Recent insights into antioxidant defenses of legume root nodules

Authors


Author for correspondence:
David A. Dalton
Tel: +1 503 5177473
Email: david.dalton@reed.edu

Abstract

Contents

 Summary960
I.Introduction961
II.Ascorbate961
III.Thiols963
IV.Ascorbate–glutathione pathway963
V.Superoxide dismutases and catalases964
VI.Thiol peroxidases: glutathione peroxidases  and peroxiredoxins966
VII.Protein disulfide reductases: thioredoxins  and glutaredoxins967
VIII.Glutathione S-transferases967
IX.Antioxidants and metal sequestration968
X.Antioxidants of nodule bacteroids969
XI.Other molecules with antioxidative properties in nodules969
XII.Antioxidants and oxidative/nitrosative signaling970
XIII.Antioxidants and oxidative/nitrosative stress971
XIV.Conclusions and perspectives972
 Acknowledgements973
 References973

Summary

Legume root nodules are sites of intense biochemical activity and consequently are at high risk of damage as a result of the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). These molecules can potentially give rise to oxidative and nitrosative damage but, when their concentrations are tightly controlled by antioxidant enzymes and metabolites, they also play positive roles as critical components of signal transduction cascades during nodule development and stress. Thus, recent advances in our understanding of ascorbate and (homo)glutathione biosynthesis in plants have opened up the possibility of enhancing N2 fixation through an increase of their concentrations in nodules. It is now evident that antioxidant proteins other than the ascorbate-glutathione enzymes, such as some isoforms of glutathione peroxidases, thioredoxins, peroxiredoxins, and glutathione S-transferases, are also critical for nodule activity. To avoid cellular damage, nodules are endowed with several mechanisms for sequestration of Fenton-active metals (nicotianamine, phytochelatins, and metallothioneins) and for controlling ROS/RNS bioactivity (hemoglobins). The use of ‘omic’ technologies has expanded the list of known antioxidants in plants and nodules that participate in ROS/RNS/antioxidant signaling networks, although aspects of developmental variation and subcellular localization of these networks remain to be elucidated. To this end, a critical point will be to define the transcriptional and post-transcriptional regulation of antioxidant proteins.

I. Introduction

Antioxidant defenses are indispensable to all aerobic life, but they are especially important for N2-fixing organisms, whether symbiotic (e.g. rhizobia in legume root nodules) or free-living (e.g. cyanobacteria). The reasons for this are not immediately obvious because the O2 sensitivity of nitrogenase mandates that a very low concentration of free O2 be maintained in the vicinity of the active enzyme. Nonetheless, several processes generate reactive oxygen species (ROS) in N2-fixing systems. ROS include the superoxide radicals and H2O2, which are produced by the high rates of respiration required to support N2 fixation, the autoxidation of the oxygenated form of leghemoglobin (Lb), and the oxidation of several proteins with strong reducing potential (e.g. nitrogenase, ferredoxin, and hydrogenase). Antioxidants in nodules include a host of enzymes and metabolites that function to eliminate ROS, generally by reducing them to less harmful forms and, in some cases, to water. However, when present at low, tightly controlled concentrations, ROS also perform useful functions in plant and nodule development and in stress perception and signaling (Section XII). As redox status is important in regulating root development, it is reasonable to expect that a similar regulation occurs in nodules, although the specifics are likely to differ substantially between these two organs (De Tullio et al., 2010). Consequently, antioxidants not only prevent cellular damage (‘oxidative stress’), but permit a fine tuning of ROS concentrations to optimize their functions in metabolism. Most of the antioxidants in legume nodules are also present in other plant organs or tissues, but the concentrations in nodules are generally higher, which suggests an important connection between N2 fixation and antioxidants.

Reactive nitrogen species (RNS), such as nitric oxide (NO) and peroxynitrite (ONOO), are also formed in nodules and other plant organs. However, much less is known about the in vivo sources of RNS compared with ROS. In fact, NO formation has been detected in the infected cells of functional nodules (Baudouin et al., 2006), but the origin of the NO is unclear. In plants, there are many potential sources of NO, both enzymatic, such as nitrate reductase and an NO synthase-like activity that still awaits identification, and nonenzymatic, such as the reduction of nitrite by ascorbate at acidic pH (for a review see Del Río et al., 2004). As with ROS, uncontrolled formation of RNS is potentially toxic and may cause cellular damage (‘nitrosative stress’), but low concentrations of RNS, especially of NO, are critical in many processes in plants, including seed germination, stomatal closure, root growth, nodule formation, and stress responses. Antioxidants can modulate RNS concentrations to avoid nitrosative stress while allowing RNS to function in plant development, metabolism, and signaling.

Oxidative challenges and defenses have been reviewed comprehensively elsewhere for both nodules (e.g. Matamoros et al., 2003; Puppo et al., 2005) and plants in general (Dalton, 1995; Noctor & Foyer, 1998; Mittler, 2002; Mittler et al., 2004). Readers are referred to those reviews for detailed background information. Here, we recapitulate briefly what is known about antioxidants in nodules and then provide an analysis of recent developments concerning the dual nature of ROS/RNS, namely, as potentially toxic and essential signaling molecules. Although rhizobia produce their own antioxidants which clearly contribute to nodule function (e.g. Muglia et al., 2008), the emphasis of this review is on the antioxidants of plant origin.

II. Ascorbate

Vitamin C (ascorbate) is a ubiquitous and abundant metabolite in plants. Ascorbate is present at concentrations of 1–2 mM in nodules (Dalton et al., 1986), 5–25 mM in leaves, and 25–50 mM in chloroplasts (Smirnoff, 2000), which is consistent with its multiple and essential functions. The steady-state concentrations of ascorbate are tightly controlled at many levels, including synthesis, degradation, transport, regeneration, and compartmentation. Ascorbate is a potent water-soluble antioxidant, acting both as a direct ROS scavenger and as a metabolite of the ascorbate-glutathione (GSH) pathway for H2O2 detoxification (Section IV), but it is also a cosubstrate of several dioxygenases involved in proline hydroxylation and in flavonoid and hormone biosynthesis (for a review see Arrigoni & De Tullio, 2002). Furthermore, the ascorbate redox state, defined as the ratio of reduced to total ascorbate (ascorbate + dehydroascorbate), affects the progression of the cell cycle (Potters et al., 2000) and is critical in the perception of stressful conditions in the apoplast (see later in this section). The essentiality of ascorbate in plants is also supported by the absence of known mutants that are completely deficient in ascorbate synthesis (De Tullio & Arrigoni, 2004).

The delay in our understanding of ascorbate physiology was largely a consequence of the difficulties in elucidating its biosynthetic pathway. A brief overview of the d-mannose/L-galactose (or Smirnoff–Wheeler) pathway is presented here as context for the nodule-related issues to follow (Table 1). Detailed reviews are available elsewhere (Ishikawa et al., 2006; Linster & Clarke, 2008). Conversion of mannose-1-P to ascorbate is a six-step process that is apparently present in virtually all plant cells (De Tullio & Arrigoni, 2004). Mannose-1-P is readily available via a one-step isomerization of fructose-6-P from glycolysis. A critical breakthrough was made possible by the identification of ascorbate-deficient mutants of Arabidopsis thaliana (Conklin et al., 2000). These mutants were named vtc (for vitamin C) and the underlying gene functions gradually identified over the next decade. VTC2, which codes for a GDP-l-galactose phosphorylase, was the last of the VTC genes to be assigned a function (Laing et al., 2007; Linster et al., 2007; Table 1). Elucidation of the complete d-mannose/l-galactose pathway has opened up the exciting prospect of using metabolic engineering to increase ascorbate production and, consequently, the capacity for N2 fixation.

