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Hydrogen peroxide signalling
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Hydrogen peroxide signalling Steven Neill*, Radhika Desikan and John Hancock Recent biochemical and genetic studies confirm that hydrogen peroxide is a signalling molecule in plants that mediates responses to abiotic and biotic stresses. Signalling roles for hydrogen peroxide during abscisic-acid-mediated stomatal closure, auxin-regulated root gravitropism and tolerance of oxygen deprivation are now evident. The synthesis and action of hydrogen peroxide appear to be linked to those of nitric oxide. Downstream signalling events that are modulated by hydrogen peroxide include calcium mobilisation, protein phosphorylation and gene expression. Calcium and Rop signalling contribute to the maintenance of hydrogen peroxide homeostasis. Addresses Centre for Research in Plant Science, Faculty of Applied Sciences, University of the West of England (UWE), Frenchay, Bristol BS16 1QY, UK *e-mail: [email protected]
Turnover of hydrogen peroxide H2O2 is continually generated from various sources during normal metabolism (Figure 1). A wide range of steady-state H2O2 concentrations (e.g. 60 µM–7 mM in Arabidopsis [4,5], and 1–2 mM in maize and rice [6,7]), has been reported, although such variation may reflect technical difficulties in quantifying H2O2. Moreover, differences in the H2O2 content of different sub-cellular compartments are not yet known. Although the precise intracellular concentrations of H2O2 that are likely to be toxic will vary, high rates of H2O2 production are normally balanced by very efficient antioxidant systems ([8,9]; Figure 1). Abiotic stresses such as dehydration, low and high temperatures, and excess irradiation can disturb this balance, such that increased H2O2 initiates signalling responses that include enzyme activation, gene expression, programmed cell death (PCD) and cellular damage.
Current Opinion in Plant Biology 2002, 5:388–395 1369-5266/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Published online 30 July 2002 Abbreviations ABA abscisic acid ABI1 ABA-INSENSITIVE1 CaM calmodulin GA gibberellin GAP GTPase-activating protein hydrogen peroxide H2O2 MAPK mitogen-activated protein kinase NO nitric oxide PCD programmed cell death PP protein phosphatase rboh respiratory burst oxidase homologue Rop Rho-like small G protein SLN1 synthetic lethal of N-end rule1 TF transcription factor
Introduction Until relatively recently, the reactive oxygen species hydrogen peroxide (H2O2) was viewed mainly as a toxic cellular metabolite. However, it is now clear that it is much more than that, and functions as a signalling molecule that mediates responses to various stimuli in both plant and animal cells [1•–3•]. The generation of H2O2 is increased in response to various stresses, implicating it as a key factor mediating the phenomena of acclimation and crosstolerance, in which previous exposure to one stress can induce tolerance of subsequent exposure to the same or different stresses [1•]. For H2O2 to act as a signalling molecule it must have regulated synthesis, specific responses and cellular targets, and there must be mechanisms for its metabolism or removal subsequent to signalling events. In this review, we focus on recent work that highlights the signalling aspects of H2O2 generation and action.
H2O2 can also be generated by specific enzymes. An oxidative burst, with rapid H2O2 synthesis and release into the apoplast, is a common response to pathogens, elicitors, wounding, heat, ultra-violet light and ozone [10–12]. Pharmacological and molecular data indicate that the enzyme responsible is similar to the superoxide (O2.–)-generating NADPH oxidase originally characterised in mammalian phagocytes ([10]; Figure 1). Several homologues of this enzyme exist in plant genomes, but until recently no firm data had been provided to confirm its activity and function. Using a novel in-gel enzyme assay, O2.–-generating (and thus H2O2-generating) activity was demonstrated in plasma membranes from tobacco and tomato. This activity was directly modulated by calcium (placing calcium upstream of H2O2 synthesis), increased by viral infection and localised with proteins reacting with an NADPH oxidase antibody [13••]. Knockout experiments demonstrated that AtrbohD and AtrbohF genes (encoding NADPH oxidase) are required for H2O2 generation during bacterial and fungal challenge in Arabidopsis [14••]. Moreover, defence responses following bacterial challenge were compromised in the knockout mutants in which these genes were not functional. Such experiments also confirm that plant NADPH oxidases differ from the mammalian version as the enzyme activity of the plasma-membrane-localised homologue is not dependent on the assembly of a complex involving cytosolic components, and the enzyme is directly activated via Ca2+ binding. As there is a large NADPH oxidase gene family in plants, and different members have differing biological activity [14••], it is imperative to ascertain their cellular patterns of expression and activity. Exciting new data emphasise the important role of Rops (Rho-like small G proteins) in regulating H2O2 production, potentially via NADPH oxidase [15••]. Previous work had demonstrated the involvement of Rop signalling in H2O2
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Figure 1 H2O2 turnover in the plant cell. H2O2 is generated in normal metabolism via the Mehler reaction in chloroplasts, electron transport in mitochondria and photorespiration in peroxisomes. Peroxisomes may also contain other systems that generate H2O2 [9]. Abiotic and biotic stresses enhance H2O2 generation via these routes and also via enzymatic sources such as plasma-membrane-localised NADPH oxidases (RBOH; [13••,14••]) or cell wall peroxidases [18•]. H2O2 diffuses freely, perhaps facilitated by movement through peroxiporin membrane channels [21]. Cellular H2O2 levels are determined by the rates of H2O2 production and metabolism via catalase and the ubiquitous ascorbate-glutathione cycle (A-G cycle; [8]), which involves ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR) and glutathione reductase (GR). H2O2 also reacts with glutathione to convert it from its reduced state (GSH) to its oxidised state (GSSG).
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generation (see [16]). Recently, oxygen deprivation was found to activate Rop signalling that in turn activated NADPH oxidase. This was attenuated by H2O2 induction of RopGAP gene expression that leads to the deactivation of Rop [15••]. Other potential enzymatic sources of H2O2 include xanthine oxidase, amine oxidase and a cell wall peroxidase [9,17,18•]. It is possible that different stimuli activate specific H2O2-generating enzymes [18•]. A cell wall peroxidase has been identified in French bean [17], and a potentially peroxidase-mediated oxidative burst has been demonstrated in Arabidopsis cultures challenged with a fungal elicitor [18•]. Arabidopsis plants transformed with an antisense bean peroxidase construct are hypersensitive to bacterial and fungal pathogens [18•] but the endogenous Arabidopsis peroxidase has yet to be identified. Under most circumstances, cellular antioxidant systems remove H2O2 efficiently. However, given that large amounts
of H2O2 are produced, any reduction in antioxidant status would have rapid and potentially disastrous effects. Indeed, several studies have demonstrated the effects of over- and under-expression of antioxidant enzymes on cell physiology (e.g. [19]). Thus, the oxidative effects of various stimuli could be mediated via reductions in the activities of antioxidant enzymes, rather than by increased H2O2 generation. For example, gibberellin (GA)-induced PCD in barley aleurone resulted from increased H2O2 effected by a reduction in antioxidant activity [20•]. It is possible that the high antioxidant status of cells prevents H2O2 from travelling far through a cell. Consequently, responses to H2O2 may be localised to microdomains (i.e. ‘H2O2 hot-spots’) within the cell. Cellular fate may then be dependent on where in the cell H2O2 synthesis is increased. Even when relatively large amounts of H2O2 are generated in the apoplast, which has only a small proportion of the cell’s antioxidant capacity,
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H2O2 and ABA signalling in guard cells. (a) Increased H2O2 generation is induced by ABA in Arabidopsis guard cells, as visualised with the H2O2-sensitive dye DCFH-2DA (2’,7’-dichlorofluorescein diacetate) and confocal microscopy. (b) H2O2-mediated ABA signalling in guard cells. ABA induces H2O2 synthesis via a plasma membrane NADPH oxidase (RBOH; [25•,41••]) or in the chloroplast [25•]. H2O2 inactivates inward K+ channels [42], causes cytosolic alkalinisation [43] and activates plasma membrane Ca2+ channels [41••]. The potential position of various signalling proteins, as deduced from studies with various mutants, is indicated. The growth control by ABA 2 (gca2) mutant responds to ABA by increasing H2O2 synthesis but Ca2+ channels are not activated during this response [40]. The guard cells of abi1 mutants do not make H2O2 but can respond to it, whereas abi2 guard cells synthesise H2O2 but do not respond to it [41••].
H2O2 will rapidly move into the cell and be metabolised. Such movement may be facilitated by ‘peroxiporins’; it has been speculated that water channels, or aquaporins, may also serve as conduits for trans-membrane H2O2 transport [21]. Various studies suggest that H2O2 can function as a mobile signal [5,22–24,25•], but whether H2O2 is the sole signal or part of a systemic response remains to be determined.
