Heribert Hirt - Publications

The Dark Side of the Salad: Salmonella typhimurium Overcomes the Innate Immune Response of Arabidopsis thaliana and Shows an Endopathogenic Lifestyle

PLoS One May 28, 2008 3(5): e2279. doi:10.1371/journal.pone.0002279 PMID: 18509467

330 Adam Schikora¹, Alessandro Carreri², Emmanuelle Charpentier³, Heribert Hirt¹,²*

¹ URGV Unité de Recherche en Génomique Végétale, INRA Institut National de la Recherche Agronomique/CNRS Centre National de la Recherche Scientifique/University of Evry Val d'Essonne, Evry, France

² Department of Plant Molecular Biology, Max F. Perutz Laboratories, Vienna, Austria

³ Department of Microbiology and Immunobiology, Max F. Perutz Laboratories, Vienna, Austria

Salmonella enterica serovar typhimurium contaminated vegetables and fruits are considerable sources of human infections. Bacteria present in raw plant-derived nutrients cause salmonellosis, the world wide most spread food poisoning. This facultative endopathogen enters and replicates in host cells and actively suppresses host immune responses. Although Salmonella survives on plants, the underlying bacterial infection mechanisms are only poorly understood. In this report we investigated the possibility to use Arabidopsis thaliana as a genetically tractable host system to study Salmonella-plant interactions. Using green fluorescent protein (GFP) marked bacteria, we show here that Salmonella can infect various Arabidopsis tissues and proliferate in intracelullar cellular compartments. Salmonella infection of Arabidopsis cells can occur via intact shoot or root tissues resulting in wilting, chlorosis and eventually death of the infected organs. Arabidopsis reacts to Salmonella by inducing the activation of mitogen-activated protein kinase (MAPK) cascades and enhanced expression of pathogenesis related (PR) genes. The induction of defense responses fails in plants that are compromised in ethylene or jasmonic acid signaling or in the MKK3-MPK6 MAPK pathway. These findings demonstrate that Arabidopsis represents a true host system for Salmonella, offering unique possibilities to study the interaction of this human pathogen with plants at the molecular level for developing novel drug targets and addressing current safety issues in human nutrition.

FWF Austrian Science Fund Press Release

Site-Specific Phosphorylation Profiling of Arabidopsis Proteins by Mass Spectrometry and Peptide Chip Analysis.

J Proteome Res. 2008 Apr 24 [Epub ahead of print] PMID: 18433157

prj75/325 de la Fuente van Bentem S ¹, Anrather D, Dohnal I, Roitinger E, Csaszar E, Joore J, Buijnink J, Carreri A ¹, Forzani C ¹, Lorkovic ZJ, Barta A, Lecourieux D ¹, Verhounig A, Jonak C, Hirt H. ¹*

¹ Department of Plant Molecular Biology, and Department of Biochemistry, Max F. Perutz Laboratories, University of Vienna,
   Dr. Bohr-Gasse 9, 1030 Vienna, Austria

* Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, 1030 Vienna, Austria

* Pepscan Presto, Zuidersluisweg 2, 8243 RC Lelystad, The Netherlands

* Department of Medical Biochemistry, Max F. Perutz Laboratories, Medical University of Vienna, Dr. Bohr-Gasse 9, 1030 Vienna, Austria

* Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Vienna Biocenter,
   Dr. Bohrgasse 3, 1030 Vienna, Austria

* URGV Plant Genomics, 2 rue Gaston Cremieux, 91057 Evry, France

An estimated one-third of all proteins in higher eukaryotes are regulated by phosphorylation by protein kinases (PKs). Although plant genomes encode more than 1000 PKs, the substrates of only a small fraction of these kinases are known. By mass spectrometry of peptides from cytoplasmic- and nuclear-enriched fractions, we determined 303 in vivo phosphorylation sites in Arabidopsis proteins. Among 21 different PKs, 12 were phosphorylated in their activation loops, suggesting that they were in their active state. Immunoblotting and mutational analysis confirmed a tyrosine phosphorylation site in the activation loop of a GSK3/shaggy-like kinase. Analysis of phosphorylation motifs in the substrates suggested links between several of these PKs and many target sites. To perform quantitative phosphorylation analysis, peptide arrays were generated with peptides corresponding to in vivo phosphorylation sites. These peptide chips were used for kinome profiling of subcellular fractions as well as H₂O₂-treated Arabidopsis cells. Different peptide phosphorylation profiles indicated the presence of overlapping but distinct PK activities in cytosolic and nuclear compartments. Among different H₂O₂-induced PK targets, a peptide of the serine/arginine-rich (SR) splicing factor SCL30 was most strongly affected. SRPK4 (SR protein-specific kinase 4) and MAPKs (mitogen-activated PKs) were found to phosphorylate this peptide, as well as full-length SCL30. However, whereas SRPK4 was constitutively active, MAPKs were activated by H₂O₂. These results suggest that SCL30 is targeted by different PKs. Together, our data demonstrate that a combination of mass spectrometry with peptide chip phosphorylation profiling has a great potential to unravel phosphoproteome dynamics and to identify PK substrates.

Protein networking: insights into global functional organization of proteomes

Proteomics 2008, 8, 799–816 DOI 10.1002/pmic.200700767 PMID: 18297653

Enrico Pieroni¹, Sergio de la Fuente van Bentem², Gianmaria Mancosu¹, Enrico Capobianco¹, Heribert Hirt², ³ and Alberto de la Fuente¹

¹ CRS4 Bioinformatica, c/o Parco Tecnologico POLARIS, Pula, Italy

² Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, Vienna, Austria

³ Plant Genomics Research Unit , Unité de Recherche en Génomique Végétale (URGV), INRA/CNRS, Evry, France

The formulation of network models from global protein studies is essential to understand the functioning of organisms. Network models of the proteome enable the application of Complex Network Analysis, a quantitative framework to investigate large complex networks using techniques from graph theory, statistical physics, dynamical systems and other fields. This approach has provided many insights into the functional organization of the proteome so far and will likely continue to do so. Currently, several network concepts have emerged in the field of proteomics. It is important to highlight the differences between these concepts, since different representations allow different insights into functional organization. One such concept is the protein interaction network, which contains proteins as nodes and undirected edges representing the occurrence of binding in large-scale protein-protein interaction studies. A second concept is the protein-signaling network, in which the nodes correspond to levels of post-translationally modified forms of proteins and directed edges to causal effects through post-translational modification, such as phosphorylation. Several other network concepts were introduced for proteomics. Although all formulated as networks, the concepts represent widely different physical systems. Therefore caution should be taken when applying relevant topological analysis. We review recent literature formulating and analyzing such networks.

Trojan Horse Strategy in Agrobacterium Transformation: Abusing MAPK Defense Signaling

Science 19 October 2007: Vol. 318. no. 5849, pp. 453 - 456 DOI: 10.1126/science.1148110 PMID: 17947581

Armin Djamei,¹* Andrea Pitzschke,¹ Hirofumi Nakagami,¹ Iva Rajh,¹ Heribert Hirt¹²

¹ Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, Dr.-Bohr-Gasse 9, 1030 Vienna, Austria.

² URGV Plant Genomics Laboratory ,  2 Rue Gaston Crémieux, 91057 Evry, France.

Nuclear import of transfer DNA (T-DNA) is a central event in Agrobacterium transformation of plant cells and is thought to occur by the hijacking of certain host cell proteins. The T-DNA–associated virulence protein VirE2 mediates this process by binding to the nuclear import machinery via the host cell factor VIP1, whose role in plants has been so far unknown. Here we show that VIP1 is a transcription factor that is a direct target of the Agrobacterium-induced mitogen-activated protein kinase (MAPK) MPK3. Upon phosphorylation by MPK3, VIP1 relocalizes from the cytoplasm to the nucleus and regulates the expression of the PR1 pathogenesis-related gene. MAPK-dependent phosphorylation of VIP1 is necessary for VIP1-mediated Agrobacterium T-DNA transfer, indicating that Agrobacterium abuses the MAPK-targeted VIP1 defense signaling pathway for nuclear delivery of the T-DNA complex as a Trojan horse.

Materials and Methods, Figs. S1 to S4, References

The Arabidopsis Mitogen-Activated Protein Kinase Kinase MKK3 Is Upstream of Group C Mitogen-Activated Protein Kinases and Participates in Pathogen Signaling

The Plant Cell, Vol. 19: 3266–3279, October 2007, www.plantcell.org © 2007 American Society of Plant Biologists

PMID: 17933903 [PubMed - as supplied by publisher]

Róbert Dóczi ¹, Günter Brader ², Aladár Pettkó-Szandtner ¹, Iva Rajh ¹, Armin Djamei ¹, Andrea Pitzschke ¹, Markus Teige ¹, and Heribert Hirt ^{1, 3 *}

¹Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, A-1030 Vienna, Austria

²Viikki Biocenter, Department of Biological and Environmental Sciences, Division of Genetics, Faculty of Biosciences, University of Helsinki, FIN-00014 Helsinki, Finland

³Unité de Recherche en Génomique Végétale, Plant Genomics Research Unit, F-91057 Evry, France

^*To whom correspondence should be addressed: heribert.hirt@univie.ac.at

Although the Arabidopsis thaliana genome contains genes encoding 20 mitogen-activated protein kinases (MAPKs) and 10 MAPK kinases (MAPKKs), most of them are still functionally uncharacterized. In this work, we analyzed the function of the group B MAPK kinase, MKK3. Transgenic ProMKK3:GUS lines showed basal expression in vascular tissues that was strongly induced by Pseudomonas syringae pv tomato strain DC3000 (Pst DC3000) infection but not by abiotic stresses. The growth of virulent Pst DC3000 was increased in mkk3 knockout plants and decreased in MKK3-overexpressing plants. Moreover, MKK3 overexpression lines showed increased expression of several PR genes. By yeast two-hybrid analysis, coimmunoprecipitation, and protein kinase assays, MKK3 was revealed to be an upstream activator of the group C MAPKs MPK1, MPK2, MPK7, and MPK14. Flagellin-derived flg22 peptide strongly activated MPK6 but resulted in poor activation of MPK7. By contrast, MPK6 and MPK7 were both activated by H2O2, but only MPK7 activation was enhanced by MKK3. In agreement with the notion that MKK3 regulates the expression of PR genes, ProPR1:GUS expression was strongly enhanced by coexpression of MKK3-MPK7. Our results reveal that the MKK3 pathway plays a role in pathogen defense and further underscore the importance and complexity of MAPK signaling in plant stress responses.

The PP2C-Type Phosphatase AP2C1 Negatively Regulates MPK4 and MPK6 and Modulates Innate Immunity and Jasmonic Acid and Ethylene Levels in Arabidopsis

The Plant Cell , published July 13, 2007

Alois Schweighofer^a, Vaiva Kazanaviciute^a, Elisabeth Scheikl^a, Markus Teige^a, Róbert Dóczi^a, Heribert Hirt^a, Manfred Schwanninger^b, Merijn Kant^c, Robert Schuurink^c, Felix Mauch^d, Antony Buchala^d, Francesca Cardinale^e, and Irute Meskiene^a,

^aMax F. Perutz Laboratories of the University of Vienna, 1030 Vienna, Austria

^bDepartment of Chemistry, University of Natural Resources and Applied Life Sciences, 1190 Vienna, Austria

^cDepartment of Plant Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, 1098 SM Amsterdam, The Netherlands

^dDépartement de Biologie, Université de Fribourg, CH-1700 Fribourg, Switzerland

^eDipartimento di Valorizzazione e Protezione delle Risorse Agroforestali, Plant Pathology, University of Turin, I-10095 Grugliasco, Italy

Summery
Wound signaling pathways in plants are mediated by mitogen-activated protein kinases (MAPKs) and stress hormones, such as ethylene and jasmonates. In Arabidopsis thaliana, the transmission of wound signals by MAPKs has been the subject of detailed investigations; however, the involvement of specific phosphatases in wound signaling is not known. Here, we show that AP2C1, an Arabidopsis Ser/Thr phosphatase of type 2C, is a novel stress signal regulator that inactivates the stress-responsive MAPKs MPK4 and MPK6. Mutant ap2c1 plants produce significantly higher amounts of jasmonate upon wounding and are more resistant to phytophagous mites (Tetranychus urticae). Plants with increased AP2C1 levels display lower wound activation of MAPKs, reduced ethylene production, and compromised innate immunity against the necrotrophic pathogen Botrytis cinerea. Our results demonstrate a key role for the AP2C1 phosphatase in regulating stress hormone levels, defense responses, and MAPK activities in Arabidopsis and provide evidence that the activity of AP2C1 might control the plant’s response to B. cinerea.