Table 1.   Enzymes involved in the biosynthesis of ascorbate in plants via the Smirnoff–Wheeler pathway
Step numberSubstrate(s)Product(s)EnzymeAbbreviation
  1. 1Cytochrome c acts as an oxidant.

1D-fructose-6-PD-mannose-6-PPhosphomannose isomerasePMI
2D-mannose-6-PD-mannose-1-PPhosphomannose mutasePMM
3D-mannose-1-P, GTPGDP-D-mannose, PPiGDP-D-Man pyrophosphorylaseGMP/VTC1
4GDP-D-mannoseGDP-L-GalGDP-D-Man 3′,5′-epimeraseGME
5GDP-L-Gal, PiL-Gal-1-P, GDPGDP-L-Gal phosphorylaseVTC2/VTC5
6L-Gal-1-PL-Gal, PiL-Gal-1-P phosphataseVTC4
7L-Gal, NAD+L-galactono 1,4-lactone, NADHL-Gal dehydrogenaseGalDH
8L-galactono-1,4-lactone1L-ascorbateL-galactono-1,4-lactone dehydrogenaseGalLDH

Ascorbate may have also regulatory roles in nodules as a major contributor to the redox state of cells. Recently, Groten et al. (2005) hypothesized that pea (Pisum sativum) nodules are unable to synthesize ascorbate and have to import it from the shoot or root through the vascular system. They also observed that the leaves, and to a lesser extent the roots, accumulated ascorbate when supplied with galactose as a precursor. The capacity to accumulate ascorbate was retained in young nodules but was lost during development. This finding could imply that the plant might regulate key aspects of nodule metabolism through the transport of ascorbate from the shoot to the nodules (Groten et al., 2005; Puppo et al., 2005). However, l-galactono-1,4-lactone dehydrogenase (GalLDH) activity was subsequently found in mitochondrial membranes of bean (Phaseolus vulgaris) nodules (Matamoros et al., 2006), as previously reported in other plant systems (Siendones et al., 1999; Bartoli et al., 2000). The expression of five genes of the Smirnoff–Wheeler pathway was also detected in Lotus japonicus and bean nodules (Colebatch et al., 2002; Matamoros et al., 2006; Loscos et al., 2008), lending further support to the functionality of the ascorbate biosynthetic pathway in nodules. Recently, the transcript of GalLDH was localized in nodules of L. japonicus and alfalfa (Medicago sativa) by in situ RNA hybridization. High GalLDH expression (mRNA and activity) and ascorbate concentration were detected in the infected zone of both types of nodule (Matamoros et al., 2006). However, many key questions still remain to be answered. The characterization of genes and enzymes will be essential to understand how ascorbate synthesis is regulated in legumes. Further studies are also necessary to ascertain the functionality in nodules of alternative pathways of ascorbate biosynthesis, such as those described in animals or in ripening strawberry (Fragaria x ananassa) fruit (Valpuesta & Botella, 2004). These pathways involve the enzymes l-gulono-1,4-lactone dehydrogenase and d-galacturonate reductase, respectively, but their relative importance is uncertain because mutants affected in the corresponding genes are yet to be isolated. However, we could not detect d-galacturonate reductase protein in legume extracts using a polyclonal antibody raised against the strawberry enzyme.

The concentrations of ascorbate in cells are also regulated by the rates of oxidation and degradation. Many physiological roles of ascorbate imply its oxidation to monodehydroascorbate or dehydroascorbate. This occurs during peroxide removal by the ascorbate-GSH pathway in the cytosol, chloroplasts, and some other organelles (Section IV), but also in the apoplast as a result of ascorbate oxidase activity, which catalyzes the oxidation of ascorbate to monodehydroascorbate. In the apoplast, ascorbate is present at millimolar concentrations (up to 10% of total ascorbate in leaf cells is in the apoplast) and ascorbate oxidase activity controls the ascorbate redox state in such a way that this compartment becomes essential in the defense and stress response of plants to abiotic and biotic stresses (Pignocchi & Foyer, 2003). Understanding of the function of this enzyme has been clouded by the complexity of the regulation of ascorbate oxidase activity, which is responsive to numerous environmental and developmental cues (Pignocchi & Foyer, 2003; Pignocchi et al., 2003). Nevertheless, information on ascorbate oxidase in plants in general and in nodules in particular is scant. Interestingly, Loscos et al. (2008) found that treatment of bean plants with jasmonic acid, a well-known stress-related compound, caused transcriptional activation of ascorbate oxidase and post-translational inhibition of dehydroascorbate reductase in nodules. These authors proposed that the combination of the two effects would increase apoplast oxidation and that this may trigger a signal by which nodules perceive and respond to stress situations.

Ascorbate is usually regenerated from its oxidation products by monodehydroascorbate reductase and dehydroascorbate reductase, which are present in several cellular compartments (Section IV). However, dehydroascorbate is unstable and can be further oxidized and hydrolyzed to many compounds, including oxalic, tartaric, and threonic acids (Hancock & Viola, 2005), if it is not rapidly reduced back to ascorbate by dehydroascorbate reductase. The route for ascorbate degradation in plants is poorly known and therefore biochemical studies on the enzymes and metabolites involved are urgently needed, especially in leaves and nodules, which show high concentrations and rapid turnover of ascorbate.

III. Thiols

The thiol tripeptide GSH (γGlu-Cys-Gly) is a major water-soluble antioxidant and redox buffer in plants, performing critical functions in cell cycle regulation, development, sulfur transport and storage, stress responses, and heavy metal detoxification (Maughan & Foyer, 2006). In legumes, homoglutathione (hGSH; γGlu-Cys-βAla) may partially or completely replace GSH (Frendo et al., 2001; Matamoros et al., 2003).

