Plant Physiol. (1998) 118: 637-650

Superoxide Dismutase in Arabidopsis: An Eclectic Enzyme Family with Disparate Regulation and Protein Localization¹

Daniel J. Kliebenstein, Rita-Ann Monde, and Robert L. Last^{2, *}

Boyce Thompson Institute for Plant Research, and Section of Genetics and Development (D.J.K., R.L.L.), and Section of Biochemistry, Molecular and Cellular Biology (R.-A.M.), Cornell University, Tower Road, Ithaca, New York 14853-1801

	ABSTRACT

Top Abstract Introduction Methods Results Discussion References

A number of environmental stresses can lead to enhanced production of superoxide within plant tissues, and plants are believed to rely on the enzyme superoxide dismutase (SOD) to detoxify this reactive oxygen species. We have identified seven cDNAs and genes for SOD in Arabidopsis. These consist of three CuZnSODs (CSD1, CSD2, and CSD3), three FeSODs (FSD1, FSD2, and FSD3), and one MnSOD (MSD1). The chromosomal location of these seven SOD genes has been established. To study this enzyme family, antibodies were generated against five proteins: CSD1, CSD2, CSD3, FSD1, and MSD1. Using these antisera and nondenaturing-polyacrylamide gel electrophoresis enzyme assays, we identified protein and activity for two CuZnSODs and for FeSOD and MnSOD in Arabidopsis rosette tissue. Additionally, subcellular fractionation studies revealed the presence of CSD2 and FeSOD protein within Arabidopsis chloroplasts. The seven SOD mRNAs and the four proteins identified were differentially regulated in response to various light regimes, ozone fumigation, and ultraviolet-B irradiation. To our knowledge, this is the first report of a large-scale analysis of the regulation of multiple SOD proteins in a plant species.

	INTRODUCTION

Top Abstract Introduction Methods Results Discussion References

All aerobic organisms are continuously subjected to potentially destructive ROS, including superoxide (O₂), lipid peroxides (ROO·), H₂O₂, and the highly reactive hydroxyl radical (OH·). These ROS are generated by metabolic processes and their concentrations can be increased by environmental stimuli. To prevent ROS from damaging cellular components, organisms have evolved multiple detoxification mechanisms, including the synthesis of low-M_r antioxidant molecules (e.g. L-ascorbic acid and glutathione) and various enzymes. O₂ is an abundant ROS that is formed by univalent electron transfer to O₂ and can contribute to the synthesis of OH·, so control of this ROS is essential (Cadenas, 1989; Halliwell and Gutteridge, 1990).

SODs catalyze the conversion of O₂ to H₂O₂. Three classes of SOD activity have been identified that differ by the active site metal cofactors (Fe, Mn, or Cu and Zn). The primary sequences of FeSOD and MnSOD apoproteins are related, whereas CuZnSOD is distinct. Fungi and animals have CuZnSOD and MnSOD, whereas some plants and bacteria have been demonstrated to contain all three forms (Bowler et al., 1992). The plant SOD isoenzymes also differ in their subcellular location. Typically, MnSOD is mitochondrial, FeSOD is plastidic, and CuZnSOD can be plastidic or cytosolic (Bowler et al., 1992). There are also reports of peroxisomal and extracellular SODs (Streller and Wingsle, 1994; Bueno et al., 1995).

The importance of SOD has been demonstrated by analysis of mutants in microbes and animals. SOD mutants in Escherichia coli, Saccharomyces cerevisiae, Neurospora crassa, and Drosophila melanogaster all exhibit increased sensitivity to methyl viologen (paraquat), a redox-active compound that enhances the production of O₂ (Carlioz and Touati, 1986; Phillips et al., 1989; Gralla and Valentine, 1991; Chary et al., 1994). SOD is also essential for DNA integrity and normal life span: the E. coli and N. crassa mutations cause an increased spontaneous mutation rate, whereas the D. melanogaster mutant has a significantly shorter life span then the wild type (Carlioz and Touati, 1986; Phillips et al., 1989; Chary et al., 1994). Mutations in human and mouse CuZnSOD have been linked to the disease familial amyotrophic lateral sclerosis, which is characterized by premature neuron death (Rosen et al., 1993). Taken together, this evidence indicates a vital role for SOD in preventing ROS-generated cell damage and death in aerobically growing organisms. SOD is also thought to be important in converting O₂ to H₂O₂ during the pathogen-induced oxidative burst in animal phagocytic immune cells and in plant cells (Desikan et al., 1996; Babior et al., 1997).

In plants exposure to photoinhibitory light, ozone, or other environmental conditions that cause oxidative stress can increase O₂ levels (Yruela et al., 1996; Runeckles and Vaartnou, 1997); however, it is not clear whether SOD plays an essential role in attenuating plant oxidative stress in these situations. To date the protective role of SOD in plants has been explored by transgenic approaches, primarily through overexpression or by correlation of SOD expression to the degree of oxidative stress resistance (Bowler et al., 1994; Alscher et al., 1997; Scandalios, 1997). Both approaches have yielded inconclusive and sometimes contradictory results about the role of SOD in plant oxidative stress responses (Bowler et al., 1994; Alscher et al., 1997; Scandalios, 1997).

The existence of three classes of SOD enzymes, each typically encoded by a small gene family, complicates the elucidation of the roles of SOD in plants. This situation is exacerbated by the fact that past work has generally focused on one member of a gene family. To circumvent this problem, we have initiated a thorough analysis of the Arabidopsis SOD genes. The availability of large numbers of cDNA and genomic DNA sequences in Arabidopsis (Newman et al., 1994; Rounsley et al., 1996; Delseny et al., 1997) has made it possible to identify the complete SOD arsenal of this plant. We describe progress toward the detailed understanding of SOD genes and their functions in Arabidopsis, and report on the sequencing of three SOD cDNAs and the map locations of seven SOD structural genes. Information is also provided on the characterization of antisera against five of the seven SOD proteins. Finally, we describe the regulation of SOD activity and mRNA and protein levels in response to ozone, UV-B light, and variations in incident light fluences.

