Draft Genome Sequence of the Plant Growth-Promoting Rhizobacterium Acinetobacter radioresistens Strain SA188 Isolated from the Desert Plant Indigofera argentea.

Genome Announc. 2017 Mar 2;5(9). pii: e01708-16. doi: 10.1128/genomeA.01708-16. PMID: 28254978
Lafi FF, Alam I, Bisseling T, Geurts R, Bajic VB, Hirt H, Saad MM.

Acinetobacter radioresistens strain SA188 is a plant endophytic bacterium, isolated from root nodules of the desert plants Indigofera spp., collected in Jizan, Saudi Arabia. Here, we report the 3.2-Mb draft genome sequence of strain SA188, highlighting characteristic pathways for plant growth-promoting activity and environmental adaptation.
Copyright © 2017 Lafi et al.

Draft Genome Sequence of Ochrobactrum intermedium Strain SA148, a Plant Growth-Promoting Desert Rhizobacterium.

Genome Announc. 2017 Mar 2;5(9). pii: e01707-16. doi: 10.1128/genomeA.01707-16. PMID: 28254977
Lafi FF, Alam I, Geurts R, Bisseling T, Bajic VB, Hirt H, Saad MM.

Ochrobactrum intermedium strain SA148 is a plant growth-promoting bacterium isolated from sandy soil in the Jizan area of Saudi Arabia. Here, we report the 4.9-Mb draft genome sequence of this strain, highlighting different pathways characteristic of plant growth promotion activity and environmental adaptation of SA148.
Copyright © 2017 Lafi et al.

Draft Genome Sequence of Enterobacter sp. Sa187, an Endophytic Bacterium Isolated from the Desert Plant Indigofera argentea.

Genome Announc. 2017 Feb 16;5(7). pii: e01638-16. doi: 10.1128/genomeA.01638-16. PMID: 28209831
Lafi FF, Alam I, Geurts R, Bisseling T, Bajic VB, Hirt H, Saad MM.

Enterobacter sp. Sa187 is a plant endophytic bacterium, isolated from root nodules of the desert plant Indigofera argentea, collected from the Jizan region of Saudi Arabia. Here, we report the genome sequence of Sa187, highlighting several genes involved in plant growth-promoting activity and environmental adaption.

Draft Genome Sequence of Plant Growth-Promoting Micrococcus luteus Strain K39 Isolated from Cyperus conglomeratus in Saudi Arabia.

Genome Announc. 2017 Jan 26;5(4). pii: e01520-16. doi: 10.1128/genomeA.01520-16. PMID: 28126944
Lafi FF, Ramirez-Prado JS, Alam I, Bajic VB, Hirt H, Saad MM

Micrococcus luteus strain K39 is an endophyte bacterium isolated from roots of the desert plant Cyperus conglomeratus collected from the Red Sea shore, Thuwal, Saudi Arabia. The draft genome sequence of strain K39 revealed a number of enzymes involved in salinity and oxidative stress tolerance or having herbicide-resistance activity.
Copyright © 2017 Lafi et al.

Draft Genome Sequence of the Plant Growth-Promoting Pseudomonas punonensis Strain D1-6 Isolated from the Desert Plant Erodium hirtum in Jordan.

Genome Announc. 2017 Jan 12;5(2). pii: e01437-16. doi: 10.1128/genomeA.01437-16 PMID: 28082490
Lafi FF, AlBladi ML, Salem NM, Al-Banna L, Alam I, Bajic VB, Hirt H, Saad MM

Pseudomonas punonensis strain D1-6 was isolated from roots of the desert plant Erodium hirtum, near the Dead Sea in Jordan. The genome of strain D1-6 reveals several key plant growth-promoting and herbicide-resistance genes, indicating a possible specialized role for this endophyte.
Copyright © 2017 Lafi et al.


Convergence of Multiple MAP3Ks on MKK3 Identifies a Set of Novel Stress MAPK Modules.

Front Plant Sci. 2016 Dec 22;7:1941. doi: 10.3389/fpls.2016.01941. eCollection 2016. PMID: 28066492
Colcombet J, Sözen C, Hirt H.

Since its first description in 1995 and functional characterization 12 years later, plant MKK3-type MAP2Ks have emerged as important integrators in plant signaling. Although they have received less attention than the canonical stress-activated mitogen-activated protein kinases (MAPKs), several recent publications shed light on their important roles in plant adaptation to environmental conditions. Nevertheless, the MKK3-related literature is complicated. This review summarizes the current knowledge and discrepancies on MKK3 MAPK modules in plants and highlights the singular role of MKK3 in green plants. In the light of the latest data, we hypothesize a general model that all clade-III MAP3Ks converge on MKK3 and C-group MAPKs, thereby defining a set of novel MAPK modules which are activated by stresses and internal signals through the transcriptional regulation of MAP3K genes.
MKK3 module; mitogen-activated protein kinases; phosphorylation cascade; stress responses; viridiplantae

Draft Genome Sequence of the Phosphate-Solubilizing Bacterium Pseudomonas argentinensis Strain SA190 Isolated from the Desert Plant Indigofera argentea.

Genome Announc. 2016 Dec 22;4(6). pii: e01431-16. doi: 10.1128/genomeA.01431-16. PMID: 28007863
Lafi FF, Alam I, Geurts R, Bisseling T, Bajic VB, Hirt H, Saad MM

Pseudomonas argentinensis strain SA190 is a plant endophytic-inhabiting bacterium that was isolated from root nodules of the desert plant Indigofera argentea collected from the Jizan region of Saudi Arabia. Here, we report the genome sequence of SA190, highlighting several functional genes related to plant growth-promoting activity, environment adaption, and antifungal activity.
Copyright © 2016 Lafi et al.

The heat shock protein/chaperone network and multiple stress resistance.

Plant Biotechnol J. 2016 Nov 10. doi: 10.1111/pbi.12659. PMID 27860233
Jacob P, Hirt H, Bendahmane A.

Crop yield has been greatly enhanced during the last century. However, most elite cultivars are adapted to temperate climates and are not well suited to more stressful conditions. In the context of climate change, stress resistance is a major concern. To overcome these difficulties, scientists may help breeders by providing genetic markers associated with stress resistance. However, multi-stress resistance cannot be obtained from the simple addition of single stress resistance traits. In the field, stresses are unpredictable and several may occur at once. Consequently, the use of single stress resistance traits is often inadequate. Although it has been historically linked with the heat stress response, the heat shock protein (HSP)/chaperone network is a major component of multiple stress responses. Among the HSP/chaperone "client proteins" many are primary metabolism enzymes and signal transduction components with essential roles for the proper functioning of a cell. HSPs/chaperones are controlled by the action of diverse heat shock factors, which are recruited under stress conditions. In this review, we give an overview of the regulation of the HSP/chaperone network with a focus on Arabidopsis thaliana. We illustrate the role of HSPs/chaperones in regulating diverse signaling pathways and discuss several basic principles that should be considered for engineering multiple stress resistance in crops through the HSP/Chaperone network. This article is protected by copyright. All rights reserved.

Draft Genome Sequence of Halomonas elongata Strain K4, an Endophytic Growth-Promoting Bacterium Enhancing Salinity Tolerance In Planta.

Genome Announc. 2016 Nov 3;4(6). pii: e01214-16. doi: 10.1128/genomeA.01214-16. PMID: 27811099
Lafi FF, Ramirez-Prado JS, Alam I, Bajic VB, Hirt H, Saad MM.

Halomonas elongata strain K4 is an endophytic bacterial strain that was isolated from roots of Cyperus conglomeratus collected at the Red Sea coast in Thuwal, Saudi Arabia. Here, we present a draft genome sequence of this strain, highlighting a number of pathways involved in plant growth promotion under salt stress.

Draft Genome Sequence of the Plant Growth-Promoting Cupriavidus gilardii Strain JZ4 Isolated from the Desert Plant Tribulus terrestris.

Genome Announc. 2016 Jul 28;4(4). pii: e00678-16. doi: 10.1128/genomeA.00678-16. PMID: 27469951
Lafi FF, Bokhari A, Alam I, Bajic VB, Hirt H, Saad MM.

We isolated the plant endophytic bacterium Cupriavidus gilardii strain JZ4 from the roots of the desert plant Tribulus terrestris, collected from the Jizan region, Saudi Arabia. We report here the draft genome sequence of JZ4, together with several enzymes related to plant growth-promoting activity, environmental adaption, and antifungal activity.

LHP1 Regulates H3K27me3 Spreading and Shapes the Three-Dimensional Conformation of the Arabidopsis Genome.

PLoS One. 2016 Jul 13;11(7):e0158936. doi: 10.1371/journal.pone.0158936. PMID: 27410265
Veluchamy A, Jégu T, Ariel F, Latrasse D, Mariappan KG, Kim SK, Crespi M, Hirt H, Bergounioux C, Raynaud C, Benhamed M.

Precise expression patterns of genes in time and space are essential for proper development of multicellular organisms. Dynamic chromatin conformation and spatial organization of the genome constitute a major step in this regulation to modulate developmental outputs. Polycomb repressive complexes (PRCs) mediate stable or flexible gene repression in response to internal and environmental cues. In Arabidopsis thaliana, LHP1 co-localizes with H3K27me3 epigenetic marks throughout the genome and interacts with PRC1 and PRC2 members as well as with a long noncoding RNA. Here, we show that LHP1 is responsible for the spreading of H3K27me3 towards the 3' end of the gene body. We also identified a subset of LHP1-activated genes and demonstrated that LHP1 shapes local chromatin topology in order to control transcriptional co-regulation. Our work reveals a general role of LHP1 from local to higher conformation levels of chromatin configuration to determine its accessibility to define gene expression patterns.

Aquaporins Link ROS Signaling to Plant Immunity.

Plant Physiol. 2016 Jul;171(3):1540. doi: 10.1104/pp.16.00433. No abstract available. PMID: 27385821
H. Hirt

Commentary on Aquaporins and Plant Immunity

Plant Growth Promoting Rhizobacteria and Silicon Synergistically Enhance Salinity Tolerance of Mung Bean.

Front Plant Sci. 2016 Jun 17;7:876. doi: 10.3389/fpls.2016.00876. PMID: 27379151
Mahmood S, Daur I, Al-Solaimani SG, Ahmad S, Madkour MH, Yasir M, Hirt H, Ali S, Ali Z.

The present study explored the eco-friendly approach of utilizing plant-growth-promoting rhizobacteria (PGPR) inoculation and foliar application of silicon (Si) to improve the physiology, growth, and yield of mung bean under saline conditions. We isolated 18 promising PGPR from natural saline soil in Saudi Arabia, and screened them for plant-growth-promoting activities. Two effective strains were selected from the screening trial, and were identified as Enterobacter cloacae and Bacillus drentensis using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry and 16S rRNA gene sequencing techniques, respectively. Subsequently, in a 2-year mung bean field trial, using a randomized complete block design with a split-split plot arrangement, we evaluated the two PGPR strains and two Si levels (1 and 2 kg ha(-1)), in comparison with control treatments, under three different saline irrigation conditions (3.12, 5.46, and 7.81 dS m(-1)). The results indicated that salt stress substantially reduced stomatal conductance, transpiration rate, relative water content (RWC), total chlorophyll content, chlorophyll a, chlorophyll b, carotenoid content, plant height, leaf area, dry biomass, seed yield, and salt tolerance index. The PGPR strains and Si levels independently improved all the aforementioned parameters. Furthermore, the combined application of the B. drentensis strain with 2 kg Si ha(-1) resulted in the greatest enhancement of mung bean physiology, growth, and yield. Overall, the results of this study provide important information for the benefit of the agricultural industry.

The Role of MAPK Modules and ABA during Abiotic Stress Signaling.

Trends Plant Sci. 2016 Aug;21(8):677-85. doi: 10.1016/j.tplants.2016.04.004. Review. PMID: 27143288
de Zelicourt A, Colcombet J, Hirt H

To respond to abiotic stresses, plants have developed specific mechanisms that allow them to rapidly perceive and respond to environmental changes. The phytohormone abscisic acid (ABA) was shown to be a pivotal regulator of abiotic stress responses in plants, triggering major changes in plant physiology. The ABA core signaling pathway largely relies on the activation of SnRK2 kinases to mediate several rapid responses, including gene regulation, stomatal closure, and plant growth modulation. Mitogen-activated protein kinases (MAPKs) have also been implicated in ABA signaling, but an entire ABA-activated MAPK module was uncovered only recently. In this review, we discuss the evidence for a role of MAPK modules in the context of different plant ABA signaling pathways.

Interview with Heribert Hirt.

Trends Plant Sci. 2016 Jan;21(1):1-2. doi: 10.1016/j.tplants.2015.11.007. PMID: 26686410
Hirt H

As a son of an engineer who traveled widely during his career, Heribert Hirt began his life in the exotic country of Iran, before receiving his high-school education in Germany and then studying biochemistry at the University of Cape Town and then later at the University of Vienna, from where he received his PhD in 1987. He then worked as a postdoctoral fellow in Vienna, Oxford, and Wageningen, before starting his own group at the University of Vienna in 1993. It was also in Vienna that he became professor of genetics in 1997, followed by vice-director of the Gregor Mendel Institute of Plant Molecular Biology, and later head of the Plant Molecular Biology Department of the University of Vienna. In 2007, he decided that it was time for new challenges and accepted an appointment in France to direct the Paris-based INRA-CNRS Plant Genomics Institute for the following 7 years. In 2014, Heribert embarked on yet another challenge by accepting the role to head up the Center for Desert Agriculture at King Abdullah University of Sciences and Technology (KAUST) in Saudi Arabia.


A SWI/SNF Chromatin Remodelling Protein Controls Cytokinin Production through the Regulation of Chromatin Architecture


PLoS One. 2015 Oct 12;10(10):e0138276. doi: 10.1371/journal.pone.0138276. eCollection 2015. PMID: 26457678
Teddy Jégu, Séverine Domenichini, Thomas Blein, Federico Ariel, Aurélie Christ, Soon-Kap Kim, Martin Crespi, Stéphanie Boutet-Mercey, Grégory Mouille, Mickaël Bourge, Heribert Hirt, Catherine Bergounioux, Cécile Raynaud, Moussa Benhamed

Chromatin architecture determines transcriptional accessibility to DNA and consequently gene expression levels in response to developmental and environmental stimuli. Recently, chromatin remodelers such as SWI/SNF complexes have been recognized as key regulators of chromatin architecture. To gain insight into the function of these complexes during root development, we have analyzed Arabidopsis knock-down lines for one sub-unit of SWI/SNF complexes: BAF60. Here, we show that BAF60 is a positive regulator of root development and cell cycle progression in the root meristem via its ability to down-regulate cytokinin production. By opposing both the deposition of active histone marks and the formation of a chromatin regulatory loop, BAF60 negatively regulates two crucial target genes for cytokinin biosynthesis (IPT3 and IPT7) and one cell cycle inhibitor (KRP7). Our results demonstrate that SWI/SNF complexes containing BAF60 are key factors governing the equilibrium between formation and dissociation of a chromatin loop controlling phytohormone production and cell cycle progression.

Plant MAPK cascades: just rapid signaling modules?




Plant Signal Behav. 2015 Aug 27:0. PMID: 26313321
Boudsocq M, Danquah A, de Zélicourt A, Hirt H, Colcombet J.

Abscisic acid (ABA) is a major phytohormone mediating important stress-related processes. We recently unveiled an ABA-activated MAPK signaling module constituted of MAP3K17/18-MKK3-MPK1/2/7/14. Unlike classical rapid MAPK activation, we showed that the activation of the new MAPK module is delayed and relies on the MAP3K protein synthesis. In this addendum, we discuss the role of this original and unexpected activation mechanism of MAPK cascades which suggests that MAPKs can regulate both early and long-term plant stress responses.
KEYWORDS: ABA signaling; Abscisic acid; delayed signaling; mitogen-activated protein kinase; protein synthesis

Identification and characterization of an ABA-activated MAP kinase cascade in Arabidopsis thaliana.

Plant J. 2015 Feb 27. doi: 10.1111/tpj.12808. PMID 25720833
- Danquah A, de Zélicourt A, Boudsocq M, Neubauer J, Frei Dit Frey N, Leonhardt N, Pateyron S, Gwinner F, Tamby JP, Ortiz-Masia D, Marcote MJ, Hirt H, Colcombet J.

Abscisic acid (ABA) is a major phytohormone involved in important stress-related and developmental plant processes. Recent phosphoproteomic analyses revealed a large set of ABA-triggered phosphoproteins as putative MAPK targets, although the evidence for MAPKs involved in ABA signalling is still scarce. Here, we identified and reconstituted in vivo a complete ABA-activated MAPK cascade, composed of the MAP3Ks MAP3K17/18, the MAP2K MKK3 and the four C group MAPKs MPK1/2/7/14. In planta, we show that ABA activation of MPK7 is blocked in mkk3-1 and map3k17mapk3k18 plants. Coherently, both mutants exhibit hypersensitivity to ABA and altered expression of a set of ABA-dependent genes. A genetic analysis further reveals that this MAPK cascade is activated by the PYR/PYL/RCAR-SnRK2-PP2C ABA core signalling module through protein synthesis of the MAP3Ks, unveiling an atypical mechanism for MAPK activation in eukaryotes. Our work provides evidence for a role of an ABA-induced MAPK pathway in plant stress signalling. This article is protected by copyright. All rights reserved.
ABA ; Arabidopsis thaliana; MAP3K18; MAPK module; MKK3; signaling pathway

Signaling Mechanisms in Pattern-Triggered Immunity (PTI).