The BRI1-Associated Kinase 1, BAK1, Has a Brassinolide-Independent Role in Plant Cell-Death Control

Current Biology 17, 1116–1122, July 3, 2007 ©2007 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2007.05.046

Birgit Kemmerling¹, Anne Schwedt¹, Patricia Rodriguez¹, Sara Mazzotta¹, Markus Frank², Synan Abu Qamar³, Tesfaye Mengiste³, Shigeyuki Betsuyaku⁴, Jane E. Parker⁴, Carsten Müssig⁵, Bart P.H.J. Thomma⁶, Catherine Albrecht⁷, Sacco C. de Vries⁷, Heribert Hirt⁸, and Thorsten Nürnberger^1*

¹Department of Plant Biochemistry, Center for Plant Molecular Biology, Eberhard-Karls-University Tübingen, 72076 Tübingen, Germany

²BASF Plant Science GmbH, BPS-LI444, 67117 Limburgerhof, Germany

³Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907-2054, USA

⁴Department of Plant-Microbe Interactions, Max-Planck-Institute for Plant Breeding Research, 50829 Köln, Germany

⁵Department of Genetics, University of Potsdam, 14476 Potsdam-Golm, Germany

⁶Laboratory of Phytopathology, Wageningen University, Wageningen 6709 PD, The Netherlands

⁷Laboratory of Biochemistry, Wageningen University, Wageningen 6703 HA, The Netherlands

⁸Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, 1030 Vienna, Austria

Summary: Programmed cell death (PCD) is a common host response to microbial infection [1–3]. In plants, PCD is associated with immunity to biotrophic pathogens, but it can also promote disease upon infection by necrotrophic pathogens [4]. Therefore, plant cell-suicide programs must be strictly controlled. Here we demonstrate that the Arabidopsis thaliana Brassinosteroid Insensitive 1 (BRI1)-associated receptor Kinase 1 (BAK1), which operates as a coreceptor of BRI1 in brassinolide (BL)-dependent plant development, also regulates the containment of microbial infectioninduced cell death. BAK1-deficient plants develop spreading necrosis upon infection. This is accompanied by production of reactive oxygen intermediates and results in enhanced susceptibility to necrotrophic fungal pathogens. The exogenous application of BL rescues growth defects of bak1 mutants but fails to restore immunity to fungal infection. Moreover, BL -insensitive and -deficient mutants do not exhibit spreading necrosis or enhanced susceptibility to fungal infections. Together, these findings suggest that plant steroid-hormone signaling is dispensable for the containment of infection-induced PCD. We propose a novel, BL-independent function of BAK1 in plant celldeath control that is distinct from its BL -dependent role in plant development.

The MAP Kinase Kinase MKK2 Affects Disease Resistance in Arabidopsis

MPMI Vol. 20, No. 5, 2007, pp. 589–596. doi:10.1094/MPMI -20-5-0589. © 2007 The American Phytopathological Society

Günter Brader¹, Armin Djamei², Markus Teige², E. Tapio Palva¹, and Heribert Hirt^2,3

¹Viikki Biocenter, Faculty of Biosciences, Department of Biological and Environmental Sciences, Division of Genetics, P.O. Box 56, FIN-00014 University of Helsinki, Finland
²Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, Dr. Bohrgasse 9, A-1030 Vienna, Austria
³Unité de Recherche en Génomique Végétale, 2 rue Gaston Crémieux, 91057 Evry cedex, France

Summary: The Arabidopsis mitogen-activated protein kinase (MAPK) kinase 2 (MKK2) was shown to mediate cold and salt stress responses through activation of the two MAP kinases MPK4 and MPK6. Transcriptome analysis of plants expressing constitutively active MKK2 (MKK2-EE plants) showed altered expression of genes induced by abiotic stresses but also a significant number of genes involved in defense responses. Both MPK4 and MPK6 became rapidly activated upon Pseudomonas syringae pv. tomato DC3000 infection and MKK2-EE plants showed enhanced levels of MPK4 activation. Although MKK2-EE plants shared enhanced expression of genes encoding enzymes of ethylene (ET) and jasmonic acid (JA) synthesis, ET, JA, and salicylic acid (SA) levels did not differ dramatically from those of wild-type or mkk2-null plants under ambient growth conditions. Upon P. syringae pv. tomato DC3000 infection, however, MKK2-EE plants showed reduced increases of JA and SA levels. These results indicate that MKK2 is involved in regulating hormone levels in response to pathogens. MKK2-EE plants were more resistant to infection by P. syringae pv. tomato DC3000 and Erwinia carotovora subsp. carotovora, but showed enhanced sensitivity to the fungal necrotroph Alternaria brassicicola. Our data indicate that MKK2 plays a role in abiotic stress tolerance and plant disease resistance.

A plastid-localized glycogen synthase kinase 3 modulatesstress tolerance and carbohydrate metabolism

The Plant Journal (2007) 49, 1076–1090 doi: 10.1111/j.1365-313X.2006.03025.x

Stefan Kempa¹, Wilfried Rozhon¹, Jozef Šamaj^2,3, Alexander Erban⁴, Frantiŝek Baluŝka³, Thomas Becker⁵, Joachim Haselmayer⁶, Enrico Schleiff⁵, Joachim Kopka⁴, Heribert Hirt⁶ and Claudia Jonak¹

¹Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Vienna Biocenter, Dr Bohrgasse 3, A-1030 Vienna, Austria

²Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademická 2, PO Box 39A, SK-950 07 Nitra, Slovak Republic

³Institute of Cellular and Molecular Botany, University of Bonn, Kirschallee 1, D-53115 Bonn, Germany

⁴Max Plank Institute of Molecular Plant Biology, Am Mühlenberg 1, D-14467 Golm, Germany

⁵Department of Biology I, Ludwig-Maximilians-University Munich, Menzinger Straße 67, D-80638 Munich, Germany

⁶Max F. Perutz Laboratories, University of Vienna, Vienna Biocenter, Dr Bohrgasse 9, A-1030 Vienna, Austria

Summary: Glycogen synthase kinase 3 (GSK-3) was originally identified as a regulator of glycogen synthesis in mammals. Like starch in plants, glycogen is a polymer of glucose, and serves as an energy and carbon store. Starch is the main carbohydrate store in plants. Regulation of starch metabolism, in particular in response to environmental cues, is of primary importance for carbon and energy flow in plants but is still obscure. Here, we provide evidence that MsK4, a novel Medicago sativa GSK-3-like kinase, connects stress signalling with carbon metabolism. MsK4 was found to be a plastid-localized protein kinase that is associated with starch granules. High-salt stress rapidly induced the in vivo kinase activity of MsK4. Metabolic profiling of MsK4 over-expressor lines revealed changes in sugar metabolism, including increased amounts of maltose, the main degradation product of starch in leaves. Plants over-expressing MsK4 showed improved tolerance to salt stress. Moreover, under high-salinity conditions, MsK4-over-expressing plants accumulated significantly more starch and showed modified carbohydrate content compared with wild-type plants. Overall, these data indicate that MsK4 is an important regulator that adjusts carbohydrate metabolism to environmental stress.

Using phosphoproteomics to reveal signalling dynamics in plants

Trends Plant Sci. 2007 Sep;12(9):404-11. Epub 2007 Aug 31. PMID: 17765599 [PubMed - in process]

Sergio de la Fuente van Bentem¹ and Heribert Hirt^{1, 2,}

¹Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, Dr. Bohr-Gasse 9, A-1030 Vienna, Austria

²URGV Plant Genomics , 2 rue Gaston Cremieux, F-91057 Evry, France

To ensure appropriate responses to stimuli, organisms have evolved signalling networks that rely on post-translational modifications of their components. Among these, protein phosphorylation has a prominent role and much research in plants has focused on protein kinases and phosphatases, which, respectively, catalyse phosphorylation and dephosphorylation of specific substrates. Technical limitations, however, have hampered the identification of these substrates. As reviewed here, novel mass spectrometry-based techniques have enabled the large-scale mapping of *in vivo* phosphorylation sites. Alternatively, methods based on peptide and protein microarrays have revealed protein kinase activities in cell extracts, in addition to kinase substrates. A combined phosphoproteomic approach of mass spectrometry and microarray technology could enhance the construction of dynamic plant signalling networks that underlie plant biology.

Activation of members of a MAPK module in β-glucan elicitor-mediated non-host resistance of soybean

Planta DOI 10.1007/s00425-006-0442-6 Received: 31 May 2006 / Accepted: 25 October 2006 © Springer-Verlag 2006

Andrea Daxberger¹, Andrea Nemak¹, Axel Mithöfer², Judith Fliegmann¹, Wilco Ligterink⁴, Heribert Hirt³, Jürgen Ebel¹

¹Department Biologie I/Botanik, Ludwig-Maximilians-Universität, Menzinger Str. 67, 80638 München, Germany

²Max-Planck-Institut für Chemische Ökologie, Bioorganische Chemie, Hans-Knöll-Str. 8, 07745 Jena, Germany

³Department für Pflanzenmolekularbiologie, Max F. Perutz Laboratories, University of Vienna, Dr. Bohrgasse 9, 1030 Vienna, Austria

⁴Laboratorium voor Plantenfysiologie, Botanisch Centrum, Arboretumlaan 4, 6703 Wageningen, The Netherlands

Abstract: Abstract Plants recognize microbial pathogens by discriminating pathogen-associated molecular patterns from self-structures. We study the non-host disease resistance of soybean (Glycine max L.) to the oomycete, Phytophthora sojae. Soybean senses a specific molecular pattern consisting of a branched heptaglucoside that is present in the oomycetal cell walls. Recognition of this elicitor may be achieved through a β-glucan-binding protein, which forms part of a proposed receptor complex. Subsequently, soybean mounts a complex defense response, which includes the increase of the cytosolic calcium concentration, the production of reactive oxygen species, and the activation of genes responsible for the synthesis of phytoalexins. We now report the identiWcation of two mitogen-activated protein kinases (MAPKs) and one MAPK kinase (MAPKK) that may function as signaling elements in triggering the resistance response. The use of speciWc antisera enabled the identiWcation of GmMPKs 3 and 6 whose activity is enhanced within the signaling pathway leading to defense reactions. Elicitor specificity of MAPK activation as well as the sensitivity against inhibitors suggested these kinases as part of the β-glucan signal transduction pathway. An upstream GmMKK1 was identiWed based on sequence similarity to other plant MAPKKs and its interaction with the MAPKs was analyzed. Recombinant GmMKK1 interacted predominantly with GmMPK6, with concomitant phosphorylation of the MAPK protein. Moreover, a preferential physical interaction between GmMKK1 and GmMPK6 was demonstrated in yeast. These results suggest a role of a MAPK cascade in mediating β-glucan signal transduction in soybean, similar to other triggers that activate MAPKs during innate immune responses in plants.

A mitogen-activated protein kinase kinase kinase mediates reactive oxygen species homeostasis in Arabidopsis *

JBC Papers in Press. Published on October 16, 2006 as Manuscript M605293200
The lates version

Hirofumi Nakagami^1,3 Hanka Soukupová², Adam Schikora1, Viktor Žárskŷ², Heribert Hirt^1,4

¹Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, Dr. Bohrgasse 9, A-1030 Vienna, Austria
² Laboratory of Cell Biology, Institute of Experimental Botany, ASCR, Rozvojová 135, Prague 6, 165 02, Czech Republic
³Plant Immunity Research Team, RIKEN Plant Science Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan
⁴Corresponding author
Running Title: MAPKKK mediates reactive oxygen species homeostasis

Mitogen-activated protein kinase kinase kinases (MAPKKKs) play key roles in intra- and extra-cellular signaling in eukaryotes. Here we report that the MAPKKK MEKK1 regulates redox homeostasis in Arabidopsis. We show that MEKK1-deficient plants are misregulated in the expression of a number of genes involved in cellular redox control and accumulate reactive oxygen species (ROS). Most strikingly, homozygous mekk1 mutant plants exhibit a lethal phenotype when developing true leaves. MEKK1 kinase activity and protein stability was regulated by H2O2 in a proteasome-dependent manner and mekk1 plants were compromised in ROS-induced MAPK MPK4 activation. Whereas mpk3 and mpk6 knock out plants showed no defects in development or changes in redox control genes, mpk4 null mutant shared several phenotypic and transcript profile features with mekk1 plants. In agreement with the concept that ROS negatively regulates auxin responses in plants, mekk1 and mpk4 mutants show reduced expression of several auxin-inducible marker genes. Overall, our data defines MPK4 as downstream target of MEKK1 and show that MEKK1 functions in integrating ROS homeostasis with plant development and hormone signaling.

Involvement of mitogen-activated protein kinases in the symbiosis Bradyrhizobium–Lupinus

Journal of Experimental Botany, Page 1 of 8 doi:10.1093/jxb/erl038

Journal of Experimental Botany Advance Access published July 25, 2006

Mercedes Fernandez-Pascual¹, M. Mercedes Lucas¹, Maria Rosario de Felipe¹, Lisardo Boscá², Heribert Hirt³ and Maria Pilar Golvano¹

¹Instituto de Recursos Naturales, Centro de Ciencias Medioambientales, Consejo Superior de Investigaciones Cientificas, Serrano, 115 dpdo, E-28006 Madrid, Spain

²Instituto de Bioquimica, Consejo Superior de Investigaciones Cientificas-Universidad Complutense de Madrid, E-28040 Madrid, Spain

³Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, Dr. Bohrgasse. 9, A -1030 Vienna, Austria

Abstract: In plants, mitogen-activated protein kinases (MAPKs) are involved in signalling to hormones, cell cycle regulation, stresses, and plant defence responses. In this work, several MAPKs were detected by immunobloting in roots and nodules of Lupinus albus produced by inoculation with Bradyrhizobium sp. (Lupinus). In vitro kinase assays showed that inoculation of seedling roots with B. sp. (Lupinus) activates salt stressinducible and stress-activated MAPKs after 5 min of incubation. By contrast, inoculation with dead B. sp. (Lupinus) or the heterologous bacteria Sinorhizobium meliloti did not induce salt stress-inducible and stress-activated MAPK activities. In vivo experiments showed that inoculation with B. sp. (Lupinus) induced the activation of MAPKs in roots. The maximal activation was in the region of the root tip with emerging hairs, which corresponds to the infection zone. The p38 MAPK inhibitors SB 202190 and SB 203580 blocked these kinase activities. Experiments with SB 202190 and the MAPKK inhibitor UO 126 altered the pattern of nodulation in the main root, decreasing the number and weight of nodules produced in the upper sites while increasing the nodule number in the younger lower root zone. These data suggest that MAPK inhibition blocks early events in the susceptible root zone to rhizobial infection, delaying nodulation, and support a role for MAPKs in the infection and nodulation of L. albus by B. sp. (Lupinus).

