- Email: [email protected]
Plant GTPases: Regulation of Morphogenesis by ROPs and ROS
Dispatch R211
no proofreading activity and are error-prone [3]. While low-fidelity polymerases may serve specific cellular needs [3], most processes involving DNA synthesis must occur faithfully to maintain genome stability. Proofreading in trans is one possible mechanism to increase the fidelity of error-prone polymerases. To date, there is good evidence that polymerase d proofreads for polymerase 3 [6] and polymerase a [9] and compelling data linking polymerase d proofreading with polymerase h [7,15,19]. Other mammalian 30 /50 exonucleases could also function as extrinsic proofreaders [4]. Specialized DNA polymerases and exonucleases may play important tissue-specific roles to suppress distinct types of spontaneous or environmentally induced mutations. An exciting challenge will be to identify these roles and the polymerase/ exonuclease partners that are involved. References 1. Drake, J.W., Charlesworth, B., Charlesworth, D., and Crow, J.F. (1998). Rates of spontaneous mutation. Genetics 148, 1667–1686. 2. Kunkel, T.A. (2004). DNA replication fidelity. J. Biol. Chem. 279, 16895–16898. 3. Bebenek, K., and Kunkel, T.A. (2004). Functions of DNA polymerases. Adv. Protein Chem. 69, 137–165. 4. Shevelev, I.V., and Hu¨bscher, U. (2002). The 30 –50 exonucleases. Nat. Rev. Mol. Cell Biol. 3, 364–376.
5. Garg, P., and Burgers, P.M. (2005). DNA polymerases that propagate the eukaryotic DNA replication fork. Crit. Rev. Biochem. Mol. Biol. 40, 115–128. 6. Morrison, A., and Sugino, A. (1994). The 30 /50 exonucleases of both DNA polymerases d and 3 participate in correcting errors of DNA replication in Saccharomyces cerevisiae. Mol. Gen. Genet. 242, 289–296. 7. Goldsby, R.E., Hays, L.E., Chen, X., Olmsted, E.A., Slayton, W.B., Spangrude, G.J., and Preston, B.D. (2002). High incidence of epithelial cancers in mice deficient for DNA polymerase d proofreading. Proc. Natl. Acad. Sci. USA 99, 15560–15565. 8. Shcherbakova, P.V., and Pavlov, Y.I. (1996). 30 /50 exonucleases of DNA polymerases 3 and d correct base analog induced DNA replication errors on opposite DNA strands in Saccharomyces cerevisiae. Genetics 142, 717–726. 9. Pavlov, Y.I., Frahm, C., McElhinny, S.A., Niimi, A., Suzuki, M., and Kunkel, T.A. (2006). Evidence that errors made by DNA polymerase a are corrected by DNA polymerase d. Curr. Biol. 16, 202–207. 10. Perrino, F.W., and Loeb, L.A. (1989). Proofreading by the 3 subunit of Escherichia coli DNA polymerase III increases the fidelity of calf thymus DNA polymerase a. Proc. Natl. Acad. Sci. USA 86, 3085–3088. 11. Perrino, F.W., and Loeb, L.A. (1990). Hydrolysis of 30 -terminal mispairs in vitro by the 30 /50 exonuclease of DNA polymerase d permits subsequent extension by DNA polymerase a. Biochemistry 29, 5226–5231. 12. Niimi, A., Limsirichaikul, S., Yoshida, S., Iwai, S., Masutani, C., Hanaoka, F., Kool, E.T., Nishiyama, Y., and Suzuki, M. (2004). Palm mutants in DNA polymerases a and h alter DNA replication fidelity and translesion activity. Mol. Cell. Biol. 24, 2734–2746. 13. Tran, H.T., Gordenin, D.A., and Resnick, M.A. (1999). The 30 /50 exonucleases of DNA polymerases d and 3 and the 50 /30 exonuclease Exo1 have major roles in postreplication mutation avoidance in
Plant GTPases: Regulation of Morphogenesis by ROPs and ROS Polarized cell growth in plants is controlled by Rho-like small GTPases (ROPs), not only through the canonical WAVE/Arp2/3 pathway, but also through newly defined plant-specific pathways involving the regulated release of reactive oxygen species (ROS). Joachim F. Uhrig and Martin Hu¨lskamp Small GTPases of the Rho family are universal master regulators, which have been shown to transmit signals from outside the cell to intracellular signalling cascades in many contexts in both animals and yeast, potentially affecting a wide variety of cellular processes.
These GTPases cycle between the active GTP-bound and inactive GDP-bound form in steps that are regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs) and guanine nucleotide dissociation inhibitors (GDIs) [1] (Figure 1). Plants do not have clear orthologues of the Rho/Rac/Cdc42
14.
