Biotechnology & Bioengineering

Characterization of the trans-resveratrol production by

different inducing factors in Monastrell grapevine cell
cultures
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Journal: Biotechnology and Bioengineering

Manuscript ID: Draft

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Wiley - Manuscript type: Article

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Date Submitted by the

Author:

Complete List of Authors: Belchi-Navarro, Sarai; University of Murcia, Plant Biology

er

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
Re

y Bioquímica
Pedreño, Maria; University of Murcia, Plant Biology

cell culture , cyclodextrin , elicitation, methyljasmonate,

Key Words:
resveratrol, Vitis vinifera
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Page 1 of 33 Biotechnology & Bioengineering

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Characterization of the trans-resveratrol production by different inducing
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Sarai Belchí-Navarro, Lorena Almagro, Diego Lijavetzky2*, Roque Bru1 and Maria A.
9 Pedreño
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11 Departamento de Biología Vegetal, Facultad de Biología, Universidad de Murcia, Campus de
12 Espinardo, 30100 Murcia, Spain; 1Departamento de Agroquímica y Bioquímica, Facultad de
13 Ciencias, Universidad de Alicante, Apartado 99, 03080 Alicante, Spain. 2Departamento de
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15 Genética Molecular de Plantas, Centro Nacional de Biotecnología, Consejo Superior de
16 Investigaciones Científicas (CSIC), Cantoblanco, 28049 Madrid, Spain;
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18 *Permanent address: Instituto de Biología Agrícola de Mendoza, Consejo Nacional de
19 Investigaciones Científicas y Técnicas, Facultad de Ciencias Agrarias, Universidad Nacional
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de Cuyo (IBAM-FCA-UNCU), Chacras de Coria (5505), Mendoza, Argentina.
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31 Corresponding author: Maria A. Pedreño (mpedreno@um.es )
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33 Department of Plant Biology, Faculty of Biology, University of Murcia, Campus de

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35 Espinardo, 30100 Murcia, Spain
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Telephone: +34868887000; Fax: +34868883963
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40 Short running title: resveratrol production by cell cultures
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 Biotechnology & Bioengineering Page 2 of 33

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3 Abstract
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6 trans-Resveratrol is one of the most thoroughly studied bioactive molecules due to its
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8 potential benefits for human health. For this reason too, new strategies based on Vitis vinifera
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cell cultures have been used to increase trans-resveratrol production. These strategies include
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13 the selection of highly productive Vitis cell lines, elicitor treatments, the addition of
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15 biosynthetic precursors and stilbene metabolic engineering. V. vinifera cell cultures have been
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used to investigate the factors involved in the induction and regulation of stilbene
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20 biosynthesis and its metabolism. One common goal of such studies has been optimisation of
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22 in vitro stilbene production through the use of biotic or abiotic elicitors, in an attempt to
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attach an economically viable technology for commercial application.
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27 In this work, the effect of different inducing factors on trans-resveratrol production in
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Monastrell grapevine cell cultures is evaluated. A detailed analysis provides the optimal
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32 concentrations of cyclodextrins and methyljasmonate and dosage UV irradiation, optimal cell
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34 density, elicitation time and sucrose content in the culture media. The results indicate that
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trans-resveratrol productivity decreases as the initial cell density increases in grapevine cells
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39 treated with cyclodextrins individually or in combination with methyljasmonate, the decrease
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observed with cyclodextrins alone being far more drastic than the combined treatment.
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44 The synergistic effect observed on extracellular trans-resveratrol accumulation provoked by
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46 the joint use of cyclodextrins and methyljasmonate is lower when these chemical compounds
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48 are combined with UV exposure. Likewise, trans-resveratrol production is dependent on
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51 levels of sucrose in the elicitation medium since, when its concentration in the culture
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53 medium is low, trans-resveratrol biosynthesis is restricted. In contrast, when the supply of
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55 carbon source is not limiting, the highest levels of trans-resveratrol are obtained by the joint
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58 action of cyclodextrins and methyljasmonate.
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3 Much of the success of this strategy is due to the use of cyclodextrins, which act not only as
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6 inducers of trans-resveratrol biosynthesis but also as promoters of adducts that remove trans-
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8 resveratrol from the medium, reducing feedback inhibition and trans-resveratrol degradation
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and allowing its accumulation in high concentrations.
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13 Keywords: cell culture; cyclodextrin; elicitation; methyljasmonate, resveratrol, Vitis vinifera.
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18 Introduction
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20 Vitaceae phytoalexins constitute a group of molecules belonging to the stilbene family
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(Langcake, and Pryce, 1977 a, b; Jeandet et al. 2002) which are derivatives of the trans-
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25 resveratrol structure (t-R, 3,5,4´-trihydroxystilbene). These compounds are known as

