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392 Research Update TRENDS in Plant Science Vol.6 No.9 September 2001 10 Smith, L.G. et al. (2001) Tangled1: a microtubule binding protein required for the spatial control of cytokinesis in maize. J. Cell Biol. 152, 231–236 11 Bichet, A. et al. (2001) BOTERO1 is required for normal orientation of cortical microtubules and anisotropic cell expansion in Arabidopsis. Plant J. 25, 137–148 12 Burk, D.H. et al. (2001) A katanin-like protein regulates normal cell wall biosynthesis and cell elongation. Plant Cell 13, 807–827 13 Furutani, I. et al. (2000) The SPIRAL genes are required for directional control of cell elongation in Arabidopsis thaliana. Development 127, 4443–4453 14 Whittington, A.T. et al. (2001) MOR1 is essential for organizing cortical microtubules in plants. Nature 411, 610–613 15 Tournebize, R. et al. (2000) Control of microtubule dynamics by the antagonistic activities of XMAP215 and XKCM1 in Xenopus egg extracts. Nat. Cell Biol. 2, 13–19 16 Charasse, S. et al. (1995) Characterization of the cDNA and pattern of expression of a new gene over-expressed in human hepatomas and colonic tumours. Eur. J. Biochem. 234, 406–413 17 Andrade, M.A. and Bork, P. (1995) HEAT repeats in the Huntingtons-disease protein. Nat. Genet. 11, 115–116 18 Groves, M.R. et al. (1999) The structure of the protein phosphatase 2A PR65/A subunit reveals the conformation of its 15 tandemly repeated HEAT motifs. Cell 96, 99–110 19 Vasquez, R.J. et al. (1994) XMAP from Xenopus eggs promotes rapid plus end assembly of microtubules and rapid microtubule polymer turnover. J. Cell Biol. 127, 985–993 20 Vasquez, R.J. et al. (1999) Phosphorylation by CDK1 regulates XMAP215 function in vitro. Cell Motil. Cytoskeleton 43, 310–321 21 Walczak, C.E. et al. (1996) XKCM1: a Xenopus kinesin-related protein that regulates microtubule dynamics during mitotic spindle assembly. Cell 84, 37–47 22 Desai, A. et al. (1999) Kin I kinesins are microtubule-destabilizing enzymes. Cell 96, 69–78 23 Popov, A.V. et al. (2001) XMAP215 regulates microtubule dynamics through two distinct domains. EMBO J. 20, 397–410 24 Spittle, C. et al. (2000) The interaction of TOGp with microtubules and tubulin. J. Biol. Chem. 275, 20748–20753 25 Heald, R. (1999) A dynamic duo of microtubule modulators. Nat. Cell Biol. 2, E11–E12 26 Kemp, B.E. and Pearson, R.B. (1990) Protein-kinase recognition sequence motifs. Trends. Biochem. Sci.15, 342–346 Patrick J. Hussey* Timothy J. Hawkins Dept of Biological Sciences, University of Durham, South Road, Durham, UK DH1 3LE. *e-mail: [email protected] Mapping out the roles of MAP kinases in plant defense Roger W. Innes Arabidopsis contains 20 MAP kinase genes, but their roles in plant physiology have remained largely unknown because of a lack of mutants. Recent papers from two groups have shed new light on the function of two different MAP kinases. The Arabidopsis MPK4 gene appears to negatively regulate salicylic acid-mediated defense responses and positively regulate jasmonic acid-induced responses. The tobacco SIPK gene (orthologous to Arabidopsis MPK6 ) appears to positively regulate programmed cell death. one has shown a complete MAPKKK–MAPKK–MAPK assembly other than by heterologous assays in yeast4,5. Equally significant, no one has identified a direct target of a plant MAPK, or determined their biological functions. The two papers I will discuss here represent important advances in our understanding of plant MAPKs in that they provide our first insights into the biological functions of two distinct MAPK family members, Arabidopsis MPK4 and tobacco SIPK. Mitogen-activated protein kinases (MAPKs) are signal transduction proteins and are found in all eukaryotes analyzed to date. They are typically involved in transducing extracellular signals1. Although MAPKs have been studied in great detail in animal systems and in yeast, little is known about their functions in plants. In animal and yeast systems, MAPKs are activated by a kinase relay consisting of an MAPKK and an MAPKKK (Ref. 1). This also appears to be true in plants2,3 (Fig. 1). Activation of MAPKKKs in animals and yeast is typically controlled by