PCD is initiated by a range of stimuli and driven by several signals including H2O2 and nitric oxide (NO) [26]. Although the execution processes of plant PCD are not firmly established, altered mitochondrial function, involving the formation of a mitochondrial permeability transition pore, is likely to be involved [26]. Interaction between NO and H2O2 was essential during PCD in soybean cultures challenged with avirulent bacteria [27••]. H2O2 was the key factor in PCD, but the NO/O2.– balance was critical in determining the rate of peroxynitrite formation. Peroxynitrite alone did not induce PCD, so the relative rates of conversion of O2.– to H2O2 or peroxynitrite may determine cellular responses. H2O2 concentrations required to effect PCD vary, perhaps reflecting differences in antioxidant capacity and competence to respond to H2O2. Thus, Arabidopsis protoplasts are considerably more H2O2-sensitive than cells (being sensitive to micromolar as opposed to millimolar concentrations), reflecting their reduced antioxidant status [1•]. Similarly, the survival of barley aleurone in 325 mM H2O2 correlated with the antioxidant capacity after treatments with GA or abscisic acid (ABA) [20•,28•]. The mechanisms by which H2O2 induces cell death are not yet clear, although several studies point to effects on mitochondria. Exposure of Arabidopsis cultures to H2O2 increased subsequent H2O2 production in mitochondria. This resulted in altered mitochondrial function and PCD [29•]. In another study, prolonged activation of mitogen-activated protein kinases (MAPKs) in Arabidopsis resulted in intracellular generation of H2O2 preceding cell death [30•]. Finally, various treatments that inhibited mitochondrial electron transport in tobacco cells, including exogenous H2O2, also increased intracellular H2O2 accumulation [31•]. It is already known that H2O2 activates MAPK pathways [32–36], and it is tempting to speculate that increased H2O2 can stimulate further mitochondrial H2O2 production, perhaps via MAPK activation, in an amplifying oxidative death cycle [37]. H2O2- and ABA-mediated stomatal closure
Although earlier studies had demonstrated the generation of H2O2 and its effects in guard cells [24,38,39], the findings that ABA activates the synthesis of H2O2 in guard cells, apparently via NADPH oxidase, and that H2O2 mediates ABA-induced stomatal closure and, at micromolar concentrations, activates plasma membrane Ca2+ channels were of major importance [40]. In subsequent work [41••], a requirement for NAD(P)H was confirmed and the two ABA-signalling protein phosphatase 2C (PP2C) enzymes, ABA-INSENSITIVE1 (ABI1) and ABI2, were positioned in the ABA and H2O2 signalling chain (Figure 2; [41••]). Mediation of ABA-induced stomatal closure by H2O2 is also seen in Vicia faba [25•], with evidence for two sources of H2O2, chloroplastic and cytoplasmic/plasma membrane, reminiscent of previous findings obtained with elicitor treatment of tobacco
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Figure 3
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H2O2 signalling in plant cells. H2O2, entering cells from the apoplast or generated internally, may diffuse only within sub-cellular microdomains, depending on the amount of H2O2 and the antioxidant status. H2O2 can oxidise or otherwise modulate signalling proteins, such as protein phosphatases (PP), protein kinases (PK) including a plasma membrane histidine kinase and MAPK cascades, TFs, and calcium channels that are located in the plasma membrane or elsewhere. Elevated cytosolic
calcium concentrations will initiate further downstream responses, via the action of calcium-binding proteins that include calmodulin (CaM), PKs and PPs. Reversible protein phosphorylation also initiates downstream signalling both in the cytoplasm and in the nucleus via effects on TFs and therefore on gene expression. The oxidation of TFs may activate them and/or induce nuclear localisation. H2O2 synthesis can be controlled by Rop signalling [15••].
epidermis [24]. Defining the sub-cellular sources of H2O2 in guard cells is an important research goal. In V. faba, H2O2 also inhibited K+ currents and induced cytosolic alkalinisation, as does ABA [42,43]. NO was recently identified as a novel component of ABA signalling in guard
cells [44•], demonstrating once more that H2O2 and NO appear to be made and to operate in tandem. Is H2O2 generation a common response to ABA or is this response confined to guard cells? ABA-induced increases
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gravitropism, respectively. Catalase had no effect on gravitropism, suggesting that an intracellular source of H2O2 is involved; the mechanism of H2O2 generation remains to be determined.
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H2O2 modulation of gene expression. H2O2 may directly oxidise transcription factors (TF) (e.g. [56]) in either the cytoplasm or the nucleus. Alternatively, H2O2-mediated activation of a signalling protein, such as a protein kinase, may activate TFs. Either way, activated TFs would subsequently interact with cognate H2O2-response elements and modulate gene expression.
in H2O2 have been reported for maize seedlings and rice roots [6,7], and earlier work demonstrated that ABAinduced expression of the catalase gene in maize was mediated by increased H2O2 [45]. On the other hand, ABA decreased the release of H2O2 from germinating radish seeds [46]. It seems likely that H2O2 responses to ABA will be tissue-specific and dependent on many factors. Recent data suggesting a role for reactive oxygen species and NO in drought-induced ABA synthesis underline such complexity [47]. Auxin, H2O2 and root gravitropism
Recent work has shown that H2O2 mediates auxin-regulated gravitropic responses [48••]. Gravity induced the asymmetric generation of H2O2 in maize roots, as did asymmetrically applied auxin. Moreover, asymmetric application of H2O2 or antioxidants promoted or inhibited
Calcium fluxes and reversible protein phosphorylation are core components of eukaryotic cell signalling and are both required for the controlled generation of H2O2 [1•]. It is likely that they are also key downstream responses (Figure 3). H2O2 activates Ca2+ channels in guard cell plasma membranes [41••] and induces a specific calcium signature in Arabidopsis seedlings (MC Rentel, University of Oxford, personal communication). Such effects on calcium fluxes, perhaps in cellular microdomains, may be a widespread response to H2O2. Recently, calcium/calmodulin (CaM) has been shown to bind to and activate plant catalases, leading to a decrease in H2O2 levels [49••]. Moreover, CaM was found in the peroxisomes, the major cellular location of catalase. This indicates that calcium has both positive and negative effects in regulating H2O2 homeostasis. H2O2 activates MAPK cascades in various tissues [32–36,48••], but how such activation is achieved remains to be determined. Constitutive activation of a H2O2-responsive MAPK afforded cross-tolerance to various environmental stresses [35]. As calcium and MAPKs regulate the activity of other signalling proteins, further downstream effects of H2O2 are likely to be uncovered. Cell signalling is best considered as an intricate web of interconnecting signal networks rather than as a collection of parallel but separate pathways. Consequently, it will be difficult to differentiate primary cellular responses to H2O2 from those that are effected via other signalling intermediates. However, if H2O2 truly is a specific signalling molecule then there must exist primary targets for H2O2. Chemically, H2O2 is a rather simple molecule, so it might be considered unlikely that there exist specific H2O2 receptors. H2O2 is a mild oxidant that can interact with cysteine residues within proteins, depending on the pKa and the molecular environment [2•,3•,50•]. For such interactions to have physiological significance, they must induce conformational changes that alter protein activity and that are sufficiently long-lasting to initiate cellular responses. Such effects can potentially be seen with the ABI1 and ABI2 enzymes [51••,52••]. H2O2 efficiently and reversibly inactivated these enzymes in vitro. ABI1 and ABI2 are negative regulators of ABA signalling [53]. Thus, one would predict that H2O2 generated as a result of ABA signalling should inactivate ABI1 and ABI2. In fact, these enzymes appear to be activated by ABA [53], and it is not yet known whether H2O2 can actually inhibit the enzymes in vivo. There are many potential PP2Cs in plants, some of which may also be susceptible to H2O2, and targets for PPs include MAPKs. Thus, MAPK activation by H2O2 may be due to the effects of H2O2 on PPs. Recent work with yeast has provided exciting evidence for the existence of a ‘peroxisensor’ [54]. Yeast mutants lacking
Hydrogen peroxide signalling Neill, Desikan and Hancock
the plasma membrane SLN1 (synthetic lethal of N-end rule1) histidine kinase were highly susceptible to H2O2. Complementation of the mutant with a functional SLN1 gene restored the ability to survive in H2O2. The Arabidopsis genome contains several genes with homology to SLN1, and it may be that one or more of the proteins encoded by these genes can function as a peroxisensor.
Acknowledgements
H2O2-regulated gene expression
1. •
H2O2 modulates the expression of various genes, including those encoding antioxidant enzymes and modulators of H2O2 production [1•,15••], indicating the complex way in which intracellular H2O2 concentrations may be monitored and maintained at a constant level [1•]. A microarray study showed that the expression of 1–2% of genes was altered in H2O2-treated Arabidopsis cultures [55••]. As expected, genes encoding antioxidant enzymes were upregulated, but so were genes encoding proteins that are potentially involved in PCD, as well as signalling proteins such as calmodulin, protein kinases and transcription factors (TFs), the latter suggesting longer-term effects. Although H2O2-responsive promoters have been identified (see [55••]), specific H2O2-regulatory DNA sequences and their cognate transcription factors have not yet been isolated and characterised. Whether H2O2regulated transcription involves oxidative changes to TFs themselves (e.g. [56]), in either the cytoplasm or the nucleus, or other modifications such as phosphorylation that are ultimately initiated by H2O2 (Figure 4), remains to be determined.
