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Structural Organization, Regulation, and Expression of the Chloroplastic Superoxide Dismutase Sod1 Gene in Maize
Archives of Biochemistry and Biophysics Vol. 391, No. 1, July 1, pp. 137–147, 2001 doi:10.1006/abbi.2001.2397, available online at http://www.idealibrary.com on
Structural Organization, Regulation, and Expression of the Chloroplastic Superoxide Dismutase Sod1 Gene in Maize Sheri P. Kernodle and John G. Scandalios 1 Department of Genetics, North Carolina State University, Raleigh, North Carolina 27695-7614
Received March 6, 2001, and in revised form April 9, 2001; published online June 6, 2001
A cDNA and genomic clone encoding maize chloroplastic Cu/Zn superoxide dismutase Sod1 were isolated. Southern blot analysis indicated little homology between the chloroplastic (Sod1) and the cytosolic (Sod2, Sod4, Sod4A) cDNAs. Sequence analysis of the genomic clone revealed a promoter, transit peptide, and partial coding sequence. The promoter contained several response elements (e.g., for light, cold temperature, xenobiotics) that may be involved in the regulation of the Sod1 gene. Sod1 expression during development and in response to physiological and chemical stressors such as temperature, xenobiotics (paraquat), and light were examined. © 2001 Academic Press Key Words: antioxidants; superoxide dismutase; gene expression; maize; oxidative stress; cis-elements; chloroplast SOD.
As aerobic and nonmotile organisms, plants are faced with the prospect of not being able to alter their environment when growing conditions become unsuitable, and therefore must adapt or perish. Environmental changes include seasonal cold or heat, drought, UV-radiation, soil contamination, and xenobiotic exposure (e.g., herbicides). The evolution of oxygenic photosynthesis altered the Earth’s atmosphere and enabled the emergence and sustenance of aerobic life. However, the incomplete reduction of oxygen to water during normal aerobic metabolism generates reactive oxygen species (ROS) that pose a serious threat to all organisms. ROS are also crucial for many physiologic processes, and usually exist in the cell in a balance with antioxidants. However, excess ROS resulting from exposure to environmental oxidants, toxicants, radiation, or numerous 1 To whom correspondence and reprint requests should be addressed. Fax: (919) 515-3355. E-mail: [email protected].
0003-9861/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
biostressors, perturbs cellular redox balance (to a more oxidized state) and disrupts normal biological functions. This condition is referred to as “oxidative stress” and may be detrimental to the organism by contributing to the pathogenesis of disease and aging, and numerous physiologic dysfunctions leading to cell death (1). Reactive oxygen species include the superoxide radical (O 2•⫺), hydroxyl radical (䡠OH) and hydrogen peroxide (H 2O 2). Superoxide dismutases (superoxide:superoxide oxidoreductase; EC 1.15, 1.1; SOD) are ubiquitous enzymes found in all aerobes and are involved in protection from oxygen toxicity (2). These metalloproteins catalyze the dismutation of the superoxide anion to molecular oxygen and hydrogen peroxide. Three types of SOD have been described based on metal content: Cu/ZnSODs are located in the chloroplast and cytosol, MnSODs in mitochondria, and FeSODs in some plant chloroplasts (3). In maize nine nuclear-encoded SOD isozymes have been described (4). The Cu/ZnSODs are associated with the chloroplast (SOD-1) and cytosol (SOD-2, SOD-4, SOD-4A, SOD-5), while a small gene family of four MnSODs (SOD-3.1, -3.2, -3.3, -3.4) is associated with the mitochondria (5). Some plant species have been reported to contain FeSOD in the chloroplast (6 – 8). The maize chloroplast-associated SOD-1 was previously identified, purified and characterized (4, 9). SOD-1 is the most abundant isozyme in green leaves and is the only SOD isozyme found in chloroplast fractions. It is a dimeric Cu/Zn metalloprotein with equal subunit molecular weights of 14.5 kDa. The molecular weight of the holoenzyme is 31–33 kDa, similar to the cytosolic Cu/ZnSOD-2 and SOD-4. SOD-1 is antigenically distinct from the cytosolic Cu/ZnSODs and all other SODs in maize. SOD-1 is also cyanide-sensitive, similar to the cytosolic Cu/ZnSODs. In addition, hydrogen peroxide sensitivity experiments indicated that SOD-1 was twice as resistant as the cytosolic SODs to 137
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inactivation. Genetic analysis indicated that SOD-1 is encoded by two codominant alleles at the Sod1 locus. The maize SODs are not tissue specific as they are detected in all tissues examined to date (4, 10 –12). However, the relative activities of the isozymes do vary depending on the tissue and developmental stage. The SODs of maize have recently been reviewed (3). This report details the isolation and characterization of the cDNA and the gene for Sod1. Expression of the SOD-1 protein and mRNA transcripts during seedling development is described. Additionally, the response of Sod1 to oxidative stress induced by temperature, herbicides (paraquat), and light are described and discussed. MATERIALS AND METHODS Seed germination. Seeds of the maize inbred line W64A, maintained by our laboratory, were used in all experiments. Seeds were surface sterilized in 1% NaOCl, rinsed in deionized water several times, and imbibed for 24 h. Seeds were then either germinated on moistened Kimpak germination paper at 25°C in the dark or planted in flats containing Metromix and grown under a 12 h/12 h light/dark (L/D) regimen. Library construction. A cDNA library from 10 dpi W64A leaves was prepared in the unidirectional vector Uni-Zap-XR (Stratagene). The cDNA was packaged using Gigapack Gold extracts (Stratagene) and plated on NZ media. Duplicate plaque lifts were made using nitrocellulose filters (Schliecher and Schuell). The filters were subsequently denatured (0.5 M NaOH, 1.5 M NaCl), neutralized (1 M Tris–HCl, pH 8.0, 1.5 M NaCl), rinsed (2⫻ SSC), air-dried, and baked (13). The filters were prewashed in 0.05 M Tris–HCL, pH 8.0, 1 M NaCl, 1 mM EDTA, and 0.1% SDS at 42°C to remove bacterial debris, and prehybridized in 6⫻ SSC, 1% SDS, and 5⫻ Denhardt’s at 50°C. The prehybridization buffer was removed and hybridization buffer added (6⫻ SSC, 1% SDS, 3⫻ Denhardt’s, 200 g/ml tRNA) containing the end-labeled 27-mer oligo, 5°-CGTCATGCGGGTGACCTGGGAAACATA-3⬘. The oligo was derived from a highly conserved area in the 5⬘ region of the tomato chloroplastic Sod cDNA. Hybridization was performed at 50°C for 16 –18 h. The filters were washed in several changes of 1⫻ SSC, 0.1% SDS, at 50°C, air-dried and exposed to Kodak XAR X-ray film. Genomic DNA was partially digested with Sau3A and ligated to the partial filled-in XhoI site of the lambda FixII vector according to manufacturer’s (Stratagene) directions. The ligated DNA was then packaged and titered in XL-1 Blue MRF’ cells. Duplicate filters were prepared as above before prehybridization in 6⫻ SSC, 5⫻ Denhardt’s, 0.05 M sodium phosphate, pH 7.0, 0.1% SDS, 200 g/ml sheared and denatured salmon sperm DNA, and 50% formamide at 42°C. The Sod1 full-length cDNA was radioactively labeled by the random primer method (14). Hybridization was carried out in a fresh aliquot of the same buffer containing labeled probe at 42°C for 24 – 48 h. Filters were sequentially washed in 2⫻ SSC, 0.1% SDS, at 42°C and 0.1⫻ SSC, 0.1% SDS at 65°C, air-dried and exposed to X-ray film as before. Positive plaques were isolated and purified to homogeneity (13). Phage and plasmid DNA isolation. Phage DNA from the purified positive plaques was isolated by a modified M13 phage DNA isolation procedure (15). Plasmid DNA was isolated by an alkaline lysis method (16). Southern blot analysis. Phage or plasmid DNA was digested with various restriction enzymes (Roche Biochemical) and electrophoresed on agarose gels of varying concentrations, depending on the size of the DNA. Denaturation, neutralization, and transfer to nitro-
