Biochemistry and Molecular Biology of Antioxidants in the Rhizobia-Legume Symbiosis

The complete reduction of molecular oxygen to water requires four electrons and is catalyzed by cytochrome oxidase in aerobic bacteria and mitochondria. However, 1% to 3% of all oxygen consumed by respiration is inevitably reduced to superoxide radicals and hydrogen peroxide (H2O2). These and other oxygen-derived molecules with moderate to very high reactivity are known as reactive oxygen species (ROS). The term includes free radicals (molecules with one or more unpaired electrons, such as the superoxide and hydroxyl radicals) and non-free radicals (molecules with no unpaired electrons, such as H2O2 and singlet oxygen). The main sources of ROS in plants under physiological conditions are respiration, photosynthesis, and N2 fixation (Table I). In addition, ROS are produced at high rates when plants are exposed to abiotic, biotic, or xenobiotic stress. Similarly, the term reactive nitrogen species (RNS) refers to nitrogen-derived molecules with variable reactivity and includes free radicals (nitric oxide) and non-free radicals (peroxynitrite). Nitric oxide is involved in many key physiological processes in animals and, as shown in recent years, also in plants (Table I). It reacts with the superoxide radicals to form peroxynitrite and probably with thiol compounds to form nitrosothiols. The investigation of RNS is at present a truly novel and important field in plant biology. Production of ROS and RNS in plants Production of ROS and RNS in plants The superoxide radical, H2O2, and nitric oxide have moderate reactivity toward biomolecules and, thus, may have some direct detrimental effects in plants. The superoxide radical inactivates dihydroxy-acid dehydratase (required for the synthesis of branched chain amino acids) and aconitase (required for the operation of the Krebs cycle) by oxidizing the iron-sulfur clusters at the active site, and ribonucleotide reductase (required for DNA synthesis) by oxidizing an essential Tyr radical. Also, H2O2 can inactivate Calvin cycle enzymes, metalloproteins such as superoxide dismutases (SODs), and hemoproteins such as nodule leghemoglobin (Dalton, 1995; Scandalios et al., 1997). However, the real threat of the superoxide radical and H2O2 is their potential to act as precursors of the hydroxyl radical. The hydroyxl radical can readily oxidize amino acid residues of proteins, fatty acids of phospholipids, and deoxy-Rib and bases in DNA (Halliwell and Gutteridge, 1999). Nitric oxide can directly inhibit iron-containing proteins (Neill et al., 2002), but its toxicity stems mainly from its ability to react with the superoxide radical to form peroxynitrite. This compound can induce lipid peroxidation, nitration of Tyr residues of proteins, oxidation of thiols, and nitration or deamination of DNA bases (Halliwell and Gutteridge, 1999). However, the same three ROS or RNS mentioned above may perform useful roles in plants. This is largely because they show moderate reactivity and are mainly generated by enzymes; hence, their rates and subcellular sites of production may be under metabolic control. The superoxide radical and H2O2 are involved in lignification of cell walls, defense against pathogen attack, and sensing of, and subsequent adaptation to, stressful conditions. H2O2 can also induce programmed cell death during the plant's hypersensitive response to infection by modulating gene expression (Neill et al., 2002). Nitric oxide also acts as a signal molecule and is involved in the control of gene expression, hypersensitive response, antioxidant defense, organogenesis, and stomatal closure (Neill et al., 2002; Lamattina et al., 2003). Plant cells contain an impressive array of antioxidant metabolites and enzymes that scavenge or prevent the formation of the most aggressive ROS and RNS, thus protecting cells from oxidative damage. In addition, antioxidant enzymes control the steadystate levels of the moderately reactive ROS and RNS, allowing them to perform important roles at specific sites, environmental conditions, or developmental stages of plants. Although antioxidants have multiple roles in diverse physiological processes in plants, we present here a restricted overview of the role of antioxidants in the rhizobia-legume symbiosis. Readers are referred elsewhere for a more general coverage of antioxidants in plants, in particular the excellent reviews by May et al. (1998) and Mittler (2002). As a result of the complex and continuous molecular interplay between the bacteria and the plant, large amounts of ROS and possibly RNS are generated during the lifetime of nodules; hence, an