We thank those colleagues who made available reprints, pre-prints and unpublished data.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest Neill SJ, Desikan R, Clarke A, Hurst RD, Hancock JT: Hydrogen peroxide and nitric oxide as signalling molecules in plants. J Exp Bot 2002, 53:1237-1247. An up-to-date comprehensive review of H2O2 signalling. 2. •
Rhee SG, Bae YS, Lee S-R, Kwon J: Hydrogen peroxide: a key messenger that modulates protein phosphorylation through cysteine oxidation. Science’s Signal Transduction Knowledge Environment, 2000. http://stke.sciencemag.org/cgi/content/full/ OC_sigtrans;2000/53/pe1 This excellent review discusses the role of cysteine oxidation in the transduction of hydrogen peroxide signalling, with particular reference to the modulation of protein phosphorylation. It highlights the importance of the molecular environment of the relevant cysteine residues, which is essential for cysteine oxidation. 3. Finkel T: Redox-dependent signal transduction. FEBS Lett 2000, • 476:52-54. This review provides a good insight into the role of reactive oxygen species in signal transduction, focussing on the regulation of intracellular production of reactive oxygen species and likely protein targets. It highlights the potential of cysteine modification as a mechanism for downstream signalling. 4.
Veljovic-Jovanovic SD, Pignocchi C, Noctor G, Foyer CH: Low ascorbic acid in the vtc-1 mutant of Arabidopsis is associated with decreased growth and intracellular redistribution of the antioxidant system. Plant Physiol 2001, 127:426-435.
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Karpinski S, Reynolds H, Karpinska B, Wingsle G, Creissen G, Mullineaux P: Systemic signaling and acclimation in response to excess excitation energy in Arabidopsis. Science 1999, 284:654-657.
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Jiang M, Zhang J: Effect of abscisic acid on active oxygen species, antioxidant defence system and oxidative damage in leaves of maize seedlings. Plant Cell Physiol 2001, 42:1265-1273.
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Lin CC, Kao CH: Abscisic acid induced changes in cell wall peroxidase activity and hydrogen peroxide level in roots of rice seedlings. Plant Sci 2001, 160:323-329.
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Noctor G, Foyer CH: Ascorbate and glutathione: keeping active oxygen under control. Annu Rev Plant Physiol Plant Mol Biol 1998, 49:249-279.
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Corpas FJ, Barroso JB, del Rio LA: Peroxisomes as a source of reactive oxygen species and nitric oxide signal molecules in plant cells. Trends Plant Sci 2001, 6:145-150.
Conclusions and future developments A signalling role for H2O2 is now firmly established, but many questions remain to be answered. What are the concentrations of H2O2 in different sub-cellular compartments? What contributions to the cellular H2O2 pool are made by the various sources? Are specific H2O2 signatures induced by different stimuli? Can H2O2 produced in one cell have an effect in others? How is H2O2 perceived by the cell? It is likely that the redox poise of the cell [57] may control cellular events through interactions with H2O2 and redox-sensitive molecules such as glutathione, thioredoxins and peroxiredoxins [2•,50•]. Sensitive intracellular imaging will be required to visualise H2O2 within specific cells and sub-cellular microdomains. The recent engineering of cysteine residues in a green fluorescent protein variant to detect modulations in the redox environment of the cell [58] may provide a useful redox reporter. The use of transgenic plants that are impaired in H2O2 generation (such as rboh mutants, or those in which specific enzymes are downregulated in discrete cells and tissues) and the isolation of H2O2-signalling mutants will be invaluable in elucidating further the biological roles of H2O2 in specific cells and in response to various stimuli. Post-genomic developments in transcriptomics and proteomics will facilitate further insights into cellular responses to H2O2. Future work will no doubt reveal novel signalling roles for H2O2 and its interaction with other signals.
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10. Bolwell GP: Role of active oxygen species and NO in plant defence responses. Curr Opin Plant Biol 1999, 2:287-294. 11. Orozco-Cardenas ML, Narvaez-Vasquez J, Ryan CA: Hydrogen peroxide acts as a second messenger for the induction of defense genes in tomato plants in response to wounding, systemin, and methyl jasmonate. Plant Cell 2001, 13:179-191. 12. Rao MV, Davis KR: The physiology of ozone-induced cell death. Planta 2001, 213:682-690. 13. Sagi M, Fluhr R: Superoxide production by plant homologues of •• the gp91phox NADPH oxidase. Modulation of activity by calcium and by tobacco mosaic virus infection. Plant Physiol 2001, 126:1281-1290. This work provides the first biochemical evidence that the plasma-membranelocalised NADPH oxidase really does generate O2.–, and hence H2O2. A novel in-gel-based assay was used to demonstrate plasma membrane NADPH-dependent and diphenylene iodonium (DPI)-sensitive O2.– generation, which was activated directly by calcium and in response to viral infection. 14. Torres MA, Dangl JL, Jones JDG: Arabidopsis gp91phox homologues •• AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response. Proc Natl Acad Sci USA 2002, 99:517-522. An excellent demonstration of the functional genomics approach. This paper confirms that rboh genes are required for H2O2 production. Distinct roles for individual members of the Atrboh gene family are revealed. AtrbohD is largely
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responsible for bacterially induced H2O2 production, whereas AtrbohF is more important for the hypersensitive response (HR). However, H2O2 production and HR following fungal challenge do not correlate, a conundrum that remains to be unravelled. rboh mutants will provide a useful resource to elucidate further the signalling role of H2O2 in various processes. 15. Baxter-Burrell A, Yang Z, Springer PS, Bailey-Serres J: •• RopGAP4-dependent Rop GTPase rheostat control of Arabidopsis oxygen deprivation tolerance. Science 2002, 296:2026-2028. The authors use Arabidopsis Rop-signalling mutants to construct a model of the role of Rop signal transduction in regulating alcohol dehydrogenase (ADH) gene expression and tolerance of oxygen deprivation. Rop signalling is activated by low O2; RopGTP activates H2O2 production (potentially via NADPH oxidase) and, in turn, this H2O2 induces the expression of ADH and RopGAP4, which encodes a Rop-inactivating protein. Negative regulation via RopGAP4 is required to control H2O2 production. These exciting data suggest that H2O2/Rop signalling may have a wide role in cellular processes. 16. Yang Z: Small GTPases: versatile signalling switches in plants. Plant Cell 2002, S375-S388. 17.
Blee KA, Jupe SC, Richard G, Bolwell GP: Molecular identification and expression of the peroxidase responsible for the oxidative burst in French bean (Phaseolus vulgaris L.) and related members of the gene family. Plant Mol Biol 2001, 47:607-620.
18. Bolwell GP, Bindschedler LV, Blee KA, Butt VS, Davies DR, • Gardner SL, Gerrish C, Minibayeva F: The apoplastic oxidative burst in response to biotic stress in plants: a three-component system. J Exp Bot 2002, 53:1367-1376. The authors present functional data that support an alternative source of H2O2 during pathogen challenge. 19. Mittler R, Herr EH, Orvar BL, van Camp W, Willekens H, Inze D, Ellis BE: Transgenic tobacco plants with reduced capability to detoxify reactive oxygen intermediates are hyperresponsive to pathogen infection. Proc Natl Acad Sci USA 1999, 96:14165-14170. 20. Fath A, Bethke PC, Jones RL: Enzymes that scavenge reactive • oxygen species are down-regulated prior to gibberellic acid-induced programmed cell death in barley aleurone. Plant Physiol 2001, 126:156-166. This work shows a causal relationship between hormones, antioxidant levels and PCD in the barley aleurone system. GA reduces catalase, ascorbate peroxidase and superoxide dismutase activity, increases H2O2 concentrations and induces PCD. ABA has the opposite effects. 21. Henzler T, Steudle E: Transport and metabolic degradation of hydrogen peroxide in Chara corallina: model calculations and measurements with the pressure probe suggest transport of H2O2 across water channels. J Exp Bot 2000, 51:2053-2066. 22. Levine A, Tenhaken R, Dixon R, Lamb C: H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell 1994, 79:583-593. 23.
Alvarez ME, Pennell RI, Meijer P-J, Ishikawa A, Dixon RA, Lamb C: Reactive oxygen intermediates mediate a systemic signal network in the establishment of plant immunity. Cell 1998, 92:773-784.
24. Allan AC, Fluhr R: Two distinct sources of elicited reactive oxygen species in tobacco epidermal cells. Plant Cell 1997, 9:1559-1572. 25. Zhang X, Zhang L, Dong F, Gao J, Galbraith DW, Song C-P: • Hydrogen peroxide is involved in abscisic acid-induced stomatal closure in Vicia faba. Plant Physiol 2001, 126:1438-1448. A detailed study of the generation of H2O2 in Vicia faba guard cells in response to ABA. Two distinct sources of H2O2 are suggested: one chloroplastic and another via a possible plasma membrane NADPH oxidase. 26. Beers EP, McDowell JM: Regulation and execution of programmed cell death in response to pathogens, stress and developmental cues. Curr Opin Plant Biol 2001, 4:561-567. 27. ••
Delledonne M, Zeier J, Marocco A, Lamb C: Signal interactions between nitric oxide and reactive oxygen intermediates in the plant hypersensitive disease resistance response. Proc Natl Acad Sci USA 2001, 98:13454-13459. A key paper demonstrating that a critical balance between H2O2 and NO regulates PCD. The reaction product of O2.– and NO, peroxynitrite, does not induce PCD. Consequently, the relative rates of conversion of O2.– to peroxynitrite or H2O2 determine the extent to which PCD occurs. 28. Bethke PC, Jones RL: Cell death of barley aleurone protoplasts is • mediated by reactive oxygen species. Plant J 2001, 25:19-29. The authors suggest that H2O2 plays a key role during developmental PCD in the barley aleurone system. High levels of H2O2 alone do not cause cell death; however, GA but not ABA treatment leads to H2O2-induced cell death.
