15th International Conference on Arabidopsis Research - TAIR

T01-067
Using microarrays to identify genes implicated in
pollen and anther development
Gema Vizcay-Barrena(1), Zoe Wilson(1)
1-Plant Sciences Division, University of Nottingham, Sutton Bonington, Loughborough. UK. LE12
5RD
The application of microarray technology along with the availability of the full
genome sequence of Arabidopsis has been demonstrated to be a powerful
tool for analysing gene expression profiling. The greatest advantage of this
approach is that around 24,000 genes can be monitored on a global scale,
compared with the traditional methods of expression analysis (e.g. northern
hybridisation) where only a few genes can be examine at a time. We have
been using the Arabidopsis Affymetrix arrays (NASC) to analyse the process
of pollen and anther development in the male sterile1 (ms1) mutant. In the
ms1 mutant the process of pollen development begins normally, with pollen
mother cells meiosis and tetrad formation progressing as in the wild type.
However, just after the microspores are released from the tetrads, the immature
pollen begins to breakdown and the anther tapetal tissue becomes abnormally
vacuolated. Degradation of the locule contents continues, resulting
in empty anthers with unviable pollen (Wilson et al., 2001). The MS1 gene is
therefore critical for the production of viable pollen.
Our analysis has focused mainly on the genes that MS1 may regulate at early
stages of pollen development. RNA extracted from floral tissue at developmental
stages during and post-MS1 expression has been used to screen the
Arabidopsis Affymetrix gene array. Evaluation of the data generated by these
experiments will be presented.
Wilson ZA, Morroll SM, Dawson J, Swarup R and Tighe P (2001). Plant Journal 28: 27-39.
T01 Development 1 (Flower, Fertilization, Fruit, Seed)
T01-068
Developmental regulation of transcription factor
genes in Arabidopsis seeds
Anna Blacha(1), Armin Schlereth(1), Tomasz Czechowski(1), Yves Gibon(1), Mark
Stitt(1), Wolf-Rüdiger Scheible(1), Michael Udvardi(1)
1-Max-Planck Institute of Molecular Plant Physiology
Seed storage compounds, such as proteins and lipids, are crucial for human
nutrition. Synthesis of storage compounds is developmentally regulated in
seeds, and controlled, at least in part, at the level of gene transcription (Ruuska
et al., 2002). Transcription factors (TFs) that orchestrate these changes
are likely to be developmentally regulated also. However, few TF genes
involved in seed metabolism have been discovered. Genetic identification of
important seed TFs may be hampered by functional redundancy and/or their
absolute requirement for seed viability. Therefore, we have embarked on a
reverse-genetics approach that utilizes a genome-scale real-time RT-PCR
platform (Czechowski et al., 2004) to identify developmentally regulated TF
genes in Arabidopsis, which may regulate storage compound synthesis.
Using gene-specific primer pairs for over 1400 TFs and SybrGreenTM to
measure cDNA amplification kinetics in real-time, we identified 56 TF genes
that are over fifty-fold more highly expressed in developing siliques than in
shoots. To facilitate identification of TFs that control seed storage compound
synthesis, we determined the timing of key metabolic transitions. Fatty acid
(20:1) was measured by gas chromatography, glycerol-3-P by an enzyme
cycling assay, and storage protein by SDS-PAGE. Measurements were made
on seed extracted from siliques numbered from the top of the plant (i.e. from
developing to mature siliques/seeds). Seed glycerol-3-P levels exhibited a
local minimum in silique number 6, which increased to a maximum at silique
10. This increase preceded, and presumably fueled 20:1 fatty acid biosynthesis,
which began in silique 10 and continued until silique 22. Synthesis
of the legumin-type and napin-type proteins began in siliques 14 and 17,
respectively. We now plan to measure transcript levels for all Arabidopsis
TF genes in developing seeds both prior to and after the onset of storage
compound synthesis. This will help us to reduce the list of TF suspects that
may control synthesis of storage lipid, protein, and other seed compounds of
nutritional interest.
Czechowski et al. (2004). Plant J. 38: 366-79.
Ruuska et al. (2002). Plant Cell 14: 1191-206.
15 th <strong>International</strong> <strong>Conference</strong> on Arabidopsis Research 2004 · Berlin