cellulose were as described (13). Hybridizations and autoradiography were performed as described above. Subcloning. Restriction fragments of interest (determined after hybridization) were ligated to appropriately digested vector pBluescriptII KS (Stratagene). Nested deletions were made using ExonucleaseIII/Nuclease S1. Transformation was by the calcium chloride method (17). The transformants were plated on LB plus ampicillin (100 g/ml) supplemented with 100 mM IPTG and 2% X-gal and incubated at 37°C overnight. Sequencing. Sequencing of the Sod1 cDNA was performed using the dideoxy chain termination method with Klenow (18). The cycle sequencing method was used to sequence the Sod1 gene as per manual (New England Biolabs). The promoter sequence was analyzed for cis-acting elements using the PLACE database (19). Chemical agent treatments. Embryos were isolated from 5 dpi (days postimbibiton) W64A kernels and placed on agar plates of MS-basal salts (20) plus B-5 vitamins, to which xenobiotic agents were added. Paraquat treatments with concentrations of 0, 10 ⫺4, 10 ⫺3, and 10 ⫺2 M were placed in constant light or constant dark at 25°C for 24 h. Other treatments, using benzyl viologen, juglone, and hydrogen peroxide agar plates, were incubated at 25°C on a 12 h/12 h L/D cycle for 24 h. In addition, 12 dpi W64A seedlings were treated hydroponically with paraquat at 25°C on a 12 h/12 h L/D cycle for 24 h. Protein isolation and electrophoresis. Tissues were homogenized with sand and polyvinylpyrrolidone (PVP) in 0.025 M glycylglycine buffer, pH 7.4, centrifuged at 12,000 rpm for 5–10 min at 4°C and the supernatant used for subsequent assays. Protein concentration was determined (21). Equal amounts of protein were electrophoresed through 9% discontinuous nondenaturing polyacrylamide gels that were stained for superoxide dismutase (SOD) activity (4). Superoxide dismutase assay. Total SOD activity was determined using the xanthine oxidase/cytochrome c method and read at an absorbance of 550 nm, as described (4). Light studies. W64A seeds were surface sterilized and imbibed as previously described, then sown in a sand/peat mixture (22). Plants were grown in environmentally controlled chambers at the Phytotron facility, North Carolina State University. Growth conditions were 26°C and 80% relative humidity. In the first experiment, plants were grown on a 12 h/12 h L/D photoperiod, in continuous light, or in continuous dark for 7 days before sampling, as described (22). Conditions and details for the light pulse and UV light experiments are described in detail elsewhere (23, 24). RNA isolation and analysis. Total RNA was extracted from maize tissues as described (25). Total RNA (20 g/lane) was electrophoresed through 1.6% formaldehyde-agarose gels (26) and transferred to Nytran nylon membranes (13). Filters were UV cross-linked and baked prior to prehybridation at 65°C in a modified Church buffer (27). Preparation of the probe was as described earlier. Fresh buffer containing 4 –5 ⫻ 10 6 cpm/ml labeled Sod1 500-bp EcoRI fragment was used for hybridization at 65°C overnight. Filters were washed in 2⫻ SSC, 0.1% SDS, and 0.1⫻ SSC, 0.1% SDS, at 65°C, air-dried, and exposed to X-ray film. The 18S ribosomal DNA fragment pHA2 was used as a loading control (28).
RESULTS
Four or five SOD isozymes are detectable by electrophoresis depending on the inbred maize line used (29). The inbred line W64A exhibits the most common phenotype, the four-isozyme pattern (Figs. 1A and 1C). The developmental expression of SOD-1 in 1–10 dpi scutella and 5–15 dpi green leaves was examined. In scutella, SOD-1 activity increased through 3 dpi and
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FIG. 1. Protein and RNA expression in maize scutella and leaves during development. (A) Expression of SOD-1 during early postgerminative development. Zymogram stained for SOD activity in 1–10 dpi developing scutella. Equal amounts of protein were electrophoresed through a 9% PAGE and stained for SOD activity. (B) RNA blot of developing scutella probed with the Sod1 500-bp EcoRI coding region fragment. Total RNA (20 g/lane) was electrophoresed through a 1.6% formaldehyde agarose gel, transferred to Nytran nylon membrane and probed. Filters were probed with the 18S ribosomal DNA fragment pHA2 as loading control. (C) Zymogram stained for SOD activity in 5–15 dpi leaves harvested from seedlings grown at 25°C on a 12 h/12 h L/D cycle. (D) Levels of the Sod1 transcript in 5–15 dpi leaves. Sc, 5 dpi scutella.
then remained fairly steady (Fig. 1A). Sod1 mRNA increased to a peak at 5 to 6 dpi before declining (Fig. 1B). In leaves of developing seedlings from 5–15 dpi, SOD-1 activity levels rose slightly over time (Fig. 1C), while Sod1 transcript levels remained fairly level (Fig. 1D). Characterization of Sod1 cDNA Two putative positive clones (pS1 and pS6) were purified, as described under Materials and Methods, and subsequently characterized by restriction enzyme analysis. Both cDNAs had the same basic restriction profile, with pS1 closest in size to the predicted full length (Fig. 2A). The 896-bp pS1 clone contains an internal EcoRI site at base-pair 500. It also contains an SstI site dividing the EcoRI fragment in half, a HindII site close to the end of the coding region and an AccI site in the middle of the 3⬘ untranslated region. Southern blots containing pS1, the cytosolic Cu/Zn Sod cDNAs and the Mn Sod3.1 cDNA probed with the pS1 cDNA indicated no cross-hybridization between pS1 and either the cytosolic Cu/Zn Sods or the mitochondrial MnSod (data not shown). The nucleotide and deduced amino acid sequence of pS1 (Fig. 2B) show two possible polyadenylation signals at base pairs 655 and 730 (AATAA). The oligo (5⬘-CGTCATGCGGGTGACCTGGGAAACATA-3⬘) used
to probe the cDNA library was derived from the coding region of the tomato chloroplast cDNA (30). Since a perfect match was not expected, hybridization and wash conditions were not very stringent. The oligo does not perfectly match the cDNA sequence at the 5⬘ end. Genomic DNA blots were hybridized with the full length (XbaI/XhoI), 5⬘/coding (EcoRI), and 3⬘ untranslated sequence fragments of pS1 (EcoRI/XhoI) (data not shown). Each band on the blots probed with the 5⬘/coding or 3⬘ untranslated fragments corresponded to a band on the blot probed with the full-length cDNA. Hybridization with the full-length cDNA resulted in two EcoRI bands, in agreement with the restriction map of the cDNA, which contained an internal EcoRI site. Therefore, it is likely that there is only one chloroplastic Sod1 gene in maize, although the presence of some faint bands on the blots could indicate an additional Sod1 gene(s). Isolation of Sod1 Gene Two genomic clones (2-1-3 and 6-1-18) that hybridize to the maize chloroplastic Sod1 cDNA pS1 were isolated from a W64A-leaf genomic library and characterized, as described. The restriction maps of the two clones overlapped. The 2-1-3 clone contained the upstream promoter sequence, the transit peptide, and a small portion of the coding region, while the 6-1-18
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FIG. 2. (A) Restriction map of the maize Sod1 cDNA. The 5⬘ EcoRI and 3⬘ XhoI sites are artificial and created during cDNA synthesis. (B) The nucleotide sequence of the Sod1 cDNA. The deduced amino acid sequence is indicated below the sequence. The oligo used to obtain the cDNA is shown above the nucleotide sequence. Possible polyadenylation signals are underlined.
clone contained a small portion of the transit peptide plus approximately one-third of the coding region. A composite restriction map of the two clones is shown (Fig. 3A). Orientation of the two clones was determined by Southern hybridization using the EcoRI/DraII fragment containing the first 170 bp of the cDNA. The EcoRI fragment from the cDNA, containing the majority of the coding region, was used as a probe to determine which restriction fragments contained sequence matching the cDNA. Nested deletions were made on the plasmid DNA for sequencing. Some small fragments (⬍600 bp) were subcloned and sequenced. Both DNA strands were se-
quenced to compensate for possible errors in sequencing. The sequence of the partial Sod1 gene is shown in Fig. 3B. Approximately 100 –150 bp of sequence in the 5⬘ region (denoted by the // mark) is not shown due to difficulties encountered while sequencing the DNA in this region. The partial sequence was found to contain four introns. The first (88 bp) and third (91 bp) introns are very short, while the second is very long at 947 bp. The 6-1-18 clone ends shortly after the beginning of intron 4. There is a putative transit peptide of 49 amino acids prior to the coding region. There are three possible ATG sites that could mark the beginning of the transit
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FIG. 3. (A) Restriction map of the Sod1 gene. Orientation of the gene was determined by Southern hybridization with a 170-bp EcoRI/DraII fragment corresponding to the beginning of the cDNA. (B) The nucleotide sequence of the Sod1 gene. The deduced amino acid sequence is indicated above the sequence. The repeated sequence “TAGAGA” is underlined. Lower case letters denotes intron sequences. “// ” denotes a region of sequence missing because of difficulties encountered in sequencing this region.
