Research articleInduction of aromatic amino acids and phenylpropanoid compounds in Scrophularia striata Boiss. cell culture in response to chitosan-induced oxidative stress
Graphical abstract
Introduction
Plants produce plenty of beneficial secondary metabolites with pharmaceutical and nutritional value as part of a final product or as a crude material, including various phenylpropanoid compounds. Phenolic acids (PAs) such as cinnamic, p-coumaric and caffeic acids are secondary metabolites that have been attributed to act as powerful antioxidant metabolites possessing diverse physiological functions in biological systems. Research data have revealed that PAs can be used as preventive and/or therapeutic agents in many diseases that are related to oxidative stress (for example, inflammatory damage, cancer, and cardiovascular diseases) (Teixeira et al., 2013). Echinacoside (ECH) which is known as caffeic acid glycoside (Murthy et al., 2014) (Fig. 1), is a natural compound in the phenylethanoid glycosides (PeGs) isolated from Cistanches herba and show neuroprotective, vasodilatory, anti-oxidative (a scavenger of nitric oxide radicals), and anti-hepatotoxic activities (Jia et al., 2009, Zhao et al., 2010, Zhu et al., 2013).
Plant cell culture technology offers an alternative approach for the production of pharmaceutical valuable secondary metabolites. This method provides many advantages such as the improvement of synthesis and accumulation of these compounds through elicitation (Gangopadhyay et al., 2011). Natural elicitors such as chitin and chitosan are exogenous biotic compounds extracted from the arthropods exoskeleton as well as the fungal cell walls often act as specific elicitors in a number of plant cell cultures for efficient induction of valuable medicinally secondary metabolites (Brasili et al., 2014, Valletta et al., 2016). Although, elicitation practice using plant cell cultures has been widely used to enhance production of medicinal compounds but there are unexpectedly fewer published reports addressing PeGs, especially ECH (Lu and Mei, 2003, Cheng et al., 2006). It is well established that, upon the challenge by elicitors, plants respond with an array of defenses including the accumulation of secondary metabolites (Dornenburg, 2004). Chitosan is known to play a key role in defense responses and it is involved in some systems of signal transduction to induce particular enzymes (Mandal, 2010). PAL is the rate limiting enzyme in production of phenylpropanoids which can be induced the response to stress (Pawlak-Sprada et al., 2011). Oxidative stress is a response of plants to elicitor treatments, which induces the activity of plant anti-oxidative enzymes to scavenge reactive oxygen species (ROS) such as hydrogen peroxide (H2O2). ROS act at low rates as signal molecules and trigger a series of cellular responses from expression of certain genes to production of secondary metabolites (Chen et al., 2008), but in high levels they can cause damage to membranes and other essential macromolecules, such as oxidation of proteins, DNA, and lipids (Ashraf and Ali, 2008). SOD catalyzes the dismutation of superoxide anion to O2 and H2O2 (Giannakoula et al., 2010). CAT is one of the most important plant anti-oxidative enzymes catalyzing the dismutation of H2O2 and is known as a moderator of oxidative damage into oxygen and water. Peroxidases are also important group of antioxidant enzymes that are able to scavenge H2O2 (Giannakoula et al., 2010).
Scrophularia striata Boiss. is a common native plant species (with common name of Tashne dari) in Iran known for its high content of secondary metabolites widely used in local healing of illnesses and wounds (Azadmehr et al., 2013). Despite such wide usage of S. striata, knowledge on its production and increase of secondary metabolites in vitro is lacking. The main aim of this study was to examine the effects of chitosan elicitation in the S. striata cell suspension cultures on the cell growth, cell viability, accumulation of aromatic amino acids (Phenylalanine [Phe] and Tyrosine [Tyr]) and phenylpropanoid compounds (PAs and ECH). Moreover, to find probable relation between plant ROS scavenging capability and secondary metabolite biosynthesis under chitosan elicitation was investigated.
Section snippets
Establishment of the cell suspension cultures
Cell suspension cultures of S. striata were prepared from stem explant-derived calli in 50 mL of MS medium (Murashige and Skoog, 1962) supplemented with 2 mg L−1 benzyl adenine (BA) and 0.5 mg L−1 naphthalene acetic acid (NAA) at pH 5.8 (Khanpour-Ardestani et al., 2014). Suspension cultures were incubated on a shaker at 110 rpm in darkness at 25 °C and subcultured every 2 weeks.
Elicitation of the cell cultures
For elicitation treatments, fresh cells (1 g) were transferred to 30 mL of the cell culture medium, supplemented with
Effect of elicitor on cell viability and growth
Chitosan reduced the growth of S. striata cell cultures in a dose-dependent manner. The concentrations of 100, 50 and 10 mg L−1 chitosan inhibited cell viability and reduced growth (as fresh weight biomass) of S. striata as much as 70, 60 and 10%, respectively (Fig. 3a and b).
Effect of the elicitor on ECH, total phenolic, flavonoids, and flavonols
HPLC analysis demonstrated that the applied concentrations of chitosan significantly stimulated the production of ECH in elicited cells as compared to the control (Fig. 3c). Accumulation of total phenolic, flavonoids, and
Discussion
Application of biotic elicitors has been an effective strategy for improving the production of valuable secondary metabolites in plant cell cultures (Komaraiah et al., 2002). It is also well-known that exogenous elicitation induces gene expression and improves biosynthesis of secondary metabolites. Many researchers have investigated the biosynthesis aspect and in vitro production of secondary metabolites (Sarfaraj Hussian et al., 2012, Hasanloo et al., 2014), but less contribution has been made
Conclusion
The current study revealed that chitosan application on cell culture of S. striata induce aromatic amino acids biosynthesis which in turn by induction of PAL can switch mode of exposed cells partially from primary to secondary metabolism. The results from this research suggest that the cell cultures of S. striata may be effective system for ECH production.
Contribution
This research paper was accomplished with the collaboration of all authors and they read and approved the manuscript. Maryam Kamalipourazad performed the experiments, analyzed data and wrote the manuscript. Mohsen sharifi and Hassan Zare Maivan designed the study and supervised the study. Mehrdad Behmanesh was the study advisor and Najmeh Ahmadian Chashmi provided consultation.
Acknowledgements
This study was fiscally supported by Tarbiat Modares University, Tehran, Iran.
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