29. Tiwari BS, Belenghi B, Levine A: Oxidative stress increased • respiration and generation of reactive oxygen species, resulting in ATP depletion, opening of mitochondrial permeability transition, and programmed cell death. Plant Physiol 2002, 128:1271-1281. This study shows that exposure to a low sustained dose of H2O2 or to a higher-dose burst of H2O2 causes an increase in endogenous H2O2 generation in mitochondria. This leads to ATP depletion, opening of the mitochondrial permeability transition pore, release of cytochrome c and PCD. 30. Ren D, Yang H, Zhang S: Cell death mediated by MAPK is • associated with hydrogen peroxide production in Arabidopsis. J Biol Chem 2002, 277:559-565. Using transgenic plants with constitutively active AtMEK4 and AtMEK5, the authors speculate that prolonged activation of MAPK pathways leads to disruption of redox balance and generation of H2O2, resulting in PCD. 31. Maxwell DP, Nickels R, McIntosh L: Evidence of mitochondrial • involvement in the transduction of signals required for the induction of genes associated with pathogen attack and senescence. Plant J 2002, 29:269-279. This study provides a clear link between mitochondria, H2O2 and gene expression. Inhibition of mitochondrial electron transport or exposure to H2O2 induces intracellular H2O2 generation and the induction of several genes that are associated with PCD. Such gene expression is inhibited by inhibition of mitochondrial pore formation, implying mitochondrion–nucleus signalling. 32. Desikan R, Clarke A, Hancock JT, Neill SJ: H2O2 activates a MAP kinase-like enzyme in Arabidopsis thaliana suspension cultures. J Exp Bot 1999, 50:1863-1866. 33. Grant JJ, Yun B-W, Loake GJ: Oxidative burst and cognate redox signalling reported by luciferase imaging: identification of a signal network that functions independently of ethylene, SA and Me-JA but is dependent on MAPKK activity. Plant J 2000, 24:569-582. 34. Samuel MA, Miles GP, Ellis BE: Ozone treatment rapidly activates MAP kinase signalling in plants. Plant J 2000, 22:367-376. 35. Kovtun Y, Chiu W-L, Tena G, Sheen J: Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants. Proc Natl Acad Sci USA 2000, 97:2940-2945. 36. Desikan R, Hancock JT, Ichimura K, Shinozaki K, Neill SJ: Harpin induces activation of the Arabidopsis mitogen-activated protein kinases AtMPK4 and AtMPK6. Plant Physiol 2001, 126:1579-1587. 37.
van Camp W, van Montagu M, Inze D: H2O2 and NO: redox signals in disease resistance. Trends Plant Sci 1998, 3:330-334.
38. McAinsh MR, Clayton H, Mansfield TA, Hetherington AM: Changes in stomatal behaviour and guard cell cytosolic free calcium in response to oxidative stress. Plant Physiol 1996, 111:1031-1042. 39
Lee S, Choi H, Suh S, Doo I-S, Oh K-Y, Choi EJ, Taylor ATS, Low PS, Lee Y: Oligogalacturonic acid and chitosan reduce stomatal aperture by inducing the evolution of reactive oxygen species from guard cells of tomato and Commelina communis. Plant Physiol 1999, 121:147-152.
40. Pei Z-M, Murata Y, Benning G, Thomine S, Klusener B, Allen GJ, Grill E, Schroeder JI: Calcium channels activated by hydrogen peroxide mediate abscisic acid signalling in guard cells. Nature 2000, 406:731-734. 41. Murata Y, Pei Z-M, Mori IC, Schroeder J: Abscisic acid activation of •• plasma membrane Ca2+ channels in guard cells requires cytosolic NAD(P)H and is differentially disrupted upstream and downstream of reactive oxygen species production in abi1-1 and abi2-1 protein phosphatase 2C mutants. Plant Cell 2001, 13:2513-2523. This follow-up study to that described in [40] implicates guard cell NADPH oxidase as a key enzyme in the generation of H2O2 that mediates ABA-induced Ca2+ currents and stomatal closure. The roles of various ABA mutants in the ABA-H2O2 signalling pathway are also elucidated. ABI1 is placed upstream of H2O2 synthesis, whereas ABI2 is placed downstream of H2O2 in the ABA signalling cascade. 42. Zhang X, Miao YC, An GY, Zhou Y, Shangguan ZP, Gao JF, Song CP: K+ channels inhibited by hydrogen peroxide mediate abscisic acid signalling in Vicia guard cells. Cell Res 2001, 11:195-202. 43. Zhang X, Dong FC, Gao JF, Song CP: Hydrogen peroxide-induced changes in intracellular pH of guard cells precede stomatal closure. Cell Res 2001, 11:37-43.
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44. Neill SJ, Desikan R, Clarke A, Hancock JT: Nitric oxide is a novel • component of abscisic acid signalling in stomatal guard cells. Plant Physiol 2002, 128:13-16. This study highlights the role of NO in ABA signalling in guard cells, and demonstrates that, in guard cells as in the pathogen response, H2O2 and NO are generated concurrently. 45. Guan LM, Zhao J, Scandalios JG: Cis-elements and trans-factors that regulate expression of the maize Cat1 antioxidant gene in response to ABA and osmotic stress: H2O2 is the likely intermediary signaling molecule for the response. Plant J 2000, 22:87-95. 46. Schopfer P, Plachy C, Frahry G: Release of reactive oxygen intermediates (superoxide radicals, hydrogen peroxide, and hydroxyl radicals) and peroxidase in germinating radish seeds controlled by light, gibberellin and abscisic acid. Plant Physiol 2001, 125:1591-1602. 47.
Zhao Z, Chen G, Zhang C: Interaction between reactive oxygen species and nitric oxide in drought-induced abscisic acid synthesis in root tips of wheat seedlings. Aust J Plant Physiol 2001, 28:1055-1061.
48. Joo JH, Bae YS, Lee JS: Role of auxin-induced reactive oxygen •• species in root gravitropism. Plant Physiol 2001, 126:1055-1060. This study demonstrates a novel role for H2O2 in auxin signalling and gravitropism. Gravistimulation and auxin induce H2O2 production in roots; H2O2 treatment induces root curvature and antioxidants inhibit root gravitropism. 49. Yang T, Poovaiah BW: Hydrogen peroxide homeostasis: activation •• of plant catalase by calcium/calmodulin. Proc Natl Acad Sci USA 2002, 99:4097-4102. This paper highlights a novel role for calmodulin (CaM) in regulating H2O2 homeostasis. In vitro, calcium/CaM binds to and activates catalase. Furthermore, CaM co-localises with catalase in peroxisomes, providing a potential negative feedback mechanism for calcium-regulation of H2O2 levels. 50. Danon A: Redox reactions of regulatory proteins: do kinetics • promote specificity? Trends Biochem Sci 2002, 27:197-203. The author reviews the role of regulatory disulphide-containing proteins in signalling events that involve perturbation of the intracellular redox poise. The mechanisms of action of these proteins are discussed with particular reference to how specificity may be ensured in such pathways.
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51. Meinhard M, Rodriguez PL, Grill E: The sensitivity of ABI2 to •• hydrogen peroxide links the abscisic acid-response regulator to redox signalling. Planta 2002, 214:775-782. This work identifies ABI2, a PP2C enzyme involved in ABA signalling, as a direct target for H2O2. Manipulation of the redox status of ABI2 in vitro indicates that cysteine modification leads to ABI2 inactivation, thus providing a link between ABA and H2O2 signalling. 52. Meinhard M, Grill E: Hydrogen peroxide is a regulator of ABI1, a •• protein phosphatase 2C from Arabidopsis. FEBS Lett 2001, 508:443-446. This work and that described in [51··] demonstrates a direct effect of H2O2 on the ABI1 and ABI2 PP2C enzymes. H2O2 reversibly inactivates these enzymes in vitro, providing a potential link between H2O2 and ABA signalling. The effects of H2O2 on in vivo ABI1 and ABI2 activities remain to be determined. 53. Merlot S, Gosti F, Guerrier D, Vavasseur A, Giraudat J: The ABI1 and ABI2 protein phosphatases 2C act in a negative feedback regulatory loop of the abscisic acid signalling pathway. Plant J 2001, 25:295-303. 54. Singh KK: The Saccharomyces cerevisiae SLN1P–SSK1P two-component system mediates response to oxidative stress and in an oxidant-specific fashion. Free Rad Biol Med 2000, 29:1043-1050. 55. Desikan R, A-H-Mackerness S, Hancock JT, Neill SJ: Regulation of •• the Arabidopsis transcriptome by oxidative stress. Plant Physiol 2001, 127:159-172. A global analysis of gene expression in response to H2O2 is described. Approximately 1% of the Arabidopsis transcriptome was found to be regulated by H2O2. Of these genes, some were also regulated by various stresses such as ultra-violet light, elicitor treatment and drought stress, highlighting overlapping and distinct responses. 56. Delaunay A, Isnard A-D, Toledano MB: H2O2 sensing through oxidation of the Yap1 transcription factor. EMBO J 2000, 19:5157-5166. 57.
Hancock JT, Desikan R, Neill SJ: Does the redox state of cytochrome c act as a fail-safe mechanism in the regulation of programmed cell death? Free Rad Biol Med 2001, 31:697-703.
