N 3d




Nitrogen Fixation




Figure 1. Expression of antioxidant enzymes during pea nodule senescence. Lanes 2 to 7: transcripts from 2-to 7-week-old nodules. Lane 3N: transcripts from 3-week-old nodules treated with nitrate for 4 days. Lane 3D: transcripts of 3-week-old nodules from plants exposed to continuous darkness for 4 days. Abbreviations: APX = ascorbate peroxidase; CuZnSODc = cytosolic CuZnSOD; CuZnSODp = plastidial CuZnSOD; GR = glutathione reductase; GSH-POX = glutathione peroxidase; Lb = leghemoglobin. Methylene blue was used to ensure uniform loading.

Table 1. Kinetic properties of the recombinant GSHS2 enzyme.


GSHS activity

hGSHS activity


Vb (nmol min^mg"1 protein)

158 ±22

3433 ±137


Vm (nmol min^mg"1 protein)

4199 ±108

4719 ±430



104 ±9

1.9 ±0.3


Specificity constant (Vm/Km)

41 ±4

2350 ±331


b Activity rate using the standard concentration (5 mM) of Gly or p Ala for the enzyme assay.

b Activity rate using the standard concentration (5 mM) of Gly or p Ala for the enzyme assay.

Table 2. Specific activities and intramitochondrial localization of antioxidant enzymes.

Enzymea Activity Latency (%) Solubilization (%)


42 ±


68 ±5

85 ±8


552 ±



11 ±6


2473 ±


46 ±3

48 ±6


104 ±


20 ±4

75 ± 1


26 ±


45 ± 10

82 ± 1


8 ±


53 ±3

81 ±5

Cytochrome c oxidase0

3984 ±


90 ± 1

3 ± 1

Malate dehydrogenase0

63 ±


86 ±2

77 ± 1

a MnSOD activity is expressed in units mg_1 and malate dehydrogenase activity in |imol mirr1 mg"1. All other enzyme activities are expressed in nmol min-1 mg_1. Latency was determined in hyposmotic and isosmotic media with or without 0.02% Triton X-100, respectively, and was calculated by the formula of Burgess et al. (1985). Values are means ± SE of 3-6 replicates, each corresponding to an independent nodule extract. b hGR activity was assayed using oxidized GSH as substrate. c Cytochrome c oxidase and malate dehydrogenase were used, respectively, as marker enzymes of the inner membrane and the matrix.

mitochondria has produced contradictory results. Prasad et al. (1995) detected membrane-bound GPX activity in maize leaf mitochondria, whereas Jiménez et al. (1997) did not find genuine GPX activity in pea leaf mitochondria. We have found GPX activity in nodule mitochondria. In fact, solubilization data indicate that 53% of the activity is in the matrix and that there is also significant activity associated with the membranes (Table 2). An interesting observation is that the mitochondrial APX is in the membrane and does not lose its activity in the absence of ascorbate. In this respect, the APX of nodule mitochondria behaves similarly to the cytosolic enzyme (Dalton 1995) but contrary to the enzymes of the chloroplasts (Amako et al. 1994), leaf peroxisomes (Jiménez et al. 1997), or potato tuber mitochondria (De Leonardis et al. 2000), which are inactivated by H2O2 in the absence of ascorbate. The zero latency of APX (Table 2) indicates that exogenous ascorbate is directly accessible to the enzyme and therefore that APX is located in the outer membrane or in the outside of the inner membrane. To localize APX more precisely, mitochondria were subjected to controlled osmotic lysis and separated into the outer membrane-enriched fraction, inner membrane-enriched fraction, and matrix. These fractionation studies revealed that most APX activity is associated with the inner membrane, with a small proportion of the activity being in the matrix. These data are consistent with the observation that only 11% of the APX activity remains in the soluble fraction (matrix) of mitochondria (Table 2).

The three other enzymes (DR, MR, GR) of the Halliwell-Asada pathway and MnSOD were found in the matrix of nodule mitochondria (Table 2). Mitochondria contained 13 ± 2 nmol of hGSH mg_1 protein, a level that is comparable to that of whole bean nodules (Matamoros et al. 1999). Therefore, the GR enzyme found in mitochondria should be functionally designated as a hGSH reductase (hGR). Because bean nodule mitochondria are devoid of the hGSH biosynthetic enzymes, y-glutamylcysteine synthetase and hGSHS, whereas the latter activity is present in the nodule cytosol, it follows that the mitochondria obtain hGSH from the cytosol and utilize it as an antioxidant metabolite in place of GSH. Oxidized hGSH is recycled by hGR using NADPH.

We propose that the H2O2 formed in the inner membrane by the electron transport chain is scavenged by the APX located in the inner membrane. The ascorbate required for APX activity can be provided by the enzyme L-galactono-y-lactone dehydrogenase, which is also localized in the inner membrane (Siendones et al. 1999). The resulting oxidized compounds, monodehydroascorbate and dehydroascorbate, can be recycled in the matrix or in the cytosol since both subcellular compartments contain MR, DR, and hGR. In the mitochondrial matrix, the H2O2 formed as a result of oxidative metabolism and of MnSOD activity could be scavenged directly by hGSH and antioxidant enzymes such as APX and GPX.

