Download PROJECT PROPOSAL for applicants for Ph.D. fellowships

PROJECT PROPOSAL
for applicants for Ph.D. fellowships
supervisor:
co-supervisor:
institution:
contact:
CV:
Katalin PICHERERNÉ GÉMESDSB, Ph.D.
Attila FEHÉR, Ph.D., D.Sc.
Institute of Plant Biology
[email protected], [email protected]
http://www.brc.hu/file/cv/plant_gemes_katalin_en.pdf
project title:
INDUCED PLANT STEM CELLS: GENETIC/EPIGENETIC
INVESTIGATION OF IN VITRO REGENERATION
PROJECT SUMMARY
Embryogenesis in plants is not restricted to the fertilized egg cell but can be induced in many
different cell types including somatic cells. In most of the experimental systems parallel response of
plant cells to auxin and stress is a key step in the induction of somatic embryogenesis. This
response includes chromatin reorganization and a release of the embryogenic programme,
otherwise blocked in vegetative cells by chromatin-mediated gene silencing. The aim of this
research project is to understand the mechanism that controls the initiation of somatic
embryogenesis in differentiated plant cells. Our studies also plan to identify and follow the dynamics
of specific genomic regions associated with this developmental transition: silenced in vegetative but
active in totipotent embryogenic cells.
BACKGROUND OF THE STUDY
Plant cells have noticeable developmental
plasticity. One of the most interesting examples
of this plasticity is somatic embryogenesis
during which differentiated somatic plant cells
regain totipotency and develop into embryos
and after plants. In order to be able to initiate
the embryogenic program, differentiated cells
have to stop their specialized functions and
reset their gene expression pattern to that of
stem cells (dedifferentiation). Although during
the last years a number of key molecular
players involved in the process of plant somatic
embryogenesis were identified, we are still far
away to understand how cellular totipotency is
regained by somatic cells and why it is
expressed only in certain cell of certain
genotypes. To answer this question we plan to
do further experiments. It is hypothesized that
specific mechanisms in the establishment and
maintenance of epigenetic information in plants
are related to the ability of somatic plant cells to
dedifferentiate and regenerate the entire plant.
Our goal is to investigate this hypothesis
studying epigenetic markers and processes
during somatic embryogenesis. While somatic
embryogenesis is widely used in certain
species for vegetative propagation, many
important species or genotypes are notoriously
recalcitrant towards the induction of embryo
formation. Our aim is to find the reason of the
differences between species regarding
epigenetic
background
and
enhance
competence for somatic embryogenesis
through the manipulation of chromatin
organization.
RELEVANT RESEARCH IN THE HOST
LABORATORY
In our laboratory, we have extensively studied
the induction of somatic embryogenesis in leaf
protoplasts of an embryogenic genotype of
Medicago (alfalfa). According to our results,
auxin and stress together cause the
reorganization of gene expression which may
release the embryogenic developmental
program silenced in vegetative cells by a
chromatin-mediated way. Our aim is to develop
an efficient somatic embryogenesis model
system in order to answer the question how the
chromatin organization and gene expression
pattern of a somatic cell are reorganized to
allow the initiation of somatic embryogenesis.
All of our experiments will be carried out on
Arabidopsis thaliana. Arabidopsis is the most
widely used model plant of plant biology and
plant developmental biology with accumulated
useful information, transgenic lines, gene
constructs and molecular markers. However
until now somatic embryogenesis research in
Arabidopsis was limited due to the absence of
an efficient and routine cell or tissue culture
system. In order to generate an efficient
experimental system for Arabidopsis thaliana to
study the initiation phase of somatic
embryogenesis, we started to use a root culture
system. We also tested under the same
conditions different Arabidopsis ecotypes using
this system and monitored the efficiency of
embryo formation during different culture
conditions (hormone concentration, time of
induction, etc.). Furthermore we also created a
root culture system to investigate local auxin
accumulation
during
the
somatic-toembryogenic transition by the constructs
introduced into all studied Arabidopsis
ecotypes.
SPECIFIC AIMS
The transient state of cells with WUS
expression but still without LEC1 expression will
be considered as a „totipotent” state and will be
in our focus. We plan to identify the dynamics of
specific genomic regions during the transition
from somatic-to-embryogenic cells fate:
silenced in vegetative but active in totipotent
embryogenic cells. It can be hypothesized that
chromatin remodeling has a primary role in reprogramming of the cells so we plan to
investigate chromatin organization, molecular
markers indicating chromatin state (posttranslational histone modifications) and the
expression of embryogenesis marker genes.
