Biology Reference
In-Depth Information
photosynthetic tissue, and (4) high organ-specifi c and developmental
plasticity in peroxisome functions. In the post-genomic era of plant
research, traditional disciplines of physiology, cell biology and bio-
chemistry can effi ciently be combined with modern “omics” meth-
ods such as genomics, proteomics, and bioinformatics to identify
and physiologically characterize unknown proteins, to describe
protein-protein interactions in functional protein complexes and to
reveal novel metabolic pathways and regulatory functions. However,
prerequisite for the application of “omics” methods generally is that
model organisms are chosen whose genome has been fully
sequenced. Traditionally, plant peroxisomes have been isolated
from specifi c tissue (endosperm, cotyledons), developmental stages
and specifi c species such as spinach, pea, castor bean, and pumpkin
[
1
-
4
]. The complete genome sequence of Arabidopsis has been the
fi rst plant genome published [
5
], predestining this plant species for
proteome analyses. Five proteome studies of Arabidopsis peroxi-
somes have been published to date for either leaf peroxisomes [
6
-
8
],
glyoxysomes [
9
], or peroxisomes from cell cultures [
10
] using
different methods for organelle purifi cation. Numerous novel
metabolic pathways and defense functions indicated by these pro-
teome analyses have been partially or fully validated experimentally
thereafter (for review
see
refs.
11
,
12
). Particular the combination of
experimental proteomics with computational prediction of peroxi-
some-targeted matrix proteins has been proven powerful to uncover
the proteome of Arabidopsis peroxisomes [
13
,
14
].
Here, we present a detailed protocol for the isolation of
Arabidopsis leaf peroxisomes from photosynthetic tissue, which is
largely based on the method described previously [
7
,
8
] with minor
improvements. To obtain relatively high yield and purity, this pro-
tocol needs to be followed tightly. Even for an experienced scientist,
peroxisome purity and yield differ signifi cantly between different
isolations, depending, for instance, on the quality of plant material,
carefulness and isolation speed and day-to-day variations. It is rec-
ommended to analyze the purity of several leaf peroxisome isolates
separately by enzymatic or immune-biochemical methods biochem-
ical to select and pool those isolates of highest purity for proteome
analyses by either standard 1D or 2D gel electrophoresis combined
with mass spectrometry.
2
Materials
2.1 Components for
Growth of Arabidopsis
thaliana Col-0
1. Arabidopsis seeds stored at room temperature in 1.5 mL
microfuge tubes.
2. Commercial plant soil mixed with perlite (ratio roughly 3:1).
3. Plastic trays and square pots (ca. 6 cm × 6 cm).
4. Fertilizer if required.