Phosphoproteomics reveals extensive in vivo phosphorylation of Arabidopsis proteins involved in RNA metabolism

Nucleic Acids Research, 2006, Vol. 34, No. 11 3267–3278 doi:10.1093/nar/gkl429 Published online June 28, 2006

Sergio de la Fuente van Bentem^*, Dorothea Anrather¹, Elisabeth Roitinger², Armin Djamei^*, Thomas Hufnagl³, Andrea Barta³, Edina Csaszar¹, Ilse Dohnal¹, David Lecourieux^* and Heribert Hirt^*

^*Department of Plant Molecular Biology and
¹Department of Biochemistry, Max F. Perutz Laboratories, University of Vienna, Dr Bohr-Gasse 9, 1030 Vienna, Austria,
²Research Institute of Molecular Pathology and 3Department of Medical Biochemistry, Max F. Perutz Laboratories, Medical University of Vienna, Dr Bohr-Gasse 9, 1030 Vienna, Austria

Most regulatory pathways are governed by the reversible phosphorylation of proteins. Recent developments in mass spectrometry-based technology allow the large-scale analysis of protein phosphorylation. Here, we show the application of immobilized metal affinity chromatography to purify phosphopeptides from Arabidopsis extracts. Phosphopeptide sequences were identified by liquid chromatography-tandem mass spectrometry (LCMS/ MS/MS). A total of 79 unique phosphorylation sites were determined in 22 phosphoproteins with a putative role in RNA metabolism, including splicing of mRNAs. Among these phosphoproteins, 12 Ser/ Arg-rich (SR) splicing factors were identified. A conserved phosphorylation site was found in most of the phosphoproteins, including the SR proteins, suggesting that these proteins are targeted by the same or a highly related protein kinase. To test this hypothesis, Arabidopsis SR protein-specific kinase 4 (SRPK4) that was initially identified as an interactor of SR proteins was tested for its ability to phosphorylate the SR protein RSp31. In vitro kinase assays showed that all in vivo phosphorylation sites of RSp31 were targeted by SRPK4. These data suggest that the plant mRNA splicing machinery is a major target of phosphorylation and that a considerable number of proteins involved in RNA metabolism may be targeted by SRPKs.

The Membrane-Anchored BOTRYTIS INDUCED KINASE1 Has Distinct Roles in Arabidopsis Resistance to Necrotrophic and Biotrophic Pathogens.

The Plant Cell, Vol. 18, 257–273, January 2006, www.plantcell.org © 2005 American Society of Plant Biologists PMID: 16339855

Paola Veronese^a, Hirofumi Nakagami^b, Burton Bluhm^a, Synan AbuQamar^a, Xi Chen^c, John Salmeron^c, Robert A. Dietrich^c, Heribert Hirt^b, and Tesfaye Mengistea^a

^aDepartment of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana 47907-2054, USA

^bGregor Mendel Institut, A-1010 Vienna, Austria

^cSyngenta Biotechnology, Research Triangle Park, North Carolina 27709, USA

Plant resistance to disease is controlled by the combination of defense response pathways that are activated depending on the nature of the pathogen. We identified the Arabidopsis thaliana BOTRYTIS-INDUCED KINASE1 (BIK1) gene that is transcriptionally regulated by Botrytis cinerea infection. Inactivation of BIK1 causes severe susceptibility to necrotrophic fungal pathogens but enhances resistance to a virulent strain of the bacterial pathogen Pseudomonas syringae pv tomato. The response to an avirulent bacterial strain is unchanged, limiting the role of BIK1 to basal defense rather than race-specific resistance. The jasmonate- and ethylene-regulated defense response, generally associated with resistance to necrotrophic fungi, is attenuated in the bik1 mutant based on the expression of the plant defensin PDF1.2 gene. bik1 mutants show altered root growth, producing more and longer root hairs, demonstrating that BIK1 is also required for normal plant growth and development. Whereas the pathogen responses of bik1 are mostly dependent on salicylic acid (SA) levels, the nondefense responses are independent of SA. BIK1 is membrane-localized, suggesting possible involvement in early stages of the recognition or transduction of pathogen response. Our data suggest that BIK1 modulates the signaling of cellular factors required for defense responses to pathogen infection and normal root hair growth, linking defense response regulation with that of growth and development.

Phosphoproteomics as a tool to unravel plant regulatory mechanisms.

Physiologia Plantarum 126: 110–119. 2006 Copyright ©Physiologia Plantarum 2006, ISSN 0031-9317

Sergio de la Fuente van Bentem^a, Elisabeth Roitinger^b, Dorothea Anrather^c, Edina Csaszar^c and Heribert Hirt^a

^aDepartment of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, Dr Bohr-Gasse 9, 1030 Vienna, Austria

^bResearch Institute of Molecular Pathology, Dr Bohr-Gasse 7, 1030 Vienna, Austria

^cDepartment of Biochemistry, Max F. Perutz Laboratories, University of Vienna, Dr Bohr-Gasse 9, 1030 Vienna, Austria

Reversible phosphorylation of proteins plays a key role in many regulatory processes that lie at the basis of life. With plants, much research has focused on protein kinases that are involved in the adaptation to different stress conditions, such as pathogen attack and cold. However, the substrates of these kinases are mostly unknown. With the recent advances in phosphoproteomic techniques, the large-scale identification of kinase substrates, including their phosphorylation sites, is finally possible. Studies in mainly non-plant systems have demonstrated the high potential of this method by uncovering numerous novel phosphorylation events. In this minireview, we focus on recent developments in the field of phosphoproteomics that are based on phosphopeptide isolation from complex mixtures by immobilized metal-affinity chromatography coupled to sequence identification by mass spectrometry. Combination of these methods with labelling techniques now allows quantitative analysis of phosphorylation between different samples. We discuss the potential of this technology to uncover entire phosphoproteomes and signalling pathways in plants in the future.

Mitogen-Activated Protein Kinases and Reactive Oxygen Species Signaling in Plants1

Plant Physiology, June 2006, Vol. 141, pp. 351–356, www.plantphysiol.org ©2006 American Society of Plant Biologists

Pitzschke, A and Hirt, H. (2006)

Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, 1030 Vienna, Austria

In plants, reactive oxygen species (ROS) can be generated by various processes occurring in different cellular compartments. Under physiological steady-state conditions, ROS are scavenged by different antioxidative components, but the balance between production and scavenging of ROS may be perturbed by a number of adverse environmental factors, giving rise to rapid increases in intracellular ROS levels. Although high concentrations of ROS can cause irreversible damage and cell death, they can also influence signaling and gene expression, indicating that cells have evolved strategies to utilize ROS to control various biological programs (Apel and Hirt, 2004). Being small and able to diffuse over short distances, ROS are ideally suited to act as signaling molecules. Among different ROS, only hydrogen peroxide (H₂O₂) can cross plant membranes and can therefore directly function in cell-to-cell signaling. Plant cells possess still-unidentified specific ROS sensors that process and translate this information into respective biological output programs. In several systems, various pathways, particularly those involving mitogen-activated protein kinases (MAPKs), are modulated by ROS and will be the focus of this review. MAPK cascades minimally consist of a MAPKKKMAPKK- MAPK module that is linked in various ways to upstream receptors and downstream targets (Nakagami et al., 2005). Receptor-mediated activation of a MAPKKK can occur through physical interaction and/or phosphorylation by either the receptor itself, intermediate bridging factors, or interlinking kinases. Activation of MAPK modules generally occurs through sequential phosphorylation of its component kinases culminating in the generation of active MAPKs, which phosphorylate a variety of substrates including transcription factors, other protein kinases, and cytoskeletonassociated proteins. Specificity of MAPK cascades functioning within the same cell and employing identical components is achieved by docking domains found in diverse components of MAPK modules and by scaffold proteins.

Reactive Oxygen Species Signaling in Plants

ANTIOXIDANTS & REDOX SIGNALING Volume 8, Numbers 9 & 10, 2006 © Mary Ann Liebert, Inc.

Pitzschke, A., Forzani, C. and Hirt, H.

Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, Vienna, Austria.

The evolution of aerobic metabolism such as respiration and photosynthesis resulted in the generation of reactive oxygen species (ROS). A common property of all ROS types is that they can cause oxidative damage to proteins, DNA, and lipids. This toxicity of ROS explains the evolution of complex arrays of nonenzymatic and enzymatic detoxification mechanisms in plants. However, increasing evidence indicates that plants also make use of ROS as signaling molecules for regulating development and various physiological responses. In this review, novel insights into the mechanisms of how plants sense and respond to ROS are discussed in the context of the biological effects and functions of ROS in plants. Antioxid. Redox Signal. 8, 1757–1764.

The MAP kinase substrate MKS1 is a regulator of plant defense responses.

The EMBO Journal (2005) 24, 2579–2589 | © 2005 European Molecular Biology Organization | All Rights Reserved 0261-4189/05 PMID: 15990873

Erik Andreasson^1,6, Thomas Jenkins^1.6, Peter Brodersen¹, Stephan Thorgrimsen¹, Nikolaj HT Petersen¹, Shijiang Zhu¹, Jin-Long Qiu¹, Pernille Micheelsen¹, Anne Rocher¹, Morten Petersen¹, Mari-Anne Newman², Henrik Bjørn Nielsen³, Heribert Hirt⁴, Imre Somssich⁵, Ole Mattsson¹ and John Mundy¹

¹Molecular Biology Institute, University of Copenhagen, Copenhagen, Denmark

²Plant Biology Institute, Royal Veterinary and Agricultural University, Frederiksberg, Denmark

³BioCentrum-DTU, Technical University of Denmark, Lyngby, Denmark

⁴Biocenter, University of Vienna, Vienna, Austria

⁵Max-Planck-Institute, Köln, Germany

Arabidopsis MAP kinase 4 (MPK4) functions as a regulator of pathogen defense responses, because it is required for both repression of salicylic acid (SA)-dependent resistance and for activation of jasmonate (JA)-dependent defense gene expression. To understand MPK4 signaling mechanisms, we used yeast two-hybrid screening to identify the MPK4 substrate MKS1. Analyses of transgenic plants and genome-wide transcript profiling indicated that MKS1 is required for full SA-dependent resistance in mpk4 mutants, and that overexpression of MKS1 in wild-type plants is sufficient to activate SA-dependent resistance, but does not interfere with induction of a defense gene by JA. Further yeast two-hybrid screening revealed that MKS1 interacts with the WRKY transcription factors WRKY25 and WRKY33. WRKY25 and WRKY33 were shown to be in vitro substrates of MPK4, and a wrky33 knockout mutant was found to exhibit increased expression of the SA-related defense gene PR1. MKS1 may therefore contribute to MPK4-regulated defense activation by coupling the kinase to specific WRKY transcription factors.

Emerging MAP kinase pathways in plant stress signalling.

TRENDS in Plant Science Vol.10 No.7 July 2005 PMID: 15953753

Nakagami, H., Pitzschke, A. Hirt, H.

Department of Genetics, Max F. Perutz Laboratories of the University of Vienna, Vienna Biocenter, Dr. Bohrgasse 9, A-1030 Vienna, Austria.

Mitogen-activated protein kinase (MAPK) pathways transfer information from sensors to cellular responses in all eukaryotes. A surprisingly large number of genes encoding MAPK pathway components have been uncovered by analysing model plant genomes, suggesting that MAPK cascades are abundant players of signal transduction. Recent investigations have confirmed major roles of defined MAPK pathways in development, cell proliferation and hormone physiology, as well as in biotic and abiotic stress signalling. Latest insights and findings are discussed in the context of novel MAPK pathways in plant stress signalling.

Plant Responses to Abiotic Stress

Publisher: Springer-Verlag

ISBN: 3-540-20037-1

Heribert Hirt, Kazuo Shinozaki

Series: Topics in Current Genetics, Vol. 4

2004, XIV, 300 p. 31 illus., 1 in colour., Hardcover

Environmental stresses represent the most limiting factors for agricultural productivity. Apart from biotic stress caused by plant pathogens, there are a number of abiotic stresses such as extremes in temperature, drought, salinity, heavy metals and radiation which all have detrimental effects on plant growth and yield. However, certain plant species and ecotypes have developed various mechanisms to adapt to such stress conditions. Recent advances in the understanding of these abiotic stress responses provided the impetus for compiling up-to-date reviews discussing all relevant topics in abiotic stress signaling of plants in a single volume. Topical reviews were prepared by selected experts and contain an introduction, discussion of the state of the art and important future tasks of the particular fields.