	MATERIALS AND METHODS

Top Abstract Introduction Methods Results Discussion References

Strains and Materials

2

1

CuZnSOD2 Microsatellite

CICYAC Pool PCR

MSD1 CAPS

RNA and Immunoblot Techniques

Construction of Fusion Protein Plasmids and Antisera Production

The MSD1, FSD1, and CSD2 cDNAs generated above were ligated into pQE32 to create pHIS-MSD1, pHIS-FSD1, and pHIS-CSD2. The CSD3 cDNA isolated above was ligated into pQE31 to create pHIS-CSD3. The PCR primers CuZnSOD1-StuI and CuZnSOD1-HindIII (Table I) were used to amplify the CSD1 ORF, which was cloned into pQE30 to create pHIS-CSD1. The His-tagged fusion proteins were purified by urea solubilization and nickel chromatography, as described by the manufacturer (Qiaexpressionist Handbook, Qiagen). All pHIS strains contained a novel, inducible SOD activity that could be seen by gel analysis, indicating that the expressed fusion proteins were active SOD enzymes (D.J. Kliebenstein and R.L. Last, unpublished results).

SOD Activity Gels

SOD Activity Fractionation

Plastid Purification

Circadian Regulation and Plastid Isolation Tissue Growth Conditions

2

1

Photoinhibition Studies

2

1

2

1

Ozone Fumigations

UV Treatments

2

1

2

1

	RESULTS

Top Abstract Introduction Methods Results Discussion References

Arabidopsis SOD Gene Family

The CSD2 cDNA contains a 651-nucleotide ORF that encodes a predicted 217-amino acid protein containing all of the conserved amino acids for a CuZnSOD protein (Bordo et al., 1994). Two lines of evidence indicate that this protein is plastid targeted. First, phylogenetic analysis indicates that the CSD2 amino acid sequence is most closely related to other known or proposed plastid-targeted CuZnSODs, as shown in Figure 1A. Second, there is an amino-terminal extension that has no homology to nonplant CuZnSOD enzymes. This region has the characteristics of typical plastid-transit peptides in that it is positively charged and rich in hydroxylated amino acids (Keegstra et al., 1989).

View larger version (21K): [in this window] [in a new window]   Figure 1. Evolutionary relationships inferred from SOD sequences. Accession numbers for the sequences utilized are listed in Table II. A, Dendrogram of plant CuZnSOD amino acid sequences. The predicted amino acid sequences were analyzed utilizing a DNASTAR (Madison, WI) program, starting at Lys-Ala-Val-Ala-Val-Leu or a homologous sequence to eliminate any amino-terminal transit sequences. B, Alignment of the first 18 amino acids and conserved carboxy-terminal amino acids of the proteins used for the dendogram in A to illustrate cladistic relationships of the plant CuZnSODs. Underlined amino acids indicate a putative peroxisomal targeting sequence. prx, Peroxisome; cyt, cytosol; ec, extracellular. C, Dendogram of FeSOD and MnSOD amino acid sequences. The amino acid sequences were analyzed from the start of homology to the E. coli FeSOD sequence.

The CSD3 cDNA contains a 465-nucleotide ORF. The deduced protein sequence is 164-amino acids long and contains all of the conserved amino acids for a functional CuZnSOD protein (Bordo et al., 1994). Additionally, the carboxyl terminus has an Ala-Lys-Leu tripeptide that can act as a peroxisomal targeting sequence (Keller et al., 1991), leading us to propose that CSD3 is a putative peroxisomal CuZnSOD. The CSD3 protein does not fall within either the cytosolic or plastidic CuZnSOD clade and forms a separate group with CuZnSOD2 from the ice plant (Fig. 1, A and B). This cladistic divergence is further illustrated by the fact that CSD3 is 70% identical to the ice plant CuZnSOD2, whereas it is at most 65% identical to individuals in either the cytosolic or plastidic groups. In contrast, the plastidic CuZnSODs are 90% identical to each other and the cytosolic CuZnSODs are 80% identical to each other. A previously published watermelon peroxisomal CuZnSOD (melon-prx) appears to be more closely related to plastidic CuZnSODs then to CSD3 (Fig. 1, A and B; Bueno et al., 1995).

As shown in Table II, published studies have identified four FeSOD cDNAs in plants, including the Arabidopsis FSD1. We identified two additional FeSOD cDNAs in the Arabidopsis DNA-sequencing projects. FSD2 is represented by an incomplete cDNA containing an ORF that encodes a protein homologous to the carboxy-terminal half of published plant FeSODs. Due to the truncated nature of the cDNA we did not completely characterize this sequence. The proposed FSD3 cDNA contains a 798-nucleotide ORF encoding a predicted 263-amino acid protein with an amino-terminal peptide extension characteristic of known plastid-transit peptides (Keegstra et al., 1989). Phylogenetic analysis indicates that the inferred FSD3 protein does not evolutionarily cluster with the other plant FeSODs, as it is only 50% identical to the known plant FeSODs, which are 70% to 75% identical to each other (Fig. 1C). However, FSD3 does contain all of the amino acids described by Lah et al. (1995) as conserved between FeSODs, strongly suggesting that it is a plastidic FeSOD protein.

The MSD1 cDNA contains a 691-nucleotide ORF that encodes a 231-amino acid hypothetical protein that is 80% to 90% identical to other plant MnSODs and contains all of the conserved amino acids required for MnSOD activity (Fig. 1C; Lah et al., 1995). Additionally, this protein contains an amino-terminal peptide that is basic and rich in hydroxylated residues, characteristic of mitochondrial transit peptides (Neupert, 1997). Therefore, MSD1 is thought to encode a mitochondrial MnSOD.