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FIG. 4. Response elements found in the Sod1 promoter. (A) Schematic diagram of putative regulatory motifs found in the Sod1 promoter. (B) Regulatory motifs, core sequences and responses (signal/factor) of the Sod1 promoter.
peptide, but only one that does not contain a stop codon. The sequence “TAGAGA” is repeated four times, 20 bp upstream of the beginning of the transit peptide. Exon/intron boundaries follow the GT . . . AG consensus sequence for splicing junctions (31). The promoter region of the Sod1 gene contains approximately 1.3 kb of sequence upstream of the transit peptide. The exact transcriptional start site is not known at this time. This sequence has been computer analyzed for various relevant regulatory elements using the PLACE signal scan program (19). A schematic diagram of the Sod1 promoter, indicating placement as well as a detailed listing of motifs, their location and signal/factor are indicated (Figs. 4A and 4B). Only those motifs that match perfectly with the consensus sequence or have 1 mismatch on the end (leaving the core sequence perfect) are listed. There are several CAAT or CCAAT boxes on the antisense strand, the closest is found ⫺268 bp from the beginning of the transit peptide (TP). No TATA boxes were found. Elements known to be involved in light regulation of some
plant genes are found in the Sod1 promoter. The 4⫻ repeated sequence, “TAGAGA”, contains three GATA elements (32). Other elements found in the Sod1 promoter that are involved in light regulation include GT-1 (33), a Gap-box (ATGAA(A/G)A) (34), and H boxes (35, 36). Elements involved in oxidative stress regulation are also found in the Sod1 promoter. The low temperature response element (LTRE) (CCGAC) has been found to control cold regulated gene expression in Arabidopsis (37). Another element that may be involved in cold tolerance in plants is the Y-box motif (GATTGG) (38). Several factors involved in the antioxidant defenses of mammals are found in the Sod1 promoter. These include a xenobiotic response element (XRE), nuclear factor B (NF-B) and AP-1 (39 – 41). The XRE is recognized by transcriptional factors of the bHLH family and is activated by dioxin and related halogenated compounds. The NFB transcription factor (GGGPuNNPyPyCC) acts as a central regulator of defensive responses that are mounted by cells against many possibly damaging environmental challenges.
STRUCTURE AND REGULATION OF THE Sod1 GENE IN MAIZE
FIG. 5. Sod1 expression in response to increased temperatures. W64A seeds were imbibed and grown at 25, 35, and 40°C. Total RNA (20 g/lane) was electrophoresed through 1.6% formaldehyde agarose gels and transferred to Nytran. RNA blots were hybridized with the coding sequence (500-bp EcoRI fragment) of the Sod1 cDNA.
AP-1 (TGAGTCA) is a mammalian antioxidant transcription factor related to the antioxidant response element (ARE) motif. Several transcriptional activation factors are also found in the Sod1 promoter (Fig. 4B). Response of Sod1 to Stressors Temperature. The effect of increasing temperature on Sod1 was examined in scutella isolated from developing seedlings (Fig. 5). W64A seeds were imbibed and germinated in the dark at 25, 35, and 40°C. The “normal” pattern for Sod1 is exhibited by the developing scutella at 25°C. At 35°C Sod1 mRNA was induced earlier and remained at higher levels than at 25°C. However at 40°C Sod1 mRNA was negligible at 1–3 dpi, increased significantly to almost normal levels at 4 –5 dpi, then decreased at 7– 8 dpi and increased again at 9 dpi, a pattern similar to the scutella at 25°C. Quality and quantity of RNA from the older (6 –10 dpi) 40°C samples were generally poor, thus 6 and 10 dpi are not included.
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Xenobiotics. Maize embryos and seedlings were treated with a diverse group of xenobiotics to determine their effect on the Sod1 transcript. The herbicide paraquat (methyl viologen; 1,1⬘-dimethyl-4,4⬘-bypyridinium chloride) can interact with the reducing site of Photosystem I by accepting electrons, thereby increasing superoxide levels (42). When maize 5 dpi embryos were treated on MS media plates supplemented with increasing concentrations (0 –10 ⫺2 M) of paraquat, SOD-1 protein and Sod1 transcript generally increased, but were inhibited slightly at 10 ⫺2 M (Fig. 6A). Seedlings (12 dpi) treated hydroponically with the same concentrations of paraquat showed a decline in Sod1 particularly at 10 ⫺2 M, although protein levels increased slightly (Fig. 6B). The leaves of seedlings treated with 10 ⫺2 M paraquat were dry and wilted. Exposure of 5 dpi embryos to a structural analog of paraquat, benzyl viologen, resulted in a steady induction of Sod1 RNA, peaking at 10 ⫺2 M (Fig. 7A), although protein levels decreased slightly. Juglone (5Hydroxy-1,4-napthoquinone), a redox-active compound unrelated to paraquat, initially induced the Sod1 transcript at low concentrations (10 ⫺5 M) followed by a decline to negligible amounts at 10 ⫺2 M (Fig. 7A). SOD-1 protein levels were not affected by juglone. Leaves from 9 dpi seedlings exposed to H 2O 2 showed an increase in Sod1 transcript, peaking at 60 mM before declining slightly at 150 mM (Fig. 7B). Light treatments. To determine if Sod1 responds to light, a series of experiments were conducted. In the first experiment, W64A kernels were germinated and grown at 25°C using either a 12 h/12 h L/D photoperiod, constant light, or constant dark. The Sod1 transcript appeared to be cycling under the 12 h/12 h L/D and constant light regimes, with no clear pattern seen in constant dark-grown plants (Fig. 8).
FIG. 6. Response of maize Sod1 to paraquat. (A) PAGE stained for SOD activity and Sod1 mRNA blot (20 g/lane) of 5 dpi embryos exposed to increasing concentrations of paraquat in the light or dark and hybridized with the Sod1 coding fragment (500-bp EcoRI fragment). (B) PAGE stained for SOD activity and Sod1 RNA blot (20 g/lane) of 12 dpi leaves harvested from seedlings treated hydroponically with increasing concentrations of paraquat.
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FIG. 7. Response of maize Sod1 to xenobiotics and hydrogen peroxide. (A) PAGE stained for SOD activity and Sod1 RNA blots (20 g/lane) from 5 dpi embryos exposed to benzyl viologen and juglone. Abbreviations: ip, in planta; C, control. (B) Sod1 RNA blot from H 2O 2-treated leaves isolated from 9 dpi seedlings.
W64A seedlings grown in constant dark for 8 days were subjected to polychromatic or monochromatic light. The seedlings were exposed to a single 15 min light pulse from white light, red light, far-red light, red light/far-red light or blue light, and then returned to the dark. One group of seedlings grown in constant darkness was used as a control. In the dark control Sod1 mRNA levels were steady through 16 h then declined over the next 16 h (Fig. 9). When the seedlings were exposed to a 15 min white light pulse or a 15 min red light pulse, the Sod1 transcript was lower initially then increased at 8 –16 h before decreasing again. Treatment with far-red light produced no differences from the control. When red light/far-red light and blue light were used, Sodl transcript levels were extremely low and no pattern discernible (data not shown). The effect of UV radiation on Sod1 was also examined using a single 15 min pulse of UV light, plus or minus white light. In addition, the three classes of UV (UV-A, UV-B, UV-C) were subtracted using the appropriate filters; for example, UV-C exclusion using a cellulose acetate filter. There were no significant differences among the treatments with or without white light (Figs. 10A and 10B), except in the full UV light plus white light samples, where the Sod1 transcript remained at steady levels longer before declining, than the same treatment minus white light. In the UV minus white light without UV-B or without UV-C, UV-B,
and most of UV-A treatment, Sod1 transcript levels seemed to be higher than their counterparts in the UV plus white light treatment. When 8 dpi 12 h/12 h L/D plants were exposed to constant UV-B light for 12 h, no change was seen in Sod1 mRNA levels as compared to control levels (Fig. 11). DISCUSSION
A cDNA (pS1) and a partial genomic clone for the maize chloroplastic Cu/ZnSod1 have been isolated and characterized. Southern hybridization indicated the cDNA was not homologous with the cytosolic Cu/ZnSods of maize. The pS1 clone was 896 bp in length and contained a short 5⬘ flanking sequence and a complete 3⬘ untranslated sequence with a poly(A) tail. The amino acid sequence showed similarities with other chloroplastic Cu/ZnSods (30, 43– 45). A genomic library was prepared and probed with pS1. The gene contained a promoter, transit peptide, approximately one-third of the coding region and four introns. Placement of the introns is similar to that of the tomato chloroplastic Cu/ZnSod gene (46); the maize cytosolic Cu/ZnSod4 and Sod4A (47), and the rice cytosolic Cu/ZnSod genes (48), indicating that this structure is conserved among plant Cu/ZnSod genes. Other genes that have been isolated for the chloroplastic Cu/ZnSOD are from tomato (46) and Arabidopsis thaliana (49), although only
FIG. 8. Sod1 expression in 7 dpi maize leaves grown at 25°C, on a 12 h/12 h L/D, a constant light, or a constant dark regimen. Samples were taken every 6 h for 2 days. RNA blots were prepared and hybridized as described.