The synthesis of GSH is accomplished in two sequential ATP-dependent reactions catalyzed by γ-glutamylcysteine synthetase (γECS) and glutathione synthetase (GSHS), whereas the synthesis of hGSH shares the same first enzyme and then requires a specific homoglutathione synthetase (hGSHS). The biochemical properties of the three thiol synthetases have been examined in several plants, but little is known about the regulation of the thiol biosynthetic pathway in legume roots and nodules. Interestingly, the hGSHS gene shows high sequence identity with the GSHS gene and probably derived from it by tandem duplication, at least in Medicago truncatula (Frendo et al., 2001) and L. japonicus (Matamoros et al., 2003). Despite this close relationship, the expression of the GSHS and hGSHS genes is strongly dependent on the legume species and tissue. For example, in M. truncatula, hGSHS can be detected in the roots and nodules and GSHS throughout the plant (Frendo et al., 1999), whereas in L. japonicus GSHS can be detected only in the nodules and hGSHS also in leaves and roots (Matamoros et al., 2003). The two genes are also differentially regulated in response to signaling compounds or stress conditions. In roots of M. truncatula, the expression of the γECS and GSHS genes, but not of the hGSHS gene, is induced by NO (Innocenti et al., 2007). Also, in roots of L. japonicus, GSHS was activated by auxins, cytokinins, and polyamines, whereas hGSHS remained unaffected (M. Becana, unpublished data). In nodules of bean plants treated with H2O2, γECS and hGSHS were up-regulated, whereas treatments with cadmium (Cd), sodium chloride, or jasmonic acid had no effect (Loscos et al., 2008). Collectively, these observations suggest the presence of gene-specific cis-regulatory elements in the GSHS promoter and/or distinct regulatory mechanisms for the GSHS and hGSHS genes, but, most importantly, they provide strong support for a different role of GSH and hGSH in plants and, particularly, in nodules. Recent studies in M. truncatula using the γECS inhibitor buthionine sulfoximine or antisense constructs of GSHS and hGSHS have shown that GSH and/or hGSH plays essential roles in nodulation; furthermore, the inhibition of nodule formation correlated with a decrease in the number of lateral roots, suggesting that thiol deficiency impairs meristem formation (Frendo et al., 2005). We propose that GSH rather than hGSH is specifically required to promote meristematic activity in nodules, based on this study and on several lines of indirect evidence: GSH is required for cell division in root tips (Vernoux et al., 2000); and GSH (and not hGSH) concentration is especially high in zones I+II of indeterminate nodules (Matamoros et al., 1999). Further research is clearly needed to identify the specific functions of GSH and hGSH in the development and stress responses of nodules.

IV. Ascorbate–glutathione pathway

The ascorbate-GSH or Halliwell–Asada pathway (Fig. 1) involves the participation of the enzymes ascorbate peroxidase (Apx), monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase in a coupled series of reactions that scavenge H2O2 by relying ultimately on the reducing power of NAD(P)H (Noctor & Foyer, 1998; Mittler et al., 2004). Isoforms of the four enzymes have been found in several cell compartments, including the cytosol, plastids, mitochondria, and peroxisomes (see Fig. 2 for relative mRNA levels of the corresponding genes), and therefore it is generally believed that the pathway is operative at multiple cellular sites. Microarray data indicate that the genes of the ascorbate-GSH pathway are expressed at high levels in nodules, as well as in other tissues (Fig. 2).

Figure 1.

 Generalized scheme showing processes for generation and removal of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in legume root nodules. ASC, ascorbate; Apx, ascorbate peroxidase; CAT, catalase; DHA, dehydroascorbate; DR, dehydroascorbate reductase; Gpx, glutathione peroxidase; GR, glutathione reductase; Grx, glutaredoxin; GSH/GSSG, reduced/oxidized glutathione; LOOH, lipid peroxide; MDHA, monodehydro-ascorbate; MR, monodehydroascorbate reductase; Prx, peroxiredoxins; SOD, superoxide dismutase; TF, transcription factor(s); Trx, thioredoxin.

Figure 2.

 Heat map showing microarray expression levels of genes related to antioxidant properties in the model legume Medicago truncatula. The color scale is based on a log2-transformed expression value obtained by robust multichip average (RMA). Numbers after the tissue designation indicate days after inoculation with rhizobia, except that Nod16* also received a high nitrate treatment at day 14. Details of methods and conditions can be found in Benedito et al. (2008) and The Medicago truncatula Gene Expression Atlas (http://mtgea.noble.org/v2/index.php). Apx, ascorbate peroxidase; DR, dehydroascorbate reductase; GalLDH, L-galactono-1,4-lactone dehydrogenase; γECS, γ-glutamylcysteine synthetase; GMP, GDP-D-mannose pyrophosphorylase; Gpx, glutathione peroxidase; GR, glutathione reductase; Grx, glutaredoxin; GSHS, glutathione synthetase; GST, glutathione S-transferase; hGSHS, homoglutathione synthetase; MR, monodehydroascorbate reductase; MT, metallothionein; NAS, nicotianamine synthase; Prx, peroxiredoxin; SOD, superoxide dismutase; Trx, thioredoxin.

The ascorbate-GSH pathway provides one of the chief antioxidant mechanisms in plants in general and was first described in nodules > 20 yr ago (Dalton et al., 1986). Since then, much evidence has been amassed demonstrating its importance for N2 fixation and the symbiotic association in general. For example, there is a close positive correlation between nodule effectiveness and the enzyme activities of the pathway (Dalton et al., 1993), and numerous parameters associated with N2 fixation and antioxidants in nodules are increased in response to an increase in the nodule ascorbate content (Table 2). Because most of this evidence goes back 5–15 yr, readers are referred to an earlier review for a comprehensive discussion (Matamoros et al., 2003). The pathway is certainly a major contributor to the antioxidant defenses in nodules but is not emphasized here because of space limitations and the goal of focusing on more emerging topics.

Table 2.   Response of N2-fixing systems to enhanced ascorbate
ParameterMaximum fold-change
Stem infusion of intact plantsExogenous application to intact plants/nodulesReconstitution system in vials (including ascorbate peroxidase)
  1. Sources: Bashor & Dalton (1999); Ross et al. (1999); Chinoy (1984); Groten et al. (2006).

  2. 1Increase, but no data given.

Nitrogenase activity4.12.35.0
Ascorbate peroxidase activity1.51.9
Lipid peroxides0.7
Leghemoglobin content1.3+1
Leghemoglobin oxygenation1.4
Nodule number2–3
Nodule weight6–12
Nodule N content3–5 
Plant total N content2 
Peroxiredoxin+1 

V. Superoxide dismutases and catalases

Superoxide dismutases (SODs) are metalloenzymes that catalyze the dismutation of superoxide radicals to H2O2 and O2. They are classified in three groups based on their metal cofactors: copper and zinc SOD (CuZnSOD), iron SOD (FeSOD), and manganese SOD (MnSOD). The three classes of enzyme occur in nodules, albeit at different subcellular locations: CuZnSOD in the cytosol, plastids, and (possibly) the periplasmic space of bacteroids; FeSOD in the cytosol, plastids, and some bacteroids; and MnSOD in bacteroids, mitochondria, and (possibly) peroxisomes. Microarray data indicate that several isoforms of CuZnSOD as well as mitochondrial MnSOD are highly expressed in nodules, whereas cytosolic FeSOD expression is considerably lower (Fig. 2).

