Abstract

In 1996, cDNA sequences referred to as plant peroxiredoxins (Prx), i.e. a 1-Cys Prx and a 2-Cys Prx, were reported from barley. Ten years of research have advanced our understanding of plant Prx as thiol-based peroxide reductases with a broad substrate specificity, ranging from hydrogen peroxide to alkyl hydroperoxides and peroxinitrite. Prx have several features in common. (i) They are abundant proteins that are routinely detected in proteomics approaches. (ii) They interact with proteins such as glutaredoxins, thioredoxins, and cyclophilins as reductants, but also non-dithiol-disulphide exchange proteins. By work with transgenic plants, their activity was shown to (iii) affect metabolic integrity, (iv) protect DNA from damage in vitro and as shown here in vivo, and (v) modulate intracellular signalling related to reactive oxygen species and reactive nitrogen species. (vi) In all organisms Prx are encoded by small gene families that are of particular complexity in higher plants. A comparison of the Prx gene families in rice and Arabidopsis thaliana supports previous suggestions on Prx function in specific subcellular and metabolic context. (vii) Prx gene expression and activity are subjected to complex regulation realized by an integration of various signalling pathways. 2-Cys Prx expression depends on redox signals, abscisic acid, and protein kinase cascades. Besides these general properties, the chloroplast Prx have acquired specific roles in the context of photosynthesis. The thioredoxin-dependent peroxidase activity can be measured in crude plant extracts and contributes significantly to the overall H2O2 detoxification capacity. Thus organellar Prx proteins enable an alternative water–water cycle for detoxification of photochemically produced H2O2, which acts independently from the ascorbate-dependent Asada–Halliwell–Foyer cycle. 2-Cys Prx and Prx Q associate with thylakoid membrane components. The mitochondrial PrxII F is essential for root growth under stress. Following a more general introduction, the paper summarizes present knowledge on plant organellar Prx, addressing Prx in signalling, and also suggests some lines for future research.

Abbreviations

    Abbreviations
  • Apx

    ascorbate peroxidase

  • Cyp

    cyclophilin

  • DTT

    dithiothreitol

  • GPx

    glutathione peroxidase

  • Grx

    glutaredoxin

  • MV

    methylviologen

  • NTR

    NADPH thioredoxin reductase

  • Prx

    peroxiredoxin

  • RNS

    reactive nitrogen species

  • ROS

    reactive oxygen species

  • Trx

    thioredoxin

Introduction

Peroxides are reactive compounds that pose an oxidation threat to cells when they accumulate to high concentrations. In principle, hydrogen peroxide is less reactive than other reactive oxygen species (ROS) such as the superoxide anion radical and the hydroxyl radical. However, in the presence of Fe2+, H2O2 is reduced in the Fenton reaction to yield the hydroxyl radical OH·. Within diffusion distance, OH·− reacts with virtually any biomolecule to abstract an electron. Due to its ability to diffuse over significant distances within and between cells, H2O2 serves a signalling function in cellular communication (Foyer and Noctor, 2000; Apel and Hirt, 2004). Also a long-distance signalling function has been attributed to H2O2, in the context of systemic acclimation to excess excitation energy (Karpinski et al., 1999). This dual role of H2O2 as a potentially damaging compound and as a messenger demands a balanced defence system. Under regular metabolic conditions H2O2 should be decomposed to a very low micromolar resting level. However, in case of a sudden and significant increase in H2O2 generation the signal must be propagated to the regulatory targets in order to allow for the appropriate activation of response reactions. H2O2 concentrations increase in response to various abiotic and biotic stresses and take part in the reactive oxygen regulatory network (Mittler et al., 2004).

Cells express a set of hydrogen peroxide-decomposing enzymes, namely catalase, ascorbate peroxidase (Apx), glutathione peroxidase (Gpx), peroxiredoxin (Prx), and type-III peroxidases (Mittler and Poulos, 2005). Gpx and Prx also decompose alkyl hydroperoxides in addition to H2O2.

Plant catalases are haem enzymes that function as tetrameric proteins with four protoporphyrin IX moieties and exhibit a high molecular efficiency but a very low Km that ranges between 10 and 140 mM H2O2 (Feierabend, 2005). Catalases are localized in peroxisomes. Only a few exceptions from this location have been reported; for example, catalase activities in the mitochondrion of maize (Scandalios et al., 1980) and the apoplast (Salguero and Böttger, 1995). Catalase is sensitive to inactivation by O2 or H2O2. Ascorbate peroxidases (Apx) and type-III peroxidases are haem enzymes. Particularly, a set of Apx proteins is known to participate in efficient decomposition of hydrogen peroxide in various subcellular compartments (Mittler and Poulos, 2005). They are commonly believed to have a predominant role in H2O2 reduction via the so-called Asada–Halliwell–Foyer cycle at the expense of ascorbate that is oxidized and regenerated either from NAD(P)H or ferredoxin via monodehydroascorbate reductase or from glutathione via dehydroascorbate reductase (Noctor and Foyer, 1998). Chloroplastic Apx are sensitive to inactivation in the presence of H2O2 when ascorbate is absent (Nakano and Asada, 1987). Glutathione peroxidases have a cytosolic and chloroplastic location (Eshdat et al., 1997; Mullineaux et al., 1998). At least some GPx seem to function as thioredoxin-dependent peroxidases (Herbette et al., 2002).

Peroxiredoxins constitute the most recently identified group of H2O2-decomposing antioxidant enzymes. In addition to the reduction of H2O2, Prx proteins also detoxify alkyl hydroperoxides and peroxinitrite, despite the fact that significant differences exist in substrate specificity and kinetic properties. Through this activity Prx is likely to modulate oxolipid-dependent and NO-related signalling (Baier and Dietz, 1999, 2005; Rhee et al., 2005). Overexpression of chloroplast 2-Cys Prx increased tolerance of yeast cells to reactive nitrogen species (Sakamoto et al., 2003). A number of recent reviews have addressed the structural and catalytic properties of the various Prx isoforms in general (Hofmann et al., 2002; Wood et al., 2003) and specifically in plants (Dietz et al., 2002; Rouhier and Jacquot, 2002, 2005; Dietz, 2003a ). Experimental evidence exists for a triple Prx function in plant cell biology as (i) antioxidant, (ii) modulator of cell signalling pathways, and (iii) redox sensor. In 1996, cDNA sequences encoding a barley 1-Cys Prx (Stacy et al., 1996) and a 2-Cys Prx (Baier and Dietz, 1996) were published and identified as Prx. Thus this paper reports on progress made during the last 10 years, focusing almost exclusively on Prx isoforms targeted to the chloroplasts and mitochondria.

