# Nanotechnology in Plant Metabolite Improvement and in Animal Welfare
## 4 Possible Mechanism of AgNPs in Plant Cells
Even though the direct biophysical and biochemical interactions between nanoparticles and the biological system have not yet been explained, it has been strongly suggested that the initial response of plants exposed to NPs might include increased levels of ROS, cytoplasmic Ca\({}^{2+}\), and mitogen-activated protein kinase (MAPK) cascades; these responses were the same as those in the biotic and abiotic stress defense mechanisms [8; 26; 56; 73]. AgNPs were reported to adhere to the surface of the primary root in the early stage of exposure. Transport of the particles in plants occurs by two mechanisms, i.e., via the intercellular transport or vascular tissue. The initial penetration of nanoparticles is at the level of the epidermal layer of the root, followed by a series of events ending in the entry of the plant vascular tissue. This mechanism enables the translocation of AgNPs in whole plants or explants [74]. AgNPs are reported to remain in nanoparticle form after intake into the plant cells, causing less severe effects than one generated by free Ag\({}^{+}\) derived from silver nitrate [75]. AgNPs could enter cells through plasmodesmata, after direct contact with the cell membrane. In this adverse condition, plants produce ROS that causes damage to the biological membrane system and to macromolecules [73; 74]. As reported in _A. thaliana_, the initial recognition of AgNPs by membrane-bound receptors triggers Ca\({}^{2+}\) burst and ROS induction [69]. Nanoparticles and silver ions were reported to stimulate changes in the secondary metabolite profile, which suggested that AgNPs affect plant metabolism via ions release in the medium. In stevia (_Stevia rebaudiana_), high doses of AgNPs were reported to accumulate in plant cells and tissues [57]. These molecules were internalized in the plant through the vascular system and translocated to neighborhood cells via apoplasts. The transfer mechanism via apoplasts requires NPs to cross a membrane; this indicates that NP uptake must be size-specific. Nanomaterials could also be transferred via plasmodesmata; this channel is approximately 40 nm in diameter, so it is possible for 40 nm-sized AgNPs to be transferred via plasmodesmata. Interestingly, nanoparticles are also reported to possibly create a pore for bigger size NPs, via NP-induced conductance mechanism [74; 76; 77; 78]. Another study demonstrated that the morphology and size of the AgNPs affected the plant response during exposure [79]. The study exhibited that smaller size AgNPs showed a more efficacious anti-microbial activity and increased response to oxidative stress. Nanoparticles also induced a ROS-mediated MAPK cascade and a calcium spike, which possibly enhanced secondary metabolite production to counteract the oxidative stress, resulting in a higher quality and quantity of secondary metabolites during AgNPs exposure [56] (Figure 1). MAPK has been known to play an important function in plant responses, triggered by ROS, through dynamic gene expression and regulations. It has been reported to be crucial for flavonoid [80] and alkalioid [81]_biosynthesis_ when plants are subjected to stress conditions. [PERSON] et al. [82] reported up-regulation of several genes involved in the biosynthesis of alkaloids, coupled with high expression of Mitogen-Activated Protein Kinase 3 (_CrMPK3_) gene on _Catharanthus roseus_ after silver exposure. [PERSON] et al. [79] reported Ca\({}^{2+}\) burst and ROS induction on the cell membrane after AgNPs exposure. Nanoparticles induced single channel-like conductance that allows Ca\({}^{2+}\) entry, resulting in ROS generation because of the cytosolic Ca\({}^{2+}\). This condition leads to the expression of MAPK genes that affects transcriptional reprogramming of secondary metabolites-related genes. Interestingly, a recent study conducted by [PERSON] et al. [49] has reported that AgNPs triggered the accumulation of alkaloids in _C. roseus_ via ROS-mediated MAPK cascade through the up-regulation of the _MAK3_ gene.