Mol Plant. 2015 Jan 9. pii: S1674-2052(15)00087-8. doi: 10.1016/j.molp.2014.12.022. PMID 25744358
Bigeard J, Colcombet J, Hirt H.


In nature, plants constantly have to face pathogen attacks. However, plant disease rarely occurs due to efficient immune systems possessed by the host plants. Pathogens are perceived by two different recognition systems that initiate the so-called pattern-triggered immunity (PTI) and effector-triggered immunity (ETI), both of which are accompanied by a set of induced defenses that usually repel pathogen attacks. Here we discuss the complex network of signaling pathways occurring during PTI, focusing on the involvement of mitogen-activated protein kinases.
MAPKs; PTI; plant defenses; plant immunity; signaling mechanisms




The Salmonella effector protein SpvC, a phosphothreonine lyase is functional in plant cells.

Front Microbiol. 2014 Oct 17;5:548. doi: 10.3389/fmicb.2014.00548. eCollection 2014. PMID: 25368608
Neumann C, Fraiture M, Hernàndez-Reyes C, Akum FN, Virlogeux-Payant I, Chen Y, Pateyron S, Colcombet J, Kogel KH, Hirt H, Brunner F, Schikora A.


Salmonella is one of the most prominent causes of food poisoning and growing evidence indicates that contaminated fruits and vegetables are an increasing concern for human health. Successful infection demands the suppression of the host immune system, which is often achieved via injection of bacterial effector proteins into host cells. In this report we present the function of Salmonella effector protein in plant cell, supporting the new concept of trans-kingdom competence of this bacterium. We screened a range of Salmonella Typhimurium effector proteins for interference with plant immunity. Among these, the phosphothreonine lyase SpvC attenuated the induction of immunity-related genes when present in plant cells. Using in vitro and in vivo systems we show that this effector protein interacts with and dephosphorylates activated Arabidopsis Mitogen-activated Protein Kinase 6 (MPK6), thereby inhibiting defense signaling. Moreover, the requirement of Salmonella SpvC was shown by the decreased proliferation of the ΔspvC mutant in Arabidopsis plants. These results suggest that some Salmonella effector proteins could have a conserved function during proliferation in different hosts. The fact that Salmonella and other Enterobacteriaceae use plants as hosts strongly suggests that plants represent a much larger reservoir for animal pathogens than so far estimated.
KEYWORDS: Salmonella; T3SS; plant infection; trans-kingdom pathogenicity

Functional analysis of Arabidopsis immune-related MAPKs uncovers a role for MPK3 as negative regulator of inducible defenses.

Genome Biol. 2014 Jun 30;15(6):R87. PMID: 24980080
- Frei Dit Frey N, Garcia AV, Bigeard J, Zaag R, Bueso E, Garmier M, Pateyron S, de Tauzia-Moreau ML, Brunaud V, Balzergue S, Colcombet J, Aubourg S, Martin-Magniette ML, Hirt H.

BACKGROUND: Mitogen-activated protein kinases (MAPKs) are key regulators of immune responses in animals and plants. In Arabidopsis, perception of microbe-associated molecular patterns (MAMPs) activates the MAPKs MPK3, MPK4 and MPK6. Increasing information depicts the molecular events activated by MAMPs in plants, but the specific and cooperative contributions of the MAPKs in these signalling events are largely unclear.
In this work, we analyze the behaviour of MPK3, MPK4 and MPK6 mutants in early and late immune responses triggered by the MAMP flg22 from bacterial flagellin. A genome-wide transcriptome analysis reveals that 36% of the flg22-upregulated genes and 68% of the flg22-downregulated genes are affected in at least one MAPK mutant. So far MPK4 was considered as a negative regulator of immunity, whereas MPK3 and MPK6 were believed to play partially redundant positive functions in defence. Our work reveals that MPK4 is required for the regulation of approximately 50% of flg22-induced genes and we identify a negative role for MPK3 in regulating defence gene expression, flg22-induced salicylic acid accumulation and disease resistance to Pseudomonas syringae. Among the MAPK-dependent genes, 27% of flg22-upregulated genes and 76% of flg22-downregulated genes require two or three MAPKs for their regulation. The flg22-induced MAPK activities are differentially regulated in MPK3 and MPK6 mutants, both in amplitude and duration, revealing a highly interdependent network.
These data reveal a new set of distinct functions for MPK3, MPK4 and MPK6 and indicate that the plant immune signalling network is choreographed through the interplay of these three interwoven MAPK pathways.

Auxin efflux by PIN-FORMED proteins is activated by two different protein kinases, D6 PROTEIN KINASE and PINOID.

Elife. 2014 Jun 19:e02860. doi: 10.7554/eLife.02860. PMID: 24948515
- Zourelidou M, Absmanner B, Weller B, Barbosa IC, Willige BC, Fastner A, Streit V, Port SA, Colcombet J, de la Fuente van Bentem S, Hirt H, Kuster B, Schulze WX, Hammes UZ, Schwechheimer C.

The development and morphology of vascular plants is critically determined by synthesis and proper distribution of the phytohormone auxin. The directed cell-to-cell distribution of auxin is achieved through a system of auxin influx and efflux transporters. PIN-FORMED (PIN) proteins are proposed auxin efflux transporters, and auxin fluxes can seemingly be predicted based on the - in many cells - asymmetric plasma membrane distribution of PINs. Here, we show in a heterologous Xenopus oocyte system as well as in Arabidopsis thaliana inflorescence stems that PIN-mediated auxin transport is directly activated by D6 PROTEIN KINASE (D6PK) and PINOID (PID)/WAG kinases of the Arabidopsis AGCVIII kinase family. At the same time, we reveal that D6PKs and PID have differential phosphosite preferences. Our study suggests that PIN activation by protein kinases is a crucial component of auxin transport control that must be taken into account to understand auxin distribution within the plant.

Protein Complexes Characterization in Arabidopsis thaliana by Tandem Affinity Purifi cation Coupled to Mass Spectrometry Analysis

Methods Mol Biol. 2014;1171:237-50. doi: 10.1007/978-1-4939-0922-3_18. PMID: 24908132
Bigeard J, Pflieger D, Colcombet J, Gérard L, Mireau H, Hirt H.


Proteins are major elements participating in all the key functions of the cells. They rarely fulfill their physiological roles in an autonomous way but rather act as part of more complex cellular machines. Indeed they can bind different types of molecules (proteins, nucleic acids, metabolites, etc.), via stable or transient interactions, depending on their nature and functions. The identification of the molecular partners of a given protein is hence essential to better understand its roles, regulation, and mechanisms of action.This chapter describes the use of a tandem affinity purification approach followed by mass spectrometry analysis to try to identify and characterize the proteins involved in protein complexes in Arabidopsis thaliana and decipher some mechanisms of regulation of the modules. Important elements to consider in such an approach are first extensively exposed in the introduction. This technique, in combination with complementary approaches like yeast two-hybrid and bimolecular fluorescence complementation, can be an interesting source of data to identify and characterize in vivo protein complexes.

Identification of Constitutively Active AtMPK6 Mutants Using a Functional Screen in Saccharomyces cerevisiae.

Methods Mol Biol. 2014;1171:67-77. doi: 10.1007/978-1-4939-0922-3_6. PMID: 24908120
Hudik E, Berriri S, Hirt H, Colcombet J.


MAPK (Mitogen-Activated Protein Kinases) mutants which are active independently of phosphorylation by upstream MAPK Kinases (MAPKKs) help to clarify signal transduction processes through MAPK modules and provide a useful tool to understand MAPK roles in the cell. The identification of such mutations is tricky. In this chapter, we provide a detailed protocol for their screening, taking advantage of a functional expression assay in yeast.

Proteomic and phosphoproteomic analyses of chromatin-associated proteins from Arabidopsis thaliana.

Proteomics. 2014 May 31. doi: 10.1002/pmic.201400072 PMID: 24889360
Bigeard J, Rayapuram N, Bonhomme L, Hirt H, Pflieger D.


The nucleus is the organelle where basically all DNA-related processes take place in eukaryotes such as replication, transcription and splicing as well as epigenetic regulation. The identification and description of the nuclear proteins is one of the requisites towards a comprehensive understanding of the biological functions accomplished in the nucleus. Many of the regulatory mechanisms of protein functions rely on their post-translational modifications amongst which phosphorylation is probably one of the most important properties affecting enzymatic activity, interaction with other molecules, localization or stability. So far, the nuclear and subnuclear proteome and phosphoproteome of the model plant Arabidopsis thaliana were the objects of very few studies. In this work, we developed a purification protocol of Arabidopsis chromatin-associated proteins and performed proteomic and phosphoproteomic analyses identifying a total of 879 proteins of which 198 were phosphoproteins that were mainly involved in chromatin remodeling, transcriptional regulation and RNA processing. From 230 precisely localized phosphorylation sites (phosphosites), 52 correspond to hitherto unidentified sites. This protocol and data thereby obtained should be a valuable resource for many domains of plant research.

Phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins.

Proteomics. 2014 May 31. doi: 10.1002/pmic.201400073. PMID: 24889195
Bigeard J, Rayapuram N, Pflieger D, Hirt H.


In eukaryotes, most of the DNA is located in the nucleus where it is organized with histone proteins in a higher-order structure as chromatin. Chromatin and chromatin-associated proteins contribute to DNA-related processes such as replication and transcription as well as epigenetic regulation. Protein functions are often regulated by post-translational modifications (PTMs) amongst which phosphorylation is one of the most abundant PTM. Phosphorylation of proteins affects important properties such as enzyme activity, protein stability or subcellular localization. We here describe the main specificities of protein phosphorylation in plants and review the current knowledge on phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins. We also outline some future challenges to further elucidate protein phosphorylation and chromatin regulation.

Salmonella enterica induces and subverts the plant immune system.

fig 1

Front Microbiol. 2014 Apr 4;5:141. eCollection 2014. PMID: 24772109 PMCID: PMC3983520
García AV, Hirt H.

Infections with Salmonella enterica belong to the most prominent causes of food poisoning and infected fruits and vegetables represent important vectors for salmonellosis. Although it was shown that plants raise defense responses against Salmonella, these bacteria persist and proliferate in various plant tissues. Recent reports shed light into the molecular interaction between plants and Salmonella, highlighting the defense pathways induced and the means used by the bacteria to escape the plant immune system and accomplish colonization. It was recently shown that plants detect Salmonella pathogen-associated molecular patterns (PAMPs), such as the flagellin peptide flg22, and activate hallmarks of the defense program known as PAMP-triggered immunity (PTI). Interestingly, certain Salmonella strains carry mutations in the flg22 domain triggering PTI, suggesting that a strategy of Salmonella is to escape plant detection by mutating PAMP motifs. Another strategy may rely on the type III secretion system (T3SS) as T3SS mutants were found to induce stronger plant defense responses than wild type bacteria. Although Salmonella effector delivery into plant cells has not been shown, expression of Salmonella effectors in plant tissues shows that these bacteria also possess powerful means to manipulate the plant immune system. Altogether, these data suggest that Salmonella triggers PTI in plants and evolved strategies to avoid or subvert plant immunity.

Salt-induced subcellular kinase relocation and seedling susceptibility caused by overexpression of Medicago SIMKK in Arabidopsis.

J Exp Bot. 2014 Jun;65(9):2335-50. doi: 10.1093/jxb/eru115. Epub 2014 Mar 19. PMID: 24648569
Ovečka M, Takáč T, Komis G, Vadovič P, Bekešová S, Doskočilová A, Smékalová V, Luptovčiak I, Samajová O, Schweighofer A, Meskiene I, Jonak C, Křenek P, Lichtscheidl I, Skultéty L, Hirt H, Samaj J.


Dual-specificity mitogen-activated protein kinases kinases (MAPKKs) are the immediate upstream activators of MAPKs. They simultaneously phosphorylate the TXY motif within the activation loop of MAPKs, allowing them to interact with and regulate multiple substrates. Often, the activation of MAPKs triggers their nuclear translocation. However, the spatiotemporal dynamics and the physiological consequences of the activation of MAPKs, particularly in plants, are still poorly understood. Here, we studied the activation and localization of the Medicago sativa stress-induced MAPKK (SIMKK)-SIMK module after salt stress. In the inactive state, SIMKK and SIMK co-localized in the cytoplasm and in the nucleus. Upon salt stress, however, a substantial part of the nuclear pool of both SIMKK and SIMK relocated to cytoplasmic compartments. The course of nucleocytoplasmic shuttling of SIMK correlated temporally with the dual phosphorylation of the pTEpY motif. SIMKK function was further studied in Arabidopsis plants overexpressing SIMKK-yellow fluorescent protein (YFP) fusions. SIMKK-YFP plants showed enhanced activation of Arabidopsis MPK3 and MPK6 kinases upon salt treatment and exhibited high sensitivity against salt stress at the seedling stage, although they were salt insensitive during seed germination. Proteomic analysis of SIMKK-YFP overexpressors indicated the differential regulation of proteins directly or indirectly involved in salt stress responses. These proteins included catalase, peroxiredoxin, glutathione S-transferase, nucleoside diphosphate kinase 1, endoplasmic reticulum luminal-binding protein 2, and finally plasma membrane aquaporins. In conclusion, Arabidopsis seedlings overexpressing SIMKK-YFP exhibited higher salt sensitivity consistent with their proteome composition and with the presumptive MPK3/MPK6 hijacking of the salt response pathway.

Identification of Novel PAMP-Triggered Phosphorylation and Dephosphorylation Events in Arabidopsis thaliana by Quantitative Phosphoproteomic Analysis.

J Proteome Res. 2014 Apr 4;13(4):2137-51. doi: 10.1021/pr401268v. Epub 2014 Mar 17. PMID: 24601666
Rayapuram N, Bonhomme L, Bigeard J, Haddadou K, Przybylski C, Hirt H, Pflieger D.


Signaling cascades rely strongly on protein kinase-mediated substrate phosphorylation. Currently a major challenge in signal transduction research is to obtain high confidence substrate phosphorylation sites and assign them to specific kinases. In response to bacterial flagellin, a pathogen-associated molecular pattern (PAMP), we searched for rapidly phosphorylated proteins in Arabidopsis thaliana by combining multistage activation (MSA) and electron transfer dissociation (ETD) fragmentation modes, which generate complementary spectra and identify phosphopeptide sites with increased reliability. Of a total of 825 phosphopeptides, we identified 58 to be differentially phosphorylated. These peptides harbor kinase motifs of mitogen-activated protein kinases (MAPKs) and calcium-dependent protein kinases (CDPKs), as well as yet unknown protein kinases. Importantly, 12 of the phosphopeptides show reduced phosphorylation upon flagellin treatment. Since protein abundance levels did not change, these results indicate that flagellin induces not only various protein kinases but also protein phosphatases, even though a scenario of inhibited kinase activity may also be possible.

The BAF60 Subunit of the SWI/SNF Chromatin-Remodeling Complex Directly Controls the Formation of a Gene Loop at FLOWERING LOCUS C in Arabidopsis.

Plant Cell. 2014 Feb;26(2):538-51. doi: 10.1105/tpc.113.114454. PMID: 24510722
Jégu T, Latrasse D, Delarue M, Hirt H, Domenichini S, Ariel F, Crespi M, Bergounioux C, Raynaud C, Benhamed M.


SWI/SNF complexes mediate ATP-dependent chromatin remodeling to regulate gene expression. Many components of these complexes are evolutionarily conserved, and several subunits of Arabidopsis thaliana SWI/SNF complexes are involved in the control of flowering, a process that depends on the floral repressor FLOWERING LOCUS C (FLC). BAF60 is a SWI/SNF subunit, and in this work, we show that BAF60, via a direct targeting of the floral repressor FLC, induces a change at the high-order chromatin level and represses the photoperiod flowering pathway in Arabidopsis. BAF60 accumulates in the nucleus and controls the formation of the FLC gene loop by modulation of histone density, composition, and posttranslational modification. Physiological analysis of BAF60 RNA interference mutant lines allowed us to propose that this chromatin-remodeling protein creates a repressive chromatin configuration at the FLC locus.
PMID: 24510722 [PubMed - in process] PMCID: PMC3967024 [Available on 2015/2/1]




Salmonella enterica flagellin is recognized via FLS2 and activates PAMP-triggered immunity in Arabidopsis thaliana.

Mol Plant. 2013 Nov 6. PMID: 24198231
Garcia AV, Charrier A, Schikora A, Bigeard J, Pateyron S, de Tauzia-Moreau ML, Evrard A, Mithöfer A, Martin-Magniette ML, Virlogeux-Payant I, Hirt H.


Infections with Salmonella enterica belong to the most prominent causes of food poisoning and infected fruits and vegetables represent important vectors for salmonellosis. Recent evidence indicates that plants recognize S. enterica and raise defense responses. Nonetheless, the molecular mechanisms controlling the interaction of S. enterica with plants are still largely unclear. Here, we show that flagellin from S. enterica represents a prominent pathogen-associated molecular pattern (PAMP) in Arabidopsis thaliana, which induces PAMP-triggered immunity (PTI) via the recognition of the flg22 domain by the receptor kinase FLS2. The Arabidopsis fls2 mutant shows reduced though not abolished PTI activation, indicating that plants rely also on recognition of other S. enterica PAMPs. Interestingly, the S. enterica type III secretion system (T3SS) mutant prgH- induced stronger defense-gene expression than wild type bacteria in Arabidopsis, suggesting that T3SS effectors are involved in defense suppression. Furthermore, we observe that S. enterica strains show variation in the flg22 epitope, which results in proteins with reduced PTI-inducing activity. Altogether these results show that S. enterica activates PTI in Arabidopsis and suggest that, in order to accomplish plant colonization, S. enterica evolved strategies to avoid or suppress PTI.