The transcripts and proteins of some SOD isoforms have been localized and their expression patterns examined in indeterminate and determinate nodules. In the indeterminate nodules of alfalfa, the expression of mitochondrial MnSOD is highest in the infected zone, whereas that of cytosolic CuZnSOD is particularly abundant in the meristem and invasion zones, suggesting distinct roles of the enzymes during nodule development (Rubio et al., 2004). In particular, colocalization of H2O2 and studies with inhibitors of CuZnSOD activity supported a role of CuZnSOD in providing H2O2 for cross-linking of highly glycosylated glycoproteins (extensins) in the extracellular matrix and in the lumen of infection threads, which is required for cell wall growth and progression of infection threads (Wisniewski et al., 2000). In determinate nodules of L. japonicus, the expression of four SOD genes, encoding cytosolic CuZnSOD, mitochondrial MnSOD, plastidic FeSOD (FeSOD1), and cytosolic FeSOD (FeSOD2), was investigated (Rubio et al., 2007). The CuZnSOD and MnSOD genes were found to be down-regulated during nodule development, whereas FeSOD2 was induced and FeSOD1 transcription was not affected. It was proposed that the two cytosolic enzymes, CuZnSOD and FeSOD2, may functionally compensate each other at the late stages of nodule development. The induction of FeSOD2 suggests a higher availability of Fe in old nodules, probably as a result of Lb degradation.

Catalases have been studied extensively in many plants where the different isoforms and genes have been characterized (see review by Scandalios et al., 1997). They are tetrameric hemoproteins (240 kDa) that catalyze the decomposition of H2O2 to O2 and water, and are mainly localized in peroxisomes and glyoxysomes. Because of the low affinity of catalases for H2O2 (Km in the molar range) compared with Apx (Km in the micromolar range), it is believed that they are only efficient at high concentrations of H2O2 and are essential for maintaining the redox balance during oxidative stress (Willekens et al., 1997). In white lupin (Lupinus albus) nodules, catalase has been immunolocalized in the peroxisomes of infected cells and found to decrease during senescence induced by nitrate (De Lorenzo et al., 1990). It is surprising that since this pioneering study almost no progress has been made in our understanding of catalases from nodule host cells. Instead, the regulation of the catalase genes of bacteroids has been examined in detail (Section X).

VI. Thiol peroxidases: glutathione peroxidases and peroxiredoxins

Thiol peroxidases include two groups of closely related enzymes, peroxiredoxins (Prxs) and glutathione peroxidases (Gpxs), that are widespread in many organisms. Both Prxs and Gpxs are small proteins (17–24 kDa) that lack heme and hence rely on external electron donors for catalytic activity. They are encoded by multigene families and the corresponding isoforms are located at multiple subcellular locations, including the cytosol, plastids, and mitochondria. Although expression levels for these genes are generally higher in leaves than in nodules, the levels in nodules are still considerable (Fig. 2).

Prxs catalyze the reduction of H2O2 or alkyl hydroperoxides (ROOH) to water or the corresponding alcohols (ROH), respectively, using preferentially thioredoxin (Trx) as an electron donor:

image

In plants there are four classes of Prxs, designated 1C-Prx, 2C-Prx, PrxQ, and PrxII, based on the number of catalytic cysteine residues and amino acid sequences (Dietz, 2003). Essentially, the reaction of the Prxs containing two catalytic cysteines (2C-Prx, PrxQ, and PrxII) is as follows. A sulfhydryl group is oxidized by the peroxide to sulfenic acid with the release of H2O, then a second sulfhydryl group attacks the sulfenic acid group forming a disulfide bridge, and finally this is reduced again to thiol groups by Trxs or alternative thiol active proteins such as glutaredoxin (Grx) or cyclophilin. Although well studied in A. thaliana, Prxs have only recently been described in N2-fixing nodules (Groten et al., 2006). Pea nodules contain at least two isoforms of Prx, located presumably in the cytosol (PrxIIB/C) and mitochondria (PrxIIF). The levels of PrxIIB/C increased with application of exogenous ascorbate and declined with nodule senescence, but those of PrxIIF remained unaffected under both conditions (Groten et al., 2006). Studies by these authors and in our laboratory failed to detect significant protein levels of the plastidic (2C-Prx and PrxQ) or nuclear (1C-Prx) isoforms in legume nodules.

The reaction catalyzed by Gpxs is usually described in the same way as that of Prxs but with GSH instead of a thiol protein as the reductant of peroxides. However, recent studies have shown that Gpxs use Trxs more efficiently, and in some cases exclusively, as electron donors (Herbette et al., 2002). Consequently, Gpxs are more appropriately designated ‘Trx peroxidases’ (Rouhier & Jacquot, 2005) and are considered a fifth class of Prxs (Navrot et al., 2006). An important difference between Gpxs and Prxs is that some Gpxs are able to reduce fatty acid and lipid hydroperoxides (but not H2O2) using GSH (Herbette et al., 2002), and this function is relevant in vivo because these enzymes protect membrane lipids from ROS-induced peroxidation.

Phylogenetic analysis of Gpxs has revealed that there are five distinct classes in vascular plants. Each of these classes is present in L. japonicus, the only N2-fixing symbiosis that has been examined in this regard (Ramos et al., 2009). Two genes, LjGpx3 and LjGpx6, which putatively encode proteins located in the cytosol or secretory pathway and in the plastids, respectively, are highly expressed in nodules. One of them, LjGpx6, was highly induced by treatment of plants with the NO-releasing compound sodium nitroprusside, suggesting that NO can modulate the function of Gpxs and that these enzymes may be, in turn, mediating the effects of NO in metabolic signaling pathways. Surprisingly, immuno-gold studies showed that at least some Gpx isoforms are associated primarily with chloroplasts, proplastids, or amyloplasts in leaves, roots, or nodules. Furthermore, the enzyme was found to be associated with starch grains (Ramos et al., 2009), a localization consistent with that reported previously for certain Trx and Prx isoforms (Balmer et al., 2006; Barajas-López et al., 2007). Because Trxs are substrates of both Gpxs and Prxs, the finding of Gpx associated with starch grains in amyloplasts suggests that H2O2 or other peroxides are formed during starch metabolism and that Gpxs may act not only as peroxide scavengers but also as signaling molecules. In fact, a dual role of the A. thaliana Gpx3 isoform, as a general ROS scavenger and specifically as an oxidative transducer in abscisic acid and drought stress signaling, has been recently demonstrated (Miao et al., 2006).

VII. Protein disulfide reductases: thioredoxins and glutaredoxins

Trxs are a family of ubiquitous small proteins (12–14 kDa) involved in redox regulation (Meyer et al., 2005). They contain a conserved reactive site (Trp-Cys-Gly-Pro-Cys) which is able to reduce disulfide bridges in target proteins. After oxidation of the thiol groups, chloroplastic and cytosolic Trxs are regenerated by ferredoxin-Trx reductase and NADPH-Trx reductase, respectively. In plants, Trxs are classified in six classes according to their sequences and localizations in the chloroplasts (m, f, x, and y), cytosol or phloem sap (h), and mitochondria (o).

The antioxidant roles of Trxs may be largely indirect as they primarily function through redox regulation of other proteins. The antioxidant properties of Trxs are not clearly understood, but it is likely that Trxs can repair other proteins that have been damaged by ROS (Vieira Dos Santos & Rey, 2006). Some of the strongest evidence supporting an antioxidant role for Trx is that transformation of a Trx-deficient mutant of yeast with the soybean (Glycine max) Trx gene confers tolerance to exogenous H2O2 (Lee et al., 2005). This gene appears to be required for nodulation in soybean as RNAi repression leads to severely impaired nodule development, and its expression in nodules increases during nodule formation and is at its highest in the central infected zone of mature nodules (Lee et al., 2005). Furthermore, two novel Trx isoforms have been found in M. truncatula and have been designated ‘s’ for ‘symbiosis’, as they function specifically in symbiotic interactions (Alkhalfioui et al., 2008). Collectively, these observations indicate that Trx is an antioxidant that is essential for proper nodule development and function.