58.. ∅stergaard H, Henriksen A, Hansen FG, Winther JR: Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein. EMBO J 2001, 21:5853-5862.
Hydrogen peroxide signalling Steven Neill*, Radhika Desikan and John Hancock Recent biochemical and genetic studies confirm that hydrogen peroxide is a signalling molecule in plants that mediates responses to abiotic and biotic stresses. Signalling roles for hydrogen peroxide during abscisic-acid-mediated stomatal closure, auxin-regulated root gravitropism and tolerance of oxygen deprivation are now evident. The synthesis and action of hydrogen peroxide appear to be linked to those of nitric oxide. Downstream signalling events that are modulated by hydrogen peroxide include calcium mobilisation, protein phosphorylation and gene expression. Calcium and Rop signalling contribute to the maintenance of hydrogen peroxide homeostasis. Addresses Centre for Research in Plant Science, Faculty of Applied Sciences, University of the West of England (UWE), Frenchay, Bristol BS16 1QY, UK *e-mail: [email protected]
Turnover of hydrogen peroxide H2O2 is continually generated from various sources during normal metabolism (Figure 1). A wide range of steady-state H2O2 concentrations (e.g. 60 µM–7 mM in Arabidopsis [4,5], and 1–2 mM in maize and rice [6,7]), has been reported, although such variation may reflect technical difficulties in quantifying H2O2. Moreover, differences in the H2O2 content of different sub-cellular compartments are not yet known. Although the precise intracellular concentrations of H2O2 that are likely to be toxic will vary, high rates of H2O2 production are normally balanced by very efficient antioxidant systems ([8,9]; Figure 1). Abiotic stresses such as dehydration, low and high temperatures, and excess irradiation can disturb this balance, such that increased H2O2 initiates signalling responses that include enzyme activation, gene expression, programmed cell death (PCD) and cellular damage.
Current Opinion in Plant Biology 2002, 5:388–395 1369-5266/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Published online 30 July 2002 Abbreviations ABA abscisic acid ABI1 ABA-INSENSITIVE1 CaM calmodulin GA gibberellin GAP GTPase-activating protein hydrogen peroxide H2O2 MAPK mitogen-activated protein kinase NO nitric oxide PCD programmed cell death PP protein phosphatase rboh respiratory burst oxidase homologue Rop Rho-like small G protein SLN1 synthetic lethal of N-end rule1 TF transcription factor
Introduction Until relatively recently, the reactive oxygen species hydrogen peroxide (H2O2) was viewed mainly as a toxic cellular metabolite. However, it is now clear that it is much more than that, and functions as a signalling molecule that mediates responses to various stimuli in both plant and animal cells [1•–3•]. The generation of H2O2 is increased in response to various stresses, implicating it as a key factor mediating the phenomena of acclimation and crosstolerance, in which previous exposure to one stress can induce tolerance of subsequent exposure to the same or different stresses [1•]. For H2O2 to act as a signalling molecule it must have regulated synthesis, specific responses and cellular targets, and there must be mechanisms for its metabolism or removal subsequent to signalling events. In this review, we focus on recent work that highlights the signalling aspects of H2O2 generation and action.
H2O2 can also be generated by specific enzymes. An oxidative burst, with rapid H2O2 synthesis and release into the apoplast, is a common response to pathogens, elicitors, wounding, heat, ultra-violet light and ozone [10–12]. Pharmacological and molecular data indicate that the enzyme responsible is similar to the superoxide (O2.–)-generating NADPH oxidase originally characterised in mammalian phagocytes ([10]; Figure 1). Several homologues of this enzyme exist in plant genomes, but until recently no firm data had been provided to confirm its activity and function. Using a novel in-gel enzyme assay, O2.–-generating (and thus H2O2-generating) activity was demonstrated in plasma membranes from tobacco and tomato. This activity was directly modulated by calcium (placing calcium upstream of H2O2 synthesis), increased by viral infection and localised with proteins reacting with an NADPH oxidase antibody [13••]. Knockout experiments demonstrated that AtrbohD and AtrbohF genes (encoding NADPH oxidase) are required for H2O2 generation during bacterial and fungal challenge in Arabidopsis [14••]. Moreover, defence responses following bacterial challenge were compromised in the knockout mutants in which these genes were not functional. Such experiments also confirm that plant NADPH oxidases differ from the mammalian version as the enzyme activity of the plasma-membrane-localised homologue is not dependent on the assembly of a complex involving cytosolic components, and the enzyme is directly activated via Ca2+ binding. As there is a large NADPH oxidase gene family in plants, and different members have differing biological activity [14••], it is imperative to ascertain their cellular patterns of expression and activity. Exciting new data emphasise the important role of Rops (Rho-like small G proteins) in regulating H2O2 production, potentially via NADPH oxidase [15••]. Previous work had demonstrated the involvement of Rop signalling in H2O2
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Figure 1 H2O2 turnover in the plant cell. H2O2 is generated in normal metabolism via the Mehler reaction in chloroplasts, electron transport in mitochondria and photorespiration in peroxisomes. Peroxisomes may also contain other systems that generate H2O2 [9]. Abiotic and biotic stresses enhance H2O2 generation via these routes and also via enzymatic sources such as plasma-membrane-localised NADPH oxidases (RBOH; [13••,14••]) or cell wall peroxidases [18•]. H2O2 diffuses freely, perhaps facilitated by movement through peroxiporin membrane channels [21]. Cellular H2O2 levels are determined by the rates of H2O2 production and metabolism via catalase and the ubiquitous ascorbate-glutathione cycle (A-G cycle; [8]), which involves ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR) and glutathione reductase (GR). H2O2 also reacts with glutathione to convert it from its reduced state (GSH) to its oxidised state (GSSG).
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generation (see [16]). Recently, oxygen deprivation was found to activate Rop signalling that in turn activated NADPH oxidase. This was attenuated by H2O2 induction of RopGAP gene expression that leads to the deactivation of Rop [15••]. Other potential enzymatic sources of H2O2 include xanthine oxidase, amine oxidase and a cell wall peroxidase [9,17,18•]. It is possible that different stimuli activate specific H2O2-generating enzymes [18•]. A cell wall peroxidase has been identified in French bean [17], and a potentially peroxidase-mediated oxidative burst has been demonstrated in Arabidopsis cultures challenged with a fungal elicitor [18•]. Arabidopsis plants transformed with an antisense bean peroxidase construct are hypersensitive to bacterial and fungal pathogens [18•] but the endogenous Arabidopsis peroxidase has yet to be identified. Under most circumstances, cellular antioxidant systems remove H2O2 efficiently. However, given that large amounts
of H2O2 are produced, any reduction in antioxidant status would have rapid and potentially disastrous effects. Indeed, several studies have demonstrated the effects of over- and under-expression of antioxidant enzymes on cell physiology (e.g. [19]). Thus, the oxidative effects of various stimuli could be mediated via reductions in the activities of antioxidant enzymes, rather than by increased H2O2 generation. For example, gibberellin (GA)-induced PCD in barley aleurone resulted from increased H2O2 effected by a reduction in antioxidant activity [20•]. It is possible that the high antioxidant status of cells prevents H2O2 from travelling far through a cell. Consequently, responses to H2O2 may be localised to microdomains (i.e. ‘H2O2 hot-spots’) within the cell. Cellular fate may then be dependent on where in the cell H2O2 synthesis is increased. Even when relatively large amounts of H2O2 are generated in the apoplast, which has only a small proportion of the cell’s antioxidant capacity,
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H2O2 and ABA signalling in guard cells. (a) Increased H2O2 generation is induced by ABA in Arabidopsis guard cells, as visualised with the H2O2-sensitive dye DCFH-2DA (2’,7’-dichlorofluorescein diacetate) and confocal microscopy. (b) H2O2-mediated ABA signalling in guard cells. ABA induces H2O2 synthesis via a plasma membrane NADPH oxidase (RBOH; [25•,41••]) or in the chloroplast [25•]. H2O2 inactivates inward K+ channels [42], causes cytosolic alkalinisation [43] and activates plasma membrane Ca2+ channels [41••]. The potential position of various signalling proteins, as deduced from studies with various mutants, is indicated. The growth control by ABA 2 (gca2) mutant responds to ABA by increasing H2O2 synthesis but Ca2+ channels are not activated during this response [40]. The guard cells of abi1 mutants do not make H2O2 but can respond to it, whereas abi2 guard cells synthesise H2O2 but do not respond to it [41••].
H2O2 will rapidly move into the cell and be metabolised. Such movement may be facilitated by ‘peroxiporins’; it has been speculated that water channels, or aquaporins, may also serve as conduits for trans-membrane H2O2 transport [21]. Various studies suggest that H2O2 can function as a mobile signal [5,22–24,25•], but whether H2O2 is the sole signal or part of a systemic response remains to be determined.
