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First published online September 25, 2003; 10.1104/pp.103.023010 Plant Physiology 133:773-782 (2003) © 2003 American Society of Plant Biologists Functional Characterization and Expression of a Cytosolic Iron-Superoxide Dismutase from Cowpea Root Nodules1,,2Departamento de Nutrición Vegetal, Estación Experimental de Aula Dei, Consejo Superior de Investigaciones Científicas, Apdo 202, 50080 Zaragoza, Spain (J.F.M., M.C.R., M.B.); Centre for High Resolution Imaging and Processing, Medical Sciences Institute/Wellcome Trust Biocentre Complex, School of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom (E.K.J.); and Department of Biochemistry, University of Nebraska, The Beadle Center, P.O. Box 880664, Lincoln, Nebraska 68588 (G.S., R.V.K.)
An iron-superoxide dismutase (FeSOD) with an unusual subcellular localization, VuFeSOD, has been purified from cowpea (Vigna unguiculata) nodules and leaves. The enzyme has two identical subunits of 27 kD that are not covalently bound. Comparison of its N-terminal sequence (NVAGINLL) with the cDNA-derived amino acid sequence showed that VuFeSOD is synthesized as a precursor with seven additional amino acids. The mature protein was overexpressed in Escherichia coli, and the recombinant enzyme was used to generate a polyclonal monospecific antibody. Phylogenetic and immunological data demonstrate that there are at least two types of FeSODs in plants. An enzyme homologous to VuFeSOD is present in soybean (Glycine max) and common bean (Phaseolus vulgaris) nodules but not in alfalfa (Medicago sativa) and pea (Pisum sativum) nodules. The latter two species also contain FeSODs in the leaves and nodules, but the enzymes are presumably localized to the chloroplasts and plastids. In contrast, immunoblots of the soluble nodule fraction and immunoelectron microscopy of cryo-processed nodule sections demonstrate that VuFeSOD is localized to the cytosol. Immunoblot analysis showed that the content of VuFeSOD protein increases in senescent nodules with active leghemoglobin degradation, suggesting a direct or indirect (free radical-mediated) role of the released Fe in enzyme induction. Therefore, contrary to the widely held view, FeSODs in plants are not restricted to the chloroplasts and may become an important defensive mechanism against the oxidative stress associated with senescence.
Superoxide dismutases (SODs; EC 1.15.1.1) are ubiquitous metalloenzymes that catalyze the dismutation of superoxide radicals and thus prevent oxidative damage in all organisms. Three classes of SODs, differing in the metals at their catalytic active site, are known in plants. The CuZnSODs are localized in the cytosol, chloroplasts, nucleus, and apoplast; the MnSODs in the mitochondria and peroxisomes; and the FeSODs in the chloroplasts (Sandalio et al., 1987  ; Van Camp et al., 1990; Bowler et al., 1994; Ogawa et al., 1996). There have been contradictory reports on the distribution of FeSODs in higher plants. Once thought to be confined to a few families of vascular plants (Bridges and Salin, 1981), it is now accepted that the FeSOD (sodB) genes are amply distributed in plants but only are expressed under certain, yet poorly defined, conditions (Van Camp et al., 1990; Bowler et al., 1994).
In an earlier study (Becana et al., 1989
Characterization of VuFeSOD and Its Derived Protein
Primers were designed against conserved sequences of FeSODs from Chlamydomonas reinhardtii and higher plants and were used to isolate a clone, VuFeSOD, from a cowpea nodule cDNA library. The same primers enabled us to isolate cDNA clones encoding FeSODs from pea (Pisum sativum; accession no. AJ496175) and soybean (accession no. AF108084) nodules. The VuFeSOD sequence was 1,043 bp long and contained an open reading frame of 738 bp, which encodes a protein of 245 amino acids, with a molecular mass of 27,411 D and a pI of 5.31. The predicted protein contains the residues thought to be essential for FeSOD activity (Tyr-58, Trp-100, and Asn-185) and metal binding (His-54, His-102, Asp-202, and His-206), as well as the residues (Ala-97, Gln-98, Trp-100, and Ala-186) proposed as primary candidates to distinguish FeSODs from MnSODs (Van Camp et al., 1990
Prediction programs such as PSORT and ChloroP indicated that VuFeSOD lacks a plastid signal peptide (Fig. 1B) and suggested a cytosolic localization. To confirm this, the enzyme was purified from cowpea nodules and leaves by anion-exchange or affinity chromatography. Two independent enzyme preparations from each plant organ were N-terminally sequenced producing identical results. Comparison of the N-terminal sequence (NVAGINLL) with the derived sequence of the precursor protein revealed the presence of seven extra amino acids (MASLGLQ; Fig. 1B). The same prediction programs suggested that soybean FeSOD (isozymes 1 and 2) are cytosolic, whereas soybean FeSOD (isozyme 3) and pea nodule FeSOD have plastid signal peptides (Fig. 1B), in agreement with the homology data mentioned above. Southern-blot analysis of cowpea genomic DNA was used to estimate the number of gene copies with a probe comprising the entire open reading frame of VuFeSOD. Two hybridization bands were observed with restriction enzymes that do not cut inside the gene (EcoRI and EcoRV) and one additional band of smaller size when using an enzyme that cuts inside the gene (HindIII). Hence, two copies of the sodB gene appear to be present in the cowpea genome (Fig. 2).