Molecular response to heavy metal stress: activation of distinct mitogen-activated protein kinase pathways by copper and cadmium.

Plant Physiology, October 2004, Vol. 136, pp. 3276–3283, www.plantphysiol.org ©2004 American Society of Plant Biologists

Claudia Jonak*, Hirofumi Nakagami, and Heribert Hirt

Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences and Institute of Microbiology and Genetics, University of Vienna, Vienna Biocenter, A–1030 Vienna, Austria

Excessive amounts of heavy metals adversely affect plant growth and development. Whereas some regions naturally contain high levels of heavy metals, anthropogenic release of heavy metals into the environment continuously increases soil contamination. The presence of elevated levels of heavy metal ions triggers a wide range of cellular responses including changes in gene expression and synthesis of metal-detoxifying peptides. To elucidate signal transduction events leading to the cellular response to heavy metal stress we analyzed protein phosphorylation induced by elevated levels of copper and cadmium ions as examples for heavy metals with different physiochemical properties and functions. Exposure of alfalfa (Medicago sativa) seedlings to excess copper or cadmium ions activated four distinct mitogen-activated protein kinases (MAPKs): SIMK, MMK2, MMK3, and SAMK. Comparison of the kinetics of MAPK activation revealed that SIMK, MMK2, MMK3, and SAMK are very rapidly activated by copper ions, while cadmium ions induced delayed MAPK activation. In protoplasts, the MAPK kinase SIMKK specifically mediated activation of SIMK and SAMK but not of MMK2 and MMK3. Moreover, SIMKK only conveyed MAPK activation by CuCl2 but not by CdCl2. These results suggest that plants respond to heavy metal stress by induction of several distinct MAPK pathways and that excess amounts of copper and cadmium ions induce different cellular signaling mechanisms in roots.

The MKK2 pathway mediates cold and salt stress signaling in Arabdiopsis.

Molecular Cell, Vol. 15, 141–152, July 2, 2004, Copyright ©2004 by Cell Press PMID: 15225555

Markus Teige^1,4,*, Elisabeth Scheikl^1,4, Thomas Eulgem^2,5, Róbert Dóczi¹, Kazuya Ichimura³, Kazuo Shinozaki³, Jeffery L. Dangl², and Heribert Hirt^1,*

¹Max F. Perutz Laboratories, University of Vienna and Gregor Mendel Institute of Molecular Plant Sciences, Austrian Academy of Sciences, Vienna Biocenter, Dr. Bohrgasse 9, A-1030 Vienna, Austria

²Department of Biology Curriculum in Genetics Department of Microbiology and Immunology University of North Carolina Chapel Hill, North Carolina 27599, USA

³Laboratory of Plant Molecular Biology RIKEN Tsukuba Institute 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074 Japan

⁴These authors contributed equally to this work.

⁵Center for Plant Cell Biology, Department of Botany and Plant Sciences, 3214 Batchelor Hall, University of California, Riverside, California 92521, USA

The Arabidopsis mitogen-activated protein kinase (MAPK) kinase 2 (MKK2) and the downstream MAPKs MPK4 and MPK6 were isolated by functional complementation of osmosensitive yeast mutants. In Arabidopsis protoplasts, MKK2 was specifically activated by cold and salt stress and by the stress-induced MAPK kinase kinase MEKK1. Yeast two-hybrid, in vitro, and in vivo protein kinase assays revealed that MKK2 directly targets MPK4 and MPK6. Accordingly, plants overexpressing MKK2 exhibited constitutive MPK4 and MPK6 activity, constitutively upregulated expression of stress-induced marker genes, and increased freezing and salt tolerance. In contrast, mkk2 null plants were impaired in MPK4 and MPK6 activation and were hypersensitive to salt and cold stress. Full genome transcriptome analysis of MKK2-overexpressing plants demonstrated altered expression of 152 genes involved in transcriptional regulation, signal transduction, cellular defense, and stress metabolism. These data identify a MAP kinase signaling cascade mediating cold and salt stress tolerance in plants.

OMTK1, a novel MAPKKK, channels oxidative stress signaling through direct MAPK binding.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 26, Issue of June 25, pp. 26959–26966, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. PMID: 15033984

Nakagami, H., Kiegerl, S. and Hirt H.

Institute of Microbiology and Genetics, Vienna Biocenter, University of Vienna, Dr. Bohrgasse 9, A-1030 Vienna, Austria.

In common with other eukaryotes, plants utilize mitogen-activated protein kinase (MAPK) cascades to mediate responses to a wide variety of stimuli. In contrast to other eukaryotes, plants have an unusually large number of MAPK components, such as more than 20 MAPKs, 10 MAPK kinases (MAPKKs), and 60 MAPKK kinases (MAPKKKs) in Arabidopsis (MAPK Group (2002) Trends Plant Sci. 7, 301-308). Presently it is mostly unknown how MAPK signaling specificity is generated in plants. Here we have isolated OMTK1 (oxidative stress-activated MAP triple-kinase 1), a novel MAPKKK from alfalfa (Medicago sativa). In plant protoplasts, OMTK1 showed basal kinase activity and was found to induce cell death. Among a panel of hormones and stresses tested, only H(2)O(2) was found to activate OMTK1. Out of four MAPKs, OMTK1 specifically activated MMK3 resulting in an increased cell death rate. Pull-down analysis between recombinant proteins indicated that OMTK1 directly interacts with MMK3 and that OMTK1 and MMK3 are part of a protein complex in vivo. These results indicate that OMTK1 plays a MAPK scaffolding role and functions in activation of H(2)O(2) -induced cell death in plants.

OXI1 kinase is necessary for oxidative burst-mediated signalling in Arabidopsis.

Nature 427, 858-861.

Maike C. Rentel¹*, David Lecourieux², Fatma Ouaked², Sarah L. Usher³, Lindsay Petersen^1,4, Haruko Okamoto¹, Heather Knight¹, Scott C. Peck⁵, Claire S. Grierson³, Heribert Hirt², Marc R. Knight¹

¹Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, UK

²Max F. Perutz Laboratories of the University of Vienna and Gregor-Mendel-Institute of Molecular Plant Biology, Austrian Academy of Sciences, Vienna Biocenter, 1030 Vienna, Austria

³School of Biological Sciences, University of Bristol, Bristol BS8 1UG, UK

⁴Department ofMolecular and Cell Biology, University of Cape Town, Private Bag Rondebosch 7701, South Africa

⁵Sainsbury Laboratory, Norwich NR4 7UH, UK

*Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143-0450, USA

Active oxygen species (AOS) generated in response to stimuli and during development can function as signalling molecules in eukaryotes, leading to specific downstream responses1,2. In plants these include such diverse processes as coping with stress (for example pathogen attack3, wounding4 and oxygen deprivation5), abscisic-acid-induced guard-cell closure6, and cellular development (for example root hair growth7). Despite the importance of signalling via AOS in eukaryotes, little is known about the protein components operating downstream of AOS that mediate any of these processes. Here we show that expression of an Arabidopsis thaliana gene (OXI1) encoding a serine/threonine kinase is induced in response to a wide range of H2O2-generating stimuli.OXI1 kinase activity is itself also induced byH2O2 in vivo. OXI1 is required for full activation of the mitogen-activated protein kinases (MAPKs) MPK3 and MPK6 after treatment with AOS or elicitor and is necessary for at least two very different AOS-mediated processes: basal resistance to Peronospora parasitica infection, and root hair growth. Thus, OXI1 is an essential part of the signal transduction pathway linking oxidative burst signals to diverse downstream responses.

Reactive Oxygen Species: Metabolism, oxidativestress and signal transduction.

Annu. Rev. Plant Biol. 2004. 55:373–99 doi: 10.1146/annurev.arplant.55.031903.141701 Copyright ©2004 by Annual Reviews. All rights reserved First published online as a Review in Advance on January 12, 2004

Klaus Apel¹ and Heribert Hirt²

¹Institute of Plant Sciences, Swiss Federal Institute of Technology (ETH) Universitätstr. 2, 8092 Zürich, Switzerland

²2Max F. Perutz Laboratories, University of Vienna, Gregor-Mendel-Institute of Molecular Plant Sciences, Austrian Academy of Sciences, Vienna Biocenter, Dr. Bohrgasse 9, 1030 Vienna, Austria

Several reactive oxygen species (ROS) are continuously produced in plants as byproducts of aerobic metabolism. Depending on the nature of the ROS species, some are highly toxic and rapidly detoxified by various cellular enzymatic and nonenzymatic mechanisms. Whereas plants are surfeited with mechanisms to combat increased ROS levels during abiotic stress conditions, in other circumstances plants appear to purposefully generate ROS as signaling molecules to control various processes including pathogen defense, programmed cell death, and stomatal behavior. This review describes the mechanisms of ROS generation and removal in plants during development and under biotic and abiotic stress conditions. New insights into the complexity and roles that ROS play in plants have come from genetic analyses of ROS detoxifying and signaling mutants. Considering recent ROS-induced genomewide expression analyses, the possible functions and mechanisms for ROS sensing and signaling in plants are compared with those in animals and yeast.

Plant PP2C Phosphatases: Emerging Functions.

Trends in Plant Science Volume 9, Issue 5, May 2004, Pages 236-243 doi:10.1016/j.tplants.2004.03.007 Copyright © 2004 Elsevier Ltd. All rights reserved. PMID: 15130549  PubMed /Registered Users Only

Alois Schweighofer, Heribert Hirt and Irute Meskiene

Institute of Microbiology and Genetics, Vienna Biocenter, University of Vienna, Dr. Bohrgasse 9, Vienna A-1030, Austria

PP2C-type protein phosphatases are monomeric enzymes present in both prokaryotes and eukaryotes. Members of this family of phosphoprotein phosphatases are involved in the regulation of several signaling pathways. A database analysis of Arabidopsis reveals PP2Cs to be the largest protein phosphatase family in plants, with 76 members, displaying high complexity, and greatly outnumbering PP2Cs in other eukaryotes. Plant PP2Cs have been found as regulators of signal transduction pathways and also involved in development. PP2C functions emphasize the existence of sophisticated signaling pathways in plants, in which protein dephosphorylation plays a crucial role towards determining specificities.

From signal to cell polarity: mitogen-activated protein kinases as sensors and effectors of cytoskeleton dynamics.

Journal of Experimental Botany, Vol. 55, No. 395, Crosstalk in Plant Signal Transduction Special Issue, pp. 189±198, January 2004 PMID: 14673033

Jozef Ŝamaj^1,3*, Frantiśek Baluśka1⁴, and Heribert Hirt²

¹Institute of Cellular and Molecular Botany, University of Bonn, Kirschallee 1, D-53115 Bonn, Germany

²Max F. Perutz Laboratories and Gregor-Mendel-Institute of Molecular Plant Sciences, Vienna Biocenter, Dr. Bohrgasse 9, A-1030 Vienna, Austria

³Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademicka 2, SK-949 01 Nitra, Slovak Republic

⁴Institute of Botany, Slovak Academy of Sciences, Dubravska cesta 14, SK-84223 Bratislava, Slovak Republic

Mitogen-activated protein kinases (MAPKs) are ubiquitous phosphorylation enzymes involved in signal transduction, gene expression and activation of diverse cytoskeletal proteins. MAPKs participate in the regulation of a broad range of crucial cellular processes including cell survival, division, polarization, stress responses, and metabolism. Phosphorylation of cytoskeletal proteins usually results in the rearrangement of cytoskeletal arrays leading to morphological changes and cell polarization. On the other hand, some cytoskeletal motor proteins, such as kinesins, could activate MAPK members and participate in signal delivery to the proper cellular destination (e.g. during cell division). Moreover, changes in the integrity of cytoskeletal elements have direct impacts on MAPK activity. Recent evidence suggests that there is bi-directional signalling between MAPK cascades and cytoskeleton. The focus here is on this cross-talk between MAPK signalling and the cytoskeleton in various eukaryotic systems including yeast, plants, and mammals and a role is proposed for MAPKs as sensors monitoring the cytoskeleton-dependent balance of forces within the cell.

Stress-induced protein phosphatase 2C is a negative regulator of a mitogen-activated protein kinase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 21, Issue of May 23, pp. 18945–18952, 2003 © 2003 by The American Society for Biochemistry and Molecular Biology, Inc. PMID: 12646559

Irute Meskiene¹, Emmanuel Baudouin¹, Alois Schweighofer¹, Aneta Liwosz¹, Claudia Jonak¹, Pedro L. Rodriguez², Heinrich Jelinek¹, and Heribert Hirt¹

¹Institute of Microbiology and Genetics, Vienna Biocenter, Dr. Bohrgasse 9, A-1030 Vienna, Austria.