Prx proteins

All Prx have a similar basic protein structure with a thioredoxin fold. Their molecular masses range between 17 and 22 kDa. Based on sequence similarities and catalytic mechanisms, four types of Prx proteins are distinguished (Rouhier and Jacquot, 2002; Dietz, 2003a). The peroxide substrates react with the N-terminally located peroxidatic Cys residue present in all Prx proteins (Fig. 1A). Its site-directed mutagenesis to Ser abolishes the peroxidase activity (König et al., 2002; Rouhier et al., 2004a; Lamkemeyer et al., 2006). In the non-mutagenized protein the peroxidatic thiol group is oxidized to the sulphenic acid form and the reduced peroxide product, i.e. H2O is released in the case of H2O2, the corresponding alcohol in the case of alkyl hydroperoxides, and nitrite in the case of ONOO. 1-Cys Prx does not possess a second catalytic Cys. The sulphenic acid derivative of its catalytic Cys is re-reduced by a yet unknown interacting partner before the next catalytic cycle begins. In a converse manner, 2-Cys Prx, PrxQ, and type-II Prx contain a second catalytic Cys residue in a distinct structural context that reacts with the sulphenic acid group of the peroxidatic Cys. Either an intramolecular disulphide bridge, in the cases of Prx Q and type-II Prx, or an intermolecular one, in the case of the 2-Cys Prx, is formed and water is produced. The disulphide formation, at least in 2-Cys Prx, is accompanied by a major structural rearrangement (König et al., 2003). For some Prx, inactivation has been demonstrated by overoxidation (2-Cys Prx) or nitrosylation (PrxII E) (Fig. 1A) (König et al., 2002; MC Romero-Puertas, M Laxa, KJ Dietz, M Delledonne, unpublished results). This may allow Prx activity to be regulated. In yeast and man, a novel enzyme named sulfiredoxin has been identified that reduces the cysteine sulphinic acid form of 2-Cys Prx under hydrolysis of ATP (Biteau et al., 2003; Chang et al., 2004). A sulphiredoxin homologue is also present in the Arabidopsis thaliana genome (At1g31170).

Fig. 1

Unified reaction cycle of peroxiredoxins (A) and comparative subcellular location of Prx in rice and Arabidopsis thaliana (B). (A) The reaction mechanism is described in the text. Note that each Prx shows specific properties as well (cf. Hofmann et al., 2002; Rouhier and Jacquot, 2002). In brief, the catalytic Cys is oxidized to sulphenic acid. A disulphide bridge is formed under a conformational switch of the protein. The disulphide-oxidized Prx is regenerated by electron donors such as Trx, Grx, Cyp, and glutathione. Nitrosylation and overoxidation to sulphinic or sulphonic acid derivatives of the catalytic Cys withdraw Prx from the cycle and are considered as regulatory and potentially signalling mechanisms. (B) Subcellular location of Prx isoforms in rice and Arabidopsis (cf. Table 2). Each species has three different types of Prx in the chloroplast. Probably due to recent gene duplication and alternative splicing in the case of Os-prxQ, the total number of plastidic Prx is four in Arabidopsis thaliana and five in O. sativa. In addition a type-II Prx resides in the mitochondrion, a 1-Cys Prx in the nucleo-cytoplasm, and at least one type-II Prx in the cytosol.

Fig. 1

Unified reaction cycle of peroxiredoxins (A) and comparative subcellular location of Prx in rice and Arabidopsis thaliana (B). (A) The reaction mechanism is described in the text. Note that each Prx shows specific properties as well (cf. Hofmann et al., 2002; Rouhier and Jacquot, 2002). In brief, the catalytic Cys is oxidized to sulphenic acid. A disulphide bridge is formed under a conformational switch of the protein. The disulphide-oxidized Prx is regenerated by electron donors such as Trx, Grx, Cyp, and glutathione. Nitrosylation and overoxidation to sulphinic or sulphonic acid derivatives of the catalytic Cys withdraw Prx from the cycle and are considered as regulatory and potentially signalling mechanisms. (B) Subcellular location of Prx isoforms in rice and Arabidopsis (cf. Table 2). Each species has three different types of Prx in the chloroplast. Probably due to recent gene duplication and alternative splicing in the case of Os-prxQ, the total number of plastidic Prx is four in Arabidopsis thaliana and five in O. sativa. In addition a type-II Prx resides in the mitochondrion, a 1-Cys Prx in the nucleo-cytoplasm, and at least one type-II Prx in the cytosol.

2-Cys Prx are functional dimers with a significant propensity to oligomerize. The 2-Cys Prx cycles between a dimeric and decameric state which has a doughnut-like shape (Wood et al., 2002). The dimer is characterized by a compact structure and parallel orientation of the characteristic β-sheets of both subunits allowing the formation of the intermolecular disulphide bridge during the catalytic reaction (König et al., 2003).

Recently, the poplar type-II Prx was crystallized (Echalier et al., 2005). The subunits within the type-II Prx dimer show perpendicular orientation of the β-sheets similar to the interface between the dimers of 2-Cys Prx in the oligomerized state. The 2-Cys Prx interface is disturbed upon transition from the reduced dithiol to the disulphide state. As a consequence of the conformational change, the oligomer disassembles upon oxidation to the disulphide form. But the oligomer is maintained if the sulphenic acid intermediate is further oxidized to sulphinic and sulphonic acid derivatives that mimic at least partly the reduced state (Echalier et al., 2005).

The catalytic activities of Prx proteins are low: they range between 1 and 300 mol substrate reduced per mol enzyme. Table 1 summarizes some published and unpublished data. The lowest activity is measured with 2-Cys Prx (Horling et al., 2003; König et al., 2003), while the highest activities are associated with type-II Prx C where rates higher than 300 mol (mol min)−1 have been observed (S Jacob, I Finkemeier, KJ Dietz, unpublished results). The table also summarizes present knowledge on reductive regeneration of Prx. In peroxide reduction assays, various plant thioredoxin isoforms have been linked to 2-Cys Prx, PrxQ, and type-II Prx regeneration (Table 1; S Jacob, unpublished results). In two studies, Grx regenerated type-II Prx, namely the poplar cytosolic PrxII and the mitochondrial At-PrxII F (Rouhier et al., 2001; Finkemeier et al., 2005). The latter also showed peroxide reduction activity with glutathione alone. Glutaredoxin (Grx) CxxC4 also regenerated PrxII C (Table 1). Finally, Cyp, a subgroup of the peptidyl-prolyl-cis-trans isomerases, which contain conserved Cys residues, were shown to reduce oxidized 2-Cys Prx A and B (M Laxa, J König, KJ Dietz, A Kandlbinder, unpublished results).