Map Positions of the Known SOD Structural Genes

View larger version (11K): [in this window] [in a new window]   Figure 2. Genetic map positions of known Arabidopsis SOD structural genes. The structural genes for the known SOD proteins were mapped to the Arabidopsis genetic map as described in the text. The numbers given are approximate positions on the February 27, 1998 Lister and Dean Recombinant Inbred map for CSD1, CSD2, CSD3, FSD1, FSD2, FSD3, and MSD1 (http://nasc.nott.ac.uk/new_ri_map.html).

To map CSD3 and the other SOD loci, PCR primers were designed that specifically amplified the SOD genes from pooled CICYAC clones in a multiplex collection of CICYAC DNA (Creusot et al., 1995). These oligonucleotides are listed in Table I. The identified CICYAC address was compared with existing Arabidopsis physical maps to obtain a genomic location. CSD3 is located on CICYAC clones 9H7, 9E10, and 9A8, which are between markers nga151 and GDH on chromosome V (Schmidt et al., 1997). This location was confirmed by genomic sequencing of the P1 element MiRG7 (accession no. AB012246). FSD1 was found on CICYACs 6B12 and 1G9, which are on chromosome IV between markers mi422 and SEP2B (Schmidt et al., 1995). FSD2 was identified on CICYACs 11F9 and 11F10, on chromosome V between nga129 and m435 (Schmidt et al., 1995). MSD1 was amplified from CICYACs 3E3, 3A3, 5D8, and 12H8, but none of these were physically mapped. To map MSD1, a CAPS marker (Konieczny and Ausubel, 1993) was identified between ecotype Columbia and Landsberg erecta within the MSD1 genomic sequence, as described in ``Materials and Methods''. This marker was mapped to chromosome III, which is 6.5 cM proximal of GAPC and 2 cM distal of nga126, using the Lister and Dean recombinant inbred lines (Lister and Dean, 1993). FSD3 was identified by genomic sequencing of the P1 clone MiKD15 (accession no. AB007648), which lies on chromosome V between markers m291 and g4556 (Kazusa Institute).

Multiple SOD mRNAs in Arabidopsis Rosette Leaves

Multiple SOD Isoenzymes in Arabidopsis

The SOD antisera were tested against Arabidopsis leaf protein extracts. As shown in Figure 3, A and B, line 1, anti-FSD1 and anti-MSD1 antiserum each detected a protein with an apparent molecular mass of 23 kD, close to the expected 22- and 23-kD sizes for FeSOD and MnSOD, respectively, as defined by the start of homology to bacterial proteins (Lah et al., 1995). At a 1:5000 antiserum dilution, nonaffinity-purified anti-CSD2 detected a protein with an apparent molecular mass of 20 kD, which agrees with the 20-kD protein predicted to be encoded by the CSD2 cDNA with the intact transit peptide (Fig. 3C). At a 1:500 antiserum dilution, affinity-purified anti-CSD1 detected a protein of 15-kD apparent molecular mass, the size predicted for the CSD1 protein based on conceptual translation of the cDNA ORF (Fig. 3F). These results suggest that the 15-kD protein is encoded by CSD1, whereas the 20-kD polypeptide corresponds to the CSD2 protein.

View larger version (50K): [in this window] [in a new window]   Figure 3. Test of SOD antisera specificity. Twenty-five micrograms of total protein from Arabidopsis rosette leaves and 12 ng of the purified 6× His-tagged MSD1, FSD1, CSD1, CSD2, and CSD3 proteins were run on SDS-PAGE and subjected to immunoblot analysis. Postions of molecular-mass markers are listed on the left of each row of gels. The antisera and dilution (v/v) used are listed at the bottom of each gel. AP, Affinity-purified antiserum.

The specificity and reactivity of these antisera was assessed further using the SOD proteins with His₆ attached to their amino termini. Consistent with our having obtained reagents of high reactivity and good specificity, each antiserum recognized the appropriate purified His₆-tagged fusion proteins when used at the high dilutions (Fig. 3, A-C, E, and F). At these low serum concentrations, we observed minimal cross-reactivity with the other His₆-tagged CuZnSOD fusion proteins (Fig. 3, C, E, and F). However, in one unique case, affinity-purified anti-CSD1 did cross-react with His₆-CSD3 at the high 1:500 dilution (Fig. 3F). The experiment was repeated with increased antisera concentrations to investigate potential cross-reactivity of the CSD antisera. As the antiserum concentration increased, all CSD antisera became fairly promiscuous, showing cross-recognition of the other CSD proteins. For example, when the CSD2 antiserum dilution was decreased from 1:5000 to 1:2000, there was cross-recognition of the His₆-CSD1 and His₆-CSD3 protein (Fig. 3D).

Several lines of evidence support the hypothesis that the 15-kD protein is encoded by CSD1 and the 20-kD polypeptide by CSD2. First, affinity purification of the CSD1 antiserum increased the specificity for the 15-kD CSD1 protein (Fig. 3, compare F and G). The second line of evidence comes from transgenic Arabidopsis plants containing an antisense CSD1 construct driven by the cauliflower mosaic virus 35S promoter, which exhibit reduced accumulation of CSD1 mRNA and the 15-kD protein (D.J. Kliebenstein, L.G. Landry, and R.L. Last, unpublished data). Transgenic Arabidopsis containing a transgene with CSD1 sense expression controlled by the cauliflower mosaic virus 35S promoter have increased levels of CSD1 mRNA and 15-kD protein.

Analysis of 35S-CSD1 sense and antisense lines also yielded circumstantial evidence for the hypothesis that the immunologically detected 20-kD protein is encoded by CSD2 (D.J. Kliebenstein, L.G. Landry, and R.L. Last, unpublished data). The CSD1 antisense lines have increased accumulation of both the CSD2 mRNA and the 20-kD protein. Furthermore, both the CSD2 mRNA and 20-kD protein are decreased in the CSD1 overexpression lines. These results also indicate that there may be a form of compensatory regulation between CSD1 and CSD2.