transmembrane receptors, although numerous other proteins are known to regulate MAPKKK activity1. Piecing together a complete pathway from a receptor to a MAPK target has not been accomplished in plants. Indeed, no First MAPK mutant in plants http://plants.trends.com The Arabidopsis genome contains 20 MAPK-like genes6, but until recently no mutations had been reported. That has changed with the publication of a transposon insertion mutation in the Arabidopsis MPK4 gene by Morten Petersen et al.7 The mpk4 mutant was identified in a standard forward-genetic screen as a severe dwarf. It was only after the disrupted gene was identified that the researchers knew that they had found an MAPK mutant. Dwarfism in plants can be caused by many different things; thus the challenge for Petersen et al. was to determine why disruption of MPK4 resulted in growth defects. Previous work had indicated that MPK4 is activated by multiple abiotic stresses, including cold, low humidity, hyper-osmolarity, touch and wounding, suggesting that its primary role might be in regulating adaptation to environmental stress8. Surprisingly, the mpk4 mutant behaved like wild-type plants in response to these stresses7. This forced Petersen et al. to scrap the abiotic stress connection and think more broadly about the possible causes of the dwarfed phenotype. ‘…the mpk4 mutation causes a greatly elevated level of salicylic acid and…the majority of the mpk4 mutant phenotypes… can be alleviated simply by reducing the endogenous levels of salicylic acid via the expression of a bacterial salicylate hydroxylase gene…’ One well known cause of dwarfism in plants is constitutive expression of defense responses associated with elevated levels of salicylic acid9,10. A quick check of defense gene mRNA levels in the mpk4 mutant revealed constitutively high levels, thus suggesting that MPK4 might directly or indirectly regulate salicylic acidmediated defenses. This led Petersen et al. into a thorough genetic and physiological analysis of defense response pathways in the mpk4 mutant. This work yielded several important conclusions. Foremost among these was that the mpk4 mutation causes a greatly elevated level of salicylic acid, and that 1360-1385/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(01)02058-1 Research Update the majority of the mpk4 mutant phenotypes (e.g. enhanced resistance to bacterial and oomycete pathogens, enhanced defense gene expression) can be alleviated simply by reducing the endogenous levels of salicylic acid via the expression of a bacterial salicylate hydroxylase gene (NahG). Significantly, NahG only partially suppressed the dwarf phenotype of the mpk4 mutant. Furthermore, induction of the defense genes PDF1.2 and THI1 by jasmonic acid is blocked in the mpk4 mutant, even in the NahG-containing line7. Thus, MPK4 must regulate more than just salicylic acid levels. It is not clear whether these three phenotypes (dwarfism, jasmonic acid insensitivity and high salicylic acid levels) are independent effects of the mpk4 mutation, or whether the defect in jasmonic acid signaling leads indirectly to elevated levels of salicylic acid and dwarfism. In support of the hypothesis the three phenotypes are independent effects of the mpk4 mutation, there are precedents from yeast and animal systems for single MAPKs functioning in two or more independent signal transduction pathways, presumably via independent protein complexes1,11. Additionally, a single MAPK pathway can have multiple targets1, thus the multiple effects of the mpk4 mutation are not particularly surprising. Although mpk4 mutant plants are dwarfed, there appears to be no gross disruption of cellular homeostasis. This is seen in the lack of lesions in mpk4 plants and by the relatively small number of genes whose expression is altered7. In addition, expression of a kinase inactive form of the MPK4 protein in the mpk4 mutant failed to rescue any of the mutant phenotypes, which argues against the possibility of ‘promiscuous’ cross-talk occurring among MAPK pathways in the absence of MPK4 protein12. These observations indicate that MPK4 plays a specific role in the regulation of defense responses, and that the