²Instituto de Biologia Molecular y Cellular de Plantas, Universidad Politecnica, SCIC, Camino de Vera, 46022 Valencia, Spain

Protein phosphatases of type 2C (PP2Cs) play important roles in eukaryotic signal transduction. In contrast to other eukaryotes, plants such as Arabidopsis have an unusually large group of 69 different PP2C genes. At present, little is known about the functions and substrates of plant PP2Cs. We have previously shown that MP2C, a wound-induced alfalfa PP2C, is a negative regulator of mitogen-activated protein kinase (MAPK) pathways in yeast and plants. In this report, we provide evidence that alfalfa salt stress-inducible MAPK (SIMK) and stress-activated MAPK (SAMK) are activated by wounding and that MP2C is a MAPK phosphatase that directly inactivates SIMK but not the wound-activated MAPK, SAMK. SIMK is inactivated through threonine dephosphorylation of the pTEpY motif, which is essential for MAPK activity. Mutant analysis indicated that inactivation of SIMK depends on the catalytic activity of MP2C. A comparison of MP2C with two other PP2Cs, ABI2 and AtP2CHA, revealed that although all three phosphatases have similar activities toward casein as a substrate, only MP2C is able to dephosphorylate and inactivate SIMK. In agreement with the notion that MP2C interacts directly with SIMK, the MAPK was identified as an interacting partner of MP2C in a yeast two-hybrid screen. MP2C can be immunoprecipitated with SIMK in a complex in vivo and shows direct binding to SIMK in vitro in protein interaction assays. Wound-induced MP2C expression correlates with the time window when SIMK is inactivated, corroborating the notion that MP2C is involved in resetting the SIMK signaling pathway.

A MAPK pathway mediates ethylene signaling in plants.

The EMBO Journal Vol. 22 No. 6 pp. 1282±1288, 2003 PMID: 12628921

Fatma Ouaked, Wilfried Rozhon, David Lecourieux and Heribert Hirt

Gregor-Mendel-Institute of Molecular Plant Sciences and Institute of Microbiology and Genetics, Vienna Biocenter, University of Vienna, Dr Bohrgasse 9, A-1030 Vienna, Austria.

Ethylene signal transduction involves ETR1, a two-component histidine protein kinase receptor. ETR1 functions upstream of the negative regulator CTR1. The similarity of CTR1 to members of the Raf family of mitogen-activated protein kinase kinase kinases (MAPKKKs) suggested that ethylene signaling in plants involves a MAPK pathway, but no direct evidence for this has been provided. Here we show that distinct MAPKs are activated by the ethylene precursor aminocyclopropane-1-carboxylic acid (ACC) in Medicago and ARABIDOPSIS: In Medicago, the ACC-activated MAPKs were SIMK and MMK3, while in Arabidopsis MPK6 and another MAPK were identified. Medicago SIMKK specifically mediated ACC-induced activation of SIMK and MMK3. Transgenic Arabidopsis plants overexpressing SIMKK have constitutive MPK6 activation and ethylene-induced target gene expression. SIMKK overexpressor lines resemble ctr1 mutants in showing a triple response phenotype in the absence of ACC. Whereas MPK6 was not activated by ACC in etr1 mutants, ein2 and ein3 mutants showed normal activation profiles. In contrast, ctr1 mutants showed constitutive activation of MPK6. These data indicate that a MAPK cascade is part of the ethylene signal transduction pathway in plants.

Protein phosphorylation and cellular information transfer: signalling by MAP kinase cascades.

Monatshefte für Chemie / Chemical Monthly 134, 1481-1487
Publisher: Springer Wien
ISSN: 0026-9247 (Paper) 1434-4475 DOI: 10.1007/s00706-003-0048-7 Issue:  Volume 134, Number 11 Date:  November 2003 Pages: 1481 - 1487

Claudia Jonak² and Heribert Hirt¹

¹Institute of Microbiology and Genetics, University of Vienna, A-1030 Vienna, Austria, AT

²Gregor Mendel Institute of Molecular Plant Sciences, Austrian Academy of Sciences, A-1030 Vienna, Austria, AT

Summary: Living cells, unicellular organisms as well as cells of multicellular organisms, are permanently exposed to a multitude of signals. Cells have to transform these external stimuli into physiological intelligible signals that are transduced from outside of the cell into the cell to induce a proper cellular response. Extracellular stimuli are perceived and internalised by various cellular receptors. Subsequently, signals are transduced by one of many protein kinase signaling cascades. Mitogen-activated protein kinases (MAPKs) belong to the evolutionary most conserved class of such molecular switches. MAPKs can change the activity of target proteins and thereby bring about physiological responses to external signals. This review discusses the basic principles of MAPK pathways in the context of cellular information processing: Cellular bioinformatics is an increasingly important interdisciplinary field with important implications for basic and applied sciences.

Involvement of MAP kinase SIMK and actin cytoskeleton in the regulation of root hair tip growth.

Cell Biology International Volume 27, Issue 3, 2003, Pages 257-259 PMID: 12681328
The Plant Cytoskeleton: Functional Diversity and Biotechnological Implications. Abstracts from a NATO Advanced Research Workshop, Kiev, Ukraine, 23-27 September 2002

Jozef Samaja^e, Miroslav Ovecka^a,b,c, Andrej Hlavacka^b, Fatma Lecourieux^a, Irute Meskiene^a, Irene Lichtscheidl^d, Peter Lenart^a, Ján Salaj^e, Dieter Volkmann^b, Laszlo Bögre^f, Frantisek Baluska^b,c and Heribert Hirt^a

^aInstitute of Microbiology and Genetics, Vienna Biocenter, Dr. Bohrgasse 9, 1030, Vienna, Austria

^bInstitute of Botany, University of Bonn, Kirschallee 1, 53115, Bonn, Germany

^cInstitute of Botany, Slovak Academy of Sciences, Dúbravská cesta 14, SK – 842 23, Bratislava, Slovak Republic

^dInstitute of Ecology, University of Vienna, Althanstrasse 14, 1091, Vienna, Austria

^eInstitute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademická 2, P. O. Box 39 A, SK – 950 07, Nitra, Slovak Republic

^fSchool of Biological Sciences, Royal Holloway, University of London, Egham, TW20 0EX, London, UK

PubMed / Registered Users Only

Complexity, Cross Talk and Integration of Plant MAP Kinase Signalling.

Claudia Jonak, László Ökrész, László Bögre, Heribert Hirt

Current Opinion in Plant Biology, Volume 5, Issue 5, October 2002, 415-424.

Opposite changes in membrane fluidity mimic cold and heat stress activation of distinct plant MAP kinase pathways.

The Plant Journal (2002) 31(5), 629±638 PMID: 12207652

Veena Sangwan¹, Björn Lárus Örvar¹, John Beyerly², Heribert Hirt² and Rajinder S. Dhindsa¹

¹Department of Biology, McGill University, 1205 Avenue Docteur Pen®eld, Montreal, Quebec, H3A 1B1, Canada

²Institute of Microbiology and Genetics, Vienna Biocenter, Dr Bohr-Gasse 9, A-1030, Vienna, Austria

Mitogen-activated protein kinases (MAPKs) appear to be ubiquitously involved in signal transduction during eukaryotic responses to extracellular stimuli. In plants, no heat shock-activated MAPK has so far been reported. Also, whereas cold activates specific plant MAPKs such as alfalfa SAMK, mechanisms of such activation are unknown. Here, we report a heat shock-activated MAPK (HAMK) immunologically related to ERK (Extracellular signal-Regulated Kinase) superfamily of protein kinases. Molecular mechanisms of heat-activation of HAMK and cold-activation of SAMK were investigated. We show that cold-activation of SAMK requires membrane rigidification, whereas heat-activation of HAMK occurs through membrane fluidization. The temperature stress- and membrane structure-dependent activation of both SAMK and HAMK is mimicked at 25 degrees C by destabilizers of microfilaments and microtubules, latrunculin B and oryzalin, respectively; but is blocked by jasplakinolide, a stabilizer of actin microfilaments. Activation of SAMK or HAMK by temperature, chemically modulated membrane fluidity, or by cytoskeleton destabilizers is inhibited by blocking the influx of extracellular calcium. Activation of SAMK or HAMK is also prevented by an antagonist of calcium-dependent protein kinases (CDPKs). In summary, our data indicate that cold and heat are sensed by structural changes in the plasma membrane that translates the signal via cytoskeleton, Ca2+ fluxes and CDPKs into the activation of distinct MAPK cascades.

Mitogen-activated protein kinase cascades in plants: a new nomenclature

Trends in Plant Science Volume 7, Issue 7, 1 July 2002, Pages 301-308

PubMed - Registered User Only

MAPK Group (Kazuya Ichimura et al.), Kazuya Ichimura^aa, Kazuo Shinozaki^a, Guillaume Tena^b, Jen Sheen^b, Yves Henry^cc, Anthony Champion^c, Martin Kreis^c, Shuqun Zhang^d, Heribert Hirt^e, Cathal Wilson^e, Erwin Heberle-Bors^e, Brian E. Ellis^f, Peter C. Morris^g, Roger W. Innes^h, Joseph R. Eckerⁱ, Dierk Scheel^j, Daniel F. Klessig^k, Yasunori Machida^l, John Mundy^m, Yuko Ohashiⁿ and John C. Walker^o

^aRIKEN Tsukuba Institute, Japan. ^bHarvard Medical School, MA, USA. ^cInstitut de Biotechnologie des Plantes, Orsay, France. ^dUniversity of Missouri –, Columbia, MO, USA. ^eVienna Biocenter, Austria. ^fUniversity of British Columbia, Vancouver, Canada. ^gHeriot–Watt University, Edinburgh, UK. ^hIndiana University, IN, USA. ⁱThe Salk Institute for Biological Studies, La Jolla, CA, USA. ^jInstitute of Plant Biochemistry, Halle, Germany. ^kBoyce Thompson Institute for Plant Research, Ithaca, NY, USA. ^lNagoya University, Japan. ^mCopenhagen University, Denmark. ⁿNational Institute of Agrobiological Sciences, Tsukuba, Japan. ^oUniversity of Missouri –, Columbia, MO, USA.

Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules in eukaryotes, including yeasts, animals and plants. These protein phosphorylation cascades link extracellular stimuli to a wide range of cellular responses. In plants, MAPK cascades are involved in responses to various biotic and abiotic stresses, hormones, cell division and developmental processes. Completion of the Arabidopsis genome-sequencing project has revealed the existence of 20 MAPKs, 10 MAPK kinases and 60 MAPK kinase kinases. Here, we propose a simplified nomenclature for Arabidopsis MAPKs and MAPK kinases that might also serve as a basis for standard annotation of these gene families in all plants.

Involvement of the mitogen-activated protein kinase SIMK in regulation of root hair tip-growth.

The EMBO Journal Vol 21 No 13 pp.3296-3306, 2002 PMID: 12093731

Jozef Ŝamaj^1,2, Miroslav Ovecka^1,3,4, Andrej Hlavacka³, Fatma Lecourieux¹, Irute Meskiene¹, Irene Lichtscheidl⁵, Peter Lenard¹, Ján Salaj², Dieter Volkmann³, Lásló Bögre⁶, Frantiśek Baluśka^3,4 and Heribert Hirt^1,7

¹Institute of Microbiology and Genetics, Vienna Biocenter, University of Vienna, Dr Bohrgasse 9, A-1030 Vienna, Austria.

²Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Nitra, Slovak Republic

³Institute of Botany, Plant Cell Biology Department, University of Bonn, Germany

⁴Institute of Botany, Slovak Academy of Sciences, Bratislava, Slovak Republic

⁵Institute of Ecology, University of Vienna, Austria

⁶School of Biological Sciences, University of London, UK

⁷Corresponding author

Mitogen-activated protein kinases (MAPKs) are involved in stress signaling to the actin cytoskeleton in yeast and animals. We have analyzed the function of the stress-activated alfalfa MAP kinase SIMK in root hairs. In epidermal cells, SIMK is predominantly nuclear. During root hair formation, SIMK was activated and redistributed from the nucleus into growing tips of root hairs possessing dense F-actin meshworks. Actin depolymerization by latrunculin B resulted in SIMK relocation to the nucleus. Conversely, upon actin stabilization with jasplakinolide, SIMK co-localized with thick actin cables in the cytoplasm. Importantly, latrunculin B and jasplakinolide were both found to activate SIMK in a root-derived cell culture. Loss of tip-focused SIMK and actin was induced by the MAPK kinase inhibitor UO 126 and resulted in aberrant root hairs. UO 126 inhibited targeted vesicle trafficking and polarized growth of root hairs. In contrast, overexpression of gain-of-function SIMK induced rapid tip growth of root hairs and could bypass growth inhibition by UO 126. These data indicate that SIMK plays a crucial role in root hair tip growth.

Convergence and divergence of stress-induced MAPK signaling pathways at the level of two distinct MAP kinase kinases.

The Plant Cell, Vol. 14, 703–711, March 2002, www.plantcell.org © 2002 American Society of Plant Biologists PMID: 11910015

Francesca Cardinale, Irute Meskiene, Fatma Ouaked, and Heribert Hirt

Institute of Microbiology and Genetics, Vienna Biocenter, University of Vienna, Dr. Bohrgasse 9, A-1030 Vienna, Austria.

