Biology Reference
In-Depth Information
roles. This includes the 80S ribosome, tRNA catabolic enzymes,
the ubiquitin-proteasome pathway, glycolysis, and associated sugar
metabolism pathways, phenylpropanoid biosynthesis, vitamin and
nucleotide metabolism, signaling and stress-responsive molecules,
and NDP-sugar biosynthesis. In addition, the identifi cation of
hundreds of proteins with unknown functions can be used to fur-
ther build on our understanding of how metabolism in the cytosol
infl uences plant function.
Cell fractionation methods in plants have been used for decades
to isolate different cell organelles. Methods for the isolation of intact
chloroplasts [
11
-
14
], mitochondria [
15
,
16
] and nuclei [
17
,
18
]
have been described. Most of these methods rely on the mechanical
disruption of plants by mincing tissue with blades, blending, or with
a mortar and pestle. However, multiple studies have established
that higher yields of organelles can be obtained when starting with
protoplasts [
11
,
19
,
20
].
Plant cell protoplasts are produced by using fungal enzymes
(e.g., cellulases and pectinases) to degrade the cell wall. Many
procedures have long been described for the production of pro-
toplasts from a variety of plant species and organ types [
21
-
24
].
The degradation of cell walls is performed in the presence of an
osmotic, usually a sugar such as mannitol, sucrose, or sorbitol. This
prevents protoplast disruption due to differences in osmolality
between the cell interior and the digestion medium. Protoplasts
have been extensively used to characterize many physiological
processes in plant cells, such as photosynthesis [
24
], guard cell sig-
naling [
25
], and protein localization [
26
] to name a few, and more
recently for the isolation of subcellular fractions [
10
,
19
,
27
].
Separation of the cytosolic fraction from plant samples requires
a method that can break apart or remove plant cell walls, while also
incurring minimal damage to organelles to prevent release of organ-
elle proteins into the cytosolic fraction. One such method used to
isolate cytosolic fractions from
Arabidopsis
cell cultures was based
on earlier fractionation studies undertaken on subcellular organelles
[
10
]. These organelle studies had demonstrated that superior yields
of intact organelles could be obtained from protoplasts and their
disruption by gentle pressure with a Potter-Elvehjem tissue grinder
resulted in maintenance of organelle integrity [
19
,
20
]. This gentle
homogenization method is followed by removal of organelles using
differential centrifugation. Extrapolating such approaches to plant
tissues, as opposed to cell cultures, requires several extra processing
steps to ensure success. When attempting the isolation of the cyto-
solic fraction from whole
Arabidopsis
seedlings, it is critical to start
with a pure sample of intact protoplasts, as many break during
digestion. Isolation of intact protoplasts from plant tissues can be
achieved by exploiting their differential fl otation density in sugar
gradients [
28
]. However, when not using a heterotrophic plant cell
culture, an additional diffi culty to isolate the cytosolic fraction from
