-unexposed CD-treated cells, while prolonged exposure to UV-light (120 min)
caused a drastic reduction in t-R accumulation and browning of the cell culture as a result of a
hypersensitive response (HR) to UV light (Figure 5). In contrast, cells treated with CDs and
MJ, followed by short or long exposure to UV light, showed lower t-R levels than UV-
unexposed cells, and HR-induced browning was aggravated by long UV exposure. Such long
UV light exposure is clearly detrimental to t-R production because of the HR induced, while
short exposures do not enhance t-R levels in the presence of CDs and when MJ is also
present, so there is an antagonism effect between MJ and UV-light.
UV light has been reported to increase the production of secondary metabolites in plant cell
cultures (Broeckling et al. 2005; Song et al. 1999; Gläβgen et al. 1998). In this sense, Ramani
and Jayabaskaran (2008) observed the enhanced production of catharanthine and vindoline
from Catharanthus roseus cell cultures, which increased their levels 3 and 12 fold,
respectively when cells were irradiated with UV-B for 5 min. Gläβgen et al. (1998) studied
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the effect of continuous irradiation with UV-containing white light (315-420 nm) on the
anthocyanin content of carrot cell cultures after 7 days of the culture. They observed that the
total anthocyanin concentration was strongly enhanced by the UV treatment (3-5 fold higher
than dark-grown cell cultures), while the strong UV induction of anthocyanin accumulation
was reduced nearly to the levels of dark-grown cells when they were treated with both UV
light and fungal elicitors. Little is known about the effect of UV irradiation on the stilbene
content of grapevine cell suspensions and most of the research related with UV light has been
directed at enhancing the stilbene content of grape berries (Adrian et al. 2000; Cantos et al,
2003) and leaves (Pezet et al. 2003). Thus, when Keller et al. (2000) studied stilbene
accumulation in Cabernet-Sauvignon callus cv irradiated with UV light, they found that only
actively growing callus was capable of producing stilbenes (including t-R), whereas old callus
had lost this ability. This response was similar to that found in ripening grape berries, which
gradually lose their potential for stilbene synthesis as they approach maturity. These results
could explain the low t-R levels found when Monastrell cell cultures were exposed to UV
light because our elicitation experiments were performed using 12-14 day old grapevine cell
suspensions which are just entering their stationary phase.
t-R production is dependent on the level of sucrose in the medium
We have studied t-R productivity in a cell line grown in G20 using elicitation media
which contained three sucrose concentrations and different MJ concentrations at a fixed CD
concentration of 50 mM. As can be observed from Figure 6, the optimal MJ concentration
suitable for the elicitation of Monastrell cell cultures is dependent on the sucrose levels in the
elicitation media. Thus, when cell suspensions were elicited in a culture medium with a low
sucrose concentration (10 g/L, gray bars in Figure 6), t-R productivity increased using the
lowest MJ dose (25 µM), reaching a maximum value of 46 µmol t-R/g FW. This value is even
lower when grapevine cell cultures are elicited with CDs and higher MJ concentrations so that
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t-R productivity is limited by this sucrose concentration (10 g/L). When sucrose levels in the
elicitation media were increased to 20 g/L and an MJ dose of 100 µM was used, maximal t-R
productivity was observed (60 µmol t-R /g FW). This maximal t-R productivity was also
observed when a MJ dose lower (50 µM) was added to elicitation media which contained 30
g/L of sucrose. In addition, the lowest t-R productivity values (16-22 µmol t-R /g FW) were
found in CD-treated cells and no significant differences were detected in t-R productivity in
this treatment using three sucrose concentrations (Figure 6).
As can be observed in Figure 1A, cell culture growth did not change much when cell
cultures were grown in culture media with different sucrose concentrations. However, the
sucrose content in the elicitation media (G20 or G30 vs G10) modified t-R productivity as is
described in Figure 6.