A 733-bp fragment coding for the mature protein was cloned into the NdeI and NotI sites of the expression vector pET-28a(+) and was used to transform E. coli BL21. Analysis of cell extracts by SDS-PAGE revealed the overproduction of a protein of approximately 30 kD (Fig. 3A). Recombinant VuFeSOD was purified to homogeneity, as judged by SDS-PAGE (Fig. 3A) and native-PAGE (Fig. 3B), by a single step of affinity chromatography using a HiTrap metal-chelating column, which selectively bound the (His)6-tagged protein. The molecular mass (29.7 kD) of the purified recombinant protein on SDS-PAGE agreed with the molecular mass calculated from its amino acid sequence and included 21 amino acids from the cloning vector at the N terminus. Thrombin removed 17 of those amino acids, including the (His)6 tag, as confirmed by SDS-PAGE (Fig. 3A) and native-PAGE (Fig. 3B). The affinity purification step was able to remove the FeSOD and other SODs of E. coli from the recombinant VuFeSOD preparation, as can be seen in Figure 4 (compare lanes 1 and 2).
Pure recombinant VuFeSOD was used to raise a monospecific polyclonal antibody. This recognized a single protein band of approximately 27 kD, which corresponds to the subunit size of VuFeSOD, in preparations of affinity-purified recombinant VuFeSOD and in extracts of cowpea nodules and leaves (Fig. 5). Immunoblots showed that recombinant VuFeSOD treated with thrombin migrated to the same position as the enzyme from nodules or leaves (Fig. 5). The presence of FeSODs in bacteroids and nodule extracts from other legumes was also investigated using the antibody. The immunoreactive protein of approximately 27 kD was absent in cowpea bacteroids or in alfalfa and pea nodules, but was clearly detectable in soybean and common bean nodules (Fig. 5). Because alfalfa and pea nodules do contain FeSOD (Rubio et al., 2001
Native-PAGE in the absence (control) or presence of inhibitors (Fig. 4) showed that the SOD isozyme composition of cowpea is similar to that of other legumes (Rubio et al., 2001 The specificity of the antibody was further tested by immunoprecipitation and subsequent analysis of SOD isozymes on native gels (Fig. 6). The FeSOD activity was greatly reduced after immunoprecipitation with 1 µg of antibody and was completely suppressed with 4 µg of antibody, whereas the MnSOD and CuZnSOD activities were unaffected.
