Research - Institute of Plant Biology - Laboratory of Functional Cell Biology

Attila FEHÉR
scientific adviser

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Katalin PICHERERNÉ GÉMES research associate
Dalma MÉNESI scientific administrator
Ildikó VALKAI scientific administrator
Dézi Bianka LAJKÓ junior research associate
Dóra BERNULA Ph.D. student
Shyamjee YADAV guest Ph.D. student
Róza NAGY laboratory assistant

FUNCTIONAL CELL BIOLOGY

Plants exhibit a remarkable developmental plasticity as a consequence of their sessile way of life. During plant ontogenesis, exogenous (environmental) and endogenous (developmental) signals converge on interlinked signaling pathways having dynamic impacts on plant form and function. We use molecular, biochemical and cellular approaches to reveal and characterize plant-specific pathways of cellular signaling that underlie this developmental plasticity.


SIGNALING THROUGH ROP GTPASES

Summary

Cell polarity plays important roles in plant development. For example, the main body axis is defined by the polarity of the single-celled zygote and the polar information is used for patterning and cell specification. ROP GTPases are key regulators of cellular polarity in plants. We aim to reveal the link of ROP GTPases to various kinases in order to have new insights into the regulation of cell polarity during morphogenesis, tip growth and fungal invasion of cells. We have shown previously that a specific class of RLCK kinases (VIA) is potential ROP effectors. As these kinases are implicated both in pollen tube growth as well as fungal susceptibility, they will be in the center of our studies. However, we also have data on the possible involvement of upstream kinases as potential ROP regulators. Therefore our investigations will include these kinases as well. We combine biochemical, structural, cellular and functional information using purified proteins, mutant and transgenic plants, yeast and chemical genomic screening systems, transient gene expression assays, confocal microscopy and in silico data analysis to compare ROP-centered kinase signaling during cell polarity (in vitro pollen tubes), morphogenesis (whole plant) and pathogenesis (fungi-infected cells).


Research questions

The main questions of the proposed research are: How the Rho-type ROP G-proteins are linked to kinase signaling in plants and what is the biological significance of these signaling pathways during cell polarity establishment related to morpho- and pathogenesis? In yeast/metazoa several types of Rho-activated kinases exist (e.g. PAKs) that are completely absent from plants. The link of Rho signaling to receptor tyrosine kinases through RhoGEFs was also revealed in these organisms. However, plants have unique Rho proteins (ROPs), unique receptor kinases (RLKs), unique RopGEFs, unique ROP effector kinases (RLCK_VIAs) and the signaling network involving these specific proteins is hardly known. Based on our preliminary data we hypothesize that all these proteins are interlinked and play together a central role in the regulation of ROP-related processes including cell polarity. We aim to prove this hypothesis by various experimental approaches. Together with our foreign partner, Ralph Hückelhoven (Technical University of Munich), we also hypothesize that compatible fungal pathogens use the plant cell polarity machinery to enter into plant cells. We aim to compare ROP- and kinase-related steps during polar cell growth (pollen tube) and fungal entrance into plant cells in addition to reveal the function of ROP-related kinases during normal morphogenesis.


Significance of the research

ROPs are the only and unique signaling-type small G-proteins in plants, but their integration into signaling networks is hardly known. In previous publications we provided the first experimental data that they can regulate kinases and they can also be regulated through phosphorylation. We aim to use the obtained knowledge to build up a general model for receptor signaling in plants; from RLKs to ROP effectors. Moreover, the data can serve as a basis for studies concerning the evolution of signaling mechanisms, as well as can give specific insights into the regulation of basic cellular processes such as polar cell growth, cell expansion, plant morphogenesis and the plants responses to fungal pathogens. This basic knowledge can be translated in long term into molecular markers and genetic technologies that can be used to alter plant shape/development and to coop with fungal plant pathogens.




Figure 1. The in vitro myelin basic protein (MyBP) phosphorylation activity of barley RBK1 (RLCK VI_A) is active ROP (CA RacB) dependent.



