Influence of in vitro growth conditions in the production of defence compounds in Mentha pulegium L.
Graphical abstract
Introduction
Throughout the course of their growth and development, plants are exposed to a variety of environmental stress factors – both biotic and abiotic – that have an enormous influence on plant primary and secondary metabolic production. Abiotic stress factors such as cold (Maruyama et al., 2009), drought (Andre et al., 2009), UV radiation (Conconi et al., 1996), heavy metals (Tumova et al., 2007) and high salinity (Nasir Khan et al., 2010) have a strong influence on gene expression and have been previously studied in many crops. However, mechanical stresses other than wounding or tissue damage due to atmospheric or pathogen attack have not been studied extensively (Bengoung et al., 2011). Mechanical impedance – defined as penetration resistance on the roots by the soil or the substrate – is present at the roots from the very beginning of plant growth, and the cellular responses of roots fundamentally influence the development of secondary morphological characteristics such as morphogenesis, root hair development, root length and diameter (Braam, 2005, Okamoto et al., 2008). Moreover, mechanical impedance is a factor to be considered when it comes to grown plants under hydroponics or non-soil in vitro culture conditions.
To our knowledge, the possible connection between mechanical impedance and the development of other secondary defence organs, i.e. glandular trichomes and leaf hairs, has not been documented to date. The morphology and phenotypic expression of roots during mechanical impedance has been shown to be strikingly similar to that observed in ethylene-treated seedlings in a number of species (Kays et al., 1975, Goeschl et al., 1996) thus suggesting that signal transduction to the rest of the plant due to hormonal production and release may be involved in the development of secondary organs in parts of the plant other than the roots. Some of these organs are of importance in plant defence (Duffey, 1986) and secondary metabolite production (Eisner et al., 1990, Wagner, 1991). Therefore, the large scale production of high-economic-value compounds through callus and in vitro culture requires an in depth understanding of the influence that such factors have on metabolite production.
Lamiaceae families are known to produce a wide array of secondary metabolites and are a rich source of economically valuable terpenes. Among them, Mentha pulegium L. (pennyroyal) is characterized by a high content of essential oils (4% of the dry weight), the main component of which is the bioactive monoterpene ketone pulegone (Thomassen et al., 1990, Aghel et al., 2004, Sampson et al., 2005, Diaz-Maroto et al., 2007). Pulegone has been related with defence roles against herbivory in wild thyme (Müller-Schwarze and Thoss, 2008) and has shown insecticidal (Liu et al., 2011, Sampson et al., 2005) antibacterial (e.g., Teixeira et al., 2013) activities, among others. Also, an increase in pulegone production in Minthostachys mollis has been observed as a consequence of leaf damage – leaf puncturing – simulating the effect of leaf miners (Banchio et al., 2007).
Pennyroyal is a well-known plant that is widely used in folk medicine in many cultures for aromatherapy, abortifacient and as a remedy for colic. The oil is also commercially used as a flavouring agent in beverages and foods. However, the essential oil has been associated also with hepatotoxicity. Cytochrome P 450-mediated oxidation of pulegone generates menthofuran (McClanahan et al., 1989) which can be further metabolized into reactive compounds that can form adducts to hepatocellular proteins (Khojasteh et al., 2012). Therefore, the interest of studying the factors affecting pulegone and essential oil production in pennyroyal and why this species was selected as a model system to study the physiological changes and variation in secondary metabolite production induced by abiotic environmental factors.
In addition to phenotype selection, an understanding of the interactions between secondary metabolic pathways and the environment is a prerequisite for the optimization of secondary metabolite production (Sharma et al., 2002). Metabolic engineering has been suggested as a promising technology for the production of phytochemicals (Croteau et al., 2000), and knowledge of the factors that influence the distribution and accumulation of secondary metabolites in plant tissue and organs is imperative. In this sense, high frequency tissue culture systems and efficient culturing systems aimed at expressing the target phytochemical(s) are attractive options for the mass production of high-value secondary metabolites (Canter et al., 2005). Tissue culture using micropropagation has been suggested as a promising technology for physiological and phytochemical studies (Murch et al., 2000); this method can also play an important role in clonal propagation of high quality plants (Sahoo and Chand, 1998, Rathore et al., 2004, Liu et al., 2006, Liu et al., 2008) and in the mass production of secondary metabolites (Prakash et al., 2002, Yoshimatsu, 2008). The use of explants cultures also ensures higher genetic stability in comparison with callus cultures, thus allowing a better standardization of secondary metabolic production.
The present study with M. pulegium L. offers some clues to understanding the physiological and phytochemical state of plants affected by culture conditions and environmental factors such as mechanical impedance (expressed through changes in the support matrix) and water levels. Knowledge about the responses of plant morphogenesis and secondary metabolic production to the culturing parameters is needed to provide potential targets for metabolic engineering. In this study we specifically investigated the effects of two culture conditions (oil and non-soil) using three different supports (agar, glass beads and soil) on: (1) biomass production and relative growth rate of shoots and roots; (2) chemical status: essential oil (pulegone) production and essential oil profiling; and (3) histochemical status of storage glands (trichomes and leaf hair density) and root hairs.
The aims of the experiments presented here were as follows: (a) to establish a correlation between the chemical (essential oil profile and production), the histological and physiological status, and (b) to determine whether growth conditions would influence the production of the allelochemical pulegone and other essential oil components.
Section snippets
Results and discussion
Micro-shoots retain morphogenetic potential over the generations and can be further multiplied or rooted. Alternatively, their potential for aseptic biomass production and mass production of secondary metabolites can also be explored and, eventually, exploited, and this is one of the main purposes of this study. Accordingly, the effects of growth conditions (with a special attention paid to mechanical impedance) on morphogenesis, chemical profile and secondary metabolite production will be
Plant material and in vitro culture
All experiments were conducted with plants grown under non-soil (in vitro) and soil (greenhouse) conditions. Original plant material was collected from M. pulegium L. growing in natural populations of North Morocco. The in vitro plants were induced from nodal explants of M. pulegium L. cultivated in MS (Murashige & Skoog) medium supplemented with 0.5 mg/L BAP (6-benzylaminopurine), 30 mg/L sucrose and 7% agar (Murashige and Skoog, 1962). Micro-shoots obtained from cultured nodal explants were
Acknowledgements
K.H.B.Z. acknowledges financial support from Ministerio de Educación y Ciencia (BOE 12/1/2007 grant A/7251/06). This research was financed by the Ministerio de Innovación, Ciencia e Tecnología (MICYT), project # AGL C009-08864(AGR) and Junta de Andalucía (Consejería de Innovación, Ciencia y Empresa, project # P07-FGM-0303). The authors also wish to thank Dr. J.G. Romagni (Bucknell University, PN) for her useful comments on the manuscript.
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