mpk4 mutant phenotype is not simply the result of pleiotropic effects caused by gross disruption of cellular functions. To fully understand the role of the MPK4 protein, the upstream and downstream components of this MAPK pathway need to be identified. There are already yeast two-hybrid and in vitro http://plants.trends.com TRENDS in Plant Science Vol.6 No.9 September 2001 393 Environmental signal H2O2, SA, cold, drought or wounding ? Ethylene Receptor ? ? ETR1 MAPKKK ANP1, 2 or 3 AtMEKK1 CTR1 MAPKK AtMKK4 MEK1 ? MAPK MPK6 MPK4 ? Targets ? ? ? ERF1? EIN3? Effects Cell death? Auxin responses SA PDF1.2 Triple response TRENDS in Plant Science Fig. 1. Representative examples from Arabidopsis of the three general classes of proposed plant mitogen-activated protein kinase (MAPK) cascades. Blue arrows indicate positive regulation and red Ts indicate negative regulation. Abbreviations: CTR1, constitutive triple response factor; EIN3, ethylene insensitive 3 protein; ERF1, ethylene response factor 1; ETR1, ethylene receptor; PDF1.2, plant defensin protein; SA, salicylic acid. data that point to the MAPKK and MAPKKK components that probably regulate MPK4 (Fig. 1)3–5, but the upstream receptor(s) and downstream targets are unknown. Likely candidates for the downstream targets would be transcription factors that bind to the PDF1.2 promoter. Although these have not been identified, the fact that PDF1.2 induction requires the ethylene signal transduction pathway13 points to the EIN3 and ERF transcription factors. These two proteins are known to constitute a transcription factor cascade leading to the induction of ethylene inducible genes14. However, it is worth noting in this context that mpk4 mutants do not display developmental defects associated with the ein3 mutation, such as lack of a triple response, nor does mpk4 suppress the constitutive triple response of ctr1 mutants (J. Mundy, pers. commun.), which seems to rule out EIN3 as a likely target, but leave open the possibility that ERF1 and/or related transcription factors are targets. There is even less to go on as far as identifying upstream receptors that regulate MPK4, or even the upstream signals that activate it. Somewhat unexpected is that neither jasmonic acid nor ABA activate MPK4 (J. Mundy, pers. commun.); thus, MPK4 is currently an MAPK in search of an activator and a target. However, it is the only plant MAPK for which we have a mutant phenotype. Potential role for tobacco SIPK In contrast with MPK4, the tobacco SIPK protein has many known activators, including avirulent pathogens15. Unfortunately, no one has been able to identify an insertional mutation in the Arabidopsis ortholog, MPK6, in spite of efforts by multiple laboratories. SIPK was first identified because it is strongly activated by wounding and by salicylic acid, and can be easily detected by in-gel protein kinase assays16. Using a transient expression assay in Arabidopsis protoplasts, Jen Sheen and colleagues recently identified a family of closely related MAPKKKs (ANP1, ANP2 and ANP3) that appear to function at the top of the SIPK/MPK6 pathway17 (Fig. 1). Perhaps significantly, these MAPKKKs also appear to regulate auxin responses and cell division17–19. This observation suggests that the failure to isolate 394 Research Update mutations in MPK6 might be because it is an essential regulator of the plant cell cycle. This possibility is important to keep in mind when interpreting the recent paper from Shuqun Zhang and colleagues, who propose that SIPK can participate directly in the induction of programmed cell death in tobacco leaves in response to pathogen elicitors20. In this work, a constitutively activated mutant version of the MAPKK protein, NtMEK2, was shown to activate SIPK in vitro and in vivo. Activation of SIPK in tobacco leaves preceded induction of several known defense genes and induction of cell death. This cell death response was apparently not dependent on salicylic acid because it was not suppressed by expression of the NahG transgene20. Although it is possible that SIPK is directly responsible for induction of cell death during a pathogen-induced hypersensitive response (HR), one must be cautious