Immunoblots were used to study the effects of stressful conditions and natural senescence (aging) on the nodule content of VuFeSOD (Fig. 7). There were either no changes, or very minor decreases, in VuFeSOD protein levels in nodules of plants exposed to salt or water stress, continuous darkness, or excess nitrate. In contrast, nodule aging had clear effects on VuFeSOD content. The protein was not detectable in very young nodules (25-d-old plants) but was relatively abundant in mature nodules (46-d-old plants) and in senescent nodules (82-d-old plants). In the latter plants, two nodule populations could be distinguished by their color ("red-brown" and "green"), which reflected increased leghemoglobin (Lb) degradation (Arredondo-Peter et al., 1997
The cytosolic localization predicted from the VuFeSOD sequence was investigated with immunoblots and immunoelectron microscopy. Cowpea nodules and leaves were extracted in iso-osmotic medium and centrifuged at 15,000g for 20 min and at 100,000g for 1 h. Immunoblots indicated that VuFeSOD was in the soluble fraction ("cytosol") and not in the membrane pellet (Fig. 8). This was confirmed by immunoelectron microscopy of high-pressure/freeze-substituted nodules, which showed significant labeling in the cytosol of both infected and uninfected ("interstitial") cells (Fig. 9). Gold particles in infected cells were counted and statistically analyzed (Table I). The low labeling (3.9 gold particles per 25 µm2) in bacteroids and negligible labeling (<0.8 gold particles per 25 µm2) in the mitochondria and peroxisomes were not significantly different from sections treated with non-immune serum. Similar counting results were obtained for uninfected cells. Additional controls, in which the antibody (1:100, v/v) was pre-adsorbed with increasing concentrations (35–700 µg protein mL–1) of VuFeSOD protein, showed a drastic (70%) reduction in labeling of infected cells (data not shown).
In this paper, we have studied in detail an FeSOD from cowpea. Comparison of the N-terminal sequence of the mature protein with the cDNA-derived sequence indicates that the precursor protein contains seven additional amino acids. Prediction programs for subcellular localization suggested that VuFeSOD is located to the cytosol. This was subsequently demonstrated by immunoblot analysis and by immunogold labeling using a monospecific antibody. Phylogenetic and immunological analyses showed that at least two types of FeSOD may exist in legumes, which are localized in the plastids (alfalfa and pea) or the cytosol (cowpea and common bean). However, soybean nodules probably contain two putative cytosolic FeSODs (isoenzymes 1 and 2, with no apparent signal peptide) and one putative plastid FeSOD (isoenzyme 3). In fact, more than three FeSOD isozymes can be detected in activity gels of soybean nodule extracts (J.F. Moran, M.C. Rubio, and M. Becana, unpublished data).
The subcellular localization of VuFeSOD is reminiscent of cyanobacteria, which contain an FeSOD in the cytosol and a MnSOD in the thylakoids (Obinger et al., 1998
Native VuFeSOD has an apparent molecular mass of 57 kD (Becana et al., 1989
Immunoblot analysis showed that there were important changes in VuFeSOD protein levels during nodule aging. The VuFeSOD protein was not detectable in young nodules but became abundant in mature and old nodules. This may be one reason why FeSOD was not previously detected in leaves of young cowpea plants (Corpas et al., 1991 In summary, this is the first conclusive report of the presence of an extra-plastidial FeSOD in higher plants. Phylogenetic and immunological analyses confirm the distinction between cytosolic and chloroplastic FeSODs. The VuFeSOD protein is more abundant in nodules with active Lb degradation, suggesting that the enzyme is induced by Fe and plays an antioxidative role with advancing senescence.
Plant Material and Treatments
Nodulated cowpea (Vigna unguiculata Walp. cv California blackeye no. 5 x Bradyrhizobium sp. [Vigna] strain 32H1) plants were grown for 46 d (vigorous vegetative stage) in pots containing 2:1 (v/v) perlite:vermiculite (three plants per pot) under controlled environment conditions (Matamoros et al., 1999
A cowpea nodule
