Protein biocargo of citrus fruit-derived vesicles reveals heterogeneous transport and extracellular vesicle populations.

Cell-derived vesicles are membrane-enclosed organelles that transport material inside and outside the cell. Plant-derived vesicles are receiving more and more attention due to their potential as nanovectors for the delivery of biologically active substances. Here, we studied the heterogeneity and protein biocargo in citrus fruit juice sac cell-derived vesicles populations. Micro- and nano-sized vesicle fractions were isolated from four citrus species, C. sinensis, C. limon, C. paradisi and C. aurantium, characterized using physicochemical methods and protein cargos were compared using label-free quantitative shotgun proteomics. In each sample approximately 600-800 proteins were identified. Orthologues of most of the top-ranking proteins have previously been reported in extracellular vesicles of mammalian origin. High expression levels of patellin-3-like, clathrin heavy chain, heat shock proteins, 14-3-3 protein, glyceraldehyde-3-phosphate dehydrogenase and fructose-bisphosphate aldolase 6 were measured in all samples while aquaporin was highly expressed only in the nanovesicle fractions. Bioinformatics revealed more than hundred protein orthologues potentially implicated in vesicular trafficking. In particular, the presence of CCV, COPI and COPII coat proteins indicates the presence of heterogeneous populations of intracellular transport vesicles. Moreover, a high number of different enzymes including hydrolases and oxidoreductases are ubiquities in citrus fruit sac cell-derived vesicles.