Redox Sensing and Signalling Associated with Reactive Oxygen in Chloroplasts, Peroxisomes and Mitochondria

Redox sensing and signalling associated with reactive oxygen in chloroplasts, peroxisomes and mitochondria

Christine H. Foyera,* and Graham Noctorb aCrop Performance and Improvement Division, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK bInstitut de Biotechnologie des Plantes, Baˆtiment 630, Universite´ Paris XI, 91405 Orsay Cedex, France *Corresponding author, e-mail: [email protected] Received 4 February 2003; revised 19 May 2003

Chloroplasts and mitochondria are the powerhouses of photo- electron transport processes with the inherent generation of synthetic cells. The oxidation-reduction (redox) cascades of superoxide, hydrogen peroxide and singlet oxygen provides a the photosynthetic and respiratory electron transport chains repertoire of additional extremely powerful signals. Accumu- not only provide the driving forces for metabolism but also lating evidence implicates the major redox buffers of plant generate redox signals, which participate in and regulate every cells, ascorbate and glutathione, in redox signal transduction. aspect of plant biology from gene expression and translation The network of redox signals from energy-generating organ- to enzyme chemistry. Plastoquinone, thioredoxin and reactive elles orchestrates metabolism to adjust energy production to oxygen have all been shown to have signalling functions. utilization, interfacing with hormone signalling to respond to Moreover, the intrinsic involvement of molecular oxygen in environmental change at every stage of plant development.

Introduction All living organisms are oxidation–reduction (redox) transduction arose around this central core. These sig- systems. They use anabolic, reductive processes to store nals exert control on nearly every aspect of plant biology energy and catabolic, oxidative processes to release it. from chemistry to development, growth and eventual Through photosynthesis, plants set this global wheel in death. Controlled production of reactive oxygen species motion. By harnessing light energy to drive biochemistry, (ROS) acts as a second messenger, alongside other medi- photosynthetic organisms have perfected the art of redox ators such as calcium, not only in plant responses to control. Redox signals are the most fundamental forms pathogens but also in hormone signalling (Pei et al. of information monitored by photosynthetic organisms 2000). Recent developments have greatly increased our (Noctor and Foyer 1998). These types of signals may understanding of how systems in organelles involved in well belong to the earliest evolved controls since photosynthesis sense redox changes, particularly those they prevent uncontrolled ‘boom and bust’ scenarios in linked to reactive oxygen in order to regulate whole cell energy availability, utilization and exchange. More redox homeostasis. complex aspects of redox control of physiology through regulation of gene expression developed with the The key role of reactive oxygen species and evolution of higher plants. It is now widely accepted antioxidants in plant redox homeostasis that redox signals are key regulators of plant metabolism, morphology and development, and it may even Photosynthetic and respiratory electron transport chains be that all intermediates and other systems of signal are the primary energy-transducing processes in eukaryotic organisms. The evolution of oxygenic photosynthesis,

Abbreviations – ABA, abscissic acid; APX, ascorbate peroxidase; DHA, dehydroascorbate; GPX, glutathione peroxidase; GRX, glutaredoxin; GSH, reduced glutathione; GSSG, glutathione disulphide; GST, glutathione S-transferase; LHC, light-harvesting complex; M-POX, Mehler-peroxidase; (i)NOS, (inducible) nitric oxide synthase; PQ, plastoquinone; PRX, peroxiredoxin; PSI, photosystem I; PSII, photosystem II; ROS, reactive oxygen species; TRX, thioredoxin.

