Production and secretion of resveratrol in hairy root cultures of peanut
Graphical abstract
Hairy root cultures of peanut were established and elicited to produce trans-resveratrol. Sodium acetate increased the levels of trans-resveratrol in the medium by almost 60-fold in comparison to non-elicited cultures.
Introduction
trans-Resveratrol 1 (trans-3,5,4′-trihydroxystilbene, Fig. 1), and its many derivatives (Larronde et al., 2005, Rimando and Barney, 2005), are naturally occurring phytoalexins produced in a select number of plant species. These plant polyphenols have received considerable interest based upon a number of associated health benefits (Baur and Sinclair, 2006, Delmas et al., 2006). Most notably, the significant levels of resveratrol 1 in red wine have been credited to the phenomenon known as “the French Paradox”, wherein low incidence of heart disease is observed among a population with a relatively high saturated fat diet and moderate wine consumption (Frankel et al., 1993, Siemann and Creasy, 1992). Over the past two decades, numerous health benefits impacting cardiovascular disease, various cancers, atherosclerosis and aging have been linked with resveratrol 1 (reviewed; Baur and Sinclair, 2006, Roupe et al., 2006). While resveratrol 1 is one of the better known and well studied phytochemicals, several other resveratrol derivatives have also been shown to have similar and/or additional health benefits. Included among these derivatives is the methylated resveratrol compound, pterostilbene 2 (Fig. 1), that has demonstrated in vivo effects for reducing cholesterol levels (Rimando et al., 2005).
A number of taxonomically unrelated plant families have been reported to produce marked levels of resveratrol 1 including: grapes, peanuts, several types of berries, some pine trees and most recently tomato fruit skin (Ragab et al., 2006). Resveratrol 1 is a phytoalexin, a class of antibiotic compounds produced as part of the plant’s defense system and believed to be the major active stilbene that confers pathogen resistance in these plants (Dixon, 2001). The terminal enzyme in the production of resveratrol 1 is resveratrol synthase, which condenses p-coumaroyl-coenzyme A and three malonyl-coenzyme A molecules to form resveratrol 1 (Schöppner and Kindl, 1984). This enzyme is highly regulated by elicitors and general plant defense compounds in an effort to protect the plant. Resveratrol 1 exists as both the trans- and cis-isomers with numerous reports suggesting trans-resveratrol to be the most bioactive form of this molecule (Roupe et al., 2006). trans-Resveratrol 1 can readily be converted to cis-resveratrol when exposed to UV light and is unstable when exposed to high pH conditions.
The use of complementary or alternative beneficial products for human health is increasing worldwide with their continued popularity in Europe and Asia and a dramatic upward trend of their use in the United States (Frost and Sullivan, 2005). Currently resveratrol 1 is primarily marketed as an herbal or dietary supplement in the form of pills, capsules, powders, and extracts from raw botanical sources (i.e. grape seeds/skins; Japanese knotweed Polygonum cuspidatum), with more recent applications beginning to incorporate this popular phytochemical into fortified food/beverage products (i.e. Old Orchard Beverage Company, Sparta, MI). While stilbenes are cost-effectively recovered from these raw materials and will continue to serve this market, these relatively crude sources of resveratrol 1 often lack the consistency and purity required for many applications in the food/pharmaceutical sectors. Furthermore, more natural product consumers and nutrition practitioners are demanding higher quality supplements that are scientifically tested and better defined in their product content (validated by third party quality assurance testing) in a desire to mitigate ineffective and/or erratic responses with these supplements.
