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Page 6 Cai et al. Extracell Vesicles Circ Nucleic Acids 2023;4:262-82 https://dx.doi.org/10.20517/evcna.2023.10

recovery. In plants, two ultracentrifugation speeds have been used to pellet EVs: 40,000 × g, which collects
the P40 fraction, and 100,000 × g, which collects the P100 fraction. For plant EVs pelleted at 100,000 × g,
[30]
92% have diameters in the range of 30-150 nm . Mammalian EVs pelleted at the same ultracentrifugation
speed have similar diameters below 150 nm and are denoted as exosomes [9,10,61] . In plants, the P100 fraction
is enriched in the exosome-like TET8-positive EVs, which contain sRNAs and RNA-binding proteins .
[79]
Most TET8-positive EVs remain in the P40 supernatant, and centrifugation of the supernatant of P40 at
100,000 × g (the P100-P40 fraction) collects mostly TET8-positive EVs [Figure 2]. The P40 fraction is
[79]
[79]
enriched in PEN1-positive EVs, which contain tiny RNAs and proteins . As well in the P40 fraction, non-
vesicular sRNAs and circular RNAs associated with RNA binding proteins co-pellet with EVs . While
[79]
circular RNAs are more resistant to degradation because of lacking free ends, it is currently unknown how
other liner non-vesicular RNAs, including sRNAs, could be protected from degradation within the plant
apoplast that contains numerous nucleases [53,54] , and whether RNA binding protein alone can completely
protect the non-vesicular RNAs from degradation. These studies using different ultracentrifugation speeds
for plant EV isolation are complementary to the discovery and characterization of distinct classes of EVs
with different sizes, densities, and cargoes.

In tandem with different centrifugation speeds, differences in the extraction methods of apoplastic washing
fluid can greatly affect the collection of different EVs or non-vesicular contents. Studies characterizing the
P40 fraction notably isolated EVs and non-vesicular RNAs/RNA-protein complexes from apoplastic
washing fluid extracted from the whole plant, rather than detached leaves [31,79] . Apoplastic washing fluid
collected by the whole plant method contains more contamination from cell debris and cytoplasmic content
compared to the extraction of apoplastic washing fluid from the detached leaves method . This is evident
[30]
through Western blotting analysis, which detected a high amount of Rubisco protein in EV fractions
isolated with the whole plant method . In comparison, the detached leaves method resulted in minimal
[30]
Rubisco protein contamination in EV preparations . It is important to consider not only extraction
[30]
methods, but also the disease or stress conditions of the plant that affect EV cargoes as well as non-vesicular
contents that co-pellet with EVs. Different methods may only capture different subsets of EVs, and it is
important to avoid generalizations about the function of all plant EV subtypes based on individual methods.

Differential centrifugation followed by gradient centrifugation can help increase the quality of isolated EVs
[80]
and reduce contaminations [29,30] . Gradient centrifugation can be done using a sucrose gradient or an
iodixanol gradient . Different plant EV subtypes are found in different density fractions, which has been
[31]
shown through the detection of EV protein markers [30,31] . PEN1-positive EVs are enriched in the iodixanol
gradient fraction from 1.029 to 1.056 g/mL . TET8-positive EVs are enriched in the iodixanol fraction of
[31]
[30]
1.08 g/mL , which is similar in density to mammalian exosomes (1.08-1.12 g/mL) [81,82] . Importantly, TET8-
positive EVs are in the same fractions of plant miRNAs and siRNAs , suggesting that TET8-positive EVs
[56]
are the major class of EVs for sRNA transport. Along with pelleting at different final ultracentrifugation
speeds, enrichment in different gradient fractions further suggests that TET8‐positive EVs and
PEN1‐positive EVs represent two distinct subpopulations with different densities. Furthermore, in
transgenic plants co-expressing fluorescently labeled TET8 and PEN1, TET8-GFP-labeled EVs and PEN1-
mCherry-labeled EVs do not colocalize, supporting the distinction between these two classes of EVs [30,56] .
Differential centrifugation, sucrose gradients, and buoyant density gradients also reduce the occurrence of
apoptotic cell fragments in the final pellet .
[83]

Methods for isolation of plant EVs, such as differential centrifugation and gradient density centrifugation,
can fractionate different classes of EVs based on size and density, but these methods cannot sufficiently
purify one specific EV subtype in isolation. The method that is able to purify one specific EV class is