Physiol. Plant. 119, 2003 355 an event that occurred two billion years ago, provided ensure control of the cellular redox state rather than to abundant oxygen and facilitated the elaboration of reac- facilitate the complete elimination of H2O2 (Foyer and tions involving O2, particularly aerobic respiration. Almost Noctor 2000). The antioxidative system determines the all life is based on the essential energy exchange reactions of lifetime of H2O2 in planta. Plant cells are strongly redox photosynthesis and respiration. The evolution of photosys- buffered and contain very large quantities of the soluble tem II first allowed use of the very high electrochemical hydrophilic antioxidants, ascorbate (10–100 mM) and potential (Em7 ¼ 1 815 mV) of the O2/H2O redox couple. glutathione (0.2–10 mM) (Noctor and Foyer 1998, Oxygenic photosynthesis and aerobic respiration today deal Hartmann et al. 2003). Most of their intracellular com- with concerted, four-electron exchange between water and partments hence have the capacity to deal with even very oxygen, without release of reactive, partially reduced inter- high fluxes of H2O2 production. Rapid compartment- mediates. However, many processes in plants (and in other specific differences in redox state (and hence signalling) organisms) catalyse only partial reduction of oxygen, and so that influence the operation of many fundamental pro- generate superoxide, H2O2, and hydroxyl radicals (Noctor cesses in plants, can be achieved by modifying ROS and Foyer 1998, Mittler 2002). In addition, a reaction (particularly H2O2) production or by repression or potentially common in the thylakoid pigment beds involves activation of antioxidant defences. Recent evidence sug- photodynamic energy transfer to ground-state triplet O2 gests that glutathione and ascorbate are key components and leads to the formation of highly reactive singlet O2. of redox signalling in plants (Baier et al. 2000, Noctor Redox signals are involved in all aspects of plant et al. 2000, Horling et al. 2003). Specific compartment- biology. They are particularly important in defence based signalling and regulation of gene expression can be responses and cross-tolerance phenomena, enabling a achieved via differential compartment-based changes in general acclimation of plants to stressful conditions. either the absolute concentrations of ascorbate and glu- H2O2 has long been recognized as a signal-transducing tathione or the ascorbate/dehydroascorbate and GSH/ molecule in the activation of defence responses in GSSG ratios, which are very high and stable in the plants. It mediates intra- and extra-cellular communica- absence of stress (Noctor et al. 2000). tion during plant reactions to pathogens and several Three factors are particularly significant in governing studies have suggested a role in systemic acquired resis- the importance of H2O2 in signalling redox status in a tance. The hypersensitive response (HR) is a widespread given compartment. The first is the absolute rate of H2O2 phenomenon that is responsible for the activation and production. Figure 1 shows rates of H2O2 production in establishment of plant immunity to disease. HR is an different compartments during photosynthesis at moder- example of plant programmed cell death as it leads ately high light and optimal temperatures. Photosynth- to rapid, localized cell death at infection sites. This esis produces superoxide as result of direct electron contributes to the limitation of the growth and spread transfer to oxygen, from which H2O2 is produced and of the invading pathogen. One of the earliest events in metabolized through the Mehler-peroxidase (M-POX) the HR is the rapid accumulation of ROS through the reaction. Assuming, however, that the M-POX reaction activation of enzyme systems, some of which are similar is not a sink for more than 10% of photosynthetic elec- to neutrophil NADPH oxidase (Keller et al. 1998). The tron flow, H2O2 is probably produced even faster in the oxidative burst is a central component of an integrated peroxisomes, at least in C3 plants, by virtue of the glycol- HR signalling system, whose function is rapid amplifica- late oxidase reaction. The second important factor is the tion of the signal. Other signalling components involved rate of scavenging by detoxifying systems. These two fac- 21 in HR are salicylic acid (SA) and cytosolic Ca .H2O2 tors are likely to be the main determinants of H2O2 con- 21 has a strong regulatory influence on fluxes through Ca centration. Third, the probability of reaction of H2O2 with channels and on Ca21 concentrations in different cellular signalling components is probably a key determinant of compartments. During HR, H2O2 is required to trigger signalling intensity. As we discuss below, early components localized host cell death but it seems that NO is in redox signalling are likely to include antioxidants and/or also needed to induce an efficient cell death response antioxidative enzymes. The influence of a given compart- (Delledonne et al. 1998). Further evidence that the ment in redox signalling may differ as a function of how H2O2 cascade interacts closely with other signalling well primed each compartment is with sensing components, systems comes from studies on hormone-mediated the nature of the signal that these components sense (e.g. stomatal movement (Pei et al. 2000), cell growth (Rodriguez H2O2 or other ROS, electron transport components, et al. 2002) and tropic responses (Joo et al. 2001). Such organic peroxides), and to what extent sensing components studies show that H2O2 is a common secondary mes- are coupled to downstream transduction factors such as senger in hormone-mediated events. kinase cascades (Kovtun et al. 2000).

