Results and Discussion

3.1. Expression of antioxidants during nodule senescence. Northern hybridization of pea nodule RNA with specific probes for key antioxidant proteins revealed up-regulation of GSH peroxidase (reported for the first time in nodules) and catalase and down-regulation of the other antioxidants during natural (aging) senescence of nodules (Figure 1). Nitrate and dark treatments decreased the transcript abundance of plastidial CuZnSOD, mitochondrial MnSOD, cytosolic APX, and leghemoglobin (used as a control). Nitrate increased the transcript of cytosolic CuZnSOD whereas dark stress decreased it; the reverse occurred with ferritin. GSH peroxidase and catalase were up-regulated in both stress conditions. These results indicate that there is differential regulation of antioxidants at the transcript level during the various types of nodule senescence. General features of senescence, however, may be a lowering of important antioxidant defenses and a significant increase of GSH peroxidase and catalase transcripts. These increases may be a response to the enhanced production of lipid hydroperoxides and H2O2 at the later stages of senescence.

3.2. Functional characterization of GSHS2 from nodules. In a previous work (Moran et al. 2000) we reported the isolation from a pea nodule library of two cDNA clones that encoded enzymes with high homology to GSHS of other plants. One of the clones, GSHS2, encoded a cytosolic protein and was chosen for complete functional characterization. Repeated attempts to produce GSHS2 using E. coli expression systems were unsuccessful. In contrast, large amounts of virtually pure protein were produced efficiently in insect cells. This is important because GSHS enzymes are labile and of low abundance in plant tissues. The kinetic analysis of pure recombinant GSHS2 is shown in Table 1. The catalytic properties of the enzyme were determined using a fixed saturating concentration of yglutamylcysteine and a range of concentrations of Gly or pAla. The enzyme showed saturation kinetics and linear double-reciprocal plots with respect to both substrates. The much higher affinity of GSHS2 for p Ala (Km =1.9 mM) than for Gly (Km = 104 mM) as well as a specificity constant of 57 for the hGSHS/GSHS ratio permit us to conclude that the GSHS2 protein is a genuine hGSHS.

3.3. Antioxidants of nodule mitochondria. Bean plants were chosen to purify mitochondria because they produce large amounts of nodules and synthesize exclusively hGSH (Matamoros et al. 1999). A main thrust of this part of the work was to demonstrate the presence in nodule mitochondria of APX, guaiacol peroxidase (GPX), and other antioxidant enzymes, and to localize them inside the mitochondria. Peroxidase activity was assayed with ascorbate (APX activity) or pyrogallol (GPX activity) as substrates in the presence of inhibitors. Both activities were clearly detected and found to be drastically inhibited by KCN or azide, as would be expected for hemoproteins. The use of pCMS was critical to discriminate APX and GPX, as this compound markedly inhibits APX but has little effect on typical GPX enzymes (Amako et al. 1994). Previous work on the presence of GPX in

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