Isolation and Characterization of Extracellular Vesicles from Arabidopsis thaliana Cell Culture and Investigation of the Specificities of Their Biogenesis

Abstract

Over recent years, extracellular vesicles (EVs), commonly termed exosomes, have gained prominence for their potential as natural nanocarriers. It has now been recognized that plants also secrete EVs. Despite this discovery, knowledge about EV biogenesis in plant cell cultures remains limited. In our study, we have isolated and meticulously characterized EVs from the callus culture of the model plant, Arabidopsis thaliana. Our findings indicate that the abundance of EVs in calli was less than that in the plant’s apoplastic fluid. This difference was associated with the transcriptional downregulation of the endosomal sorting complex required for transport (ESCRT) genes in the calli cells. While salicylic acid increased the expression of ESCRT components, it did not enhance EV production. Notably, EVs from calli contained proteins essential for cell wall biogenesis and defense mechanisms, as well as microRNAs consistent with those found in intact plants. This suggests that plant cell cultures could serve as a feasible source of EVs that reflect the characteristics of the parent plant species. However, further research is essential to determine the optimal conditions for efficient EV production in these cultured cells.

Plant-Derived Vesicle-like Nanoparticles: The Next-Generation Drug Delivery Nanoplatforms

“…In the EXPO pathway, EXPOs, rather than MVBs, fuse with PMs and release its internal vesicles into the cell wall. Different from MVBs, EXPOs are independent of the endocytic pathway and are unaffected by inhibitors of the secretory and endocytic pathways [17,18]. Additionally, the central vacuole in Arabidopsis has been reported to externally release antibacterial proteins by fusing with plasma membranes [8].…”

Plant Extracellular Vesicles: Current Landscape and Future Directions

“…Callus culture is the technique of growing plant tissues or cells in vitro in an artificial medium, usually containing relatively high auxin concentrations or a combination of auxin and cytokinin [ 47 ]. Attempts have also been made to isolate vesicles from plant callus cultures of two species, tobacco [ 22 ] and Arabidopsis thaliana [ 48 ]. In these studies, the harvested calli were homogenised, and vesicles were isolated from the homogenisation buffer.…”

“…In these studies, the harvested calli were homogenised, and vesicles were isolated from the homogenisation buffer. A comparative analysis shows fewer callus-derived vesicles (1.8 × 10 10 ) compared to apoplastic-fluid-derived AVs (2.9 × 10 10 particles g −1 fresh weight) in Arabidopsis [ 48 ]. Based on scanning electron microscopy (SEM) and nanoparticle tracking analysis (NTA), the callus-derived vesicles showed a rather broad size distribution, and the expression levels of endosomal sorting complex (ESCRT) related TET8 and PEN genes were found to be significantly lower too.…”