The role of ABA and MAPK signaling pathways in plant abiotic stress responses.

Biotechnol Adv. 2013 Sep 30. pii: S0734-9750(13)00162-6. doi: 10.1016/j.biotechadv.2013.09.006. PMID: 24091291
Danquah A, de Zelicourt A, Colcombet J, Hirt H.


As sessile organisms, plants have developed specific mechanisms that allow them to rapidly perceive and respond to stresses in the environment. Among the evolutionarily conserved pathways, the ABA (abscisic acid) signaling pathway has been identified as a central regulator of abiotic stress response in plants, triggering major changes in gene expression and adaptive physiological responses. ABA induces protein kinases of the SnRK family to mediate a number of its responses. Recently, MAPK (mitogen activated protein kinase) cascades have also been shown to be implicated in ABA signaling. Therefore, besides discussing the role of ABA in abiotic stress signaling, we will also summarize the evidence for a role of MAPKs in the context of abiotic stress and ABA signaling.



New checkpoints in stomatal defense.


Trends Plant Sci. 2013 Apr 10. pii: S1360-1385(13)00060-5. doi: 10.1016/j.tplants.2013.03.007. PMID: 23582764
Montillet JL, Hirt H.

  • An oxylipin-dependent signaling pathway controls stomatal defense.
  • Abscisic acid modulates the biotic stress signal in guard cells.
  • Distinct mitogen-activated protein kinase modules regulate biotic and abiotic stresses.
  • Distinct NADPH oxidase isoforms control reactive oxygen species in response to biotic and abiotic stresses.
  • Salicylic acid is a key signaling step required for stomatal defense.

Recent reports have revealed new guard cell signaling elements that function in stomatal defense in Arabidopsis thaliana (Arabidopsis). We discuss here the role of oxylipins, salicylic acid (SA), and abscisic acid (ABA) in stomatal immunity in response to the bacterial pathogen Pseudomonas syringae.


Regulation of the heat stress response in Arabidopsis by MPK6-targeted phosphorylation of the heat stress factor HsfA2

PeerJ Published 2 April 2013 DOI 10.7717/peerj.59
Alexandre Evrard, Mukesh Kumar, David Lecourieux, Jessica Lucks Pascal, von Koskull-Doring and Heribert Hirt

So far little is known on the functional role of phosphorylation in the heat stress response of plants. Here we present evidence that heat stress activates the Arabidopsis mitogen-activated protein kinase MPK6. In vitro and in vivo evidence is provided that MPK6 specifically targets the major heat stress transcription factor HsfA2. Activation of MPK6 results in complex formation with HsfA2. MPK6 phosphorylates HsfA2 on T249 and changes its intracellular localisation. Protein kinase and phosphatase inhibitor studies indicate that HsfA2 protein stability is regulated in a phosphorylation-dependent manner, but this mechanism is independent of MPK6. Overall, our data show that heat stress-induced targeting of HsfA2 by MPK6 participates in the complex regulatory mechanism how plants respond to heat stress.

An abscisic Acid-independent oxylipin pathway controls stomatal closure and immune defense in Arabidopsis.

PLoS Biol. 2013 Mar;11(3):e1001513. doi: 10.1371/journal.pbio.1001513. Epub 2013 Mar 19. PMID: 23526882
Montillet JL, Leonhardt N, Mondy S, Tranchimand S, Rumeau D, Boudsocq M, Garcia AV, Douki T, Bigeard J, Laurière C, Chevalier A, Castresana C, Hirt H.

Plant stomata function in innate immunity against bacterial invasion and abscisic acid (ABA) has been suggested to regulate this process. Using genetic, biochemical, and pharmacological approaches, we demonstrate that (i) the Arabidopsis thaliana nine-specific-lipoxygenase encoding gene, LOX1, which is expressed in guard cells, is required to trigger stomatal closure in response to both bacteria and the pathogen-associated molecular pattern flagellin peptide flg22; (ii) LOX1 participates in stomatal defense; (iii) polyunsaturated fatty acids, the LOX substrates, trigger stomatal closure; (iv) the LOX products, fatty acid hydroperoxides, or reactive electrophile oxylipins induce stomatal closure; and (v) the flg22-mediated stomatal closure is conveyed by both LOX1 and the mitogen-activated protein kinases MPK3 and MPK6 and involves salicylic acid whereas the ABA-induced process depends on the protein kinases OST1, MPK9, or MPK12. Finally, we show that the oxylipin and the ABA pathways converge at the level of the anion channel SLAC1 to regulate stomatal closure. Collectively, our results demonstrate that early biotic signaling in guard cells is an ABA-independent process revealing a novel function of LOX1-dependent stomatal pathway in plant immunity.

Rhizosphere Microbes as Essential Partners for Plant Stress Tolerance


Mol. Plant (2013) doi: 10.1093/mp/sst028 First published online: March 9, 2013, PMID: 23475999
Axel de Zelicourt, Mohamed Al-Yousif and Heribert Hirt

Ever since plants colonized land, they evolved mechanisms to respond to changing environmental conditions and settle in extreme habitats. Recent studies show that several plant species require microbial associations for stress tolerance and survival. Although many plants lack the adaptive capability to adapt to stress conditions, the ability of a variety of plants to adapt to stress conditions often appears to depend on their association with certain microbes, raising a number of questions: What distinguishes the microbes and plants that can adapt to extreme environmental conditions? Can all plants improve stress tolerance when associated with appropriate microbial partners? Answers to these questions should modify our concepts of plant physiology and could lead to new ways towards a sustainable agriculture.

[Role of micro-organisms in adapting plants to environmental stress conditions].
Rôle des micro-organismes bénéfiques pour aider les plantes à aquérir une tolérance aux environnementaux

Biol Aujourdhui. 2012;206(4):285-90. doi: 10.1051/jbio/2012028. Epub 2013 Feb 19. PMID: 23419255
Hirt. H

Due to their sessile nature, plants have always been confronted to various abiotic and biotic stresses in their immediate environment. As a consequence, the survival of plants depended on their ability to adjust rapidly their physiology, development and growth to escape or mitigate the impacts of stress. All plants are known to perceive and respond to stress signals such as drought, heat, salinity, attacks by herbivores and pathogens. Some biochemical processes are common to all plant stress responses including the production of certain stress proteins and metabolites, as well as the modification of the reactive oxygen species (ROS) metabolism. Although there has been extensive research in the plant stress response field, it is not yet known which factors are responsible for conferring to some plant species the capacity to colonize extreme habitats. Although considerable progress has been made in our understanding of plant stress physiology, the contribution of the plant-associated microbial community in the soil, commonly called the rhizosphere, has only recently received enhanced attention. Recent studies showed that some plant species in natural habitats require microbial associations for stress tolerance and survival. Since plants have colonized land, they have evolved mechanisms to respond to changing environmental conditions and settle in extreme habitats. Although many plants lack the adaptive capability to adapt to stress conditions, the ability of a variety of plants to adapt to stress conditions appears to depend on the association with microbes, raising a number of questions: can all plants improve stress tolerance when associated with their appropriate microbial partners? Did we miss identifying the right partners for a given plant species or variety? What distinguishes the microbes and plants that are adapted to extreme environmental conditions from those living in temperate zones? Answers to these questions are likely to revolutionize plant biology and could lead to new methods for a sustainable agriculture.
© Société de Biologie, 2013.

Brassinosteroid-regulated GSK3/shaggy-like kinases phosphorylate MAP kinase kinases, which control stomata development in Arabidopsis thaliana

JBC The Journal of Biological Chemistry in Press. Published on January 22, 2013 as Manuscript M112.384453, PMID: 2334168
Mamoona Khan, Wilfried Rozhon, Jean Bigeard, Delphine Pflieger, Sigrid Husar, Andrea Pitzschke, Markus Teige, Claudia Jonak, Heribert Hirt and Brigitte Poppenberger

Brassinosteroids (BRs) are steroid hormones, which coordinate fundamental developmental programs in plants. In this study we show that in addition to the well-established roles of BRs in regulating cell elongation and cell division events, BRs also govern cell fate decisions during stomata development in Arabidopsis thaliana (arabidopsis). In wild-type arabidopsis stomatal distribution follows the one-cell spacing rule, that is adjacent stomata are spaced by at least one intervening pavement cell. This rule is interrupted in BR-deficient and BR signaling-deficient arabidopsis mutants resulting in clustered stomata. We demonstrate that BIN2 and its homologues, GSK3/shaggy-like kinases involved in BR signaling, can phosphorylate the MAPK kinases MKK4 and MKK5, which are members of the MAPK module YODA-MKK4/5-MPK3/6 that controls stomata development and patterning. BIN2 phosphorylates a GSK3/shaggy-like kinase recognition motif in MKK4, which reduces MKK4 activity against its substrate MPK6 in vitro. In vivo we show that MKK4 and MKK5 act down-stream of BR signaling, since their over-expression rescued stomata patterning defects in BR-deficient plants. A model is proposed in which GSK3-mediated phosphorylation of MKK4 and MKK5 enables for a dynamic integration of endogenous or environmental cues signaled by BRs, into cell fate decisions governed by the YODA-MKK4/5-MPK3/6 module.


Nucleic Acids Res. 2013 Jan 21, PMID 23341037
Latrasse D, Jégu T, Meng PH, Mazubert C, Hudik E, Delarue M, Charon C, Crespi M, Hirt H, Raynaud C, Bergounioux C, Benhamed M.

Because regulation of its activity is instrumental either to support cell proliferation and growth or to promote cell death, the universal myo-inositol phosphate synthase (MIPS), responsible for myo-inositol biosynthesis, is a critical enzyme of primary metabolism. Surprisingly, we found this enzyme to be imported in the nucleus and to interact with the histone methyltransferases ATXR5 and ATXR6, raising the question of whether MIPS1 has a function in transcriptional regulation. Here, we demonstrate that MIPS1 binds directly to its promoter to stimulate its own expression by locally inhibiting the spreading of ATXR5/6-dependent heterochromatin marks coming from a transposable element. Furthermore, on activation of pathogen response, MIPS1 expression is reduced epigenetically, providing evidence for a complex regulatory mechanism acting at the transcriptional level. Thus, in plants, MIPS1 appears to have evolved as a protein that connects cellular metabolism, pathogen response and chromatin remodeling.


The role of the kinase OXI1 in cadmium and copper induced molecular responses in Arabidopsis thaliana.

Plant Cell Environ. 2012 Dec 19. doi: 10.1111/pce.12056. PMID: 23278806
Smeets K, Opdenakker K, Remans T, Forzani C, Hirt H, Vangronsveld J, Cuypers A.

The hypothesis that MAPK (mitogen-activated protein kinase) signalling is important in plant defences against metal stress has become accepted in recent years. To test the role of OXI1 (oxidative signal inducible kinase) in metal-induced oxidative signalling, the responses of oxi1 knock-out lines to environmentally realistic cadmium (Cd) and copper (Cu) concentrations were compared to those of wild type plants. A relationship between OXI1 and the activation of lipoxygenases and other initiators of oxylipin production was observed under these stress conditions, suggesting that lipoxygenase-1 may be a downstream component of OXI1 signalling. Metal-specific differences in OXI1 action were observed. For example, OXI1 was required for the up-regulation of antioxidative defences such as catalase in leaves and Fe-superoxide dismutase in roots, following exposure to Cu, processes that may involve the MEKK1-MKK2-WRKY25 cascade. Moreover, the induction of CuZn superoxide dismutases in Cu-exposed leaves was regulated by OXI1 in a manner that involves fluctuations in the expression of miRNA398. These observations contrast markedly with the responses to Cd exposure, which also involves OXI1-independent pathways but rather involves changes in components mediating intracellular communication.

Constitutively active MPK4 helps to clarify its role in plant immunity


Plant Signal Behav. 2012 Dec 6;8(2). PMID: 23221776
Colcombet J, Berriri S, Hirt H.

Mitogen-Activated Protein Kinase (MAPK) modules are often involved in stress responses and plant developmental processes. Among these MAPKs, MPK4 has a complex role in biotic stress signaling, cell division control and cytoskeletal organization. mpk4 knockout (KO) plants are dwarfed and very sick, making it difficult to distinguish between cause and effect of its phenotype. To overcome this difficulty, we developed mutations triggering constitutive MPK4 activity and created transgenic lines allowing phenotypic studies on a WT-like plant. By this approach, we confirmed that MPK4 functions as a negative regulator of pathogen defense, but our work also suggests that MPK4 interferes with stress signaling pathways at several distinct steps in pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) as well as in effector-triggered immunity (ETI). This study shows that CA mutations are valuable complementary tools to study MAPK signaling pathways in planta.

Constitutively Active Mitogen-Activated Protein Kinase Versions Reveal Functions of Arabidopsis MPK4 in Pathogen Defense Signaling.

Plant Cell. 2012 Oct 31 PMID: 23115249
Berriri S, Garcia AV, Frei Dit Frey N, Rozhon W, Pateyron S, Leonhardt N, Montillet JL, Leung J, Hirt H, Colcombet J.


Plant mitogen-activated protein kinases (MAPKs) are involved in important processes, including stress signaling and development. In a functional yeast screen, we identified mutations that render Arabidopsis thaliana MAPKs constitutively active (CA). Importantly, CA-MAPKs maintain their specificity toward known activators and substrates. As a proof-of-concept, Arabidopsis MAPK4 (MPK4) function in plant immunity was investigated. In agreement with the phenotype of mpk4 mutants, CA-MPK4 plants were compromised in pathogen-induced salicylic acid accumulation and disease resistance. MPK4 activity was found to negatively regulate pathogen-associated molecular pattern-induced reactive oxygen species production but had no impact on callose deposition, indicating that CA-MPK4 allows discriminating between processes regulated by MPK4 activity from processes indirectly affected by mpk4 mutation. Finally, MPK4 activity was also found to compromise effector-triggered immunity conditioned by the Toll Interleukin-1 Receptor-nucleotide binding (NB)-Leu-rich repeat (LRR) receptors RPS4 and RPP4 but not by the coiled coil-NB-LRR receptors RPM1 and RPS2. Overall, these data reveal important insights on how MPK4 regulates plant defenses and establishes that CA-MAPKs offer a powerful tool to analyze the function of plant MAPK pathways.

Automated phosphopeptide identification using multiple MS/MS fragmentation modes.

J Proteome Res. 2012 Oct 24 PMID: 23094866
Vandenbogaert M, Hourdel V, Jardin-Mathé O, Bigeard J, Bonhomme L, Legros V, Hirt H, Schwikowski B, Pflieger D.


Phosphopeptide identification is still a challenging task because fragmentation spectra obtained by mass spectrometry do not necessarily contain sufficient fragment ions to establish with certainty the underlying amino acid sequence and the precise phosphosite. To improve upon this, it has been suggested to acquire pairs of spectra from every phosphorylated precursor ion using different fragmentation modes, for example CID, ETD and/or HCD. The development of automated tools for the interpretation of these paired spectra has however, until now, lagged behind. Using phosphopeptide samples analyzed by an LTQ-Orbitrap instrument, we here assess an approach in which, on each selected precursor, a pair of CID spectra, with or without Multistage Activation (MSA or MS2, respectively), are acquired in the linear ion trap. We applied this approach on phosphopeptide samples of variable proteomic complexity obtained from Arabidopsis thaliana. We present a straightforward computational approach to reconcile sequence and phosphosite identifications provided by the database search engine Mascot on the spectrum pairs, using two simple filtering rules, at the amino acid sequence and phosphosite localization levels. If multiple sequences and/or phosphosites are likely, they are reported in the consensus sequence. Using our program FragMixer, we could assess that on samples of moderate complexity, it was worth combining the two fragmentation schemes on every precursor ion to help efficiently identify amino acid sequences and precisely localize phosphosites. FragMixer can be flexibly configured, independently of the Mascot search parameters, and can be applied to various spectrum pairs, such as MSA/ETD and ETD/HCD, to automatically compare and combine the information provided by these more differing fragmentation modes. The software is openly accessible and can be downloaded from our Web site at http://proteomics.fr/FragMixer.

Role of AGC kinases in plant growth and stress responses.

Cell Mol Life Sci. 2012 Jul 31. PMID: PMID: 22847330
Garcia AV, Al-Yousif M, Hirt H.

AGC kinases are important regulators of cell growth, metabolism, division, and survival in mammalian systems. Mutation or deregulation of members of this family of protein kinases contribute to the pathogenesis of many human diseases, including cancer and diabetes. Although AGC kinases are conserved in the plant kingdom, little is known about their molecular functions and targets. Some of the best-studied plant AGC kinases mediate auxin signaling and are thereby involved in the regulation of growth and morphogenesis. Furthermore, certain members are regulated by lipid-derived signals via the 3-phosphoinositide-dependent kinase 1 (PDK1) and the kinase target of rapamycin (TOR), similar to its animal counterparts. In this review, we discuss recent findings on plant AGC kinases that unravel important roles in the regulation of plant growth, immunity and cell death, and connections to stress-induced mitogen-activated protein kinase signaling cascades.