Grxs are also small proteins closely related to Trxs which are encoded by multigene families. They are present in the same tissue and cellular locations as Trxs, including the phloem sap, reinforcing the view that they have overlapping functions (Meyer et al., 1999). Grxs participate in oxidative stress protection in several ways. They directly reduce peroxides and regenerate ascorbate from dehydroascorbate, act as electron donors for some Prxs, and are involved in the protection of thiol groups through glutathionylation/deglutathionylation reactions. Therefore, Grxs may have an important role as redox regulators in plant tissues (Rouhier et al., 2006). However, virtually nothing is known about the presence of Grxs in nodules.

VIII. Glutathione S-transferases

Glutathione S-transferases (GSTs) are ubiquitous enzymes best known for their role in detoxifying xenobiotics, especially herbicides such as atrazine. They accomplish this by conjugating the target molecules to (h)GSH, thus facilitating their metabolism, sequestration, or removal (Edwards et al., 2000). However, GSTs may also act as antioxidants via at least two mechanisms. First, GSTs may act as a Gpx to directly scavenge peroxides. Secondly, lipid peroxidation end products such as alkenals, 4-hydroxynonenal, and other α,β-unsaturated aldehydes may be conjugated to GSH and targeted for removal (Edwards et al., 2000; Dalton et al., 2009).

GSTs constitute a large gene family in plants, with 25 members in soybean and 42 in maize (Zea mays) (McGonigle et al., 2000). The importance of GSTs in N2-fixing nodules is indicated by the observation that soybean nodules contain at least 14 isoforms of GSTs with variable, though substantial, levels of expression (Dalton et al., 2009; Fig. 2). Down-regulation by RNAi technology of the most prevalent form (GST9) results in substantial decreases in nitrogenase (acetylene reduction) activity (Dalton et al., 2009). The GST-suppressed nodules also showed increased oxidative damage of proteins. Furthermore, there was a marked organ specificity for GSTs in soybean as the relative abundance of isoforms is different in nodules compared with uninfected roots or leaves. The abundance of GST9 transcript was similar to that of Apx, one of the most abundant proteins in nodules.

Elucidation of the role of GSTs in nodules is complicated not only by the abundance of different isoforms, but also by the fact that the normal target molecules have not been specifically identified and are likely to be equally diverse. Another factor to consider is that the host cells of nodules of some legume species, such soybean and bean, contain hGSH, which partially or completely replaces GSH, thus adding a further level of complexity. For instance, for some substrates, hGSH is conjugated more readily than GSH (McGonigle et al., 1998).

IX. Antioxidants and metal sequestration

Metal homeostasis is central in nodules because they contain abundant metalloproteins essential for N2 fixation or ROS protection. However, the Fe or Cu of their prosthetic groups or cofactors can be released by proteases during senescence or under stress conditions. These metals are potentially pro-oxidants at trace (‘catalytic’) amounts, giving rise to highly oxidizing hydroxyl radicals (˙OH) according to Fenton chemistry:

image

Therefore, a strict control of the intracellular concentrations of Fe and Cu is critical to avoid oxidative damage (Halliwell & Gutteridge, 2007). Protection against metal-promoted toxicity is largely based on mechanisms to remove metals by sequestration into storage proteins or by chelation to specific polypeptides or metabolites. Both mechanisms operate in nodules and will be now briefly described.

Ferritin is a spherical protein complex of 24 subunits, capable of concentrating and storing up to 4500 atoms of Fe in the form of hydrated ferric oxide in a large central compartment (reviewed by Liu & Theil, 2005). Not only is such stored Fe Fenton inactive, but the creation of the oxide also results in removal of O2, further enhancing the antioxidant properties. Ferritin plays a critical role in nodules because of the high Fe requirement and the associated high risk of oxidative damage. Ferritin is localized primarily in the plastids and amyloplasts of nodules, as well as in the bacteroids, and is associated with effectiveness (Ko et al., 1987; Lucas et al., 1998). Ferritin mRNA and protein increase markedly early in maturation of soybean nodules, at the same stage that Lb synthesis starts (Ragland & Theil, 1993). Immunolabeling studies showed that ferritin decreased in the infected cells of senescing soybean and white lupin nodules and was also lower in the senescent zone of alfalfa nodules (Lucas et al., 1998). Subsequent work by Strozycki et al. (2007) showed that, in mature nodules of yellow lupin (Lupinus luteus), ferritin polypeptides accumulate in a layer of cells between the meristem and the bacteroid tissue, which is reminiscent of the interzone II/III of typical indeterminate nodules. These authors also found that ferritin expression is correlated with development of yellow lupin nodules, suggesting that this protein is transcriptionally regulated and takes part in a mechanism by which nodule function is prolonged in indeterminate nodules (Strozycki et al., 2007).

Another protective mechanism against metal toxicity is chelation by cysteine-rich polypeptides or proteins, which include two major groups: phytochelatins (PCs) and metallothioneins (MTs). Both of these are present in nodules. PCs have a general structure (γGlu-Cys)2-11-Gly and are synthesized from GSH by phytochelatin synthase (Cobbett & Goldsbrough, 2002) according to the reaction:

image

In some legumes hGSH can replace GSH, producing homophytochelatins (hPCs) of general structure (γGlu-Cys)2-11-βAla (Grill et al., 1986; Klapheck et al., 1995). However, phytochelatin synthase has a higher specificity for GSH than for hGSH. Both types of polypeptides, PCs and hPCs, are able to chelate certain metals (Cu, Zn, Cd, mercury, and lead) and metalloids (arsenic). The resulting complexes are transported into the vacuoles, avoiding cellular toxicity. Ramos et al. (2007) found that exposure of L. japonicus plants to Cd caused accumulation of PCs and hPCs in roots and nodules. This and a follow-up study (Ramos et al., 2008) revealed that L. japonicus contain three functional phytochelatin synthase genes that are differentially regulated in response to metals and have different abundances in roots and nodules. However, phyto-chelatin synthases also fulfill other functions. In A. thaliana, PC synthesis is required for the degradation of glutathione conjugates (Blum et al., 2007) and for the homeostasis of Zn (Tennstedt et al., 2009). In addition to the role of PCs in avoiding the pro-oxidant effects of metals, the high thiol content of these polypeptides suggests that they may interact with ROS/RNS and, in fact, nitrosylated PCs have been recently detected in vivo (De Michele et al., 2009). The possibility that these PC nitrosothiols modulate NO concentrations awaits detailed investigation.

