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Resveratrol MURCIA

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Biotechnology & BioengineeringCharacterization of the trans-resveratrol production by different inducing factors in Monastrell grapevine cell culturesManuscript ID:Wiley - Manuscript type:Date Submitted by the Author: Complete List of Authors:Key Words:r FoJournal: DraftBiotechnology and BioengineeringArticleBelchi-Navarro, Sarai; University of Murcia, Plant Biology Almagro, Lorena; University of Murcia, Plant Biology Lijavetzky, Diego; Universidad Nacional de Cuyo, Instituto

Text of Resveratrol MURCIA

Biotechnology & Bioengineering

Characterization of the trans-resveratrol production by different inducing factors in Monastrell grapevine cell cultures

Manuscript ID:

Wiley - Manuscript type:

Date Submitted by the Author: Complete List of Authors:

Key Words:

r FoJournal: Draft

Biotechnology and Bioengineering

Article

Belchi-Navarro, Sarai; University of Murcia, Plant Biology Almagro, Lorena; University of Murcia, Plant Biology Lijavetzky, Diego; Universidad Nacional de Cuyo, Instituto de Biologa Agrcola de Mendoza, Consejo Nacional de Investigaciones Cientficas y Tcnicas Bru, Roque; University of Alicante, 1Departamento de Agroqumica y Bioqumica Pedreo, Maria; University of Murcia, Plant Biology 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 culturesSarai Belch-Navarro, Lorena Almagro, Diego Lijavetzky2*, Roque Bru1 and Maria A. Pedreo Departamento de Biologa Vegetal, Facultad de Biologa, Universidad de Murcia, Campus de Espinardo, 30100 Murcia, Spain; 1Departamento de Agroqumica y Bioqumica, Facultad de Ciencias, Universidad de Alicante, Apartado 99, 03080 Alicante, Spain. 2Departamento de Gentica Molecular de Plantas, Centro Nacional de Biotecnologa, Consejo Superior de Investigaciones Cientficas (CSIC), Cantoblanco, 28049 Madrid, Spain; *Permanent address: Instituto de Biologa Agrcola de Mendoza, Consejo Nacional de Investigaciones Cientficas y Tcnicas, Facultad de Ciencias Agrarias, Universidad Nacional de Cuyo (IBAM-FCA-UNCU), Chacras de Coria (5505), Mendoza, Argentina.

Corresponding author: Maria A. Pedreo (mpedreno@um.es ) 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 transresveratrol 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 transresveratrol 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; Lpez-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 g 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 mol/g dried weight (DW). However, the amount of stilbenes secreted to the medium is negligible. Similar experiments were carried

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