An image classification approach to analyze the suppression of plant immunity by the human pathogen Salmonella Ty-phimurium.

BMC Bioinformatics. 2012 Jul 19;13(1):171. PMID: 22812426
Schikora M, Neupane B, Madhogaria S, Koch W, Cremers D, Hirt H, Kogel KH, Schikora A.

The enteric pathogen Salmonella is the causative agent of the majority of food-borne bacterial poisonings.Resent research revealed that colonization of plants by Salmonella is an active infection process. Salmonellachanges the metabolism and adjust the plant host by suppressing the defense mechanisms. In this report wedeveloped an automatic algorithm to quantify the symptoms caused by Salmonella infection on Arabidopsis.
The algorithm is designed to attribute image pixels into one of the two classes: healthy and unhealthy. Thetask is solved in three steps. First, we perform segmentation to divide the image into foreground andbackground. In the second step, a support vector machine (SVM) is applied to predict the class of each pixelbelonging to the foreground. And finally, we do refinement by a neighborhood-check in order to omit allfalsely classified pixels from the second step. The developed algorithm was tested on infection with thenon-pathogenic E. coli and the plant pathogen Pseudomonas syringae and used to study the interactionbetween plants and Salmonella wild type and T3SS mutants. We proved that T3SS mutants of Salmonellaare unable to suppress the plant defenses. Results obtained through the automatic analyses were furtherverified on biochemical and transcriptome levels.
This report presents an automatic pixel-based classification method for detecting "unhealthy" regions in leafimages. The proposed method was compared to existing method and showed a higher accuracy. We usedthis algorithm to study the impact of the human pathogenic bacterium Salmonella Typhimurium on plantsimmune system. The comparison between wild type bacteria and T3SS mutants showed similarity in theinfection process in animals and in plants. Plant epidemiology is only one possible application of theproposed algorithm, it can be easily extended to other detection tasks, which also rely on color information,or even extended to other features.

Plants as alternative hosts for Salmonella.

Trends Plant Sci. 2012 Apr 16. PMID: 22513107
Schikora A, Garcia AV, Hirt H.
Institute for Plant Pathology and Applied Zoology, Research Centre for BioSystems, Land Use and Nutrition, JL University Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany.

Recent findings show that many human pathogenic bacteria can use multiple host organisms. For example, Salmonella Typhimurium can use plants as alternative hosts to humans and other animals. These bacteria are able to adhere to plant surfaces and actively infect the interior of plants. Similarly to the infection of animal cells, S. Typhimurium suppresses plant defense responses by a type III secretion mechanism, indicating that these bacteria possess a dedicated multi-kingdom infection strategy, raising the question of host specificity. In addition, evidence is accumulating that the interaction of Salmonella with plants is an active process with different levels of specificity, because different Salmonella serovars show variations in pathogenicity, and different plant species reveal various levels of resistance towards these bacteria.


Improvement of stress tolerance in plants by genetic manipulation of mitogenactivated protein kinases

Biotechnology Advances 2011, Dec 06 PMID: 22198202
Samajova O,Plihal O,Yousif MA,Hirt H,Samaj J

Plant stress tolerance depends on many factors among which signaling by mitogen-activated protein-kinase (MAPK) modules plays a crucial role. Reversible phosphorylation of MAPKs, their upstream activators and downstream targets such as transcription factors can trigger a myriad of transcriptomic, cellular and physiological responses. Genetic manipulation of abundance and/or activity of some of these modular MAPK components can lead to better stress tolerance in Arabidopsis and crop plant species such as tobacco and cereals. The main focus of this review is devoted to the MAPK-related signaling components which show the most promising biotechnological potential. Additionally, recent studies identified MAPK components to be involved both in plant development as well as in stress responses, suggesting that these processes are tightly linked in plants.

Conservation of Salmonella Infection Mechanisms in Plants and Animals


PLoS One September 2011 Volume 6 Issue 9, e24112 PMID: 21915285
Adam Schikora, Isabelle Virlogeux-Payant, Eduardo Bueso, Ana V. Garcia, Theodora Nilau, Amélie Charrier, Sandra Pelletier, Pierrette Menanteau, Manuela Baccarini, Philippe Velge, Heribert Hirt

Salmonella virulence in animals depends on effectors injected by Type III Secretion Systems (T3SSs). In this report we demonstrate that Salmonella mutants that are unable to deliver effectors are also compromised in infection of Arabidopsis thaliana plants. Transcriptome analysis revealed that in contrast to wild type bacteria, T3SS mutants of Salmonella are compromised in suppressing highly conserved Arabidopsis genes that play a prominent role during Salmonella infection of animals. We also found that Salmonella originating from infected plants are equally virulent for human cells and mice. These results indicate a high degree of conservation in the defense and infection mechanism of animal and plant hosts during Salmonella infection.

Press Release Sept 09, 2011:
Salmonella uses similar mechanism to infect plants and humans
INRA - CNRS - Université d'Evry

AGC kinases in plant development and defense

Plant Signaling & Behavior 6:7, 1-4; July 2011; © 2011 Landes Bioscience PMID: 22005000
Heribert Hirt, Ana V. Garcia, Ralf Oelmüller

More than 100,000 publications demonstrate that AGC kinases are important regulators of growth, metabolism, proliferation, cell divison, survival and apoptosis in mammalian systems.1 Mutation and/or dysregulation of these kinases contribute to the pathogenesis of many human diseases, including cancer and diabetes. Although AGC kinases are also present in plants, little is known about their functions. We demonstrated that the AGC kinase OXIDATIVE SIGNAL-INDUCIBLE1 (OXI1/AGC2-1) regulate important developmental processes and defense responses in plants. The summary of recent progress also demonstrates that we are only beginning to understand the role of this kinase pathway in plants.

The OXI1 Kinase Pathway Mediates Piriformospora indica-Induced Growth Promotion in Arabidopsis

PLOS Pathogens May 2011 doi:10.1371/journal.ppat.1002051 PMID: 21625539
Iris Camehl, Corinna Drzewiecki, Jyothilakshmi Vadassery, Bationa Shahollari, Irena Sherameti, Celine Forzani, Teun Munnik, Heribert Hirt, Ralf Oelmüller

Piriformospora indica is an endophytic fungus that colonizes roots of many plant species and promotes growth and resistance to certain plant pathogens. Despite its potential use in agriculture, little is known on the molecular basis of this beneficial plant-fungal interaction. In a genetic screen for plants, which do not show a P. indica- induced growth response, we isolated an Arabidopsis mutant in the OXI1 (Oxidative Signal Inducible1) gene. OXI1 has been characterized as a protein kinase which plays a role in pathogen response and is regulated by H2O2 and PDK1 (3-PHOSPHOINOSITIDE-DEPENDENT PROTEIN KINASE1). A genetic analysis showed that double mutants of the two closely related PDK1.1 and PDK1.2 genes are defective in the growth response to P. indica. While OXI1 and PDK1 gene expression is upregulated in P. indica-colonized roots, defense genes are downregulated, indicating that the fungus suppresses plant defense reactions. PDK1 is activated by phosphatidic acid (PA) and P. indica triggers PA synthesis in Arabidopsis plants. Under beneficial co-cultivation conditions, H2O2 formation is even reduced by the fungus. Importantly, phospholipase D (PLD)a1 or PLDd mutants, which are impaired in PA synthesis do not show growth promotion in response to fungal infection. These data establish that the P. indica-stimulated growth response is mediated by a pathway consisting of the PLD-PDK1-OXI1 cascade.

Isolation and characterization of plant protein complexes by mass spectrometry.

Proteomics. 2010 Nov 2. doi: 10.1002/pmic.201000635. PMID: 21472857
Pflieger D, Bigeard J, Hirt H.

The components that enable cells and organisms to fulfill a plethora of chemical and physical reactions, including their ability to metabolize, replicate, repair and communicate with their environment are mostly based on the functioning of highly complex cellular machines which are to a large extent composed of proteins. With the development of MS techniques compatible with the analysis of minute amounts of biological material, it has become more and more feasible to dissect the composition and modification of these protein machineries. Indeed, new purification methods of protein complexes followed by MS analysis together with the genomic sequencing of various organisms - and in particular of crop species - now provide unforeseen insight to understand biological processes at a molecular level. We here review the current state of the art of in vivo protein complex isolation and their MS-based analytical characterization, emphasizing on the tandem affinity purification approach.

Linking the proteins-Elucidation of proteome-scale networks using mass spectrometry.

Mass Spectrom Rev. 2011 Mar;30(2):268-97. doi: 10.1002/mas.20278. Epub 2010 May 24. PMID: 21337599
Pflieger D, Gonnet F, de la Fuente van Bentem S, Hirt H, de la Fuente A.
Laboratoire Analyse et Modélisation pour la Biologie et l'Environnement, Université d'Evry Val d'Essonne, CNRS UMR 8587, Evry, France

Fig 1

Proteomes are intricate. Typically, thousands of proteins interact through physical association and post-translational modifications (PTMs) to give rise to the emergent functions of cells. Understanding these functions requires one to study proteomes as "systems" rather than collections of individual protein molecules. The abstraction of the interacting proteome to "protein networks" has recently gained much attention, as networks are effective representations, that lose specific molecular details, but provide the ability to see the proteome as a whole. Mostly two aspects of the proteome have been represented by network models: proteome-wide physical protein-protein-binding interactions organized into Protein Interaction Networks (PINs), and proteome-wide PTM relations organized into Protein Signaling Networks (PSNs). Mass spectrometry (MS) techniques have been shown to be essential to reveal both of these aspects on a proteome-wide scale. Techniques such as affinity purification followed by MS have been used to elucidate protein-protein interactions, and MS-based quantitative phosphoproteomics is critical to understand the structure and dynamics of signaling through the proteome. We here review the current state-of-the-art MS-based analytical pipelines for the purpose to characterize proteome-scale networks. © 2010 Wiley Periodicals, Inc., Mass Spec Rev 30:268-297, 2011.

The Arabidopsis protein kinase PTI1-4 is a common target of the oxidative signal-inducible1 (OXI1) and MAP kinases.

FEBS J. 2011 Jan 29. doi: 10.1111/j.1742-4658.2011.08033.x. PMID: 21276203
Forzani C, Carreri A, de la Fuente van Bentem S, Lecourieux D, Lecourieux F, Hirt H.
Max Perutz Laboratories, Dr. Bohrgasse 9, A-1030 Vienna, Austria
URGV Plant Genomics, INRA-CNRS-University of Evry, 2 rue Gaston Cremieux, F-91057

Fig 2

In Arabidopsis thaliana the serine/threonine protein kinase oxidative signal-inducible1 (OXI1), mediates oxidative stress signalling. Its activity is required for the full activation of the mitogen-activated protein kinases (MAPKs), MPK3 and MPK6 in response to oxidative stress. In addition, the serine/threonine protein kinase PTI1-2 has been positioned downstream from OXI1 but whether PTI1-2 signals through MAPK cascades is unclear. Using a yeast two-hybrid screen we show that OXI1 also interacts with PTI1-4. OXI1 and PTI1-4 are stress-responsive genes and are expressed in the same tissues. Therefore, studies were undertaken to determine whether PTI1-4 is positioned in the OXI1/MAPK signalling pathway. The interaction between OXI1 and PTI1-4 was confirmed by using in vivo co-immunoprecipitation experiments. OXI1 as well as PTI1-4 were substrates of MPK3 and MPK6 in vitro. Whereas no direct interaction was detected between MPK3, MPK6 and OXI1, in vitro binding studies showed an interaction between MPK3, MPK6 and PTI1-4. In addition, PTI1-4 and MPK6 were found in vivo in the same protein complex. These results demonstrate that PTI1-4 signals via OXI1 and MPK6 signalling cascades.
Journal compilation © 2011 Federation of European Biochemical Societies.


The MAP Kinase MPK4 Is Required for Cytokinesis in Arabidopsis thaliana

Plant Cell Advance Online Publication Published on November 23, 2010; 10.1105/tpc.110.077164 PMID: 21098735
Ken Kosetsu (a), Sachihiro Matsunaga (b), Hirofumi Nakagami (c), Jean Colcombet (d), Michiko Sasabe (a), Takashi Soyano (a), Yuji Takahashi (a), Heribert Hirt (c, d) and Yasunori Machida (a, 3)
(a) Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan
(b) Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan
(c) Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, A-1030 Vienna, Austria
(d) Unité de Recherche en Génomique Végétale Plant Genomics, Institut National de la Recherche Agronomique, Centre National de la Recherche Scientifique, Université d’Evry, 91057 Evry, France
(3) Address correspondence to yas@bio.nagoya-u.ac.jp


Cytokinesis in plants is achieved by the formation of the cell plate. A pathway that includes mitogen-activated protein (MAP) kinase kinase kinase and MAP kinase kinase (MAPKK) plays a key role in the control of plant cytokinesis. We show here that a MAP kinase, MPK4, is required for the formation of the cell plate in Arabidopsis thaliana. Single mutations in MPK4 caused dwarfism and characteristic defects in cytokinesis, such as immature cell plates, which became much more prominent upon introduction of a mutation in MKK6/ANQ, the MAPKK for cytokinesis, into mpk4. MKK6/ANQ strongly activated MPK4 in protoplasts, and kinase activity of MPK4 was detected in wild-type tissues that contained dividing cells but not in mkk6/anq mutants. Fluorescent protein–fused MPK4 localized to the expanding cell plates in cells of root tips. Expansion of the cell plates in mpk4 root tips appeared to be retarded. The level of MPK11 transcripts was markedly elevated in mpk4 plants, and defects in the mpk4 mpk11 double mutant with respect to growth and cytokinesis were more severe than in the corresponding single mutants. These results indicate that MPK4 is the downstream target of MKK6/ANQ in the regulation of cytokinesis in Arabidopsis and that MPK11 is also involved in cytokinesis.

New insights into an old story: Agrobacterium-induced tumour formation in plants by plant transformation.

EMBO J. 2010 Feb 11. PMID: 20150897
Andrea Pitzschke (1) and Heribert Hirt (2,3,*)
(1) Department of Applied Genetics and Cell Biology, University of Natural Resources and Applied Life Sciences, Muthgasse 18, Vienna, Austria,
(2) Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, Dr-Bohr-Gasse 9, Vienna, Austria and
(3) URGV Plant Genomics, INRA-University of Evry, 2 Rue Gaston Cremieux, Evry, France


Agrobacterium tumefaciens causes tumour formation in plants. Plant signals induce in the bacteria the expression of a range of virulence (Vir) proteins and the formation of a type IV secretion system (T4SS). On attachment to plant cells, a transfer DNA (T-DNA) and Vir proteins are imported into the host cells through the bacterial T4SS. Through interaction with a number of host proteins, the Vir proteins suppress the host innate immune system and support the transfer, nuclear targeting, and integration of T-DNA into host cell chromosomes. Owing to extensive genetic analyses, the bacterial side of the plant-Agrobacterium interaction is well understood. However, progress on the plant side has only been achieved recently, revealing a highly complex molecular choreography under the direction of the Vir proteins that impinge on multiple processes including transport, transcription, and chromosome status of their host cells.

Mechanism of MAPK-targeted gene expression unraveled in plants.

Cell Cycle. 2010 Jan 1;9(1):18-9. Epub 2010 Jan 14. PMID: 20016264
Andrea Pitschke (1, +), Heribert Hirt (1, 2)
(1) Department of Plant Molecular Biology; Max F. Perutz Laboratories; University of Vienna; Vienna, Austria;
(2) URGV Plant Genomics Laboratory; Evry, France
(+) Present address: Department of Applied Genetics and Cell Biology; University of Natural Resources and Applied Life Sciences; Vienna, Austria

Mitogen-activated protein kinase (MAPK) cascades—phosphorelay modules minimally composed of a MAPK kinase kinase, a MAPK kinase and a MAPK—are key players in eukaryotic cell signaling, linking developmental or environmental stimulus perception to alteration/adaptation of gene expression. Their prominent role in mammalian cancer development, but also in regulating plant development and stress adaptation are well-documented (reviewed in refs. 1–4). Through their kinase activity, MAPK cascades translate incoming environmental signals into post-translational modification of target proteins, e.g., transcription factors, to ultimately reorganize gene expression and stress adaptation.


Bioinformatic and systems biology tools to generate testable models of signalling pathways and their targets

Plant Physiology Preview. Published on November 13, 2009, as DOI:10.1104/pp.109.149583, PMID: 19915012
Andrea Pitzschke (a) and Heribert Hirt (b,c)
(a) Department of Applied Genetics and Cell Biology; University of Natural Resources and Applied Life Sciences, Muthgasse 18, 1190 Vienna, Austria
(b) Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, Dr.-Bohr-Gasse 9, 1030 Vienna, Austria
(c) URGV Plant Genomics Laboratory, 2 Rue Gaston Cremieux, 91057 Evry, France

Microarray comparison Response to signals

Over the last years a number of bioinformatic software programs have been developed in the area of molecular biology. The application of these bioinformatic tools to the wealth of existing transcriptomic and proteomic data can be used to predict the structure and hierarchy of signalling pathways and gene networks. In genetically tractable model organisms such as Arabidopsis thaliana, these hyphotheses can be validated experimentally and modified in reiterative cycles, giving hypothesis-driven research high feasibility. These predictive systems biology approaches significantly reduce the scale, time and manpower usually required in classical approaches. Here, we provide an overview on the use of currently available tools in deciphering signalling pathways in Arabidopsis research.