Table 1

Catalytic efficiency of plant Prx as selectively compiled from the literature or obtained as unpublished results

Peroxiredoxin type Species Cell location Redox potential Molar efficiency [mol (mol min)−1]
 
Reference 
    DTT Thioredoxin Glutaredoxin  
2-Cys Prx At-2-Cys Prx A Plastid −307 mV 1 (H2O27 (H2O2)a − Horling et al., 2003 
 At-2-Cys Prx B Plastid −322 mV 6 (H2O28 (H2O2)a − Horling et al., 2003 
 Hordeum vulgare Plastid −315 mV    König et al., 2002 
 At-2-Cys Prx Plastid   0.32 (CDSP)  Broin et al., 2002 
Prx Q At-PrxQ Plastid  1.5 (H2O224 (H2O2)b − Lamkemeyer et al., 2006 
 AtPrxQ Plastid   34 (BOOH)c  Collin et al., 2004 
 Gentiana triflora Plastid  − 2 (H2O2− Kiba et al., 2005 
 Populus tremula× tremuloides Plastid −325 mV  176 (H2O2)d − Rouhier et al., 2004b 
     54 (BOOH)   
     141 (COOH)   
Type-II Prx At-PrxII C Cytosol  155 (H2O2  Horling et al., 2002 
    309 (H2O2 22 (H2O2S Jacob, unpublished 
    287(BOOH)    
    49 (COOH)    
    −(LOOH)    
 At-PrxII E Plastid −288 mV 57 (H2O2  Horling et al., 2003 
 At-PrxII F Mitochondrion −307 mV 40 (H2O220 (H2O217 (H2O2Finkemeier et al., 2005 
    20 (BOOH)    
    17 (COOH)    
    6 (PLOOH)    
    1 (LOOH)    
Peroxiredoxin type Species Cell location Redox potential Molar efficiency [mol (mol min)−1]
 
Reference 
    DTT Thioredoxin Glutaredoxin  
2-Cys Prx At-2-Cys Prx A Plastid −307 mV 1 (H2O27 (H2O2)a − Horling et al., 2003 
 At-2-Cys Prx B Plastid −322 mV 6 (H2O28 (H2O2)a − Horling et al., 2003 
 Hordeum vulgare Plastid −315 mV    König et al., 2002 
 At-2-Cys Prx Plastid   0.32 (CDSP)  Broin et al., 2002 
Prx Q At-PrxQ Plastid  1.5 (H2O224 (H2O2)b − Lamkemeyer et al., 2006 
 AtPrxQ Plastid   34 (BOOH)c  Collin et al., 2004 
 Gentiana triflora Plastid  − 2 (H2O2− Kiba et al., 2005 
 Populus tremula× tremuloides Plastid −325 mV  176 (H2O2)d − Rouhier et al., 2004b 
     54 (BOOH)   
     141 (COOH)   
Type-II Prx At-PrxII C Cytosol  155 (H2O2  Horling et al., 2002 
    309 (H2O2 22 (H2O2S Jacob, unpublished 
    287(BOOH)    
    49 (COOH)    
    −(LOOH)    
 At-PrxII E Plastid −288 mV 57 (H2O2  Horling et al., 2003 
 At-PrxII F Mitochondrion −307 mV 40 (H2O220 (H2O217 (H2O2Finkemeier et al., 2005 
    20 (BOOH)    
    17 (COOH)    
    6 (PLOOH)    
    1 (LOOH)    

CDSP, chloroplast drought stress protein of 32 kDa; BOOH, tertiary butylhydroperoxide; COOH, cumene hydroperoxide; LOOH, lipid hydroperoxide; PLOOH, phospholipid hydroperoxide.

a

Escherichia coli Trx;

b

At-Trx x;

c

At-Trx-y2;

d

Pt-Trx h3.

Prx gene families in rice, A. thaliana, and two cyanobacteria

Comprehensive identification of all Prx members through genome-wide searches provides some evolutionary clues on a potential link between Prx function and photosynthesis. The A. thaliana genome encodes 10 Prx genes (Dietz et al., 2002; Dietz, 2003a). The rice genome was investigated by BLAST searches against nine At-prx sequences and allowed the identification of (at least) eight Os-prx genes (Table 2). In addition, two peculiar sequences with Prx-like domains were identified that need to be verified. This comparison of a monocot and a dicot reveals conserved as well as variable features of the Prx gene families. Common to both plants is the presence of at least one gene coding for a 2-Cys Prx-, PrxQ-, PrxII E-, PrxII F-, and a cytosolic Prx II-like protein (Fig. 1B). Most likely recent gene duplication events gave rise to the existence of two highly sequence-similar copies of the Os-1-Cys Prx and Os-PrxII E in the rice genome. As a result, at least four Prx proteins are predicted to be targeted to the rice chloroplast as in A. thaliana. In order to get further insight into the Prx gene family associated with oxygenic photosynthesis, other genome-wide searches identified five and six prx genes, respectively, in the two cyanobacteria Synechocystis PCC 6803 and Synechococcus elongatus PCC 7942 (Stork et al., 2005). The study also revealed significant similarity as well as dissimilarity within the Prx gene families in cyanobacteria. While Synechocystis expresses a type-II Prx and two Prx Q-like proteins, Synechococcus expresses four Prx Q proteins and lacks a PrxII. An extended stress-related expressional analysis showed distinct regulation and suggested that one of the Prx Q took over the function of the type-II Prx in Synechococcus. The highest degree of amino acid conservation is seen in the group of 1-Cys Prx and 2-Cys Prx and the least for type-II Prx and some PrxQ such as slr0241 of Synechocystis sp. PCC 6803 (Stork et al., 2005).

Table 2

Compilation and comparison of prx-sequences in the genomes of Oryza sativa and Arabidopsis thaliana

graphic 
graphic 

Organellar location of Prx in plant cells

In A. thaliana and probably also in Oryza sativa four prx gene products are targeted to the plastids. Gene expression studies in A. thaliana basically revealed similar responses of 2-Cys Prx A and 2-Cys Prx B to environmental and developmental clues, but also some variation in response strength and kinetics (Horling et al., 2003). Despite the distinct features, in part, it appears allowable for the time being to assume that the At-2-Cys Prx A and the At-2-Cys Prx B, on the one hand, and the Os-PrxII E1 and the Os-Prx II E2, on the other hand, are potentially redundant proteins with identical functions arising from recent gene duplications. Based on this consideration and the gene Prx families in cyanobacteria, the minimum set of plastid Prx is suggested to comprise one 2-Cys Prx, one Prx Q, and one type-II Prx. Suborganellar distribution analyses by fractionation and subsequent western blot analysis (Dietz et al., 2006) and immunocytochemistry (Dietz et al., 2005) show that Prx Q is associated with thylakoid membranes (Lamkemeyer et al., 2006). The 2-Cys Prx switches between the stroma soluble dimeric form and a thylakoid attached oligomeric form (König et al., 2002, 2003). PrxII E is found in the soluble phase (Dietz et al., 2006).