We also plan to characterize the effect of
different treatments on somatic embryogenesis
induction and chromatin reorganization. It will
be monitored what is the proper
Supported by the
TÁMOP 4.1.1.C -13/1/KONV.2014-0001
project
timing/dynamics of chromatin reorganization
and gene expression changes during the
initiation of somatic embryogenesis and which
chromatin modifying protein complexes
participate in the acquisition of totipotency or
pluripotency in differentiated somatic plant cells.
Our studies also aim to enhance competence
for somatic embryogenesis in recalcitrant
genotypes/explants through the manipulation of
overall chromatin organization in target cells.
MATERIAL AND METHODS
 Arabidopsis cell and tissue culture
 Transgenic
plant
production
and
characterization
 Chromatin immunoprecipitation followed by
gene identification (ChIP-Seq)
 Stereo
and
confocal
fluorescence
microscopic techniques
 RNA isolation, mRNA purification, cDNA
synthesis
 Real-time polymerase chain reaction (RTQPCR)
 Western blot analysis
 In silico sequence analysis
SUGGESTED READINGS
Radoeva T, et al.: A roadmap to embryo identity in plants.
Trends Plant Sci, 1-8(2014)
Su YH, et al.: The hormonal control of regeneration in
plants. Curr Top Dev Biol, 108:35-69(2014)
Engelhorn J, et al.: Gene activation and cell fate control
in plants: a chromatin perspective. Cell Mol Life Sci,
71:3119-37(2014)
Rose RJ, et al.: Developmental biology of somatic
embryogenesis. Eds.: E.C. Pua, M.R. Davey, Plant Dev
Biol - Biotechnol Perspect, Springer Berlin Heidelberg, 326(2010)
Yang X, et al.: Regulation of Somatic Embryogenesis in
Higher Plants. CRC Crit Rev Plant Sci, 29:36-57(2010)
Sugimoto K, et al.: Arabidopsis regeneration from
multiple tissues occurs via a root development pathway.
Developmental Cell, 18:463-471(2010)
Tanaka M, et al.: The Arabidopsis histone deacetylases
HDA6 and HDA19 contribute to the repression of
embryonic properties after germination. Plant Physiol,
146:149-161(2008)
Ikeuchi M, et al.: Plant callus: mechanisms of induction
and repression. Plant Cell, 25:3159-73(2013)
Márton L, et al.: Facile transformation of Arabidopsis.
Plant Cell Rep, 10:235-239(1991)
Wenck RA, et al.: Large scale protoplast isolation and
plant regeneration of Arabidopsis thaliana. Bio
Techniques, 18:640-643(1995)
SNAPSHOTS FROM THE HOST LABORATORY
Significant publications
Fehér A.: Somatic embryogenesis - Stress-induced remodelling of plant cell fate. Biochim Biophys Acta, 1849:385402(2015)
Fehér A, et al.: Transition of somatic plant cells to an embryogenic state. Plant Cell Tissue Organ Cult, 74:201-228(2003)
Feher A.: Why Somatic Plant Cells Start to form Embryos? Eds.: A. Mujib, J. Samaj, Somat. Embryog, Plant Cell, SpringerVerlag, Berlin Heidelberg, pp 85–101(2005)
Ötvös K, et al.: Nitric oxide is required for and promotes auxin-mediated activation of cell division and embryogenic cell
formation but does not influence cell cycle progression in alfalfa cell cultures. Plant J., 43:849-860(2005)
Pasternak TP, et al.: The role of auxin, pH, and stress in the activation of embryogenic cell division in leaf protoplastderived cells of alfalfa. Plant Physiol., 129:1807-1819(2002)
Representative recent research grants
“Chromatin-regulated transcriptional reprogramming in somatic plant cells during the acquisition of totipotency” (OTKA
K108802, 2014 – 2017)
Some of the latest students in the laboratory
Bernula D, Ph.D., 2014-2017; “Chromatin-regulated transcriptional reprogramming in somatic plant cells during the
acquisition of totipotency”
Domoki M, Ph.D., 2002-2009; “Identification of genes associated with the induction of embryogenic competence in leafprotoplast-derived alfalfa cells and characterization of the „Oxprot” gene”
Ötvös K, Ph.D., 2001-2006; “New insights into the biological activity of nitric oxide: A gas regulating cell division and
differentiation in alfalfa”
Supported by the
TÁMOP 4.1.1.C -13/1/KONV.2014-0001
project