15th International Conference on Arabidopsis Research - TAIR

T09-047
Expression of nuclear genes for organellar RNA
polymerases in Arabidopsis
Carola Emanuel(1), Andreas Weihe(1), Thomas Börner(1)
1-Humboldt-University Berlin, Institute of Biology/Genetics, Chausseestr.117, 10115 Berlin
The Arabidopsis thaliana nuclear genome encodes three organellar phagetype
RNA polymerases (RpoTm, RpoTmp, RpoTp). While we could show that
RpoTm is targeted to mitochondria and the amino terminus of RpoTp targets
the proteins to plastids, RpoTmp is dually targeted to both mitochondria and
plastids.
To gain first insights into the division of labor among the different organellar
RNA polymerases, we have investigated RpoT transcript abundance in
Arabidopsis tissues by quantitative real-time RT-PCR. Relative determination
of transcript levels revealed a higher abundance of RpoTp mRNA in leaf
tissue, whereas RpoTm transcripts levels were higher in root, stem and
flower tissue. All RpoT transcripts were found to accumulate to higher levels
in young tissues.
Histochemical GUS assays of transgenic Arabidopsis plants showed
differences in RpoT promoter activity, especially in seedlings and flowers.
Whereas promoter activities of RpoTm and RpoTmp were detectable in some
meristematic tissues like root tips and young primary leaves, in leaf veins,
stigmatic papillae and carpels, RpoTp promoter GUS activity was only visible
in leaf tissue and sepals. In cross sections of stems, RpoTp promoter activity
showed high GUS staining intensity predominantly in the primary cortex,
while detection of RpoTm and RpoTmp GUS signals were restricted to the
stele. Analyses of transcript levels of the RpoT genes using in situ hybridization
corroborate the findings obtained by GUS staining.
We generated transgenic Arabidopsis plants expressing RpoT antisense transcripts.
The lower expression of the RpoT genes affects the transcription of
the organellar genes in these plants. RpoTm- and RpoTmp-antisense plants
were green and showed retarded growth, whereas RpoTp-antisene plants
displayed bleached leave tissue.
T09 Genetic Mechanisms (Transcriptional and Chromatin Regulation)
T09-048
Analysis of a suppressor mutant of the
immunophilin-like twisted dwarf1 (twd1) gene
mutation
Claudia Moeller(1), Dierk Wanke(2), Burkhard Schulz(1)
1-University of Tuebingen, ZMBP, Plant Physiology
2-University of Cologne, Botanical Institute
Null mutations of the recessive Arabidopsis FKBP-like immunophilin gene
TWISTED DWARF1 (TWD1) cause a pleiotropic phenotype characterised by
reduction of cell elongation and disorientated growth caused by impairment
of polar auxin transport. This results in plants with a severe dwarf phenotype
and twisted organs above and below ground.
Screens for suppressor mutants of the twd1 mutation resulted in the isolation
of a plant that shows an intermediate phenotype between wild-type (wt) and
the dwarf phenotype of the null mutation. The size of most organs is only
slightly reduced whereas the stems and siliques still show twisted growth
behavior. This plant was called twd-sup.
The suppressor screens has been performed with a null mutant induced by
T-DNA insertion in the fourth intron. This insertion leads to a complete knockout
of the TWD1 gene and results in the twd1 phenotype. Complementation
analysis between twd1 mutants and twd-sup plants revealed genetic
dominance of twd-sup over twd1 mutation. Analysis of more than 30 000
offspring of a cross between wt and twd-sup revealed tight genetic linkage
between the twd1 mutation and the twd-sup mutation. Intragenic suppressor
mutation cannot be ruled out. Surprisingly, no sequence alteration has been
found in the TWD1 gene sequences of twd1 and twd-sup plants. No structural
rearrangements of the inserted T-DNA could be found in both lines.
A striking difference between twd1 and twd-sup, however, can be found in
the methylation pattern of the NPTII gene of the T-DNA which serves as a
selection marker of T-DNA transformed plants. In twd-sup plants we found
silencing of the NPTII gene which leads to sensitivity to kanamycin. Twd1
plants that have the identical genetic makeup concerning the T-DNA insertion
are completely kanamycin resistant. Silencing of the NPTII gene has never
been observed for this line. The silencing of the NPTII gene can be shown
by RT-PCR. Treating twd-sup plants with 5-aza-cytidine reactivated the
expression of the silenced NPTII gene and resulted in kanamycin resistant
twd-sup plants.
Kamphausen et al., (2002) Plant J. 32, 263
Geisler et al., (2003) MBC 14, 4238
Matzke et al., (1989) EMBO J. 8, 643
15 th <strong>International</strong> <strong>Conference</strong> on Arabidopsis Research 2004 · Berlin