Rates of H2O2 production in different compartments Redox signalling from the chloroplast during C photosynthesis 3 The chloroplast is an excellent model for understanding Plants are more tolerant of H2O2 than animals, and their redox-regulated plant gene expression (Pfannschmidt antioxidant systems appear to have been designed to et al. 1999). The light-driven chemistry of photosynthesis

356 Physiol. Plant. 119, 2003 Chloroplast Peroxisome Mitochondrion

Photorespiration Glycolate Electron CO fixation transport 2 H O chain 4030 2 2 –2 –1 nmol.m .s Glyoxylate 10000 –2 –1 NADH nmol.m .s H2O2 DARK H2O2 <182 nmol.m–2.s–1 TCA Reductant cycle Shuttle

Electron NADH Electron transport transport chain Serine H2O2 LIGHT NADH <216 nmol.m–2.s–1 Glycine

Fig. 1. Typical rates of H2O2 production in the organelles of mesophyll cells during C3 photosynthesis. Numerous other ROS-generating reactions are not considered – the above processes are presumed to be the fastest ROS-generating reactions in photosynthetic cells, at least in the absence of an oxidative burst at the plasmalemma. Values are necessarily approximate and are supposed to compare likely typical rates of H2O2 production in the different compartments. These are derived using established models of C3 photosynthesis (eg. Noctor et al. (2002)), in which TCA cycle activity in the light is taken to be 50% of the rate in the dark and 50% of the NADH produced by glycine decarboxylase is 2 1 oxidized by the mitochondrial electron transport chain. Rates of net photosynthetic CO2 assimilation (19 mmol m leaf surface s ) are taken 2 1 to be about 10 times faster than total respiratory O2 uptake in the dark (2 mmol m leaf surface s ). Chloroplastic and mitochondrial H2O2 production are calculated assuming likely upper limits of electron leakage to superoxide in optimal conditions (10% of total photosynthetic electron flow and 5% of total respiratory electron flow – for further discussion, see Foyer and Noctor (2000)). Peroxisomal H2O2 production through reactions other than glycollate oxidation is considered to be relatively minor in C3 leaves in the light, and is not considered.

consists of a series of redox steps involving structural signal-transducing components. Thus far, redox signals components or functionally coupled pools of redox- have been shown to control post-translational modifica- active compounds, such as thioredoxin (TRX), ascorbate tion of proteins via phosphorylation, redox modulation and glutathione. Changes in the redox state of these of assimilatory reactions and control of gene transcrip- components regulate the expression of both plastome- tion and translation (Somanchi and Mayfield 1999, Link and nuclear-encoded chloroplast proteins. This redox 1999, Pfannschmidt et al. 1999). Photosynthetic control information co-ordinates expression in both compart- of gene expression can now be described for the genes ments (Allen and Pfannschmidt 2000). To date, genetic located in chloroplasts themselves, both at transcrip- and biochemical analysis has identified plastoquinone tional and post-transcriptional levels (Pfannschmidt (PQ), ROS, ascorbate and the ferredoxin/TRX system et al. 1999). Redox signals also leave the chloroplast to as redox-active signalling components (Danon and provide a decisive input into transcriptional control Mayfield 1994, Escoubas et al. 1995, Karpinski et al. 1997, in the cell nucleus. Allen (1993) suggested that redox Pfannschmidt et al. 1999, Schu¨ rmann and Jacquot 2000, signalling is a key function of the cytoplasmic genomes Kiddle et al. 2003). There is little doubt that other redox found in the chloroplast and mitochondria, with the signalling molecules remain to be discovered. For example, exchange of redox information between these organelles the membrane-bound peroxiredoxins (PRX), whose and the nucleus functioning within the global network main role may be protection against lipid peroxidation, regulating plastid and mitochondrial metabolism and have all the necessary molecular characteristics of thiol development.