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Saccharomyces cerevisiae. Mol. Cell. Biol. 19, 2000–2007. Johansson, E., Garg, P., and Burgers, P.M. (2004). The Pol32 subunit of DNA polymerase d contains separable domains for processive replication and proliferating cell nuclear antigen (PCNA) binding. J. Biol. Chem. 279, 1907–1915. Cleaver, J.E. (2005). Cancer in xeroderma pigmentosum and related disorders of DNA repair. Nat. Rev. Cancer 5, 564–573. Prakash, S., Johnson, R.E., and Prakash, L. (2005). Eukaryotic translesion synthesis DNA polymerases: specificity of structure and function. Annu. Rev. Biochem. 74, 317–353. Gibbs, P.E., McDonald, J., Woodgate, R., and Lawrence, C.W. (2005). The relative roles in vivo of Saccharomyces cerevisiae Pol h, Pol z, Rev1 protein and Pol32 in the bypass and mutation induction of an abasic site, T-T (6-4) photoadduct and T-T cis-syn cyclobutane dimer. Genetics 169, 575–582. Friedberg, E.C., Lehmann, A.R., and Fuchs, R.P. (2005). Trading places: how do DNA polymerases switch during translesion DNA synthesis? Mol. Cell 18, 499–505. McCulloch, S.D., Kokoska, R.J., Chilkova, O., Welch, C.M., Johansson, E., Burgers, P.M., and Kunkel, T.A. (2004). Enzymatic switching for efficient and accurate translesion DNA replication. Nucl. Acids Res. 32, 4665–4675. Catalano, C.E., and Benkovic, S.J. (1989). Inactivation of DNA polymerase I (Klenow fragment) by adenosine 20 ,30 -epoxide 50 -triphosphate: evidence for the formation of a tight-binding inhibitor. Biochemistry 28, 4374–4382.
Departments of 1Pathology and 2 Pediatrics, University of Washington, 1959 NE Pacific Street, Seattle, Washington 98195, USA. E-mail: [email protected]
DOI: 10.1016/j.cub.2006.02.031
GTPases, but they do have a distinct class of Rho-like proteins known as Rho-like small GTPases (ROPs) [2]. The regulators of ROPs in plants are also markedly different, with the exception of three GDI homologs which have been identified by sequence similarity and shown to interact with ROPs [3]. Most ROP-GAPs contain a distinctive Cdc42/Rac-interactive binding (CRIB) domain. CRIB domains are also found in Cdc42/ Rac effectors, where they mediate binding to the active GTPase. How ROPs are activated by GEFs had been unclear until very recently, as the Arabidopsis genome does not appear to encode GEFs of the usual type — with the characteristic tandem arrangement
Current Biology Vol 16 No 6 R212
R K PRONE-GEFs ROP (GDP)
ROP (GTP) CRIB-GAPs
GDI
ROP (GDP)
RIC3 NAP, Abi, HSPC300, WAVE, PIR/Sra
RIC4
ROS
RIC1 Ca2+
Ordered F-actin microtubules assembly
F-actin disassembly
Vesicle fusion
Ca2+ flux
Cell wall weakening
ARP2/3
Actin assembly
Current Biology
Figure 1. The role of ROPs in plant cell morphogenesis. The top half of the figure shows the GDP–GTP cycle of ROPs and its regulators. The bottom half highlights four pathways for regulating local cell growth. Proteins or protein complexes marked in red are found in both animals and plants; those labelled in green are plant-specific.