important asset of antioxidant enzymes is expected in both symbiotic partners. These and other molecular studies of the symbiosis are greatly facilitated by selecting Medicago truncatula or Lotus japonicus as model legumes, respectively, for indeterminate or determinate nodulation (Udvardi, 2001). Both legume species have a small diploid genome, are autogamous, have a short generation time and large seed production, and are amenable to transformation and mutant screening. In addition, the chloroplastic genome of L. japonicus and the genomes of Sinorhizobium meliloti and Mesorhizobium loti (the bacterial components of the symbioses) have been entirely sequenced, and the nuclear genomes of M. truncatula and L. japonicus are being sequenced at a fast pace. Ascorbate (vitamin C) is a water-soluble reductant that can be found in nodules at concentrations of 1 to 2 mm. Ascorbate is required for the progression of the cell cycle and for cell elongation. The latter effect has been attributed to its participation as cofactor of prolyl hydroxylase (required for the synthesis of Hyp-rich proteins of the cell wall) and to the ability of apoplastic ascorbate to alter the properties of the plasma membrane or to inhibit the cross-linking of Hyp-rich proteins by phenols (Horemans et al., 2000). However, the best known functions of ascorbate are based on its properties as an antioxidant. Ascorbate regenerates the α-tocopherol oxidized by ROS at the membrane-cytosol interface, is a direct scavenger of most ROS, and is the substrate of ascorbate peroxidase (APX). The major pathway for ascorbate synthesis has been elucidated (Wheeler et al., 1998). The last step, catalyzed by l-galactono-γ-lactone dehydrogenase, occurs in the inner membrane of mitochondria (Horemans et al., 2000). The thiol tripeptide GSH (γGlu-Cys-Gly) is also an abundant metabolite of plants, where it performs multiple functions, including transport and storage of sulfur, control of cell redox status, progression of the cell cycle, protection of protein thiol groups, and detoxification of heavy metals and xenobiotics (May et al., 1998). GSH is an important antioxidant in its own right but also as a substrate for glutathione reductase and glutathione peroxidase (GSH-PX). However, in some legumes, homoglutathione (γGlu-Cys-βAla) may partially or completely replace GSH. Homoglutathione is the major tripeptide in nodules of soybean (Glycine max), common bean (Phaseolus vulgaris), and mung bean (Vigna radiata), whereas GSH is predominant in nodules of pea (Pisum sativum), alfalfa (Medicago sativa), and cowpea (Vigna unguiculata). In each case, the major thiol is present at concentrations of 0.5 to 1 mm. The synthesis of GSH and homoglutathione proceeds through two ATP-dependent steps catalyzed, respectively, by γ-glutamylcysteine synthetase and a specific glutathione or homoglutathione synthetase (Fig. 1). The enzymes from pea, mung bean, and tobacco (Nicotiana tabacum) leaves have been partially purified and localized to the cytosol and plastids (Rennenberg, 1997). The biochemical properties of such enzymes, along with the information gained for the nodule enzymes using molecular approaches, are summarized in Table II. Genomic and cDNA clones for all three enzymes have been isolated, and gene structures have been determined. The γecs gene of L. japonicus contains 15 exons with identical size and high sequence homology (78% identity) to that of Arabidopsis (Matamoros et al., 2003). In both M. truncatula and L. japonicus, the gshs and hgshs genes have 12 exons of identical size (except for the first ones, which are very close in size), show high sequence homology (83% identity between the coding sequences of the two genes), and are tandemly arranged (and with the same orientation) in the genome. These observations indicate that the two genes originated by duplication (Frendo et al., 2001; Matamoros et al., 2003). In L. japonicus, the genes are separated by only 8 kb, appear to be present as single copies, and encode proteins with putative plastid signal peptides. The expression patterns of gshs and hgshs are clearly different in the two model legumes. In M. truncatula, hgshs is preferentially expressed in the roots and nodules and gshs in the leaves (Frendo et al., 2001), whereas in L. japonicus, hgshs is expressed in the roots and leaves and gshs in the nodules (Matamoros et al., 2003). Why the hgshs gene was recruited during evolution exclusively in the legume family and is only expressed in some species or organs remain unsolved questions but the differential expression of gshs and hgshs do suggest specific roles for their enzymatic products. Three