Physiol. Plant. 119, 2003 357 Coupling of photosynthetic electron transport and has led to the integration of signals from such inevitable redox poise to the expression of genes encoding chloro- side reactions into cellular control mechanisms involving plast proteins enables plants to respond to diverse envir- profound changes in gene function (Allen and Raven onmental conditions. Of the latter, light quality and 1996, Foyer and Noctor 2000, Noctor et al. 2000). quantity have been most intensely studied. The expres- Because of the potentially high capacity of photosynth- sion of plastid-encoded photosynthetic proteins is pre- esis for the production of superoxide, hydrogen peroxide cisely co-ordinated with that of their nuclear-encoded and singlet oxygen, the intracellular levels of these partners (Kettunen et al. 1997, Surpin and Chory oxidants are tightly buffered and controlled by the 1997). The light-activated increases in chloroplast pro- detoxifying antioxidant system, comprising a network tein synthesis are governed by redox-modulated tran- of enzymatic and non-enzymatic components (Noctor scriptional and translational controls (Rochaix 1996). and Foyer 1998, Mittler 2002). Major detoxification For example, the activity of the psbA-RNA binding reactions associated with photosynthesis are mediated, protein complex is subject to redox-regulation, probably first, by catalase (in the peroxisomes) and, second (in all via TRX (Danon and Mayfield 1994). In this system, the compartments), by reductive processes involving the flux of electrons through the electron transport chain major redox buffers of plant cells, ascorbate and gluta- induces a change in the sulfhydryl status of TRX and thione pools (Noctor and Foyer 1998). hence the RNP complex, thereby allowing binding to the Two notable, fairly common features of the oxidative psbA mRNA and formation of a translation initiation stress response are that (1) the most strongly induced complex. genes are not necessarily those on the front line of the A second light-modulated system, which has been antioxidant defence system: genes markedly induced are described in detail, involves the reversible redox those encoding, for example, pathogenesis-related pro- activation of membrane protein kinases. The kinases teins and heat-shock proteins, rather than classical anti- phosphorylate two distinct groups of proteins. The first oxidants; and (2) antioxidant genes that are induced are the light-harvesting chlorophyll a/b antenna system often tend to encode cytosolic rather than chloroplastic of PSII. The reversible activation of the light-harvesting antioxidative proteins, even in conditions in which the antenna complex II (LHCII) kinase(s) is related to major stress is predicted to be located in the chloroplast. the regulation and optimization of energy transfer For example, cytosolic APX transcripts, as well as the between PSII and PSI. Activation of the LHCII protein promoter activity of this nuclear gene, were shown to be kinase occurs via dynamic conformational changes in the influenced by the redox state of the photosynthetic elec- cytochrome b6f complex taking place during plasto- tron transport chain (Karpinski et al. 1997). quinol oxidation. De-activation of the kinase involves its re-association with an oxidized cytochrome complex. Maximal phosphorylation of components such as Lhcb1 Peroxisomes and reactive oxygen and nitrogen species and Lhcb2 proteins is only observed at irradiances far below those experienced by the leaves during growth. Peroxisomes are known to release signals that regulate Such observations are explained by the presence of a nuclear gene expression. Signals arising from peroxi- redox (probably TRX)-dependent regulatory loop that somes regulate photomorphogenesis, plant development inhibits the activation of the kinase at high light via peroxisomal biogenesis, light signalling and stress reduction of protein disulphide groups (Gal et al. 1997). responses (Hu et al. 2002). Redox signals are very likely Recently, a thylakoid protein kinase with these proper- important in this process since peroxisomes produce ties has been cloned from Chlamydomonas (Depe` ge et al. H2O2 at high rates through several reactions, including 2003). The second group of phosphorylated thylakoid b-oxidation of long chain fatty acids and glycollate oxi- proteins are the PSII core proteins (D1 and D2 reaction dase. The latter reaction means that during moderate to centre proteins, CP43 internal antenna protein and the high rates of photosynthesis, the peroxisomes of C3 9 kDa psbH gene product). However, it is noteworthy plants are the site of massive light-dependent generation that the thylakoid protein kinase that phosphorylates of H2O2 (Fig. 1). To cope with this, these organelles have CP43 (but not LHCII) shows both redox-dependent a very high antioxidant capacity, notably including cata- and redox-independent regulation (Vink et al. 2000). lase but also APX and other enzymes of the ascorbate/ With midpoint redox potentials as low as 0.9 V (pri- glutathione system (Jime´ nez et al. 1997). Catalase is mary acceptors of photosystem I), the photosynthetic clearly necessary for photorespiratory H2O2 processing electron transport chain generates reductants with poten- (Smith et al. 1984, Willekens et al. 1997) and insufficient tials far lower than those involved in the mitochondrial activity of this enzyme causes perturbation of cellular respiratory chain. This and other aspects of the essential redox state when photorespiration is rapid, notably asso- photoelectrochemistry of photosynthesis first evolved in ciated with a marked accumulation of oxidized gluta- an anaerobic world, where there was probably little thione. Photorespiration-linked perturbation of the selective pressure against such low potential components redox states of both ascorbate and glutathione can (Allen 1993). Electron flow from these highly reducing occur transiently in plants with a full complement of components is thermodynamically favourable for reduc- catalase (Noctor et al. 2002), and the glycollate oxidase tion of oxygen to superoxide (Em7 ¼0.33 V). Evolution reaction could possibly be one way in which signals