CHROMATIN-REGULATED TRANSCRIPTIONAL REPROGRAMMING IN SOMATIC PLANT CELLS DURING THE ACQUISITION OF TOTIPOTENCY

Summary

The goal of the current research project is to understand the mechanism that controls the initiation of the process of somatic embryogenesis (SE) in differentiated plant cells. We plan to establish and use an efficient embryogenic tissue/cell culture system of the model plant Arabidopsis thaliana in order to identify and follow the dynamics of specific genomic regions associated with this developmental transition: silenced in vegetative but active in totipotent embryogenic cells. To achieve this goal, we will focus on the investigation of markers and regulators of chromatin organization. We aim to define and investigate the transitory “totipotent” state of induced cells when they already express pluripotency transcription factors but still not express those associated with embryo development. Our studies may also serve to enhance competence for somatic embryogenesis in recalcitrant genotypes/explants through the manipulation of overall chromatin organisation in target cells using various chromatin-related drugs.

Research question

Plant cells possess remarkable developmental plasticity. One of the most intriguing examples of this plasticity is somatic embryogenesis (SE) during which differentiated plant cells regain totipotency and develop into embryos. Despite the fact that SE is widely used for in vitro plant propagation, the biological background of this plant-specific phenomenon is hardly known. In 2005, a special issue of the Science magazine listed among the 125 most important scientific questions the one: “How does a single somatic cell become a whole plant?”. The answer for this question is important not only to understand the mechanisms behind the remarkable regeneration capability of plants. The similarities between plant and animal stem cells are more and more evident. Therefore the knowledge accumulating about somatic plant cells regaining totipotency might be integrated into a generalized model on the nature of eukaryotic stem cells.

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 experiments aim to investigate this hypothesis studying epigenetic markers and processes during the somatic-to-embryogenic transition of Arabidopsis cells when they transiently regain totipotency.

Furthermore, while SE is widely used in certain species for vegetative propagation, many important species or genotypes are recalcitrant towards the induction of embryo formation. We aim to find clues to explain this difference and to potentially extend the use of SE to those explants/genotypes where it is currently not possible.

Significance of the research

Arabidopsis is the most widely used model plant of plant developmental biology with accumulated useful information, lines, and markers. However, this species was until know not readily amenable for studying somatic embryogenesis (SE) due to the absence of an efficient and routine SE system. Based on some old, almost forgotten results, we can establish a really efficient SE system for Arabidopsis. This system can be used to answer the main question of SE initiation unanswered until now: how the chromatin and the gene expression pattern of a somatic cell is reorganized to allow the initiation of a new embryogenic program?

This knowledge will also serve to compare the mechanisms governing the formation and maintenance of animal and plant stem cells.

Additionally, the research has potential practical applications. While SE is widely used in certain species for vegetative propagation, many important species or genotypes are notoriously recalcitrant towards the induction of embryo formation. Elaboration of efficient SE for a wide range of plant genotypes would provide the advantage to preserve and quickly and efficiently propagate (“clone”) superior germplasms (e.g. with hybrid genomes) with economic importance avoiding genetic recombination associated with sexual propagation. Furthermore, in vitro plant regeneration is the prerequisite for the genetic modification of plants therefore the range of genotypes amenable for gene technology could be widened if we could extend SE to presently recalcitrant genotypes.




Figure 2. Hypotetical model on the role chromatin remodelling in auxin and stress induced somatic embryogenesis influenced by the genotype and the developmental state of the explant.


Selected publications

A. Feher A , DB. Lajko, Signals fly when kinases meet Rho ¬of ¬plants (ROP) small G¬ proteins. Plant Science 237 (2015) 93.

T. Reiner, C. Hoefle, C. Huesmann, D. Ménesi, A. Fehér, R. Hückelhoven, The Arabidopsis ROP-activated receptor-like cytoplasmic kinase RLCK VI_A3 is involved in control of basal resistance to powdery mildew and trichome branching. Plant Cell Rep. In press (2015). doi:10.1007/s00299-014-1725-1.