STRUCTURE AND REGULATION OF THE Sod1 GENE IN MAIZE
FIG. 9. Accumulation of Sod1 mRNA in leaves of 8 dpi dark-grown maize seedlings subjected to a 15-min light pulse of white light, red light, or far-red light. Plants grown in total darkness were used as a control. After treatment plants were returned to constant darkness. Leaf material was harvested immediately before the light pulse (DC, dark control), immediately after the light treatment (0), and every 4 h for 32 h. RNA blots were prepared and hybridized as described.
the tomato gene was studied in some detail. The Sod1 transit peptide was 49 amino acids long, a length similar to that of rice (45). A comparison of the transit peptides of the maize Sod1 and tomato chloroplastic Cu/ZnSod gene resulted in no large areas of homology, although both contain many serine residues. As a general rule, transit peptides do not contain any similar amino acid sequences, but are characterized by containing more hydroxylated and small hydrophobic amino acids, lacking acidic amino acids and having a positive charge (50). The maize Sod1 promoter contains several putative cis-acting elements for light regulation. The GATA motif (I-box) is a common cis-acting regulatory element found in many plant promoters, both light regulated and non-light regulated (32). A group of three GATA motifs were found in tandem shortly before the putative start of transcription of Sod1. Groups of GATA
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boxes are often found near the TATA box in promoters from light-regulated genes such as CAB and RbcS (51, 52). In this case, the maize Sod1 promoter does not contain a TATA box. The 12 h/12 h light/dark and constant light regimens appeared to cause a cycling effect, while constant dark treatment did not result in a definable cycle, indicating that Sod1 may be regulated by light. To date, no other Cu/ZnSod has been reported to be under circadian regulation. In Arabidopsis an FeSod was reported to be influenced by a circadian rhythm (7). Some of the other light treatments examined also had an effect on the Sod1 transcript. For example, a 15-min white light or red light pulse delayed induction of the Sod1 transcript in dark-grown plants. A UV light pulse also led to changes in Sod1 mRNA expression, although treatment of 12 h/12 h L D seedlings with or without constant UV-B did not result in an increase of SOD-1 in the leaves. In Arabidopsis, it was also found that UV-B did not alter chloroplastic Cu/ZnSod expression (7). In view of the results that different light treatments affect Sod1, functional analysis of light elements present in the maize Sod1 promoter may lead to information regarding the role of these elements. In light studies involving other plant species, it was found that although transgenic tobacco plants containing the tomato chloroplastic Cu/ZnSod gene respond to light, no cis-elements involved in light regulation were found in the truncated (285 bp) promoter sequence reported (46). In tomato, the chloroplastic and cytosolic Cu/ZnSod transcripts increased after exposure to white light (53). Although UV light did not result in dramatic changes in the maize Sod1 transcript, it was shown that ROS levels, as a result of
FIG. 10. Accumulation of Sod1 mRNA in leaves of 8 dpi dark-grown seedlings subjected to a 15-min pulse of UV light plus (A) or minus (B) white light. Filters were used to exclude UV-C (cellulose acetate), UV-C and UV-B (polyester), or UV-C, UV-B, and most of UV-A (PVC). After treatment, plants were returned to the dark. Leaf material was harvested immediately before the light pulse (C, dark control), immediately after the light treatment (0), and every 4 h for 48 h. RNA blots were prepared and hybridized as described.
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FIG. 11. Sod1 expression in 8 dpi 12 h/12 h L/D-grown plants exposed to plus or minus UV-B light. Seedlings grown at 25°C on a 12 h/12 h L/D photoperiod were transferred to plus constant UV-B light or minus constant UV-B light. Samples were harvested every 2 h. Lane C, control sample taken immediately prior to treatment.
UV-B exposure, play a role in the regulation of some chloroplast-associated genes in peas, with greater increases seen in protein activity of SOD, ascorbate peroxidase (Apx), and glutathione reductase (GR) in etiolated and green buds after UV-B exposure, and smaller increases in Sod and Apx transcript levels (54). Many environmental challenges have been shown to increase ROS levels in cells (3). Previous studies of the maize SOD proteins showed no significant increase in total SOD activity in response to ozone (O 3) and SO 2 (55), although the chloroplastic Sod1 transcript was reduced after acute and chronic O 3 exposure, while the cytosolic Cu/ZnSods and MnSod3 transcripts increased slightly (56). Increases in temperature adversely affect the metabolic processes of plants resulting in induction of heat shock proteins and repression of normal cellular protein synthesis (57). Previous work from this laboratory showed that seedlings germinated and grown at 40°C had lower total SOD activity through 3 dpi, but total activity then rose to levels similar to seedlings germinated and grown at 25°C (58). Sod1 transcript levels in scutella isolated from seedlings grown at 40°C followed the same pattern as the SOD-1 protein, indicating that no permanent damage was done to the cell’s ability to produce SOD-1. Bipyridyl herbicides (e.g., paraquat), act by accepting an electron from Photosystem I to become reduced free radicals, which are then immediately re-oxidized, transferring an electron to oxygen generating superoxide (42). Paraquat is highly toxic in the light, but can also cause damage in the dark by accepting electrons from other sources such as the mitochondrial transport chain. It was previously shown, in 10 dpi maize leaves, that the chloroplastic, cytosolic, and mitochondrial SODs increased in response to short-term treatment with low concentrations of paraquat and juglone (59). In this study, treatment of 5 dpi embryos with paraquat resulted in an increase of both SOD-1 protein and Sod1 mRNA levels, while treatment of 12 dpi seedlings resulted in a decrease of Sod1 mRNA in leaves at higher concentrations but an increase in SOD-1 protein. Benzyl viologen, a paraquat analog, also induced Sod1 in 5 dpi scutella as concentration increased, although previously no effect was observed on maize total SOD protein levels (59). Treatment with juglone, a redox
compound unrelated to paraquat that also produces superoxide, resulted in initially elevated Sod1 mRNA levels at low concentrations but reduced levels at high concentrations. In pea leaves, it was shown that sensitivity to paraquat was dependent on cultivar and leaf age (60). Transgenic plants that overexpress the pea chloroplastic Cu/ZnSOD showed enhanced resistance to paraquat damage as well as to chilling and moderate light intensity (61). Hydrogen peroxide treatment did not inhibit the Sod1 transcript. This correlates with previous results (9) that the SOD-1 protein is more resistant to H 2O 2 than the cytosolic Cu/ZnSODs. This may be due to naturally higher levels of H 2O 2 in the chloroplast and, in order to function properly, SOD-1 is more resistant. In conclusion, the maize chloroplastic Cu/ZnSod1 gene has been isolated and characterized. The promoter contains several responsive elements that may play a significant role(s) in its response to various environmental insults known to increase ROS levels in the chloroplast. Deletion analyses of the Sod1 promoter are currently underway, using transgenic maize plants, to elucidate those elements involved in Sod1 regulation. Additional studies should help to further define the mechanisms by which Sod1 perceives signals to initiate the antioxidant response cascade. ACKNOWLEDGMENTS Research was supported, in part, by Grants from the USEPA, NSF, and USDA. We thank Stephanie Ruzsa for commenting on the manuscript.