Plants also contain small proteins (1–2 kDa), called MTs, that chelate metals and protect cells against oxidative stress. MTs are encoded by large families of closely related genes and this complexity has precluded in-depth studies of their function (Cobbett & Goldsbrough, 2002). In yeast and mammals, MTs are involved in the homeostasis of essential metals (Cu and Zn) and the detoxification of heavy metals (Cd). There is also evidence supporting a role of MTs in Cu homeostasis and tolerance in plants. However, an additional feature of MTs is their ability to efficiently scavenge ROS, including superoxide and hydroxyl radicals (Kumari et al., 1998; Wong et al., 2004). In this respect, it is worth noting that Clement et al. (2008) identified two genes, encoding ferritin and MT, that were markedly up-regulated in soybean nodules in response to drought, a common cause of oxidative stress. The mRNA levels of both genes were particularly high in the infected cells. This finding, which was somewhat expected in the case of ferritin, also lends further indirect support to an antioxidant role of MTs in nodules through chelating potentially Fenton-active Cu, through directly scavenging ROS, or via both mechanisms.

Another important metal chelator in plants is nicotianamine. This compound has a high binding affinity for Fe2+ and forms complexes that are poor Fenton reagents, which supports a role of nicotianamine in protecting cells from oxidative damage (von Wirén et al., 1999). Nicotianamine is synthesized by nicotianamine synthase (NAS) in a one-step reaction with three molecules of S-adenosyl-l-methionine as the sole substrate. Very little is known about nicotianamine or NAS in nodules beyond the recent, single report that there are two forms of NAS in nodules of L. japonicus (Hakoyama et al., 2009). The gene encoding one of these (LjNAS2) was specifically expressed in nodules, whereas that encoding the other form (LjNAS1) was expressed mainly in leaves, stems, and cotyledons. Expression of LjNAS2 in nodules was highest 24 d after inoculation with rhizobia. A mutant deficient in LjNAS2 formed ineffective nodules. Although an antioxidant role for nicotianamine is still plausible, the observation that LjNAS2 mRNA was detected only in vascular bundles suggests that Fe transport may account for the observed phenotype of this mutant. Microarray data indicate that at least one form of NAS (and of MT) is highly expressed in nodules (Fig. 2).

X. Antioxidants of nodule bacteroids

The antioxidants discussed up to this point are all of plant origin, but bacteroids also contain metabolites (GSH) and enzymes (MnSOD, catalase, Prx, and glutathione reductase) that fulfill antioxidant roles and may be involved in redox regulation. These functions are beyond the scope of this review, but a few examples deserve mention, especially to the extent that they influence plant responses or processes. Although nodule host cells make their own GSH, some of this critical antioxidant needs to be produced by the bacterial partner to achieve optimal N2 fixation, as evidenced by the observation that rhizobia deficient in GSHS formed nodules with early senescence and diminished symbiotic performance (Harrison et al., 2005; Muglia et al., 2008). Catalase is another interesting example of how alterations of antioxidant enzymes of bacteroids can dramatically affect N2 fixation. In Sinorhizobium (Ensifer) meliloti there are three catalase genes encoding two monofunctional (KatA and KatC) and one bifunctional (KatB) catalase-peroxidase enzyme (Jamet et al., 2003). KatA is inducible by H2O2 and is constitutively expressed in bacteria and bacteroids, whereas KatB are KatC are expressed in bacteria within the infection threads. The single katA or katC mutants nodulate normally, but the katAkatC or katBkatC double mutants produce nodules with a drastic reduction in N2-fixing activity. The katAkatB double mutant is not viable. Therefore, the three catalases are required for symbiosis, although possibly at different stages of infection and nodule development (Sigaud et al., 1999; Jamet et al., 2003). However, this situation may be different in other rhizobial species such as Rhizobium etli, in which only one catalase gene, katG, encoding a dual catalase-peroxidase, is detectable (Vargas et al., 2003).

Another case in which bacterial antioxidants can be manipulated deserves attention. The overexpression in bacteroids of flavodoxin, an antioxidant that is not normally present in either rhizobia or plants, was found to delay senescence of M. truncatula nodules, using as markers the decline in N2-fixing activity and the structural alteration of nodule components (Redondo et al., 2009). In this case, flavodoxin may promote a favorable redox balance or perhaps even detoxify ROS. A follow-up study demonstrated that the flavodoxin-expressing bacteroids even ameliorated Cd-induced damage in alfalfa nodules (Shvaleva et al., 2010).

XI. Other molecules with antioxidative properties in nodules

Nodules contain other metabolites and enzymes that can obliterate ROS/RNS, or modulate their concentrations, at least when assayed in vitro. However, the biological significance of these molecules in vivo requires further investigation. Two examples are uric acid, an abundant metabolite of nodules of warm-season legumes (e.g. soybean) and an efficient scavenger of peroxynitrite, and liposoluble antioxidants such as tocopherols, ubiquinol, or flavonoids, which protect membrane fatty acids from peroxidation. None of these compounds has been studied in nodules in connection with ROS/RNS metabolism. More information is available, although still clearly insufficient, with respect to other molecules of nodules with antioxidative properties. We will briefly describe some of them because of their considerable interest for future studies. These metabolites or enzymes can also be considered as ‘antioxidants’ in broad terms because of their abilities to modulate ROS/RNS concentrations, and include polyamines, heme oxygenase, and hemoglobins (Hbs). Because of their important roles in signaling, Hbs will be described in the next section.

Polyamines are polycationic compounds widespread in many organisms and particularly in plants, where they play as yet poorly defined roles in developmental processes and stress responses (Bouchereau et al., 1999). Legume nodules accumulate polyamines to concentrations that are 5- to 10-fold higher than those in the roots or leaves (Fujihara et al., 1994). In nodules of L. japonicus, the expression of genes involved in the synthesis of spermidine, spermine, and putrescine is induced early in nodule development and declines with aging, whereas polyamines accumulate steadily during nodule maturation, suggesting that they are involved in nodule cell division and expansion, but also in other functions related to N2 fixation (Flemetakis et al., 2004; Efrose et al., 2008). Exogenous addition of polyamines delays senescence (Lahiri et al., 1992) and this effect may be ascribed at least in part to their ROS-scavenging properties (Bors et al., 1989; Bouchereau et al., 1999). In addition, polyamines can give rise to H2O2 as substrates of diamine and polyamine oxidases (see next section) and there is strong evidence that they are also precursors of NO (Yamasaki & Cohen, 2006), further suggesting an important role of these compounds in ROS/RNS metabolism.