A. Fehér, Somatic embryogenesis - Stress-induced remodeling of plant cell fate. Biochim. Biophys. Acta. Special issue: Stress as a fundamental theme in cell plasticity. In press (2015). doi:10.1016/j.bbagrm.2014.07.005.

M. Domoki, A. Szűcs, K. Jäger, S. Bottka, B. Barnabás, A. Fehér, Identification of genes preferentially expressed in wheat egg cells and zygotes. Plant Cell Rep. 32 (2013) 339–48.

J. Bíró, I. Farkas, M. Domoki, K. Otvös, S. Bottka, V. Dombrádi, et al. The histone phosphatase inhibitory property of plant nucleosome assembly protein-related proteins (NRPs). Plant Physiol. Biochem. 52 (2012) 162–8.

C. Huesmann, T. Reiner, C. Hoefle, J. Preuss, M.E. Jurca, M. Domoki, et al. Barley ROP binding kinase1 is involved in microtubule organization and in basal penetration resistance to the barley powdery mildew fungus. Plant Physiol. 159 (2012) 311–20.

C. Fodor-Dunai, I. Fricke, M. Potocký, D. Dorjgotov, M. Domoki, M.E. Jurca, et al. The phosphomimetic mutation of an evolutionarily conserved serine residue affects the signaling properties of Rho of plants (ROPs). Plant J. 66 (2011) 669–79.

D. Dorjgotov, M.E. Jurca, C. Fodor-Dunai, A. Szűcs, K. Ötvös, É. Klement, et al. Plant Rho-type (Rop) GTPase-dependent activation of receptor-like cytoplasmic kinases in vitro. FEBS Lett. 583 (2009) 1175–82.

B. Barnabas, K. Jager, A. Fehér, The effect of drought and heat stress on reproductive processes in cereals. Plant. Cell Environ. 31 (2008) 11–38.

A. Fehér, K. Ötvös, T. Pasternak, A. Pettkó Szandtner, The involvement of reactive oxygen species (ROS) in the cell cycle activation (G 0 -to-G 1 transition) of plant cells, Plant Signal. Behav. 3 (2008) 823–826.

A. Fehér, The initiation phase of somatic embryogenesis: What we know and what we don’t, Acta Biol. Szeged. 52 (2008) 53–56.

M.E. Jurca, S. Bottka, A. Fehér, Characterization of a family of Arabidopsis receptor-like cytoplasmic kinases (RLCK class VI). Plant Cell Rep. 27 (2008) 739–48.

T.P. Pasternak, K. Ötvös, M. Domoki, A. Fehér, Linked activation of cell division and oxidative stress defense in alfalfa leaf protoplast-derived cells is dependent on exogenous auxin, Plant Growth Regul. 51 (2007) 109–117.

M. Domoki, J. Gyorgyey, J. Biro, T.P. Pasternak, A. Zvara, S. Bottka, et al. Identification and characterization of genes associated with the induction of embryogenic competence in leaf-protoplast-derived alfalfa cells. Biochim. Biophys. Acta. 1759 (2006) 543–51.

A. Fehér, Why Somatic Plant Cells Start to form Embryos?, in: A. Mujib, J. Samaj (Eds.), Somat. Embryog. Plant Cell, Springer-Verlag, Berlin Heidelberg, 2005: pp. 85–101.

K. Ötvös, T.P. Pasternak, P. Miskolczi, M. Domoki, D. Dorjgotov, A. Szűcs, et al. Nitric oxide is required for, and promotes auxin-mediated activation of, cell division and embryogenic cell formation but does not in uence cell cycle progression in alfalfa cell cultures. Plant J. 43 (2005) 849–860.

A. Fehér, T.P. Pasternak, D. Dudits, Transition of somatic plant cells to an embryogenic state. Plant Cell, Tissue, Organ Cult. 74 (2003) 201–228.

T.P. Pasternak, E. Prinsen, F. Ayaydin, G. Potters, H. Asard, H.A. Van Onckelen, et al. The Role of Auxin, pH, and Stress in the Activation of Embryogenic Cell Division in Leaf Protoplast-Derived Cells of Alfalfa, Plant Physiol. 129 (2002) 1807–1819.