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Structural Organization, Regulation, and Expression of the Chloroplastic Superoxide Dismutase Sod1 Gene in Maize Sheri P. Kernodle and John G. Scandalios 1 Department of Genetics, North Carolina State University, Raleigh, North Carolina 27695-7614
Received March 6, 2001, and in revised form April 9, 2001; published online June 6, 2001
A cDNA and genomic clone encoding maize chloroplastic Cu/Zn superoxide dismutase Sod1 were isolated. Southern blot analysis indicated little homology between the chloroplastic (Sod1) and the cytosolic (Sod2, Sod4, Sod4A) cDNAs. Sequence analysis of the genomic clone revealed a promoter, transit peptide, and partial coding sequence. The promoter contained several response elements (e.g., for light, cold temperature, xenobiotics) that may be involved in the regulation of the Sod1 gene. Sod1 expression during development and in response to physiological and chemical stressors such as temperature, xenobiotics (paraquat), and light were examined. © 2001 Academic Press Key Words: antioxidants; superoxide dismutase; gene expression; maize; oxidative stress; cis-elements; chloroplast SOD.
As aerobic and nonmotile organisms, plants are faced with the prospect of not being able to alter their environment when growing conditions become unsuitable, and therefore must adapt or perish. Environmental changes include seasonal cold or heat, drought, UV-radiation, soil contamination, and xenobiotic exposure (e.g., herbicides). The evolution of oxygenic photosynthesis altered the Earth’s atmosphere and enabled the emergence and sustenance of aerobic life. However, the incomplete reduction of oxygen to water during normal aerobic metabolism generates reactive oxygen species (ROS) that pose a serious threat to all organisms. ROS are also crucial for many physiologic processes, and usually exist in the cell in a balance with antioxidants. However, excess ROS resulting from exposure to environmental oxidants, toxicants, radiation, or numerous 1 To whom correspondence and reprint requests should be addressed. Fax: (919) 515-3355. E-mail: [email protected].
0003-9861/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
biostressors, perturbs cellular redox balance (to a more oxidized state) and disrupts normal biological functions. This condition is referred to as “oxidative stress” and may be detrimental to the organism by contributing to the pathogenesis of disease and aging, and numerous physiologic dysfunctions leading to cell death (1). Reactive oxygen species include the superoxide radical (O 2•⫺), hydroxyl radical (䡠OH) and hydrogen peroxide (H 2O 2). Superoxide dismutases (superoxide:superoxide oxidoreductase; EC 1.15, 1.1; SOD) are ubiquitous enzymes found in all aerobes and are involved in protection from oxygen toxicity (2). These metalloproteins catalyze the dismutation of the superoxide anion to molecular oxygen and hydrogen peroxide. Three types of SOD have been described based on metal content: Cu/ZnSODs are located in the chloroplast and cytosol, MnSODs in mitochondria, and FeSODs in some plant chloroplasts (3). In maize nine nuclear-encoded SOD isozymes have been described (4). The Cu/ZnSODs are associated with the chloroplast (SOD-1) and cytosol (SOD-2, SOD-4, SOD-4A, SOD-5), while a small gene family of four MnSODs (SOD-3.1, -3.2, -3.3, -3.4) is associated with the mitochondria (5). Some plant species have been reported to contain FeSOD in the chloroplast (6 – 8). The maize chloroplast-associated SOD-1 was previously identified, purified and characterized (4, 9). SOD-1 is the most abundant isozyme in green leaves and is the only SOD isozyme found in chloroplast fractions. It is a dimeric Cu/Zn metalloprotein with equal subunit molecular weights of 14.5 kDa. The molecular weight of the holoenzyme is 31–33 kDa, similar to the cytosolic Cu/ZnSOD-2 and SOD-4. SOD-1 is antigenically distinct from the cytosolic Cu/ZnSODs and all other SODs in maize. SOD-1 is also cyanide-sensitive, similar to the cytosolic Cu/ZnSODs. In addition, hydrogen peroxide sensitivity experiments indicated that SOD-1 was twice as resistant as the cytosolic SODs to 137
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inactivation. Genetic analysis indicated that SOD-1 is encoded by two codominant alleles at the Sod1 locus. The maize SODs are not tissue specific as they are detected in all tissues examined to date (4, 10 –12). However, the relative activities of the isozymes do vary depending on the tissue and developmental stage. The SODs of maize have recently been reviewed (3). This report details the isolation and characterization of the cDNA and the gene for Sod1. Expression of the SOD-1 protein and mRNA transcripts during seedling development is described. Additionally, the response of Sod1 to oxidative stress induced by temperature, herbicides (paraquat), and light are described and discussed. MATERIALS AND METHODS Seed germination. Seeds of the maize inbred line W64A, maintained by our laboratory, were used in all experiments. Seeds were surface sterilized in 1% NaOCl, rinsed in deionized water several times, and imbibed for 24 h. Seeds were then either germinated on moistened Kimpak germination paper at 25°C in the dark or planted in flats containing Metromix and grown under a 12 h/12 h light/dark (L/D) regimen. Library construction. A cDNA library from 10 dpi W64A leaves was prepared in the unidirectional vector Uni-Zap-XR (Stratagene). The cDNA was packaged using Gigapack Gold extracts (Stratagene) and plated on NZ media. Duplicate plaque lifts were made using nitrocellulose filters (Schliecher and Schuell). The filters were subsequently denatured (0.5 M NaOH, 1.5 M NaCl), neutralized (1 M Tris–HCl, pH 8.0, 1.5 M NaCl), rinsed (2⫻ SSC), air-dried, and baked (13). The filters were prewashed in 0.05 M Tris–HCL, pH 8.0, 1 M NaCl, 1 mM EDTA, and 0.1% SDS at 42°C to remove bacterial debris, and prehybridized in 6⫻ SSC, 1% SDS, and 5⫻ Denhardt’s at 50°C. The prehybridization buffer was removed and hybridization buffer added (6⫻ SSC, 1% SDS, 3⫻ Denhardt’s, 200 g/ml tRNA) containing the end-labeled 27-mer oligo, 5°-CGTCATGCGGGTGACCTGGGAAACATA-3⬘. The oligo was derived from a highly conserved area in the 5⬘ region of the tomato chloroplastic Sod cDNA. Hybridization was performed at 50°C for 16 –18 h. The filters were washed in several changes of 1⫻ SSC, 0.1% SDS, at 50°C, air-dried and exposed to Kodak XAR X-ray film. Genomic DNA was partially digested with Sau3A and ligated to the partial filled-in XhoI site of the lambda FixII vector according to manufacturer’s (Stratagene) directions. The ligated DNA was then packaged and titered in XL-1 Blue MRF’ cells. Duplicate filters were prepared as above before prehybridization in 6⫻ SSC, 5⫻ Denhardt’s, 0.05 M sodium phosphate, pH 7.0, 0.1% SDS, 200 g/ml sheared and denatured salmon sperm DNA, and 50% formamide at 42°C. The Sod1 full-length cDNA was radioactively labeled by the random primer method (14). Hybridization was carried out in a fresh aliquot of the same buffer containing labeled probe at 42°C for 24 – 48 h. Filters were sequentially washed in 2⫻ SSC, 0.1% SDS, at 42°C and 0.1⫻ SSC, 0.1% SDS at 65°C, air-dried and exposed to X-ray film as before. Positive plaques were isolated and purified to homogeneity (13). Phage and plasmid DNA isolation. Phage DNA from the purified positive plaques was isolated by a modified M13 phage DNA isolation procedure (15). Plasmid DNA was isolated by an alkaline lysis method (16). Southern blot analysis. Phage or plasmid DNA was digested with various restriction enzymes (Roche Biochemical) and electrophoresed on agarose gels of varying concentrations, depending on the size of the DNA. Denaturation, neutralization, and transfer to nitro-