Heme oxygenase catalyzes the breakdown of heme according to the following reaction:

image

Although the release of free Fe3+ could have pro-oxidant consequences, the process is considered to provide substantial defense against ROS as a result of the antioxidant properties of biliverdin (Ryter & Tyrrell, 2000; Yannarelli et al., 2006). Once heme oxygenase opens up the porphyrin ring, biliverdin is reduced to bilirubin by a NADPH-dependent biliverdin reductase. This produces bilirubin, an antioxidant that scavenges ROS with the concomitant regeneration of biliverdin. The reductase then functions to regenerate bilirubin in a continuing cycle that protects cells from up to a 10 000-fold excess of H2O2. The operation of this cycle in nodules is still speculative because only the first enzyme, heme oxygenase, has been reported. Expression of the heme oxygenase 1 (HO1) gene was enhanced in nodules in comparison to leaves and uninfected roots, and was highest in mature nodules (Baudouin et al., 2004). In contrast to the situation in mammals, pro-oxidants such as H2O2 and paraquat did not induce expression, an observation that suggests that heme oxygenase is not involved in antioxidant protection in nodules. By contrast, more recent studies have shown that, under oxidative conditions induced by Cd (Balestrasse et al., 2005) or salt (Zilli et al., 2008) stress, there is a marked increase in heme oxygenase expression (mRNA and protein) in nodules, providing credence to an antioxidative role. Furthermore, both UV irradiation and application of exogenous H2O2 caused oxidative damage and up-regulation of heme oxygenase in soybean leaves (Yannarelli et al., 2006). These treatments also increased Apx and catalase activities, making it tempting to include heme oxygenase in the list of antioxidants. The second enzyme of the heme degradation pathway, biliverdin reductase, has been found in A. thaliana (Gisk et al., 2010). Its presence in nodules has not been reported, but seems likely because of the high heme content of those organs. The fact that heme oxygenase is encoded by a small gene family, with four putative members in A. thaliana, argues further that the reactions of heme degradation have an importance in plant physiology that has not previously been appreciated (Gisk et al., 2010).

XII. Antioxidants and oxidative/nitrosative signaling

In plants and other organisms, antioxidants prevent the potentially deleterious effects of ROS (‘oxidative stress’) and RNS (‘nitrosative stress’). However, these reactive molecules also perform critical functions at low controlled concentrations by acting in certain cellular locations, developmental stages, or stressful conditions. Antioxidants are able to modulate ROS/RNS concentrations and thereby are likely to affect signaling transduction cascades. This has led to the concept of ‘oxidative signaling’, which emphasizes the multiple useful roles of ROS in plants, especially in redox signaling (Foyer & Noctor, 2005). This concept is very appropriate in the light of several facts: some ROS are second messengers implicated in signaling pathways that are activated in the plants in response to developmental and environmental cues; ROS can modify gene expression in an ROS-specific manner; and the production of ROS is, in many cases, genetically programmed. This can be exemplified in the nodulation process. In the early stages of infection, superoxide radicals and H2O2 are produced by the root cells in response to rhizobia, which suggests that the symbiotic bacteria are initially perceived as invaders (Santos et al., 2001). Furthermore, H2O2 accumulation has been detected in the invasion zone of alfalfa and pea nodules, in association with infection threads (Santos et al., 2001; Rubio et al., 2004). This H2O2 is required for inter- and intra-molecular cross-linking of extensins (Section V) and may be produced by CuZnSOD activity (Rubio et al., 2004), diamine oxidase activity using putrescine as a substrate (Wisniewski et al., 2000), and/or a germin-like protein with SOD activity (Gucciardo et al., 2007). The concentration of H2O2 in the infection threads may be also modulated by the catalase activity of bacteroids, as shown by experiments with S. meliloti mutants overexpressing KatB (Jamet et al., 2007). Collectively, these data indicate that controlled ROS production is essential for the onset of symbiosis. However, how the plant’s defense response is suppressed is not completely clear. Rhizobial mutant strains defective in exopolysaccharides, lipopolysaccharides, or cyclic β-glucans are unable to infect root cells and activate defense reactions, which is strong evidence for a signaling role of these complex carbohydrates during the symbiotic interaction (see review by Mithöfer, 2002). Similar experiments with incompatible rhizobia or with S. meliloti nodC mutants have shown that Nod factors are implicated in suppressing the plant’s defense response (Bueno et al., 2001). Also, application of compatible Nod factors to M. truncatula slowed the rate of H2O2 efflux from excised root segments (Shaw & Long, 2003), and similar studies in bean showed a transient increase of ROS, within seconds, at the tip of actively growing root hair cells (Cárdenas et al., 2008).

Redox signaling can be also mediated by RNS, for example, via post-translational modification of antioxidant proteins or transcription factors. Thus, RNS can cause nitrosylation (addition of an NO group) or nitration (addition of an NO2 group) of cysteine or tyrosine residues, respectively. For example, in nodules of M. truncatula, NO has been shown to activate two genes encoding proteins involved in H2O2 metabolism (a peroxidase and a germin-like oxalate oxidase), suggesting a cross-talk between ROS and RNS signaling (Ferrarini et al., 2008). In this context, it would then appear logical to extend the concept of ‘oxidative signaling’ to the participation of RNS (‘nitrosative signaling’) in signal transduction pathways.

An important case of modulation and signaling by NO and other RNS is closely related to the function of some Hbs. Three types are known and may coexist in plants: nonsymbiotic, symbiotic, and truncated Hbs. The first group is classified, in turn, into class 1 Hbs (with very high O2 affinity) and class 2 Hbs (with lower O2 affinity and a primary sequence more similar to those of symbiotic Hbs). Class 1 Hbs are expressed under hypoxia, cold, and osmotic stress, upon treatment with NO, and during rhizobial infection. In hypoxic conditions, these Hbs are part of an NO dioxygenase system, converting NO to nitrate. This system consumes NAD(P)H and mantains ATP concentrations, allowing plant survival (Igamberdiev & Hill, 2004). In L. japonicus, a class 1 Hb controls the plant’s defense response during the early stages of the rhizobial interaction, by modulating NO concentration, and overexpression of this protein enhances symbiotic N2 fixation (Shimoda et al., 2009). By contrast, very little is known about class 2 and truncated Hbs, albeit recent data suggest that at least some of them can also modulate NO concentrations and are expressed in nodules (Vieweg et al., 2005).

Symbiotic Hbs include Lbs and Hbs from some actinorhizal plants. In addition to the role of Lbs in facilitating O2 diffusion to symbiosomes, these abundant proteins can form complexes with NO and thus modulate NO bioactivity. The nitrosyl complexes (LbNO) are very stable and can be detected in intact nodules by electron paramagnetic resonance (Mathieu et al., 1998; Meakin et al., 2007). The NO bound to Lb may have originated in the host cells (Baudouin et al., 2006), in the bacteroids (Meakin et al., 2007), or in both nodule compartments. It can be argued that the presence of LbNO, decreasing O2 buffering in the cytoplasm, is potentially detrimental to nitrogenase. However, LbNO complexes are most abundant at the early stages of nodule development, which suggests a beneficial role of Lb as an NO reservoir or as part of a mechanism to detoxify RNS or prevent rejection of symbiotic rhizobia. This hypothesis is supported by the enhanced expression of Lb before active N2 fixation. Recent in vitro experiments have demonstrated that ferrous Lb (in the oxygenated form) can scavenge NO and peroxynitrite, and also that these RNS can reduce ferryl-Lb, an inactive form produced by oxidation of Lb with H2O2 (Herold & Puppo, 2005). Taken together, these observations suggest that nonsymbiotic Hbs and Lbs are involved in metabolism, transport, and signaling by RNS.