The majority of resveratrol-containing dietary supplements are composed of unknown/unidentified botanical components wherein resveratrol 1 and resveratrol derivatives only make up a small fraction of the product. While chemically-synthesized resveratrol 1 may address this issue, natural sources often contain derivatives, co-factors and other phytonutrients that provide added or synergistic benefits to the nutraceutical product and are often preferred by the consumer (Wallace, 1998). Recent studies showing anti-aging benefits of resveratrol 1 (Baur et al., 2006) further accelerate interest in a natural, food-grade, source of enriched resveratrol/resveratrol derivatives that delivers a more defined and consistent product composition and ensures a stable supply chain, several biotic production strategies targeting recombinant plants, yeast and bacteria have been advanced (Becker et al., 2003, Paiva and Hipskind, 2005, Watts et al., 2006). While these approaches potentially offer a more consistent, concentrated resveratrol 1 source, widespread use of these strategies have not been adopted due in part to natural product consumers’ negative perception of genetically modified organisms and issues with associated production efficiency/costs. Grape cell suspension cultures for resveratrol 1 production avoid some of these recombinant issues and provide potential production of a suite of resveratrol compounds (Bru et al., 2006, Liu et al., 2003), however, cell suspension production systems have reported issues of genetic instability and losses of secondary metabolite production following elicitation (Gossens et al., 2003) or repetitive subculturing (Chattopadhyay et al., 2002).
To this end, plant hairy roots offer a novel and sustainable tissue-based system that preserves the multiple specialized cell types believed important in maintaining better consistency in the synthesis of bioactive secondary molecules. Tissue-based systems more accurately reflect the metabolic phenotype and performance of the host plant in comparison to plant cell cultures and further the potential of producing various combinations of valued products from a single production line (Guillon et al., 2006a, Guillon et al., 2006b, Sevon and Oksman-Caldentey, 2002). Recent advances with large scale production have successfully produced ginseng roots in a 10,000 l bioreactor establishing the feasibility of the root system to accommodate industrial processes (Sivakumar et al., 2006).
Here we report the development of peanut hairy root lines for sustained and reproducible production of a naturally-derived source of resveratrol and resveratrol derivatives. These phytochemicals, can be readily recovered from the culture medium in relatively enriched form, detected and quantitated. The use of plant elicitors to enhance secretion of resveratrol 1 and other resveratrol derivatives support our efforts in exploring the commercial potential of this scalable bioprocessing system for valued botanical compounds with nutraceutical properties such as resveratrol and its associated derivatives.
Section snippets
Establishment of peanut hairy root lines
With numerous studies demonstrating that peanut is among a divergent group of plants with endogenously high levels of resveratrol 1 (Chen et al., 2002, Liu et al., 2003, Sanders et al., 2000, Sobolev and Cole, 1999), we targeted a runner peanut cultivar for establishing hairy root lines as a sustainable bioproduction platform in the delivery of a well-defined and enriched resveratrol 1 product. In initial experiments with 21-day old seedlings of cv. Andru II, a minimum of one explant of each
Concluding remarks
The hairy root culture platform is a unique bioproduction system for generating well-defined, highly-enriched fractions of resveratrol 1 and other beneficial stilbene compounds. In capturing the spatio-temporal organization of the source plant, this tissue-based culture system may better preserve the natural metabolic processes as they occur in nature. Maintenance of tissue integrity likely supports the distinctive genetic and biosynthetic stability of hairy roots and enables their fast growth
Establishment of hairy root cultures
Seeds of peanuts (Arachis hypogaea) cv. Andru II (kindly provided by Dr. Daniel Gorbet, University of Florida) were surface sterilized as follows. Seeds were presoaked for 2 min in sterile water containing 0.003% Ivory™ detergent; immersed for 15 min in sterilization solution (50% Clorox™, 0.003% Ivory™ detergent) and rinsed in sterile water. To minimize Chlorox™ damage to the embryo, the testa was aseptically removed and seeds were further rinsed in two changes of sterile water over a 15 min
Acknowledgements
We acknowledge Dr. Daniel Gorbet (University of Florida) for providing seeds of peanut cv. Andru II for these studies. Funding for this research was provided by the Arkansas Biosciences Institute at Arkansas State University. Additional funding through Nature West Inc. was provided by the Arkansas Science and Technology Authority (ASTA).
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