cellulose were as described (13). Hybridizations and autoradiography were performed as described above. Subcloning. Restriction fragments of interest (determined after hybridization) were ligated to appropriately digested vector pBluescriptII KS (Stratagene). Nested deletions were made using ExonucleaseIII/Nuclease S1. Transformation was by the calcium chloride method (17). The transformants were plated on LB plus ampicillin (100 g/ml) supplemented with 100 mM IPTG and 2% X-gal and incubated at 37°C overnight. Sequencing. Sequencing of the Sod1 cDNA was performed using the dideoxy chain termination method with Klenow (18). The cycle sequencing method was used to sequence the Sod1 gene as per manual (New England Biolabs). The promoter sequence was analyzed for cis-acting elements using the PLACE database (19). Chemical agent treatments. Embryos were isolated from 5 dpi (days postimbibiton) W64A kernels and placed on agar plates of MS-basal salts (20) plus B-5 vitamins, to which xenobiotic agents were added. Paraquat treatments with concentrations of 0, 10 ⫺4, 10 ⫺3, and 10 ⫺2 M were placed in constant light or constant dark at 25°C for 24 h. Other treatments, using benzyl viologen, juglone, and hydrogen peroxide agar plates, were incubated at 25°C on a 12 h/12 h L/D cycle for 24 h. In addition, 12 dpi W64A seedlings were treated hydroponically with paraquat at 25°C on a 12 h/12 h L/D cycle for 24 h. Protein isolation and electrophoresis. Tissues were homogenized with sand and polyvinylpyrrolidone (PVP) in 0.025 M glycylglycine buffer, pH 7.4, centrifuged at 12,000 rpm for 5–10 min at 4°C and the supernatant used for subsequent assays. Protein concentration was determined (21). Equal amounts of protein were electrophoresed through 9% discontinuous nondenaturing polyacrylamide gels that were stained for superoxide dismutase (SOD) activity (4). Superoxide dismutase assay. Total SOD activity was determined using the xanthine oxidase/cytochrome c method and read at an absorbance of 550 nm, as described (4). Light studies. W64A seeds were surface sterilized and imbibed as previously described, then sown in a sand/peat mixture (22). Plants were grown in environmentally controlled chambers at the Phytotron facility, North Carolina State University. Growth conditions were 26°C and 80% relative humidity. In the first experiment, plants were grown on a 12 h/12 h L/D photoperiod, in continuous light, or in continuous dark for 7 days before sampling, as described (22). Conditions and details for the light pulse and UV light experiments are described in detail elsewhere (23, 24). RNA isolation and analysis. Total RNA was extracted from maize tissues as described (25). Total RNA (20 g/lane) was electrophoresed through 1.6% formaldehyde-agarose gels (26) and transferred to Nytran nylon membranes (13). Filters were UV cross-linked and baked prior to prehybridation at 65°C in a modified Church buffer (27). Preparation of the probe was as described earlier. Fresh buffer containing 4 –5 ⫻ 10 6 cpm/ml labeled Sod1 500-bp EcoRI fragment was used for hybridization at 65°C overnight. Filters were washed in 2⫻ SSC, 0.1% SDS, and 0.1⫻ SSC, 0.1% SDS, at 65°C, air-dried, and exposed to X-ray film. The 18S ribosomal DNA fragment pHA2 was used as a loading control (28).
RESULTS
Four or five SOD isozymes are detectable by electrophoresis depending on the inbred maize line used (29). The inbred line W64A exhibits the most common phenotype, the four-isozyme pattern (Figs. 1A and 1C). The developmental expression of SOD-1 in 1–10 dpi scutella and 5–15 dpi green leaves was examined. In scutella, SOD-1 activity increased through 3 dpi and
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FIG. 1. Protein and RNA expression in maize scutella and leaves during development. (A) Expression of SOD-1 during early postgerminative development. Zymogram stained for SOD activity in 1–10 dpi developing scutella. Equal amounts of protein were electrophoresed through a 9% PAGE and stained for SOD activity. (B) RNA blot of developing scutella probed with the Sod1 500-bp EcoRI coding region fragment. Total RNA (20 g/lane) was electrophoresed through a 1.6% formaldehyde agarose gel, transferred to Nytran nylon membrane and probed. Filters were probed with the 18S ribosomal DNA fragment pHA2 as loading control. (C) Zymogram stained for SOD activity in 5–15 dpi leaves harvested from seedlings grown at 25°C on a 12 h/12 h L/D cycle. (D) Levels of the Sod1 transcript in 5–15 dpi leaves. Sc, 5 dpi scutella.
then remained fairly steady (Fig. 1A). Sod1 mRNA increased to a peak at 5 to 6 dpi before declining (Fig. 1B). In leaves of developing seedlings from 5–15 dpi, SOD-1 activity levels rose slightly over time (Fig. 1C), while Sod1 transcript levels remained fairly level (Fig. 1D). Characterization of Sod1 cDNA Two putative positive clones (pS1 and pS6) were purified, as described under Materials and Methods, and subsequently characterized by restriction enzyme analysis. Both cDNAs had the same basic restriction profile, with pS1 closest in size to the predicted full length (Fig. 2A). The 896-bp pS1 clone contains an internal EcoRI site at base-pair 500. It also contains an SstI site dividing the EcoRI fragment in half, a HindII site close to the end of the coding region and an AccI site in the middle of the 3⬘ untranslated region. Southern blots containing pS1, the cytosolic Cu/Zn Sod cDNAs and the Mn Sod3.1 cDNA probed with the pS1 cDNA indicated no cross-hybridization between pS1 and either the cytosolic Cu/Zn Sods or the mitochondrial MnSod (data not shown). The nucleotide and deduced amino acid sequence of pS1 (Fig. 2B) show two possible polyadenylation signals at base pairs 655 and 730 (AATAA). The oligo (5⬘-CGTCATGCGGGTGACCTGGGAAACATA-3⬘) used
to probe the cDNA library was derived from the coding region of the tomato chloroplast cDNA (30). Since a perfect match was not expected, hybridization and wash conditions were not very stringent. The oligo does not perfectly match the cDNA sequence at the 5⬘ end. Genomic DNA blots were hybridized with the full length (XbaI/XhoI), 5⬘/coding (EcoRI), and 3⬘ untranslated sequence fragments of pS1 (EcoRI/XhoI) (data not shown). Each band on the blots probed with the 5⬘/coding or 3⬘ untranslated fragments corresponded to a band on the blot probed with the full-length cDNA. Hybridization with the full-length cDNA resulted in two EcoRI bands, in agreement with the restriction map of the cDNA, which contained an internal EcoRI site. Therefore, it is likely that there is only one chloroplastic Sod1 gene in maize, although the presence of some faint bands on the blots could indicate an additional Sod1 gene(s). Isolation of Sod1 Gene Two genomic clones (2-1-3 and 6-1-18) that hybridize to the maize chloroplastic Sod1 cDNA pS1 were isolated from a W64A-leaf genomic library and characterized, as described. The restriction maps of the two clones overlapped. The 2-1-3 clone contained the upstream promoter sequence, the transit peptide, and a small portion of the coding region, while the 6-1-18
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FIG. 2. (A) Restriction map of the maize Sod1 cDNA. The 5⬘ EcoRI and 3⬘ XhoI sites are artificial and created during cDNA synthesis. (B) The nucleotide sequence of the Sod1 cDNA. The deduced amino acid sequence is indicated below the sequence. The oligo used to obtain the cDNA is shown above the nucleotide sequence. Possible polyadenylation signals are underlined.
clone contained a small portion of the transit peptide plus approximately one-third of the coding region. A composite restriction map of the two clones is shown (Fig. 3A). Orientation of the two clones was determined by Southern hybridization using the EcoRI/DraII fragment containing the first 170 bp of the cDNA. The EcoRI fragment from the cDNA, containing the majority of the coding region, was used as a probe to determine which restriction fragments contained sequence matching the cDNA. Nested deletions were made on the plasmid DNA for sequencing. Some small fragments (⬍600 bp) were subcloned and sequenced. Both DNA strands were se-
quenced to compensate for possible errors in sequencing. The sequence of the partial Sod1 gene is shown in Fig. 3B. Approximately 100 –150 bp of sequence in the 5⬘ region (denoted by the // mark) is not shown due to difficulties encountered while sequencing the DNA in this region. The partial sequence was found to contain four introns. The first (88 bp) and third (91 bp) introns are very short, while the second is very long at 947 bp. The 6-1-18 clone ends shortly after the beginning of intron 4. There is a putative transit peptide of 49 amino acids prior to the coding region. There are three possible ATG sites that could mark the beginning of the transit
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FIG. 3. (A) Restriction map of the Sod1 gene. Orientation of the gene was determined by Southern hybridization with a 170-bp EcoRI/DraII fragment corresponding to the beginning of the cDNA. (B) The nucleotide sequence of the Sod1 gene. The deduced amino acid sequence is indicated above the sequence. The repeated sequence “TAGAGA” is underlined. Lower case letters denotes intron sequences. “// ” denotes a region of sequence missing because of difficulties encountered in sequencing this region.