types of peroxidases that can be found in legume nodules. The scheme depicts gene structures, proteins, and activities catalyzed by representative enzymes of each type: APX of pea leaf cytosol, GPX of horseradish roots, and GSH-PX of L. japonicus nodules. Gene diagrams show exons (except untranslated regions [UTRs]) in red, introns in yellow, and UTRs in blue. Numbers are length in base pairs. Protein diagrams show: a, in APX and GPX, the distal and proximal His residues (H) that bind the heme groups (in red); b, in GPX, the N- and C-terminal signal peptides, the four conserved disulfide bridges, and one of the eight glycosylated Asn residues (N*); and c, in GSH-PX, the plastid signal peptide and some important residues of the three typical domains (“signatures”). Numbers are length in amino acid residues. ASC, ascorbate; MDHA, monodehydroascorbate; RH2, artificial reductant. Antioxidant proteins of legume nodules Molecular mass from cDNA for the nodule for the of and molecular mass for plant enzymes (Rennenberg, 1997). molecular mass from cDNA sequences of the pea and bean nodule Antioxidant proteins of legume nodules Molecular mass from cDNA for the nodule for the of and molecular mass for plant enzymes (Rennenberg, 1997). molecular mass from cDNA sequences of the pea and bean nodule also have high GSH concentrations to their own γ-glutamylcysteine synthetase and glutathione synthetase et al., 2000). homoglutathione but amounts of homoglutathione are found in bean nodule as a result of from the cells et al., 2000). The GSH may be consumed by in metabolic and of redox being to the plant et al., 2001). a mutant of has been which is in glutathione synthetase and contains only 3% of the GSH present in the et al., 2000). This mutant is to acids and to and oxidative and the of GSH the to to the mutant can form nodules on bean, but it is by the that GSH is important for and the symbiotic et al., 2000). to the of and the reduction of H2O2 to water by In APX has been found in the cytosol and mitochondria et al., et al., 2001), but probably in and as occurs in leaves et al., 1997). APX has been purified from soybean cDNA clones isolated, and and The most important properties of APX are in Table II. The is in the of to the chloroplastic and is also from amino acid sequence has homology with peroxidases but homology with cytochrome peroxidase and The genes for APX of pea and Arabidopsis have exons and introns (Fig. 1). The first is in the and may have an effect on expression and This with the gene the two and of chloroplastic APX et al., 2002). or are peroxidases found in the and have been in a of including defense against and oxidative stress. compounds as and are with artificial In they as multiple but of them have been and also but at However, are by the thiol because they contain free whereas horseradish contain four conserved disulfide The of such is the for an to between and et al., The two types of peroxidases with the of the and the do The are also important at the gene The and of introns of or are very different from of (Fig. 1). are peroxidases that the reduction of H2O2, and lipid to water by GSH. to be present only in animals and it that is also present in plants. The first from plants, which found to contain GSH-PX et al., This was clearly different from glutathione of which may also GSH-PX and was by stress. cDNA clones enzymes have been in pea and other plants et al., 1998). of them are to contain or at their active of the found in plant are to be in the putative and found in et al., 1999). also have cDNA and clones that are expressed in nodules of L. japonicus (Fig. 1). The proteins contain the three conserved found in and plant and of subcellular indicate that are cytosol and plastid genes have been completely sequenced and found to exons of identical (except for the first but different gene structures for a the three types of nodule peroxidases (Fig. 1). The of APX is free which to ascorbate and and are reduced to ascorbate by specific using and the by reductase is reduced to GSH by glutathione reductase using the pathway four enzymes in to H2O2 at the of the of or (Fig. Antioxidant enzymes of legume nodules. ASC, cytosol plastid γ-glutamylcysteine transport glutathione reduced oxidized MDHA, monodehydroascorbate; oxidative APX was hence, we on the other enzymes of the pathway (Fig. are found in the cytosol, and but are probably present also in nodule plastids because the is abundant in and plastids and and because cDNA clones a putative plastid glutathione reductase have been and It is that the enzymes of nodule mitochondria and plastids are for by a single as occurs with the