In both yeast and man, at least one Prx is targeted to the mitochondrion (Pedrajas et al., 2000; Wood et al., 2003). Likewise, one Prx, i.e. PrxII F, is sorted to the mitochondrion in both A. thaliana and O. sativa (Table 2). Thus it is tempting to conclude at the moment that organelles containing their own DNA depend on the presence of nuclear-encoded Prx, and that photosynthesis may be related to the presence of three different types of Prx.

Prx protein abundance

Peroxiredoxins are frequently identified as significant spots in proteomic approaches using two-dimensional gel separations; for example, from chloroplast (Peltier et al., 2002; Majeran et al., 2005) or mitochondria fractions (Kruft et al., 2001; Sweetlove et al., 2002). Alternatively, through immunological quantification in extracts using calibration against heterologously expressed purified protein, the plastid 2-Cys Prx protein was estimated to amount to ∼0.6% of the chloroplast protein, being equivalent to ∼60 μM concentration (König et al., 2002; Dietz et al., 2005). Likewise, chloroplast Prx Q was estimated to represent ∼0.3% of the chloroplast protein (Lamkemeyer et al., 2006).

Prx function in peroxide detoxification in photosynthesis

The redox state of the photosynthetic electron transport carriers needs to be poised in order to avoid over-reduction and damage to the photosynthetic apparatus. In the Mehler reaction, ferredoxin-mediated electron transfer to O2 provides a drainage reaction if other acceptors such as CO2 and NO2 are in short supply. Generated O2 is dismutated by superoxide dismutase to produce H2O2. H2O2 is decomposed by peroxidases to yield H2O (Fig. 2). In the overall reaction, electrons abstracted from H2O through the oxygen-evolving complex of photosystem II are transferred to O2 again and a water–water cycle is established. Concomitantly, light energy is safely dissipated as heat. Depending on the environmental condition, as little as a few per cent to >40% of the electrons are suggested to be diverted into the water–water cycle, although the precise figures are a matter of open discussion (Heber, 2002). It is frequently reported that thylakoid bound and stromal Apx take over the peroxidase function (Asada, 2000). However, other peroxidases, particularly the Prx, can partly replace Apx to establish an alternative pathway for the water–water cycle (Dietz et al., 2002) (Fig. 2). Attempts to quantify the relative contribution of Prx in extracts to the overall water–water cycle activity are lacking since an enzymatic test has not been established. This is mainly due to the need for reductive regeneration of oxidized Prx after each catalytic cycle. Cyclophilin (Cyp), Grx, thioredoxin (Trx), and glutathione function as regenerants, however, with highly varying efficiency. Further on, various Trx isoforms are present in the plant cells. Their regeneration capacity, for instance with 2-Cys Prx and Prx Q, varies strongly (Collin et al., 2003, 2004). In vitro the artificial reductant dithiothreitol (DTT) is often used as a substitute for the natural regenerants. However, DTT is efficient only with some Prx, particularly the type-II Prx, while it is inefficient with At-2-Cys PrxB and is hardly effective with At-2-Cys PrxA and 2-Cys Prx from other species and PrxQ. In an attempt to quantify the contribution of thiol-linked H2O2 detoxification in leaves and chloroplasts a Trx-dependent assay was established with Escherichia coli Trx as regenerant linked to thioredoxin reductase and NADPH (Fig. 3A). Both in crude leaf and chloroplast extracts peroxidatic activity was reliably detected as NADPH oxidation in the presence of the complete system. Related to leaf as well as chloroplast protein contents, about 50 μmol H2O2 were reduced g−1 protein min−1 corresponding to about 30 μmol H2O2 mg−1 chlorophyll h−1. Typical C3 photosynthesis rates amount to 100 μmol CO2 mg−1 chlorophyll h−1. Thus Trx-dependent peroxidases are important players in the chloroplast antioxidant defence system. For comparison, Apx activities were quantified as well (Fig. 3B). While Apx activity in leaf extracts was 7-fold higher than thiol-dependent reduction rates, in chloroplasts the Apx activity was only about twice that of the Trx-dependent peroxidatic activity. It should be noted that E. coli Trx is electron donor to 2-Cys Prx and plastidial PrxQ, but inefficient to some type-II Prx. Trx also activates GPx (Herbette et al., 2002). Thus the established test only gives a rough estimate of the Trx-dependent peroxidase activity. Due to the inherent susceptibility of chloroplast Apx to oxidative inactivation in the absence of ascorbate, for instance under stress, Apx activity may be limiting in protecting photosynthesis (Yabuta et al., 2002). Also Prx are robust enzymes; for example, PrxII E still has a high activity at 50 °C (Fig. 3C). Additional evidence for the contribution of Prx to H2O2 reduction comes from antisense suppression of 2-Cys Prx in transgenic Arabidopsis thaliana. The plants had a more oxidized ascorbate pool (Baier et al., 2000), suggesting a partial shift of the burden from peroxide detoxification through the Prx- to the Apx-dependent pathway. These results and considerations strongly support the view that Prx-mediated peroxide reduction significantly contributes to the detoxification of Mehler-derived H2O2 in the chloroplast (Fig. 2B) and should be taken into account when discussing the chloroplast ROS detoxification network.

Fig. 2

Prx-mediated alternative water–water cycle. The superoxide anion radical generated in the light reactions from excess electron flux is dismutated to H2O2. According to reviews and textbooks, ascorbate peroxidase is commonly considered to exclusively detoxify H2O2. In this water–water cycle, monodehydroascorbate is reduced by monodehydroascorbate reductase linked to ferredoxin or, after disproportionation of two molecules, monodehydroascorbate reductase to ascorbate and dehydroascorbate via dehydroascorbate reductase linked to glutathione (not shown). GPx and Prx can function in an alternative water–water cycle. This is exemplified in the figure for 2-Cys Prx. Prx Q, and PrxII E function as well, in particular since their catalytic efficiency is even higher than that of 2-Cys Prx (Lamkemeyer et al., 2006; Table 1). Thioredoxin or other reductants reduce oxidized Prx and need to be regenerated themselves, for example by ferredoxin-dependent thioredoxin reductase (FTR).