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27 viniferins and have been found in both grapevine tissue and cell cultures as the result of
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30 infection or stress, displaying in some cases, strong antifungal activity (Pezet et al. 2004). Due
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32 to their potential benefits for human health, t-R especially has been thoroughly studied. Thus,
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34 multiple lines of compelling evidence indicate its beneficial effects on neurological (Okawara
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37 et al. 2007) and cardiovascular systems (Bradamante et al. 2004). One of the most striking
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39 biological activities of t-R investigated during recent years has been its anticancer activity and
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it has been seen to prevent carcinogenesis in the stages of tumour initiation, promotion and
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44 progression (Pervaiz, 2003). More recent data provide interesting insights into the effect of
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46 this compound on the lifespan of yeast, worms and flies, suggesting that t-R could be
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regarded as a potential antiaging agent in treating age-related human diseases (De la Lastra
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51 and Villegas, 2005). In addition, effects described in mice subjected to a high-calorie diet
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53 (Baur et al. 2006) point to new approaches for treating not only age-related diseases but also
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56 obesity-related disorders (Kaeberlein and Rabinovitch, 2006). That is why new strategies
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58 based on the use of Vitis vinifera cell cultures have been used to increase the level of t-R
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60 production. These strategies include the selection of highly producing Vitis cell lines, elicitor

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3 treatments, the addition of biosynthetic precursors and stilbene metabolic engineering. V.
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6 vinifera cell cultures have been used in several studies to investigate the factors involved in
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8 the induction and regulation of stilbene biosynthesis and its metabolism (Waffo Teguo et al.
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1998; Krisa et al. 1999; Decendit et al. 2002; Commun et al. 2003). One common goal of
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13 such studies has been the optimisation of the in vitro stilbene production through the use of
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15 biotic or abiotic elicitors such as UV light irradiation (Langcake, and Pryce, 1977b; Adrian et
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al. 2000), aluminium ions (Dercks, and Creasy, 1989; López-Serrano et al. 1997), glucans
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20 (Aziz et al. 2003), fungal products (Calderon et al. 1993) or oligosaccharides (Darvill et al.
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22 2003). However, differences found in the stilbene production depend on methodology, Vitis
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species and cultivars used. Thus, in response to UV-C irradiation, American Vitis species
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27 showed a higher capacity for t-R biosynthesis than susceptible species. This response was
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particularly high for V. rupestris which provided up to 750 µg t-R /g fresh weight (FW) within
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32 48 h of irradiation. The production of t-R was dependent on the both time dose of UV light
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34 and the time elapsing after treatment (Douillet-Breuil et al. 1999).
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Jasmonic acid and its more active derivative methyljasmonate (MJ) are signal
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39 molecules that act as key compounds of the signal transduction pathway involved in the
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induction of the biosynthesis of secondary metabolites which take part in plant defence
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44 reactions (Gundlach et al. 1992; Creelman, and Mullet, 1997; Staswick 1998, Chung et al.
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46 2003). Thus, the production of secondary metabolites increases when plant cell cultures are
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48 elicited with jasmonates (Gundlach et al. 1992; Blechert et al. 1995; Zhao et al. 2005;
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51 Vasconsuelo and Boland, 2007). In V. vinifera, Krisa et al. (1999) showed that Cabernet
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53 Sauvignon cell cultures respond to MJ with an increase in the total piceids (resveratrol
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55 glycosylated forms) content when MJ is added at the beginning of exponential growth phase,
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58 being the maximum level reached around 30 µmol/g dried weight (DW). However, the
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60 amount of stilbenes secreted to the medium is negligible. Similar experiments were carried

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3 out in V. vinifera cv Barbera cell cultures responding to MJ (Tassoni et al. 2005). In this case,
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6 the endogenous t-R accumulation was around 100 nmol/g DW and the t-R secreted to the
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8 medium was over 30 nmol/g DW. Kiselev et al. (2007) were able to produce up to 138 µmol
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/g DW of endogenous t-R using transgenic rolB V. amurensis callus cultures.
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13 Many researchers have investigated the mechanism controlling the influence of sugar
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15 on anthocyanin and stilbene accumulation (Larronde et al. 1998). Thus, after adding sucrose
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to the culture medium of grapevine cell cultures, anthocyanin accumulation is increased,
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20 while stilbene levels were only slightly affected (Larronde et al. 1998). Moreover, sugars are
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22 essential to induce the expression of chalcone synthase gene, the level of which coincides
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with the intracellular level of sugars, in particular sucrose and glucose (Tsukaya et al. 1991;
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27 Takeuchi et al. 1994).
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Amongst the different strategies known to increase the production of t-R using
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32 grapevine cell cultures, we have chosen elicitation with β-cyclodextrins (CDs) since these
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34 compounds chemically resemble to the alkyl-derived pectic oligosaccharides naturally
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37 released from the cell walls during fungal attack (Bru et al. 2006). Recently, Zamboni et al.
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39 (2009) reported that CDs trigger a signal transduction cascade which activates different
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families of transcription factors in grapevine cells, inducing a halt in cell division,

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44 reinforcement of the cell wall and the production of t-R and defence-related proteins. Thus,
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46 the productivity of t-R in the spent medium of Monastrell cell cultures elicited with CDs was
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around 300 µmol /g DW (Lijavetzky et al. 2008). This innovative method (Bru and Pedreño.
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51 2003) differs from those that use other elicitors not only in the high levels of t-R produced but
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53 also in the extraction process of this compound. Thus, in traditional elicitation methods, t-R
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extraction from elicited cells gives low yields, whereas in this new process, t-R is secreted as
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58 it is produced by cells and recovered directly from the spent media with no biomass
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60 destruction. In addition, the high levels of t-R accumulated in the spent medium were seen to