PCD is initiated by a range of stimuli and driven by several signals including H2O2 and nitric oxide (NO) [26]. Although the execution processes of plant PCD are not firmly established, altered mitochondrial function, involving the formation of a mitochondrial permeability transition pore, is likely to be involved [26]. Interaction between NO and H2O2 was essential during PCD in soybean cultures challenged with avirulent bacteria [27••]. H2O2 was the key factor in PCD, but the NO/O2.– balance was critical in determining the rate of peroxynitrite formation. Peroxynitrite alone did not induce PCD, so the relative rates of conversion of O2.– to H2O2 or peroxynitrite may determine cellular responses. H2O2 concentrations required to effect PCD vary, perhaps reflecting differences in antioxidant capacity and competence to respond to H2O2. Thus, Arabidopsis protoplasts are considerably more H2O2-sensitive than cells (being sensitive to micromolar as opposed to millimolar concentrations), reflecting their reduced antioxidant status [1•]. Similarly, the survival of barley aleurone in 325 mM H2O2 correlated with the antioxidant capacity after treatments with GA or abscisic acid (ABA) [20•,28•]. The mechanisms by which H2O2 induces cell death are not yet clear, although several studies point to effects on mitochondria. Exposure of Arabidopsis cultures to H2O2 increased subsequent H2O2 production in mitochondria. This resulted in altered mitochondrial function and PCD [29•]. In another study, prolonged activation of mitogen-activated protein kinases (MAPKs) in Arabidopsis resulted in intracellular generation of H2O2 preceding cell death [30•]. Finally, various treatments that inhibited mitochondrial electron transport in tobacco cells, including exogenous H2O2, also increased intracellular H2O2 accumulation [31•]. It is already known that H2O2 activates MAPK pathways [32–36], and it is tempting to speculate that increased H2O2 can stimulate further mitochondrial H2O2 production, perhaps via MAPK activation, in an amplifying oxidative death cycle [37]. H2O2- and ABA-mediated stomatal closure
Although earlier studies had demonstrated the generation of H2O2 and its effects in guard cells [24,38,39], the findings that ABA activates the synthesis of H2O2 in guard cells, apparently via NADPH oxidase, and that H2O2 mediates ABA-induced stomatal closure and, at micromolar concentrations, activates plasma membrane Ca2+ channels were of major importance [40]. In subsequent work [41••], a requirement for NAD(P)H was confirmed and the two ABA-signalling protein phosphatase 2C (PP2C) enzymes, ABA-INSENSITIVE1 (ABI1) and ABI2, were positioned in the ABA and H2O2 signalling chain (Figure 2; [41••]). Mediation of ABA-induced stomatal closure by H2O2 is also seen in Vicia faba [25•], with evidence for two sources of H2O2, chloroplastic and cytoplasmic/plasma membrane, reminiscent of previous findings obtained with elicitor treatment of tobacco
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Figure 3
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H2O2 signalling in plant cells. H2O2, entering cells from the apoplast or generated internally, may diffuse only within sub-cellular microdomains, depending on the amount of H2O2 and the antioxidant status. H2O2 can oxidise or otherwise modulate signalling proteins, such as protein phosphatases (PP), protein kinases (PK) including a plasma membrane histidine kinase and MAPK cascades, TFs, and calcium channels that are located in the plasma membrane or elsewhere. Elevated cytosolic
calcium concentrations will initiate further downstream responses, via the action of calcium-binding proteins that include calmodulin (CaM), PKs and PPs. Reversible protein phosphorylation also initiates downstream signalling both in the cytoplasm and in the nucleus via effects on TFs and therefore on gene expression. The oxidation of TFs may activate them and/or induce nuclear localisation. H2O2 synthesis can be controlled by Rop signalling [15••].
epidermis [24]. Defining the sub-cellular sources of H2O2 in guard cells is an important research goal. In V. faba, H2O2 also inhibited K+ currents and induced cytosolic alkalinisation, as does ABA [42,43]. NO was recently identified as a novel component of ABA signalling in guard
cells [44•], demonstrating once more that H2O2 and NO appear to be made and to operate in tandem. Is H2O2 generation a common response to ABA or is this response confined to guard cells? ABA-induced increases
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gravitropism, respectively. Catalase had no effect on gravitropism, suggesting that an intracellular source of H2O2 is involved; the mechanism of H2O2 generation remains to be determined.
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H2O2 modulation of gene expression. H2O2 may directly oxidise transcription factors (TF) (e.g. [56]) in either the cytoplasm or the nucleus. Alternatively, H2O2-mediated activation of a signalling protein, such as a protein kinase, may activate TFs. Either way, activated TFs would subsequently interact with cognate H2O2-response elements and modulate gene expression.
in H2O2 have been reported for maize seedlings and rice roots [6,7], and earlier work demonstrated that ABAinduced expression of the catalase gene in maize was mediated by increased H2O2 [45]. On the other hand, ABA decreased the release of H2O2 from germinating radish seeds [46]. It seems likely that H2O2 responses to ABA will be tissue-specific and dependent on many factors. Recent data suggesting a role for reactive oxygen species and NO in drought-induced ABA synthesis underline such complexity [47]. Auxin, H2O2 and root gravitropism
Recent work has shown that H2O2 mediates auxin-regulated gravitropic responses [48••]. Gravity induced the asymmetric generation of H2O2 in maize roots, as did asymmetrically applied auxin. Moreover, asymmetric application of H2O2 or antioxidants promoted or inhibited
Calcium fluxes and reversible protein phosphorylation are core components of eukaryotic cell signalling and are both required for the controlled generation of H2O2 [1•]. It is likely that they are also key downstream responses (Figure 3). H2O2 activates Ca2+ channels in guard cell plasma membranes [41••] and induces a specific calcium signature in Arabidopsis seedlings (MC Rentel, University of Oxford, personal communication). Such effects on calcium fluxes, perhaps in cellular microdomains, may be a widespread response to H2O2. Recently, calcium/calmodulin (CaM) has been shown to bind to and activate plant catalases, leading to a decrease in H2O2 levels [49••]. Moreover, CaM was found in the peroxisomes, the major cellular location of catalase. This indicates that calcium has both positive and negative effects in regulating H2O2 homeostasis. H2O2 activates MAPK cascades in various tissues [32–36,48••], but how such activation is achieved remains to be determined. Constitutive activation of a H2O2-responsive MAPK afforded cross-tolerance to various environmental stresses [35]. As calcium and MAPKs regulate the activity of other signalling proteins, further downstream effects of H2O2 are likely to be uncovered. Cell signalling is best considered as an intricate web of interconnecting signal networks rather than as a collection of parallel but separate pathways. Consequently, it will be difficult to differentiate primary cellular responses to H2O2 from those that are effected via other signalling intermediates. However, if H2O2 truly is a specific signalling molecule then there must exist primary targets for H2O2. Chemically, H2O2 is a rather simple molecule, so it might be considered unlikely that there exist specific H2O2 receptors. H2O2 is a mild oxidant that can interact with cysteine residues within proteins, depending on the pKa and the molecular environment [2•,3•,50•]. For such interactions to have physiological significance, they must induce conformational changes that alter protein activity and that are sufficiently long-lasting to initiate cellular responses. Such effects can potentially be seen with the ABI1 and ABI2 enzymes [51••,52••]. H2O2 efficiently and reversibly inactivated these enzymes in vitro. ABI1 and ABI2 are negative regulators of ABA signalling [53]. Thus, one would predict that H2O2 generated as a result of ABA signalling should inactivate ABI1 and ABI2. In fact, these enzymes appear to be activated by ABA [53], and it is not yet known whether H2O2 can actually inhibit the enzymes in vivo. There are many potential PP2Cs in plants, some of which may also be susceptible to H2O2, and targets for PPs include MAPKs. Thus, MAPK activation by H2O2 may be due to the effects of H2O2 on PPs. Recent work with yeast has provided exciting evidence for the existence of a ‘peroxisensor’ [54]. Yeast mutants lacking
Hydrogen peroxide signalling Neill, Desikan and Hancock
the plasma membrane SLN1 (synthetic lethal of N-end rule1) histidine kinase were highly susceptible to H2O2. Complementation of the mutant with a functional SLN1 gene restored the ability to survive in H2O2. The Arabidopsis genome contains several genes with homology to SLN1, and it may be that one or more of the proteins encoded by these genes can function as a peroxisensor.
Acknowledgements
H2O2-regulated gene expression
1. •
H2O2 modulates the expression of various genes, including those encoding antioxidant enzymes and modulators of H2O2 production [1•,15••], indicating the complex way in which intracellular H2O2 concentrations may be monitored and maintained at a constant level [1•]. A microarray study showed that the expression of 1–2% of genes was altered in H2O2-treated Arabidopsis cultures [55••]. As expected, genes encoding antioxidant enzymes were upregulated, but so were genes encoding proteins that are potentially involved in PCD, as well as signalling proteins such as calmodulin, protein kinases and transcription factors (TFs), the latter suggesting longer-term effects. Although H2O2-responsive promoters have been identified (see [55••]), specific H2O2-regulatory DNA sequences and their cognate transcription factors have not yet been isolated and characterised. Whether H2O2regulated transcription involves oxidative changes to TFs themselves (e.g. [56]), in either the cytoplasm or the nucleus, or other modifications such as phosphorylation that are ultimately initiated by H2O2 (Figure 4), remains to be determined.