Apart from their funtion in controlling ROS/RNS concentration, antioxidants themselves may act as signals, as can be illustrated with two examples. Studies with A. thaliana mutants with ascorbate deficiency (vtc1) have shown that ascorbate influences plant growth and development by modulating expression of genes involved in defense and abscisic acid signaling (Pastori et al., 2003). Another major case of a signaling function for antioxidants follows from studies with animal systems and points to Prxs as components of redox signaling cascades in plants. In A. thaliana, nitrosylation of a specific cysteine residue of PrxIIE inhibits the capacity of the enzyme to detoxify peroxynitrite (Romero-Puertas et al., 2007). This post-translational modification of PrxIIE causes a dramatic increase in nitrotyrosine formation, modulating tyrosine kinase signaling pathways, and is biologically relevant. Although similar information does not exist in legume nodules, the involvement of redox signaling by Prxs in the first steps of symbiosis and in nodule operation could be anticipated.

XIII. Antioxidants and oxidative/nitrosative stress

The findings mentioned above clearly illustrate that ROS/RNS are produced in plants, and particularly in nodules, with useful purposes, one of the most important being redox signaling. Other studies also support the concept of ‘oxidative/nitrosative signaling’. For example, using proteomic analysis and detection with an antibody against nitrotyrosine, only 21 nitrated proteins were identified in sunflower (Helianthus annuus) hypocotyls (Chaki et al., 2009), suggesting that nitration is specifically targeted in cells rather than an indiscriminate phenomenon. However, this term may not apply to all circumstances, especially in nodules at the later stages of senescence or under stressful conditions. Nodule natural senescence (aging) is a complex and programmed process, which shares some features with stress-induced senecence, such as a decrease of N2-fixing activity and Lb content and an increase of proteolytic activity and ROS production. In aging soybean nodules, Evans et al. (1999) found an increase of ROS (mainly organic peroxides), catalytic Fe, oxidized homoglutathione, and oxidatively modified proteins and DNA bases, but no changes in ascorbate or tocopherol, concluding that these nodules were suffering from oxidative stress. Lipid peroxidation was also found to be elevated in nodules of pigeonpea (Cajanus cajan) and bean with advancing age (Swaraj et al., 1995; Loscos et al., 2008).

Similarly, in nodules of several legumes exposed to drought (Gogorcena et al., 1995), nitrate (De Lorenzo et al., 1994; Escuredo et al., 1996), prolonged darkness (Gogorcena et al., 1997; Hernández-Jiménez et al., 2002), or pro-oxidants such as Cd or H2O2 (Loscos et al., 2008), there was accumulation of lipid peroxides or oxidized proteins concomitant with a decline in antioxidant protection. In some cases, an increase in hydroxyl radical production and catalytic Fe was detected (Becana & Klucas, 1992; Gogorcena et al., 1995). These observations were also interpreted in terms of oxidative damage in nodules as a result of an increase in ROS production and/or a decrease in antioxidant defenses. Recent work indicated that the application to pea roots of paraquat, a compound that exacerbates formation of superoxide radicals, caused similar effects to those produced by drought (Marino et al., 2006), lending indirect support to the participation of ROS in the deleterious consequences of stress on N2 fixation. Indeed, drought induced the expression of several antioxidant genes and caused oxidative damage in alfalfa nodules (Naya et al., 2007). However, results were different in nodulated plants exposed to salt stress. In soybean or bean nodules exposed to high salinity, no symptoms of oxidative stress could be found, although antioxidant enzyme activities were induced (Comba et al., 1998; Loscos et al., 2008). The up-regulation of antioxidant enzymes, and particularly of SOD, was also seen in several other studies, suggesting that plants are perceiving an increase in ROS production and that antioxidants contribute to salt tolerance (Tejera et al., 2004; Jebara et al., 2005; Nandwal et al., 2007). Therefore, the data published to date indicate that the plant’s response, in terms of antioxidants and oxidative damage, is dependent on the type of stress and the legume species. The complexity of the interaction between the two symbiotic partners, probably differing in stress tolerance, and the structural and biochemical differences between indeterminate and determinate nodules make it difficult, if not impossible, to establish a general model for stress-induced nodule senescence.

XIV. Conclusions and perspectives

Nitrogen-fixing nodules have a high potential for production of ROS/RNS and hence require powerful antioxidant protection. Our knowledge of the most prominent of these defenses, the ascorbate-GSH pathway, has matured considerably since its initial description nearly 30 yr ago. Indeed, nodules may not function without it, a situation similar to that in chloroplasts, which are also sites of concentrated biochemical activity prone to generation of ROS/RNS. In recent years, the prospects for enhancing the activity of the ascorbate-GSH pathway, and concomitantly N2 fixation, have been improved by advances in our understanding of the ascorbate biosynthetic pathway. The goal of increasing N2 fixation has been touted for many years as a sort of holy grail that has been used to justify countless grants and research careers without much practical success. Such a goal may now be within reach, especially considering the numerous studies in which metabolic engineering has been used to enhance the ascorbate content, and thus stress tolerance, of nonfixing plants (see Ishikawa et al., 2006).

It may be useful to consider the various strategies of plants to protect against potentially toxic ROS/RNS concentrations while allowing them to perform essential functions in growth and metabolism. These strategies may be used at several levels that vary temporally and functionally. An initial, preventative strategy is to minimize ROS/RNS formation by restricting concentrations of ‘catalytic’ Fe, O2, or NO by binding to ferritin, Lbs, or class 1 Hbs, respectively. A second line of defense includes ROS scavenging by enzymes such as SODs, peroxidases, and catalases, which provide a more conventional class of antioxidant protection. Finally, a third defense is provided by GSTs, which remove the toxic by-products of ROS action. Control of ROS/RNS concentrations by antioxidant enzymes and metabolites will permit the plant cells, for instance, to utilize these reactive molecules as components of a redox transduction pathways in which appropriate responses are mediated through regulation of gene expression.

The use of genomic and proteomic technologies has greatly expanded the list of known antioxidants in plants and, in some cases, in nodules. The list now includes dozens of new entries, including multiple isoforms of the enzymes of the ascorbate-GSH pathway, SODs, GSTs, Trxs, and Gpxs. It is now necessary that future research will focus on more precisely defining the physiological roles of the various components, their interactions (‘antioxidant network’), and their post-translational modifications (nitrosylation, nitration, glutathionylation, and others), which may modulate their antioxidant activities and signaling functions in vivo. New roles are also emerging for numerous metabolites or proteins, which may be considered as ‘antioxidants’ in broad terms because they operate by metal sequestration or by modulating ROS/RNS bioactivity. It is also critical to ascertain the contributions and interactions of ROS/RNS in signal transduction pathways (‘signaling network’) associated with the onset or breakdown of the rhizobial symbiosis and with the response of nodules to stressful conditions. Finally, but probably most importantly, these networks need to be placed into a spatio-temporal context (nodule tissues and cells, gene and protein expression, and metabolite distribution) from rhizobial root infection to nodule senescence.

Acknowledgements

We are very grateful to Dr Yuhong Tang (Noble Foundation) for generating the heat map of gene expression, Dr Xinbin Dai (Noble Foundation) for mapping the Affymetrix probe-sets to IMGAG v. 3.5 gene identifiers, and Dr Carmen Pérez-Rontomé (CSIC) for assistance with drawing Fig. 1. The research described here was supported by a grant from the National Science Foundation (IOS-0517688) to D.A.D., and a grant from the Spanish Ministry of Science and Innovation-FEDER (AGL2008-01298) and Government of Aragón (group A53) to M.B.

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