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3 have no toxic effect on the cell lines, allowing successful subcultures. This innovative
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6 elicitation process is mainly based on the characteristics of CDs.
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8 Cyclodextrins are cyclic oligosaccharides derived from starch with 6, 7 or 8 glucose
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residues linked by α (1
4) glycosidic bonds, namely α, β and γ cyclodextrins. They have a
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13 hydrophilic external surface and hydrophobic central cavity that can trap apolar compounds,
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15 including resveratrol (Morales et al. 1998). This hydrophobic cavity forms inclusion
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18 complexes, which, in the case of CDs with t-R, is of the 1:1 type, altering its physicochemical
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20 behaviour and making it a highly water-soluble compound.
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In a previous work, we analysed the effects of MJ, CDs and a combination of both on
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25 t-R production and the expression of stilbene biosynthetic genes in grapevine cell
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27 suspensions. MJ and CDs significantly but transiently induced the expression of stilbene
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biosynthetic genes when used independently to treat grapevine cells. Such expression
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32 correlated with t-R production in CD-treated cells but not in MJ-treated cells. In the combined
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34 treatment involving CDs and MJ, t-R production was almost one order of magnitude higher
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37 (1400 µmol/g DW) and was correlated with the maximum expression values for stilbene
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39 biosynthetic genes, demonstrating the synergistic effect of the combination of MJ with a true
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and strong elicitor like CDs (Lijaveztky et al. 2008).

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44 The final goal of our research is to choose the best operating conditions to achieve
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46 high t-R levels and to increase t-R productivity evaluating the effect of different inducing
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factors on Monastrell grapevine cell cultures. For this, a detailed analysis of these factors in t-
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51 R production will provide the optimal concentrations of CDs and MJ and UV irradiation
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53 dosage, optimal cell density, time elicitation and sucrose content in the culture media.
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Materials and Methods
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58 Establishment of callus and suspension cell cultures
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3 V. vinifera L. cv. Monastrell albino calli were established in our laboratory in 1990 as
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6 described by Calderon et al. (1993). Since then, calli have been maintained at 25 ºC in
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8 darkness in 250 ml flasks containing 100 ml of growth medium (Gamborg B5 medium
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supplemented with Morel vitamins (Morel. 1970), 0.25 g/L casein hydrolysate, 20 g/L
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13 sucrose, 0.2 mg/L kinetin, 0.1mg/L 1-naphthaleneacetic acid (NAA)), 8 g/L agar as
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15 solidifying agent and pH adjusted at 6.0. Grapevine calli were subcultured on solid growth
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medium every month.
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20 Grapevine cell suspensions were initiated by inoculating friable callus pieces in 250
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22 mL Erlenmeyer flasks containing 100 mL of liquid growth medium adjusted to pH 6.0 and,
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maintained in a rotary shaker (110 rpm) at 25 °C in the dark. The cell suspension was
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27 routinely maintained by periodical subcultures every 14-16 days by diluting with one volume
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of fresh growth medium and then distributing into two flasks.

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32 Elicitor treatments and effect of sucrose content on t-R production in grapevine cell
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34 cultures
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Elicitation experiments were performed in triplicate using 12-14 day old grapevine
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39 cell suspensions. For this, 20 g of washed cells (FW) were transferred to a 250 ml Erlenmeyer
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flask and suspended in 100 ml of sterile fresh growth medium supplemented with either
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44 methylated CDs (CAVASOL® W7M, Wacker) or MJ or a combination of both CDs and MJ,
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46 followed by at least 96 h of incubation at 25 °C in darkness in a rotary shaker (110 rpm).
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48 Control cultures without elicitors were always run in parallel. After elicitation, cells were
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51 separated from the culture medium under a gentle vacuum, rapidly washed with cold distilled
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53 water, weighed and frozen at -80 ºC until use. The spent medium was used for measuring the
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55 t-R content.
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58 Suspension cell cultures were also elicited under UV light at short and long time
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60 exposition in the presence or in the absence of the above chemical elicitors. For this, flasks

