Elsevier

Plant Physiology and Biochemistry

Volume 107, October 2016, Pages 374-384
Plant Physiology and Biochemistry

Research article
Induction of aromatic amino acids and phenylpropanoid compounds in Scrophularia striata Boiss. cell culture in response to chitosan-induced oxidative stress

https://doi.org/10.1016/j.plaphy.2016.06.034Get rights and content

Highlights

  • Echinacoside accumulation was increased in Scrophularia striata Boiss. cells after chitosan feeding.

  • Chitosan treatment altered the concentration of phenylalanine and tyrosine in S. striata cells.

  • Phenylalanine ammonia-lyase (PAL) gene expression and its activity were enhanced in S. striata cells by chitosan.

  • H2O2 as a signaling molecule and the enzymatic antioxidant system activated in S. striata cells under chitosan elicitation.

Abstract

Manipulation of cell culture media by elicitors is one of most important strategies to inducing secondary metabolism for the production of valuable metabolites. In this investigation, inducing effect of chitosan on physiological, biochemical, and molecular parameters were investigated in cell suspension cultures of Scrophularia striata Boiss. The results showed that chitosan concentration and time of elicitation are determinants of the effectiveness of the elicitor. Accumulation of aromatic amino acids (phenylalanine [Phe] and tyrosine [Tyr]), phenylpropanoid compounds (phenolic acids [PAs] and echinacoside [ECH]), hydrogen peroxide (H2O2) production, phenylalanine ammonia-lyase (PAL) activity and gene expression, and antioxidant enzymes (superoxide dismutase [SOD], peroxidase [POX], catalase [CAT]) activities were altered by changing the exposure time of elicitation. Results showed that, upon elicitation with chitosan, oxidative events were induced, antioxidant responses of S. striata cells were boosted through enhanced activity of an effective series of scavenging enzymes (SOD, CAT, and POX), and biosynthesis of non-enzymatic antioxidants (ECH and PAs [cinnamic, p-coumaric and, caffeic acids]). The increase in amino acid content and PAL activity at early days of exposure to chitosan was related with rises in phenolic compounds. These results provide evidence that chitosan by up-regulation of PAL gene differentially improves the production of phenylpropanoid compounds, which are of medical commercial value with good biotechnological prospects.

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.

References (54)

  • P. Komaraiah et al.

    Elicitor enhanced production of plumbagin in suspension cultures of Plumbago rosea L.

    Enzyme Microb. Technol.

    (2002)
  • R.W. Owen et al.

    Isolation, structure elucidation and antioxidant potential of the major phenolic and flavonoid compounds in brined olive drupes

    Food Chem. Toxicol.

    (2003)
  • R.W. Owen et al.

    Phenolic compounds and squalene in olive oils: the concentration and antioxidant potential of total phenols, simple phenols, secoiridoids, lignans and squalene

    Food Chem. Toxicol.

    (2000)
  • V. Velikova et al.

    Oxidative stress and some antioxidant systems in acid rain-treated bean plants. Protective role of exogenous polyamines

    Plant Sci.

    (2000)
  • J. Wang et al.

    Enhanced production of flavonoids by methyl jasmonate elicitation in cell suspension culture of Hypericum perforatum

    Bioresour. Bioprocess.

    (2015)
  • J.W. Wang et al.

    Involvement of nitric oxide in oxidative burst, phenylalanine ammonia-lyase activation and Taxol production induced by low-energy ultrasound in Taxus yunnanensis cell suspension cultures

    Nitric Oxide

    (2006)
  • Q. Zhao et al.

    Neurotrophic and neurorescue effects of echinacoside in the subacute MPTP mouse model of parkinson’s disease

    Brain Res.

    (2010)
  • S. Ahmadi-Sakha et al.

    Bioproduction of phenylethanoid glycosides by plant cell culture of Scrophularia striata Boiss.: from shake-flasks to bioreactor

    Plant Cell Tiss. Org.

    (2015)
  • R.G. Alscher et al.

    Role of superoxide dismutases (SODs) in controlling oxidative stress in plants

    J. Exp. Bot.

    (2002)
  • I. Apostol et al.

    Rapid stimulation of an oxidative burst during elicitation of cultured plant cells: role in defense and signal transduction

    Plant Physiol.

    (1989)
  • A. Azadmehr et al.

    Antioxidant and neuroprotective effects of Scrophularia striata extract against oxidative stress-induced neurotoxicity

    Cell Mol. Neurobiol.

    (2013)
  • M. Babar Ali et al.

    Phenolics metabolism and lignin synthesis in root suspension cultures of Panax ginseng in response to copper stress

    Plant Sci.

    (2006)
  • C.J. Baker et al.

    An improved method for monitoring cell death in cell suspension and leaf disc assays using Evans Blue

    Plant Cell Tiss. Org.

    (1994)
  • M. Biermann et al.

    Simultaneous analysis of the non-canonical amino acids norleucine and norvaline in biopharmaceutical-related fermentation processes by a new ultra high performance liquid chromatography approach

    Amino Acids

    (2013)
  • E. Brasili et al.

    A non-targeted metabolomics approach to evaluate the effects of biomass growth and chitosan elicitation on primary and secondary metabolism of Hypericum perforatum in vitro roots

    Metabolomics

    (2014)
  • U. Chakraborty et al.

    Evaluation of heat acclimation and salicylic acid treatments as potent inducers of thermotolerance in Cicer arietinum L.

    Curr. Sci. India

    (2005)
  • J. Chen et al.

    Identification of hydrogen peroxide responsive ESTs involved in phenylethanoid glycoside biosynthesis in Cistanche salsa cell culture

    Biol. Plant.

    (2015)
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