At a 1:1000 dilution, anti-CSD3 detected several high-molecular-mass proteins from whole leaf extracts, but no protein in the 15- to 20-kD range as predicted by DNA sequence analysis of CSD3 (Fig. 3E). At higher concentrations anti-CSD3 occasionally detected proteins of 20-, 17-, and 15-kD apparent molecular mass, similar to the other CSD antisera (D.J. Kliebenstein and R.L. Last, unpublished results). Two lines of evidence indicate that CSD3 may encode the 17-kD protein. First, anti-CSD3 was highly specific for the His₆-CSD3 fusion protein and recognized the 17-kD protein more strongly than either anti-CSD1 or anti-CSD2. Second, affinity purification of the CSD3 antiserum decreased detection of the 15- and 20-kD proteins but not the 17-kD protein. However, even after affinity purification, the 15- and 20-kD signals were still stronger than the faint 17-kD signal. We believe that this faint signal was due to low endogenous levels of the protein, because we were able to easily detect 4 ng of the purified His₆-CSD3 protein. This is consistent with the low levels of detectable mRNA for CSD3. Regardless of the reason, we found inconsistent detection of the 17-kD protein in Arabidopsis leaf extracts and did not further characterize the CSD3 antiserum.

To confirm the specificity of the SOD antisera, total leaf protein extract was separated by two-dimensional PAGE. As shown in Figure 4, anti-CSD1 detects three different isoelectric variants, each with an apparent molecular mass of 15 kD, and anti-CSD2 recognizes one isoelectric variant of the 20-kD protein. Two-dimensional-PAGE analysis with the CSD1 antisense and overexpression transgenic lines were consistent with the 15- and 20-kD variants being derived from the CSD1 and CSD2 genes, respectively (D.J. Kliebenstein and R.L. Last, unpublished results). Anti-MSD1 identified only one 23-kD isoelectric variant in Arabidopsis leaf extracts, whereas the antiserum generated against GST-FSD1 recognizes three isoelectric variants at a 23-kD apparent molecular mass (Fig. 4). The available data do not allow us to assess whether these three isoelectric variants are encoded by distinct genes, so we refer to this antiserum as anti-FSD.

View larger version (47K): [in this window] [in a new window]   Figure 4. SOD isoelectric variants in Arabidopsis rosette leaves. Twenty-five micrograms of total protein from Arabidopsis rosette leaves was separated by IEF in the first dimension and by SDS-PAGE in the second dimension prior to immunoblot analysis. Arrowheads indicate the SOD isoelectric variants consistently detected.

Four Major SOD Activities in Arabidopsis Rosettes

View larger version (36K): [in this window] [in a new window]   Figure 5. Characterization of the major Arabidopsis SOD activities. A, Forty micrograms of total protein from Arabidopsis rosette tissue was fractionated on a nondenaturing PAGE gel and stained for SOD activity (clear gel regions). Gels were preincubated with KCN (which inhibits CuZnSOD) or H2O2 (which inhibits both CuZnSOD and FeSOD) to facilitate identification of the different activities. Asterisks mark the location of two minor activities that were seen only occasionally. B, Graphic representation of the experiment shown in Figure 5C. SOD activity gels were cut into slices, the slices were boiled in SDS loading buffer to elute and denature proteins, and aliquots were run on SDS-PAGE and immunodetected. C, Immunoblots of SOD activity gel fractions as illustrated in B. Antisera used are listed to the left.

To confirm the provisional identification based upon SOD inhibitor sensitivity the activity gel was fractionated, and the proteins were separated by SDS-PAGE and subjected to immunoblot analysis (the experiment is shown schematically in Fig. 5B). Anti-MSD1 and anti-FSD recognized proteins from the expected regions of the activity gel, lending further support for the identification of the two activities as MnSOD and FeSOD (Fig. 5C). The 20-kD CSD2 protein is enriched in the slower-migrating section of the diffuse CuZnSOD activity doublet, whereas the 15-kD CSD1 protein is more prominent in the faster-migrating area.

Arabidopsis Plastids Contain FeSOD and CuZnSOD2

View larger version (69K): [in this window] [in a new window]   Figure 6. Copurification of CSD2 and FSD proteins with Arabidopsis chloroplasts. Plastids were purified from rosette leaves, run on SDS-PAGE, and immunodetected with the antisera listed to the left. Cp, Intact chloroplast fraction; Total, total cell extract; APX, ascorbate peroxidase; ASA, anthranilate synthase alpha. Equal protein was loaded based on the level of Rubisco large subunit protein.

FSD1 mRNA Is under Circadian Control

View larger version (31K): [in this window] [in a new window]   Figure 7. Circadian oscillation of FSD1 mRNA. Total RNA was isolated from tissue collected between 8 AM and midnight from plants grown for 21 d in a 9-h photoperiod (lights were on from 8 AM to 5 PM) and subjected to RNA blot hybridization analysis conducted using an FSD1 cDNA probe. The open bar at the top of the figure represents samples harvested during the illuminated period; the dark bar represents darkness. The time is presented on a 24-h time scale.

SOD Accumulation in Response to Growth under Differing Light Fluences

2

1

2

1

2

1

2

1

View larger version (70K): [in this window] [in a new window]   Figure 8. Regulation of SOD mRNAs and proteins in response to growth under varied light fluences. Arabidopsis seeds were germinated for 2 d in constant light at 100 µmol m2 s1 PAR, and then moved to a different chamber, where the incident light was adjusted to 60, 120, 250, or 500 µmol m2 s1 PAR with cheesecloth and nylon mesh. The plants were grown under this fluence for 2 weeks, and tissue was collected for analysis. A, RNA-blot hybridization using the cDNAs listed on the left as probes. B, Immunoblots of protein extracted from plants grown in the same pots that were used for the RNA blots in A. The proteins were immunodetected using the antisera listed.