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3 were opened in a laminar flow hood and exposed to UV-C light (254 nm) for either 15 or 120
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6 minutes, at an irradiation distance of 15 cm. During this time and after irradiation, flasks were
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8 kept in continuous agitation for at least 96 h.
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The effect of sucrose content on grapevine cell cultures was assessed in two different
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13 ways: either by transferring cells into sterile fresh Gamborg B5 medium with sucrose at 10 or
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15 20 g/L at the moment the elicitation experiments were carried out, in the presence or in the
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absence of the above chemical elicitors or by maintaining suspension cell cultures for at least
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20 three subcultures in fresh growth media with sucrose at 10 or 20 g/L before carrying out the
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Determination of cell growth and viability
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27 A time course of cell growth was followed by measuring the packed cell volume
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(PCV, expressed in percentage). For this, 2 mL of cell suspension homogeneous aliquots were
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32 periodically withdrawn and centrifuged at 100 x g for 5 min. The conductivity and the pH
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34 were also determined in the spent medium using a Crison micro CM 2100 conductivity meter
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and a Crison 2002 pHmeter respectively. Cell viability was assessed by incubating the cells
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39 for 1-2 min in fresh Gamborg B5 medium containing 100 µg ml-1 fluorescein diacetate (DAF)
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(Huang et al. 1986) and observing the fluorescence emission by living cells with a DMRB
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44 Leica optical microscope (λexc = 490 nm, λemi = 520 nm) using a Leica filter I3.
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46 Analysis of t-R in the spent medium
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48 Aliquots of the spent medium were diluted with water and methanol to a final
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51 concentration of 80% methanol (v/v). 20 µl of diluted and filtered (Anopore 0.2µm) samples
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53 were analyzed in a HPLC (Waters 600E) with Diode Array Detector system (Waters 996).
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Sample separation was performed using a Spherisorb ODS2 C-18 column (250 x 4.6 mm,
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58 particle size 5µ) and they were eluted with a mobile phase of 1% acetic acid (A)-acetonitrile
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60 (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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3 15 min, 65% A; 17-25 min, 10 % A; 25-30 min, 65% A, 30-35 min, 85% A. The t-R was
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6 identified and quantified by comparison with authentic t-R of >99% purity (Sigma-Aldrich,
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8 Madrid, Spain).
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Results and Discussion
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13 Characterization of Monastrell cell culture growth
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15 Prior to the elicitation treatments, Monastrell cell culture growth was characterized by
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measuring PCV, pH and conductivity in Gamborg B5 medium supplemented with two
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20 different sucrose concentrations (10 or 20 g/l, namely G10 and G20, respectively). As shown
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22 in Figure 1A, these grapevine cell suspensions exhibited a typical sigmoid-shaped growth
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curve with no significant differences in the lag or exponential phases. Differences existed in
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27 the stationary phases of the cell lines grown in G20 and G10. Thus, the kinetic parameters of
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cells growing in G20 pointed to a maximum biomass of 85% (PCVmax) and a biomass yield of
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32 2.35 (∆PCV/g sucrose), 16 days being necessary to reach the stationary phase.
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34 The corresponding values for cell suspensions grown in G10 were lower, and the
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37 maximum PCV was around 78% (Figure 1A). As can be observed from Figures 1A and 1C,
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39 the trend of the conductivity in the spent medium was contrary to that of PCV. The decrease
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in conductivity was directly related with the depletion of mineral ions in the culture medium
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44 since the cell culture uses them for growth.
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46 The pH of the spent medium (Figure 1B), which was initially adjusted to 6.0,
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underwent acidification during the first 48 hours and then increased before reaching a plateau
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51 (from day 11) which was maintained until the end of the exponential growth phase. The pH of
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53 the spent medium is conditioned by the cell metabolism and it is tightly related to the
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consumption of the nitrogen source and the energetic state of cells (McDonald and Jackman,
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58 1989).
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60 Cell growth and t-R production is dependent on MJ dose and CDs

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3 The effect of the elicitors on cell growth was checked by determining cell FW
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6 (initially 20 g in all treatments) after 96 hours of treatment. As shown in Figure 2, the FW of
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8 MJ-treated cells remains constant at first or decreased slightly (16.7%) while in the combined
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CDs + MJ treatment, cell growth was clearly reduced (27.1%). These results show that the
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13 joint action of CDs and MJ leads to a reduction of cell growth, at least during the first 96 h of
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15 treatment. The use of chemical compounds like jasmonates to induce the production of
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stilbenes in grapevine cell cultures has been associated to modifications in the pattern of cell
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20 growth (Krisa et al. 1999). Tassoni et al. (2005) described the undesirable effect of a
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22 reduction of cell growth in MJ treated V. vinifera cv Barbera cell cultures. Kiselev et al.
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(2007) also described the inhibition of cell growth in V. amurensis callus cultures as a result
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27 of treatment with different elicitors. This effect of MJ on cell growth has been suggested to be
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caused by the diversion of the metabolic flux (carbon allocation), favouring the activation of
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32 the secondary metabolism over the primary metabolism (Larronde et al. 2003; Sivakumar and
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34 Paek, 2005), although a direct effect of MJ on the repression of cell multiplication and/or
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growth has recently been reported by Pauwels et al. (2008). Although cell growth was halted
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39 in elicitor treated-cells, cell viability, as assessed by fluorescence microscopy of cells treated
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with DAF (Figure 3), seems to be unaffected.

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44 Figure 2 also shows the effect of different concentrations of MJ on t-R productivity by
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46 Monastrell cell cultures elicited during 96 h, in the absence or in the presence of CDs (50
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48 mM). No t-R was detected in cells treated with ethanol, the solvent in which MJ is delivered
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51 to the culture (data not shown). As can be observed in Figure 2, the maximum level of t-R
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53 produced and secreted by cells to the medium was reached when cell suspensions were
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55 incubated in the joint presence of CDs and 100 µM MJ, this level being around 4.3-fold more
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58 (1447.78 µmol t-R/g DW, Figure 2, solid line) than when cells were treated only with CDs
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60 (338.60 µmol t-R/g DW). However, when cell cultures were elicited only with MJ, very low