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FIG. 4. Response elements found in the Sod1 promoter. (A) Schematic diagram of putative regulatory motifs found in the Sod1 promoter. (B) Regulatory motifs, core sequences and responses (signal/factor) of the Sod1 promoter.
peptide, but only one that does not contain a stop codon. The sequence “TAGAGA” is repeated four times, 20 bp upstream of the beginning of the transit peptide. Exon/intron boundaries follow the GT . . . AG consensus sequence for splicing junctions (31). The promoter region of the Sod1 gene contains approximately 1.3 kb of sequence upstream of the transit peptide. The exact transcriptional start site is not known at this time. This sequence has been computer analyzed for various relevant regulatory elements using the PLACE signal scan program (19). A schematic diagram of the Sod1 promoter, indicating placement as well as a detailed listing of motifs, their location and signal/factor are indicated (Figs. 4A and 4B). Only those motifs that match perfectly with the consensus sequence or have 1 mismatch on the end (leaving the core sequence perfect) are listed. There are several CAAT or CCAAT boxes on the antisense strand, the closest is found ⫺268 bp from the beginning of the transit peptide (TP). No TATA boxes were found. Elements known to be involved in light regulation of some
plant genes are found in the Sod1 promoter. The 4⫻ repeated sequence, “TAGAGA”, contains three GATA elements (32). Other elements found in the Sod1 promoter that are involved in light regulation include GT-1 (33), a Gap-box (ATGAA(A/G)A) (34), and H boxes (35, 36). Elements involved in oxidative stress regulation are also found in the Sod1 promoter. The low temperature response element (LTRE) (CCGAC) has been found to control cold regulated gene expression in Arabidopsis (37). Another element that may be involved in cold tolerance in plants is the Y-box motif (GATTGG) (38). Several factors involved in the antioxidant defenses of mammals are found in the Sod1 promoter. These include a xenobiotic response element (XRE), nuclear factor B (NF-B) and AP-1 (39 – 41). The XRE is recognized by transcriptional factors of the bHLH family and is activated by dioxin and related halogenated compounds. The NFB transcription factor (GGGPuNNPyPyCC) acts as a central regulator of defensive responses that are mounted by cells against many possibly damaging environmental challenges.
STRUCTURE AND REGULATION OF THE Sod1 GENE IN MAIZE
FIG. 5. Sod1 expression in response to increased temperatures. W64A seeds were imbibed and grown at 25, 35, and 40°C. Total RNA (20 g/lane) was electrophoresed through 1.6% formaldehyde agarose gels and transferred to Nytran. RNA blots were hybridized with the coding sequence (500-bp EcoRI fragment) of the Sod1 cDNA.
AP-1 (TGAGTCA) is a mammalian antioxidant transcription factor related to the antioxidant response element (ARE) motif. Several transcriptional activation factors are also found in the Sod1 promoter (Fig. 4B). Response of Sod1 to Stressors Temperature. The effect of increasing temperature on Sod1 was examined in scutella isolated from developing seedlings (Fig. 5). W64A seeds were imbibed and germinated in the dark at 25, 35, and 40°C. The “normal” pattern for Sod1 is exhibited by the developing scutella at 25°C. At 35°C Sod1 mRNA was induced earlier and remained at higher levels than at 25°C. However at 40°C Sod1 mRNA was negligible at 1–3 dpi, increased significantly to almost normal levels at 4 –5 dpi, then decreased at 7– 8 dpi and increased again at 9 dpi, a pattern similar to the scutella at 25°C. Quality and quantity of RNA from the older (6 –10 dpi) 40°C samples were generally poor, thus 6 and 10 dpi are not included.
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Xenobiotics. Maize embryos and seedlings were treated with a diverse group of xenobiotics to determine their effect on the Sod1 transcript. The herbicide paraquat (methyl viologen; 1,1⬘-dimethyl-4,4⬘-bypyridinium chloride) can interact with the reducing site of Photosystem I by accepting electrons, thereby increasing superoxide levels (42). When maize 5 dpi embryos were treated on MS media plates supplemented with increasing concentrations (0 –10 ⫺2 M) of paraquat, SOD-1 protein and Sod1 transcript generally increased, but were inhibited slightly at 10 ⫺2 M (Fig. 6A). Seedlings (12 dpi) treated hydroponically with the same concentrations of paraquat showed a decline in Sod1 particularly at 10 ⫺2 M, although protein levels increased slightly (Fig. 6B). The leaves of seedlings treated with 10 ⫺2 M paraquat were dry and wilted. Exposure of 5 dpi embryos to a structural analog of paraquat, benzyl viologen, resulted in a steady induction of Sod1 RNA, peaking at 10 ⫺2 M (Fig. 7A), although protein levels decreased slightly. Juglone (5Hydroxy-1,4-napthoquinone), a redox-active compound unrelated to paraquat, initially induced the Sod1 transcript at low concentrations (10 ⫺5 M) followed by a decline to negligible amounts at 10 ⫺2 M (Fig. 7A). SOD-1 protein levels were not affected by juglone. Leaves from 9 dpi seedlings exposed to H 2O 2 showed an increase in Sod1 transcript, peaking at 60 mM before declining slightly at 150 mM (Fig. 7B). Light treatments. To determine if Sod1 responds to light, a series of experiments were conducted. In the first experiment, W64A kernels were germinated and grown at 25°C using either a 12 h/12 h L/D photoperiod, constant light, or constant dark. The Sod1 transcript appeared to be cycling under the 12 h/12 h L/D and constant light regimes, with no clear pattern seen in constant dark-grown plants (Fig. 8).
FIG. 6. Response of maize Sod1 to paraquat. (A) PAGE stained for SOD activity and Sod1 mRNA blot (20 g/lane) of 5 dpi embryos exposed to increasing concentrations of paraquat in the light or dark and hybridized with the Sod1 coding fragment (500-bp EcoRI fragment). (B) PAGE stained for SOD activity and Sod1 RNA blot (20 g/lane) of 12 dpi leaves harvested from seedlings treated hydroponically with increasing concentrations of paraquat.
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FIG. 7. Response of maize Sod1 to xenobiotics and hydrogen peroxide. (A) PAGE stained for SOD activity and Sod1 RNA blots (20 g/lane) from 5 dpi embryos exposed to benzyl viologen and juglone. Abbreviations: ip, in planta; C, control. (B) Sod1 RNA blot from H 2O 2-treated leaves isolated from 9 dpi seedlings.
W64A seedlings grown in constant dark for 8 days were subjected to polychromatic or monochromatic light. The seedlings were exposed to a single 15 min light pulse from white light, red light, far-red light, red light/far-red light or blue light, and then returned to the dark. One group of seedlings grown in constant darkness was used as a control. In the dark control Sod1 mRNA levels were steady through 16 h then declined over the next 16 h (Fig. 9). When the seedlings were exposed to a 15 min white light pulse or a 15 min red light pulse, the Sod1 transcript was lower initially then increased at 8 –16 h before decreasing again. Treatment with far-red light produced no differences from the control. When red light/far-red light and blue light were used, Sodl transcript levels were extremely low and no pattern discernible (data not shown). The effect of UV radiation on Sod1 was also examined using a single 15 min pulse of UV light, plus or minus white light. In addition, the three classes of UV (UV-A, UV-B, UV-C) were subtracted using the appropriate filters; for example, UV-C exclusion using a cellulose acetate filter. There were no significant differences among the treatments with or without white light (Figs. 10A and 10B), except in the full UV light plus white light samples, where the Sod1 transcript remained at steady levels longer before declining, than the same treatment minus white light. In the UV minus white light without UV-B or without UV-C, UV-B,
and most of UV-A treatment, Sod1 transcript levels seemed to be higher than their counterparts in the UV plus white light treatment. When 8 dpi 12 h/12 h L/D plants were exposed to constant UV-B light for 12 h, no change was seen in Sod1 mRNA levels as compared to control levels (Fig. 11). DISCUSSION
A cDNA (pS1) and a partial genomic clone for the maize chloroplastic Cu/ZnSod1 have been isolated and characterized. Southern hybridization indicated the cDNA was not homologous with the cytosolic Cu/ZnSods of maize. The pS1 clone was 896 bp in length and contained a short 5⬘ flanking sequence and a complete 3⬘ untranslated sequence with a poly(A) tail. The amino acid sequence showed similarities with other chloroplastic Cu/ZnSods (30, 43– 45). A genomic library was prepared and probed with pS1. The gene contained a promoter, transit peptide, approximately one-third of the coding region and four introns. Placement of the introns is similar to that of the tomato chloroplastic Cu/ZnSod gene (46); the maize cytosolic Cu/ZnSod4 and Sod4A (47), and the rice cytosolic Cu/ZnSod genes (48), indicating that this structure is conserved among plant Cu/ZnSod genes. Other genes that have been isolated for the chloroplastic Cu/ZnSOD are from tomato (46) and Arabidopsis thaliana (49), although only
FIG. 8. Sod1 expression in 7 dpi maize leaves grown at 25°C, on a 12 h/12 h L/D, a constant light, or a constant dark regimen. Samples were taken every 6 h for 2 days. RNA blots were prepared and hybridized as described.