of B-cell lymphoma homology (DH) and pleckstrin homology (PH) domains — and the plant SPIKE protein [4], which looks like an unconventional GEFs with a Dock-homology region (DHR), has not been been definitively shown to have GEF activity. But a plantspecific family of GEFs, defined by the presence of a so-called PRONE domain, has recently been identified [5] and these proteins have been shown to control ROP-dependent polar growth [6]. Rho GTPases in animals, yeast and plants are important regulators of local cell growth. In plants, it has become evident that ROP dependent cell growth involves not only the well known WAVE/Arp2/3 pathway, but also newly identified signal transduction routes via ‘ROP-interactive CRIB motifcontaining’ (RIC) proteins, as well as signalling cascades that are used in animals for pathogen defence but not thought to be involved in cell morphogenesis [7,8] (Figure 1). The WAVE/Arp2/3 pathways of plants and animals are generally similar, though significant differences have been found, as discussed elsewhere [9]. A plant homologue of the Rho effector Sra,
which is on this pathway, has been identified, but homologues of other Rho effectors that directly mediate cell growth in animals do not appear to be encoded by the Arabidopsis genome. Plants seem to rely instead on RICs, which form a novel plant-specific family of ROP effector proteins. Two different RIC-dependent pathways have been described so far. RIC3 regulates morphogenesis by influencing Ca2+ fluxes, while RIC1 and RIC4 coordinate actin and microtubule organization so that growing regions accumulate actin filaments and are devoid of microtubules [7,10]. In addition to the established functions of ROP-GTPases in actin- or microtubule-dependent morphogenetic processes, another quite different role in cell differentiation and cell growth regulation is emerging. Reactive oxygen species (ROS), initially appreciated mainly for their cytotoxic potential, now are increasingly recognized as versatile second messengers for processes as diverse as pathogen defence, responses to abiotic stresses and regulation of cell elongation [11]. In plants, ROPs are associated with several signalling
pathways leading to the spatially regulated production of ROS, and recent results suggest ROPs have an essentialy function in the regulation of superoxideproducing NADPH oxidases [8]. As small GTPases do in animal phagocytes, ROPs regulate release of ROS in plant defence responses [12]. Furthermore, a recent study of an Arabidopsis mutant with defective ROP-GDI provided evidence for ROP regulation of ROS production in the context of polarized cell expansion [8], substantiating earlier evidence of ROP involvement in root hair development [13]. While the molecular mechanisms of assembly and activation of the plant NADPH oxidase are not yet know, two possible ways that ROS release might lead to local cell expansion can be envisioned: release of ROS might cause cell wall loosening by non-enzymatic cleavage of cell wall polysaccharides [14]; alternatively, ROS might act as second messengers activating Ca2+ channels, and there is increasing evidence for this [15,16]. Comparison with the analogous Rac GTPase-dependent assembly and activation of NADPH oxidase in phagocytes provides a framework for understanding the molecular basis of ROP function and reveals similarities and differences between animals and plants (Figure 2). Phagocytes are components of the innate immune system which are able to engulf microorganisms and kill them by the release of microbicidal agents such as ROS. Recognition of microbial pathogens and phagocytosis trigger the assembly and activation of NADPH oxidase (NOX2), a large enzyme complex composed of the membranebound heterodimeric cytochrome b559 and the cytosolic proteins p67phox, p47phox, p40phox and the small GTPase Rac2 [17]. A crucial step in this process is the activation of Rac2 by dissociation from RhoGDI followed by translocation to the plasma membrane and Rho-GEF-dependent guanine exchange [18,19]. Activated Rac2 binds directly to p67phox and this interaction is essential for NADPH oxidase activity [20].
Dispatch R213
While the overall scheme for ROP/Rac-dependent activation of NADPH oxidase leading to localized release of ROS is similar for animals and plants, the molecular realization appears to be markedly different. In addition to the above-mentioned differences between the small GTPases and their regulators in plant and animal cells, there are striking differences in the structure and the composition of the NADPH oxidase complexes. The cytochrome b559 catalytic subunit of the plant NADPH oxidase, Atrboh, in contrast to its animal counterpart, NOX2, has two EF-hand domains, known in other contexts for their ability to bind Ca2+ ions. Here, a regulatory loop could be envisioned, in which ROS triggers Ca2+ influx, and the Ca2+ ions in turn regulate the activity of NADPH oxidase via binding to the EF-hands. Additionally, phagocyte NADPH oxidase activity is strictly dependent on the presence of p47phox and p67 phox, the latter being the direct target of activated Rac2. Homologues of these two proteins have not been found in plants; it therefore remains an open question what the direct downstream targets of ROP might be, and how exactly assembly and activation of NADPH oxidase is accomplished in plants. References 1. Hall, A. (1998). Rho GTPases and the actin cytoskeleton. Science 279, 509–514. 2. Zheng, Z.L., and Yang, Z. (2000). The Rop GTPase: an emerging signaling switch in plants. Plant Mol. Biol. 44, 1–9. 3. Bischoff, F., Vahlkamp, L., Molendijk, A., and Palme, K. (2000). Localization of AtROP4 and AtROP6 and interaction with the guanine nucleotide dissociation inhibitor AtRhoGDI1 from Arabidopsis. Plant Mol. Biol. 42, 515–530. 4. Qiu, J.L., Jilk, R., Marks, M.D., and Szymanski, D.B. (2002). The Arabidopsis SPIKE1 gene is required for normal cell shape control and tissue development. Plant Cell 14, 101–118. 5. Berken, A., Thomas, C., and Wittinghofer, A. (2005). A new family of RhoGEFs activates the Rop molecular switch in plants. Nature 436, 1176–1180. 6. Gu, Y., Li, S., Lord, E.M., and Yang, Z. (2006). Members of a novel class of Arabidopsis Rho guanine nucleotide exchange factors control Rho GTPase-dependent polar growth. Plant Cell, in press. 7. Fu, Y., Gu, Y., Zheng, Z., Wasteneys, G., and Yang, Z. (2005). Arabidopsis interdigitating cell growth requires two antagonistic pathways with opposing action on cell morphogenesis. Cell 120, 687–700. 8. Carol, R.J., Takeda, S., Linstead, P., Durrant, M.C., Kakesova, H.,
Plants Pathogen recognition, abiotic stresses
ROP-GDP
ROP-GTP
Animals Pathogen recognition, phagocytosis
Developmental signals
ROP-GDP
ROP-GTP
RAC-GTP
RAC-GDP
Effectors?