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3 amounts of t-R were detected in the spent medium (16.36 µmol t-R/g DW with 100 µM MJ).
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6 Krisa et al. (1999) described how the amount of total stilbenes secreted to the culture medium
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8 was negligible in both MJ and control cultures of three V. vinifera cultivars. In addition, they
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reported that stilbene accumulation was notably induced in grape cell CS4 (Cavernet
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13 Sauvignon) cultivar when 25 µM MJ was added to the induction medium on day 6. In these
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15 conditions and after 8 days of treatment, they obtained 771 µmol/L in 27.8 g DW /L, which
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means 27.73 µmol t-R /g DW. In the presence of 25 µM MJ, we detected similar levels of t-R
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20 in the spent medium (15.86 µmol t-R /g DW). However, t-R production levels increased 58.8
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22 times (932.46 µmol t-R /g DW) when 25 µM MJ and 50 mM CDs were jointly used as
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inducers.
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27 On the other hand, Kiselev et al. (2007) reported high t-R production by V. amurensis
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callus cultures transformed with the rolB gene of Agrobacterium rhizogenes. These callus
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32 cultures accumulated up to 138.16 µmol t-R/g DW (3.15 % DW of t-R). This value, which is
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34 two orders of magnitude higher that reported for other resveratrol-producing plant cell
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cultures (Ku et al. 2005; Tassoni et al. 2005), is 10.4 times lower than that obtained in our
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39 best conditions.
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t-R production is dependent on elicitation time

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44 The time course of t-R accumulation in Monastrell cell suspensions treated with CDs
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46 and the optimal concentration of MJ (100 µM), separately or in combination, was followed by
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48 periodically analyzing the t-R concentration in the spent medium (Table 1). Only a negligible
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51 amount of t-R was detected in control and MJ treated-cells (data not shown). As can be
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53 observed in Table 1, in both cases (using only CDs or a combination of CDs and MJ) a
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55 continuous accumulation of t-R (measured as t-R production) was observed. The higher
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58 accumulation rate in the combined treatment compared with the CD treatment (>4.5-fold) and
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60 the larger accumulation with time are the driving forces behind the greater overall production

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3 of t-R. Since t-R production in the combined treatment was higher than the sum of the
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6 individual treatments (due to the low production in MJ treated-cells), a synergistic effect
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8 between both elicitors is assumed, as described Lijaveztky et al. (2008). Zamboni et al. (2006)
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analysed variations in the response to CDs in four different grape genotypes. Thus, cell
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13 cultures obtained from the crossing between V. riparia and V. berlandieri and V. amurensis
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15 cell lines were capable of producing more t-R (911.25 and 225.22 mg/L, respectively) than
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grapevine cell cultures of Pinot Noir (0.51 mg/L) or Merzling (4.31 mg/L) after 48 h of CD-
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20 treatment. The results obtained by these authors using high-producer cell lines are in
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22 accordance with those obtained from Monastrell cell cultures (Table 1). Differences in t-R
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levels amongst Vitis genotypes could be related with their different levels of defence
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27 response. This variability of the response of Vitis cultures to elicitation in presence of CDs
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under the same conditions demonstrates that is very important to select genotypes which have
30
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32 the ability to produce the highest levels of t-R.
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34 t-R production is dependent on initial cell density
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36
Once the optimal MJ concentration and time elicitation was fixed for the different
37
ev

38
39 treatments, we analyzed the influence of initial cell density (expressed as % PCV) on t-R
40
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iew

production. As can be observed in Figure 4, the specific productivity of t-R decreased as the
42
43
44 initial cell density increased in grapevine cells treated with CDs (black bars) and CDs in
45
46 combination with MJ (white bars). As the response of cell cultures to elicitation depends on
47
48 cell density, and since the maximum levels of t-R are reached with a low cell density, it would
49
50
51 seem that this need to work at low cell density would make the scaling up process and the
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53 transition to bioreactors would not be economically viable. Despite this fact, the levels of t-R
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55 in all the combined treatments (CDs and MJ) are higher than those in the absence of MJ
56
57
58 (white bars vs black bars in Figure 4). In fact, normalization of the values in relation to the
59
60 specific productivity with a cell density of 25% PCV clearly shows that the decrease in t-R

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Page 13 of 33 Biotechnology & Bioengineering

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3 productivity is more dramatic for the treatment with CD alone (plaid bars) than for the
4
5
6 combined treatment with CDs and MJ (striped bars). For this reason, we decided to work at
7
8 intermediate cell density (50% PCV), using both CDs and MJ and to explore the possibility of
9
10
increasing t-R productivity through the addition of a third elicitor, in this case, UV light.
11
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13 Effect of UV light exposure and both CDs and MJ on t-R production
14
15 UV light has been described as a physical elicitor of stilbene biosynthesis in grapevine
16
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18
(Douillet-Breuil et al. 1999; González-Barrio et al. 2006). For this reason, the effect of the
19
20 exposure of grapevine cells to UV light in the presence of CDs, separately or in combination
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22 with MJ, on t-R productivity was studied (Figure 5).
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Cells treated with both white and UV-light, with and without MJ, produced a
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26
27 negligible amount of t-R (data not shown). In addition, cell suspensions elicited only with
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CDs and exposed for short times to UV light (15 min) showed a similar increase in t-R levels
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32 as seen in UV-unexposed CD-treated cells, while prolonged exposure to UV-light (120 min)
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34 caused a drastic reduction in t-R accumulation and browning of the cell culture as a result of a
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hypersensitive response (HR) to UV light (Figure 5). In contrast, cells treated with CDs and
37
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38
39 MJ, followed by short or long exposure to UV light, showed lower t-R levels than UV-
40
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iew