The 5'-RACE procedure was used to verify the start codon for cowpea and soybean FeSOD cDNAs. This was found not to be necessary for the plastid FeSOD of pea nodules. RNA was extracted from cowpea nodules as described by Verwoerd et al. (1989
DNA sequencing was performed by the dideoxy method using an ABI Prism 377 automated sequencer and AmpliTaqDNA Polymerase (Applied Biosystems, Foster City, CA). Homology searches were performed with BLAST (Altschul et al., 1997
Primers (5'-GCATATGAATGTGGCTGGCATCA-3' and 5'-CGTCGACTATCATGCACTTAAAGCC-'3) containing, respectively, NdeI and SalI restriction sites (underlined), were designed to amplify by PCR a 700-bp fragment (encoding the mature protein) using the nodule cDNA library as a template. The PCR mix contained 0.2 µM primers, 240 µM dNTPs, 1.5 mM MgCl2, 0.05% (v/v) W-1 detergent (Invitrogen), and 1.25 units of Taq polymerase in a final volume of 25 µL of the PCR buffer (20 mM Tris-HCl, pH 8.4, and 50 mM KCl). PCR conditions were as follows: 95°C for 3 min; 35 cycles of 61°C for 45 s, 72°C for 1 min, and 94°C for 45 s; and a final extension at 72°C for 5 min. The PCR product was cloned into the linearized pCRII vector (Invitrogen), and the pCRII::VuFeSOD construct was digested with NdeI and SalI restriction enzymes and separated on agarose gels. The 700-bp fragment was gel extracted using the Geneclean kit and was directionally subcloned in the pET-28a(+) vector (Novagen, Madison, WI) into the NdeI and SalI sites. Cells of Escherichia coli DH5
SOD isozymes were separated in 15% (w/v) native-PAGE gels and activity bands were visualized by the method of Beauchamp and Fridovich (1971
Transformed E. coli BL21(DE3) cells, containing the pET-28a(+)::VuFeSOD construct, were grown at 37°C in Luria-Bertani broth containing kanamycin (100 µg mL–1) until the A600 reached 0.5. IPTG was then added at a final concentration of 1 mM. Cells were further incubated for 4 h, pelleted by centrifugation, and stored at –80°C until used. The cell paste was thawed on ice and resuspended in 10 mL of buffer containing 20 mM sodium phosphate (pH 7.5), 500 mM NaCl, 0.1% (v/v) Triton X-100, DNase I (25 µg mL–1), RNase (50 µg mL–1), pepstatin (1.25 µg mL–1), and leupeptin (1.25 µg mL–1). The cell suspension was incubated with lysozyme (2 µg mL–1) at 4°C for 15 min with gentle stirring, sonicated, and cleared by centrifugation at 48,000g at 4°C for 15 min. The resulting supernatant was chromatographed on a 5-mL HiTrap Chelating column (Amersham Biosciences, Uppsala), loaded with Ni, and equilibrated with 20 mM sodium phosphate (pH 7.5), 500 mM NaCl, and 0.1% (v/v) Triton X-100. The column was successively washed with 30 mL each of equilibrating buffer containing 0, 10, and 50 mM imidazole. A linear gradient from 50 to 500 mM imidazole (120 mL in total) was applied, and recombinant VuFeSOD was eluted at 160 mM imidazole. This was removed with Centricon C-10 filters, and fractions were directly stored at 4°C or made up to 0.5 mM EDTA and 50% (v/v) glycerol and stored at –80°C. The fused recombinant VuFeSOD containing a (His)6-tag was digested overnight at 4°C with thrombin (Sigma-Aldrich, St. Louis). Protein purification was monitored on native-PAGE gels (stained for SOD activity) and SDS-PAGE gels (stained with Coomassie Brilliant Blue R-250). Protein was determined by a dye-binding assay (Bio-Rad Laboratories, Hercules, CA) using bovine serum albumin as a standard.
For purification of native VuFeSOD, 60 g of cowpea leaves was homogenized at 4°C using an Omnimixer (Sorvall, Wilmington, DE) with 400 mL of an optimized SOD extraction buffer consisting of 50 mM potassium phosphate (pH 7.8), 0.1 mM EDTA, 0.1% (v/v) Triton X-100, and 1% (w/v) polyvinylpyrrolidone-10. The extract was filtered through cheesecloth and centrifuged at 12,000g at 4°C for 20 min to remove cell debris. Proteins were precipitated at 4°C between 35% and 65% (w/v) ammonium sulfate and resuspended in 25 mL of 10 mM potassium phosphate (pH 7.8). The suspension was dialyzed overnight at 4°C against the same buffer, centrifuged to remove precipitated proteins, and chromatographed on a DEAE cellulose column (2.5 x 8.5 cm; DE52, Whatman, Maidstone, Kent, UK) equilibrated with the same buffer. After loading the sample and washing the column with equilibrating buffer, the proteins were eluted with a linear gradient (400 mL in total) of 10 to 100 mM potassium phosphate (pH 7.8) at 1.2 mL min–1. Fractions