Effectors?
p67phox interaction, membrane translocation
NADPH oxidase (AtrbohD/AtrbohF)
NADPH oxidase (AtrbohC)
NADPH oxidase (NOX2)
ROS
ROS
ROS
Ca2+ flux
e.g. HR, cell death
Cell wall loosening
K+ flux
Ca2+ flux
Polar cell expansion
Destruction of ingested bacteria Current Biology
Figure 2. ROP/Rac-regulated pathways leading to ROS production. Plants and animals employ analogous ROP/Rac-dependent pathways to regulate the activity of NADPH oxidase for the localized production and release of reactive oxygen species (ROS). However, despite the overall similarity of the pathways, there are differences in the molecular components and mechanisms, the incoming signals and the physiological output.
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Derbyshire, P., Drea, S., Zarsky, V., and Dolan, L. (2005). A RhoGDP dissociation inhibitor spatially regulates growth in root hair cells. Nature 438, 1013–1016. Deeks, M.J., and Hussey, P.J. (2005). Arp2/3 and SCAR: plants move to the fore. Nat. Rev. Mol. Cell Biol. 6, 954–964. Gu, Y., Fu, Y., Dowd, P., Li, S., Vernoud, V., Gilroy, S., and Yang, Z. (2005). A Rho family GTPase controls actin dynamics and tip growth via two counteracting downstream pathways in pollen tubes. J. Cell Biol. 169, 127–138. Torres, M.A., and Dangl, J.L. (2005). Functions of the respiratory burst oxidase in biotic interactions, abiotic stress and development. Curr. Opin. Plant Biol. 8, 397–403. Agrawal, G.K., Iwahashi, H., and Rakwal, R. (2003). Small GTPase ‘Rop’: molecular switch for plant defense responses. FEBS Lett. 546, 173–180. Molendijk, A.J., Bischoff, F., Rajendrakumar, C.S., Friml, J., Braun, M., Gilroy, S., and Palme, K. (2001). Arabidopsis thaliana Rop GTPases are localized to tips of root hairs and control polar growth. EMBO J. 20, 2779–2788. Liszkay, A., van der Zalm, E., and Schopfer, P. (2004). Production of reactive oxygen intermediates (O(2)(.-), H(2)O(2), and (.)OH) by maize roots and their role in wall loosening and elongation growth. Plant Physiol. 136, 3114–3123. Foreman, J., Demidchik, V., Bothwell, J.H., Mylona, P., Miedema, H., Torres, M.A., Linstead, P., Costa, S., Brownlee, C., Jones, J.D., et al. (2003). Reactive oxygen
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species produced by NADPH oxidase regulate plant cell growth. Nature 422, 442–446. Mori, I.C., and Schroeder, J.I. (2004). Reactive oxygen species activation of plant Ca2+ channels. A signaling mechanism in polar growth, hormone transduction, stress signaling, and hypothetically mechanotransduction. Plant Physiol. 135, 702–708. Nauseef, W.M. (2004). Assembly of the phagocyte NADPH oxidase. Histochem. Cell Biol. 122, 277–291. Abo, A., Webb, M.R., Grogan, A., and Segal, A.W. (1994). Activation of NADPH oxidase involves the dissociation of p21rac from its inhibitory GDP/GTP exchange protein (rhoGDI) followed by its translocation to the plasma membrane. Biochem. J. 298, 585–591. Bokoch, G.M., Bohl, B.P., and Chuang, T.H. (1994). Guanine nucleotide exchange regulates membrane translocation of Rac/ Rho GTP-binding proteins. J. Biol. Chem. 269, 31674–31679. Diekmann, D., Abo, A., Johnston, C., Segal, A.W., and Hall, A. (1994). Interaction of Rac with p67phox and regulation of phagocytic NADPH oxidase activity. Science 265, 531–533.