unexposed cells, and HR-induced browning was aggravated by long UV exposure. Such long
42
43
44 UV light exposure is clearly detrimental to t-R production because of the HR induced, while
45
46 short exposures do not enhance t-R levels in the presence of CDs and when MJ is also
47
48 present, so there is an antagonism effect between MJ and UV-light.
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50
51 UV light has been reported to increase the production of secondary metabolites in plant cell
52
53 cultures (Broeckling et al. 2005; Song et al. 1999; Gläβgen et al. 1998). In this sense, Ramani
54
55 and Jayabaskaran (2008) observed the enhanced production of catharanthine and vindoline
56
57
58 from Catharanthus roseus cell cultures, which increased their levels 3 and 12 fold,
59
60 respectively when cells were irradiated with UV-B for 5 min. Gläβgen et al. (1998) studied

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 Biotechnology & Bioengineering Page 14 of 33

1
2
3 the effect of continuous irradiation with UV-containing white light (315-420 nm) on the
4
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6 anthocyanin content of carrot cell cultures after 7 days of the culture. They observed that the
7
8 total anthocyanin concentration was strongly enhanced by the UV treatment (3-5 fold higher
9
10
than dark-grown cell cultures), while the strong UV induction of anthocyanin accumulation
11
12
13 was reduced nearly to the levels of dark-grown cells when they were treated with both UV
14
15 light and fungal elicitors. Little is known about the effect of UV irradiation on the stilbene
16
17
18
content of grapevine cell suspensions and most of the research related with UV light has been
19
20 directed at enhancing the stilbene content of grape berries (Adrian et al. 2000; Cantos et al,
Fo

21
22 2003) and leaves (Pezet et al. 2003). Thus, when Keller et al. (2000) studied stilbene
23
24
accumulation in Cabernet-Sauvignon callus cv irradiated with UV light, they found that only
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27 actively growing callus was capable of producing stilbenes (including t-R), whereas old callus
28
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had lost this ability. This response was similar to that found in ripening grape berries, which
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32 gradually lose their potential for stilbene synthesis as they approach maturity. These results
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33
34 could explain the low t-R levels found when Monastrell cell cultures were exposed to UV
35
36
light because our elicitation experiments were performed using 12-14 day old grapevine cell
37
ev

38
39 suspensions which are just entering their stationary phase.
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t-R production is dependent on the level of sucrose in the medium

42
43
44 We have studied t-R productivity in a cell line grown in G20 using elicitation media
45
46 which contained three sucrose concentrations and different MJ concentrations at a fixed CD
47
48 concentration of 50 mM. As can be observed from Figure 6, the optimal MJ concentration
49
50
51 suitable for the elicitation of Monastrell cell cultures is dependent on the sucrose levels in the
52
53 elicitation media. Thus, when cell suspensions were elicited in a culture medium with a low
54
55 sucrose concentration (10 g/L, gray bars in Figure 6), t-R productivity increased using the
56
57
58 lowest MJ dose (25 µM), reaching a maximum value of 46 µmol t-R/g FW. This value is even
59
60 lower when grapevine cell cultures are elicited with CDs and higher MJ concentrations so that

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Page 15 of 33 Biotechnology & Bioengineering

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3 t-R productivity is limited by this sucrose concentration (10 g/L). When sucrose levels in the
4
5
6 elicitation media were increased to 20 g/L and an MJ dose of 100 µM was used, maximal t-R
7
8 productivity was observed (60 µmol t-R /g FW). This maximal t-R productivity was also
9
10
observed when a MJ dose lower (50 µM) was added to elicitation media which contained 30
11
12
13 g/L of sucrose. In addition, the lowest t-R productivity values (16-22 µmol t-R /g FW) were
14
15 found in CD-treated cells and no significant differences were detected in t-R productivity in
16
17
18
this treatment using three sucrose concentrations (Figure 6).
19
20 As can be observed in Figure 1A, cell culture growth did not change much when cell
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22 cultures were grown in culture media with different sucrose concentrations. However, the
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sucrose content in the elicitation media (G20 or G30 vs G10) modified t-R productivity as is
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27 described in Figure 6.
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Larronde et al. (1998) analyzed the effect of sucrose (100-200 mM) on the polyphenol
30
31
32 content in Gamay Fréaux cell suspensions. Their results indicated that anthocyanin
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34 accumulation was strongly stimulated by sucrose while this sugar only had a slight effect on
35
36
the production of stilbenes. Belhadj et al. (2008) studied the effect of MJ treatment (20 µM) in
37
ev

38
39 combination with added sucrose (80 mM) on stilbene accumulation in the culture medium.
40
41
iew