VIP1 response elements mediate mitogen-activated protein kinase 3-induced stress gene expression.

Proc Natl Acad Sci U S A. 2009 Oct 9. PMID: 19820165
Pitzschke A, Djamei A, Teige M, Hirt H.
Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, Dr.-Bohr-Gasse 9, 1030 Vienna, Austria.
URGV Plant Genomics Laboratory, 2 Rue Gaston Crémieux, 91057 Evry, France

Fig. 1. Indirect induction of PR1 by VIP1. (A) bZIP residue Lys-212 is essential for
VIP1-induced PR1 expression. Protoplasts were transfected with the PR1::GUS
reporter construct alone or in combination with constructs for overexpression of
VIP1-Myc or VIP1 K212-Myc mutant variants. Given aremeanvalues and standard
deviations of GUS activity (nmol 4-MU) min1 (mg protein)1; n  6. Transgene
expression was visualized by immunoblotting with anti-Myc antibody. Equal
loading was visualized by staining of themembranewith PonceauS. (B) VIP1 does
not bind to the PR1 promoter (EMSA). The indicated PR1 promoter fragments or
the as-1 element were biotin-labeled and incubated without or with recombinant
VIP1 protein. Nonbound DNA fragments are indicated by arrows. No band
shift was observed.

The plant pathogen Agrobacterium tumefaciens transforms plant cells by delivering its T-DNA into the plant cell nucleus where it integrates into the plant genome and causes tumor formation. A key role of VirE2-interacting protein 1 (VIP1) in the nuclear import of T-DNA during Agrobacterium-mediated plant transformation has been unravelled and VIP1 was shown to undergo nuclear localization upon phosphorylation by the mitogen-activated protein kinase MPK3. Here, we provide evidence that VIP1 encodes a functional bZIP transcription factor that stimulates stress-dependent gene expression by binding to VIP1 response elements (VREs), a DNA hexamer motif. VREs are overrepresented in promoters responding to activation of the MPK3 pathway such as Trxh8 and MYB44. Accordingly, plants overexpressing VIP1 accumulate high levels of Trxh8 and MYB44 transcripts, whereas stress-induced expression of these genes is impaired in mpk3 mutants. Trxh8 and MYB44 promoters are activated by VIP1 in a VRE-dependent manner. VIP1 strongly enhances expression from a synthetic promoter harboring multiple VRE copies and directly interacts with VREs in vitro and in vivo. Chromatin immunoprecipitation assays of the MYB44 promoter confirm that VIP1 binding to VREs is enhanced under conditions of MPK3 pathway stimulation. These results provide molecular insight into the cellular mechanism of target gene regulation by the MPK3 pathway.

MAP KINASE PHOSPHATASE1 and PROTEIN TYROSINE PHOSPHATASE1 Are Repressors of Salicylic Acid Synthesis and SNC1-Mediated Responses in Arabidopsis

Plant Cell Advance Online Publication
Published on September 29, 2009; 10.1105/tpc.109.067678 PMID: 19789277
Sebastian Bartels (1), Jeffrey C. Anderson (2), Marina A. González Besteiro (3), Alessandro Carreri (4), Heribert Hirt (5), Antony Buchala (6), Jean-Pierre Métraux (6), Scott C. Peck (2), and Roman Ulm (7*)
(1) Faculty of Biology, Institute of Biology II, University of Freiburg, D-79104 Freiburg, Germany
(2) Department of Biochemistry, University of Missouri, Columbia, Missouri 65211
(3) Faculty of Biology, Institute of Biology II, University of Freiburg, D-79104 Freiburg, Germany; Spemann Graduate School of Biology and Medicine, University of Freiburg, D-79104 Freiburg, Germany
(4) Max F. Perutz Laboratories, University of Vienna, A-1030 Vienna, Austria
(5) Max F. Perutz Laboratories, University of Vienna, A-1030 Vienna, Austria; URGV - Plant Genomics, INRA, CNRS, University Evry, F-91057 Evry Cedex, France
(6) Department of Biology, University of Fribourg, CH-1700 Fribourg, Switzerland
(7) Faculty of Biology, Institute of Biology II, University of Freiburg, D-79104 Freiburg, Germany; Centre for Biological Signaling Studies (bioss), University of Freiburg, D-79104 Freiburg, Germany
(*) To whom correspondence should be addressed: roman.ulm@biologie.uni-freiburg.de

MKP1 and PTP1 Repress Defense Responses

Mitogen-activated protein (MAP) kinase phosphatases are important negative regulators of the levels and kinetics of MAP kinase activation that modulate cellular responses. The dual-specificity phosphatase MAP KINASE PHOSPHATASE1 (MKP1) was previously shown to regulate MAP KINASE6 (MPK6) activation levels and abiotic stress responses in Arabidopsis thaliana. Here, we report that the mkp1 null mutation in the Columbia (Col) accession results in growth defects and constitutive biotic defense responses, including elevated levels of salicylic acid, camalexin, PR gene expression, and resistance to the bacterial pathogen Pseudomonas syringae. PROTEIN TYROSINE PHOSPHATASE1 (PTP1) also interacts with MPK6, but the ptp1 null mutant shows no aberrant growth phenotype. However, the pronounced constitutive defense response of the mkp1 ptp1 double mutant reveals that MKP1 and PTP1 repress defense responses in a coordinated fashion. Moreover, mutations in MPK3 and MPK6 distinctly suppress mkp1 and mkp1 ptp1 phenotypes, indicating that MKP1 and PTP1 act as repressors of inappropriate MPK3/MPK6-dependent stress signaling. Finally, we provide evidence that the natural modifier of mkp1 in Col is largely the disease resistance gene homolog SUPPRESSOR OF npr1-1, CONSTITUTIVE 1 (SNC1) that is absent in the Wassilewskija accession. Our data thus indicate a major role of MKP1 and PTP1 in repressing salicylic acid biosynthesis in the autoimmune-like response caused by SNC1.

MAPK cascade signalling networks in plant defence.

Curr Opin Plant Biol. 2009 Jul 14. PMID: 19608449
Pitzschke A, Schikora A, Hirt H.
Max F. Perutz Laboratories, University of Vienna, Dr. Bohrgasse 9, 1030 Vienna, Austria.
URGV Plant Genomics, INRA-CNRS-University of Evry, 2 rue Gaston Cremieux, 91057 Evry, France

The sensing of stress signals and their transduction into appropriate responses is crucial for the adaptation and survival of plants. Kinase cascades of the mitogen-activated protein kinase (MAPK) class play a remarkably important role in plant signalling of a variety of abiotic and biotic stresses. MAPK cascade-mediated signalling is an essential step in the establishment of resistance to pathogens. Here, we describe the most recent insights into MAPK-mediated pathogen defence response regulation with a particular focus on the cascades involving MPK3, MPK4 and MPK6. We also discuss the strategies developed by plant pathogens to circumvent, inactivate or even 'hijack' MAPK-mediated defence responses.

Transgenerational stress memory is not a general response in Arabidopsis.

PLoS ONE. 2009;4(4):e5202. Epub 2009 Apr 21. PMID: 19381297
Pecinka A (1), Rosa M (1), Schikora A (2), Berlinger M (3), Hirt H (2), Luschnig C (3), Mittelsten Scheid O (1)
(1) Gregor Mendel Institute of Molecular Plant Biology (GMI), Austrian Academy of Sciences, Vienna, Austria,
(2) INRA – URGV, Plant Genomics Research Unit, Evry, France
(3) University of Natural Resources and Applied Life Sciences (BOKU), Vienna, Austria

Expression of genes involved in homologous recombination in treated S0 and untreated S1 and S2 generations

Adverse conditions can trigger DNA damage as well as DNA repair responses in plants. A variety of stress factors are known to stimulate homologous recombination, the most accurate repair pathway, by increasing the concentration of necessary enzymatic components and the frequency of events. This effect has been reported to last into subsequent generations not exposed to the stress. To establish a basis for a genetic analysis of this transgenerational stress memory, a broad range of treatments was tested for quantitative effects on homologous recombination in the progeny. Several Arabidopsis lines, transgenic for well-established recombination traps, were exposed to 10 different physical and chemical stress treatments, and scored for the number of somatic homologous recombination (SHR) events in the treated generation as well as in the two subsequent generations that were not treated. These numbers were related to the expression level of genes involved in homologous recombination and repair. SHR was enhanced after the majority of treatments, confirming previous data and adding new effective stress types, especially interference with chromatin. Compounds that directly modify DNA stimulated SHR to values exceeding previously described induction rates, concomitant with an induction of genes involved in SHR. In spite of the significant stimulation in the stressed generations, the two subsequent non-treated generations only showed a low and stochastic increase in SHR that did not correlate with the degree of stimulation in the parental plants. Transcripts coding for SHR enzymes generally returned to pre-treatment levels in the progeny. Thus, transgenerational effects on SHR frequency are not a general response to abiotic stress in Arabidopsis and may require special conditions.

Disentangling the complexity of mitogen-activated protein kinases and reactive oxygen species signaling.

Plant Physiol. 2009 Feb;149(2):606-15. [PubMed - in process] PMID: 19201916
Andrea Pitzschke (1) and Heribert Hirt (1, 2)
(1) Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, 1030 Vienna, Austria (A.P., H.H.)
(2) URGV Plant Genomics, INRA-CNRS-Université d'Evry, 91057 Evry, France (H.H.)

For about 2 million years, molecular oxygen arising from photosynthetic processes has become pivotal to almost all organisms. Reactive oxygen species (ROS), the partially reduced or activated derivatives of oxygen (hydrogen peroxide [H₂O₂], HO·, ¹O₂, O₂–), are the highly reactive by-products of aerobic metabolism. They arise from various chemical reactions and can lead to oxidative damage of cells.
Plants possess a sophisticated ROS network, comprising antioxidative enzymes, antioxidants, and ROS-producing enzymes, which allow them to keep ROS levels under tight control. Moreover, as research of the past few years has shown, plants have developed efficient strategies for targeted production of ROS. For instance, ROS play a role in programmed cell death (PCD), development, and stress response. Mitogen-activated protein kinase (MAPK) cascades are key players in ROS signaling. Several studies have shown that MAPK signaling pathways are not only induced by ROS but can also regulate ROS production. MAPK cascades are signaling modules that minimally consist of a MAPK kinase kinase (MAPKKK/MEKK), a MAPK kinase (MAPKK/MKK), and MAPK. Upon a stimulus-triggered activation of a MAPKKK, the signal is transduced via phosphorylation-mediated activation of a corresponding downstream MAPKK, which in turn phosphorylates and thereby activates a specific MAPK. The Arabidopsis (Arabidopsis thaliana) genome contains more than 60 MAPKKKs, 20 MAPKs, and 10 MAPKs, which can, depending on the environmental stimulus or developmental stage, engage in different MAPK modules. With the characterization of mutants affected in pathogen response as well as the development and dynamics of stomata, the network of MAPK cascade activation and ROS is being disentangled. Here, we discuss the most recent insights into ROS production and perception involving MAPK-mediated signaling.

Protein tyrosine phosphorylation in plants: more abundant than expected?

Trends Plant Sci. 2009 Jan 20. [Epub ahead of print] PMID: 19162527
de la Fuente van Bentem S (1), Hirt H. (1,2)
(1) Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, Dr. Bohr-Gasse 9, 1030 Vienna, Austria
(2) Unité de Recherche en Génomique Végétale, INRA-CNRS-UEVE, 2 rue Gaston Crémieux, 91057 Evry Cédex, France

Protein phosphorylation in eukaryotes predominantly occurs on serine (Ser) and threonine (Thr) residues, whereas phosphorylation on tyrosine (Tyr) residues is less abundant. Plants lack classic Tyr kinases, such as the epidermal growth factor receptor, that govern Tyr phosphorylation in animals. A long-standing debate questions whether plants have any Tyr-specific kinases and, although several protein kinases with both Ser/Thr and Tyr specificities exist, data supporting the existence of other such kinases are scarce. As we discuss here, mass-spectrometry-based analyses now indicate that Tyr phosphorylation is as extensive in plants as it is in animals. However, careful inspection of available data indicates that these promising mass spectrometry studies have to be interpreted with caution before current ideas on Tyr phosphorylation in plants are revised.

A Major Role of the MEKK1–MKK1/2–MPK4 Pathway in ROS Signalling

Molecular Plant Advance Access published January 6, 2009 | Molecular Plant • Pages 1–18, 2008 PMID: 19529823
Andrea Pitzschke (a), Armin Djamei (a,b,) Frédérique Bitton (c) and Heribert Hirt (a,c,1)
(a) Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, Dr.-Bohr-Gasse 9, 1030 Vienna, Austria
(b) Present address: Max-Planck-Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse, 35043 Marburg, Germany
(c) URGV Plant Genomics Laboratory, 2 Rue Gaston Cre´ mieux, 91057 Evry, France
(1) To whom correspondence should be addressed. E-mail hirt@evry.inra.fr

molecular plant

ABSTRACT Over the last few years, it has become evident that reactive oxygen species (ROS) signalling plays an important role in various physiological responses, including pathogen defense and stomatal opening/closure. On the other hand, ROS overproduction is detrimental for proper plant growth and development, indicating that the regulation of an appropriate redox balance is essential for plants. ROS homeostasis in plants involves the mitogen-activated protein kinase (MAPK) pathway consisting of the MAPK kinase kinase MEKK1 and the MAPK MPK4. Phenotypic and molecular analysis revealed that the MAPK kinases MKK1 and MKK2 are part of a cascade, regulating ROS and salicylic acid (SA) accumulation. Gene expression analysis shows that of 32 transcription factors reported to be highly responsive to multiple ROS-inducing conditions, 20 are regulated by the MEKK1, predominantly via the MEKK1–MKK1/2–MPK4 pathway. However, MEKK1 also functions on other as yet unknown pathways and part of the MEKK1-dependent MPK4 responses are regulated independently of MKK1 and MKK2. Overall, this analysis emphasizes the central role of this MAPK cascade in oxidative stress signalling, but also indicates the high level of complexity revealed by this signalling network


Towards functional phosphoproteomics by mapping differential phosphorylation events in signaling networks.

Proteomics. 2008 Oct 29;8(21):4453-4465. PMID: 18972525
de la Fuente van Bentem S, Mentzen WI, de la Fuente A, Hirt H.
Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, Vienna, Austria.

Protein phosphorylation plays a central role in many signal transduction pathways that mediate biological processes. Novel quantitative mass spectrometry-based methods have recently revealed phosphorylation dynamics in animals, yeast, and plants. These methods are important for our understanding of how differential phosphorylation participates in translating distinct signals into proper physiological responses, and shifted research towards screening for potential cancer therapies and in-depth analysis of phosphoproteomes. In this review, we aim to describe current progress in quantitative phosphoproteomics. This emerging field has changed numerous static pathways into dynamic signaling networks, and revealed protein kinase networks that underlie adaptation to environmental stimuli. Mass spectrometry enables high-throughput and high-quality analysis of differential phosphorylation at a site-specific level. Although determination of differential phosphorylation between treatments is analogous to detecting differential gene expression, the large body of statistical techniques that has been developed for analysis of differential gene expression is not generally applied for detecting differential phosphorylation. We suggest possible improvements for analysis of quantitative phosphorylation by increasing the number of biological replicates and adapting statistical tests used for gene expression profiling and widely implemented in freely available software tools.

Possible involvement of MAP kinase pathways in acquired metal-tolerance induced by heat in plants.

Planta. 2008 Aug;228(3):499-509. Epub 2008 May 28. doi 10.1007/s00425-008-0753-x PMID: 18506480
Po-Yu Chen (1), Kuo-Ting Lee (1), Wen-Chang Chi (1), Heribert Hirt (2), Ching-Chun Chang (3) and Hao-Jen Huang (1)
(1) Department of Life Sciences, National Cheng Kung University, Tainan, 701, Taiwan, ROC
(2) Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, Dr. Bohr-Gasse 9, 1030 Vienna, Austria
(3) Institute of Biotechnology, National Cheng Kung University, Tainan, 701, Taiwan, ROC

fig 2 b

Cross tolerance is a phenomenon that occurs when a plant, in resisting one form of stress, develops a tolerance to another form. Pretreatment with nonlethal heat shock has been known to protect cells from metal stress. In this study, we found that the treatment of rice roots with more than 25 muM of Cu(2+) caused cell death. However, heat shock pretreatment attenuated Cu(2+)-induced cell death. The mechanisms of the cross tolerance phenomenon between heat shock and Cu(2+) stress were investigated by pretreated rice roots with the protein synthesis inhibitor cycloheximide (CHX). CHX effectively block heat shock protection, suggesting that protection of Cu(2+)-induced cell death by heat shock was dependent on de novo protein synthesis. In addition, heat pretreatment downregulated ROS production and mitogen-activated protein kinase (MAPK) activities, both of which can be greatly elicited by Cu(2+) stress in rice roots. Moreover, the addition of purified recombinant GST-OsHSP70 fusion proteins inhibited Cu(2+)-enhanced MAPK activities in an in vitro kinase assay. Furthermore, loss of heat shock protection was observed in Arabidopsis mkk2 and mpk6 but not in mpk3 mutants under Cu(2+) stress. Taken together, these results suggest that the interaction of OsHSP70 with MAPKs may contribute to the cellular protection in rice roots from excessive Cu(2+) toxicity.

