## 2 Mechanism of Nano-Elicitation  
Since little is known about most of plant secondary metabolism, the effects of elicitors on plant cells or tissue cultures are therefore difficult to gauge. The mechanism employed by elicitors is complex and distinctive. Elicitors act as a signal which is received by the elicitor specific receptors located on plasma membranes [27]. Several elicitor-binding sites have been identified in cell plasma membranes for a range of elicitors. Transmembrane receptor-like protein kinases (RLKs) are one of the well-known categories of receptors which might be concerned with the perception of different types of stimuli [28]. NPs can directly bind receptors (e.g., chitosan-NPs) and/or trigger the production of the second endogenous messenger that could bind to these receptors [20].  
After the binding of receptor-NPs, the initial response to NPs involves an active exchange of ions, i.e., Cl\({}^{+}\) and K\({}^{+}\) efflux and Ca\({}^{2+}\) influx occur across the cell membrane into the cytosol, as shown in Figure 3. Ca\({}^{2+}\) influx is an important step during the elicitation process as it further modulates various physiological and cellular pathways. Cytoplasm acidification occurs in response to H+-ATPase inactivation and an increase in extracellular pH. Cytoplasmic acidification is important for signal transduction, leading to the oxidative burst and biosynthesis of plant secondary metabolites.  
These events lead to reversible phosphorylation and dephosphorylation of plasma membrane proteins and cytosolic proteins and the activation of protein kinases (PK) and mitogen-activated protein kinases (MAPK). The involvement of GTP binding proteins (G-proteins) during elicitation is also reported. G-proteins probably respond by  
Figure 3: Representation of a proposed mechanism involved in nano-elicitation of commercially important flavonoids in various in vitro systems of plants (Note: dashed arrows indicate indirect reactions and the continuous arrows indicate direct ones; ROS: reactive oxygen species; MAPKs: mitogen-activated protein kinases; NPs: nanoparticles; JA: jasmonate; MeJA: methyl! jasmonate; SA: salicylic acid).  
activating phospholip, leading to the downstream production of plant secondary metabolites [29; 30; 31].  
The PLC (phospholipase-C)/IP3-DAG (Inositol trisphosphate- diacylglycerol)/PKC (protein kinase C) cascade also plays a key role during elicitation. The second calcium influx occurs due to PLC activation and inositol triphosphate (IP3) released Ca\({}^{2+}\) from intracellular calcium stores, such as the vacuoles, Golgi apparatus, and endoplasmic reticulum [32]. The increase in cytoplasmic calcium levels activates the defense response, either directly by calcium or through calcium sensors, such as calmodulins and Ca\({}^{2+}\)-dependent protein kinases (CDPKs). Increased intracellular Ca\({}^{2+}\) level activates NADPH oxidase and the subsequent generation of ROS (reactive oxygen species) [20]. ROS induces the expression of many defense genes and secondary metabolite biosynthetic genes, such as phenylalanine ammonia lyase (PAL), and the induction of signaling molecules, such as salicylic acid (SA), ethylene (EA) and jasmonate (JA). These signaling molecules further activate the pathways of secondary metabolite production in plant cells [33].  
Several factors such as the concentration of an elicitor, the morphology of an elicitor, exposure time, the type of in vitro culture, nutrient composition, growth regulators and plant species influence the production of secondary metabolites [25].