After 18 h of treatment, the t-R accumulated in the culture medium reached a maximal value
42
43
44 of 52 nmol t-R/g FW. Working in similar experimental conditions (25 µM MJ and 60 mM
45
46 sucrose) but in the presence of CDs, we obtained 45 µmol t-R/g FW that is, 865-fold higher
47
48 than those obtained by Belhadj et al. (2008). We have also carried out elicitation experiments
49
50
51 in a culture medium which contained 30 g/L (black bars and dotted line in Figure 6). In these
52
53 conditions, the maximal level of t-R (55.2 µmol t-R/g FW) is obtained in the combined
54
55 treatment of CDs and 50 µM MJ and no significant differences were found when CDs and
56
57
58 100 µM MJ were added (53.8 µmol t-R/g FW). These results show that t-R productivity only
59
60 depends on the carbon source when its concentration in the culture medium is low (10 g/L,

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 Biotechnology & Bioengineering Page 16 of 33

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3 around 30 mM) because this restricts t-R biosynthesis. In contrast, when the supply of carbon
4
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6 source is not limiting, the highest levels of t-R are obtained by the joint action of MJ and CDs,
7
8 and the use of a higher concentration of sucrose (30 g/L) in the elicitation media does not
9
10
substantially increase t-R productivity (dotted line in Figure 6).
11
12
13 We also studied the dependence of t-R productivity on both MJ dose and cell lines
14
15 growing in G10 and G20. For this, cell lines were grown and maintained during at least three
16
17
18
subcultures in G10 and G20 and then, they were elicited with different MJ concentrations in
19
20 the presence of 50 mM CDs (Figure 7). As can be observed in this figure, the lowest MJ
Fo

21
22 concentration required to obtain the highest t-R productivity (46.8 µmol t-R/g FW) in the cell
23
24
line grown in G10 was 50 µM. Although cell growth is not affected by the shortage of sucrose
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27 in the culture medium (dashed line in Figure 1A), t-R productivity is still limited by the
28
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carbon source (dashed line in Figure 7). However, the highest t-R productivity value was
30
31
32 reached when 100 µM MJ was used in the cell line grown in G20. In addition, no significant
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34 differences were found in t-R productivity (40-50 µmol t-R/g FW) in both cell lines elicited at
35
36
other MJ concentrations, except in the cell line grown in G10 elicited with 25 µM of MJ (30.8
37
ev

38
39 µmol t-R/g FW, grey bars in Figure 7). It is worth noting that in the combined treatments of
40
41
iew

CDs and 270 µM MJ, a HR was observed, perhaps due to the high MJ concentration.
42
43
44 In conclusion, in order to achieve high t-R levels and to increase t-R productivity
45
46 using a highly productive Monastrell cell line, the best operating conditions would involve
47
48 elicitation of these cell cultures using a combination of CDs and MJ. The CD concentration
49
50
51 was fixed at 50 mM since a previous work showed that the use of this concentration led to the
52
53 highest t-R production in this cell line. Moreover, CDs do not only act as elicitors but also
54
55 form inclusion complexes (adducts) with t-R, favouring the substantial accumulation of this
56
57
58 compound and also preventing the toxic and/or inhibitory effect of t-R on grapevine cells. The
59
60 optimal MJ concentration found for the production of t-R depended on the sucrose

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Page 17 of 33 Biotechnology & Bioengineering

1
2
3 concentration of the culture medium in which elicitation was carried out. When the sucrose
4
5
6 concentration is not limiting, in order to obtain the same t-R productivity, the higher the
7
8 concentration of sugar, the lower the concentration of MJ needed. t-R productivity increased
9
10
as cell density decreased. However, the fall in t-R productivity was less drastic when
11
12
13 suspension cultured-cells were elicited with CDs and MJ together. The use of a third elicitor
14
15 (UV light) did not improve t-R productivity in any case.
16
17
18
19
20 Acknowledgements
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22 Almagro L. has a fellowship from the MICINN. We thank José Martínez Parra and Francisco
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24
Fernández-Pérez for the identification and quantification of trans-resveratrol by HPLC and
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27 Pepita Alemán for help in maintaining Monastrell cell cultures. This work has been partially
28
29
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supported by the Consejería de Educación, Ciencia e Investigación de la Región de Murcia

30
31
32 (BIO BVA 07 01 0003), by Programa de Generación de Conocimiento Científico de
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34 Excelencia de la Fundación Séneca, Agencia de Ciencia y Tecnología de la Región de Murcia
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36
en el marco del II PCTRM 2007-10 (08799/PI/08) and by the Ministerio de Ciencia e
37
ev

38
39 Innovación (BIO2008-02941).
40
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iew

42
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44 Abbreviations: CDs, methylated β-cyclodextrins; DAF, fluorescein diacetate, DW, dry
45
46 weight; FW, fresh weight; G20 and G10, Gamborg B5 medium supplemented with 10 and 20
47
48
49 g/l of sucrose respectively; HR, hypersensitive response; MJ, methyljasmonate; PCV, Packed
50
51 cell volume, trans-resveratrol, t-R.
52
53
54
55 References
56
57 Adrian M, Jeandet P, Douillet-Breuil AC, Tesson L, Bessis R. 2000. Stilbene content of
58
59 mature Vitis vinifera berries in response to UV-C elicitation. J Agr Food Chem 48:6103-6105.
60