Arabidopsis MAPKs: a complex signalling network involved in multiple biological processes.

Figure 1

Biochem J. 2008 Jul 15;413(2):217-26. PMID: 18570633
prj77/335 Colcombet J, Hirt H.

Many changes in environmental conditions and hormones are mediated by MAPK (mitogen-activated protein kinase) cascades in all eukaryotes, including plants. Studies of MAPK pathways in genetic model organisms are especially informative in revealing the molecular mechanisms by means of which MAPK cascades are controlled and modulate cellular processes. The present review highlights recent insights into MAPK-based signalling in Arabidopsis thaliana (thale cress), revealing the complexity and future challenges to understanding signal-transduction networks on a global scale.

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 ext 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 ext, 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 ext,  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, ext 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

Fig A B

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 PMID: 17630279

Alois Schweighofera, Vaiva Kazanaviciutea, Elisabeth Scheikla, Markus Teigea, Róbert Dóczia, Heribert Hirta, Manfred Schwanningerb, Merijn Kantc, Robert Schuurinkc, Felix Mauchd, Antony Buchalad, Francesca Cardinalee, and Irute Meskienea,

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


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 PMID: 17583510

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 Nürnberger1*

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 PMID: 17506336

Günter Brader1, Armin Djamei2, Markus Teige2, E. Tapio Palva1, and Heribert Hirt2,3

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 PMID: 17319843

Stefan Kempa1, Wilfried Rozhon1, Jozef Šamaj2,3, 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 Hirt1, 2,

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

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

Early phosphorylation events during flagellin signalling in Arabidopsis

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

Use of MAPK-specific antisera

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 *

A mitogen-activated protein kinase

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

Hirofumi Nakagami1,3 Hanka Soukupová2, Adam Schikora1, Viktor Žárskŷ2, Heribert Hirt1,4

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

Activation of SIMK and SAMK in primary lupin roots inoculated with rhizobia.

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

Identification of a conserved phosphorylation motif within RNA

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 Veronesea, Hirofumi Nakagamib, Burton Bluhma, Synan AbuQamara, Xi Chenc, John Salmeronc, Robert A. Dietrichc, Heribert Hirtb, and Tesfaye Mengisteaa

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

Root growth phenotypes of wild-type and bik1 plants.

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 Bentema, Elisabeth Roitingerb, Dorothea Anratherc, Edina Csaszarc and Heribert Hirta

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

Functional classification of intracellular phosphoproteins identified

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

ROS signaling in stomatal closure.

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 Andreasson1,6, Thomas Jenkins1.6, 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

MKS1 interaction, phosphorylation and localization in vivo. (D) MPK4-GFP, (E) MKS1-GFP and (F) GUS-GFP fusion proteins. Cyt: cytoplasm; Nuc: nuclei; Chl: chloroplast (orange autofluorescent); —10 mm size bar.

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

Plant Responses to Abiotic Stress Book Cover

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

Excess levels of CuCl2 induce hydrogen peroxide accumulation.

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.

Profiling of MKK2 Triggered Gene

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

Markus Teige1,4,*, Elisabeth Scheikl1,4, Thomas Eulgem2,5, Róbert Dóczi1, Kazuya Ichimura3, Kazuo Shinozaki3, Jeffery L. Dangl2, and Heribert Hirt1,*

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.

Schematic representation of MMK3 activation in different

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.

OXI1 is necessary for basal resistance to P. parasitica.

Nature 427, 858-861.

Maike C. Rentel1*, David Lecourieux2, Fatma Ouaked2, Sarah L. Usher3, Lindsay Petersen1,4, 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 ext

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 Ŝamaj1,3*, 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 Samajae, Miroslav Oveckaa,b,c, Andrej Hlavackab, Fatma Lecourieuxa, Irute Meskienea, Irene Lichtscheidld, Peter Lenarta, Ján Salaje, Dieter Volkmannb, Laszlo Bögref, Frantisek Baluskab,c and Heribert Hirta

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

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

Complexity, Cross Talk and Integration of Plant MAP Kinase Signalling Book Cover

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

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MAPK Group (Kazuya Ichimura et al.), Kazuya Ichimuraaa, Kazuo Shinozakia, Guillaume Tenab, Jen Sheenb, Yves Henrycc, Anthony Championc, Martin Kreisc, Shuqun Zhangd, Heribert Hirte, Cathal Wilsone, Erwin Heberle-Borse, Brian E. Ellisf, Peter C. Morrisg, Roger W. Innesh, Joseph R. Eckeri, Dierk Scheelj, Daniel F. Klessigk, Yasunori Machidal, John Mundym, Yuko Ohashin and John C. Walkero

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 Ŝamaj1,2, Miroslav Ovecka1,3,4, Andrej Hlavacka3, Fatma Lecourieux1, Irute Meskiene1, Irene Lichtscheidl5, Peter Lenard1, Ján Salaj2, Dieter Volkmann3, Lásló Bögre6, Frantiśek Baluśka3,4 and Heribert Hirt1,7

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.

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

Claudia Jonaka, László Ökrészb, László Bögrec and Heribert Hirta

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.

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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

MAP Kinases in Plant Signal Transduction Book Cover

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


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.

Book Cover 97

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 Kiegerla, Francesca Cardinalea, Christine Siligana, Andrea Grossb, Emmanuel Baudouina, Aneta Liwosza, Staffan Eklöfa, Sandra Tilla, László Bögrea, Heribert Hirta, and Irute Meskienea

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

Frantisek Balnska1,2, 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 p53WIG , we show that p53WIG kinase is activated immediately after wounding. Wound-induced activation of p53WIG kinase is a post-translational process, because the concentrations of p53WIG protein do not change in intact and wounded leaves, and inhibition of transcription or translation does not block activation by wounding. However, inactivation of p53WIG 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 Biolgy 42

Plant Molecular Biology, Volume 42, Number 6 ext / 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 ext

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 Romeisa, Pedro Piedrasa, Shuqun Zhangb, Daniel F. Klessigb, Heribert Hirtc and Jonathan D.G. Jonesa

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 Bögrea, Ornella Calderinia,b, Pavla Binarovac, Markus Mattaucha, Sandra Tilla, Stefan Kiegerla, Claudia Jonaka, Christina Pollascheka, Patrick Barkerd, Neville S. Huskissond, Heribert Hirta and Erwin Heberle-Borsa

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

MAP kinases in plant signal transduction

CMLS, Cellular and Molecular Life Sciiences 55 (1999) 204–213, 1420-682X:99:020204-10 $ 1.500.20:0 © Birkhäuser Verlag, Basel, 1999

C. Jonak, W. Ligterink and H. Hirt

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

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.


MP2C, a plant protein phosphatase 2C, functions as a negative regulator of mitogen-activated protein kinase pathways in yeast and plants

Proceedings of the National Academy of Sciences, PNAS 1998;95;1938-1943, doi:10.1073/pnas.95.4.1938 PMID: 9465121

Irute Meskiene, Laszlo Bögre, Walter Glaser, Judit Balog, Markus Brandstätter, Karin Zwerger, Gustav Ammerer, and Heribert Hirt

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

By interference of the yeast pheromone mitogen-activated protein kinase (MAPK) pathway with an alfalfa cDNA expression library, we have isolated the MP2C gene encoding a functional protein phosphatase type 2C. Epistasis analysis in yeast indicated that the molecular target of the MP2C phosphatase is Ste11, a MAPK kinase kinase that is a central regulator of the pheromone and osmosensing pathways. In plants, MP2C functions as a negative regulator of the stress-activated MAPK (SAMK) pathway that is activated by cold, drought, touch, and wounding. Although activation of the SAMK pathway occurs by a posttranslational mechanism, de novo transcription and translation of protein factor(s) are necessary for its inactivation. MP2C is likely to be this or one of these factors, because wound-induced activation of SAMK is followed by MP2C gene expression and recombinant glutathione S-transferase-MP2C is able to inactivate extracts containing wound-induced SAMK. Wound-induced MP2C expression is a transient event and correlates with the refractory period, i.e., the time when restimulation of the SAMK pathway is not possible by a second stimulation. These data suggest that MP2C is part of a negative feedback mechanism that is responsible for resetting the SAMK cascade in plants.

The SAM kinase pathway: An integrated circuit for stress signaling in plants.

J. Plant Res. 111, 339-344.

Meskiene, W. Ligterink, L. Bögre, C. Jonak, S. Kiegerl, J. Balog, S. Eklöf, G. Ammerer, and H. Hirt (1998)


Nod factor-induced cell cycle activation in root cortical cells.

Kluwer Acad. Publ. pp. 189-192.

Kondorosi, E., H. Trinh, F. Roudier, F. Foucher, D. Vaubert, A. Cebolla, A. Lodeiro, A. Feher, Z. Keleman, J. Györgyey, P. Mergaert, A. Kereszt, D. Dudits, H. Hirt, and A. Kondorosi (1997)

(eds. C. Elmerich, A. Kondorosi, W.E: Newton)

In: Biological Nitrogen Fixation for the 21st Century

Cell cycle regulation and nodule development.

(H. Bothe, ed.) NATO ASI series, Springer Verlag, Vol. G39, 63-65.

Meskiene, W.C. Yang, C. deBlank, L. Bögre, K. Zwerger, M. Brandstätter, M. Mattauch, T. Bisseling, and H. Hirt (1997)

In: Biological fixation of nitrogen for ecology and sustainable agriculture

Receptor-mediated activation of a MAP kinase in pathogen defense of plants.

Science 276, 2054-2057. PMID: 9197271 ext

Ligterink, T. Kroj, U. zur Nieden, H.. Hirt, and D. Scheel (1997)

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

Parsley cells recognize the fungal plant pathogen Phytophthora sojae through a plasma membrane receptor. A pathogen-derived oligopeptide elicitor binds to this receptor and thereby stimulates a multicomponent defense response through sequential activation of ion channels and an oxidative burst. An elicitor-responsive mitogen-activated protein (MAP) kinase was identified that acts downstream of the ion channels but independently or upstream of the oxidative burst. Upon receptor-mediated activation, the MAP kinase is translocated to the nucleus where it might interact with transcription factors that induce expression of defense genes.

The cdc2Ms kinase is differently regulated in the cytoplasm and in the nucleus.

Plant Physiol. 113, 841-852. PMID: 12223648

Bögre, K. Zwerger, I. Meskiene, P. Binarova, V. Csizmadia, C. Planck, E. Wagner, H. Hirt, and E. Heberle-Bors (1997)

Vienna Biocenter, lnstitute of Microbiology and Genetics, University of Vienna, Austria
Institute of Experimental Botany, Norman Borlaug Center for Plant Science, De Montfort University, Czech Republic
Vienna Biocenter, lnstitute of Molecular Pathology, Vienna, Austria

To study a cyclin-dependent kinase (CDK) from alfalfa (Medicago sativa L.), an antibody was raised against the C-terminal 16 amino acids of the protein cdc2aMs. The cdc2Ms protein was immunopurified with this antibody and its histone kinase activity was measured. The cdc2Ms kinase is activated at the G1/S transition when phosphate-starved cells from the G0 phase re-enter the cell cycle and remain active as cells transit the S, G2, and M phases, indicating that the same CDK regulates all of these phases in alfalfa. In contrast, when cdc2Ms kinase was purified by binding to p13suc1, it was active only in the G2 and M phases. In immunoblots the C-terminal antibody detected an equal amount of the cdc2Ms protein in the cytoplasm and in the nucleus. By indirect immunofluorescence, however, the cytoplasmic form of cdc2Ms could not be found in the S phase of the cells, indicating that the epitope for the cdc2 antibody is not accessible. Binding of putative inhibitor proteins to cdc2 was shown by inactivation of purified plant CDK when cell extracts were added. Furthermore, purified CDK inhibitors, such as the mouse p27kip1 and the yeast p40sic1, blocked the purified plant CDK activity.

Wounding induces the rapid and transient activation of a specific MAP kinase pathway.

The Plant Cell, Vol. 9, 75-83, January 1997 © 1997 American Society of Plant Physiologists PMID: 12237344

László Bögrea, Wilco Ligterinka, lrute Meskienea, Patrick J. Barkerb, Erwin Heberle-Borsa, Neville S. Huskissonb, and Heribert Hirta

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

b Microchemical Facility, Babraham Institute, Cambridge CB2 4AT, United Kingdom

Mechanical injury in plants induces responses that are involved not only in healing but also in defense against a potential pathogen. To understand the intracellular signaling mechanism of wounding, we have investigated the involvement of protein kinases. Using specific antibodies, we showed that wounding alfalfa leaves specifically induces the transient activation of the p44MMK4 kinase, which belongs to the family of mitogen-activated protein kinases. Whereas activation of the MMK4 pathway is a post-translational process and was not blocked by [alpha]-amanitin and cycloheximide, inactivation depends on de novo transcription and translation of a protein factor(s). After wound-induced activation, the MMK4 pathway was subject to a refractory period of 25 min, during which time restimulation was not possible, indicating that the inactivation mechanism is only transiently active. After activation of the p44MMK4 kinase by wounding, transcript levels of the MMK4 gene increased, suggesting that the MMK4 gene may be a direct target of the MMK4 pathway. In contrast, transcripts of the wound-inducible MsWIP gene, encoding a putative proteinase inhibitor, were detected only several hours after wounding. Abscisic acid, methyl jasmonic acid, and electrical activity are known to mediate wound signaling in plants. However, none of these factors was able to activate the p44MMK4 kinase in the absence of wounding, suggesting that the MMK4 pathway acts independently of these signals.

Multiple roles of MAP kinases in plant signal transduction.

Trends in Plant Science Volume 2, Issue 1, January 1997, Pages 11-15

Hirt (1997)

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

The MAP kinases are important mediators of intracellular signal transduction, and regulation involving them has been implicated in a wide variety of physiological processes. Studies in yeast and mammals have identified subgroups of these enzymes that have different substrate specificities and are regulated by different extracellular stimuli. Recently, studies in plants have revealed that there is a similar divergence of MAP kinase functions, and pathways involving MAP kinases have been linked to signal transduction caused by wounding, pathogens and abiotic stresses, as well as the plant hormones abseisic acid, auxin and ethylene. Thus, pathways involving MAP kinases are a common mechanism for signal transduction in all eukaryotes, and have been adapted to couple distinct stimuli to specific physiological responses.


Mechanosensors in plants.

Nature 383, 489-490.

Bögre, W. Ligterink, E. Heberle-Bors, and H. Hirt (1996)

PMID: 8849721

A unified nomenclature for plant A-, B-, and D-type cyclins based on sequence organisation.

Plant Mol Biol. 1996 Dec;32(6):1003-18. PMID: 9002599

Renaudin, J.H. Doonan, D. Freeman, J. Hashimoto, H. Hirt, D. Inze, T. Jacobs, H. Kouchi, P. Rouze, M. Sauter, A. Savoure, D.A. Sorrell, V. Sundaresan, And J.A.H. Murray (1996).

Laboratory of Plant Biochemistry and Physiology, INRA/ENSAM/CNRS, Montpellier, France.

The comparative analysis of a large number of plant cyclins of the A/B family has recently revealed that plants possess two distinct B-type groups and three distinct A-type groups of cyclins. Despite earlier uncertainties, this large-scale comparative analysis has allowed an unequivocal definition of plant cyclins into either A or B classes. We present here the most important results obtained in this study, and extend them to the case of plant D-type cyclins, in which three groups are identified. For each of the plant cyclin groups, consensus sequences have been established and a new, rational, plant-wide naming system is proposed in accordance with the guidelines of the Commission on Plant Gene Nomenclature. This nomenclature is based on the animal system indicating cyclin classes by an upper-case roman letter, and distinct groups within these classes by an arabic numeral suffix. The naming of plant cyclin classes is chosen to indicate homology to their closest animal class. The revised nomenclature of all described plant cyclins is presented, with their classification into groups CycA1, CycA2, CycA3, CycB1, CycB2, CycD1, CycD2 and CycD3.

Stress signalling in plants: A MAP kinase pathway is activated by cold and drought.

Proceedings of the National Academy of Sciences, PNAS 1996;93;11274-11279 doi:10.1073/pnas.93.20.11274 PMID: 8855346

Jonak, S. Kiegerl, W. Ligterink, P. J. Barker, N.S. Huskisson, and H. Hirt (1996)

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

Yeast and animals use mitogen-activated protein (MAP) kinase cascades to mediate stress and extracellular signals. We have tested whether MAP kinases are involved in mediating environmental stress responses in plants. Using specific peptide antibodies that were raised against different alfalfa MAP kinases, we found exclusive activation of p44MMK4 kinase in drought- and cold-treated plants. p44MMK4 kinase was transiently activated by these treatments and was correlated with a shift in the electrophoretic mobility of the p44MMK4 protein. Although transcript levels of the MMK4 gene accumulated after drought and cold treatment, no changes in p44MMK4 steady state protein levels were observed, indicating a posttranslational activation mechanism. Extreme temperatures, drought, and salt stress are considered to be different forms of osmotic stress. However, high salt concentrations or heat shock did not induce activation of p44MMK4, indicating the existence of distinct mechanisms to mediate different stresses in alfalfa. Stress adaptation in plants is mediated by abscisic acid (ABA)-dependent and ABA-independent processes. Although ABA rapidly induced the transcription of an ABA-inducible marker gene, MMK4 transcript levels did not increase and p44MMK4 kinase was not activated. These data indicate that the MMK4 kinase pathway mediates drought and cold signaling independently of ABA.