We thank those colleagues who made available reprints, pre-prints and unpublished data.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest Neill SJ, Desikan R, Clarke A, Hurst RD, Hancock JT: Hydrogen peroxide and nitric oxide as signalling molecules in plants. J Exp Bot 2002, 53:1237-1247. An up-to-date comprehensive review of H2O2 signalling. 2. •
Rhee SG, Bae YS, Lee S-R, Kwon J: Hydrogen peroxide: a key messenger that modulates protein phosphorylation through cysteine oxidation. Science’s Signal Transduction Knowledge Environment, 2000. http://stke.sciencemag.org/cgi/content/full/ OC_sigtrans;2000/53/pe1 This excellent review discusses the role of cysteine oxidation in the transduction of hydrogen peroxide signalling, with particular reference to the modulation of protein phosphorylation. It highlights the importance of the molecular environment of the relevant cysteine residues, which is essential for cysteine oxidation. 3. Finkel T: Redox-dependent signal transduction. FEBS Lett 2000, • 476:52-54. This review provides a good insight into the role of reactive oxygen species in signal transduction, focussing on the regulation of intracellular production of reactive oxygen species and likely protein targets. It highlights the potential of cysteine modification as a mechanism for downstream signalling. 4.
Veljovic-Jovanovic SD, Pignocchi C, Noctor G, Foyer CH: Low ascorbic acid in the vtc-1 mutant of Arabidopsis is associated with decreased growth and intracellular redistribution of the antioxidant system. Plant Physiol 2001, 127:426-435.
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Karpinski S, Reynolds H, Karpinska B, Wingsle G, Creissen G, Mullineaux P: Systemic signaling and acclimation in response to excess excitation energy in Arabidopsis. Science 1999, 284:654-657.
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Jiang M, Zhang J: Effect of abscisic acid on active oxygen species, antioxidant defence system and oxidative damage in leaves of maize seedlings. Plant Cell Physiol 2001, 42:1265-1273.
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Lin CC, Kao CH: Abscisic acid induced changes in cell wall peroxidase activity and hydrogen peroxide level in roots of rice seedlings. Plant Sci 2001, 160:323-329.
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Noctor G, Foyer CH: Ascorbate and glutathione: keeping active oxygen under control. Annu Rev Plant Physiol Plant Mol Biol 1998, 49:249-279.
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Conclusions and future developments A signalling role for H2O2 is now firmly established, but many questions remain to be answered. What are the concentrations of H2O2 in different sub-cellular compartments? What contributions to the cellular H2O2 pool are made by the various sources? Are specific H2O2 signatures induced by different stimuli? Can H2O2 produced in one cell have an effect in others? How is H2O2 perceived by the cell? It is likely that the redox poise of the cell [57] may control cellular events through interactions with H2O2 and redox-sensitive molecules such as glutathione, thioredoxins and peroxiredoxins [2•,50•]. Sensitive intracellular imaging will be required to visualise H2O2 within specific cells and sub-cellular microdomains. The recent engineering of cysteine residues in a green fluorescent protein variant to detect modulations in the redox environment of the cell [58] may provide a useful redox reporter. The use of transgenic plants that are impaired in H2O2 generation (such as rboh mutants, or those in which specific enzymes are downregulated in discrete cells and tissues) and the isolation of H2O2-signalling mutants will be invaluable in elucidating further the biological roles of H2O2 in specific cells and in response to various stimuli. Post-genomic developments in transcriptomics and proteomics will facilitate further insights into cellular responses to H2O2. Future work will no doubt reveal novel signalling roles for H2O2 and its interaction with other signals.
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10. Bolwell GP: Role of active oxygen species and NO in plant defence responses. Curr Opin Plant Biol 1999, 2:287-294. 11. Orozco-Cardenas ML, Narvaez-Vasquez J, Ryan CA: Hydrogen peroxide acts as a second messenger for the induction of defense genes in tomato plants in response to wounding, systemin, and methyl jasmonate. Plant Cell 2001, 13:179-191. 12. Rao MV, Davis KR: The physiology of ozone-induced cell death. Planta 2001, 213:682-690. 13. Sagi M, Fluhr R: Superoxide production by plant homologues of •• the gp91phox NADPH oxidase. Modulation of activity by calcium and by tobacco mosaic virus infection. Plant Physiol 2001, 126:1281-1290. This work provides the first biochemical evidence that the plasma-membranelocalised NADPH oxidase really does generate O2.–, and hence H2O2. A novel in-gel-based assay was used to demonstrate plasma membrane NADPH-dependent and diphenylene iodonium (DPI)-sensitive O2.– generation, which was activated directly by calcium and in response to viral infection. 14. Torres MA, Dangl JL, Jones JDG: Arabidopsis gp91phox homologues •• AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response. Proc Natl Acad Sci USA 2002, 99:517-522. An excellent demonstration of the functional genomics approach. This paper confirms that rboh genes are required for H2O2 production. Distinct roles for individual members of the Atrboh gene family are revealed. AtrbohD is largely
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responsible for bacterially induced H2O2 production, whereas AtrbohF is more important for the hypersensitive response (HR). However, H2O2 production and HR following fungal challenge do not correlate, a conundrum that remains to be unravelled. rboh mutants will provide a useful resource to elucidate further the signalling role of H2O2 in various processes. 15. Baxter-Burrell A, Yang Z, Springer PS, Bailey-Serres J: •• RopGAP4-dependent Rop GTPase rheostat control of Arabidopsis oxygen deprivation tolerance. Science 2002, 296:2026-2028. The authors use Arabidopsis Rop-signalling mutants to construct a model of the role of Rop signal transduction in regulating alcohol dehydrogenase (ADH) gene expression and tolerance of oxygen deprivation. Rop signalling is activated by low O2; RopGTP activates H2O2 production (potentially via NADPH oxidase) and, in turn, this H2O2 induces the expression of ADH and RopGAP4, which encodes a Rop-inactivating protein. Negative regulation via RopGAP4 is required to control H2O2 production. These exciting data suggest that H2O2/Rop signalling may have a wide role in cellular processes. 16. Yang Z: Small GTPases: versatile signalling switches in plants. Plant Cell 2002, S375-S388. 17.
Blee KA, Jupe SC, Richard G, Bolwell GP: Molecular identification and expression of the peroxidase responsible for the oxidative burst in French bean (Phaseolus vulgaris L.) and related members of the gene family. Plant Mol Biol 2001, 47:607-620.
18. Bolwell GP, Bindschedler LV, Blee KA, Butt VS, Davies DR, • Gardner SL, Gerrish C, Minibayeva F: The apoplastic oxidative burst in response to biotic stress in plants: a three-component system. J Exp Bot 2002, 53:1367-1376. The authors present functional data that support an alternative source of H2O2 during pathogen challenge. 19. Mittler R, Herr EH, Orvar BL, van Camp W, Willekens H, Inze D, Ellis BE: Transgenic tobacco plants with reduced capability to detoxify reactive oxygen intermediates are hyperresponsive to pathogen infection. Proc Natl Acad Sci USA 1999, 96:14165-14170. 20. Fath A, Bethke PC, Jones RL: Enzymes that scavenge reactive • oxygen species are down-regulated prior to gibberellic acid-induced programmed cell death in barley aleurone. Plant Physiol 2001, 126:156-166. This work shows a causal relationship between hormones, antioxidant levels and PCD in the barley aleurone system. GA reduces catalase, ascorbate peroxidase and superoxide dismutase activity, increases H2O2 concentrations and induces PCD. ABA has the opposite effects. 21. Henzler T, Steudle E: Transport and metabolic degradation of hydrogen peroxide in Chara corallina: model calculations and measurements with the pressure probe suggest transport of H2O2 across water channels. J Exp Bot 2000, 51:2053-2066. 22. Levine A, Tenhaken R, Dixon R, Lamb C: H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell 1994, 79:583-593. 23.
Alvarez ME, Pennell RI, Meijer P-J, Ishikawa A, Dixon RA, Lamb C: Reactive oxygen intermediates mediate a systemic signal network in the establishment of plant immunity. Cell 1998, 92:773-784.
24. Allan AC, Fluhr R: Two distinct sources of elicited reactive oxygen species in tobacco epidermal cells. Plant Cell 1997, 9:1559-1572. 25. Zhang X, Zhang L, Dong F, Gao J, Galbraith DW, Song C-P: • Hydrogen peroxide is involved in abscisic acid-induced stomatal closure in Vicia faba. Plant Physiol 2001, 126:1438-1448. A detailed study of the generation of H2O2 in Vicia faba guard cells in response to ABA. Two distinct sources of H2O2 are suggested: one chloroplastic and another via a possible plasma membrane NADPH oxidase. 26. Beers EP, McDowell JM: Regulation and execution of programmed cell death in response to pathogens, stress and developmental cues. Curr Opin Plant Biol 2001, 4:561-567. 27. ••
Delledonne M, Zeier J, Marocco A, Lamb C: Signal interactions between nitric oxide and reactive oxygen intermediates in the plant hypersensitive disease resistance response. Proc Natl Acad Sci USA 2001, 98:13454-13459. A key paper demonstrating that a critical balance between H2O2 and NO regulates PCD. The reaction product of O2.– and NO, peroxynitrite, does not induce PCD. Consequently, the relative rates of conversion of O2.– to peroxynitrite or H2O2 determine the extent to which PCD occurs. 28. Bethke PC, Jones RL: Cell death of barley aleurone protoplasts is • mediated by reactive oxygen species. Plant J 2001, 25:19-29. The authors suggest that H2O2 plays a key role during developmental PCD in the barley aleurone system. High levels of H2O2 alone do not cause cell death; however, GA but not ABA treatment leads to H2O2-induced cell death.