containing most FeSOD activity (monitored by gel SOD staining as described above) were pooled, concentrated, and loaded on a preparative (2-mm thick) 15% (w/v) native-PAGE gel. The FeSOD activity band was cut out, and the protein was eluted, concentrated, and loaded on a 10% (w/v) SDS-PAGE gel. The protein was blotted onto a polyvinylidene difluoride membrane (Immobilon-Psq, Millipore, Bedford, MA) at 15 mV for 20 min with a Milliblot II graphite electroblotter (Millipore), and the 27-kD band was N-terminal sequenced. At later stages of the work, VuFeSOD was purified from 8 to 10 g of nodules by ammonium sulfate fractionation and anion-exchange chromatography, as described above. The protein, concentrated to 2.4 mL of 100 mM Tris-HCl (pH 7.8), was loaded on an affinity column (Actigel ALD, Sterogene, Carlsbad, CA), prepared as follows. Approximately 1.5 mg of anti-VuFeSOD antibody was coupled to 2 mL of resin in 100 mM potassium phosphate (pH 7.8). The coupling reagent was 100 mM NaCNBH3 and the reaction was left to proceed for 2 h at room temperature. The coupled resin was placed in a column and equilibrated with 100 mM Tris-HCl (pH 7.8) containing 1 M NaCl. After loading the sample, the column was washed with three column volumes of the same buffer. The protein was eluted with 2.5 mL of 200 mM Gly (pH 3.0), immediately buffered with 500 mM Tris-HCl (pH 8.0), concentrated, loaded on 10% (w/v) SDS-PAGE, and transferred to Immobilon-Psq using a mini-transblot unit (Bio-Rad Laboratories) for subsequent N-terminal sequencing.
Affinity-purified recombinant VuFeSOD (>3 mg) was used to raise polyclonal monospecific antibodies in rabbits following conventional immunization protocols (BioGenes, Berlin). The specificity of the anti-VuFeSOD antibody was assessed by immunoprecipitation (Miller et al., 1987
Genomic DNA was isolated from cowpea leaves using the cetyltrimethyl ammonium bromide method (Saghai-Maroof et al., 1984
Proteins were extracted in SOD medium, separated in 10% (w/v) SDS-PAGE, and electroblotted to polyvinylidene difluoride membranes. In some cases, 15% (w/v) native-PAGE gels were used for immunoblots. Membranes were blocked with 5% (w/v) skim milk in 20 mM Tris-buffered saline, at 4°C overnight. The primary antibody was anti-VuFeSOD (1:1,000, v/v) or anti-Lb (1:1,500, v/v). The secondary antibody was goat anti-rabbit IgG alkaline phosphatase conjugate (1:30,000, v/v; Sigma-Aldrich) or horseradish peroxidase conjugate (1:20,000, v/v; Sigma-Aldrich). Immunoreactive bands were visualized with the nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate system (Sigma-Aldrich) or with a chemiluminescent substrate (Pierce Chemical, Rockford, IL), respectively. Controls were run with rabbit serum to check for nonspecific adsorption of primary antibody.
To determine whether VuFeSOD is in the soluble fraction or associated with membranes, cowpea nodules (1 g) were homogenized with a mortar and pestle at 4°C with 5 mL of an iso-osmotic medium consisting of 0.3 M mannitol, 1 mM EDTA, and 20 mM potassium phosphate (pH 7.8). The extract was filtered through Miracloth and centrifuged successively at 15,000g for 20 min ("extract"), and at 100,000g for 1 h ("cytosol"). The pellet ("membranes") was washed with iso-osmotic medium and was resuspended in 20 mM potassium phosphate (pH 7.8), 1 mM EDTA, and 0.1% (v/v) Triton X-100. Immunoblot analysis of the three nodule fractions was performed after 10% (w/v) SDS-PAGE as described above.
For immunogold localization of VuFeSOD by electron microscopy, mature cowpea nodules were fixed in 2.5% (v/v) glutaraldehyde in 100 mM sodium cacodylate buffer (pH 7.0). Nodule slices (200 µm) were taken on a Vibratome 1000 (Agar Scientific, Stansted, UK). One-millimeter-diameter discs were punched from the slices, and air was extracted from the discs under mild vacuum while they were immersed in 1-hexadecene (Leica, Wetzlar, Germany) The discs were then high-pressure frozen according to Studer et al. (2001
Upon request, all novel materials described in this publication will be made available in a timely manner, and in limited amounts, for noncommercial research purposes, subject to the requisite permission from any third-party owners of all or parts of the material. Obtaining any permissions will be the responsibility of the requestor.