Larronde et al. (1998) analyzed the effect of sucrose (100-200 mM) on the polyphenol
content in Gamay Fréaux cell suspensions. Their results indicated that anthocyanin
accumulation was strongly stimulated by sucrose while this sugar only had a slight effect on
the production of stilbenes. Belhadj et al. (2008) studied the effect of MJ treatment (20 µM) in
combination with added sucrose (80 mM) on stilbene accumulation in the culture medium.
After 18 h of treatment, the t-R accumulated in the culture medium reached a maximal value
of 52 nmol t-R/g FW. Working in similar experimental conditions (25 µM MJ and 60 mM
sucrose) but in the presence of CDs, we obtained 45 µmol t-R/g FW that is, 865-fold higher
than those obtained by Belhadj et al. (2008). We have also carried out elicitation experiments
in a culture medium which contained 30 g/L (black bars and dotted line in Figure 6). In these
conditions, the maximal level of t-R (55.2 µmol t-R/g FW) is obtained in the combined
treatment of CDs and 50 µM MJ and no significant differences were found when CDs and
100 µM MJ were added (53.8 µmol t-R/g FW). These results show that t-R productivity only
depends on the carbon source when its concentration in the culture medium is low (10 g/L,
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around 30 mM) because this restricts t-R biosynthesis. In contrast, when the supply of carbon
source is not limiting, the highest levels of t-R are obtained by the joint action of MJ and CDs,
and the use of a higher concentration of sucrose (30 g/L) in the elicitation media does not
substantially increase t-R productivity (dotted line in Figure 6).
We also studied the dependence of t-R productivity on both MJ dose and cell lines
growing in G10 and G20. For this, cell lines were grown and maintained during at least three
subcultures in G10 and G20 and then, they were elicited with different MJ concentrations in
the presence of 50 mM CDs (Figure 7). As can be observed in this figure, the lowest MJ
concentration required to obtain the highest t-R productivity (46.8 µmol t-R/g FW) in the cell
line grown in G10 was 50 µM. Although cell growth is not affected by the shortage of sucrose
in the culture medium (dashed line in Figure 1A), t-R productivity is still limited by the
carbon source (dashed line in Figure 7). However, the highest t-R productivity value was
reached when 100 µM MJ was used in the cell line grown in G20. In addition, no significant
differences were found in t-R productivity (40-50 µmol t-R/g FW) in both cell lines elicited at
other MJ concentrations, except in the cell line grown in G10 elicited with 25 µM of MJ (30.8
µmol t-R/g FW, grey bars in Figure 7). It is worth noting that in the combined treatments of
CDs and 270 µM MJ, a HR was observed, perhaps due to the high MJ concentration.
In conclusion, in order to achieve high t-R levels and to increase t-R productivity
using a highly productive Monastrell cell line, the best operating conditions would involve
elicitation of these cell cultures using a combination of CDs and MJ. The CD concentration
was fixed at 50 mM since a previous work showed that the use of this concentration led to the
highest t-R production in this cell line. Moreover, CDs do not only act as elicitors but also
form inclusion complexes (adducts) with t-R, favouring the substantial accumulation of this
compound and also preventing the toxic and/or inhibitory effect of t-R on grapevine cells. The
optimal MJ concentration found for the production of t-R depended on the sucrose
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concentration of the culture medium in which elicitation was carried out. When the sucrose
concentration is not limiting, in order to obtain the same t-R productivity, the higher the
concentration of sugar, the lower the concentration of MJ needed. t-R productivity increased
as cell density decreased. However, the fall in t-R productivity was less drastic when
suspension cultured-cells were elicited with CDs and MJ together. The use of a third elicitor
(UV light) did not improve t-R productivity in any case.
