Open Access Research Article

Elicitation, A Mechanistic Approach to Change the Metabolic Pathway of Plants to Produce Pharmacological Important Compounds in In-vitro Cell Cultures

Muhammad Naeem Bajwa*1, Amna Bibi2, Muhammad Zaeem Idrees 2, Gohar Zaman2, Umar Farooq1, Talha Tufail Bhatti2

1Department of Biotechnology Quaid-I-Azam University Islamabad

2Department of Chemistry University of Sialkot, Sialkot

Corresponding Author

Received Date: June 22, 2021;  Published Date: July 12, 2021


Plant’s secondary metabolites, produced usually under stress, are one of the promising sources for food additives, pharmaceuticals, food flavors and other industrial materials. The comprehensive probing of metabolite’s production mechanism, stress signal transduction pathway, would be great push toward in commercial production. Higher plants inevitably encounter stresses and sustain themselves by producing various secondary metabolites which, the secondary metabolites, have various industrial application that’s why are promising candidates for commercialization. Due to certain limitations of natural plant extraction, plant cell/tissue culture is considered a best alternative way for in-vitro production of bioactive secondary metabolites. Elicitation can be employed to overcome the constraints of plant cell technology that retard the process of commercialization. A way to enhance the secondary metabolite’s production in plants is Elicitation. In which an exogeneous elicitor, biotic or abiotic, is exposed in growth medium to trigger the production of secondary metabolite. During this phenomenon, several defense/ non-defenses related genes, activated/ deactivated. Furthermore, transient phosphorylation or dephosphorylation of proteins, expression of enzymes occurs through which biosynthetic pathways of several secondary metabolites can be ascertained. Additionally, a push toward advancement of metabolic engineering and gene manipulation to increase the productivity of secondary metabolites can be gained through integration of proteomics, transcriptomics, and metabolomics with system biology.


Plants are the best reservoir of medicinally important compounds present in their roots, stem, leaves and fruits. These pharmaceutical important phytochemicals produce a protective response against many diseases and illness of humans as well animals [1]. Due to their medicinal uses, they have been used by many cultures throughout the word in traditional herbal remedy against a lot of diseases and illness [2]. Due to rapid development of technologies pharmaceutical industries are showing interest in exploring of such valuable bioactive compounds from plants. Plants possess variety of such pharmaceutically important compounds including flavonoids, terpenoids, saponins and phenolics in various plant parts [2]. These phytochemicals possess specific phytochemical properties against cancer, tumor repression, microbes, and viruses [3]. With the advent of modernization, industrialization, and revolution in medicinal field use of pants and their extracts were reduced due to the availability of synthetic products available for diseases [1]. But due to the hazardous effects of these synthetic drugs trend is changing towards the medicinal plants for treating these dieses. The medicinal plants are safe, cheap and have no side effects on the body as compared to modern synthetic drugs [4]. Reports showed that no. of diseases increased to a large no. due to use of synthetic drugs. Antibiotic resistance is also one of the reasons to go for newer medicines to combat pathogens. several medicinal plants and their pathogens have been used against pathogenic resistant microbes [5].

For large scale production of such highly valuable medicinally important compounds from plants in-vitro culture techniques are used. These invitro culture techniques have advantage over wild grown plants that they have no geographical or seasonal restrictions and have a yield with minimum production time. For large scale industrial production of such compounds in-vitro culture techniques are used in which wild plants are grown under optimal growth conditions and for large biomass production all type of stress to the pants are removed. As a result the secondary metabolite production and therapeutic activities are highly reduced [6].

The plants ‘defense system produces chemicals that cope with the infections caused by internal or external stress. These chemical compounds are the products of secondary metabolism. It means that this process can be enhanced by applying any external stress to increase yield of these compounds, known as secondary metabolites [7]. Various approaches are considered in in-vitro cultures for up gradation or high yield of such vital phytochemicals. One of the most important and efficient strategies is ―Elicitation‖, in which the metabolic pathways are triggered by incorporating agents for optimum production of secondary metabolites. It is an efficient tool usually employed to stimulate phytochemicals yield [8, 9]. For instance, many reports are available on the application of elicitors to employ plants defense mechanisms to enhance the production of these compounds during in-vitro cultures [10]. The elicitors are identified by the cell membrane bounded receptors that activate the signal transduction pathways network by distinct genes, to enhance secondary metabolism [11]. It can also be adopted to characterize and examine the role of different agents on plants by using in-vitro plant cell culture as model system. The agents employed in the process are known as ―Elicitors‖ which generally classified into two; of abiotic or biotic nature [11].