The same tissue was analyzed for variation in SOD protein accumulation. Levels of CSD1 protein significantly increased between the 60 and 125 µmol m² s¹ PAR samples, reflecting the mRNA, but showed no further increase at the higher light levels (Fig. 8B). The CSD2 protein did not mirror the CSD2 mRNA, as the protein showed no striking differences in accumulation between the four fluences (Fig. 8B). The FSD polypeptides increased slightly in the 250 µmol m² s¹ PAR sample and to a greater extent at 500 µmol m² s¹ PAR (Fig. 8B). As predicted from the mRNA, levels of MSD1 protein did not change throughout the experiment (Fig. 8B). Based upon activity gels, the SOD activities showed no significant variation throughout the experiment (D.J. Kliebenstein and R.L. Last, unpublished results).

Short-Term High-Light Treatment Causes Changes in SOD mRNA Accumulation

2

1

2

1

1

View larger version (48K): [in this window] [in a new window]   Figure 9. Regulation of SODs in response to photoinhibitory light. Plants were grown for 2 weeks in 70 µmol m2 s1 PAR and exposed to 1750 µmol m2 s1 PAR for 4 h. Controls were placed in the same chamber covered with filters to maintain the incident light at 70 µmol m2 s1 PAR. All plants were returned to the original chamber for the recovery period. Shown are RNA-blot hybridizations using the cDNAs listed on the left as probes. A, Atmospheric conditions (CO2 at 335-360 µg mL1); B, CO2 at 2250 µg mL1.

In contrast to CSD3, mRNA for the plastidic CSD2 only gradually increased following the high-light pulse (Fig. 9A). Of the two plastidic FeSODs tested, FSD2 mRNA displayed a strong increase only at the 12-h time point, whereas the diurnal rhythm of FSD1 mRNA was unaltered by the high-light pulse (Fig. 9A). MSD1 and CSD1 mRNA showed no significant variation throughout the experiment (Fig. 9A). Again, the SOD proteins and activities did not alter throughout this treatment (D.J. Kliebenstein and R.L. Last, unpublished results).

SOD Regulation in Response to Ozone-Mediated Oxidative Stress.

View larger version (84K): [in this window] [in a new window]   Figure 10. Regulation of SOD isoenzyme expression in response to ozone fumigation. Plants were fumigated with 330 ppb ozone for 8 h. C represents unfumigated control plants maintained in charcoal-filtered air. The ozone-treated plants were returned to charcoal-filtered air during the recovery period. A, RNA-blot hybridization using the cDNAs listed on the left as probes. B, Immunoblots of protein extracted from plants grown in the same pots as used for the RNA blots in A. The proteins were immunodetected using the antisera listed on the left.

In contrast, mRNAs for two plastid-targeted SODs decreased in response to ozone. FSD1 mRNA decreased within 4 h after the start of the fumigation and recovered to nearly prefumigation levels between 48 and 72 h after the fumigation. This decrease is even more dramatic compared with the 4-h control, because this should be near the maximum of the circadian oscillation in untreated plants. The CSD2 gene, which also encodes a plastidic protein, showed a pattern similar to FSD1 (Fig. 10A). In contrast, the mRNAs detected with the FSD2 and MSD1 probes did not show any reproducible changes in response to ozone exposure (Fig. 10A).

The same tissue was used to analyze the regulation of the SOD proteins and enzyme activities in response to ozone fumigation. CSD1 protein began to increase during the fumigation, reaching a plateau 48 h after the start of the ozone treatment (Fig. 10B). In contrast, CSD2 protein showed no response until an increase in the protein level at the 48- and 72-h time points, whereas the FSD protein showed a slight decrease in protein accumulation 24 to 48 h after the onset of the ozone fumigation (Fig. 10B). MSD1 protein did not vary throughout the experiment, as predicted from the mRNA results (Fig. 10B). None of the SOD activities showed any significant changes throughout the experiment (D.J. Kliebenstein and R.L. Last, unpublished results).

Differential Response of SODs to UV-B Exposure

2

1

View larger version (74K): [in this window] [in a new window]   Figure 11. Regulation of SOD isoenzyme expression in response to UV-B. UV-B-treated plants were exposed to 15 kJ of UV-B light. Controls were grown under Mylar filters. Both the controls and the UV-B-treated plants were protected from UV-C by Pyrex glass filters placed over the pots. Samples were harvested directly before starting the UV-B treatment and after 1, 2, and 3 d of treatment. C, UV-B control samples; UV, UV-B-treated samples. A, RNA-blot hybridization using the cDNAs listed on the left as probes. B, Immunoblots of protein extracted from plants grown in the same pots as used for the RNA blots in A. The proteins were immunodetected using the antisera listed on the left.

The same tissue was tested for levels of SOD protein and activities (Fig. 11B; D.J. Kliebenstein and R.L. Last, unpublished results). CSD1 protein showed a gradual increase in the control samples throughout the experiment, presumably due to environmental changes caused by covering the pots with Pyrex glass filters. However, the UV-B treatment caused increases in CSD1 protein levels above the controls on d 2 and 3 (Fig. 11B). CSD2 showed the opposite response, with decreased protein accumulation on d 1 and 2 in response to the UV-B treatment. Protein levels recovered to control levels by d 3 (Fig. 11B). FSD protein was higher in the UV-B-treated samples then in the controls on all days of the experiment. Additionally, the FSD and CSD1 protein in UV-B-treated samples steadily increased from d 1 to 3 (Fig. 11B). Consistent with the mRNA analysis, MSD1 protein showed no variation in response to UV-B (Fig. 11B). The MnSOD and CuZnSOD activities showed no significant variation throughout this experiment (D.J. Kliebenstein and R.L. Last, unpublished results). FeSOD activity was extremely low throughout the experiment and did not measurably increase.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

Multiple SOD Genes in Arabidopsis

Analysis of the DNA sequences for CSD2, FSD1, FSD3, and MSD1 revealed the presence of amino-terminal transit peptides. CSD2 clusters within the plastidic CuZnSOD clade (Fig. 1, A and B), suggesting that it is plastidic. Other studies have typically identified plant FeSODs as plastidic and MnSODs as mitochondrial, suggesting that FSD1 and FSD3 are plastidic and MSD1 is mitochondrial (Bowler et al., 1994; Alscher et al., 1997; Scandalios, 1997). Our cellular-fractionation studies showed that Arabidopsis chloroplasts contain CSD2 and FeSOD protein, consistent with the analysis of FSD1, FSD3, and CSD2 cDNA sequences.