Acknowledgements
Almagro L. has a fellowship from the MICINN. We thank José Martínez Parra and Francisco
Fernández-Pérez for the identification and quantification of trans-resveratrol by HPLC and
Pepita Alemán for help in maintaining Monastrell cell cultures. This work has been partially
supported by the Consejería de Educación, Ciencia e Investigación de la Región de Murcia
(BIO BVA 07 01 0003), by Programa de Generación de Conocimiento Científico de
Excelencia de la Fundación Séneca, Agencia de Ciencia y Tecnología de la Región de Murcia
en el marco del II PCTRM 2007-10 (08799/PI/08) and by the Ministerio de Ciencia e
Innovación (BIO2008-02941).
Abbreviations: CDs, methylated β-cyclodextrins; DAF, fluorescein diacetate, DW, dry
weight; FW, fresh weight; G20 and G10, Gamborg B5 medium supplemented with 10 and 20
g/l of sucrose respectively; HR, hypersensitive response; MJ, methyljasmonate; PCV, Packed
cell volume, trans-resveratrol, t-R.
References
Adrian M, Jeandet P, Douillet-Breuil AC, Tesson L, Bessis R. 2000. Stilbene content of
mature Vitis vinifera berries in response to UV-C elicitation. J Agr Food Chem 48:6103-6105.
Page 17 of 33
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18
Aziz A, Poinssot B, Daire X, Adrian M, Bezier A, Lambert B, Joubert JM, Pugin A. 2003.
Laminarin elicits defense responses in grapevine and induces protection against Botrytis
cinerea and Plasmopara viticola. Molecular Plant-Microbe Interact 16:1118-1128.
Baur JA, Pearson KJ, Price NL, Jamieson HA, Lerin C, Kalra A, Prabhu VV, Allard JS,
Lopez-Lluch G, Lewis K. 2006. Resveratrol improves health and survival of mice on a high-
calorie diet. Nature 444:337-342.
Belhadj A, Telef N, Saigne C. 2008. 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 46:493–499.
Blechert S, Brodschelm W, Holder S, Kammerer L, Kutchan TM, Mueller MJ, Xia Z, Zenk
MH. 1995. The octadecanoic pathway: signal molecules for the regulation of secondary
pathways. Proc Natl Acad Sci U S A 92:4099-4105.
Bradamante S, Barenghi L, Villa A. 2004. Cardiovascular protective effects of resveratrol.
Cardiovasc Drug Rev 22:169 –188.
Broeckling CD, Huhman DV, Farag MA, Smith JT, May GD, Mendes P, Dixon RA, Sumner
LW. 2005. Metabolic profiling of Medicago trunculata cell cultures reveals the effects of
biotic and abiotic elicitors on metabolism. J Exp Bot 56:323–326.
Bru R, Pedreño MA. Method for the production of resveratrol in cell cultures. PCT Patent
WO/2003/062406. 2003.
Bru R, Selles S, Casado-Vela J, Belchi-Navarro S, Pedreño MA. 2006. Modified
cyclodextrins are chemically defined glucan inducers of defense responses in grapevine cell
cultures. J Agr Food Chem 54:65–71.
Calderón AA, Zapata JM, Muñoz R, Pedreño MA, Ros Barceló A. 1993. Resveratrol
production as a part of the hypersensitive-like response of grapevine cells to an elicitor from
Trichoderma viride. New Phytol 124:455-463.
Page 18 of 33
John Wiley & Sons
Biotechnology & Bioengineering
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Peer Review
19
Cantos E, Espín JC, Fernández MJ, Oliva J, Tomás-Barberán FA. 2003. Postharvest UV-C
irradiated grapes as potential source for producing stilbene-enriched red wines. J Agric Food
Chem 51:1208–1214.
Chung I, Park MR, Chun JC, Yun SJ. 2003. Resveratrol accumulation and resveratrol
synthase gene expression in response to abiotic stresses and hormones in peanut plants. Plant
Science 164:103–109.
Commun K, Mauro MC, Chupeau Y, Boulay M, Burrus M, Jeandet P. 2003. Phytoalexin
production in grapevine protoplasts during isolation and culture. Plant Physiol and Biochem.