Types of Elicitors

Elicitors can be either physical or chemical in nature. Elicitors can be either biotic or Abiotic according to the nature. The biotic elicitors are such elicitors that are biological nature derived from plants or pathogens while Abiotic elicitors have non biological nature and can be either physical agents or chemicals [7]. In in-vitro plant cell cultures elicitation is the best strategy for the process of fermentation of antibiotics or other fermented products. Elicitation triggers the membrane specific receptors of the metabolic pathway for the enhanced production of secondary metabolites. This strategy can be applied for the large-scale industrial production of commercially viable secondary metabolites. The detailed classification is given in the flow chart Figure 1.


Role of Elicitors in Plant Metabolic Engineering

Biosynthetic pathway for efficient production of secondary metabolites is a challenging issue. Only few metabolic pathways for viable synthesis of secondary metabolites have been discovered so for including flavonoid pathway, terpenoid pathway, indole alkaloid pathway and iso quinoline alkaloid pathways including berberine, morphine production pathways [12-14]. These metabolic pathways have been discovered after an extensive and laborious research work in in-vitro plant cultures. The changing of metabolic pathways in Eukaryotic cells like plants is difficult to attain because these metabolic pathways are complex and triggered by various enzymes which are substrate specific and present in a minute quantity. The changing of metabolic pathway for production of such enzymes is accomplished through metabolic engineering of [15] regulatory genes and their transcription factors [16,17]. So, for most of the studies of plant metabolomics have been carried out mostly to primary metabolites while secondary metabolite production is complex and more challenging due to their highly divergent chemical structures and sensitivities in extraction conditions [15,16].

How elicitors change metabolic pathways

First of all, receptors present on the plasma membrane detect the elicitors and show a signaling response. The effect of elicitors varies from species to species due to their chemical or physical nature [18]. different plant receptors detect the response and show a signal to produce secondary metabolites to minimize the effect of that stress e.g. AVR plant resistance gene products for pathogen avirulent gene [19]. Studies shows that a same elicitor could show response in several plant species it means that different plant species have common receptors for that specific elicitor [20]. The plasma membrane receptors show a signal transduction pathway where various secondary messengers in cell like active oxygen species, free calcium, nitrogen oxide, cGMP, cytosolic PH and cADPR interact in a branched way. As a result, changes in Krebs cycle and Pentose phosphate pathway are signs of serious stress effects on the behaviour of cells and activation of defence responses to minimize that stress [20] phytoalexin and pathogenesis related proteins (PR) are produced in the cell by the activation of the signal transduction pathways [20].

The process of signal transduction is somehow complex and followed by a cascade of reaction where receptors for secondary messenger molecules like active oxygen species, free calcium molecules, nitrogen oxide, PH of cytosol, cADPR and cGMP interact with each other. As a result, changes in Krebs cycle and pentose phosphate pathway occurs that indicates cells behaviour and defence response to minimize that stress [20]. As a result of signal transduction pathways, the plants defence system genes activated that produce some specific compounds like pathogenesis proteins, phytoalexins, and calluses deposition in cell wall to strengthen it [13-21]. These responses are highly specific by various messengers for secondary metabolism and activation and production of target specific proteins [19]. These responses are highly specific and differ from cell to cell and among species [19,24]. Elicitation is mainly used to activate plant defence system as well as production of commercially viable secondary metabolites for cosmetic, food and pharmaceutical industries [25]. According to Giri & Zaheer [26] secondary metabolite production though elicitation of in-vitro cultures can be enhanced up to 1-2230-fold. Moreover, in postharvest treatments, elicitors can increase nutritional value in grapes (enhanced antioxidant properties) [27] or shelf life, in horticultural [28,29] as well as in ginseng roots [30] (Figure 2).


Elicitation is a very complex process carried out by thousands of intertwined events. The mechanism and mode of action of elicitors change with respect to its origin, concentration and specificity nutritional, physiochemical environment, and growth uptake of plants. In the mechanism of elicitation, mitogen-activated protein kinase (MAPK) phosphorylation, reactive oxygen species (ROS) burst, calcium flux are mostly initial events activated in majority of the elicitor to plant cell interactions [31]. Later on, activation of signalling pathways as well as transcription factors that lead to the plant secondary metabolism pathway are being reported [32,33]. The receptors present on plasma membrane recognizes and bind that elicitor and initiates the cascade of events like ion fluxes NADPH oxidase activation , ROS burst, Ca2+ burst, MAPK phosphorylation, cytoplasmic acidification and G-protein activation [10]. Initially the plant responses by the exchange of ions for example K+/Cl− effluxes and Ca2+/H+ influxes. The most important event is Ca2+ influx that involves the physiological processes of the cell [34,35]. This Ca2+ signals produce conformational changes in several Ca2+-binding proteins such as calmodulin like proteins, calcium- dependent kinases (CDPKs) calmodulin and phospholipases as well as by secondary messengers like diacylglycerol (DAG) and inositol 1,4,5- triphosphate IP3 [36]. Ca2+/Calmodulin-mediated pathways show stimulus towards physiological responses of plants and cellular processes like regulation of the oxidative burst, hormonal signalling and gene expression and protein phosphorylation [37]. Another important phenomenon in plant defense system is ROS generation that is produced by oxidases like NADPH oxidase as well as Ca2+ [35, 38]. According to different studies G-proteins play a role in the stimulation of ion channels as well as ROS, phospholipase A, phospholipase C and cell death [36, 39]. The levels of DAG, cAMP, IP3 are stimulated by activated G-proteins that triggers the targeted PKA and PKC. These proteins activate the phosphorylation of MAPKs, that results in transcription, and translation of the gene that leads to enzymatic reactions, which in turn reprogram the pathway of secondary metabolite production.