Plants respond to biotic and abiotic stresses by inducing overlapping sets of mitogen-activated protein kinases (MAPKs) and response genes. To define the mechanisms of how different signals can activate a common signaling pathway, upstream activators of SIMK, a salt stress- and pathogen-induced alfalfa MAPK, were identified. Here, we compare the properties of SIMKK, a MAPK kinase (MAPKK) that mediates the activation of SIMK by salt stress, with those of PRKK, a distantly related novel MAPKK. Although both SIMKK and PRKK show strongest interaction with SIMK, SIMKK can activate SIMK without stimulation by upstream factors. In contrast, PRKK requires activation by an upstream activated MAPKK kinase. SIMKK mediates pathogen elicitor signaling and salt stress, but PRKK transmits only elicitor-induced MAPK activation. Of four tested MAPKs, PRKK activates three of them (SIMK, MMK3, and SAMK) upon elicitor treatment of cells. However, PRKK is unable to activate any MAPK upon salt stress. In contrast, SIMKK activates SIMK and MMK3 in response to elicitor, but it activates only SIMK upon salt stress. These data show that (1) MAPKKs function as convergence points for stress signals, (2) MAPKKs activate multiple MAPKs, and (3) signaling specificity is obtained not only through the inherent affinities of MAPKK-MAPK combinations but also through stress signal-dependent intracellular mechanisms.

A new blueprint for plant pathogenresistance.

Nature Biotechnology Vol 20, 450-451. May 2002 PMID: 11981555

Heribert Hirt

Institute of Microbiology and Genetics, Vienna Biocenter, University of Vienna, Dr. Bohrgasse 9, A-1030 Vienna, Austria.

A signaling cascade downstream of a leucine-rich repeat receptor kinase identified in Arabidopsis offers new options for engineering crop disease resistance.

Every year, a large fraction of worldwide crop productions falls prey to viral, bacterial, or fungal infection. Such infections not only cause severe losses in world foof production, but also can damage human health by contaminating crops with potent carcinogens and toxins. Traditionally, farmers have controlled crop disease through the application of fungicides and pesticides, but these chemicals pose environmental and health problems in themselves if used indiscriminately. Genetic modification allows disease-resistance traits to be introduced into crop plants, usually by overexpression of antimicrobial proteins (e.g., chitinase) or by induction of key plant defense pathways through signaling molecules (e.g., salicyclic acid, jasmonic acid, or ethylene). A paper published recently in Nature by Sheen an dcolleagues describes an additional signaling pathway, the flagellin mitogen-activated protein kinase (MAPK) cascade, that has considerable potential for the engineering of crops with broad-spectrum resistance against fungal and bacterial pathogens.

Complexity, cross talk and integration of plant MAP kinase signalling.

PubMed / Registered Users Only

Current Opinion in Plant Biology Volume 5, Issue 5, 1 October 2002, Pages 415-424 PMID: 12183180

Claudia Jonak^a, László Ökrész^b, László Bögre^c and Heribert Hirt^a

^aInstitute of Microbiology and Genetics, Vienna Biocenter, Dr. Bohrgasse 9, 1030, Vienna, Austria

^bInstitute of Plant Biology, Biological Research Center of Hungarian Academy of Sciences,P. O. B. 521, H-6701, Szeged, Hungary

^cSchool of Biological Sciences, Royal Holloway University of London, Egham TW20 OEX, UK

Mitogen-activated protein kinases (MAPKs) link information transfer from external stimuli-activated sensors to cellular responses. The completed Arabidopsis genome sequence revealed an extraordinary complexity in MAPK-signalling components in plants. Information obtained from Arabidopsis provides a framework for a unified nomenclature and the assembly and function of MAPK-signalling pathways. Strategies and tools are evolving to connect MAPK pathways and to determine their function. As a result, MAPK signalling modules emerged, one of which appears to antagonistically regulate stress- and growth-responses and another that regulates cytokinesis.

Glycogen synthase kinase 3/shaggy-like kinases in plants: an emerging family with novel functions.

Registered Users Only

Trends in Plant Science, Volume 7, Issue 10, 1 October 2002, Pages 457-461 PMID: 12399181

Claudia Jonak and Heribert Hirt

Institute of Microbiology and Genetics, Vienna Biocenter, Dr. Bohrgasse 9, 1030, Vienna, Austria

Animal glycogen synthase kinase 3 (GSK-3)/SHAGGY kinases have been studied for more than 20 years, whereas plant glycogen synthase kinase 3/SHAGGY-like kinases (GSKs) have only recently entered the scene. Present evidence indicates that plant GSKs are involved in different processes, such as flower development, brassinosteroid signaling, NaCl stress and wound responses. In contrast to mammals, which contain two genes, plants have a multigene family of GSKs. Analysis of the Arabidopsis genome revealed the existence of ten GSK genes that fall into four distinct subfamilies. We discuss the functions and mechanisms of GSK action in plants and other organisms.

Plant glycogen synthase kinase 3/SHAGGY-like kinases (GSKs) are important regulators of development, stress and hormone signaling. The identification of the nuclear proteins BES1 and BZR1 as substrates for BIN2 points to an important role for plant GSKs in transcriptional regulation.

Phosphatidic acid activates a wound-activated SIMK-like MAP kinase in Glycine max

The Plant Journal (2001) 26(5), 479±486 PMID: 11439134

Sumin Lee¹, Heribert Hirt² and Youngsook Lee¹

¹Division of Molecular Life Sciences, Pohang University of Science and Technology, Pohang, 790-784, Korea.

²Insitute of Microbiology and Genetics, Vienna Biocenter, Dr. Bohrgasse 9, 1030 Vienna, Austria

Many plant species demonstrate a systemic increase in phosphatidic acid (PA) levels after being wounded (Lee et al., 1997). To understand the role of PA in wound signal transduction, we investigated if PA can activate protein kinases in soybean (Glycine max L.). We found that a MAPK is activated in soybean seedlings in both wounded and neighboring unwounded leaves. The wound-activated soybean kinase is specifically recognized by an antibody against the alfalfa MAPK, SIMK. When PA production is inhibited with n-butanol, an inhibitor of phospholipase D, the wound-induced activation of the MAPK is suppressed, suggesting that an elevation in PA levels is essential for its activation. Supporting this is the observation that exogenous PA activates the MAPK in suspension-cultured soybean cells. Activation of the 49 kDa MAPK occurs almost exclusively by PA, as other lipids are unable to or can only weakly activate the kinase. PA-induced activation of the MAPK is not a direct effect on the kinase but is mediated by upstream kinases. Our results suggest that PA acts as a second messenger in wound-induced MAPK signaling in plants.

Plant MAP kinase pathways: how many and what for?

Biol Cell. 2001 Sep;93(1-2):81-7. PMID: 11730326

Wrzaczek, M., and Hirt. H. (2001)

Institute of Microbiology and Genetics, Vienna Biocenter, Austria.

Mitogen activated protein kinases (MAPK) are important mediators in signal transmission, connecting the perception of external stimuli to cellular responses. MAPK cascades are involved in signalling various biotic and abiotic stresses, like wounding and pathogen infection, temperature stress or drought, but also some plant hormones, such as ethylene and auxin. Moreover, MAPKs have been implicated in cell cycle and developmental processes. In Arabidopsis mutant screens and in vivo assays several components of plant MAPK cascades have been identified. This review compares results obtained from functional analyses of MAPK cascades in plants with recent data obtained from searching the complete Arabidopsis genome. This analysis reveals that plants have an overall of 24 MAPK pathways of which only a small subset has been studied so far.

Recent advances in plant MAP kinase signalling.

Biol. Chem. 382, 1123-1131. PMID: 11592393

Zwerger, K., and Hirt, H. (2001)

Institute of Microbiology and Genetics, Vienna Biocenter, Austria.

Mitogen activated protein kinases (MAPK) are important mediators in signal transmission, connecting the perception of external stimuli to cellular responses. MAPK cascades are involved in signalling various biotic and abiotic stresses, like wounding and pathogen infection, temperature stress or drought, but are also involved in mediating the action of some plant hormones, such as ethylene and auxin. Moreover, MAPKs have been implicated in cell cycle and developmental processes. In Arabidopsis mutant screens and in vivo assays several components of plant MAPK cascades have been identified. This review gives an update of recent advances in plant MAPK signalling and discusses the emerging mechanisms of some selected MAPK pathways.

MAP kinase pathways in plants: versatile signaling tools.

International Review of Cytology, Volume 201, 2001, Pages 209-275

Ligterink, W., and Hirt, H. (2001)

Institute of Microbiology and Genetics, Vienna Biocenter, Austria.

Mitogen-activated protein kinases (MAPKs) are important signaling tools in all eukaryotes, and function in mediating an enormous variety of external signals to appropriate cellular responses. MAPK pathways have been studied extensively in yeast and mammalian cells, and a large body of knowledge on their functioning has accumulated, which is summarized briefly. Plant MAPK pathways have attracted increasing interest, resulting in the isolation of a large number of different components of MAPK cascades. Studies on the functions of these components have revealed that MAPKs play important roles in the response to a broad variety of stresses, as well as in the signaling of most plant hormones and in developmental processes. Finally, the involvement of various plant phosphatases in the inactivation of MAPKs is discussed.

MAP Kinases in Plant Signal Transduction

Series: Results and Problems in Cell Differentiation , Vol. 27 Hirt, Heribert (Ed.) 2000, X, 167 pp. 26 figs., 5 tabs., Hardcover ISBN: 978-3-540-65625-8

Springer Science + Business Media

Preview

Mitogen-activated protein kinase (MAPK) pathways are modules involved in the transduction of extracellular signals to intracellular targets in all eukaryotes. Distinct MAPK pathways are regulated by different extracellular stimuli and are implicated in a wide variety of biological processes. In plants, there is evidence for MAPKs playing a role in the signaling of abiotic stresses, pathogens, plant hormones, and cell cycle cues. The large number and divergence of plant MAPKs indicates that this ancient mechanism of bioinformatics is extensively used in plants and may provide new molecular hands on old questions.

Receptor-mediated signal transduction in plant defense.

In: Biology of Plant-Microbe Interactions, Vol. 2 (2000) International Society for Molecular Plant-Microbe Interactions, St. Paul, U.S.A., pp. 131-135. PMID: 10533200

Scheel, D., Blume, B., Brunner, F., Fellbrich, G., Dalbøge, H., Hirt, H., Kauppinen, S., Kroj, T., Ligterink, W., Nürnberger, T., Tschöpe, M., Zinecker, H., zur Nieden, U.

Institute of Microbiology und Genetics, Wien, Austria.

Plants mount a complex array of defense reactions in response to attack by pathogens. Initiation of these events depends on perception and signal transduction of elicitors, which are plant-derived or pathogen-derived signals, that give rise to transcriptional activation of defense-related genes as well as to changes in activities of enzymes involved in cell wall reinforcement and oxygen radical formation. An oligopeptide, identified within a 42 kDa glycoprotein elicitor from Phythophthora sojae, activates in parsley cells typical plant defense reactions, enabling researchers to study plant-pathogen interaction at the single cell level. The oligopeptide elicitor was found to be necessary and sufficient to stimulate a complex defense response in parsley cells, comprising H+/Ca2+ influxes, K+/Cl- effluxes, activation of a mitogen-activated protein (MAP) kinase, an oxidative burst, defense-related gene activation, and phytoalexin formation.

MAP kinases in Plant Signal Transduction

Kluwer Academic Publishers, 67-79.

Jonak, C. Kiegerl, S., Ligterink, W., Siligan, C., Baudouin, E. Beyerly, J., Cardinale, F., Hausl, C., Zwerger, K., Meskiene, I., and Hirt, H. (2000)

Versatile tools for signaling stress, cell cycle, and more.

J.H. Cherry et al. (eds.)

Plant Tolerance to Abiotic Stresses:

Role of genetic Engineering,

Receptor-mediated MAP kinase activation in plant defense.

Springer, Heidelberg, Vol 27, 85-92.

Hirt, H., and Scheel, D. (2000)

In: MAP kinases in plant signal transduction .

Series: Results and Problems in Cell Differentiation (H. Hirt, ed.)

MAP kinases in plant signal transduction.

Springer, Heidelberg, Vol 27, 1-9.

Hirt, H. (2000)

Series: Results and Problems in Cell Differentiation (H. Hirt, ed.)

Differential activation of four specific MAPK pathways by distinct elicitors.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 47, Issue of November 24, pp. 36734–36740, © 2000 by The American Society for Biochemistry and Molecular Biology, Inc. PMID: 10973984

Francesca Cardinale¹, Claudia Jonak¹, Wilco Ligterink¹, Karsten Niehaus², Thomas Boller³, and Heribert Hirt¹

¹ Institute of Microbiology and Genetics, Vienna Biocenter, University of Vienna, Dr. Bohrgasse 9, A-1030 Vienna, Austria.