University of Ko¨ln, Botanical Institute III, Gyrhofstr. 15, D-50931, Ko¨ln, Germany. E-mail: [email protected]; [email protected] DOI: 10.1016/j.cub.2006.02.028
no proofreading activity and are error-prone [3]. While low-fidelity polymerases may serve specific cellular needs [3], most processes involving DNA synthesis must occur faithfully to maintain genome stability. Proofreading in trans is one possible mechanism to increase the fidelity of error-prone polymerases. To date, there is good evidence that polymerase d proofreads for polymerase 3 [6] and polymerase a [9] and compelling data linking polymerase d proofreading with polymerase h [7,15,19]. Other mammalian 30 /50 exonucleases could also function as extrinsic proofreaders [4]. Specialized DNA polymerases and exonucleases may play important tissue-specific roles to suppress distinct types of spontaneous or environmentally induced mutations. An exciting challenge will be to identify these roles and the polymerase/ exonuclease partners that are involved. References 1. Drake, J.W., Charlesworth, B., Charlesworth, D., and Crow, J.F. (1998). Rates of spontaneous mutation. Genetics 148, 1667–1686. 2. Kunkel, T.A. (2004). DNA replication fidelity. J. Biol. Chem. 279, 16895–16898. 3. Bebenek, K., and Kunkel, T.A. (2004). Functions of DNA polymerases. Adv. Protein Chem. 69, 137–165. 4. Shevelev, I.V., and Hu¨bscher, U. (2002). The 30 –50 exonucleases. Nat. Rev. Mol. Cell Biol. 3, 364–376.
5. Garg, P., and Burgers, P.M. (2005). DNA polymerases that propagate the eukaryotic DNA replication fork. Crit. Rev. Biochem. Mol. Biol. 40, 115–128. 6. Morrison, A., and Sugino, A. (1994). The 30 /50 exonucleases of both DNA polymerases d and 3 participate in correcting errors of DNA replication in Saccharomyces cerevisiae. Mol. Gen. Genet. 242, 289–296. 7. Goldsby, R.E., Hays, L.E., Chen, X., Olmsted, E.A., Slayton, W.B., Spangrude, G.J., and Preston, B.D. (2002). High incidence of epithelial cancers in mice deficient for DNA polymerase d proofreading. Proc. Natl. Acad. Sci. USA 99, 15560–15565. 8. Shcherbakova, P.V., and Pavlov, Y.I. (1996). 30 /50 exonucleases of DNA polymerases 3 and d correct base analog induced DNA replication errors on opposite DNA strands in Saccharomyces cerevisiae. Genetics 142, 717–726. 9. Pavlov, Y.I., Frahm, C., McElhinny, S.A., Niimi, A., Suzuki, M., and Kunkel, T.A. (2006). Evidence that errors made by DNA polymerase a are corrected by DNA polymerase d. Curr. Biol. 16, 202–207. 10. Perrino, F.W., and Loeb, L.A. (1989). Proofreading by the 3 subunit of Escherichia coli DNA polymerase III increases the fidelity of calf thymus DNA polymerase a. Proc. Natl. Acad. Sci. USA 86, 3085–3088. 11. Perrino, F.W., and Loeb, L.A. (1990). Hydrolysis of 30 -terminal mispairs in vitro by the 30 /50 exonuclease of DNA polymerase d permits subsequent extension by DNA polymerase a. Biochemistry 29, 5226–5231. 12. Niimi, A., Limsirichaikul, S., Yoshida, S., Iwai, S., Masutani, C., Hanaoka, F., Kool, E.T., Nishiyama, Y., and Suzuki, M. (2004). Palm mutants in DNA polymerases a and h alter DNA replication fidelity and translesion activity. Mol. Cell. Biol. 24, 2734–2746. 13. Tran, H.T., Gordenin, D.A., and Resnick, M.A. (1999). The 30 /50 exonucleases of DNA polymerases d and 3 and the 50 /30 exonuclease Exo1 have major roles in postreplication mutation avoidance in
Plant GTPases: Regulation of Morphogenesis by ROPs and ROS Polarized cell growth in plants is controlled by Rho-like small GTPases (ROPs), not only through the canonical WAVE/Arp2/3 pathway, but also through newly defined plant-specific pathways involving the regulated release of reactive oxygen species (ROS). Joachim F. Uhrig and Martin Hu¨lskamp Small GTPases of the Rho family are universal master regulators, which have been shown to transmit signals from outside the cell to intracellular signalling cascades in many contexts in both animals and yeast, potentially affecting a wide variety of cellular processes.
These GTPases cycle between the active GTP-bound and inactive GDP-bound form in steps that are regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs) and guanine nucleotide dissociation inhibitors (GDIs) [1] (Figure 1). Plants do not have clear orthologues of the Rho/Rac/Cdc42
14.
15.
16.
17.
18.