41:317-323.
Creelman RA, Mullet JE. 1997. Biosynthesis and action of jasmonates in plants. Annu
Rev Plant Physiol Plant Mol Biol 48:355- 381.
Darvill A, Augur C, Bergmann C, Carlson RW, Cheong JJ, Eberhard S, Hahn MG, Lo VM,
Marfa V, Meyer B. 2003. Oligosaccharins-oligosaccharides that regulate growth,
development and defence responses in plants. Glycobiol 2:181-198.
De la Lastra and Villegas. 2005. Resveratrol as an anti-inflammatory and anti-aging agent:
mechanisms and clinical implications. Mol Nutr Food Res 49:405–430.
Decendit A , Waffo-Teguo P , Richard T , Krisa S , Vercauteren J, Monti JP, Deffieux G ,
Merillon JM. 2002. Galloylatedc atechinsa nd stilbene diglucosidesin Vitis vinifera cell
suspension cultures. Phytochemistry 60:795-798.
Dercks W, Creasy LL. 1989. The significance of stilbene phytoalexins in the Plasmopara
viticola-grapevine interation. Physiol Mol Plant Pathol 34:189-202.
Douillet-Breuil AC, Jeandet P, Adrian M, Bessis R. 1999. Changes in the phytoalexin content
of various Vitis spp. in response to ultraviolet C elicitation. J Agric Food Chem 47:4456-61.
Page 19 of 33
John Wiley & Sons
Biotechnology & Bioengineering
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Peer Review
20
Gläβgen WE, Rose A, Madlung J, Koch W, Gleitz J, Seitz Hu. 1998. Regulation of enzymes
involved in anthocyanin biosynthesis in carrot cell cultures in response to treatment with
ultraviolet light and fungal elicitors. Planta 204:490–498.
González-Barrio R, Beltrán D, Cantos E, Gil MI, Espín JC, Tomás-Barberán FA. 2006.
Comparison of Ozone and UV-C Treatments on the Postharvest Stilbenoid Monomers,
Dimers and Trimers Induction in Var. ’Superior’ White Table Grapes. J Agr Food Chem
54:4222-4228.
Gundlach H, Müller MJ, Kutchan TM and Zenk MH. 1992. Jasmonic acid is a signal
transducer in elicitor-induced plant cell cultures. Proc Natl Acad Sci USA 89:2389–2393.
Huang CN, Cornejo MJ, Bush DS, Jones RL. 1986. Estimating viability of plant protoplasts
using double and single staining. Protoplasma 135: 80-87.
Jeandet P, Douillet-Breuil AC, Bessis R, Debord S, Sbaghi M, Adrian M. 2002. Phytoalexins
from the Vitaceae: biosynthesis, phytoalexin gene expression in transgenic plants, antifungal
activity, and metabolism. J Agric Food Chem 50:2731-2741.
Kaeberlein M, Rabinovitch PS. 2006. Medicine: grapes versus gluttony. Nature 444: 280-281.
Keller M, Steel CC, Creasy GL. 2000. Stilbene accumulation in grapevine tissues:
Developmental and environmental effects. Acta Hortic 514:275-286.
Kiselev KV, Dubrovina AS, Vuslova MV, Bulgakov VP, Fedoreyev SA, Zhuravlev YN.
2007. The rolB gene-induced overproduction of resveratrol in Vitis amurensis transformed
cells. J. Biotechnol. 128:681-692.
Krisa S, Larronde F, Budzinski H, Decendit A, Deffieux G, Mérillon JM. 1999. Stilbene
Production by Vitis vinifera Cell Suspension Cultures: Methyl Jasmonate Induction and 13C
Biolabeling. J Nat Prod 62:1688–1690.
