15th International Conference on Arabidopsis Research - TAIR

T10-017
The essentials of tissue-specific protein and
metabolite profiling - Laser Microdissection, LC/MS/
MS and GCMS
Martina Schad(1), Richard D. Smith(2), Patrick Giavalisco(1), Oliver Fiehn(1),
Stefanie Wienkoop(1), Wolfram Weckwerth(1), Julia Kehr(1)
1-Max Planck Institute for Molecular Plant Physiology, Department Willmitzer, Am Mühlenberg 1,
14476 Golm, Germany
2-Pacific Northwest National Laboratory, P.O. Box 999, Richland, WA 99352, USA
The vascular bundle is a tissue system of vital importance for higher plants.
It appears to have much more functions than only the transport of water and
metabolites, since also proteins, nucleic acids and signal substances are
transported.
A powerful method for contamination-free collection of specific cell types
from tissue sections is Laser Microdissection (LM) coupled to Laser Pressure
Catapulting (LPC). This technique has been routinely used in mammals and is
just starting to spread in plant sciences.
To demonstrate the applicability of LMPC for protein and metabolite analysis,
we collected samples from different stem tissues from Arabidopsis thaliana.
One strategy we used for the analysis of proteins was separation of intact
proteins by classical two-dimensional polyacrylamide gel electrophoresis
(2D-PAGE) coupled to mass spectrometry. After tryptic digest, proteins were
identified by matrix assisted laser desorption/ ionization (MALDI TOF MS) or
hybrid mass spectrometry (QTOF MSMS). A drawback of 2D-PAGE for small
sample amounts is the relatively high amount of material needed. Regarding
the limited sample amount obtainable by microdissection, the combination
of protease digest of the complex protein mixtures with HPLC separation and
subsequent tandem mass spectrometry (LC/MS/MS) provides an alternative,
promising tool for tissue specific proteome analysis.
Additionally, we optimise our sample preparation procedures to allow not
only proteome analyses but also investigations of tissue-specific metabolite
concentrations and even enzyme activities in microdissected samples.
15 th <strong>International</strong> <strong>Conference</strong> on Arabidopsis Research 2004 · Berlin
T10-018
AtGenExpress ¯ Expression atlas of Arabidopsis
Development
Markus Schmid(1), Stefan Henz(1), Timothy Davison(1, 2), Utz Pape(3), Martin
Vingron(3), Bernhard Schölkopf(2), Detlef Weigel(1, 4), Jan U. Lohmann(1)
1-Max Planck Institute for Developmental Biology, Spemannstr. 37-39, 72076 Tübingen, Germany
2-Max Planck Institute for Biological Cybernetics, Spemannstr. 38, 72076 Tübingen, Germany
3-Max Planck Institute for Molecular Genetics, Ihnestraße 73 14195 Berlin, Germany
4-Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 9203, USA
The activity of genes and their encoded products can be regulated in
several ways, but transcription is the primary level, since all other modes of
regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a
gene being transcribed in the first place. The importance of transcriptional
regulation has been underscored by the recent flood of global expression
analyses, which have confirmed that transcriptional co-regulation of genes
that act together is the norm, not the exception. Moreover, many studies
suggest that evolutionary change is driven in large part by modifications of
transcriptional programs.
An essential first step toward deciphering the transcriptional code is to
determine the expression pattern of all genes. With this goal in mind, an
international effort to develop a gene expression atlas of Arabidopsis has
been underway since fall 2003. This project, dubbed AtGenExpress, is funded
by the DFG and the MPG, and will provide the Arabidopsis community with
free access to a comprehensive set of Affymetrix microarray data. As part of
this collaboration, we have generated expression data from 79 samples in
triplicate focusing on development of wild-type Columbia (Col-0) and various
mutants. Samples consist of a wide range of Arabidopsis tissues, including
microscopically dissected organs, at various developmental stages. A metaanalysis
of the data set will be presented.
T10 Novel Tools, Techniques and Resources