STRUCTURE AND REGULATION OF THE Sod1 GENE IN MAIZE
FIG. 9. Accumulation of Sod1 mRNA in leaves of 8 dpi dark-grown maize seedlings subjected to a 15-min light pulse of white light, red light, or far-red light. Plants grown in total darkness were used as a control. After treatment plants were returned to constant darkness. Leaf material was harvested immediately before the light pulse (DC, dark control), immediately after the light treatment (0), and every 4 h for 32 h. RNA blots were prepared and hybridized as described.
the tomato gene was studied in some detail. The Sod1 transit peptide was 49 amino acids long, a length similar to that of rice (45). A comparison of the transit peptides of the maize Sod1 and tomato chloroplastic Cu/ZnSod gene resulted in no large areas of homology, although both contain many serine residues. As a general rule, transit peptides do not contain any similar amino acid sequences, but are characterized by containing more hydroxylated and small hydrophobic amino acids, lacking acidic amino acids and having a positive charge (50). The maize Sod1 promoter contains several putative cis-acting elements for light regulation. The GATA motif (I-box) is a common cis-acting regulatory element found in many plant promoters, both light regulated and non-light regulated (32). A group of three GATA motifs were found in tandem shortly before the putative start of transcription of Sod1. Groups of GATA
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boxes are often found near the TATA box in promoters from light-regulated genes such as CAB and RbcS (51, 52). In this case, the maize Sod1 promoter does not contain a TATA box. The 12 h/12 h light/dark and constant light regimens appeared to cause a cycling effect, while constant dark treatment did not result in a definable cycle, indicating that Sod1 may be regulated by light. To date, no other Cu/ZnSod has been reported to be under circadian regulation. In Arabidopsis an FeSod was reported to be influenced by a circadian rhythm (7). Some of the other light treatments examined also had an effect on the Sod1 transcript. For example, a 15-min white light or red light pulse delayed induction of the Sod1 transcript in dark-grown plants. A UV light pulse also led to changes in Sod1 mRNA expression, although treatment of 12 h/12 h L D seedlings with or without constant UV-B did not result in an increase of SOD-1 in the leaves. In Arabidopsis, it was also found that UV-B did not alter chloroplastic Cu/ZnSod expression (7). In view of the results that different light treatments affect Sod1, functional analysis of light elements present in the maize Sod1 promoter may lead to information regarding the role of these elements. In light studies involving other plant species, it was found that although transgenic tobacco plants containing the tomato chloroplastic Cu/ZnSod gene respond to light, no cis-elements involved in light regulation were found in the truncated (285 bp) promoter sequence reported (46). In tomato, the chloroplastic and cytosolic Cu/ZnSod transcripts increased after exposure to white light (53). Although UV light did not result in dramatic changes in the maize Sod1 transcript, it was shown that ROS levels, as a result of
FIG. 10. Accumulation of Sod1 mRNA in leaves of 8 dpi dark-grown seedlings subjected to a 15-min pulse of UV light plus (A) or minus (B) white light. Filters were used to exclude UV-C (cellulose acetate), UV-C and UV-B (polyester), or UV-C, UV-B, and most of UV-A (PVC). After treatment, plants were returned to the dark. Leaf material was harvested immediately before the light pulse (C, dark control), immediately after the light treatment (0), and every 4 h for 48 h. RNA blots were prepared and hybridized as described.
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FIG. 11. Sod1 expression in 8 dpi 12 h/12 h L/D-grown plants exposed to plus or minus UV-B light. Seedlings grown at 25°C on a 12 h/12 h L/D photoperiod were transferred to plus constant UV-B light or minus constant UV-B light. Samples were harvested every 2 h. Lane C, control sample taken immediately prior to treatment.
UV-B exposure, play a role in the regulation of some chloroplast-associated genes in peas, with greater increases seen in protein activity of SOD, ascorbate peroxidase (Apx), and glutathione reductase (GR) in etiolated and green buds after UV-B exposure, and smaller increases in Sod and Apx transcript levels (54). Many environmental challenges have been shown to increase ROS levels in cells (3). Previous studies of the maize SOD proteins showed no significant increase in total SOD activity in response to ozone (O 3) and SO 2 (55), although the chloroplastic Sod1 transcript was reduced after acute and chronic O 3 exposure, while the cytosolic Cu/ZnSods and MnSod3 transcripts increased slightly (56). Increases in temperature adversely affect the metabolic processes of plants resulting in induction of heat shock proteins and repression of normal cellular protein synthesis (57). Previous work from this laboratory showed that seedlings germinated and grown at 40°C had lower total SOD activity through 3 dpi, but total activity then rose to levels similar to seedlings germinated and grown at 25°C (58). Sod1 transcript levels in scutella isolated from seedlings grown at 40°C followed the same pattern as the SOD-1 protein, indicating that no permanent damage was done to the cell’s ability to produce SOD-1. Bipyridyl herbicides (e.g., paraquat), act by accepting an electron from Photosystem I to become reduced free radicals, which are then immediately re-oxidized, transferring an electron to oxygen generating superoxide (42). Paraquat is highly toxic in the light, but can also cause damage in the dark by accepting electrons from other sources such as the mitochondrial transport chain. It was previously shown, in 10 dpi maize leaves, that the chloroplastic, cytosolic, and mitochondrial SODs increased in response to short-term treatment with low concentrations of paraquat and juglone (59). In this study, treatment of 5 dpi embryos with paraquat resulted in an increase of both SOD-1 protein and Sod1 mRNA levels, while treatment of 12 dpi seedlings resulted in a decrease of Sod1 mRNA in leaves at higher concentrations but an increase in SOD-1 protein. Benzyl viologen, a paraquat analog, also induced Sod1 in 5 dpi scutella as concentration increased, although previously no effect was observed on maize total SOD protein levels (59). Treatment with juglone, a redox
compound unrelated to paraquat that also produces superoxide, resulted in initially elevated Sod1 mRNA levels at low concentrations but reduced levels at high concentrations. In pea leaves, it was shown that sensitivity to paraquat was dependent on cultivar and leaf age (60). Transgenic plants that overexpress the pea chloroplastic Cu/ZnSOD showed enhanced resistance to paraquat damage as well as to chilling and moderate light intensity (61). Hydrogen peroxide treatment did not inhibit the Sod1 transcript. This correlates with previous results (9) that the SOD-1 protein is more resistant to H 2O 2 than the cytosolic Cu/ZnSODs. This may be due to naturally higher levels of H 2O 2 in the chloroplast and, in order to function properly, SOD-1 is more resistant. In conclusion, the maize chloroplastic Cu/ZnSod1 gene has been isolated and characterized. The promoter contains several responsive elements that may play a significant role(s) in its response to various environmental insults known to increase ROS levels in the chloroplast. Deletion analyses of the Sod1 promoter are currently underway, using transgenic maize plants, to elucidate those elements involved in Sod1 regulation. Additional studies should help to further define the mechanisms by which Sod1 perceives signals to initiate the antioxidant response cascade. ACKNOWLEDGMENTS Research was supported, in part, by Grants from the USEPA, NSF, and USDA. We thank Stephanie Ruzsa for commenting on the manuscript.
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