Developmental and cell cycle regulation of alfalfa nucMs1, a plant homolog of the yeast Nsr1 and mammalian nucleolin.

The Plant Cell, Vol. 8, 417-428, March 1996 O 1996 American Society of Plant Physiologists PMID: 8721748

L. Bögrea, C. Jonaka, M. Minka, I. Meskienea, J. Traasb, D.T.C. Haa, I. Swobodaa, C. Plankc, E. Wagnerc, E. Heberle-Borsa, and H. Hirta

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

b Laboratoire de Biologie Cellulaire, INRA, Route de St. Cyr, F 78026 Versailles, France

c lnstitute of Molecular Pathology, University of Vienna, Vienna Biocenter, Dr. Bohrgasse 9, 1030 Vienna, Austria

We report here the isolation and characterization of the nucMs1 alfalfa cDNA, whose predicted amino acid sequence structurally resembles the yeast Nsr1 protein and animal nucleolins. These proteins consist of an N-terminal acidic domain, centrally located RNA recognition motifs (RRMs), and a C-terminal glycine- and arginine-rich domain. In comparison with animal nucleolins that contain four RRMs, NucMs1 more closely resembles the yeast Nsr1 protein, which contains only two RRMs. A NucMs1 C-terminal peptide antibody specifically recognized a 95-kD nucleolar protein in alfalfa cells that changed its localization in a cell cycle-dependent manner. The nucMs1 transcript and p95nucMs1 protein levels correlated with cell proliferation, and nucMs1 gene expression was found to be induced in the G1 phase upon mitogenic stimulation of G0-arrested leaf cells. In situ hybridization analysis of different alfalfa organs during various developmental stages showed that nucMs1 gene expression is highest in root meristematic cells, but it is also found in other meristematic cells of the plant body. nucMs1 expression is tightly linked to cell proliferation but does not depend on a particular cell cycle phase. No nucMs1 expression was observed in cells that had exited the cell cycle and were undergoing differentiation or polar growth, indicating that nucMs1 may not be necessary for processes other than cell proliferation.

In and out of the plant cell cycle.

Plant Mol. Biol. 31, 459-464. (1996)

PMID: 8790280


The D-type alfalfa cyclin gene cycMs4 complements G1 cyclin deficient yeast and is induced in the G1 phase of the cell cycle.

The Plant Cell, Vol. 7, 1847-1857, November 1995 © 1995 American Society of Plant Physiologists PMID: 8535138

Marlis Dahl, lrute Meskiene, Laszlo Bögre, Dang Thi Cam Ha, lnes Swoboda, Rainer Hubmann, Heribert Hirt, and Erwin Heberle-Bors

Institute of Microbiology and Genetics, Vienna Biocenter, Austria

Cyclins are key regulators of the cell cycle in all eukaryotes. In alfalfa, we have previously isolated three B-type cyclins. The closely related cycMs1 and cycMs2 genes are expressed primarily during the G2 and M phases and are most likely mitotic cyclins; expression of the cycMs3 gene is induced in the G0-to-G1 transition, when cells reenter the cell cycle. By complementation of G1 cyclin-deficient yeast cells, a novel alfalfa cyclin, designated cycMs4, was isolated. The predicted amino acid sequence of the cycMs4 gene is most similar to that of the Arabidopsis cyclin delta 3 gene. CycMs4 and cyclin delta 3 belong to the class of D-type cyclins and contain PEST-rich regions and a retinoblastoma binding motif. When comparing expression levels in different organs, cycMs4 transcripts were present predominantly in roots. Whereas expression of the cycMs4 gene was cell cycle-regulated in suspension-cultured cells, transcription in roots was observed to depend also on the positional context of the cell. When differentiated G0-arrested leaf cells were induced to resume cell division by treatment with plant hormones, cycMs4 transcription was induced before the onset of DNA synthesis. Whereas this induction was preceded by that of the cycMs3 gene, cycMs2 expression occurred later and at the same time as mitotic activity. These data suggest that cycMs4 plays a role in the G1-to-S transition and provide a model to investigate the plant cell cycle at the molecular level.

Tyrosine phosphatase signalling in a lower plant: cell cycle and oxidative stress-regulated expression of the Chlamydomonas eugametos VH-PTP13 gene.

The Plant Journal (1995) 7(6) 981-988 PMID: 7599654

Michael A. Haring1, Marco Siderius1, Claudia Jonak2, Heribert Hirt2, Kevin M. Walton3, and Alan Musgrave1

1 Biocentrum Amsterdam, University of Amsterdam, The Netherlands.

2 Institute of Microbiology and Genetics, Vienna Biocenter, Austria

3 The University of Michigan Medical School, Dept of Biological Chemistry, Ann Arbor, MI, USA

The first evidence for tyrosine phosphatase signalling pathways in plants is presented by characterizing a putative protein tyrosine phosphatase gene from the unicellular green alga Chlamydomonas eugametos. This cDNA, referred to as VH-PTP13, contains an open reading frame specifying a protein with a molecular weight of 30.3 kDa, that has significant homology with a distinct group of dual-specificity phosphatases. The highest homology is found with CL-100, a human stress-response gene that regulates MAPkinase activity. The purified VH-PTP13 protein expressed in E. coli had phosphatase activity and inactivated MAPkinases from alfalfa and tobacco. Nondividing C. eugametos gametes did not express the VH-PTP13 gene whereas synchronously dividing vegetative cells only expressed VH-PTP13 in the early G1-phase of the cycle, implying a function there. When vegetative cells were subjected to oxidative stress, expression of the VH-PTP13 gene was strongly induced, analogous to the human CL-100 gene. Its potential role in plant signalling pathways is discussed.

cycMs3 , a novel B-type alfalfa cyclin gene, is induced in the G0-to-G1 transition of the cell cycle.

The Plant Cell, Vol. 7, 759-771, June 1995 © 1995 American Society of Plant Physiologists PMID: 7647566

lrute Meskienea, László Bögrea, Marlis Dahla, Manfred Pircka, Dang Thi Cam Haa, lnes Swobodaa, Erwin Heberle-Borsa, Gustav Ammererb, and Heribert Hirta

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

b Vienna Biocenter, lnstitute of Biochemistry and Molecular Cell Biology, Dr. Bohrgasse 9, A-1030 Vienna, Austria

Cyclins are key regulators of the cell cycle in all eukaryotes. We have previously isolated two B-type cyclin genes, cycMs1 and cycMs2, from alfalfa that are primarily expressed during the G2-to-M phase transition and are most likely mitotic cyclin genes. Here, we report the isolation of a novel alfalfa cyclin gene, termed cycMs3 (for cyclin Medicago sativa), by selecting for mating type alpha-pheromone-induced cell cycle arrest suppression in yeast. The central region of the predicted amino acid sequence of the cycMs3 gene is most similar to the cyclin box of yeast B-type and mammalian A- and B-type cyclins. In situ hybridization showed that cycMs3 mRNA can be detected only in proliferating cells and not in differentiated alfalfa cells. When differentiated G0-arrested cells were induced to reenter the cell cycle in the G1 phase and resume cell division by treatment with plant hormones, cycMs3 transcript levels increased long before the onset of DNA synthesis. In contrast, histone H3-1 mRNA and cycMs2 transcripts were not observed before DNA replication and mitosis, respectively. In addition, cycMs3 mRNA was found in all stages of the cell cycle in synchronously dividing cells, whereas the cycMs2 and histone H3-1 genes showed a G2-to-M phase- or S phase-specific transcription pattern, respectively. These data suggest that the role of cyclin CycMs3 differs from that of CycMs1 and CycMs2. We propose that CycMs3 helps control reentry of quiescent G0-arrested cells into the G1 phase of the cell cycle.

MMK2, a novel alfalfa MAP kinase, specifically complements the yeast MPK1 function.

Mol. Gen. Genet. 248, 686-694. PMID: 7476871

Jonak, S. Kiegerl, C. Lloyd, J. Chan, and H. Hirt (1995).

Institute of Microbiology and Genetics, Biocenter Vienna, Austria.

Mitogen-activated protein (MAP) kinases are serine/threonine protein kinases that are activated in response to a variety of stimuli. Here we report the isolation of an alfalfa cDNA encoding a functional MAP kinase, termed MMK2. The predicted amino acid sequence of MMK2 shares 65% identity with a previously identified alfalfa MAP kinase, termed MMK1. Both alfalfa cDNA clones encode functional kinases when expressed in bacteria, undergoing autophosphorylation and activation to phosphorylate myelin basic protein in vitro. However, only MMK2 was able to phosphorylate a 39 kDa protein from the detergent-resistant cytoskeleton of carrot cells. The distinctiveness of MMK2 was further shown by complementation analysis of three different MAP kinase-dependent yeast pathways; this revealed a highly specific replacement of the yeast MPK1(SLT2) kinase by MMK2, which was found to be dependent on activation by the upstream regulators of the pathway. These results establish the existence of MAP kinases with different characteristics in higher plants, suggesting the possibility that they could mediate different cellular responses.

The function of the hypusine-containing proteins of yeast and other eukaryotes is well conserved.

Mol. Gen. Genet. 244, 646-652. PMID: 7969034

Magdolen, H. Klier, T. Wöhl, F. Klink, H. Hirt, J. Hauber, and F. Lottspeich. (1995).

Max-Planck-Institut für Biochemie, Martinsried, Germany.

The hypusine-containing protein (Hypp) is highly conserved in evolution, from man to archaebacteria, but is not found in eubacteria. Hypp is essential for the viability for yeast cells, where two forms are encoded by the genes HYP1 and HYP2. The hypusine-containing protein Hyp2p, encoded by the HYP2 gene in yeast, is present under both aerobic and anaerobic conditions, whereas Hyp1p synthesis is restricted to anaerobiosis. hyp1 disruption mutants grown under anaerobic conditions reveal no detectable alteration in phenotype relative to wild-type strains. We demonstrate that either Hyp1p or Hyp2p alone is sufficient for normal growth under both metabolic conditions. Moreover, Hypp from various eukaryotic species (slime mold, alfalfa and man) carries the lysine to hypusine modification when expressed in yeast and can substitute functionally for Hyp2p in strains disrupted for HYP2, indicating a highly conserved function of this protein. In contrast, the archaebacterial Hypp expressed in yeast is neither modified by hypusine, nor does it allow growth of cells deficient for yeast Hypp.

Inflorescence-specific expression of AtK-1, a novel Arabidopsis thaliana homologue of shaggy/glycogen synthase kinase-3.

Plant Mol Biol. 1995 Jan;27(1):217-21. PMID: 7865793

Jonak C, Heberle-Bors E, Hirt H.

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

We report here the isolation of the Arabidopsis thaliana gene AtK-1. The predicted protein sequence of AtK-1 shows 70% identity to the Arabidopsis ASK and alfalfa MsK kinases that are homologs of the Drosophila shaggy and rat GSK-3 serine/threonine protein kinases playing an important role in signal transduction processes in animals. Northern analysis of different organs revealed exclusive expression in inflorescences suggesting an involvement of the AtK-1 kinase in reproduction-specific processes.


Rhizobium Nod factors reactivate the cell cycle during infection and nodule formation, but the cycle is only completed in primordium formation.

The Plant Cell, Vol. 6, 1415-1426, October 1994 © 1994 American Society of Plant Physiologists PMID: 7994175

W.Yanga, C. de Blanka, I. Meskieneb, H. Hirtb, J. Bakkera, A. van Kammena, H. Franssena, and T. Bisselinga

a Department of Molecular Biology, Wageningen Agricultura1 University, Dreijenlaan 3, 6703HA, Wageningen, The Netherlands

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

Rhizobia induce the formation of root nodules on the roots of leguminous plants. In temperate legumes, nodule organogenesis starts with the induction of cell divisions in regions of the root inner cortex opposite protoxylem poles, resulting in the formation of nodule primordia. It has been postulated that the susceptibility of these inner cortical cells to Rhizobium nodulation (Nod) factors is conferred by an arrest at a specific stage of the cell cycle. Concomitantly with the formation of nodule primordia, cytoplasmic rearrangement occurs in the outer cortex. Radially aligned cytoplasmic strands form bridges, and these have been called preinfection threads. It has been proposed that the cytoplasmic bridges are related to phragmosomes. By studying the in situ expression of the cell cycle genes cyc2, H4, and cdc2 in pea and alfalfa root cortical cells after inoculation with Rhizobium or purified Nod factors, we show that the susceptibility of inner cortical cells to Rhizobium is not conferred by an arrest at the G2 phase and that the majority of the dividing cells are arrested at the G0/G1 phase. Furthermore, the outer cortical cells forming a preinfection thread enter the cell cycle although they do not divide.

Isolation and characterization of phophoprotein phosphatase 1 from alfalfa.

Mol. Gen. Genet. 244, 176-182. PMID: 7519721

Pay, M. Pirck, L. Bögre, H. Hirt, and E. Heberle-Bors(1994).

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

Protein phosphatases are central regulatory components of diverse processes in eukaryotes and are among the most highly conserved proteins known. In this paper, we report the cloning and sequencing of a type 1 protein phosphatase (pp1Ms) cDNA from alfalfa. Southern analysis indicates the presence of a gene family of PP1 proteins in alfalfa. The pp1Ms open reading frame is very similar to one of five predicted Arabidopsis type 1 protein phosphatases, indicating that different subtypes are individually conserved. Expression of the alfalfa pp1Ms in a temperature-sensitive Schizosaccharomyces pombe PP1 mutant, dis2-11, revealed no complementation, suggesting that PP1Ms is not involved in mitotic regulation. In different plant organs, different pp1Ms transcript levels were observed; in contrast, mRNA levels remained constant in all phases of the cell cycle and in logarithmically growing cells. However, when cells entered stationary phase pp1Ms transcript levels decreased considerably.

The plant transcription factor TGA1 stimulates expression of the CaMV 35S promoter in Saccharomyces cerevisiae.

Plant Mol. Biol. 25, 323-328. PMID: 8018880

Rüth, R.J. Schweyen, and H. Hirt (1994).

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

We have previously shown that two CRE elements situated on a 31 bp region of the cauliflower mosaic virus (CaMV) 35S promoter activate gene expression in the yeast Saccharomyces cerevisiae and are regulated by cAMP. Studies with the yeast transcription factors GCN4, SKO1 and YAP1, which bind CRE-like sequences, showed no influence on expression of the 35S promoter indicating that a yet unknown factor is involved in activation. Band shift experiments with the 31 bp promoter region revealed binding of similar factors in yeast and plant protein extracts. In a previous study this promoter region was shown to confer tissue-specific expression in plants and to interact with the transcription factor TGA1. To test whether expression of TGA1 in yeast also stimulates transcription of the 35S promoter, we co-transformed yeast cells with a cDNA clone of this transcription factor and a 35S promoter/reporter gene construct. Promoter activity studies revealed that TGA1 confers enhanced expression of a reporter gene under the control of the 35S promoter in yeast cells. Yeast cells that were transformed with a 35S promoter construct that containing a mutated TGA1-binding site showed that both TGA1 and the intact binding site are necessary for this activation. These results suggest that stimulation of the 35S promoter by TGA1 is mediated by competition with an endogenous down-regulating yeast factor that is modulated by the nutritional state of the cells.

The cDNA sequence encoding an annexin from Medicago sativa

Plant Physiol. (1994) 104: 1463-1464

Manfred Pirck, Heribert Hirt, and Erwin Heberle-Bors

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

Annexins are a family of at least 13 calcium-binding proteins in higher eukaryotes. They share the common features of binding phospholipids in a Ca2+-dependent manner and contain a 4- or 8-fold repeated sequence of about 70 amino acid residues termed the annexin repeat. An N-terminal domain is of greater variability and may confer specific functions for each type. At least some of the annexins seem to be differentially expressed with respect to cell proliferation (Schlaepfer and Haigler, 1990). However, the biological function of the annexins is still not clearly defined. Proposed roles include exocytosis and membrane trafficking (Creutz, 1992; Gruenberg and Emans, 1993), inhibition of phospholipase Az (Haigler et al., 1987), and mitogenic signaling (Haigler et al., 1987).

Cell cycle regulation in higher plants

Sem. Dev. Biol. 5, 147-154.

Hirt, and E. Heberle-Bors (1994).

MAP kinases: universal multi-purpose signaling tools.

Plant Mol. Biol. 24, 407-416. PMID: 8123784

Jonak, E. Heberle-Bors, and H. Hirt (1994).

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

MAP (mitogen-activated protein) kinases are serine/threonine protein kinases and mediate intracellular phosphorylation events linking various extracellular signals to different cellular targets. MAP kinase, MAP kinase kinase and MAP kinase kinase kinase are functional protein kinase units that are conserved in several signal transduction pathways in animals and yeasts. Isolation of all three components was also shown in plants and suggests conservation of a protein kinase module in all eukaryotic cells. In plants, MAP kinase modules appear to be involved in ethylene signaling and auxin-induced cell proliferation. Therefore, coupling of different extracellular signals to different physiological responses is mediated by MAP kinase cascades and appears to have evolved from a single prototypical protein kinase module which has been adapted to the specific requirements of different organisms.


cdc2MsB , a cognate cdc2 gene from alfalfa, complements the G1/S but not the G2/M transition of budding yeast cdc28 mutants.