An Ala-Lys-Leu tripeptide was identified within the CSD3-coding region, which is sufficient for peroxisomal targeting in yeast (Keller et al., 1991). Such carboxyl-terminal peroxisomal targeting sequences have been shown to be functionally conserved between plants and yeast (Gould et al., 1990). The peroxisomal CuZnSOD from watermelon groups phylogenetically with plastidic CuZnSODs and not CSD3 (Bueno et al., 1995; Fig. 1, A and B), suggesting that there could be several classes of peroxisomal CuZnSODs in plants.

Analysis of Multiple SOD Isoenzymes in Arabidopsis

Published reports have also identified multiple SOD activities in Arabidopsis. Pan and Yau (1992) cataloged three CuZnSOD activities, one FeSOD activity, and one MnSOD activity, and Ormrod et al. (1995) found that Arabidopsis contained four CuZnSOD activities, no FeSOD activity, and one MnSOD activity. Our identification of the slowest-migrating activity as MnSOD and the fastest-migrating activities as CuZnSODs agrees with these reports. However, Pan and Yau (1992) identified no SOD activity in a similar location to the activity we identified as FeSOD, whereas Ormrod et al. (1995) identified a CuZnSOD activity in this region. It is possible that these inconsistencies are caused by physiological variations due to different growing conditions or genetic backgrounds. The four major activities we detected are present in all organs analyzed (juvenile, adult and cauline leaves, siliques, stems, and roots) throughout the life cycle of the plant, and the minor activities do not significantly increase in any organ or developmental stage (D.J. Kliebenstein and R.L. Last, unpublished results).

Differential Regulation of Multiple mRNAs for Plastidic SODs

Ozone fumigation and UV-B illumination also differentially affect the mRNAs encoding plastidic SODs. Ozone fumigation caused a transient decrease in the accumulation of the CSD2 and FSD1 mRNAs, although no reproducible effect was seen on the FSD2 mRNA. In contrast, UV-B led to a dramatic increase in accumulation of the FSD2 mRNA but had no effect on the accumulation of CSD2 or FSD1 mRNA. These results indicate that CSD2, FSD1, and FSD2 are independently regulated and raises the possibility that they are responsible for protecting Arabidopsis plastids against different oxidative stresses.

Although the mRNAs encoding plastidic SODs showed significant regulation in response to the various stresses, the corresponding activities and proteins typically did not. SOD activity analysis is complicated by the possibility that more then one isoenzyme could be present in each activity band. Therefore, any change in the level of activity from one isoenzyme could be masked by the other isoenzymes present in the activity band. Additionally, comparing the anti-FSD results with any specific FSD mRNA is not valid, because we do not know whether the antiserum specifically detects only one of the three known FeSOD isoenzymes. However, we believe that the anti-CSD2 is a specific antiserum. The fact that the CSD2 protein does not mirror the pattern of CSD2 mRNA regulation suggests that there is a translational or posttranslational level of control for CSD2 protein. Madmanchi and coworkers (1994) also found evidence for posttranscriptional regulation of plant SODs.

Regulation of mRNA for the Putative Peroxisomal CSD3

Regulation of Cytosolic CSD1 by Oxidative Stress

MnSOD Is Unresponsive to the Environmental Stimuli Tested

We are currently using a genetic approach to further our understanding of why plants contain multiple SODs within the same subcellular compartments. Our goal is to identify Arabidopsis mutants deficient in specific SODs to test the role of individual SODs in ROS protection. We are also screening for mutants that alter the accumulation of multiple SODs as a way to understand the molecular basis for their regulation. Through a combination of biochemical, molecular, and mutant analysis, we hope to obtain a better understanding of the roles of SOD in plants.

	FOOTNOTES

   Received May 21, 1998; accepted July 21, 1998.

	ABBREVIATIONS

Abbreviations: CAPS, cleaved amplified polymorphic sequence. CICYAC, CEPH/Institut National de la Recherche Agronomique/Centre National de la Recherche Scientifique yeast artificial chromosome . EST, expressed sequence tag. GST, glutathione-S-transferase tag. His₆, six-His residue tag. ORF, open reading frame. ROS, reactive oxygen species. SOD, superoxide dismutase. TAMUBAC, Texas A&M University bacterial artificial chromosome.

	ACKNOWLEDGMENTS

The authors thank Drs. Katherine Denby and Patricia Conklin (Boyce Thompson Institute for Plant Research) for helpful comments concerning the manuscript; Dr. Paul King for help with the high-CO₂ experiment; the Arabidopsis Biological Resource Center for all cDNAs used in this research; and Drs. Kenneth Dewar and Joseph Ecker for performing the BAC library hybridization analysis.