19.
20.
Saccharomyces cerevisiae. Mol. Cell. Biol. 19, 2000–2007. Johansson, E., Garg, P., and Burgers, P.M. (2004). The Pol32 subunit of DNA polymerase d contains separable domains for processive replication and proliferating cell nuclear antigen (PCNA) binding. J. Biol. Chem. 279, 1907–1915. Cleaver, J.E. (2005). Cancer in xeroderma pigmentosum and related disorders of DNA repair. Nat. Rev. Cancer 5, 564–573. Prakash, S., Johnson, R.E., and Prakash, L. (2005). Eukaryotic translesion synthesis DNA polymerases: specificity of structure and function. Annu. Rev. Biochem. 74, 317–353. Gibbs, P.E., McDonald, J., Woodgate, R., and Lawrence, C.W. (2005). The relative roles in vivo of Saccharomyces cerevisiae Pol h, Pol z, Rev1 protein and Pol32 in the bypass and mutation induction of an abasic site, T-T (6-4) photoadduct and T-T cis-syn cyclobutane dimer. Genetics 169, 575–582. Friedberg, E.C., Lehmann, A.R., and Fuchs, R.P. (2005). Trading places: how do DNA polymerases switch during translesion DNA synthesis? Mol. Cell 18, 499–505. McCulloch, S.D., Kokoska, R.J., Chilkova, O., Welch, C.M., Johansson, E., Burgers, P.M., and Kunkel, T.A. (2004). Enzymatic switching for efficient and accurate translesion DNA replication. Nucl. Acids Res. 32, 4665–4675. Catalano, C.E., and Benkovic, S.J. (1989). Inactivation of DNA polymerase I (Klenow fragment) by adenosine 20 ,30 -epoxide 50 -triphosphate: evidence for the formation of a tight-binding inhibitor. Biochemistry 28, 4374–4382.
Departments of 1Pathology and 2 Pediatrics, University of Washington, 1959 NE Pacific Street, Seattle, Washington 98195, USA. E-mail: [email protected]
DOI: 10.1016/j.cub.2006.02.031
GTPases, but they do have a distinct class of Rho-like proteins known as Rho-like small GTPases (ROPs) [2]. The regulators of ROPs in plants are also markedly different, with the exception of three GDI homologs which have been identified by sequence similarity and shown to interact with ROPs [3]. Most ROP-GAPs contain a distinctive Cdc42/Rac-interactive binding (CRIB) domain. CRIB domains are also found in Cdc42/ Rac effectors, where they mediate binding to the active GTPase. How ROPs are activated by GEFs had been unclear until very recently, as the Arabidopsis genome does not appear to encode GEFs of the usual type — with the characteristic tandem arrangement
Current Biology Vol 16 No 6 R212
R K PRONE-GEFs ROP (GDP)
ROP (GTP) CRIB-GAPs
GDI
ROP (GDP)
RIC3 NAP, Abi, HSPC300, WAVE, PIR/Sra
RIC4
ROS
RIC1 Ca2+
Ordered F-actin microtubules assembly
F-actin disassembly
Vesicle fusion
Ca2+ flux
Cell wall weakening
ARP2/3
Actin assembly
Current Biology
Figure 1. The role of ROPs in plant cell morphogenesis. The top half of the figure shows the GDP–GTP cycle of ROPs and its regulators. The bottom half highlights four pathways for regulating local cell growth. Proteins or protein complexes marked in red are found in both animals and plants; those labelled in green are plant-specific.