Fig. 2

Prx-mediated alternative water–water cycle. The superoxide anion radical generated in the light reactions from excess electron flux is dismutated to H2O2. According to reviews and textbooks, ascorbate peroxidase is commonly considered to exclusively detoxify H2O2. In this water–water cycle, monodehydroascorbate is reduced by monodehydroascorbate reductase linked to ferredoxin or, after disproportionation of two molecules, monodehydroascorbate reductase to ascorbate and dehydroascorbate via dehydroascorbate reductase linked to glutathione (not shown). GPx and Prx can function in an alternative water–water cycle. This is exemplified in the figure for 2-Cys Prx. Prx Q, and PrxII E function as well, in particular since their catalytic efficiency is even higher than that of 2-Cys Prx (Lamkemeyer et al., 2006; Table 1). Thioredoxin or other reductants reduce oxidized Prx and need to be regenerated themselves, for example by ferredoxin-dependent thioredoxin reductase (FTR).

Fig. 3

Activities of Trx-dependent peroxidase (Prx and possibly GPx) and ascorbate peroxidase in leaf and chloroplast extracts and temperature-dependent activity of PrxII E. Total leaf extracts were prepared from 5-week-old plants in 100 mM TRIS-HCl pH 7.4. Chloroplasts were isolated according to Lamkemeyer et al. (2006). The protein contents of the extracts were quantified spectrophotometrically by using the Bio-Rad Protein reagent. (A) Trx-dependent peroxidase: 80 mM TRIS-HCl, pH 7.4, 10 μM NADPH, 4 μM E. coli Trx a, 1.8 μM E. coli NTR, 340 μM H2O2, 50–100 μg protein. The reaction was started by addition of H2O2. Decrease in absorbance at 340 nm was recorded over a 1 min time period, εNADPH=6.22 mM−1 cm−1. No NADPH oxidation was observed without addition of Trx and NTR. (B) Ascorbate peroxidase: 80 mM TRIS-HCl, pH 7.4, 10 μM NADPH, 250 μM ascorbate, 500 μM H2O2, 50–100 μg protein. The reaction was started by addition of H2O2. The decrease in absorbance at 290 nm was recorded over a 1 min time period, εascorbate=2.8 mM−1 cm−1. (C) Temperature-dependency of PrxII E activity: PrxII E was incubated at the indicated temperatures for 20 min. Subsequently its activity towards H2O2 was monitored at 30 °C using the xylene orange test for quantification of the residual peroxide as described before (Collin et al., 2004). The activity was calculated from the slope between 20 s and 80 s.

Fig. 3

Activities of Trx-dependent peroxidase (Prx and possibly GPx) and ascorbate peroxidase in leaf and chloroplast extracts and temperature-dependent activity of PrxII E. Total leaf extracts were prepared from 5-week-old plants in 100 mM TRIS-HCl pH 7.4. Chloroplasts were isolated according to Lamkemeyer et al. (2006). The protein contents of the extracts were quantified spectrophotometrically by using the Bio-Rad Protein reagent. (A) Trx-dependent peroxidase: 80 mM TRIS-HCl, pH 7.4, 10 μM NADPH, 4 μM E. coli Trx a, 1.8 μM E. coli NTR, 340 μM H2O2, 50–100 μg protein. The reaction was started by addition of H2O2. Decrease in absorbance at 340 nm was recorded over a 1 min time period, εNADPH=6.22 mM−1 cm−1. No NADPH oxidation was observed without addition of Trx and NTR. (B) Ascorbate peroxidase: 80 mM TRIS-HCl, pH 7.4, 10 μM NADPH, 250 μM ascorbate, 500 μM H2O2, 50–100 μg protein. The reaction was started by addition of H2O2. The decrease in absorbance at 290 nm was recorded over a 1 min time period, εascorbate=2.8 mM−1 cm−1. (C) Temperature-dependency of PrxII E activity: PrxII E was incubated at the indicated temperatures for 20 min. Subsequently its activity towards H2O2 was monitored at 30 °C using the xylene orange test for quantification of the residual peroxide as described before (Collin et al., 2004). The activity was calculated from the slope between 20 s and 80 s.

DNA protective function of Prx in organelles

Two biochemical protection assays led to the initial identification of Prx, the assays for glutamine synthase activity and DNA integrity. Glutamine synthase is rapidly inactivated upon exposure to ROS while nucleic acids undergo strand breakage (Kim et al., 1988). The sensitive biomolecules of interest were exposed to the so-called mixed function oxidation assay with Fe2+, O2, and DTT. Glutamine synthase inactivation and DNA strand nicking occurred under control conditions while the presence of Prx proteins prevented damage development. This allowed yeast protein fractions to be screened for the presence of protector protein and was also used in plants later on (Stacy et al., 1996; Cheong et al., 1999; Haslekas et al., 2003). Subsequently, it was realized that DTT in the test solution regenerated oxidized Prx enabling the reaction cycle to go on (Rhee et al., 2005). Trx was identified as physiological electron donor to (some) Prx that were subsequently named thioredoxin peroxidase until the early 1990s, when peroxiredoxin was suggested as the proper denomination since not all Prx accept electrons from Trx (Rhee et al., 2005).

Recently, the mitochondrial PRDX 5 of man was also linked to DNA protection in vivo (Banmeyer et al., 2005). Transfected Chinese hamster ovary cells overexpressing or underexpressing PRDX 5 showed increased and decreased mitochondrial DNA degradation, respectively, upon exposure to methylviologen (MV). A similar function was investigated for plant Prx Q (Fig. 4). Leaf discs of wild-type and Prx Q KO plants were tested for intactness of plastid DNA following long-term 16 h exposure to 0.25 μM MV or for 32 h to 0.4 mM H2O2 under illumination with 150 μmol quanta m−2 s−1. DNA was extracted from the leaf tissue and analysed for the presence of intact plastome DNA employing long-run PCR to amplify a 11.4 kbp fragment of the plastome located between ndhF and rps15 (Fig. 4). While the DNA from wild-type plants showed similar signal strength in the controls and the MV- and the H2O2-treated samples, significantly less product was measured in the samples that were obtained from Prx Q knockout plants after oxidative stress (V Tognetti, K-J Dietz, unpublished results). The data reveal a genoprotective role of Prx Q in plants. This type of experiment needs to be extended to other members of the plant Prx family in order to distinguish specific and general functions of plant Prx, in particular, of the three plastid Prx isoforms.

Fig. 4

Genoprotective effect of the presence of wild-type levels of Prx Q in A. thaliana leaves. Longrun PCR amplifications were performed for the DNA region between ndhF and rps15 of the A. thaliana plastid genome. Leaf discs excised from leaves of 4-week-old WT and Prx Q knockout plants (Lamkemeyer et al., 2006) were exposed to either 0.25 μM MV for 16 h or to 0.4 mM H2O2 for 32 h at 150 μmol quanta m−2 s−1. Untreated discs were maintained as controls. Genomic DNA was extracted and PCR performed with primers located in the ndhF and rps15 genes. The products were visualized by ethidium bromide fluorescence after separation on 1% agarose gels. Similar results were seen repeatedly.