358 Physiol. Plant. 119, 2003 linked to photosynthesis are exported from the chloro- and is involved in the determination of cell survival in plast. Such a mechanism may be important in certain oxidative conditions (Robson and Vanlerberghe 2002, conditions such as drought, other stresses that decrease Vanlerberghe et al. 2002). stomatal conductance, or increased temperature. Under It should be noted that the relative rates shown in other conditions, such as low temperature, peroxisomal Fig. 1 will probably change differentially as a function H2O2 formation will become less important whereas the of the type of stress applied, and in certain conditions the probability (although perhaps not the absolute rate) of mitochondrial contribution may be significant, even in mitochondrial and chloroplastic production may tend to the light. Even under conditions in which the mitochon- increase in these conditions. It should also be noted that dria contribute only a fraction of total cellular ROS peroxisomes can generate significant amounts of super- production, the mitochondrial oxidative load could be oxide from which H2O2 is produced by a peroxisomal crucial in influencing and setting the cellular redox-state, superoxide dismutase (for review, see del Rı´ o et al. 2002). either because of the presence of specific signalling com- It is possible that peroxisomes are a major site of nitric ponents or because detoxification capacity is relatively oxide (NO) synthesis in plants (Corpas et al. 2001). low in comparison with the chloroplast and peroxisome. There is some evidence to suggest that NO synthase Like these other organelles, however, mitochondria (NOS), a key enzyme involved in mammalian macro- house both enzymic and non-enzymic antioxidants phage action, is conserved in plants (Ninnemann and (Rasmusson and Møller 1990, Jime´ nez et al. 1997), Maier 1996, Beligni and Lamattina 2001). An NADPH- including a TRX system (Laloi et al. 2001), and are the and Ca21-dependent NOS activity was found in pea site of ascorbic acid biosynthesis. The final step of peroxisomes (Barroso et al. 1999). Moreover, a 130-kDa ascorbic acid biosynthesis is catalysed by a galactono- peroxisomal pea protein was recognized by a polyclonal g-lactone dehydrogenase located in the inner mito- antibody raised against 14 residues from the C-terminus chondrial membrane (Bartoli et al. 2000). Evidence has of iNOS (Barroso et al. 1999). Whereas the sequencing recently been obtained that this enzyme is an intrinsic of the complete Arabidopsis genome did not reveal the component of complex I, and that respiratory electron presence of putative NOS genes, a complementary flow may exert significant control on ascorbate synthesis DNA sequence with high homology to a protein NOS (Millar et al. 2003). inhibitor, has been described in plants (Jaffrey and Evidence that perturbation of the mitochondria redox Snyder 1996). state has important consequences for whole cell redox homeostasis has been provided by studies on a Nicotiana sylvestris mutant, CMSII, which lacks functional com- Redox signalling associated with mitochondrial plex I and has hence lost a major NADH sink. Substan- respiration tial re-adjustments in antioxidant defence and related changes in stress tolerance are observed in the mutant Like chloroplasts, mitochondria also originated as (Dutilleul et al. 2003a). When complex I function is bacterial endosymbionts, and they retain a specialized perturbed, signalling is initiated which resets the antox- genome. Redox signalling as the function of the idative capacity throughout the cell. This includes mitochondrial genome has wide implications. The ‘free enhanced expression of mitochondrial antioxidants (AOX radical’ and ‘mitochondrial’ theories of ageing are central and Mn-SOD) and also of chloroplastic Fe-SOD, perox- tenets to animal and human biology but have not been isomal catalase, and cytosolic APX. Mitochondrial sig- explored in plants (Allen 1993). Mitochondria have not nalling hence acts to lower cellular H2O2 and allows traditionally been regarded as a major source of ROS in soluble cellular antioxidants (ascorbate, glutathione) to leaves, although it has been known for many years that retain a high reduction state. This redox re-adjustment is reactions associated with complexes I and III produce associated with the inability of the mutant to use its superoxide (for review, see Møller 2001). Indeed, various photosynthetic capacity as efficiently as the wild type mitochondrial enzymes, such as aconitase and enzymes (Dutilleul et al. 2003b), probably due to perturbations containing lipoic acid, are susceptible to oxidative inacti- in NAD(P)H shuttling between intracellular compart- vation. From a whole leaf point of view, however, at ments (for reviews, see Gardestro¨ m et al. 2002, Scheibe least in C3 plants at moderate to high light intensities, 2003). These shuttles represent another example of the peroxisomal and chloroplastic H2O2 production may be important interactions between chloroplasts and mito- up to 30–100 times faster than formation of H2O2 in the chondria, and could be crucial in adjusting the rate of mitochondria (Fig. 1). Interestingly, calculations suggest ROS production in both compartments. Reductant export that mitochondrial ROS production is not likely to be from the chloroplast to the mitochondria might act to greatly different in the light and dark, since total O2 lower ROS production in the chloroplast by relieving consumption is less affected by light than tricarboxylic electron pressure, while increasing the probability of ROS acid cycle activity. However, the probability of super- formation in the respiratory chain. At the origin of oxide production by the respiratory chain could be mitochondrial redox signalling may be changes in the changed on illumination, notably if light affects alternative redox state of ubiquinone or components of the cyto- oxidase (AOX) capacity (Dutilleul et al. 2003a). This chrome bc1 complex, by analogy to the signalling linked enzyme influences ROS generation (Maxwell et al. 1999) to the chloroplast plastoquinone pool. Such changes could

