15th International Conference on Arabidopsis Research - TAIR

T03-035
Identification of SNARE molecules involved in the
post-Golgi network pathways in Arabidopsis
Tomohiro Uemura(1), Takashi Ueda(3), Akihiko Nakano(3, 4), Kunio Takeyasu(1),
Masa H. Sato(2)
1-Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake-cho, Sakyo-ku, Kyoto,
Japan
2-Graduate School of Human and Environmental Studies, Kyoto University, Yoshidanihonmatsu,
Sakyo-ku, Kyoto, Japan
3-Department of Biological Sciences, Graduate school of Science, University of Tokyo, 7-3-1,
Hongo, Bunkyo-ku, Tokyo, Japna
4-Molecular Membrane Biology Laboratory, RIKEN Discovery Research Institute, 2-1 Hirosawa,
Wako, Saitama, Japan
In all eucaryotic cells, specific vesicle fusion during vesicular transport is
mediated by membrane-associated proteins called SNAREs (soluble N-ethylmaleimide
sensitive factor attachment protein receptors). Sequence analyses
revealed 54 SNARE genes (18 Qa-SNAREs/Syntaxins, 11 Qb-SNAREs, 8
Qc-SNAREs, 14 R-SNAREs/VAMPs and 3 SNAP-25) in the Arabidopsis
genome. RT-PCR analyses revealed that all SNARE genes but AtYKY62 were
differentially expressed among tissues. A series of transient expression
assays using GFP (green fluorescent protein) fused proteins revealed the
subcellular localization of the SNARE proteins involved in various vesicular
transport pathways; 6 ER-localized SNAREs, 9 Golgi apparatus-localized
SNAREs, 7 TGN-localized SNAREs, 2 endosome-localized SNAREs, 18 PMlocalized
SNAREs and 9 vacuole-localized SNAREs. The data showed that the
trans-Golgi network (TGN) was an independent organelle separated from the
Golgi apparatus. The results obtained in this study suggest that combinations
of SNARE molecules are involved in each transport pathway, indicating the
complexity of the post-Golgi transport pathways.
T03 Cell Biology
T03-036
In vitro evolution of telomerase-deficient tissue
cultures of Arabidopsis thaliana
Petra Bulankova(1), Matthew J. Watson(2), Karel Riha(3), Dorothy E. Shippen(2),
Boris Vyskot(1)
1-Laboratory of Plant Developmental Genetics, Institute of Biophysics, Academy of Sciences of the
Czech Republic, Kralovopolska 135, CZ-612 65 Brno, Czech Republic
2-Departement of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843-
2128, USA
3-Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Rennweg 14,
A-1030 Vienna, Austria
Telomerase is the reverse transcriptase responsible for the extension of
telomeric repeat sequences in most species studied. Telomerase inactivation
causes telomere shortening and results in the loss of the telomere’s protective
function, which in mammals leads to cell cycle arrest and apoptosis.
Experiments performed on Arabidopsis thaliana mutants lacking telomerase
activity showed their great tolerance for genome instability. Plants survived
up to ten generations of selfing, but during the last five generations they suffered
from developmental defects and genome rearrangements manifested
by anaphase bridges, nuclear fragmentation, and aneuploidy.
In this study we present karyological analysis of two in vitro cell lines (A-G8
and B-G8) derived from seeds of the 8th generation of telomerase-deficient
A. thaliana. On slides prepared by enzymatic squashing of fixed tissue cultures,
nuclear cytometry and FISH with centromeric, 25S-rDNA, and telomeric
probes was performed. As expected, in wild type in vitro cultures, genomes
were stable. In the two G8 cell lines the absence of telomeric repeats
(TTTAGGG) at the ends of chromosomes was demonstrated by Southern blot
analysis and FISH with a telomeric PNA probe. The B-G8 line, which was cultured
for 2 years in vitro, suffered from severe growth irregularities, including
a loss of typical callus morphology (friable structure) and a high mortality in
numerous callus sectors. This line also harboured frequent anaphase bridges
and a high variation in chromosome number. A great proportion of nuclei
were polyploid and displayed an increased number of 25S-rDNA signals. The
other line A-G8 was cultured for 3 years in vitro survived a growth crisis, and
is now phenotypically stable and fast-growing. Despite being cultured longer
in vitro, the A-G8 line possesses the standard callus phenotype, and is much
more homogenous in all parameters (ploidy level, number of FISH signals).
These findings imply its genome is stabilised. However anaphases bridges
were still detected in A-G8 samples. We speculate that a partial stabilization
of the A-G8 genome occurred from restructuring chromosome terminal
regions to provide chromosome ends with partial, but not complete protection
from end-joining activities.
Acknowledgements: This research was supported by the Czech Academy of
Sciences (A6004304).
Riha et al., Science 291: 1797, 2001
Siroky et al., Chromosoma 112: 116, 2003
15 th <strong>International</strong> <strong>Conference</strong> on Arabidopsis Research 2004 · Berlin