We thank Carroll Vance for kindly supplying the pea nodule cDNA library and two anonymous reviewers and Frank Minchin for helpful comments and English correction. Received March 5, 2003; returned for revision April 11, 2003; accepted May 17, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.023010.
1 This work was supported by the Dirección General de Investigación (Ministerio de Ciencia y Tecnología, Spain; grant nos. PB98–0522 and AGL2002–02876). J.F.M. and M.C.R. were the recipients, respectively, of a postdoctoral contract and a postdoctoral fellowship funded by the Ministerio de Ciencia y Tecnología-European Union (I3P program).
2 In memoriam of Prof. Robert V. Klucas, our mentor and friend.
3 Present address: Departamento de Ciencias del Medio Natural, Universidad Pública de Navarra, 31006 Pamplona, Spain.
4 Present address: U.S. Department of Agriculture-Agricultural Research Service, University of Nebraska, Lincoln, NE 68583–0939. * Corresponding author; e-mail becana{at}eead.csic.es; fax 34–976–716145.
Almansa MS, del Río LA, Sevilla F (1994) Characterization of an iron-containing superoxide dismutase from a higher plant, Citrus limonum. Physiol Plant 90: 339–347[CrossRef]
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389–3402 Arredondo-Peter R, Moran JF, Sarath G, Klucas RV (1997) Analysis of Vigna unguiculata leghemoglobin genes. R Bras Fisiol Veg 9: 143–149 Beauchamp C, Fridovich I (1971) Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 44: 276–287[CrossRef][Web of Science][Medline]
Becana M, Klucas RV (1992) Transition metals in legume root nodules: iron-dependent free radical production increases during nodule senescence. Proc Natl Acad Sci USA 89: 8958–8962
Becana M, Paris FJ, Sandalio LM, del Río LA (1989) Isoenzymes of superoxide dismutase in nodules of Phaseolus vulgaris L., Pisum sativum L., and Vigna unguiculata (L) Walp. Plant Physiol 90: 1286–1292 Bowler C, Van Camp W, Van Montagu M, Inzé D (1994) Superoxide dismutase in plants. Crit Rev Plant Sci 13: 199–218
Bridges SM, Salin ML (1981) Distribution of iron-containing superoxide dismutase in vascular plants. Plant Physiol 68: 275–278
Clare DA, Blum J, Fridovich I (1984) A hybrid superoxide dismutase containing both functional iron and manganese. J Biol Chem 259: 5932–5936
Claros MG (1995) MitoProt, a Macintosh application for studying mitochondrial proteins. Comput Appl Biosci 11: 441–447 Corpas FJ, Sandalio LM, Palma JM, Leidi EO, Hernández JA, Sevilla F, del Río LA (1991) Subcellular distribution of superoxide dismutase in leaves of ureide-producing leguminous plants. Physiol Plant 82: 285–291[CrossRef]
Crowell DN, Amasino RM (1991) Induction of specific mRNAs in cultured soybean cells during cytokinin or auxin starvation. Plant Physiol 95: 711–715
Droillard MJ, Paulin A (1990) Isozymes of superoxide dismutase in mitochondria and peroxisomes isolated from petals of carnation (Dianthus caryophyllus) during senescence. Plant Physiol 94: 1187–1192 James EK, Iannetta PPM, Nixon PJ, Whiston AJ, Peat L, Crawford RMM, Sprent JI, Brewin NJ (1996) Photosystem II and oxygen regulation in Sesbania rostrata stem nodules. Plant Cell Environ 19: 895–910[CrossRef]
Kliebenstein DJ, Monde RA, Last RL (1998) Superoxide dismutase in Arabidopsis: an eclectic enzyme family with disparate regulation and protein localization. Plant Physiol 118: 637–650 Kurepa J, Bueno P, Kampfenkel K, Van Montagu M, Van den Bulcke, Inzé D (1997) Effects of iron deficiency on iron superoxide dismutase expression in Nicotiana tabacum. Plant Physiol Biochem 35: 467–474 Luan P, Aréchaga-Ocampo E, Sarath G, Arredondo-Peter R, Klucas RV (2000) Analysis of a ferric leghemoglobin reductase from cowpea (Vigna unguiculata) root nodules. Plant Sci 154: 161–170[Medline]
