Book of Abstracts - Geyseco

FESPB 2010 - XVII Congress of the Federation of European Societies of Plant Biology
P12
Epigenetics
Posttranslational modifications of core histones are one of the
most important mechanisms utilized by eukaryotic cells to regulate
gene expression at transcriptional level. Modifications,
such as acetylation, methylation or phosphorylation, can modulate
chromatin state by altering interactions between histones
and DNA or by recruitment of protein factors specifically
recognizing modified residues. Many histone-binding proteins
have been described in yeast and mammals, however, there is
still poor understanding of such interactions in plants. One of
the basic methods for identifying proteins that bind histones in a
modification-dependent manner is peptide pull-down. This method
utilizes synthetic histone peptides carrying a selected modification.
After immobilization on solid support, peptides are used
to precipitate interacting proteins from nuclear extract. Upon elution,
bound proteins are resolved on SDS-PAGE, excised from
gel and identified using mass spectrometry techniques. Method
described above has been used for identification of histone H3
partners in yeast and mammals. In our study, we aimed at adapting
this method for plant material. Optimalization of the extraction
of native nuclear proteins from Arabidopsis thaliana, as well
as the conditions of peptide pull-down, enabled identification of
proteins interacting with histone H3 peptides methylated at lysine
4. Application of our protocol resulted in precipitation of AL1
and AL7, a known H3K4me2-binding, PHD-containing proteins
and their close homologue AL6. This result indicates, that peptide
pull-down can be applied for identification of Arabidopsis
histone H3-interacting proteins.
P12-001: POSTTRANSLATIONAL MODIFICATIONS OF
HISTONE H1 IN TOBACCO (NICOTIANA THABACUM L.
Kotlinski, M 1 * - Dadlez, M. 2 - Jerzmanowski, A. 1
1
Laboratory of Plant Mol. Biol. Warsaw University / Institute of
Biochemistry and Biophysics, PAS
2
Institute of Biochemistry and Biophysics, PAS
*Corresponding author, e-mail: mkotl@ibb.waw.pl
Posttranslational modifications of core histones are well characterized
and known to play significant role in regulation of transcription
in Eucaryota. The knowledge about posttranslational
modifications of linker (H1) histones is much more limited and
so far restricted to mammals and Drosophila.
Here we present the first study of posttranslational modifications
of plant (tobacco) H1. We optimized protocols of H1 purification
and digestion for analyzes with mass spectrometry. The use
of hybrid linear ion trap-orbitrap mass spectrometer allowed to
obtain high mass accuracy spectra, for both the precursor and
product ions. We utilized two different peptide fragmentation
techniques: collision induced dissociation (CID) and electron
transfer dissociation (ETD). The ETD technique provided good
quality fragmentation spectra of multiply charged peptides frequently
occurring in enzymatic digests of basic proteins such
as histone H1. Plants are known to have many non-allelic H1
variants differing in domain structure and functions. We determined
the patterns of posttranslational modifications for all of the
6 H1 variants occurring in tobacco, mapping numerous sites of
acetylation, methylation and phosphorylation. We also observed
differences in H1 modification patterns between different tissues.
P12-002: THE APPLICATION OF PEPTIDE PULL-DOWN
TECHNIQUE FOR IDENTYFICATION OF ARABIDOP-
SIS HISTONE H3 – INTERACTING PROTEINS
Buszewicz, D. 1 *- Palusinski, A. 2 - Prymakowska-Bosak, M. 3 -
Jerzmanowski A 1
1
(Laboratory of Plant Molecular Biology, Institute of Biochemistry
and Biophysics, PAS)
2
(Warsaw University of Life Sciences)
3
(Laboratory of Plant Molecular Biology, University of Warsaw)
*Corresponding author, e-mail: dbuszewicz@gmail.com
P12-003: FUNCTIONAL ANALYSIS OF SLEZ2, A TOMA-
TO ENHANCER OF ZESTE PROTEIN
Boureau, L.*- How Kit, A. - Stammitti, L. - Moing, A. - Maucourt,
M. - Deborde, C. - Bernillon, S. - Teyssier, E. - Gallusci, P.
UMR 619 Biologie du fruit - Centre INRA Bordeaux-Aquitaine
*Corresponding author, e-mail: lisa.b33@hotmail.fr
Polycomb (PcG) proteins, initially identified in Drosophila,
have been characterized in plants and play essential functions in
the control of plant development and reproduction. Among the
different classes of PcG proteins, three have been analyzed in
Arabidopsis: the Enhancer of Zeste (E(z)), the Extra Sex Com
(ESC), the Suppressor of Zeste (12) (Su(z)12). These proteins
are components of Polycomb Repressive Complexes 2 (PRC2)
that maintain chromatin in a closed state and control the transition
between plant developmental phases. In order to unravel
the function of the E(z) protein in the control of tomato fruit and
plant development, we have characterized three E(z) encoding
genes, namely SlEz1, SlEz2 and SlEZ3. SlEZ21 and SlEZ2 encode
functional proteins, whereas SlEZ3 is most likely a pseudo
gene. Whereas both SlEZ1 and SlEZ2 tomato E(z) genes are
similarly expressed in vegetative tissue, they present contrasted
expression patterns in tomato flowers and fruits. Analysis of
SlEZ2 RNAi lines indicates that this protein is involved in tomato
plant growth but also participates to the control of flower and
fruit development. Phenotypes include abnormal flowers, fruit
development abortion, altered fruit colour and texture, reduced
seed germination and plant of reduced size. Fruit metabolomic
analyzes suggest an alteration of the metabolite content of fruits
of transgenic plants.
P12-004: COORDINATED SILENCING AND REACTIVA-
TION OF TWO TANDEM REPORTER GENES IN VEGE-
TATIVELY PROPAGATED POTATO LINES
Fischer, L.* - Nocarova, E.
Charles University in Prague, Faculty of Science
*Corresponding author, e-mail: lukasf@natur.cuni.cz
Instability of transgene expression inseparably accompanies preparation
of transgenic plants. Although most changes in transgene
expression occur early after transformation, the transgenes
can be silenced long time after their integration, which complicates
the use of transgenic plants in both basic research and agriculture.
To study long-term changes in transgene expression in
potato (Solanum tuberosum), we monitored the activity of two
reporter genes, encoding green fluorescent protein (GFP) and
neomycin phosphotransferase (NPTII), in a set of 17 transgenic
lines during five years of vegetative propagation in vitro. Decrease
in transgene expression was observed preferentially in lines
with higher number of T-DNA insertions and higher initial GFP
expression level. The silencing was observed in four lines, all of
which successively silenced both reporter genes, furthermore the
loss of GFP fluorescence preceded the loss of kanamycin resistance
(silencing of NPTII gene) indicating interconnections between
silencing of the two loci. This successiveness in silencing
of the two genes was also reproduced in the silenced lines after
reactivation of the silenced transgenes by a DNA demethylating
drug 5-azacytidine. The possible mechanism of the coordinated
silencing is discussed in the respect of a switch of GFP silencing
from posttranscriptional to transcriptional level that was indica-