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3 Aziz A, Poinssot B, Daire X, Adrian M, Bezier A, Lambert B, Joubert JM, Pugin A. 2003.
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8 cinerea and Plasmopara viticola. Molecular Plant-Microbe Interact 16:1118-1128.
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Baur JA, Pearson KJ, Price NL, Jamieson HA, Lerin C, Kalra A, Prabhu VV, Allard JS,
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Belhadj A, Telef N, Saigne C. 2008. Effect of methyl jasmonate in combination with
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44 Bru R, Pedreño MA. Method for the production of resveratrol in cell cultures. PCT Patent
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55 Calderón AA, Zapata JM, Muñoz R, Pedreño MA, Ros Barceló A. 1993. Resveratrol
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3 Cantos E, Espín JC, Fernández MJ, Oliva J, Tomás-Barberán FA. 2003. Postharvest UV-C
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González-Barrio R, Beltrán D, Cantos E, Gil MI, Espín JC, Tomás-Barberán FA. 2006.
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27 the CHS-A gene for chalcone synthase from petunia in transgenic Arabidopsis. Plant Physiol
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97:1414–1421.
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32 Vasconsuelo A, Boland R. 2007. Molecular aspects of the early stages of elicitation of
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34 secondary metabolites in plants. Plant Sci. 172:861-875.
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Waffo-Teguo P, Fauconneau B, Deffieux G, Huguet F, Vercauteren J, Merillon JM. 1998.
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39 Isolation, identification, and antioxidant activity of three stilbene glucosides newly extracted
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from Vitis Vinifera cell cultures. J Nat Prod 61:655- 657.

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44 Zamboni A, Vrhovsek U, Kassemeyer HH, Mattivi F, Velasco R. 2006. Elicitor-induced
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46 resveratrol production in cell cultures of different grape genotypes (Vitis spp.). Vitis 45:63–
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48 68.
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51 Zamboni A, Gatto P, Cestaro A, Pilati S, Viola R, Mattivi F, Moser C, Velasco R. 2009.
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53 Grapevine cell early activation of specific responses to DIMEB, a resveratrol elicitor, BMC
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55 Genomics 10:363-376.
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58 Zhao J, Davis LC, Verpoorte R. 2005. Elicitor signal transduction leading to production of
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60 plant secondary metabolites. Biotech Adv 23:283-333.

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3 Table I. Effect of elicitation time on t-R production in Monastrell cell cultures treated
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6 with 50 mM CD individually and in combination with 100 µM MJ.
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10 Time CDs CDs+MJ
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12 (hours) t-R (mg/L)
13 4 9.0 ± 0.1 2.1 ± 0.3
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15 24 127.6 ± 0.1 325.2 ± 3.9
16 72 499.5 ± 9.9 1403.9 ± 3.1
17 96 539.3 ± 9.7 1742.7 ± 8.1
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19 120 522.2 ± 8.1 2032.9 ± 8.8
20 144 646.9 ± 2.1 2686.4 ± 9.5
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168 737.0 ± 4.6 3047.6 ± 9.4
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3 List of Figures
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6 Figure 1. Time course of growth of Monastrell cell culture, measuring PCV (A), pH (B) and
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8 conductivity (C), in G20 (solid line) and G10 (dashed line). Experiments were repeated twice.
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Data are the mean ±SD of one experiment with three replicates.
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13 Figure 2. Effect of different MJ concentrations on growth (measured as FW) and t-R
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15 productivity in Monastrell cell cultures elicited for 96 h, in the absence or in the presence of
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50 mM CDs. White bars represent FW and t-R productivity is represented as µmol/g DW
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20 (solid line). The initial cell FW in all treatments was 20 g. Control cell FW after treatments
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22 was 22 ± 0.92. Experiments were repeated twice with similar results. Data are the mean ±SD
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of one experiment with three replicates. A Fisher test was applying as data statistic treatment.
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Materials and Methods.

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34 Monastrell cell cultures elicited for 96 h with CDs (black bars) or with a combination of CDs
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and MJ (white bars). Experiments were repeated twice. Data are the mean ±SD of one
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39 experiment with three replicates. The values were normalized considering the specific
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productivity of a cell density of 25% PCV as 100%. Plaid bars represent CD treatment and
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44 striped bars represent the combined treatment with CDs and MJ.
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46 Figure 5. Effect of the exposure of Monastrell cell cultures to UV light (15 and 120 minutes)
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48 in presence of CDs individually or in combination with MJ in Monastrell cell cultures elicited
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51 for 96 h. Values are given as the mean ± SD of three replicates. Bars represent µmol t-R/g FW
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53 and solid line µmol t-R/g DW.
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55 Figure 6. Effect of sucrose concentration in the elicitation medium on t-R productivity in
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58 Monastrell cell cultures elicited for 96 h. Experiments were repeated three times. Data are the
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3 corresponding to 10 g/L, striped bars to 20 g/L and black bars to 30 g/L) and lines µmol t-R/g
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6 DW (dashed line corresponding to 10 g/L, solid line to 20 g/L and dotted line to 30 g/L).
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8 Figure 7. Effect of sucrose on t-R productivity of two different Monastrell cell lines grown
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13 50 mM CDs and different MJ concentrations. Experiments were repeated three times. Data
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15 are the mean ±SD of one experiment with three replicates. Bars represent µmol t-R/g FW
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(grey colour corresponding to 10 g/L and striped bars to 20 g/L) and lines µmol t-R/g DW
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