# Plant Extracellular Vesicles: Investigating Their Utilization as Beneficial Nutrients in Diet
## 2 Structure and Biological Composition of P-EVs  
Extracellular vesicles are a heterogeneous group of membrane-enclosed particles that are different in size and shape [20]. Their membrane consists of a phospholipid bilayer with integrated proteins, which protects the molecular content from enzymatic degradation, as well as from environmental conditions (e.g., extreme temperatures, pH, salinity) [2]. The lipid composition of P-EVs varies depending on the plant species and the physiological state of the producing cells. However, the P-EVs membrane typically contains a mixture of phospholipids, glycolipids, and phytosterols [21]. Lipidomic analyses have revealed that phosphatidic acid, phosphatidylcholine, and phosphatidylethanolamine account for most of the phospholipid content [2]. In particular, phosphatidic acid is a major component in ginger- and grape-derived vesicles, and it has been demonstrated its involvement in the mechanism of membrane fusion [22]. Moreover, since it is a cell-signaling lipid involved in the activation of the mammalian target of rapamycin (mTOR), as well as mitogen-activated protein kinase (MAPK) pathways, phosphatidic acid can be considered the main effector on mammalian cells growth and proliferation upon treatment with P-EVs [8]. Besides their role in vesicle internalization, lipids may also contribute to P-EVs' intrinsic therapeutic properties [23]. For instance, phosphatidylcholine- and phosphatidylethanolamine-enriched graprefruit-derived vesicles have shown antioxidant, anticolitic, and anti-inflammatory activities [24]. Differences in the composition of the lipid bilayer can affect vesicle uptake by specific gut bacteria, suggesting that specific lipids could signal for preferential uptake by specific cells, playing a key role in the reported  
Figure 1: Schematic representation of P-EVs biogenesis. MVBs fuse with the plasma membrane and release exosome-like vesicles into the extracellular space. Plant-EVs are also released in the apoplast by EXPOs and vacuolar vesicles, which also fuse with the cell membrane. These vesicles contain not only bioactive molecules but also defense proteins and sRNAs to counteract pathogen infections. therapeutic activities of these vesicles [25]. The main lipid families found in P-EVs are reported in Table 1.  
Similarly to lipids, P-EVs' protein profile depends on the origin of vesicles and the physiological conditions of the plant, and the main role of the proteins is in facilitating plant vesicle uptake by animal cells [22]. P-EVs contain significantly lower protein content compared to mammalian vesicles, and the main proteins present belong to the families of aquaporins, involved in the regulation of cell turgor; heat-shock proteins (HSPs), related to biotic and abiotic stress responses and plant growth; metabolic enzymes; and annexins, implicated in intracellular vesicle transport and exosome secretion [11] (Table 2). Plant vesicles contain a considerable number of small RNAs (sRNAs), including microRNAs (miRNA), which are short non-coding RNA of about 15 to 27 nucleotides, capable of regulating gene expression by binding and cleaving target mRNA molecules [26]. In the case of pathogen infections, plants use EVs to secrete miRNA into the host and silence virulence-related genes. Emerging data demonstrate that EVs deriving from edible plants are also able to control target gene expression in the mammalian genome via cross-kingdom interactions, by transferring sRNAs [4]. Consistent with these findings, plant miRNAs are naturally modified at their 3' ends by methylation to acquire greater stability and protection against degradation [7]. [PERSON] et al. [27] proposed that ingested plant miRNAs are absorbed by the intestinal epithelial cells of consuming animals and are then packaged into extracellular vesicles. These structures protect miRNAs from degradation and deliver them to distant cells, where they interact with endogenous RNAs and regulate their expression. Several studies also show that these molecules can prevent human diseases.