Matamoros MA, Moran JF, Iturbe-Ormaetxe I, Rubio MC, Becana M (1999) Glutathione and homoglutathione synthesis in legume root nodules. Plant Physiol 121: 879–888
Miller SS, Boylan KLM, Vance CP (1987) Alfalfa root nodule carbon dioxide fixation: III. Immunological studies of nodule phosphoenolpyruvate carboxylase. Plant Physiol 84: 501–508 Nakai K, Kanehisa M (1992) A knowledge base for predicting protein localization sites in eukaryotic cells. Genomics 14: 897–911[CrossRef][Web of Science][Medline] Obinger C, Regelsberger G, Pircher A, Strasser G, Peschek GA (1998) Scavenging of superoxide and hydrogen peroxide in blue-green algae (cyanobacteria). Physiol Plant 104: 693–698[CrossRef]
Ogawa KI, Kanematsu S, Asada K (1996) Intra- and extra-cellular localization of "cytosolic" CuZn-superoxide dismutase in spinach leaf and hypocotyl. Plant Cell Physiol 37: 790–799 Roberts IM (2002) Iso-butanol saturated water: a simple procedure for increasing staining intensity of resin sections for light and electron microscopy. J Microsc 207: 97–107[Medline] Rubio MC, Ramos J, Webb KJ, Minchin FR, Gonzalez E, Arrese-Igor C, Becana M (2001) Expression studies of superoxide dismutases in nodules and leaves of transgenic alfalfa reveal abundance of iron-containing isozymes, posttranslational regulation, and compesation of isozymes activities. Mol Plant-Microbe Interact 14: 1178–1188[Medline]
Saghai-Maroof MA, Soliman KM, Jorgensen RA, Allard RW (1984) Ribosomal DNA spacer-length polymorphisms in barley: Mendelian inheritance, chromosomal location, and population dynamics. Proc Nat Acad Sci USA 81: 8014–8018 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY Sandalio LM, Palma JM, del Río LA (1987) Localization of manganese-superoxide dismutase in peroxisomes isolated from Pisum sativum L. Plant Sci 51: 1–8 Srivalli B, Khanna-Chopra R (2001) Induction of new isoforms of superoxide dismutase and catalase enzymes in the flag leaf of wheat during monocarpic senescence. Biochem Biophys Res Commun 288: 1037–1042[CrossRef][Web of Science][Medline] Studer D, Graber W, Al-Amoudi A, Eggli P (2001) A new approach for cryofixation by high pressure freezing. J Microsc 203: 285–294[Web of Science][Medline]
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22: 4673–4680
Tsang EWT, Bowler C, Hérouart D, Van Camp W, Villarroel R, Genetello C, Van Montagu M, Inzé D (1991) Differential regulation of superoxide dismutases in plants exposed to environmental stress. Plant Cell 3: 783–792
Van Camp W, Bowler C, Villarroel R, Tsang EWT, Van Montagu M, Inzé D (1990) Characterization of iron superoxide dismutase cDNAs from plants obtained by genetic complementation in Escherichia coli. Proc Natl Acad Sci USA 87: 9903–9907 VandenBosch KA, Newcomb EH (1988) The occurrence of leghemoglobin protein in the uninfected interstitial cells of soybean root nodules. Planta 175: 442–451
Verwoerd TC, Dekker BMM, Hoekema A (1989) A small scale procedure for the rapid isolation of plant RNAs. Nucleic Acids Res 17: 2362 This article has been cited by other articles:
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-galactoside (IPTG) induction (60 µg of protein). Lane 4, Recombinant VuFeSOD after affinity-chromatography purification (5 µg of protein). Lane 5, Same as lane 4 treated with thrombin (5 µg of protein). B, Native gel (15% [w/v] acrylamide) stained with Coomassie. Lane 1, Wild-type BL21 cells (60 µg of protein). Lane 2, Transformed cells after 1-h induction with IPTG (60 µg of protein). Lane 3, Purified recombinant VuFeSOD (5 µg of protein). Lane 4, Same as lane 3 treated with thrombin (5 µg of protein).
gt11 cDNA library (Luan et al., 2000
(BD Biosciences Clontech, Palo Alto, CA) and BL21(DE3) (Invitrogen) were transformed with the resultant pET-28a::VuFeSOD construct. Positive colonies were selected with kanamycin (100 µg mL–1) in Luria-Bertani plates. 
