15th International Conference on Arabidopsis Research - TAIR

T03-053
Global transcription analysis of Arabidopsis core cell
cycle regulators in suspension-cultured cells and
plants reveals multiple and highly specific profiles of
gene expression
Margit Menges(1), James A.H. Murray(1)
1-University of Cambridge, Institute of Biotechnology, Tennis Court Road, CB2 1QT, UK
Dispersed plant suspension cultures allow cell proliferation and growth to be
analysed in the absence of developmental processes. We have established
synchronisation procedures for two Arabidopsis cell lines MM1 and MM2d
and used these to analyse cell cycle regulated gene expression by Gene-
Chip arrays, Massively Parallel Signature Sequencing (MPSS) of cDNAs on
immobilised microbead arrays and real-time reverse transcriptase PCR. Gene
expression was analysed in cell line MM2d during synchronous re-entry of
the cell cycle, during cell cycle progression from a G1/ S phase block and
during normal growth from sub-culture to stationary phase. Comparative
analysis of results shows that around 1100 genes show significant cell cycle
regulation, and these are involved in a wide range of cellular processes. To
further extend this analysis, we used synchronised samples of the alternative
light-grown cell line MM1 for transcript profiling analysis using MPSS
and Affymetrix GeneChip arrays to confirm the expression patterns seen
previously.
From this analysis we present the expression profiles of key regulators of
the plant cell cycle. These show distinctive and specific patterns throwing
new light on their likely roles in the cell cycle. Almost 90% of core cell cycle
regulators are expressed at detectable levels in cell suspension. Most D-type
cyclins show different timing suggesting distinct roles. In contrast, five of six
groups of A and B type cyclins show highly similar expression with a peak
in G2/M, including two previously unrecognised B-type cyclins. The CYCA3
group alone shows a peak in S phase. 80 genes share the G2/M regulatory
pattern, about half being new candidate mitotic genes of previously unknown
function. This unified and global model of transcriptional timing of all plant
cell cycle regulators reveals new patterns of cell cycle regulation, identifies
novel candidate mitotic genes and reveals CYCA3 as S phase cyclins. These
data strongly suggest that cell cultures can be used to model many cellular
processes.
In addition, available transcript profiling analysis results of core cell cycle
genes using various plant tissues is currently being subjected to bioinformatic
analysis to identify tissue-specific gene expression of key regulators, which
will be presented.
15 th <strong>International</strong> <strong>Conference</strong> on Arabidopsis Research 2004 · Berlin
T03-054
AraPerox: A Database of plant peroxisomal proteins
Sigrun Reumann(1), Changle Ma(1), Steffen Lemke(1), Lavanya Babujee(1)
1-University of Göttingen, Dept. Plant Biochemistry, Justus-von-Liebig Weg 11, D-37077 Göttingen,
email: sreuman@gwdg.de
Our knowledge on plant peroxisomal metabolism is mostly limited to the most
abundant enzymes that play a role in photorespiration and fatty acid beta
oxidation. Most matrix proteins from peroxisomes are targeted to the organelle
by a peroxisome targeting signal type 1 or type 2 (PTS1 or PTS2): The
PTS1 is a C-terminal tripeptide of the prototype SKL>, whereas the PTS2 is a
nonapeptide of four conserved residues (e.g., RLx5HL) located close to the Nterminal
end of the protein. We applied a bioinformatics approach to specify
the PTS peptides for plants by analyzing semi-quantitatively plant ESTs that
are homologous to PTS-targeted plant peroxisomal proteins for the nature of
their PTS. Accordingly, nine major PTS1 and two major PTS2 peptides have
been defined that are considered to indicate targeting to peroxisomes with
high probability. These PTS peptides including the minor PTS peptides were
applied to screen the Arabidopsis genome for unknown peroxisomal matrix
proteins. About 220 and 60 proteins were identified that carry a putative
PTS1 or PTS2, respectively, of which about 80% are unknown. To further
support postulated targeting to peroxisomes, several prediction programs
were applied and the putative targeting domains analyzed for properties
conserved in peroxisomal proteins and for PTS conservation in homologous
plant ESTs. Novel non-hypothetical proteins include several enzymes involved
in &#946;-oxidation of unsaturated fatty acids and branched amino acids, 2hydroxy
acid oxidases with a predicted function in fatty acid beta oxidation as
well as NADP-dependent dehydrogenases and reductases. In addition, large
protein families with many putative peroxisomal isoforms were recognized,
including a large number of mostly unknown acyl-activating enzymes and
largely unknown GDSL lipases and small thioesterases. Putative regulatory
proteins of plant peroxisomes include protein kinases, small heat-shock
proteins, and proteases. The information on targeting prediction, homology
and in silico expression analysis for these Arabidopsis proteins has been
compiled in the public database “AraPerox” to accelerate discovery and
experimental investigation of novel metabolic and regulatory pathways of
plant peroxisomes.
T03 Cell Biology