The Figure 3 shows the molecular mechanism of elicitation: how the plasma membrane- bound receptors recognizes the elicitors that results in ion fluxes, ROS burst, cytoplasmic acidification NADPH oxidase activation, Ca2+ burst,G- protein activation, and mitogen-activated protein kinase phosphorylation. It also activates downstream signalling pathway messengers like salicylic acid, jasmonic acid and methyl jasmonate. Messengers activate transcription factors and gene expression, which lead to reprogramming secondary metabolism [40].

Factors affecting the elicitation process

Elicitation is a complex process that is regulated by several factors [18,25]. the plants produce defence responses to a given elicitor. The concentration of an elicitor play a major role in the production of secondary metabolites [41]. Salicylic Acid (SA) (0,75-5 mM) produced drought tolerance in Eucalyptus globulus and this effect was linked with the concentration of elicitor, showing highest dosage produced tremendous effects. Singh & Usha [42] studied the similar effects in wheat seedlings where water stress conditions were linked to decreased transpiration and improved photosynthesis. Elicitation of chitosan to Basil plant also resulted in lower transpiration that improves plant behaviour under drought conditions [43]. The time of exposure for elicitor is also an important factor for the process of elicitation. The type of cell culture as well as the conditions for the growth room are also the important factors in the process of elicitation [44-47].

Future Perspective

Potential application of nanotechnology

Nano particles are emerging a new class of abiotic elicitors. Studies shows the production of secondary metabolites elicited by different nano particles e.g., the Artemisia annua was elicited for high production of artemisinin through silver oxide nano particles [48]. whereas in in-vitro callus cultures of Calendula officinalis the saponin and carotenoid content was highly increased while anthocyanin and flavonoid contents were decreased by the elicitation of silver nano particles [49]. In in-vitro cultures of Satureja khuzestanica, biosynthesis of secondary metabolites as well as antioxidant capacity was increased by the elicitation of multiwalled carbon nanotubes [50]. Biosynthesis of hypericin and hyperforin production was enhanced through elicitation by zinc and iron nano- oxides nano particles in cell suspension cultures of H. perforatum [51]. Besides the elicitation by metallic nano particles the signaling compounds like SA and MeJA and JA, encapsulated in biodegradable polymers having as that of nano particles for sustained and slow release of signaling molecules for sustainable biosynthesis of secondary metabolites in in-vitro. cell cultures . In cultures of A. Thaliana, metallic nanoparticles elicitation for biosynthesis secondary metabolites were also reported [52,53]. This capacity of secondary metabolites to be adsorbed exhibited linear relation with surface coverage of TiO2 revealing the interrelation of quercetin adsorption with functional surface. By deliberating over this phenomenon, a novel technique, nano trapping strategies for various secondary metabolites, can be established in near future.

A road to drug discovery

Analysis of the new compounds extracted from cells exposed to elicitors can provide us novel drugs and pattern of their bioactivities; bioactivity-guided fractionation can also be employed. Despite the alluring possibility of producing variety of bioactive secondary metabolites(compounds) via tissue cultures and H. perforatum cell through elicitation, commercial implementation of elicitation- based changes in secondary metabolism and pharmacological properties is still in its early stages. To gain substantial amount of aseptic biomass for elicitation is the striking issue currently. In regard to this, small-scale bioreactors of in vitro cultures for obtaining active compounds have been reported [54]. Recently, large scale bioreactor comprising of adventitious root culture for production of H. perforatum phytochemicals have been developed [55,56]. The correct culture vessels selection along with the resolve of exogenous signals required for in vitro production of biomass and optimization of elicitation measures are crucial elements for the further development in this field.


Plants are the reservoir of many useful compounds for nutraceutical, pharmacological and industrial products. Production of such secondary metabolites for industrial scale production in in-vitro cultures is lowered. For production and enhancement of such valuable compounds various techniques are used, elicitation is an effective strategy. The production of secondary metabolites through elicitation varies by the types of cultures, nature and concentration of elicitors, physical conditions of growth chamber and other factors. So, the research is required to optimize the best methods for the optimum production of secondary metabolites. In this study we have discussed how metabolic pathways are changed for secondary metabolite production through changes in genes and production of some specific enzymes.



Conflict of Interest

The authors declared no conflict of interest.


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