Biology Reference
In-Depth Information
considered a dynamic compartment that plays a critical role not
only in determining cell shape and growth but also in interacting
with environmental factors including those required for nutrition
and defense against abiotic stress and pathogen attacks [
19
,
20
].
Despite the central role of extracellular proteins in most processes
that control growth, development [
21
], and even defense
responses, the extracellular proteome (secretome) is less well char-
acterized than other subcellular compartments. In fact, the
Arabidopsis genome shows that about 17 % of the genome (i.e.,
5,000 genes) encodes for proteins with a predicted signal peptide
that targets them towards the cell wall [
22
], but the function of
most remains unknown. Different proteomic tools are used in
order to investigate and understand the functions of cell wall pro-
teins. Thus, the use of two-dimensional electrophoresis, the avail-
ability of complete genome sequences, and the recent progress
made in mass spectrometry technology allow the complete identi-
fi cation of entire extracellular proteome and the assignment of new
functions. Compared with other methods for isolating extracellular
proteins (cell wall isolation, intercellular washing fl uid extraction,
amongst others), the spent medium of an SCC provides a conve-
nient, continuous, and unique source of extracellular proteins, eas-
ily obtained without cell disruption and so without any cytosolic
contamination [
23
-
26
]. In fact, the spent medium of SCC consti-
tutes a useful model system of reduced complexity that contains all
proteins involved in the different aspects of cell wall metabolism,
including those involved in defense. Indeed, pathogenesis-related-
proteins (PR-proteins), specifi cally PR-1 and -5, endochitinases,
chitinases, peroxidases, and other hydrolytic enzymes, are often
presented in the extracellular proteome of SCC from
Zinnia ele-
gans
,
Cycas revoluta
,
Taxus baccata
[
27
],
Capsicum
sp. [
25
,
28
],
Nicotiana tabacum
[
24
], and
Solanum lycopersicum
[
26
]. Most of
these proteins are involved in the dynamic modifi cation of the cell
wall architecture during cell culture growth and in the prevention
of cell wall degradation by microbial attacks.
For these reasons, the main goal of this chapter is focused on
the description of all procedures needed, from the initiation of an
SCC, obtaining and purifying proteins from extracellular medium
until the analysis and identifi cation of the extracellular proteome.
2
Materials
Immature berries from
Vitis vinifera
.
2.1
Plant Material
1. Laminar air-fl ow chamber.
2. Autoclave.
3. Plant growth chamber.
2.2 Equipment for
Plant Cell Cultures