² Universität Bielefeld, P.O. Box 100131, D-33501 Bielefeld, Germany

³ Friedrich Miescher-Institut, P.O. Box 2543, CH-4002 Basel, Switzerland

Plant cells respond to elicitors by inducing a variety of defense responses. Some of these reactions are dependent on the activity of protein kinases. Recently, mitogen-activated protein kinases (MAPKs) have been identified to be activated by fungal and bacterial elicitors as well as by pathogen infection. In gel kinase assays of alfalfa cells treated with yeast cell wall-derived elicitor (YE) revealed that 44- and 46-kDa MAPKs are rapidly and transiently activated. Immunokinase assays with specific MAPK antibodies revealed that YE mainly activated the 46-kDa SIMK and the 44-kDa MMK3 and to a lesser extent the 44-kDa MMK2 and SAMK. When cells were treated with chemically defined elicitors potentially contained in the YE (chitin and N-acetylglucosamine oligomers, beta-glucan, and ergosterol), the four MAPKs were found to be activated to different levels and with different kinetics. Whereas SIMK and SAMK have been found to be activated by a number of diverse stimuli, MMK3 is activated during mitosis and was therefore assumed to participate in cell division (). No physiological process could be associated with MMK2 activity so far. This is the first report that MMK2 and MMK3 can be activated by external stimuli. Overall, our findings indicate that plant cells can sense different cues of a given microorganism through the activation of multiple MAPKs.

SIMKK, a mitogen-activated protein kinase (MAPK) kinase, is a specific activator of the salt stress-induced MAPK SIMK.

The Plant Cell, Vol. 12, 2247–2258, November 2000, www.plantcell.org © 2000 American Society of Plant Physiologists PMID: 11090222

Stefan Kiegerl^a, Francesca Cardinale^a, Christine Siligan^a, Andrea Gross^b, Emmanuel Baudouin^a, Aneta Liwosz^a, Staffan Eklöf^a, Sandra Till^a, László Bögre^a, Heribert Hirt^a, and Irute Meskiene^a

^a Institute of Microbiology and Genetics, Vienna Biocenter, University of Vienna, 1030 Vienna, Austria.

^b Institute of Genetics, Faculty of Biology, D-33501 Bielefeld, Germany

In eukaryotes, mitogen-activated protein kinases (MAPKs) play key roles in the transmission of external signals, such as mitogens, hormones, and different stresses. MAPKs are activated by MAPK kinases through phosphorylation of MAPKs at both the threonine and tyrosine residues of the conserved TXY activation motif. In plants, several MAPKs are involved in signaling of hormones, stresses, cell cycle, and developmental cues. Recently, we showed that salt stress-induced MAPK (SIMK) is activated when alfalfa cells are exposed to hyperosmotic conditions. Here, we report the isolation and characterization of the alfalfa MAPK kinase SIMKK (SIMK kinase). SIMKK encodes an active protein kinase that interacts specifically with SIMK, but not with three other MAPKs, in the yeast two-hybrid system. Recombinant SIMKK specifically activates SIMK by phosphorylating both the threonine and tyrosine residues in the activation loop of SIMK. SIMKK contains a putative MAPK docking site at the N terminus that is conserved in mammalian MAPK kinases, transcription factors, and phosphatases. Removal of the MAPK docking site of SIMKK partially compromises but does not completely abolish interaction with SIMK, suggesting that other domains of SIMKK also are involved in MAPK binding. In transient expression assays, SIMKK specifically activates SIMK but not two other MAPKs. Moreover, SIMKK enhances the salt-induced activation of SIMK. These data suggest that the salt-induced activation of SIMK is mediated by the dual-specificity protein kinase SIMKK.

Phytophthora parasitica elicitor-induced reactions in cells of Petroselinum crispum.

Plant Cell Physiol. 2000 Jun;41(6):692-701. PMID: 10945338

Fellbrich, G., Blume, B., Brunner, F., Hirt, H., Kroj, T., Ligterink, W., Romanski, A., and Nürnberger, T. (2000)

Institute of Plant Biochemistry, Department of Stress and Developmental Biology, Halle/Saale, Germany.

Cultured parsley (Petroselinum crispum) cells respond to treatment with elicitors derived from different species of the genus Phytophthora with transcript accumulation of defense-associated genes and the production of furanocoumarin phytoalexins. Pep-25, an oligopeptide fragment of a Phytophthora sojae 42-kDa cell wall protein, and a cell wall elicitor preparation derived from Phytophthora parasitica (Pp-elicitor) stimulate accumulation of the same gene transcripts and formation of the same pattern of furanocoumarins. Treatment of cultured cells and protoplasts with proteinase-digested Pp-elicitor identified proteinaceous constituents as active eliciting compounds in parsley. Similar to Pep- 25, Pp-elicitor induced effluxes of K+ and Cl- and influxes of protons and Ca2+. Concomitantly, as monitored in aequorin-transgenic parsley cell lines both elicitors induced an immediate increase in the cytoplasmic Ca2+ concentration up to sustained levels of 175 nM (Pp-elicitor) or 300 nM (Pep-25), respectively. The signature of the Ca2+ response differed greatly between the two elicitors tested. Extracellular Ca2+ proved essential for activation of an oxidative burst, MAP kinase activity and phytoalexin production by either elicitor. While Pp-elicitor induced a qualitatively similar spectrum of defense responses as did Pep-25, elicitor-specific quantitative differences in response intensity and kinetics suggest activation of a conserved signaling cascade through separate ligand binding sites.

Salt stress induces changes in amounts and localisation of the mitogen-activated protein kinase SIMK in alfalfa roots.

Protoplasma Volume 212, Numbers 3-4 / September 2000, 262-267.

Frantisek Balnska^1,2, Miroslav Ovecka² and Heribert Hirt³

¹ Institute of Botany, University of Bonn, Bonn

² Slovak Academy of Sciences, Institute of Botany, Bratislava

³ Institute of Microbiology and Genetics, University of Vienna, Vienna Biocenter, Dr.-Bohr-Gasse 9, A-1030 Vienna, Austria

Summary SIMK is an alfalfa mitogen-activated protein kinase (MAPK) that is activated by salt stress and shows a nuclear localization in suspension-cultured cells. We investigated the localization of SIMK in alfalfa (Medicago sati a) roots. Although SIMK was expressed in most tissues of the root apex, cells of the quiescent center and statocytes showed much lower SIMK protein amounts. In cells of the elongation zone, SIMK was present in much higher amounts in epidermal than in cortex cells. In dividing cells of the root tip, SIMK revealed a cell cycle phase-dependent localization, being predominantly nuclear in interphase but associating with the cell plate and the newly formed cell wall in telophase and early G1 phase. In dividing cells, salt stress resulted in an association of part of the SIMK with the preprophase band. Generally, salt stress resulted in much higher amounts of SIMK in dividing cells of the root apex and epidermal cells of the elongation zone. These data demonstrate that amounts and subcellular localization of SIMK in roots is highly regulated and sensitive to environmental stress.

Wound-induced expression and activation of WIG, a novel glycogen synthase kinase 3.

The Plant Cell, Vol. 12, 1467–1475, August 2000, www.plantcell.org © 2000 American Society of Plant Physiologists PMID: 10948263

Claudia Jonak, Dieter Beisteiner, John Beyerly, and Heribert Hirt

Institute of Microbiology and Genetics, Vienna Biocenter, University of Vienna, Dr. Bohrgasse 9, 1030 Vienna, Austria

Glycogen synthase kinase 3 (GSK-3) is involved in the regulation of several physiological processes, including glycogen metabolism, protein synthesis, transcription factor activity, and developmental control. Although GSK-3–like genes have been isolated from plants, no function for any of these kinases has been defined. We report here that the alfalfa wound-induced gene (WIG, for wound-induced GSK-3), lencoding a functional plant GSK-3–like kinase, is activated when the alfalfa leaves are wounded. Although WIG transcripts are hardly detectable in mature leaves, WIG mRNA accumulates rapidly after wounding. Using a peptide antibody that specifically recognizes p53^WIG , we show that p53^WIG kinase is activated immediately after wounding. Wound-induced activation of p53^WIG kinase is a post-translational process, because the concentrations of p53^WIG protein do not change in intact and wounded leaves, and inhibition of transcription or translation does not block activation by wounding. However, inactivation of p53^WIG kinase, which usually occurs within 60 min after wounding, is dependent on transcription and translation of one or more protein factors. These data suggest that the WIG kinase is involved in wound signaling in plants.

Connecting oxidative stress, auxin, and cell cycle regulation through a plant mitogen-activated protein kinase pathway.

PNAS Proceedings of the National Academy of Sciences, March 14, 2000 vol. 97 no. 6 u 2405–2407 PMID: 10716978

Heribert Hirt

Institute of Microbiology and Genetics, Vienna Biocenter, University of Vienna, Dr. Bohrgasse 9, 1030 Vienna, Austria

Like all living organisms, plants must respond to many external stimuli. Mitogen-activated protein kinases (MAPKs) mediate signal transduction of stress, cell cycle, and growth control in all eukaryotes. MAPKs perform their function as part of protein kinase modules, which in addition to other components are composed of MAPKs, MAPKKs (MAPK kinases), and MAPKKKs (MAPKK kinases) (for reviews, see refs. 1 and 2). MAPKs are serineythreonine protein kinases with a two-lobed structure. The active site is found at the domain interface and contains the MAPK-specific TXY (threonine-Xtyrosine) motif that is targeted by MAPKKs, dual-specificity protein kinases that activate MAPKs by phosphorylation of both the threonine and tyrosine residue of the TXY motif. MAPKKs are activated themselves by phosphorylation of two conserved serine or threonine residues (SyTXXXSyT) by MAPKKKs. According to their divergent structures MAPKKKs can be classified into different subfamilies. MAPKKKs contain different regulatory motifs, including pleckstrin homology domains, prolinerich sequences involved in Src homology 3 binding, zinc finger motifs, leucine zippers, and binding sites for G proteins. MAPKKKs can be activated by a wide range of stimuli and by different mechanisms, such as phosphorylation and interaction with G proteins or receptors.

Microbial elicitors induce activation and dual phosphorylation of the Arabidopsis thaliana MAPK6.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 11, Issue of March 17, pp. 7521–7526, © 2000 by The American Society for Biochemistry and Molecular Biology, Inc. PMID: 10713056

Thomas S. Nühse¹, Scott C. Peck¹, Heribert Hirtsup>2, and Thomas Boller¹

¹ Friedrich-Miescher-Institut, Maulbeerstr. 66, 4058 Basel, Switzerland.

² Institute of Microbiology and Genetics, Vienna Biocenter, University of Vienna, Dr. Bohrgasse 9, 1030 Vienna, Austria

Protein kinases related to the family of mitogen-activated kinases (MAPKs) have been established as signal transduction components in a variety of processes in plants. For Arabidopsis thaliana, however, although one of the genetically best studied plant species, biochemical data on activation of mitogen-activated protein kinases are lacking. A. thaliana MAPK 6 (AtMPK6) is the Arabidopsis orthologue of a tobacco MAPK termed salicylate-induced protein kinase, which is activated by general and race-specific elicitors as well as by physical stress. Using a C terminus-specific antibody, we show that AtMPK6 is activated in elicitor-treated cell cultures of A. thaliana. Four different elicitors from bacteria, fungi, and plants lead to a rapid and transient activation of AtMPK6, indicating a conserved signaling pathway. The induction was equally rapid as medium alkalinization, one of the earliest elicitor response observed in cell cultures. A similarly rapid activation of AtMPK6 was observed in elicitor-treated leaf strips, demonstrating that recognition of the elicitors and activation of the MAPK pathway occurs also in intact plants. We demonstrate by in vivo labeling that AtMPK6 is phosphorylated on threonine and tyrosine residues in elicited cells.

Hyperosmotic stress stimulates phospholipase D activity and elevates the levels of phosphatidic acid and diacylglycerol pyrophosphate.

The Plant Journal (2000) (22)2 147-154 PMID: 10792830

Teun Munnik¹, Harold J.G. Meijer¹, Heribert Hirt², Wolfgang Frank³, Dorothea Bartels⁴ and Alan Musgrave¹

¹ Institute for Molecular Cell Biology, BioCentrum Amsterdam, University of Amsterdam, Kruislaan 318, NL-1098 SM Amsterdam, The Netherlands

² Institute of Microbiology and Genetics, Vienna Biocenter, University of Vienna, Dr. Bohrgasse 9, 1030 Vienna, Austria

³ Fraunhofer Institut für Umweltchemie und Ökotoxikologie, Schmallenberg, Germany

⁴ Max Planck-Institut für Züchtungsforschung, Cologne, Germany

In mammalian cells, phospholipase D (PLD) and its product phosphatidic acid (PA) are involved in a number of signalling cascades, including cell proliferation, membrane trafficking and defence responses. In plant cells a signalling role for PLD and PA is also emerging. Plants have the extra ability to phosphorylate PA to produce diacylglycerol pyrophosphate (DGPP), a newly discovered phospholipid whose formation attenuates PA levels, but which could itself be a second messenger. Here we report that increases in PA and its conversion to DGPP are common stress responses to water deficit. Increases occur within minutes of treatment and are dependent on the level of stress. Part of the PA produced is due to PLD activity as measured by the in vivo transphosphatidylation of 1-butanol, and part is due to diacylglycerol kinase activity as monitored via 32P-PA formation in a differential labelling protocol. Increases in PA and DGPP are found not only in the green alga Chlamydomonas moewusii and cell-suspension cultures of tomato and alfalfa when subjected to hyperosmotic stress, but also in dehydrated leaves of the resurrection plant Craterostigma plantagineum. These results provide further evidence that PLD and PA play a role in plant signalling, and provide the first demonstration that DGPP is formed during physiological conditions that evoke PA synthesis.