The Plant Journal (1993) 4(1), 61-69 PMID: 8220475

Heribert Hirt, Anniko Pay, Laszlo Bögre, Irute Meskiene and Erwin Heberle-Bors

Institute of Microbiology and Genetics, University of Vienna, Austria

The product of the cdc2 gene encodes the p34cdc2 protein kinase that controls entry of yeast cells into S phase and mitosis. In higher eukaryotes, at least two cdc2-like genes appear to be involved in these processes. A cdc2 homologous gene has previously been isolated from alfalfa and shown to complement a fission yeast cdc2ts mutant. Here the isolation of cdc2MsB, a cognate cdc2 gene from alfalfa (Medicago sativa) is reported. Southern blot analysis shows that cdc2MsA and cdc2MsB are present as single copy genes in different tetraploid Medicago species. cdc2MsB encodes a slightly larger mRNA (1.5 kb) than cdc2MsA (1.4 kb). Both genes were found to be expressed at similar steady state levels in different alfalfa organs. Expression levels of both cdc2Ms genes correlate with the proliferative state of the organs. Complementation studies revealed that in contrast to cdc2MsA, cdc2MsB was not able to rescue a cdc2ts fission yeast mutant. cdc2MsB was also unable to rescue a G2/M-arrested cdc28ts budding yeast mutant which could be rescued by expression of the cdc2MsA gene. Conversely, cdc2MsB but not cdc2MsA was found to complement the G1/S block of another cdc28ts budding yeast mutant. These results suggest that cdc2MsA and cdc2MsB function at different control points in the cell cycle.

The plant homologue of MAP kinase is expressed in a cell cycle-dependent and organ-specific manner.

The Plant Journal (1993) 3(4), 611-617 PMID: 8220466

Claudia Jonak, Aniko Pay, Laszlo Bögre, Heribert Hirt and Erwin Heberle-Bors

Institute of Microbiology and Genetics, University of Vienna, Austria

In animals, MAP kinase plays a key role in growth factor-stimulated signalling and in mitosis. The isolation of a Medicago sativa cDNA clone MsK7 which shows 52% identity to animal MAP kinases is reported. The deduced protein sequence shows all the important structural features of MAP kinases and also contains the highly conserved Thr-183 and Tyr-185 residues. Northern analysis of synchronized alfalfa cells showed that the MsK7 kinase gene is expressed at low levels in G1 phase but at higher levels in S and G2 phases of the cell cycle. In the plant, only stems and roots were found to contain MAP kinase MsK7 mRNA. Southern and PCR analyses indicated that alfalfa contains at least four highly related MAP kinase genes.

Isolation and characterization of phosphoprotein phosphatase type 2a from alfalfa.

Mol. Gen. Genet. 240, 126-131. PMID: 8393512

Pirck, A. Pay, L. Bögre, E. Heberle-Bors and H. Hirt (1993).

Institute of Microbiology and Genetics, Vienna Biocenter, Austria.

Phosphoprotein phosphatases are central regulatory components of the cell cycle in eukaryotes. We report the cloning and sequencing of an alfalfa phosphoprotein phosphatase type 2A (pp2aMs) cDNA. The predicted protein sequence shows high similarity to PP2A from Brassica napus, rabbit and Drosophila. No changes in pp2aMs mRNA abundance during the cell cycle were found. During growth of a batch cell culture, mRNA levels decreased gradually. In planta, all organs contained pp2a transcripts but maximal mRNA levels were detected in stems. Since Southern analysis indicated the presence of a small pp2a gene family in alfalfa, it appears that different subtypes may have specialized roles in various tissues and developmental situations which await characterization.

The MsK family of alfalfa protein kinase genes encodes homologs of shaggy/glycogen synthase kinase-3 and shows differential expression patterns in plant organs and development.

The Plant Journal (1993) 3(6), 847-856 PMID: 8401615

Anika Pay, Claudia Jonak, Laszlo Bögre, Irute Meskiene, Erwin Heberle-Bors and Heribert Hirt

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

This paper reports on the isolation of a novel class of plant serine/threonine protein kinase genes, MsK-1, MsK-2 and MsK-3. They belong to the superfamily of cdc2-like genes, but show highest identity to the Drosophila shaggy and rat GSK-3 proteins (65-70%). All of these kinases share a highly conserved catalytic protein kinase domain. Different amino-terminal extensions distinguish the different proteins. The different plant kinases do not originate from differential processing of the same gene as is found for shaggy, but are encoded by different members of a gene family. Similarly to the shaggy kinases, the plant kinases show different organ-specific and stage-specific developmental expression patterns. Since the shaggy kinases play an important role in intercellular communication in Drosophila development, the MsK kinases are expected to perform a similar function in plants.


Alfalfa cyclins: differential expression during the cell cycle and in planta.

The Plant Cell 4, Dec 1992, 1531-1538. PMID: 1307238

Heribert Hirt, M. Mink, M. Pfosser, L. Bögre, J. Györgyey, C. Jonak, A. Gartner, D. Dudits and E. Heberle-Bors (1992).

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

Cell division in eukaryotes is mediated by the action of the mitosis promoting factor, which is composed of the CDC2 protein kinase and one of the various mitotic cyclins. We have recently isolated a cdc2 gene from alfalfa. Here, we report the isolation of two cyclin genes, cycMs1 and cycMs2, from alfalfa. The cycMs2 gene shows highest similarity to type B cyclins. In contrast, the predicted amino acid sequence of the cycMs1 gene shows similar homology scores to cyclins of all types (25 to 35%). Both genes are expressed in dividing suspension cultured cells but cease to be expressed when the cells enter stationary phase. In synchronized alfalfa suspension cultured cells, the mRNAs of cycMs1 and cycMs2 show maximal expression in the G2 and M phases. Transcripts of cycMs2 are found only in late G2 and M phase cells, an expression pattern typical for cyclin B genes, whereas cycMs1 appears with the onset of G2. This pattern indicates that alfalfa cycMs1 and cycMs2 belong to different classes of cyclins. In young leaves, expression of both genes is high, whereas in mature leaves no transcripts can be detected, indicating that the two cyclin genes are true cell division markers at the mRNA level. In other organs, a more complex expression pattern of the two cyclin genes was found.

The 35S Cauliflower Mosaic Virus Promoter is regulated by cAMP in Saccharomyces cerevisiae

Mol. Gen. Genet. 235, (1992) 365-372. PMID: 1334531

Rüth, H. Hirt and R. Schweyen .

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

The cauliflower mosaic virus 35S promoter confers strong gene expression in plants, animals and fission yeast, but not in budding yeast. On investigating this paradox, we found that in budding yeast the promoter acts through two domains. Whereas the upstream domain acts as a silencer, the downstream domain couples expression to the nutritional state of the cells via the RAS/cAMP pathway. Point mutations indicate that two boxes with similarity to the cAMP regulated element (CRE) of mammalian cells mediate this response. Gel retardation assays show that, in both yeast and plant protein extracts, factors bind to this promoter element. Therefore, transcriptional activation appears to be highly conserved at the level of transcription factors and specific DNA target elements in eukaryotes. This offers new ways to investigate gene regulation mechanisms of higher eukaryotes, which are not as amenable to genetic analysis as yeast.

An alfalfa cDNA encodes a protein with similarity to human translationally controlled tumor protein.

Plant Mol. Biol. (1992) 19, 501-503. PMID: 1623194

A. Pay, E. Heberle-Bors and H. Hirt

An alfalfa cDNA encodes a protein with similarity to mammalian snRNP-E proteins

Nucleic Acids Research, (1992), Vol. 20, No. 3 613

H. Hirt, A. Gartner and E. Heberle-Bors

Institute of Microbiology and Genetics, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria

Small nuclear ribonucleoproteins (snRNPs) are complexes composed of discrete sets of proteins associated with the small nuclear RNAs Ul, U2, U5 and U4/U6. These snRNAs have been shown to be required for a variety of RNA processing reactions in eukaryotic cells (1, 2). Ul snRNP acts at the 5' splice site, U2 snRNP interacts with the branch point and U5 snRNP probably associates with the 3' splice site (3). The specific roles of the individual snRNA associated proteins in RNA processing are still unclear. However, at least some of the snRNA associated proteins appear to be necessary for specific interaction of the snRNA with the pre-mRNA. A subset of these proteins is recognized by autoantibodies from patients with the autoimmune disease systemic lupus erythematosus (SLE) (4, 5). These autoantibodies react with the Sm epitope and can precipitate Ul, U2, U4, U5 and U6 snRNPs from cell extracts (6). This indicates that these proteins must have been highly conserved during evolution. Therefore, investigation of the primary structure of the proteins from distantly related organisms should yield valuable information on important protein domains and contribute to an understanding of the function of these proteins in RNA processing.


A novel method for in situ screening of yeast colonies with the ß-glucuronidase reporter gene.

Curr. Genet. 20, 437-439. PMID: 1807836

Hirt (1991)

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

Expression of the beta-galactosidase gene in yeast has served as a screening marker for many purposes. Here it is shown that in two yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe, the beta-glucuronidase (GUS) gene can be used as an alternative marker. Since the histochemical substrate can not be taken up by yeast cells, direct colony screening of plates was found to be impossible. However, by a replica plating technique, GUS expression became visibly detectable within 10 min when the GUS gene was strongly expressed. The staining method could still be performed for expression at a 100-fold lower level, but incubation times of several hours were needed. Furthermore, specific GUS expression levels of yeast protein extracts could be quantified by a fluorometric assay which is both very simple to perform and highly sensitive. Since the GUS gene can also tolerate large N-terminal fusions, this method should be particularly attractive for studying such diverse problems as transcriptional and translational regulation or subcellular localization in yeast.

Isolation and characterization of the plant homologue of the eukaryotic initiation factor 4D from alfalfa, Medicago sativa

Plant Mol. Biol. 17, 927-929. PMID: 1912507

A. Pay, E. Heberle-Bors and H. Hirt (1991).

Alfalfa heat shock genes are differentially expressed during somatic embryogenesis.

Plant Mol. Biol. 16, 999-1007. PMID: 1863771

Györgyey, A. Gartner, K. Nemeth, Z. Magyar, H. Hirt, E. Heberle-Bors and D. Dudits (1991).

Institute of Plant Physiology, Hungarian Academy of Sciences, Szeged.

We have isolated two cDNA clones (Mshsp18-1; Mshsp18-2) from alfalfa (Medicago sativa L.) which encode for small heat shock proteins (HSPs) belonging to the hsp17 subfamily. The predicted amino acid sequences of the two alfalfa proteins are 92% identical and a similar degree of homology (90%) can be detected between Mshsp18-2 and the pea hsp17. In comparison to various members of small HSPs from soybean amino acid sequence similarities of 80-86% were identified. The alfalfa HSPs share a homologous stretch of amino acids in the carboxy terminal region with hsp22, 23, 26 from Drosophila. This region contains the GVLTV motif which is characteristic of several members of small HSPs. At room temperature alfalfa hsp18 mRNAs were not detectable in root and leaf tissues but northern analysis showed a low level of expression in microcallus suspension (MCS). The transcription of Mshsp18 genes is induced by elevated temperature, CdCl2 treatment and osmotic shock in cultured cells. In alfalfa somatic embryos derived from MCS a considerable amount of hsp18 mRNA can be detected during the early embryogenic stages under normal culture conditions. The differential expression of these genes during embryo development suggests a specific functional role for HSPs in plant cells at the time of the developmental switch in vitro.

Complementation of yeast cell cycle mutant by alfalfa cDNA encoding a protein kinase homolog of the p34 cdc2 .

Proc. Natl. Acad. Sci. 88, 1636-1640. PMID: 2000373

H. Hirt, A. Pay, J. Györgyey, L. Bako, L. Bögre, R. Schweyen, E. Heberle-Bors and D. Dudits (1991).

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

The cdc2 protein kinase plays a central role in control of the eukaryotic cell cycle of animals and yeasts. We have isolated a cDNA clone (cdc2Ms) from alfalfa (Medicago sativa L.) that is homologous to the yeast cdc2/CDC28 genes. The encoded protein is 64% identical to the yeast and mammalian counterparts and shows all the prominent structural features known from these organisms. Antibody raised against a 16-amino acid synthetic peptide with crossreactivity against p34 proteins recognized a 34-kilodalton protein in extracts of alfalfa cells. When transferred into a fission yeast, the plant cdc2 homolog can complement a temperature-sensitive cdc2 mutant. Northern analysis revealed higher transcript levels in shoots and suspension cultures than in roots. In addition to the dominant transcript of 1.4 kilobases detected in the poly(A)+fraction, 2.5- and 1.2-kilobase transcripts were detected in total RNA preparations from shoots or somatic embryos. Suspension cultures that were induced to form somatic embryos by an auxin (2,4-dichlorophenoxyacetic acid) showed fluctuations in transcription pattern during the induction period and embryogenesis.


Evolutionary conservation of transcription machinery between yeast and plants as shown by the efficient expression from the CaMV 35S promoter and 35S terminator.

Curr. Genet. 17, 473-479. PMID: 2202523

H. Hirt, M. Kögl, T. Murbacher and E. Heberle-Bors (1990).

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

Complementation of fission yeast mutants by plant genomic libraries could be a promising method for the isolation of novel plant genes. One important prerequisite is the functioning of plant promoters and terminators in Schizosaccharomyces pombe and Saccharomyces cerevisiae. Therefore, we studied the expression of the bacterial beta-glucuronidase (GUS) reporter gene under the control of the Cauliflower Mosaic Virus (CaMV) 35S promoter and 35S terminator. We show here that S. pombe initiates transcription at exactly the same start site as was reported for tobacco. The 35S CaMV terminator is appropriately recognized leading to a polyadenylated mRNA of the same size as obtained in plant cells transformed with the same construct. Furthermore, the GUS-mRNA is translated into fully functional GUS protein, as determined by an enzymatic assay. Interestingly, expression of the 35S promoter in the budding yeast S. cerevisiae was found to be only moderate and about hundredfold lower than in S. pombe. To investigate whether different transcript stabilities are responsible for this enormous expression difference in the two yeasts, the 35S promoter was substituted by the ADH (alcohol dehydrogenase) promoter from fission yeast. In contrast to the differential expression pattern of the 35S promoter, the ADH promoter resulted in equally high expression rates in both fission and budding yeast, comparable to the 35S promoter in S. pombe. Since the copy number of the 35S-GUS constructs differs only by a factor of two in the two yeasts, it appears that differential recognition of the 35S promoter is responsible for the different transcription rates.

Effects of Cadmium on Tobacco: Synthesis and Regulation of Cadmium-Binding Peptides.

Biochem. Physiol. Pflanzen (1990) 186, 153-163.

H. Hirt, K. Sommergruber and A. Barta .


Cadmium-enhanced gene expression in suspension culture cells of tobacco.

Planta (1989) 179, 414-420.

H. Hirt, G. Casari and A. Barta


The human growth hormone gene locus: structure, evolution and allelic variants.

H. Hirt, J. Kimelman, M. Birnbaum, E. Chen, P. Seeburg, N. Eberhardt and A. Barta (1987).

Plectin and High Mol Weight MAPs: Versatile connecting Links of the Cytoskeleton.

IRL Press, Oxford, 107-117.

Wiche, R. Foisner, H. Herrmann, H. Hirt and G. Weizer.(1987)

ICSU symp. ser. vol. 8. (Macchioni R. and Arrechaga J., eds.),

In: The Cytoskeleton in Cell Differentiation and Development ,

Human growth hormone genes: structure, evolution, expression and hormonal regulation.

In: Selected Topics in Mol. Endocrin ., 58-67.

Eberhardt, P. Cattini, H. Hirt, T. Anderson, L. Peritz, J. Baxter, P. Mellon, R. Isaacs, E. Slater and A. Barta (1987).


Differential distribution of microtubule-associated proteins MAP- 1 and MAP-2 in neurons of rat brain and association of MAP-1 with microtubules of neuroblastoma cells (N 2 A).

The EMBO Journal, Vol 2, 11, pp 1915-1920. August 1983 PMID: 6641705

G. Wiche, E. Briones, H. Hirt, R. Krepler, U. Artlieb and H. Denk

Institute of Biochemistry, and Division of Gastroenterologic Pathology and Molecular Pathology (Hans Popper-Labortory), Department of Pathology, School of Medicine, University of Vienna, 1090 Vienna, Austria

To study the individual location of the microtubule proteins MAP-1 and MAP-2 in neuronal tissues and cells, antisera to electrophoretically purified MAP-1 and MAP-2 components were raised in rabbits. When frozen sections through rat brain were examined by immunofluorescence microscopy the antibodies to MAP-1 strongly stained a variety of nerve cells including dendrites and myelinated axons in the cerebrum and cerebellum. Antibodies to MAP-2 showed similar staining patterns, except that myelinated axons were unstained. These results were confirmed by immunoelectron microscopy of frozen sections through cerebellum using the peroxidase technique. Thereby, the association of MAP-1 with microtubules was also clearly demonstrated. When cultured mouse neuroblastoma N2A cells were examined by immunofluorescence microscopy the antiserum to MAP-1 brightly stained filamentous structures resembling microtubules, whereas relatively weak and diffuse staining of the cytoplasm was observed with the antiserum to MAP-2. In agreement with the immunolocalization, MAP-1, but not MAP-2, was found as a prominent component of microtubules proteins polymerized in vitro by taxol from soluble N2A cell extracts. Together these results indicate that neuronal microtubules are preferentially associated with distinct high mol. wt. polypeptides. Therefore, they support the concept that different complements of associated proteins determine distinct functions of microtubules.