Page 20 of 33
John Wiley & Sons
Biotechnology & Bioengineering
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21
Ku KL, Chang PS, Cheng YC, Lien CY. 2005. Production of stilbenoids from the callus of
Arachis hypogaea: a novel source of the anticancer compound piceatannol. J Agr Food
Chem 53:3877-3881.
Langcake P, Pryce RJ. 1997a. A new class of phytoalexins from grapevines. Experientia
15;33:151-152.
Langcake P, Pryce RJ. 1977b. The production of resveratrol and the viniferins by grapevines
in response to ultraviolet irradiations. Phytochemistry 16:1193-1196.
Larronde F, Gaudillere JP, Krisa S, Decendit A, Deffieux G, Merillon JM. 2003. Airborne
methyl jasmonate induces stilbene accumulation in leaves and berries of grapevine plants. Am
J Enol Vit 54:63-66.
Larronde F, Krisa S, Decendit A, Cheze C, Merillon JM. 1998. Regulation of polyphenol
production in Vitis vinifera cell suspension cultures by sugars. Plant Cell Rep 17: 946–950.
Lijavetzky D, Almagro L, Belchi–Navarro S, Martinez–Zapater JM, Bru R, Pedreño MA.
2008. Synergistic effect of methyljasmonate and cyclodextrin on stilbene biosynthesis
pathway gene expression and resveratrol production in Monastrell grapevine cell cultures.
BMC Res Notes 1:132-140.
López-Serrano M, Ferrer MA, Pedreño MA, Ros Barceló A. 1997. Ca 2+
and Mg 2+
ions
counteract the reduction by fosetyl-Al (aluminum tris [ethyl phosphonate]) of peroxidase
activity from suspension-cultured grapevine cells. Plant Cell Tissue Organ Cult 47:207-212.
Okawara M, Katsuki H, Kurimoto E, Shibata H, Kume T, Akaike A. 2006. Resveratrol
protects dopaminergic neurons in midbrain slice culture from multiple insults, Biochem.
Pharmacol 73:550–560.
McDonald KA, Jackman AP. 1989. Bioreactor studies of growth and nutrient utilization in
alfalfa suspension cultures. Plant Cell Rep 8:455-458.
Page 21 of 33
John Wiley & Sons
Biotechnology & Bioengineering
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Peer Review
22
Morales M, Bru R, García-Carmona F, Ros Barceló A, Pedreño MA, 1998. Effect of
dimethyl-β-cyclodextrins on resveratrol metabolism in Gamay grapevine cell cultures before
and after inoculation with shape Xylophilus ampelinus. Plant Cell Tissue Organ Cult 53:179–
187.
Morel G. 1970. Le problème de la transformation tumorale chez les végétaux. Physiol.
Végétale 1970, 8:189–191.
Pauwels L, Morreel K, De Witte E, Lammertyn F, Van Montagu M, Boerjan W, Inze´ D and
Goossens A. 2008. Mapping methyl jasmonate-mediated transcriptional reprogramming of
metabolism and cell cycle progression in cultured Arabidopsis cells. PNAS 105:1380-1385.
Pervaiz S. 2003. Resveratrol: from grapevines to mammalian biology. FASEB J., 17:1975–
1985.
Pezet R, Perret C, Jean-Denis JB, Tabacchi R, Gindro K, Viret O. 2003. δ-Viniferin, a
resveratrol dehydrodimer: One of the major stilbenes synthesized by stressed grapevine
leaves. J Agric Food Chem 51:5488–5492.
Pezet R, Gindro K, Viret O, Richter H. 2004. Effects of resveratrol and pterostilbene on
Plasmopara viticola zoospore mobility and disease development. Vitis 43:145–148.
Ramani S, Jayabaskaran C. 2008. Enhanced catharanthine and vindoline production in
suspension cultures of Catharanthus roseus by ultraviolet-B light. J Mol Signal 3:9-14.