29. Tiwari BS, Belenghi B, Levine A: Oxidative stress increased • respiration and generation of reactive oxygen species, resulting in ATP depletion, opening of mitochondrial permeability transition, and programmed cell death. Plant Physiol 2002, 128:1271-1281. This study shows that exposure to a low sustained dose of H2O2 or to a higher-dose burst of H2O2 causes an increase in endogenous H2O2 generation in mitochondria. This leads to ATP depletion, opening of the mitochondrial permeability transition pore, release of cytochrome c and PCD. 30. Ren D, Yang H, Zhang S: Cell death mediated by MAPK is • associated with hydrogen peroxide production in Arabidopsis. J Biol Chem 2002, 277:559-565. Using transgenic plants with constitutively active AtMEK4 and AtMEK5, the authors speculate that prolonged activation of MAPK pathways leads to disruption of redox balance and generation of H2O2, resulting in PCD. 31. Maxwell DP, Nickels R, McIntosh L: Evidence of mitochondrial • involvement in the transduction of signals required for the induction of genes associated with pathogen attack and senescence. Plant J 2002, 29:269-279. This study provides a clear link between mitochondria, H2O2 and gene expression. Inhibition of mitochondrial electron transport or exposure to H2O2 induces intracellular H2O2 generation and the induction of several genes that are associated with PCD. Such gene expression is inhibited by inhibition of mitochondrial pore formation, implying mitochondrion–nucleus signalling. 32. Desikan R, Clarke A, Hancock JT, Neill SJ: H2O2 activates a MAP kinase-like enzyme in Arabidopsis thaliana suspension cultures. J Exp Bot 1999, 50:1863-1866. 33. Grant JJ, Yun B-W, Loake GJ: Oxidative burst and cognate redox signalling reported by luciferase imaging: identification of a signal network that functions independently of ethylene, SA and Me-JA but is dependent on MAPKK activity. Plant J 2000, 24:569-582. 34. Samuel MA, Miles GP, Ellis BE: Ozone treatment rapidly activates MAP kinase signalling in plants. Plant J 2000, 22:367-376. 35. Kovtun Y, Chiu W-L, Tena G, Sheen J: Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants. Proc Natl Acad Sci USA 2000, 97:2940-2945. 36. Desikan R, Hancock JT, Ichimura K, Shinozaki K, Neill SJ: Harpin induces activation of the Arabidopsis mitogen-activated protein kinases AtMPK4 and AtMPK6. Plant Physiol 2001, 126:1579-1587. 37.
van Camp W, van Montagu M, Inze D: H2O2 and NO: redox signals in disease resistance. Trends Plant Sci 1998, 3:330-334.
38. McAinsh MR, Clayton H, Mansfield TA, Hetherington AM: Changes in stomatal behaviour and guard cell cytosolic free calcium in response to oxidative stress. Plant Physiol 1996, 111:1031-1042. 39
Lee S, Choi H, Suh S, Doo I-S, Oh K-Y, Choi EJ, Taylor ATS, Low PS, Lee Y: Oligogalacturonic acid and chitosan reduce stomatal aperture by inducing the evolution of reactive oxygen species from guard cells of tomato and Commelina communis. Plant Physiol 1999, 121:147-152.
40. Pei Z-M, Murata Y, Benning G, Thomine S, Klusener B, Allen GJ, Grill E, Schroeder JI: Calcium channels activated by hydrogen peroxide mediate abscisic acid signalling in guard cells. Nature 2000, 406:731-734. 41. Murata Y, Pei Z-M, Mori IC, Schroeder J: Abscisic acid activation of •• plasma membrane Ca2+ channels in guard cells requires cytosolic NAD(P)H and is differentially disrupted upstream and downstream of reactive oxygen species production in abi1-1 and abi2-1 protein phosphatase 2C mutants. Plant Cell 2001, 13:2513-2523. This follow-up study to that described in [40] implicates guard cell NADPH oxidase as a key enzyme in the generation of H2O2 that mediates ABA-induced Ca2+ currents and stomatal closure. The roles of various ABA mutants in the ABA-H2O2 signalling pathway are also elucidated. ABI1 is placed upstream of H2O2 synthesis, whereas ABI2 is placed downstream of H2O2 in the ABA signalling cascade. 42. Zhang X, Miao YC, An GY, Zhou Y, Shangguan ZP, Gao JF, Song CP: K+ channels inhibited by hydrogen peroxide mediate abscisic acid signalling in Vicia guard cells. Cell Res 2001, 11:195-202. 43. Zhang X, Dong FC, Gao JF, Song CP: Hydrogen peroxide-induced changes in intracellular pH of guard cells precede stomatal closure. Cell Res 2001, 11:37-43.
Hydrogen peroxide signalling Neill, Desikan and Hancock
44. Neill SJ, Desikan R, Clarke A, Hancock JT: Nitric oxide is a novel • component of abscisic acid signalling in stomatal guard cells. Plant Physiol 2002, 128:13-16. This study highlights the role of NO in ABA signalling in guard cells, and demonstrates that, in guard cells as in the pathogen response, H2O2 and NO are generated concurrently. 45. Guan LM, Zhao J, Scandalios JG: Cis-elements and trans-factors that regulate expression of the maize Cat1 antioxidant gene in response to ABA and osmotic stress: H2O2 is the likely intermediary signaling molecule for the response. Plant J 2000, 22:87-95. 46. Schopfer P, Plachy C, Frahry G: Release of reactive oxygen intermediates (superoxide radicals, hydrogen peroxide, and hydroxyl radicals) and peroxidase in germinating radish seeds controlled by light, gibberellin and abscisic acid. Plant Physiol 2001, 125:1591-1602. 47.
Zhao Z, Chen G, Zhang C: Interaction between reactive oxygen species and nitric oxide in drought-induced abscisic acid synthesis in root tips of wheat seedlings. Aust J Plant Physiol 2001, 28:1055-1061.
48. Joo JH, Bae YS, Lee JS: Role of auxin-induced reactive oxygen •• species in root gravitropism. Plant Physiol 2001, 126:1055-1060. This study demonstrates a novel role for H2O2 in auxin signalling and gravitropism. Gravistimulation and auxin induce H2O2 production in roots; H2O2 treatment induces root curvature and antioxidants inhibit root gravitropism. 49. Yang T, Poovaiah BW: Hydrogen peroxide homeostasis: activation •• of plant catalase by calcium/calmodulin. Proc Natl Acad Sci USA 2002, 99:4097-4102. This paper highlights a novel role for calmodulin (CaM) in regulating H2O2 homeostasis. In vitro, calcium/CaM binds to and activates catalase. Furthermore, CaM co-localises with catalase in peroxisomes, providing a potential negative feedback mechanism for calcium-regulation of H2O2 levels. 50. Danon A: Redox reactions of regulatory proteins: do kinetics • promote specificity? Trends Biochem Sci 2002, 27:197-203. The author reviews the role of regulatory disulphide-containing proteins in signalling events that involve perturbation of the intracellular redox poise. The mechanisms of action of these proteins are discussed with particular reference to how specificity may be ensured in such pathways.
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51. Meinhard M, Rodriguez PL, Grill E: The sensitivity of ABI2 to •• hydrogen peroxide links the abscisic acid-response regulator to redox signalling. Planta 2002, 214:775-782. This work identifies ABI2, a PP2C enzyme involved in ABA signalling, as a direct target for H2O2. Manipulation of the redox status of ABI2 in vitro indicates that cysteine modification leads to ABI2 inactivation, thus providing a link between ABA and H2O2 signalling. 52. Meinhard M, Grill E: Hydrogen peroxide is a regulator of ABI1, a •• protein phosphatase 2C from Arabidopsis. FEBS Lett 2001, 508:443-446. This work and that described in [51··] demonstrates a direct effect of H2O2 on the ABI1 and ABI2 PP2C enzymes. H2O2 reversibly inactivates these enzymes in vitro, providing a potential link between H2O2 and ABA signalling. The effects of H2O2 on in vivo ABI1 and ABI2 activities remain to be determined. 53. Merlot S, Gosti F, Guerrier D, Vavasseur A, Giraudat J: The ABI1 and ABI2 protein phosphatases 2C act in a negative feedback regulatory loop of the abscisic acid signalling pathway. Plant J 2001, 25:295-303. 54. Singh KK: The Saccharomyces cerevisiae SLN1P–SSK1P two-component system mediates response to oxidative stress and in an oxidant-specific fashion. Free Rad Biol Med 2000, 29:1043-1050. 55. Desikan R, A-H-Mackerness S, Hancock JT, Neill SJ: Regulation of •• the Arabidopsis transcriptome by oxidative stress. Plant Physiol 2001, 127:159-172. A global analysis of gene expression in response to H2O2 is described. Approximately 1% of the Arabidopsis transcriptome was found to be regulated by H2O2. Of these genes, some were also regulated by various stresses such as ultra-violet light, elicitor treatment and drought stress, highlighting overlapping and distinct responses. 56. Delaunay A, Isnard A-D, Toledano MB: H2O2 sensing through oxidation of the Yap1 transcription factor. EMBO J 2000, 19:5157-5166. 57.
Hancock JT, Desikan R, Neill SJ: Does the redox state of cytochrome c act as a fail-safe mechanism in the regulation of programmed cell death? Free Rad Biol Med 2001, 31:697-703.
58.. ∅stergaard H, Henriksen A, Hansen FG, Winther JR: Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein. EMBO J 2001, 21:5853-5862.