4. References

Amako K et al. (1994) Plant Cell Physiol. 35, 497-504 Becana M et al. (1998) Plant Soil 201, 137-147 Becana M et al. (2000) Physiol. Plant. 109, 372-381 Burgess Nei al. (1985) Planta 166, 151-155

Dalton DA (1995) In Ahmad S (ed), Oxidative Stress and Antioxidant Defenses in Biology, pp. 298-355, Chapman and Hall, New York De Leonardis S et al. (2000) Plant Physiol. Biochem. 38, 773-779 Gogorcena Y et al. (1997) Plant Physiol. 113, 1193-1201 Jiménez A et al. (1997) Plant Physiol. 114, 275-284 Klapheck S (1988) Physiol. Plant. 74, 727-732 Matamoros MA et al. (1999) Plant Physiol. 121, 879-888 Millar AH et al. (1995) Plant Cell Environ. 18, 715-726 Moran JF et al. (2000) Plant Physiol. 124, 1381-1392 Prasad TK et al. (1995) Plant Physiol. 108, 1597-1605

Sambrook J et al. (1989) Molecular Cloning, CSH Laboratory Press, New York

Santos R et al. (2001) Mol. Plant-Microb. Interact. 14, 86-89

Siendones E et al. (1999) Plant Physiol. 120, 907-912

Struglics A et al. (1993) Physiol. Plant. 88, 19-28

Verwoerd TC et al. (1989) Nucleic Acid Res. 17, 2362

5. Acknowledgements

We thank David Dalton and Frank Minchin for helpful comments. This work was funded by grant PB98-0522 from the Dirección General de Enseñanza Superior e Investigación Científica (Spain).


D. Hérouart1, E. Baudouin1, R. Santos2, C. Mathieu1, S. Sigaud1, A. Jamet1, P. Evans3, P. Frendo1, G. Van de Sype1, M.J. Davies4, B. Halliwell3, D. Touati2, A. Puppo1

'Lab. Biol. Végétale. Microbiol. CNRS FRE 2294, U Nice-Sophia Antipolis, Nice, France 2Inst. Jacques Monod CNRS UMR 7592, U Paris 6-7, Paris, France 3Pharmacology Group, King's College, London, UK 4Heart Research Inst., Camperdown, Sydney, Australia

1. Introduction

Reduction of molecular oxygen proceeds through four steps, thus generating several active oxygen species (ROS) (Elstner 1982). The reaction chain requires initiation at the first step whereas subsequent steps are exothermic and can occur spontaneously, either catalyzed or not.

The first step in oxygen reduction produces superoxide anion (02~), which can cause lipid peroxidation and oxidize specific amino acids, such as histidine, methionine and tryptophan (Fridovich 1995). The second reduction step generates hydrogen peroxide (H202), a relatively long-lived molecule that can diffuse from its site of production (Levine et al. 1994). H202 is toxic through oxidation of SH groups. The toxicity of H202 can be enhanced in the presence of ferrous iron via the Fenton reaction.

The last species generated by this series of reductions is the hydroxyl radical (OH'). It reacts with biological molecules at its site of production with almost diffusion-controlled rates.

To protect against the damage caused by ROS, cells possess a number of antioxidant enzymes and low molecular weight antioxidants, such as superoxide dismutases (SOD), catalases and peroxidases, glutathione and ascorbate (Halliwell 1990). When an imbalance between production and removal of ROS occurs, an oxidative stress appears.

There are several known sources of ROS in plants. Some of the most common include the leakage of electrons to 02 from electron transport chains in the chloroplasts (Foyer et al. 1994), mitochondria (Elstner, Osswald 1994) and peroxisomes (Corpas et al. 2001). Other sources include cell wall oxidases and peroxidases (Bolwell, Wojtaszek 1997). These ROS, generated by the reduction of molecular oxygen in plant systems, can have deleterious effects and lead to a situation of oxidative stress but are also, in contrast, interesting candidates for cell signaling (Dat et al. 2000). This dual role for ROS appears to be very important in legume nodule development.

Legume root nodules, which are characterized by an early senescence, are especially at risk from oxidative damage by ROS. Leghemoglobin autoxidation has been shown to produce 02", which disproportionates to form H202 (Puppo et al. 1981). Additionally, the reaction of leghemoglobin with H202 generates oxidizing species such as ferryl heme proteins and protein radicals (Davies, Puppo 1992). H202 can also cause protein degradation to release "catalytic" iron, i.e. iron in a molecular form that can promote lipid peroxidation and OH' generation (Puppo, Halliwell 1988). The formation of ROS may additionally occur as a result of the strong reducing conditions required for nitrogen fixation, and the action of several proteins including ferredoxin, uricase and hydrogenase (Dalton et al. 1991). Thus, a question arises: "Does oxidative stress occur during nodule senescence?"

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