Fig. 4

Genoprotective effect of the presence of wild-type levels of Prx Q in A. thaliana leaves. Longrun PCR amplifications were performed for the DNA region between ndhF and rps15 of the A. thaliana plastid genome. Leaf discs excised from leaves of 4-week-old WT and Prx Q knockout plants (Lamkemeyer et al., 2006) were exposed to either 0.25 μM MV for 16 h or to 0.4 mM H2O2 for 32 h at 150 μmol quanta m−2 s−1. Untreated discs were maintained as controls. Genomic DNA was extracted and PCR performed with primers located in the ndhF and rps15 genes. The products were visualized by ethidium bromide fluorescence after separation on 1% agarose gels. Similar results were seen repeatedly.

Peroxiredoxins in the C4 plant maize

Prx function has rarely been studied in plants with special metabolic traits such as C4 plants. Recently, a comparative proteomics analysis of mesophyll and bundle sheath chloroplasts was performed in order to identify novel proteins needed to establish the functional difference between the two chloroplast types in C4 photosynthesis (Majeran et al., 2005). Both 2-Cys Prx and PrxII E were less abundant in the bundle sheath than in mesophyll chloroplasts with a ratio of 0.4 each. While C4 photosynthesis in mesophyll cells employs photosystems II and I and generates ROS, bundle sheath cells are believed to encounter less oxidative stress due to decreased activity of photosystem II and increased acceptor availability through the CO2 concentrating mechanism (Kingston-Smith and Foyer, 2000). The preferential expression of Prx in mesophyll cells is in line with an increased requirement for antioxidant capacity. Elucidation of Prx functions in a special metabolic context such as C4 plants is expected to facilitate our understanding of Prx beyond its role in antioxidant defence.

Prx in pathogen defence

In two recent investigations, Prx Q was associated with pathogen defence. Prx Q transcript levels increased upon infection of Populus trichocarpa leaves with Melampsora larici populina (Rouhier et al., 2004b). Gentiana triflora Gt-Prx Q was isolated in a screen for proteins with antifungal activity against Botrytis cinerea. The IC50 was 180–200 μg ml−1 corresponding to a concentration of about 10 μmol l−1 (Kiba et al., 2005). Gt-Prx Q exhibited a rather low Trx-dependent peroxidase activity of 1.9 μmol g−1 protein corresponding to a molar activity of 2 mol substrate reduced mol−1 Prx Q min−1. Leaves from tobacco overexpressing the Prx Q gene GtAFP1 showed slightly enhanced resistance to paraquat treatment and, upon Botrytis cinerea inoculation, lesions with decreased diameters. The protective effect was small compared with other antifungal proteins such as thionins and defensins (Kiba et al., 2005). Expression of GtAFP1 was induced by treatment with salicylic acid. The authors suggest that ROS detoxification suppresses disease development and that GtAFP1 might be useful to engineer enhanced disease resistance. The Prx Q effect on fungal infection needs to be quantitatively assessed in comparison with other defence genes in order to test this hypothesis. Interestingly, enhanced accumulation of Prx Q transcript was not seen in response to the incompatible interaction of Phytophthora infestans with A. thaliana (Fig. 6). Since Prx Q is localized to the plastids (Rouhier et al., 2004b; Lamkemeyer et al., 2006) it will be interesting to unravel the role of the chloroplast in pathogen defence. Increasing evidence suggests that pathogen and abiotic stress defence employ similar signalling routes including photoinhibitory ROS production.

Expressional regulation of organellar Prx

Prx gene expression is under the control of both developmental and environmental stimuli (Baier and Dietz, 1997; Horling et al., 2002, 2003; Kandlbinder et al., 2004). Expression of each of the Prx isoforms appears to respond specifically to overlapping and discrete signalling pathways. The best analysed prx gene so far is the 2-cys prx A of A. thaliana (Baier et al., 2004). The current model suggests that abscisic acid suppresses promoter activity. Oxidative stimuli may counteract ABA-mediated suppression at least partly through a mitogen-activated kinase pathway, while reducing conditions rapidly switch off promoter activity (Horling et al., 2001; Baier et al., 2004; Baier and Dietz, 2005). The four chloroplast prx genes tentatively responded similarly to light, dark, ascorbic acid treatment, and salt treatment (Horling et al., 2003), but they partly reacted in a distinct manner to nutrient deficiency, and peroxide and diamide treatments (Horling et al., 2003; Kandlbinder et al., 2004). Figure 5 shows the age-dependent alterations in transcript abundance as taken from publicly available array data. Generally, the highest signal intensities were seen for 2-cys prxA, followed by prxQ. 2-cys prxB intensity was similar to prxII E, while the least intensity was seen for mitochondrial prxII F. However, it should be noted that the relationship between transcript abundance and signal intensity also depends on DNA sequence properties. Transcripts of both organellar type-II prx remained constant until senescence, while both 2-cys prx isoforms and prxQ declined during the last step of ageing, probably correlated with chlorophyll loss. From this, prxII E and F appear to have a housekeeping antioxidant function in general cell metabolism, while the 2-cys prxA and B and prxQ appear to be correlated with photosynthetic activity. This has also been suggested previously from transcript regulation (Dietz et al., 2005) and biochemical data (Baier and Ditz, 1997; König et al., 2002; Lamkemeyer et al., 2006). Essentially, the same parallel regulation of the 2-cys prx A, B and prxQ transcripts is seen in response to biotic and abiotic stressors (Fig. 6), with some deviation under high light and hypoxia, where prxII E mRNA decreased. The most striking opposite regulation was seen for P. infestans inoculation where the plastid prx transcripts strongly declined while the mitochondrial prxII F increased, suggesting a role of PrxII F in protecting the mitochondrion during pathogen infection.

Fig. 5

Leaf age-dependent accumulation of organellar prx transcripts. Transcript abundances of the plastidic Prx 2-cys prxA, 2-cys prxB, prxQ, and prxII E and the mitochondrial prxII F in wild-type plants of different developmental stages were obtained from the microarray database and analysis toolbox ‘Genevestigator’ (Zimmermann et al., 2004, http://www.genevestigator.ethz.ch) that contains microarray hybridization results from more than 1800 experiments. Age groups were combined. Each data point represents hybridization results from at least 41 chips. The signal intensities during different stages of development were analysed with the ‘Gene Chronologer’.

Fig. 5

Leaf age-dependent accumulation of organellar prx transcripts. Transcript abundances of the plastidic Prx 2-cys prxA, 2-cys prxB, prxQ, and prxII E and the mitochondrial prxII F in wild-type plants of different developmental stages were obtained from the microarray database and analysis toolbox ‘Genevestigator’ (Zimmermann et al., 2004, http://www.genevestigator.ethz.ch) that contains microarray hybridization results from more than 1800 experiments. Age groups were combined. Each data point represents hybridization results from at least 41 chips. The signal intensities during different stages of development were analysed with the ‘Gene Chronologer’.

