Abstract
While human extracellular vesicles (EVs) have attracted a big deal of interest and have been extensively characterized over the last years, plant-derived EVs and nanovesicles have earned less attention and have remained poorly investigated. Although a series of investigations already revealed promising beneficial health effects and drug delivery properties, adequate (pre)clinical studies are rare. This fact might be caused by a lack of sources with appropriate qualities. Our study introduces plant cell suspension culture as a new and well controllable source for plant EVs. Plant cells, cultured in vitro, release EVs into the growth medium which could be harvested for pharmaceutical applications. In this investigation we characterized EVs and nanovesicles from distinct sources. Our findings regarding secondary metabolites indicate that these might not be packaged into EVs in an active manner but enriched in the membrane when lipophilic enough, since apparently lipophilic compounds were associated with nanovesicles while more hydrophilic structures were not consistently found. In addition, protein identification revealed a possible explanation for the mechanism of EV cell wall passage in plants, since cell wall hydrolases like 1,3-β-glucosidases, pectinesterases, polygalacturonases, β-galactosidases and β-xylosidase/α-L-arabinofuranosidase 2-like are present in plant EVs and nanovesicles which might facilitate cell wall transition. Further on, the identified proteins indicate that plant cells secrete EVs using similar mechanisms as animal cells to release exosomes and microvesicles.
Highlights
Multivesicular bodies (MVBs) [4] and apoptotic bodies [5] have been observed in plants and even though we found no reports of plasma membrane-derived microvesicles, we assume that the mechanisms of extracellular vesicles (EVs) formation [6] can be transferred from the animal kingdom to plants, or even to eukaryotes in general
Lacking knowledge on marker proteins of plant EVs or nanovesicles at the current state, we imaged the samples that were supposed to contain nanometer-scaled vesicles, using transmission electron microscopy (TEM) in order to confirm the success of the isolation
Besides the mainly used isolation technique of differential centrifugation, plant EVs have been concentrated by tangential flow filtration (TFF, HansaBioMed Life Sciences, Tallinn, Estonia) from cell culture medium (Figure 2d)
Summary
Plant nanovesicles are closely related to mammalian extracellular vesicles (EVs) but, compared to the latter, they have been relegated to the background. Scientific interest has been increasingly paid to the plant equivalent of exosomes and microvesicles lately [1,2]. Structure and density of plant nanovesicles are comparable with exosomes from mammals [3]. Not all of those “exosome-like nanoparticles”, that can be isolated from (homogenized) plant materials like juices, are certainly of extracellular origin. We recommend using the term “nanovesicles” for nanometer-scaled (1–999 nm) membrane-delimited vesicles, as long as the selected raw material does not ensure that EVs are concentrated. Isolated nanovesicles from apoplastic fluid can be assumed to be EVs. The scheme in Figure 1 provides an overview on present key issues in plant nanovesicle research
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