15th International Conference on Arabidopsis Research - TAIR

T04-035
Identification of sugar-regulated genes in Arabidopsis
thaliana using high-throughput RT-PCR and
Affymetrix gene chips.
Daniel Osuna(1), Rosa Morcuende(1), Wolf-Rüdiger Scheible(1), Mark Stitt(1)
1-Max-Planck-Institute for Molecular Plant Physiology, Am Mühlenberg 1, 14476 Golm, Germany
We used Affymetrix ATH1 GeneChips and a real-time RT-PCR platform,
comprising primer pairs of over 1400 transcription factors (Czechowski et
al., 2004), to identify sugar responsive transcripts in Arabidopsis seedlings
grown in liquid-culture before and in a time course after addition of sucrose
and glucose. A total of 67 transcription factors were identified as sugar-regulated.
Five transcription factors that were repressed after two days of sugar
starvation were up-regulated after 3 hours of sucrose re-addition and nine
were identified like fast-response genes (30 min after sucrose readdition).
To identify regulatory genes involved in both, sucrose- and glucose-specific
sensing and signalling pathways in Arabidopsis we also carried out comparisons
of global transcript expression patterns obtained after 3h of sucrose
and glucose re-addition using MAPMAN software (Thimm et al., 2004). In
addition, we have identified genes that specifically respond to low (15 mM
D-glucose) and high sugar concentration (100 mM D-glucose) and these are
involved in hormone metabolism, signalling and redox response. Transcription
factors identified belong to diverse families including the AP2-EREBP,
bZIP, C2C2(Zn)-CO-like, HB, MADS box, Myb, C2H2 zinc finger, C3H zinc
finger, NAC domain and Triple-Helix families. Other genes that are involved
in cysteine and phenylalanine synthesis, phenolpropanoids metabolism and
development also showed specific response.
To study response-specificity to sugars, our interest was focused on those
genes that are differentially expressed in comparison with other regimes
(nitrate, phosphate and sulphate readdition) and several regulatory genes
were selected. We are studying if these proteins have a main function like
transacting regulators in the coordinated regulation of metabolic pathways
induced or repressed by sucrose. For this purpose we have now available
inducible and constitutive overexpression constructs/lines as well as T-DNA
knockout mutants for some of them. It will allow us to elucidate new sucrose
signalling pathways, to identify target genes involved in these processes and
to study changes in the expression of these target genes. Also phenotypic
analyses will be done.
Czechowski et al. (2004). Plant J. 38, 366-79.
Thimm et al. (2004). Plant J. 37, 914-39.
T04 Interaction with the Environment 1 (Abiotic)
T04-036
Arabidopsis thaliana dehydrins ERD 14, LTI 29 and
COR 47 protect thylakoid membranes during freezing
Vladan Bozovic(1), Janne Svensson(2), Jürgen M. Schmitt(1), Carsten A. Köhn(1)
1-Freie Universität Berlin, Institut für Pflanzenphysiologie, Königin-Luise-Str. 12 - 16a, 14195
Berlin, Germany
2-Department of Botany and Plant Sciences, University of California, Riverside, California
92521¯0124, USA
In general dehydrins occur in plants as multi-gene families. As the name
dehydrins implicates these proteins are typically expressed in response to dehydration.
Dehydration can be caused by drought, osmotic stress or freezing
temperatures. In the last case water is withdrawn from the cell by ice formation
in the apoplast leading to a drastic increase of the solute concentration
in the cell. The membranes of the cell are thought to be a primary site of
freezing damage in plants. We tested four Arabidopsis dehydrins (ERD 14,
RAB 18, LTI 29 and COR 47) for protection of thylakoid membranes during
a freeze thaw cycle in vitro. ERD 14, LTI 29 and COR 47 had cryoprotective
activity while RAB 18 did not protect the thylakoid membranes. The cryoprotective
activity reached a maximum of 50% to 60 % at protein concentrations
of 140-250 μg/ml in the assay. The cryoprotctive activity could not be
increased further by increasing the protein concentration. This is in contrast
to an other cryoprotective protein, Cryoprotectin (Hincha et al., 2001), which
can protect the thylakoids up to 100%. To get a hint whether different
dehydrins might protect membranes by different molecular mechanisms we
tested for additive effects. The activity of dehydrins seems to be additive, but
the overall cryoprotective activity could not be increased over the maximum
cryoprotective activity seen for the single dehydrins. This suggests that the
dehydrins protect by the same mechanism. A contribution of dehydrins to
freezing tolerance in vivo is supported by the observation of Nylander et al.
(2001) that LTI29 and COR 47 are cold induced on mRNA and protein level.
On the other hand the non cryoprotective dehydrin RAB 18 is neither induced
at mRNA level nor protein level during cold acclimation.
Hincha et al. (2001): Plant Physiol. 125 (2), 835-846.
Nylander et al. (2001): Plant Mol. Biol. 45 (3), 263-279
15 th <strong>International</strong> <strong>Conference</strong> on Arabidopsis Research 2004 · Berlin