Fig. 6

Expression of organellar prx transcripts under various stress treatments. The data sets were derived from the microarray database and analysis toolbox ‘Genevestigator’ (Zimmermann et al., 2004, http://www.genevestigator.ethz.ch) (cf. Fig. 5). The signal intensities under stress treatment were extracted from the ‘Response Viewer’, the displayed ratios are calculated from the mean signal obtained from the treated samples divided by the mean signal intensities of the controls. The numbers of evaluated chips was as follows: inoculation with P. infestans, six; ozone, three; light intensity, six; hypoxia, two; salt stress, 12.

Fig. 6

Expression of organellar prx transcripts under various stress treatments. The data sets were derived from the microarray database and analysis toolbox ‘Genevestigator’ (Zimmermann et al., 2004, http://www.genevestigator.ethz.ch) (cf. Fig. 5). The signal intensities under stress treatment were extracted from the ‘Response Viewer’, the displayed ratios are calculated from the mean signal obtained from the treated samples divided by the mean signal intensities of the controls. The numbers of evaluated chips was as follows: inoculation with P. infestans, six; ozone, three; light intensity, six; hypoxia, two; salt stress, 12.

Towards understanding the function of three distinct Prx in chloroplasts

Three different types of Prx reside in the plastids of rice and A. thaliana. The degree of conservation and the distinct suborganellar location suggest specific roles in the antioxidant defence and signalling of plastids (Fig. 7A). PrxII E to a major extent is a soluble protein in the stroma and is likely to function as an enzymatic antioxidant to protect sensitive protein stromal targets or proteins associated with the inner envelope membrane from oxidation by peroxides escaping from the thylakoids or invading from the cytoplasm. Its endogenous electron donor still has to be identified since in vitro only the artificial electron donor DTT sustained PrxII E activity, while E. coli Trx, two putative chloroplast At-Grx, Cyp20-3, as well as glutathione were inactive (M Laxa, J König, KJ Dietz, A Kandlbinder, I Finkemeier, unpublished results). However, it should be noted that a variant of poplar GrxC4, where Ser substituted for the resolving Cys, covalently linked to A. thaliana PrxII E as well as the cytosolic PrxII B and the mitochondrial PrxII F, emphasizing that the catalytic interaction between PrxII E and Grx needs further attention (Rouhier et al., 2005).

Fig. 7

Suborganellar distribution (A) and possible signalling function of chloroplast Prx (B). (A) The three different types of Prx have specific properties and suborganellar distribution. While PrxII E is soluble in the stroma, Prx Q is mostly associated with the thylakoids, while 2-Cys Prx cycles between a soluble dimeric and thylakoid-associated oligomeric form. These distinct properties imply specific function in antioxidant defence and signalling. (B) Potential functions of Prx in chloroplast signalling. Prx reduces a broad range of peroxides. By modifying their level, Prx may indirectly affect the activity of peroxide-dependent signalling pathways. Oxidized Prx accepts electrons from a set of donors (Trx y1, Trx x, CDSP32, and a new form of thioredoxin reductase, NTRC) including cyclophilin Cyp20-3. Oxidation of Cyp20-3 affects rotamase activity and probably also binding properties as chaperone (M Laxa, J König, KJ Dietz, A Kandlbinder, unpublished results) and, thereby, could mediate signalling. Prx isoforms also interact with protein partners and could trigger or modulate signal pathways through that route. In fact Prx may act as redox sensor or signal molecule by itself (Dietz et al., 2003b, 2006).

Fig. 7

Suborganellar distribution (A) and possible signalling function of chloroplast Prx (B). (A) The three different types of Prx have specific properties and suborganellar distribution. While PrxII E is soluble in the stroma, Prx Q is mostly associated with the thylakoids, while 2-Cys Prx cycles between a soluble dimeric and thylakoid-associated oligomeric form. These distinct properties imply specific function in antioxidant defence and signalling. (B) Potential functions of Prx in chloroplast signalling. Prx reduces a broad range of peroxides. By modifying their level, Prx may indirectly affect the activity of peroxide-dependent signalling pathways. Oxidized Prx accepts electrons from a set of donors (Trx y1, Trx x, CDSP32, and a new form of thioredoxin reductase, NTRC) including cyclophilin Cyp20-3. Oxidation of Cyp20-3 affects rotamase activity and probably also binding properties as chaperone (M Laxa, J König, KJ Dietz, A Kandlbinder, unpublished results) and, thereby, could mediate signalling. Prx isoforms also interact with protein partners and could trigger or modulate signal pathways through that route. In fact Prx may act as redox sensor or signal molecule by itself (Dietz et al., 2003b, 2006).

By contrast to PrxII E, PrxQ is associated with thylakoids and was recovered in thylakoid membrane fractions enriched in photosystem II as well as a core photosystem II complex preparation (Lamkemeyer et al., 2006). The absence or presence of Prx Q in transgenic A. thaliana affected chlorophyll a fluorescence parameters suggesting a role in protecting photosynthesis. The increase in relative photosystem II fluorescence emission at 77 K observed in Prx Q knockout plants could be reversed in vitro when thylakoids of these plants were reconstituted with heterologously expressed Prx Q (Lamkemeyer et al., 2006). The authors speculate on a possible function of Prx Q in protecting photosystem II from H2O2 escaping from photosystem I. Reduction of oxidized Prx Q is achieved with Trx y1 and with a lower degree of efficiency with Trx x and Trx m4 (Collin et al., 2004).

The third chloroplast Prx belongs to the group of 2-Cys Prx and probably is the Prx that has been studied most intensely on the biochemical, physiological, as well as genetic level so far (Baier and Dietz, 1996, 1997; Baier et al., 2000). 2-Cys Prx exists in two conformational states, the reduced or overoxidized oligomer and the disulphide dimer (König et al., 2002, Bernier-Villamor et al., 2004). The oligomer forms a doughnut-like structure (Wood et al., 2002). In plants, the dimer is present in the stroma, while the decamer attaches to the thylakoid membrane (Fig. 7A) (König et al., 2002, 2003). The switch between two conformations and subsequent suborganellar re-localization were suggested to have a regulatory function and to mediate redox-dependent signalling (König et al., 2003; Dietz et al., 2006). 2-Cys Prx is efficiently regenerated by Trx x (Collin et al., 2003). 2-Cys Prx and Prx Q were identified as targets of another Trx-like protein, the chloroplast drought-induced stress protein CDSP32 (Broin et al., 2002). Potato plants lacking CDSP32 were exposed to MV and revealed increased chloroplast lipid peroxidation (Broin and Rey, 2003). Apparently, CDSP32 protected chloroplasts from oxidative damage. Surprisingly, photosystem II activity of potato plants overexpressing CDSP32 was more susceptible to oxidative stress upon exposure to MV (Rey et al., 2005). With <1 mol (mol−1 min−1) the peroxidatic activity of 2-Cys Prx in conjunction with CDSP32 is very low (Table 1). It could be proposed that the interaction between CDSP32 and 2-Cys Prx plays a role in redox-dependent regulation by association with targets or trapping of proteins and not by catalytic peroxide detoxification. A recently identified bifunctional chloroplast protein with Trx and NADPH thioredoxin reductase (NTR) domains (Serrato et al., 2004) was shown to regenerate 2-Cys Prx with much higher catalytic efficiency (Pérez-Ruiz et al., 2005).