Physiol. Plant. 119, 2003 359 be linked to increased ROS generation through electron H2O2 concentration is not greatly increased relative to leakage to superoxide. the wild type but the glutathione pool is massively perturbed (Noctor et al. 2002). Increased availability of ROS may therefore be sensed by the cell as increased ROS and redox signal perception and transduction oxidative flux through key components, rather than (or as well as) marked increases in ROS concentration To date no ROS receptor has been unambiguously iden- (Fig. 2). This view suggests a dynamic system constructed tified in plants, and thus a key question is: how is to allow acclimatory changes through components of the increased ROS production sensed? One simple possibility antioxidative system that are plugged into signalling net- is direct modification of transcription factors with redox- works. It would allow appropriate responses to occur as sensitive groups (Tron et al. 2002), but there are very a result of increased flux to ROS, even in the absence of likely much more complex signal transduction routes. marked changes in ROS concentration. Furthermore, This must be the case when redox changes in organelles changes in ROS trigger profound modifications in gene are signalled to the nucleus. Important sensing compo- expression that extend far beyond the antioxidative sys- nents may be found in the antioxidative system itself. tem (Kovtun et al. 2000, Desikan et al. 2001, Vranova This system is a strong buffer against ROS, maintaining et al. 2002). This confirms the view that the defence relatively low oxidant concentrations under most condi- system comprising pathogen responses, defence against tions. Although localized increases in ROS can occur in xenobiotics, stabilizing components, and antioxidants certain circumstances, such as during the oxidative burst forms an integrated network with extensive crosstalk at the plasmalemma, in many cases redox balance can be that can be triggered by ROS. Moreover, priming of markedly perturbed without large changes in H2O2 con- different components can be triggered by low ROS centration. This is, perhaps, not suprising given the concentrations such that the system responds more plethora of components capable of scavenging H2O2.In rapidly and/or effectively to subsequent assault (Vranova catalase mutants placed in conditions where the leaf can et al. 2002). We suggest that some antioxidative compo- no longer cope with photorespiratory H2O2 production, nents have a dual function of scavenging and signalling