in interpreting the above data. Sustained activation of SIPK by the mutant MAPKK could be causing pleiotropic effects (e.g. activation of inappropriate cell division), which then cause activation of programmed cell death via a pathway independent of the pathogen-induced HR. However, even with this caution, this work is significant because it convincingly shows that NtMEK2 activates SIPK in vivo. Given the data mentioned above from Sheen and colleagues17,18, one would predict that NPK1 (tobacco ortholog of the Arabidopsis ANP genes) functions as the MAPKKK that activates NtMEK2 (Fig. 1). Thus, it should now be possible to assemble the full MAPKKK–MAPKK–MAPK cascade for SIPK/MPK6, which would be a first for any MAPK pathway in plants. Based on two-hybrid and complementation analyses performed in yeast4,5, the full triad for MPK4 can also be predicted (Fig. 1). Significantly, the MAPKKKs predicted to regulate MPK4 and MPK6 belong to distinct subclasses17. Three general classes of MAPK cascades in plants Three subclasses of MAPKKKs have been defined in plants, the MEKK1 class, the ANP class and the Raf class17. Members of all three subclasses have now been implicated in the regulation of disease resistance because the Raf-like http://plants.trends.com TRENDS in Plant Science Vol.6 No.9 September 2001 MAPKKK gene EDR1 has recently been shown to negatively regulate plant defense responses, including programmed cell death21. In addition, a second Raf-like gene CTR1 functions to negatively regulate ethylene inducible genes22, including the PDF1.2 gene. Thus the picture that emerges is one of competing MAPK pathways that positively and negatively regulate plant defenses (Fig. 1). Acknowledgements I thank John Mundy and his laboratory members for permission to discuss unpublished results. Work discussed from my laboratory was supported by National Institutes of Health grant R01-GM46451 and a grant from Novartis Agricultural Biotechnology Research, Inc. References 1 Widmann, C. et al. (1999) Mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human. Physiol. Rev. 79, 143–180 2 Ligterink, W. (2000) MAP kinases in plant signal transduction: how many, and what for? In MAP Kinases in Plant Signal Transduction (Vol. 27) (Hirt, H., ed.), pp. 11–27, Springer-Verlag 3 Huang, Y. et al. (2000) ATMPK4, an Arabidopsis homolog of mitogen-activated protein kinase, is activated in vitro by AtMEK1 through threonine phosphorylation. Plant Physiol. 122, 1301–1310 4 Ichimura, K. et al. (1998) Isolation of ATMEKK1 (a MAP kinase kinase kinase)-interacting proteins and analysis of a MAP kinase cascade in Arabidopsis. Biochem. Biophys. Res. Commun. 253, 532–543 5 Mizoguchi, T. et al. 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Plant Cell 9, 809–824 17 Kovtun, Y. et al. (2000) Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants. Proc. Natl. Acad. Sci. U. S. A. 97, 2940–2945 18 Kovtun, Y. et al. (1998) Suppression of auxin signal transduction by a MAPK cascade in higher plants. Nature 395, 716–720 19 Nishihama, R. and Machida, Y. (2000) The MAP kinase cascade that includes MAPKKK-related protein kinase NPK1 controls a mitotic process in plant cells. Results Probl. Cell Differ. 27, 119–130 20 Yang, K-Y. et al. (2001) Activation of mitogenactivated protein kinase pathway is involved in disease resistance in tobacco. Proc. Natl. Acad. Sci. U. S. A. 98, 741–746 21 Frye, C.A. et al. (2001) Negative regulation of defense responses in plants by a conserved MAPKK kinase. Proc. Natl. Acad. Sci. U. S. A. 98, 373–378 22 Kieber, J.J. et al. (1993) CTR1, a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the raf family of protein kinases. Cell 72, 427–441 Roger W. Innes Dept of Biology, Indiana University, Bloomington, IN 47405-3700, USA. e-mail: [email protected] Pictures in Plant Science Have you generated images that are not only visually stunning, but also provide a real insight into the molecular understanding of plant science? Please send these images*, plus a short explanation of the background to the work, to our editorial office: [email protected] *Please contact the editorial office for details of the correct electronic format before sending any images.