	LITERATURE  CITED

Top Abstract Introduction Methods Results Discussion References

Alscher RG, Donahue JL, Cramer CL (1997) Reactive oxygen species and antioxidants: relationships in green cells. Physiol Plant 100: 224-233 [CrossRef]

Alscher RG, Hess JL (1993) Antioxidants in Higher Plants. CRC Press, Boca Raton, FL

Babior BM, El Benna J, Chanock SJ, Smith RM (1997) The NADPH oxidase of leukocytes: The respiratory burst oxidase. In JG Scandalios, eds, Oxidative Stress and the Molecular Biology of Antioxidant Defenses, Vol 34. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 737-783

Beauchamp C, Fridovich I (1971) Superoxide dismutase: improved assays and assay applicable to acrylamide gels. Anal Biochem 44: 276-287 [CrossRef][Web of Science][Medline]

Bell CJ, Ecker JR (1994) Assignment of 30 microsatellite loci to the linkage map of Arabidopsis. Genomics 19: 137-144 [CrossRef][Web of Science][Medline]

Bordo D, Djinovic K, Bolognesi M (1994) Conserved patterns in the Cu, Zn superoxide dismutase family. J Mol Biol 238: 366-386 [CrossRef][Web of Science][Medline]

Bowler C, Van Camp W, Van Montagu M, Inzé D (1994) Superoxide dismutase in plants. Crit Rev Plant Sci 13: 199-218

Bowler C, Van Montagu M, Inzé D (1992) Superoxide dismutase and stress tolerance. Annu Rev Plant Physiol Mol Biol 43: 83-116 [CrossRef][Web of Science]

Bueno P, Varela J, Gimenez GG, Del Rio LA (1995) Peroxisomal copper, zinc superoxide dismutase: characterization of the isoenzyme from watermelon cotyledons. Plant Physiol 108: 1151-1160 [Abstract]

Cadenas E (1989) Biochemistry of oxygen toxicity. Annu Rev Biochem 58: 79-110 [CrossRef][Web of Science][Medline]

Caldwell CR (1993) Ultraviolet-induced photodegradation of cucumber (Cucumis sativus L.) microsomal and soluble protein tryptophanyl residues in vitro. Plant Physiol 101: 947-953 [Abstract]

Carlioz A, Touati D (1986) Isolation of superoxide dismutase mutants in Escherichia coli: is superoxide dismutase necessary for aerobic life? EMBO 5: 623-630 [Web of Science][Medline]

Chary P, Dillon D, Schroeder AL, Natvig DO (1994) Superoxide dismutase (sod-1) null mutants of Neurospora crassa: oxidative stress sensitivity, spontaneous mutation rate and response to mutagens. Genetics 137: 723-730 [Abstract]

Choi S, Creelman RA, Mullet JE, Wing RA (1995) Construction and characterization of a bacterial artificial chromosome library in Arabidopsis thaliana. Weeds World 2: 17-20

Conklin PL, Last RL (1995) Differential accumulation of antioxidant mRNAs in Arabidopsis thaliana exposed to ozone. Plant Physiol 109: 203-212 [Abstract]

Conklin PL, Williams EH, Last RL (1996) Environmental stress sensitivity of an ascorbic acid-deficient Arabidopsis mutant. Proc Natl Acad Sci USA 93: 9970-9974 [Abstract/Free Full Text]

Creusot F, Fouilloux E, Dron M, Lafleuriel J, Picard G, Billault A, Le P-D, Cohen D, Chaboute ME, Durr A, and others (1995) The CIC library: a large insert YAC library for genome mapping in Arabidopsis thaliana. Plant J 8: 763-770 [CrossRef][Web of Science][Medline]

Delseny M, Cooke R, Raynal M, Grellet F (1997) The Arabidopsis thaliana cDNA sequencing projects. FEBS Lett 405: 129-132 [CrossRef][Web of Science][Medline]

Desikan R, Hancock JT, Coffey MJ, Neill SJ (1996) Generation of active oxygen in elicited cells of Arabidopsis thaliana is mediated by a NADPH oxidase-like enzyme. FEBS Lett 382: 213-217 [CrossRef][Web of Science][Medline]

Durrant I, Fowler S (1994) Chemiluminescent detection systems for protein blotting. In BS Dunbar, eds, Protein Blotting, Vol 140. Oxford University Press, Oxford, UK, pp 141-152

Gould S, Keller JGA, Schneider M, Howell SH, Garrard LJ, Goodman JM, Distel B, Tabak H, Subramani S (1990) Peroxisomal protein import is conserved between yeast, plants, insects and mammals. EMBO J 9: 85-90 [Web of Science][Medline]

Gralla EB, Valentine JS (1991) Null mutants of Saccharomyces cerevisiae Cu, Zn superoxide dismutase: characterization and spontaneous mutation rates. J Bacteriol 173: 5918-5920 [Abstract/Free Full Text]

Halliwell B (1987) Oxidative damage, lipid peroxidation and antioxidant protection in chloroplasts. Chem Phys Lipids 44: 327-340 [CrossRef]

Halliwell B, Gutteridge J (1990) Role of free radicals and catalytic metal ions in human disease: an overview. Methods Enzymol 186: 1-85 [CrossRef][Medline]

Heath RL (1994) Possible mechanisms for the inhibition of photosynthesis by ozone. Photosynth Res 39: 439-451 [CrossRef]

Hindges R, Slusarenko A (1992) cDNA and derived amino acid sequence of a cytosolic Cu, Zn superoxide dismutase from Arabidopsis thaliana (L.) Heyhn. Plant Mol Biol 18: 123-125 [Medline]

Jarvis P, Lister C, Szabo V, Dean C (1994) Integration of CAPS markers into the RFLP map generated using recombinant inbred lines of Arabidopsis thaliana. Plant Mol Biol 24: 685-687 [CrossRef][Web of Science][Medline]

Keegstra K, Olsen LJ, Theg SM (1989) Chloroplastic precursors and their transport across the envelope membranes. Annu Rev Plant Physiol Mol Biol 40: 471-502 [CrossRef][Web of Science]

Keller GA, Krisans S, Gould SJ, Sommer JM, Wang CC, Schliebs W, Kunau W, Brody S, Subramani S (1991) Evolutionary conservation of a microbody targeting signal that targets proteins to peroxisomes glyoxysomes and glycosomes. Cell Biol 114: 893-904

Konieczny A, Ausubel FM (1993) A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based markers. Plant J 4: 403-410 [CrossRef][Web of Science][Medline]