A Optimal conditions Fig. 2. A model for the Detox-scavenging perception of increased ROS production via the antioxidant system. In optimal conditions (A), ROS are produced by Photosynthesis many metabolic reactions and Photorespiration [ROS] are efficiently removed by Respiration detoxification processes (‘Detox-scavenging’). Stress conditions (B) cause increased Sensor-scavenging production of ROS or decreased antioxidant activity, which can cause an increase in ROS concentration. This could ––lead to increased oxidation of specific ‘sensor-scavenging’ B Stress conditions antioxidant components locked Detox-scavenging Other defences into signal transduction pathways. Stimulation of transduction pathways occurs + + via kinase cascades and other second messengers, and leads to Photosynthesis Acclimatory Cell death OR either acclimation (up- Photorespiration [ROS] pathway pathway regulation of ROS Respiration detoxification capacity, induction of other defences) or cell death pathways involving defence withdrawal. The Sensor-scavenging Kinase cascades decisive factors that determine (Thiol oxidation? either acclimatory or cell death Glutathionylation?) responses are not known, but could include location of the initial signal or signal intensity. Control of cell fate by signal intensity would be a molecular application to plants of the aphorism formulated, more than a century ago, by the philosopher Nietszche: That which does not kill me, makes me stronger.

360 Physiol. Plant. 119, 2003 (Fig. 2: ‘sensor-scavenging’ components). These could novel, specific chloroplastic TRX isoform (Collin et al. perhaps be relatively low in antioxidative capacity com- 2003). A PRX localized in poplar phloem can function paredtoclassicaldetoxification-scavenging(Fig. 2:‘Detox- with either the TRX h/NADPH-TRX reductase or GRX/ scavenging’) components such as catalase or chloroplas- glutathione/GR systems (Rouhier et al. 2001). It is possible tic APX. that the primary role of at least some of these thiol pro- What could be the nature of such ‘sensor-scavenging’ teins, particularly those with relatively low capacity, is not components)? It is perhaps noteworthy that catalases to detoxify peroxides but to sense their increased produc- and APX are haem-based whereas many other tion, that is, their most important function may be as signal antioxidant components operate through thiol–disulphide or signal-scavenging components (Fig. 2). exchange reactions. As well as the soluble thiol gluta- Net oxidation of the glutathione pool, accompanied thione, the plant cell contains numerous proteins with by increased total glutathione, is a clearly established redox-active thiol groups, some of which have been response to many stresses or to insufficient detoxification shown to have activity against peroxides. These include capacity (Smith et al. 1984, Sen Gupta et al. 1991, both chloroplastic and cytosolic glutathione peroxidases Willekens et al. 1997, Noctor et al. 2002). A mechanism (GPX: Eshdat et al. 1997, Mullineaux et al. 1998), chloro- important in sensing this redox perturbation may be plastic and cytosolic PRX (Baier and Dietz 1999, Rouhier protein glutathionylation, in which glutathione forms a et al. 2001, Horling et al. 2003), and glutaredoxins mixed disulphide with a target protein. In yeast and (GRX). Specific TRX are also found in several compart- animals, glutathionylation has been shown to modify ments of the photosynthetic cell (Rivera-Madrid et al. the activity of enzymes and transcription factors and is 1995). There is considerable heterogeneity within these considered likely to play an important role in redox families: for PRX, there are monomeric and dimeric signalling and protection of protein structure and func- enzymes, and differences in the number of Cys involved tion (Klatt and Lamas 2000). For example, gluthathio- in catalysis. Similarly, several sequences predicted to nylation of aldolase and triose phosphate isomerase has encode TRX-like proteins and both dithiol and mono- recently been reported in Arabidopsis leaves (Ito et al. thiol GRX-like proteins (Vlamis-Gardikas and Holmgren 2003). Spontaneous formation of mixed disulphides may 2002) are found in the Arabidopsis genome. Although require decreases in GSH/GSSG that are not thought to the exact roles of most of these plant thiol proteins be physiologically relevant, at least in most mammalian remain to be elucidated, it