of B-cell lymphoma homology (DH) and pleckstrin homology (PH) domains — and the plant SPIKE protein [4], which looks like an unconventional GEFs with a Dock-homology region (DHR), has not been been definitively shown to have GEF activity. But a plantspecific family of GEFs, defined by the presence of a so-called PRONE domain, has recently been identified [5] and these proteins have been shown to control ROP-dependent polar growth [6]. Rho GTPases in animals, yeast and plants are important regulators of local cell growth. In plants, it has become evident that ROP dependent cell growth involves not only the well known WAVE/Arp2/3 pathway, but also newly identified signal transduction routes via ‘ROP-interactive CRIB motifcontaining’ (RIC) proteins, as well as signalling cascades that are used in animals for pathogen defence but not thought to be involved in cell morphogenesis [7,8] (Figure 1). The WAVE/Arp2/3 pathways of plants and animals are generally similar, though significant differences have been found, as discussed elsewhere [9]. A plant homologue of the Rho effector Sra,
which is on this pathway, has been identified, but homologues of other Rho effectors that directly mediate cell growth in animals do not appear to be encoded by the Arabidopsis genome. Plants seem to rely instead on RICs, which form a novel plant-specific family of ROP effector proteins. Two different RIC-dependent pathways have been described so far. RIC3 regulates morphogenesis by influencing Ca2+ fluxes, while RIC1 and RIC4 coordinate actin and microtubule organization so that growing regions accumulate actin filaments and are devoid of microtubules [7,10]. In addition to the established functions of ROP-GTPases in actin- or microtubule-dependent morphogenetic processes, another quite different role in cell differentiation and cell growth regulation is emerging. Reactive oxygen species (ROS), initially appreciated mainly for their cytotoxic potential, now are increasingly recognized as versatile second messengers for processes as diverse as pathogen defence, responses to abiotic stresses and regulation of cell elongation [11]. In plants, ROPs are associated with several signalling
pathways leading to the spatially regulated production of ROS, and recent results suggest ROPs have an essentialy function in the regulation of superoxideproducing NADPH oxidases [8]. As small GTPases do in animal phagocytes, ROPs regulate release of ROS in plant defence responses [12]. Furthermore, a recent study of an Arabidopsis mutant with defective ROP-GDI provided evidence for ROP regulation of ROS production in the context of polarized cell expansion [8], substantiating earlier evidence of ROP involvement in root hair development [13]. While the molecular mechanisms of assembly and activation of the plant NADPH oxidase are not yet know, two possible ways that ROS release might lead to local cell expansion can be envisioned: release of ROS might cause cell wall loosening by non-enzymatic cleavage of cell wall polysaccharides [14]; alternatively, ROS might act as second messengers activating Ca2+ channels, and there is increasing evidence for this [15,16]. Comparison with the analogous Rac GTPase-dependent assembly and activation of NADPH oxidase in phagocytes provides a framework for understanding the molecular basis of ROP function and reveals similarities and differences between animals and plants (Figure 2). Phagocytes are components of the innate immune system which are able to engulf microorganisms and kill them by the release of microbicidal agents such as ROS. Recognition of microbial pathogens and phagocytosis trigger the assembly and activation of NADPH oxidase (NOX2), a large enzyme complex composed of the membranebound heterodimeric cytochrome b559 and the cytosolic proteins p67phox, p47phox, p40phox and the small GTPase Rac2 [17]. A crucial step in this process is the activation of Rac2 by dissociation from RhoGDI followed by translocation to the plasma membrane and Rho-GEF-dependent guanine exchange [18,19]. Activated Rac2 binds directly to p67phox and this interaction is essential for NADPH oxidase activity [20].
Dispatch R213
While the overall scheme for ROP/Rac-dependent activation of NADPH oxidase leading to localized release of ROS is similar for animals and plants, the molecular realization appears to be markedly different. In addition to the above-mentioned differences between the small GTPases and their regulators in plant and animal cells, there are striking differences in the structure and the composition of the NADPH oxidase complexes. The cytochrome b559 catalytic subunit of the plant NADPH oxidase, Atrboh, in contrast to its animal counterpart, NOX2, has two EF-hand domains, known in other contexts for their ability to bind Ca2+ ions. Here, a regulatory loop could be envisioned, in which ROS triggers Ca2+ influx, and the Ca2+ ions in turn regulate the activity of NADPH oxidase via binding to the EF-hands. Additionally, phagocyte NADPH oxidase activity is strictly dependent on the presence of p47phox and p67 phox, the latter being the direct target of activated Rac2. Homologues of these two proteins have not been found in plants; it therefore remains an open question what the direct downstream targets of ROP might be, and how exactly assembly and activation of NADPH oxidase is accomplished in plants. References 1. Hall, A. (1998). Rho GTPases and the actin cytoskeleton. Science 279, 509–514. 2. Zheng, Z.L., and Yang, Z. (2000). The Rop GTPase: an emerging signaling switch in plants. Plant Mol. Biol. 44, 1–9. 3. Bischoff, F., Vahlkamp, L., Molendijk, A., and Palme, K. (2000). Localization of AtROP4 and AtROP6 and interaction with the guanine nucleotide dissociation inhibitor AtRhoGDI1 from Arabidopsis. Plant Mol. Biol. 42, 515–530. 4. Qiu, J.L., Jilk, R., Marks, M.D., and Szymanski, D.B. (2002). The Arabidopsis SPIKE1 gene is required for normal cell shape control and tissue development. Plant Cell 14, 101–118. 5. Berken, A., Thomas, C., and Wittinghofer, A. (2005). A new family of RhoGEFs activates the Rop molecular switch in plants. Nature 436, 1176–1180. 6. Gu, Y., Li, S., Lord, E.M., and Yang, Z. (2006). Members of a novel class of Arabidopsis Rho guanine nucleotide exchange factors control Rho GTPase-dependent polar growth. Plant Cell, in press. 7. Fu, Y., Gu, Y., Zheng, Z., Wasteneys, G., and Yang, Z. (2005). Arabidopsis interdigitating cell growth requires two antagonistic pathways with opposing action on cell morphogenesis. Cell 120, 687–700. 8. Carol, R.J., Takeda, S., Linstead, P., Durrant, M.C., Kakesova, H.,
Plants Pathogen recognition, abiotic stresses
ROP-GDP
ROP-GTP
Animals Pathogen recognition, phagocytosis
Developmental signals
ROP-GDP
ROP-GTP
RAC-GTP
RAC-GDP
Effectors?