2-Cys Prx also accepts electrons from Cyp20-3 (Bernier-Villamor et al., 2004; M Laxa, J König, KJ Dietz, A Kandlbinder, unpublished results). Cyp20-3 is a peptidyl-prolyl cis-trans isomerase which has four conserved Cys residues. In the oxidized state the peptidyl-prolyl cis-trans isomerase of Cyp20-3 is inhibited. Its activity is recovered upon reduction by Trx (Motohashi et al., 2003). The function of Cyp20-3 in the chloroplast remains elusive. Cyclophilins act as foldases and chaperones and have acknowledged functions in protein assembly and signalling (Min et al., 2005). The relative rates of Trx-dependent reduction and Prx-mediated oxidation will determine the oxidation state of the regulatory Cys residues, and thus the activity and biological function of Cyp20-3. It may be hypothesized that altered chaperone or foldase activity triggers changes in metabolism or signalling events (Fig. 7B). In this case, the 2-Cys Prx would act as a kinetic peroxide and redox sensor (Dietz, 2003b). Considering all these specific redox interactions, it may be allowable to propose that Prx are knot points in the redox regulation networks and antioxidant defence of chloroplasts.

Some recent evidence supports the view that chloroplast Prx proteins function in a specific metabolic context and signalling events. In 2000, Baier et al. quantified transcript levels of nuclear encoded genes involved in antioxidant synthesis and defence in transgenic A. thaliana plants with decreased 2-Cys Prx levels. Genes related to ascorbate metabolism were up-regulated while gene products involved in glutathione metabolism were unaltered (Baier et al., 2000). In a converse manner, Prx Q knockout plants alter transcripts of glutathione-related genes (Lamkemeyer et al., 2006). These results tentatively suggest that a lack of either 2-Cys Prx or Prx Q in chloroplasts induces distinct changes in metabolism and signalling, respectively, and are not redundant proteins. Apparently, distinct routes of retrograde signals from the chloroplast to the nucleus are activated (Baier and Dietz, 2005) that need to be further investigated, in direct comparison with Prx overexpressing and knockdown plants since growth conditions and plant developmental stages were not uniform in both experiments.

Prx function in mitochondrial metabolism

Mitochondria of all eukaryotic organisms appear to contain at least one Prx (Pedrajas et al., 2000; Wood et al., 2003; Banmeyer et al., 2005). In the case of higher plants, PrxII F is targeted to the mitochondrion. In vitro, PrxII F decomposed peroxides in the order of efficiency H2O2>t-butylhydroperoxide>cumenehydroperoxide>lipid peroxides (Finkemeier et al., 2005). Oxidized PrxII F was regenerated by glutaredoxin and with lower efficiency by glutathione (Finkemeier et al., 2005, Rouhier et al., 2005). Two A. thaliana T-DNA insertion lines without PrxII F protein were analysed in detail for alterations in biochemical properties and nuclear gene expression. Under regular growth conditions, no phenotypical differences to wild type were apparent. On the biochemical level, up-regulation of ascorbate- and glutathione-dependent peroxidase activity in the KO lines was suggestive of successful compensation for PrxII F deficiency. This conclusion is supported by increased accumulation of cytosolic prxII B and grx-isogene transcripts. However, root growth of PrxII F knockout plants was significantly more inhibited than the wild type in the presence of 7.5 μM Cd and 25 μM salicylhydroxamic acid, an inhibitor of alternative oxidase (Finkemeier et al., 2005). Both treatments impose oxidative stress on the cells. Under this stress condition, cDNA array analyses revealed altered regulation of transcripts related to redox metabolism. These data suggest that PrxII F is involved in cellular redox homeostasis under regular growth conditions. Its function can be compensated by metabolic and genetic adjustment. However, compensation is insufficient under stress, leading to severe growth defects.

Outlook

Despite 10 years of progress, Prx functions in plants are far from being understood comprehensively. Several lines of research may be suggested to elucidate further the role of Prx in antioxidant and redox metabolism as well as redox and NO signalling. (i) Detailed comparative analyses of transgenic plants with elevated or suppressed expression of single or multiple prx genes will elucidate redundancy and specificity of Prx proteins. As indicated by the available data and selectively summarized in this review, chloroplast Prx function in a specific redox context. These Prx functions will have to be differentiated from that of other antioxidant enzymes with similar activities such as Apx and GPx. To this end, both the biochemical disturbances and the alterations in nuclear gene expression in the mutants will provide meaningful readouts to identify sensitive targets and the signalling pathways involved. (ii) It has been established that Prx interact with various target proteins. It is unlikely that these screens were exhaustive. Most studies were restricted to dithiol–disulphide exchange reactions (Dietz, 2005). Novel insight is expected from identifying non-covalent binding partners. For instance the molecular basis of the thylakoid attachment needs to be understood in order to asses the site-specific function of 2-Cys Prx and Prx Q in photosynthesis. (iii) In addition to detoxifying peroxides, Prx may be considered as peroxide sensors transmitting the presence of peroxides to upstream redox proteins such as Trx and Cyp whose cellular activities may be altered. Transgenic plants expressing site-directed mutagenized variants of Prx and target proteins will allow such causal relationships to be addressed. (iv) Recent data suggest that Prx proteins modulate signalling pathways that involve ROS and reactive nitrogen species (RNS). ROS and RNS regulate developmental processes, adaptation to biotic and abiotic stress, and programmed cell death (Neill et al., 2002; Romero-Puertas et al., 2004). At least some Prx decompose peroxides and peroxinitrite. Furthermore, the activities and properties of some Prx are regulated by overoxidation and nitrosylation. Thus, Prx are likely to play an important role in modulating the ROS–RNS regulation networks and the intensity of the systems' response.

The work cited by the authors was supported by the DFG (FOR 387 TP3, Di 346/6-5+6).

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