Sivakumar G, Paek K. 2005. Methyl Jasmonate Induce Enhanced Production of Soluble
Biophenols in Panax ginseng Adventitious Roots from Commercial Scale Bioreactors. Chem
Nat Comp 41:669-673.
Song XQ, Xia GM, Zhou AF, Bao XZ, Chen HM. 1999. Hybrid plant regeneration from
interfamiliar somatic hybridization between grapevine (Vitis vinifera L.) and thorowax
(Bupleurum scorzonerifolium Willd). Chin Sci Bull 44:1878–1882.
Page 22 of 33
John Wiley & Sons
Biotechnology & Bioengineering
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Peer Review
23
Staswick P. 1998. Discovering the signaling role of jasmonic acid in plant biology. In:
Discoveries in Plant Biology. Edited by Shain-Dow Kung S-FY. Singapore: World Scientific
Publishing.
Takeuchi A, Matsumoto S, Hayatsu M. 1994. Chalcone synthase from Camellia sinensis:
isolation of the cDNAs and the organ-specific and sugar-responsive expression of the genes.
Plant Cell Physiol 35:1011–18.
Tassoni A, Fornale S, Franceschetti M, Musiani F, Micheal AJ, Perry B, Bagni N. 2005.
Jasmonates and Na-orthovanadate promote resveratrol production in Vitis vinifera L. cv.
Barbera cell cultures. New Phytol 166:895-905.
Tsukaya H, Ohshima T, Naito S, Chino M, Komeda Y. 1991. Sugar dependent expression of
the CHS-A gene for chalcone synthase from petunia in transgenic Arabidopsis. Plant Physiol
97:1414–1421.
Vasconsuelo A, Boland R. 2007. Molecular aspects of the early stages of elicitation of
secondary metabolites in plants. Plant Sci. 172:861-875.
Waffo-Teguo P, Fauconneau B, Deffieux G, Huguet F, Vercauteren J, Merillon JM. 1998.
Isolation, identification, and antioxidant activity of three stilbene glucosides newly extracted
from Vitis Vinifera cell cultures. J Nat Prod 61:655- 657.
Zamboni A, Vrhovsek U, Kassemeyer HH, Mattivi F, Velasco R. 2006. Elicitor-induced
resveratrol production in cell cultures of different grape genotypes (Vitis spp.). Vitis 45:63–
68.
Zamboni A, Gatto P, Cestaro A, Pilati S, Viola R, Mattivi F, Moser C, Velasco R. 2009.
Grapevine cell early activation of specific responses to DIMEB, a resveratrol elicitor, BMC
Genomics 10:363-376.
Zhao J, Davis LC, Verpoorte R. 2005. Elicitor signal transduction leading to production of
plant secondary metabolites. Biotech Adv 23:283-333.
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Table I. Effect of elicitation time on t-R production in Monastrell cell cultures treated
with 50 mM CD individually and in combination with 100 µM MJ.
CDs CDs+MJ Time
(hours) t-R (mg/L)
4 9.0 ± 0.1 2.1 ± 0.3
24 127.6 ± 0.1 325.2 ± 3.9
72 499.5 ± 9.9 1403.9 ± 3.1
96 539.3 ± 9.7 1742.7 ± 8.1
120 522.2 ± 8.1 2032.9 ± 8.8
144 646.9 ± 2.1 2686.4 ± 9.5
168 737.0 ± 4.6 3047.6 ± 9.4
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List of Figures
Figure 1. Time course of growth of Monastrell cell culture, measuring PCV (A), pH (B) and
conductivity (C), in G20 (solid line) and G10 (dashed line). Experiments were repeated twice.
Data are the mean ±SD of one experiment with three replicates.
Figure 2. Effect of different MJ concentrations on growth (measured as FW) and t-R
productivity in Monastrell cell cultures elicited for 96 h, in the absence or in the presence of
50 mM CDs. White bars represent FW and t-R productivity is represented as µmol/g DW
(solid line). The initial cell FW in all treatments was 20 g. Control cell FW after treatments
was 22 ± 0.92. Experiments were repeated twice with similar results. Data are the mean ±SD
of one experiment with three replicates. A Fisher test was applying as data statistic treatment.