Effectors?
p67phox interaction, membrane translocation
NADPH oxidase (AtrbohD/AtrbohF)
NADPH oxidase (AtrbohC)
NADPH oxidase (NOX2)
ROS
ROS
ROS
Ca2+ flux
e.g. HR, cell death
Cell wall loosening
K+ flux
Ca2+ flux
Polar cell expansion
Destruction of ingested bacteria Current Biology
Figure 2. ROP/Rac-regulated pathways leading to ROS production. Plants and animals employ analogous ROP/Rac-dependent pathways to regulate the activity of NADPH oxidase for the localized production and release of reactive oxygen species (ROS). However, despite the overall similarity of the pathways, there are differences in the molecular components and mechanisms, the incoming signals and the physiological output.
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Derbyshire, P., Drea, S., Zarsky, V., and Dolan, L. (2005). A RhoGDP dissociation inhibitor spatially regulates growth in root hair cells. Nature 438, 1013–1016. Deeks, M.J., and Hussey, P.J. (2005). Arp2/3 and SCAR: plants move to the fore. Nat. Rev. Mol. Cell Biol. 6, 954–964. Gu, Y., Fu, Y., Dowd, P., Li, S., Vernoud, V., Gilroy, S., and Yang, Z. (2005). A Rho family GTPase controls actin dynamics and tip growth via two counteracting downstream pathways in pollen tubes. J. Cell Biol. 169, 127–138. Torres, M.A., and Dangl, J.L. (2005). Functions of the respiratory burst oxidase in biotic interactions, abiotic stress and development. Curr. Opin. Plant Biol. 8, 397–403. Agrawal, G.K., Iwahashi, H., and Rakwal, R. (2003). Small GTPase ‘Rop’: molecular switch for plant defense responses. FEBS Lett. 546, 173–180. Molendijk, A.J., Bischoff, F., Rajendrakumar, C.S., Friml, J., Braun, M., Gilroy, S., and Palme, K. (2001). Arabidopsis thaliana Rop GTPases are localized to tips of root hairs and control polar growth. EMBO J. 20, 2779–2788. Liszkay, A., van der Zalm, E., and Schopfer, P. (2004). Production of reactive oxygen intermediates (O(2)(.-), H(2)O(2), and (.)OH) by maize roots and their role in wall loosening and elongation growth. Plant Physiol. 136, 3114–3123. Foreman, J., Demidchik, V., Bothwell, J.H., Mylona, P., Miedema, H., Torres, M.A., Linstead, P., Costa, S., Brownlee, C., Jones, J.D., et al. (2003). Reactive oxygen
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species produced by NADPH oxidase regulate plant cell growth. Nature 422, 442–446. Mori, I.C., and Schroeder, J.I. (2004). Reactive oxygen species activation of plant Ca2+ channels. A signaling mechanism in polar growth, hormone transduction, stress signaling, and hypothetically mechanotransduction. Plant Physiol. 135, 702–708. Nauseef, W.M. (2004). Assembly of the phagocyte NADPH oxidase. Histochem. Cell Biol. 122, 277–291. Abo, A., Webb, M.R., Grogan, A., and Segal, A.W. (1994). Activation of NADPH oxidase involves the dissociation of p21rac from its inhibitory GDP/GTP exchange protein (rhoGDI) followed by its translocation to the plasma membrane. Biochem. J. 298, 585–591. Bokoch, G.M., Bohl, B.P., and Chuang, T.H. (1994). Guanine nucleotide exchange regulates membrane translocation of Rac/ Rho GTP-binding proteins. J. Biol. Chem. 269, 31674–31679. Diekmann, D., Abo, A., Johnston, C., Segal, A.W., and Hall, A. (1994). Interaction of Rac with p67phox and regulation of phagocytic NADPH oxidase activity. Science 265, 531–533.
University of Ko¨ln, Botanical Institute III, Gyrhofstr. 15, D-50931, Ko¨ln, Germany. E-mail: [email protected]; [email protected] DOI: 10.1016/j.cub.2006.02.028