Kramer GF, Norman HA, Krizek DT, Mirecki RM (1991) Influence of UV-B radiation on polyamines, lipid peroxidation and membrane lipids in cucumber. Phytochemistry 30: 2101-2108 [CrossRef]

Kreps JA, Kay SA (1997) Coordination of plant metabolism and development by the circadian clock. Plant Cell 9: 1235-1244 [CrossRef][Web of Science][Medline]

Lah MS, Dixon MM, Pattridge KA, Stallings WC, Fee JA, Ludwig ML (1995) Structure-function in Escherichia coli iron superoxide dismutase: comparisons with the manganese enzyme from Thermus thermophilus. Biochemistry 34: 1646-1660 [CrossRef][Medline]

Landry LG, Chapple CCS, Last RL (1995) Arabidopsis mutants lacking phenolic sunscreens exhibit enhanced ultraviolet-B injury and oxidative damage. Plant Physiol 109: 1159-1166 [Abstract]

Lister C, Dean C (1993) Recombinant inbred lines for mapping RFLP and phenotypic markers in Arabidopsis thaliana. Plant J 4: 745-750 [CrossRef][Web of Science]

Madamanchi NR, Donahue JL, Cramer CL, Alscher RG, Pedersen K (1994) Differential response of Cu, Zn superoxide dismutases in two pea cultivars during a short-term exposure to sulfur dioxide. Plant Mol Biol 26: 95-103 [CrossRef][Web of Science][Medline]

Meinke D, Koornneef M (1997) Community standards for Arabidopsis genetics. Plant J 12: 247-253 [CrossRef][Web of Science]

Mishkind ML, Greer KL, Schmidt GW (1987) Cell-free reconstitution of protein transport into chloroplasts. Methods Enzymol 148: 274-294

Neupert W (1997) Protein import into mitochondria. Annu Rev Biochem 66: 863-917 [CrossRef][Web of Science][Medline]

Newman T, de Bruijn FJ, Green P, Keegstra K, Kende H, McIntosh L, Ohlrogge J, Raikhel N, Somerville S, Thomashow M, and others (1994) Genes galore: a summary of methods for accessing results from large-scale partial sequence of anonymous Arabidopsis cDNA clones. Plant Physiol 106: 1241-1255 [Abstract]

Ormrod DP, Landry LG, Conklin PL (1995) Short-term UV-B radiation and ozone exposure effects on aromatic secondary metabolite accumulation and shoot growth of flavonoid-deficient Arabidopsis mutants. Physiol Plant 93: 602-610 [CrossRef]

Pan S-M, Yau Y-Y (1992) Characterization of superoxide dismutase in Arabidopsis. Plant Cell Physiol 37: 58-66

Phillips JP, Campbell SD, Michaud D, Charbonneau M (1989) Null mutation of copper/zinc superoxide dismutase in Drosphila confers hypersensitivity to paraquat and reduced longevity. Proc Natl Acad Sci USA 86: 2761-2765 [Abstract/Free Full Text]

Rosen DR, Siddique T, Patterson D, Figlewicz DA, Sapp P, Hentati A, Donaldson D, Goto J, O'Regan JP, Deng H-X, and others (1993) Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 362: 59-62 [CrossRef][Medline]

Rounsley SD, Glodek A, Sutton G, Adams MD, Somerville CR, Venter JC, Kerlavage AR (1996) The construction of Arabidopsis expressed sequence tag assemblies. Plant Physiol 112: 1177-1183 [Abstract]

Runeckles VC, Vaartnou M (1997) EPR evidence for superoxide anion formation in leaves during exposure to low levels of ozone. Plant Cell Environ 20: 306-314

Scandalios JG (1997) Molecular genetics of superoxide dismutases in plants. In JG Scandalios, eds, Oxidative Stress and the Molecular Biology of Antioxidant Defenses, Vol 34. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 527-568

Schmidt R, Love K, West J, Lenehan Z, Dean C (1997) Description of 31 YAC contigs spanning the majority of Arabidopsis thaliana chromosome 5. Plant J 11: 563-572 [CrossRef][Web of Science][Medline]

Schmidt R, West J, Love K, Lenehan Z, Lister C, Thompson H, Bouchez D, Dean C (1995) Physical map and organization of Arabidopsis thaliana chromosome 4. Science 270: 480-483 [Abstract/Free Full Text]

Sharma YK, Davis KR (1994) Ozone-induced expression of stress-related genes in Arabidopsis thaliana. Plant Physiol 105: 1089-1096 [Abstract]

Smith DB, Johnson KS (1988) Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67: 31-40 [CrossRef][Web of Science][Medline]

Somerville CR, Ogren WL (1981) Photorespiration-deficient mutants of Arabidopsis thaliana lacking mitochondrial serine transhydroxymethylase activity. Plant Physiol 67: 666-671 [Abstract/Free Full Text]

Streller S, Wingsle G (1994) Pinus sylvestris L. needles contain extracellular CuZn superoxide dismutase. Planta 192: 195-201 [CrossRef][Web of Science][Medline]

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 [Abstract/Free Full Text]

Van Camp W, Willekens H, Bowler C, Van Montagu M, Inzé D, Reupold-Popp P, Sandermann H Jr., Langebartels C (1994) Elevated levels of superoxide dismutase protect transgenic plants against ozone damage. Biotechniques 12: 165-168

Yruela I, Pueyo JJ, Alonso PJ, Picorel R (1996) Photoinhibition of photosystem II from higher plants: effect of copper inhibition. J Biol Chem 271: 27408-27415 [Abstract/Free Full Text]

Zhao J, Last RL (1995) Immunological characterization and chloroplast localization of the tryptophan biosynthetic enzymes of the flowering plant Arabidopsis thaliana. J Biol Chem 270: 6081-6087 [Abstract/Free Full Text]

Copyright Clearance Center:   0032-0889/98/118//14
 
© 1998 American Society of Plant Physiologists