enzymes of pea leaves et al., such as of bean and homoglutathione the is a homoglutathione are of plants that in and In two have been and at one of them is with the cell levels are in the cytosol et al., it that of ascorbate through the pathway in the cytosol is mainly by whereas reductase may be involved in of apoplastic synthesis of Hyp-rich proteins, and lignification of cell is known in plants and, in in nodules (Table reductase is a protein with active thiol groups and has been localized to the cytosol and mitochondria of nodule cells et al., et al., 2001). The pathway to be in nodule other the The four enzymes of the pathway have been in nodule mitochondria et al., et al., 2001). model has been et al., for bean nodule in which H2O2 generated in the inner membrane is by and the ascorbate oxidation are to ascorbate by and homoglutathione in the or the cytosol (Fig. The enzymes of the pathway have also been found in pea leaf et al., thus, the pathway is probably in nodule of show that the pathway is for nodule (Dalton, 1995; et al., 1998). The and of the key of the are very abundant in in the and cells (Fig. In the APX leghemoglobin and other proteins from H2O2, in the nodule cell the the may in the operation of the oxygen This has been to be for the most in the nodule and oxygen the The cells have only high levels of APX but also of ascorbate and (Fig. we that the cells oxygen to the by their et al., 1998). The of the H2O2 be by allowing for H2O2 to act as a signal molecule for the or of the oxygen et al., 1998). of and H2O2 in alfalfa nodules. of levels are in the and in a of cells in the nodule from et al., 1998). of is in the nodule respiration with the oxygen and probably with the of APX protein shown in et al., 1998). In of is most abundant in the nodule which the and In of is most abundant in the and in the cells and of nodule was with and for The of H2O2 is by the of which can be in the and of infection in and in the cell and of the in that H2O2 can be also the bacteria the in are of the of the pathway for N2 The activities of all four enzymes are to in nodules in The activities and thiol are also to in in nodules. Also, of plants with nitrogen N2 fixation with three activities of the that is a between N2 fixation and antioxidant The most for the between antioxidants and N2 fixation from observations that direct of ascorbate stems of soybean plants to an in leghemoglobin a in rates of N2 and a in nodule and 1999). of ascorbate and purified APX in an in leghemoglobin and in of leghemoglobin and to a in N2 fixation et al., 1999). and other observations have that antioxidants an important role in protecting and N2 are a family of that the of superoxide radicals molecular oxygen and Three of in their metals at the active site, may in plants, and all of them have been found in the nodule plant The subcellular and biochemical properties of the and of nodules are in Table II. the proteins and of and have been localized in alfalfa and pea nodules and M. The is predominant in the nodule (Fig. in the infection cytosol to cell walls, and the is abundant in the in the cells (Fig. the plastid is localized to the whereas is also found in the and bacteria infection The of and suggest specific functions for the two The may be with cell in the infection and and with the plant's response to bacterial The a role to the protection and of symbiotic in nodules. The structures of the genes and of L. japonicus have been determined. The gene of eight the first is in the as occurs for the pea The gene has exons with no The are the most of in the gene was to be or only in a of plants. The when appear to be localized exclusively in the have found in nodules of most and for some types of clearly the typical localized in the plastids of alfalfa and pea nodules and an localized in the cytosol of cowpea nodules et al., 2003). a in the and a in the These enzymes are by the bacterial and The of meliloti high amino acid sequence with bacterial and is a in other it active when the is by et al., 1999). the is to H2O2 of the at the active The mutant of meliloti to and et al., 2000). The of meliloti is expressed during infection et al., as a response of the bacteria to the superoxide radicals produced by the plant H2O2 to water and molecular oxygen and, thus, may plant cells with an to H2O2 et al., 1997). However, have a for H2O2 and are expected to be active only at subcellular sites where H2O2 or concentrations are very such as the from nodule was purified and to be to other plant It is a specific occurs in nodule as was for leaves et al., 1997). The production of oxygen by appear to be in the of because and other