is clear there may be consider- cells, but in the absence of marked accumulation of GSSG able divergence of function within each class. the reaction can be catalysed by GRX (Starke et al. 2003). In mammals, where TRX exists as mitochondrial Reversal of glutathionylation may be carried out (TRX2) and cytosolic/nuclear (TRX1) forms, expression by dithiol or monothiol GRX, as well as the dithiol protein, is influenced by an ARE (antioxidant response element) TRX. It is interesting to note that these components belong cis-acting factor, and TRX1 mediates numerous defence to a large super-family of proteins, and that proteins related processes, including Ref-1 activity and the nuclear versus to classical TRX, but with a monocysteinic active site cytosolic localization of the transcription factor NF-kB motif, also exist in plants. Considerable work will be (Vlamis-Gardikas and Holmgren 2002). Recently, it has required to elucidate the functions of many of these pro- been shown in yeast that the redox transduction sensor, teins. It is possible that whereas only the dithiol enzymes YAP-1, interacts in a complex fashion with both TRX have activity against protein disulphide bonds, the mono- and non-selenium (i.e. plant-type) GPX (Delauney et al. thiol GRX may be the most important in deglutathionylat- 2002). YAP-1 is a basic zipper-type transcription factor ing mixed disulphides to protein-SH and GSH (Shenton that induces several genes in response to peroxides. Per- et al. 2002, Vlamis-Gardikas and Holmgren 2002). oxides enhance the nuclear accumulation of YAP-1 by Another important group of thiol proteins may be oxidizing two Cys residues to form an intramolecular certain subclasses of the GST super-family that are active disulphide bond that appears to act to trap YAP-1 in in reducing peroxides or dehydroascorbate (DHA), or in the nucleus, thereby increasing its activity (Kuge et al. catalysing thiol transferase (i.e. GRX) activity (Bartling 1997). Oxidation of YAP-1 is not mediated directly, et al. 1993, Dixon et al. 2002). Some GSTs are strongly however, but occurs via H2O2 reduction by a GPX-like induced by oxidative stress, including that linked to the protein, which is then reduced by YAP-1 (Delauney et al. pathogen response (Conklin and Last 1995, Desikan et al. 2002). Re-reduction of YAP-1 may occur by a TRX- 2001, Dixon et al. 2002). Until recently considered as dependent pathway (Delauney et al. 2002). Although exclusively cytosolic enzymes, GSTs form a large gene the Arabidopsis genome does not appear to contain family in Arabidopsis, but the physiological functions of sequences homologous to YAP-1, there may well be many of these genes remain unclear. Lately, it has been functionally similar elements in the initial perception of shown that in two subclasses of the GST family found in changes in redox state in plants. PRX are often alterna- Arabidopsis, the classical GST active site Ser is replaced tively termed TRX peroxidase and chloroplastic PRX by Cys (Dixon et al. 2002). This confers both DHA purified from Chlamydomonas has been shown to reduce reductase and GRX activity on the first class, and peroxides using reductant from TRX (Goyer et al. 2002). DHA reductase activity on the second: in each class, This enzyme system is therefore closely linked to the photo- gene sequences predicted at least one chloroplastic and synthetic electron transport chain in vivo, probably via a one cytosolic product (Dixon et al. 2002).

Physiol. Plant. 119, 2003 361 Integration of redox signalling and plant development: superoxide. Moreover, there are numerous interactions interactions between ROS and phytohormones between redox and hormonal controls. These situate ROS and antioxidants at the heart of the fine control of While redox sensing in plants may have some similarities plant development and acclimation to external conditions, with that observed in yeast, downstream signalling is and suggest that elucidating the details of the signalling probably more intricate, because the photosynthetic, networks will be a challenging and fascinating task. multicellular nature of plants necessitates integration of a complex array of internal and external signals during development. MAP kinase cascades are implicated in the References H2O2 response in plants (Kovtun et al. 2000). 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Edited by P. Gardestro¨ m

364 Physiol. Plant. 119, 2003