# Plant Extracellular Vesicles: Investigating Their Utilization as Beneficial Nutrients in Diet
## 1 Introduction  
The beneficial functions of food and its bioactive constituents on human health are widely recognized; consumption of fruits and vegetables is associated with a risk reduction in a number of leading human diseases, likely due to plant antioxidant content [1]. Recently, it has been reported that food-derived vesicles containing bioactive molecules enhance the efficacy of natural compounds by improving their bioavailability, due to biological protection of the content obtained by the presence of a membrane [2]. Plants secrete a multitude of molecules in the extracellular environment, facilitating cell-to-cell signaling, which is crucial to plant defense and immunity. Based on current knowledge, secreted extracellular vesicles (EVs) represent a major way to achieve this crosstalk [3]. Furthermore, it is known that they mediate different mechanisms of interspecies communication, gaining increasing attention as a valuable therapeutic tool [4]. Mammalian EVs are categorized as exosomes, microcwesicles, and apoptotic bodies (known and classical EVs), and autophagic EVs, stressed EVs, and matrix vesicles (recently considered as new EVs types) [5]. Since it is very difficult to categorize the different types of EVs, as specific markers to distinguish them are lacking, the International Society for Extracellular Vesicles (ISEV) recommends the use of the term \"extracellular vesicles\" (EVs) to generically refer to particles delimited by a lipid bilayer naturally released from mammalian cells. The classification of EVs can be based on size, biogenesis mechanism, and biochemical composition. In line with guidelines reported in [PERSON] et al., 2018, the authors are recommended to consider (a) physical characteristics of EVs, such as size (\"small EVs\" (sEVs) and \"medium/large EVs\" (m/EVs)), with ranges defined, for instance, respectively,\(<\)100 nm or \(<\)200 nm (small), or \(>\)200 nm (large and/or medium) or density (low, middle, high, with each range defined); (b) biochemical composition (CD63+/CD81+ EVs, Annexin AS-stained EVs, etc. ); or (c) descriptions of conditions or cell of origin [6]. However, in general, the experts have recommended calling exosomes as small EVs (sEV) originating from the endosomal compartment regardless of the generating cells, while for EVs budding from plasma membrane and known as microvesicles (MVs) or ectosomes or microparticles, it is recommended to use the term large EVs (IEVs). Apoptosis-derived vesicles, which are characterized by their large size range from 800 nm to 5 \(\upmu\)m, result from cell death and contain a mixture of damaged or unnecessary cellular components [5]. Similar to mammalian ones, plant-derived extracellular vesicles (P-EVs) are small vesicles surrounded by a lipid-enriched membrane, transporting a variety of bioactive substances such as nucleic acids, vitamins, antioxidant molecules, proteins, and metabolites [7]. Due to current insufficient knowledge, it is still difficult to classify plant EVs with the established nomenclature used for mammalian EVs [8]. In order to standardize the literature, the term \"extracellular vesicles\" was proposed to include all vesicles isolated from extracellular compartments (e.g., apoplastic fluid or growth medium), and \"plant-derived vesicles\" for vesicular fractions obtained by disrupting plant tissues, when natural release into the apoplast cannot be established [9]. Moreover, the name \"exosomes, widely used for mammalian vesicles, is barely adopted for plants due to limited knowledge of surface protein markers [10]. Although the biogenesis pathway of plant EVs is not completely clarified, it is believed that these vesicles are mainly produced by three specific organelles: MVBs, exosome-positive organelles (EXPO), and vacuolar bodies [11]. In the plant, exosomes are secreted by the fusion of MVBs with the plasma membrane [12]. A specific marker for these vesicles type has been identified in the integral protein tetraspanin-8 (TET8), which is the orthologue for the human exosomal marker CD63 [13].