proteins with high redox potential are readily by it is that do contain peroxidases but have meliloti and other have three two and and one is by H2O2 and expressed in whereas and are expressed by the bacteria the infection et al., 2003). or are in N2 whereas the single no the of during the infection et al., 2003). antioxidant enzymes are to plant such as the and of et al., and the APX of mitochondria et al., 2001). These enzymes from ROS but may have useful reductase can superoxide common with the chloroplastic which may be by the plant as a signal Plant are mainly against lipid and other types of oxidative by small molecules such as and is found at concentrations of 15 in both and soybean nodules et al., 1999). (the reduced form of and acid are abundant in of mitochondria and other where they act as of lipid However, they have been in nodules. and other are found in nodules at concentrations of to mm. of compounds have important antioxidant protecting by lipid radicals et al., 1997). are also abundant in nodules and inhibit lipid in probably by their ability to with and et al., Plant cells also have an protection against in the free form or to small is because it can formation of hydroxyl The to be acid and compounds that are to in a form and may inhibit oxidative of and proteins in et al., 1997). The of free be because plants a for the synthesis of and some and 1997). This be against the potential toxicity of The protein to of in a form that the effects of its for metabolic Plant are of a by a of identical have an active and abundant which is localized in plastids and the of leaves that is localized exclusively in the et al., Matamoros et al., 1999). The protein and very in in nodule the protein its with the in and leghemoglobin This that is a of and it for and leghemoglobin in protein and are expression may be and The during and of can be found on legume species and nodule This is most probably to of expression by the during of leghemoglobin and other proteins et al., Matamoros et al., 1999). to pathogen infection with a hypersensitive an of which is the and production of ROS and 1997). of legume roots by also a hypersensitive the first nodule have been an of infection in a cells in which both and cells The hypersensitive may be of a the plant infection and, thus, nodulation et al., As in the of by cells to infection with an production of superoxide and H2O2 et al., 2001; et al., 2002; et al., 2002). It has been that is a oxidative but the that H2O2 is restricted to the very stages of nodule formation in one of the genes more by or to encode a peroxidase and has in its that may be to H2O2 is to in the of ROS may act in the signal pathway et al., 2002). In et al. and et al. have that nodulation requires the production of H2O2 is of an oxidative in stages of nodule H2O2 may be more to cell formation and cross-linking of both of which are required for However, an as unsolved is some have during infection and form nodules. It is during may or inhibit the This has been attributed to the bacterial et al., The enzymes for ROS formation during infection and nodule have been The superoxide radicals are in the infection et al., 2001), possibly by a the superoxide generation during the oxidative in sources for H2O2 are cell and et al., 2000). have found that H2O2 in the and of infection bacteria the and in the of the nodule (Fig. mainly on we that is a potential of This peroxide may be important for the cross-linking of cell proteins in the and of the in the infection et al., 2000). signal molecules may be important for nodule acid may be in the stages of infection because inhibit the of acid in the et al., 1998). Nitric oxide be signal molecule because both nitric oxide et al., and nitric oxide et al., have been in nodules. et al. have found nitric oxide and its in pea leaf However, has the nitric as a of the protein of the complex et al., 2003). It be of to nitric oxide is produced in the nodule and to its In case, it is that the of RNS in nodules is an of is a in the lifetime of nodules by an production of ROS and probably amounts of H2O2 in the cells and in the of soybean nodules et al., and in the of alfalfa and pea nodules and M. In the nodule is a major in antioxidant oxidative of leghemoglobin to and processes Matamoros et al., 1999). These are all to ROS and RNS As a oxidative of proteins, and DNA has been in nodules during et al., and and Matamoros et al., Similarly, the of and in the cells and biochemical may be in of a from a to an oxidative which may be a general of plant and are most to for excellent with and for are also to all and have directly or to the information in to we to because of