MAP kinase pathways: molecular plug-and-play chips for the cell.

Plant Molecular Biology, Volume 42, Number 6 / April 2000 pp. 791-806 PMID: 10890528

Irute Meskiene, Heribert Hirt

Institute of Microbiology and Genetics, Vienna Biocenter, University of Vienna, Austria.

Mitogen-activated protein kinase (MAPK) pathways transduce a eukaryotes, and plants. In recent years, plant MAPK pathways have attracted increasing interest resulting in the isolation of a large number of different components. Studies on the function of these components have revealed that MAPKs play important roles in the response to a broad variety of stresses, but also in the signaling of plant hormones and the cell cycle. Besides giving an update on recent results, the success and logic of MAPK-based signal transduction cascades is discussed.

Stressing the role of MAP kinases in mitogenic stimulation.

Plant Molecular Biology 43, 705-718. 2000 August PMID: 11089871

Bögre, L., Meskiene, I., Heberle-Bors, E., and Hirt, H.

School of Biological Sciences, Royal Holloway and Bedford New College, University of London, Egham, Surrey, UK.

In yeast and animal cells, distinct subfamilies of mitogen-activated protein kinases (MAPKs) have evolved for transmitting different types of signals, such as the extracellular signal-regulated kinase (ERK) for mitogenic stimuli and differentiation, p38 and JUN kinase (JNK) for stress factors. Based on sequence analysis, the presently known plant MAPKs are most similar to ERKs, even though compelling evidence implies a role in various forms of biotic and abiotic stress responses. However, knowledge of their involvement in controlling proliferation is just emerging. A subgroup of the plant MAPKs, containing the alfalfa MMK3 and tobacco NTF6, are only active in mitotic cells and their localisation to the cell plate suggests a role in cytokinesis. An upstream regulator of MAPKs, the tobacco NPK1, appears to be also activated during mitosis. NPK1 might be associated and regulated by a microtubule motor protein. The localisation of NPK1 to the cell plate and its mitosis-specific activation suggest that together with NTF6 it could constitute a mitotic MAPK signalling module in tobacco. NPK1 appears to have a second role in repression of auxin-induced gene expression. MAPKs might also be involved in signalling within the meristems as suggested by the recruitement of a small G-protein to the CLAVATA 1 receptor-like protein kinase upon activation. In animal and yeast cells some of the small G-proteins relay signals from receptors to MAPK pathways.

Vicia faba germination: Synchronized cell growth and localisation of nucleolin and ?-tubulin.

Seed Sci. Res. 9, 297-304.

Fujikura Y., Dolezel, J., Cihalikova, J., Bögre, L., Heberle-Bors, E. Hirt, H., and Binarova, P. (1999)

The first cell cycle of Vicia faba L. seeds, which begins upon imbibition of dry seeds and is completed at the first mitosis after radicle protrusion, was characterised by the flow cytometry and immunodetection of nucleolin and tubulins in root tip meristems. Flow cytometry revealed highly synchronised profiles from the quiescent G(1) phase to the late G(2) phase, indicating uniform cell cycle progression within a root tip until the first mitosis. Using immunoblotting, nucleolin was detected in two distinct bands with the apparent molecular masses of 89 and 99 kD; the former was detected only in seeds imbibed at 4 degrees C for 1 day whereas the latter was found at all stages examined, suggesting that the 89 kD nucleolin may be seed-specific. Unusual localization of nucleolin in cold- imbibed seeds, undetectable in half of the cells and present in nucleoplasm, was revealed by immunofluorescence microscopy. While alpha- and beta-tubulin were detected at all stages and no significant changes in accumulation of the proteins were observed, few microtubules were detected at the beginning of germination when cells were still in the G(1) phase, suggesting that microtubules may be depolymerized in the dry seeds.

Unsaturated fatty acids inhibit MP2C, a protein phosphatase 2C involved in the wound-induced MAP kinase pathway regulation.

The Plant Journal (1999) 20(3), 343-348 PMID: 10571894

Baudouin, E., Meskiene, I., and Hirt, H.

Institute of Microbiology and Genetics, Vienna Biocenter, University of Vienna, Austria

When mechanically injured, plants develop multiple defense systems including the activation of specific genes. These responses are triggered by a complex network of signalling events that include Ca2+ fluxes, the production of free fatty acids from membrane lipids, as well as the activation of mitogen-activated protein kinases (MAPK). In the present paper, we address the question of the regulation of the MAPK pathway by wound-induced Ca2+ and fatty acid signals. We report that MP2C, a serine/threonine protein phosphatase 2C from alfalfa involved in MAPK pathway inactivation, is inhibited specifically in vitro by long-carbon-chain polyunsaturated fatty acids, and alpha-linolenic acid, the primary product of the octadecanoid pathway, was found to be the most potent inhibitor. Ca2+ also inhibits MP2C, but only at high concentrations, and other divalent cations show similar inhibitory effect, making it unlikely that Ca2+ is involved in the regulation of MP2C in vivo. Overall, our data suggest that cross-talk between wound-induced MAPK and octadecanoid pathways may occur at the level of protein phosphatase 2C and linolenic acid.

Distinct osmosensing protein kinase pathways are involved in signaling moderate and severe hyperosmotic stress.

The Plant Journal (1999) 20(4), 381-388 PMID: 10607291

Teun Munnik¹, Wilco Ligterink², Irute Meskiene², Ornella Calderini², John Beyerly², Alan Musgrave¹ and Heribert Hirt²

¹ Institute of Molecular Cell Biology, Biocentrum Amsterdam, The Netherlands

² Institute of Microbiology and Genetics, Vienna Biocenter, Austria

Plant growth is severely affected by hyper-osmotic salt conditions. Although a number of salt-induced genes have been isolated, the sensing and signal transduction of salt stress is little understood. We provide evidence that alfalfa cells have two osmo-sensing protein kinase pathways that are able to distinguish between moderate and extreme hyper-osmotic conditions. A 46 kDa protein kinase was found to be activated by elevated salt concentrations (above 125 mM NaCl). In contrast, at high salt concentrations (above 750 mM NaCl), a 38 kDa protein kinase, but not the 46 kDa kinase, became activated. By biochemical and immunological analysis, the 46 kDa kinase was identified as SIMK, a member of the family of MAPKs (mitogen-activated protein kinases). SIMK is not only activated by NaCl, but also by KCl and sorbitol, indicating that the SIMK pathway is involved in mediating general hyper-osmotic conditions. Salt stress induces rapid but transient activation of SIMK, showing maximal activity between 8 and 16 min before slow inactivation. When inactive, most mammalian and yeast MAPKs are cytoplasmic but undergo nuclear transloca- tion upon activation. By contrast, SIMK was found to be a constitutively nuclear protein and the activity of the kinase was not correlated with changes in its intra-cellular compartmentation, suggesting an intra-nuclear mechanism for the regulation of SIMK activity.

Rapid Avr-9 and Cf-9-dependent activation of MAP kinases in tobacco cell cultures and leaves: convergence of resistance gene, elicitor, wound, and salicylate responses.

The Plant Cell, Vol. 11, 273-287, February 1999, www.plantcell.org © 1999 American Society of Plant Physiologists PMID: 9927644

Tina Romeis^a, Pedro Piedras^a, Shuqun Zhang^b, Daniel F. Klessig^b, Heribert Hirt^c and Jonathan D.G. Jones^a

^a Sainsbury Laboratory, John Innes Centre, Norwich, United Kingdom

^b Waksman Institute and Department of Molecular Biology and Biochemistry, Rutgers, New Jersey, USA

^c Institute of Microbiology and Genetics, Vienna Biocenter, Austria

The Cf-9 resistance (R) gene from tomato confers resistance to the fungal pathogen Cladosporium fulvum expressing the corresponding, pathogen-derived avirulence gene product Avr9. To understand how an initial R/Avr recognition event is transmitted and triggers the induction of plant defenses, we investigated early Avr9/Cf-9-dependent activation of protein kinases in transgenic tobacco expressing the Cf-9 gene. We identified two protein kinases of 46 and 48 kD, using myelin basic protein as substrate, that became rapidly activated in a strictly gene-for-gene manner within 2 to 5 min after Avr9 elicitation in both Cf9 tobacco plants and derived cell cultures. Studies with pharmacological inhibitors and effectors revealed that Ca2+ influx and a phosphorylation event(s) are required for kinase activation, but neither enzyme is involved in the Avr9-dependent synthesis of active oxygen species. The activation of both kinases is achieved via post-translational mechanisms, and the activation but not inactivation step includes tyrosine phosphorylation. Using specific antibodies, we found that the 46- and 48-kD kinases were similiar to WIPK (for wound-induced protein kinase) and SIPK (for salicylic acid-induced protein kinase), two previously characterized mitogen-activated protein (MAP) kinases from tobacco. In addition, Cf9 tobacco plants and cell cultures showed an Avr9-dependent accumulation of the WIPK transcript. Cf9 tobacco suspension cultures are thus a unique system in which to analyze the earliest events in R gene function. These data indicate that (1) the R/Avr-mediated induction of plant defense is accomplished via several parallel signaling mechanisms, and (2) R/Avr-dependent signal transduction pathways are interlinked at MAP kinases with responses of plants not only to non-race-specific elicitors but also to abiotic stimuli, such as wounding and mechanical stress.

A MAP kinase is activated late in plant mitosis and becomes localized to the plane of cell division.

The Plant Cell, Vol. 11, 101-113, January 1999, www.plantcell.org © 1999 American Society of Plant Physiologists PMID: 9878635

László Bögre^a, Ornella Calderini^a,b, Pavla Binarova^c, Markus Mattauch^a, Sandra Till^a, Stefan Kiegerl^a, Claudia Jonak^a, Christina Pollaschek^a, Patrick Barker^d, Neville S. Huskisson^d, Heribert Hirt^a and Erwin Heberle-Bors^a

^a Vienna Biocenter, Institute of Microbiology and Genetics, University of Vienna, Dr. Bohrgasse 9, A-1030 Vienna, Austria

^b Instituto di Recerche sul Miglioramento Genetico Plante Foraggere CNR, Perugia, Italy

^c Norman Borlaug Center for Plant Science, Prague, Czech Republic

^d Microchemical Facility, Babraham Institute, Cambridge, U.K.

In eukaryotes, mitogen-activated protein kinases (MAPKs) are part of signaling modules that transmit diverse stimuli, such as mitogens, developmental cues, or various stresses. Here, we report a novel alfalfa MAPK, Medicago MAP kinase 3 (MMK3). Using an MMK3-specific antibody, we detected the MMK3 protein and its associated activity only in dividing cells. The MMK3 protein could be found during all stages of the cell cycle, but its protein kinase activity was transient in mitosis and correlated with the timing of phragmoplast formation. Depolymerization of microtubules by short treatments with the drug amiprophosmethyl during anaphase and telophase abolished MMK3 activity, indicating that intact microtubules are required for MMK3 activation. During anaphase, MMK3 was found to be concentrated in between the segregating chromosomes; later, it localized at the midplane of cell division in the phragmoplast. As the phragmoplast microtubules were redistributed from the center to the periphery during telophase, MMK3 still localized to the whole plane of division; thus, phragmoplast microtubules are not required to keep MMK3 at this location. Together, these data strongly support a role for MMK3 in the regulation of plant cytokinesis.

MAP kinase activation in plant defense.

Biologia 54, 56-57.

Cardinale, F. Ligterink, W., Baudouin, E., Beyerly, J., Hausl, C., Jonak, C., Kiegerl, S., Meskiene, I., Siligan, C., Zwerger, K., and Hirt, H. (1999)

Transcriptional upregulation of signaling pathways: more complex than anticipated ?

Hirt, H. (1999)

Trends in Plant Science, Volume 4, Issue 1, 1 January 1999, Pages 7-8
sciencedirect.com / Registered Users Only

MAP kinases as transducers in plant signalling.

Cell. Mol. Life Sci. 55, 204-213.

Jonak, C., Ligterink, W., and H. Hirt (1999)

Mitogen-activated protein kinase (MAPK) pathways are modules involved in the transduction of extracellular signals to intracellular targets in all eukaryotes. Distinct MAPK pathways are regulated by different extracellular stimuli and are implicated in a wide variety of biological processes. In plants there is evidence for MAPKs playing a role in the signaling of abiotic stresses, pathogens and plant hormones. The large number and divergence of plant MAPKs indicates that this ancient mechanism of bioinformatics is extensively used in plants and may provide a new molecular handle on old questions.

Cellular and Molecular Life Sciences (CMLS)
Publisher: Birkhäuser Basel
ISSN:  1420-682X (Paper) 1420-9071 (Online)
DOI:  10.1007/s000180050285
Issue:  Volume 55, Number 2
Date:  February 1999
Pages:  204 - 213

Functional isolation and selection of plant cell cycle genes in yeast.

Portland Press Ltd, London, 129-143.

Hirt, L. Bögre, I. Meskiene, M. Dahl, K. Zwerger, and E. Heberle-Bors (1998)

(D. Dudits, D. Francis, and D. Inze, eds.)

In: Molecular and Cell Biology of the Plant Cell Cycle

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