Figure 3. Cell viability of Monastrell cell cultures assessed with DAF, as described in
Materials and Methods.
Figure 4. Effect of different initial cell densities (measured as %PCV) on t-R productivity in
Monastrell cell cultures elicited for 96 h with CDs (black bars) or with a combination of CDs
and MJ (white bars). Experiments were repeated twice. Data are the mean ±SD of one
experiment with three replicates. The values were normalized considering the specific
productivity of a cell density of 25% PCV as 100%. Plaid bars represent CD treatment and
striped bars represent the combined treatment with CDs and MJ.
Figure 5. Effect of the exposure of Monastrell cell cultures to UV light (15 and 120 minutes)
in presence of CDs individually or in combination with MJ in Monastrell cell cultures elicited
for 96 h. Values are given as the mean ± SD of three replicates. Bars represent µmol t-R/g FW
and solid line µmol t-R/g DW.
Figure 6. Effect of sucrose concentration in the elicitation medium on t-R productivity in
Monastrell cell cultures elicited for 96 h. Experiments were repeated three times. Data are the
mean ±SD of one experiment with three replicates. Bars represent µmol t-R/g FW (grey bars
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corresponding to 10 g/L, striped bars to 20 g/L and black bars to 30 g/L) and lines µmol t-R/g
DW (dashed line corresponding to 10 g/L, solid line to 20 g/L and dotted line to 30 g/L).
Figure 7. Effect of sucrose on t-R productivity of two different Monastrell cell lines grown
during at least, three subcultures in G10 and G20, elicited for 96 h in the same medium with
50 mM CDs and different MJ concentrations. Experiments were repeated three times. Data
are the mean ±SD of one experiment with three replicates. Bars represent µmol t-R/g FW
(grey colour corresponding to 10 g/L and striped bars to 20 g/L) and lines µmol t-R/g DW
(dashed line corresponding to 10 g/L and solid line to 20 g/L).
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Figure 1
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Treatments
MJ
0
MJ
5
MJ2
5
MJ5
0
MJ
10
0
MJ
27
0
CD
MJ
0
CD
MJ
5
CD
MJ2
5
CD
MJ5
0
CD
MJ
10
0
CD
MJ
27
0
Fre
sh
Weig
ht
(g)
0
5
10
15
20
25
t-R
(µµ µµ
mo
l/g
DW
)
0
500
1000
1500
2000
Figure 2
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Figure 3
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Figure 4
% PCV
25 50 75
t-R
(µµ µµ
mo
l/g
DW
)
0
400
800
1200
1600
%
0
20
40
60
80
100
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Treatments
CD
CD
UV
15
CD
UV
12
0
CD
MJ
CD
MJU
V1
5
CD
MJ
UV
12
0
t-R
(µµ µµ
mo
l/g
FW
)
0
10
20
30
40
50
60
70
t-R
(µµ µµ
mo
l/g
DW
)
0
200
400
600
800
1000
1200
1400
Figure 5
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For Peer Review
8
Treatments
0 25 50 100 270
t-R
(µµ µµ
mo
l/g
FW
)
0
10
20
30
40
50
60
70
t-R
(µµ µµ
mo
l/g
DW
)
200
400
600
800
1000
1200
1400
1600
Figure 6
Page 32 of 33
John Wiley & Sons
Biotechnology & Bioengineering
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Peer Review
9
Treatments
0 25 50 100 270
t-R
(µµ µµ
mo
l/g
FW
)
0
20
40
60
80
t-R
(µµ µµ
mo
l/g
DW
)
0
200
400
600
800
1000
1200
1400
1600
Figure 7
Page 33 of 33
John Wiley & Sons
Biotechnology & Bioengineering
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