15th International Conference on Arabidopsis Research - TAIR

T10-031
A novel approach to dissect the abscission process
in Arabidopsis.
Gonzalez-Carranza, Zinnia H.(1), Roberts, Jeremy A.(1)
1-The University of Nottingham, Division of Plant Sciences. Sutton Bonington Campus, Loughborough,
Lecies. UK.
Isolation and characterization of genes from a particular tissue sample has
been used widely as a method to understand and study any process in
the development of a plant. However, the origin of the transcripts isolated
may have come from a mixture of specialised cells and not only from those
involved in a unique process. Recently the use of laser capture microdissection
technology and the production of GFP protoplasts, which are sorted
by fluorescence, have been reported to resolve this difficulty. The use of the
latest technology overcomes the problem of mixture of transcripts, however,
manipulation of tissue either to embed the samples for microdissection or the
generation of protoplasts may alter the transcript profile in the samples. In
previous studies we demonstrated that a polygalacturonase (PGAZAT) from
Arabidopsis (Gonzalez, et al., 2002) was expressed specifically in abscission
zone cells by fusing the promoter of this gene to the reporter GFP. Using
these transgenic lines we have now developed a strategy to generate cDNA
and yeast libraries specifically from abscission zone cells of floral organs of
Arabidopsis using the GFP tag. This approach has overcome some of the
problems previously faced when studying development and it is enabling us
to dissect the abscission process in a unique way.
Gonzalez-Carranza, et al. 2002. Plant Physiol. 128 (2): 534¯543.
T10 Novel Tools, Techniques and Resources
T10-032
Enrichment of phosphorylated proteins from A.
thaliana
Florian Wolschin(1), Wolfram Weckwerth(1)
1-Max Planck Institute of Molecular Plant Physiology
Phosphorylation of proteins is one of the most important regulatory mechanism
in living organisms. However, mainly due to the low abundance of
phosphorylated proteins (about 10 % at any given timepoint) the analysis of
protein phosphorylation is difficult.
The enrichment of these low abundance proteins is therefore highly desirable
when trying to get a glance at the phosphorylation status.
We compared different methods for the enrichment of phosphoproteins out
of sub-stoichiometric mixtures. The methods were evaluated with mixtures of
model proteins and one of the methods was used to enrich phosphoproteins
out of a complex leaf extract from A. thaliana. The successful enrichment was
confirmed by staining an sds-gel with pro-q diamond stain, which selectively
stains phosphorylated proteins.
From a digested gel band we identified the small subunit of rubisco as a
phosphorylated protein in A. thaliana.
Furthermore, the method was used to enrich the phosphorylated peptides out
of a tryptic digest from alpha-casein. The eluate contained only phosphorylated
peptides as confirmed by beta-elimination of the phosphate-group. Thus,
the proposed method demonstrates applicability even for the enrichment of
phosphopeptides.
15 th <strong>International</strong> <strong>Conference</strong> on Arabidopsis Research 2004 · Berlin