2 Present address: Scottish Crop Research Institute, Invergowrie, Dun- dee DD2 5DA, Scotland, United Kingdom. ... A Unicam SP 1800. Ultraviolet ...
Plant Physiol. (1987) 85, 1016-1020 0032-0889/87/85/10 16/05/$0 1.00/0
Superoxide Dismutase as an Anaerobic Polypeptide' A KEY FACTOR IN RECOVERY FROM OXYGEN DEPRIVATION IN IRIS PSEUDACORUS? Received for publication April 3, 1987 and in revised form July 27, 1987
LORNA S. MONK*2, KURT V. FAGERSTEDT, AND ROBERT M. M. CRAWFORD Department of Plant Biology and Ecology, University of St. Andrews, St Andrews, Fife KY16 9AL, Scotland, United Kingdom ABSTRACT
The perennating organ, the rhizome, was chosen for examination of response to anoxia in the species Iris pseudacorus L., Iris germanica L. var Quechei, and Glyceria maxima (Hartm.) Holmberg. These monocots are known to differ in their tolerance of anoxia. Intact rhizomes were subjected to periods of prolonged anoxia of up to 28 days and superoxide dismutase (SOD) activity was determined in a 48 hour postanoxic recovery phase. Tests were performed to ensure the accuracy of the measured enzyme activities. In the most anoxia tolerant species, I. pseadaorus, SOD activity rose continuously during the period of imposed anoxia, and levels were maintained in the postanoxic recovery phases: 28 days brought about a 13-fold increase to 1576 U SOD per milligram protein. Small increases were found in the less anoxia tolerant I. germanica during anoxic/postanoxic phases, while a drop in activity was recorded in the least anoxia tolerant G. maxima. However, initial levels in G. maxima were more than twice as high as in the other two species. Experiments applying cycloheximide to anoxic rhizome slices of I. pseudacorus inhibited the increase in SOD activity. This indicates that SOD is, paradoxically, induced under anoxia and we suggest that in this species SOD is one of the enzymes identified as anaerobic polypeptides. The significance of the induction of an 'oxygen-protecting' enzyme during complete oxygen deprivation is discussed with regard to a possible critical role duning recovery from anoxic stress.
Superoxide dismutase (EC 1.15.1.1.) plays a central role in the protection against oxygen toxicity in aerobic organisms (7, 8), catalyzing the disproportionation of the superoxide radical to hydrogen peroxide and dioxygen. In higher plants SOD3 has been isolated in maiy tissues (9, 21), and three distinct metalloenzymes have been characterized. The Cu,Zn-SOD and Mn-SOD forms of the enzyme could be expected to be present in nonphotosynthetic rhizomes, the tissue which is the subject of this investigation. The third form, Fe-SOD, appears to be associated only with the chloroplast in higher plants (2). There are a number of cellular reactions which proceed by a single electron transfer from the substrate onto each molecule of oxygen used, producing superoxide. Halliwell ( 11) has cited the enzymes nitropropane dioxygenase, galactose oxidase, and xan-
thine oxidase as being among those that produce superoxide in plant tissues. If there is sufficient superoxide production, reactions such as the iron-catalyzed Haber-Weiss reaction may take place leading to the generation of hydroxyl radicals (12). Prerequisites for the reaction are traces of a transition metal ion. Hydroxyl radicals will react indiscriminately with cellular compounds, and are capable ofabstracting hydrogen from membrane lipids, which triggers the chain reaction of lipid peroxidation. If uncontrolled, the lipid peroxidation process may affect membrane integrity, since lipid peroxides and some of their degradation products cause extensive damage to membrane-bound enzymes and to the lipid bilayer itself, producing a decrease in electrical resistance and membrane fluidity (26). The nonenzymic defense against such oxidative injury has been the subject of recent studies (4) and may comprise small molecular compounds such as ascorbate, a-tocopherol, and reduced glutathione. Superoxide as well as other reactive oxygen species are scavenged by these antioxidants. It has been observed that plants completely deprived ofoxygen survive a period of imposed anoxia, only to die on reexposure to air (25), suggesting oxidative damage during the recovery phase. Since anoxic stress is a major factor in the flooded environment (3), a similar sequence of events could be expected to occur in a plant which has undergone a period of waterlogging. A significant rise in the lipid peroxidation product, malondialdehyde, has previously been reported in rhizomes of the anoxia and flooding sensitive species Iris germanica on return to air after oxygen deprivation. In contrast, levels remained the same in the related anoxia tolerant wetland species Iris pseudacorus (15). Therefore, experiments with three species of differing tolerance of anoxia were carried out to examine SOD levels during a recovery phase following prolonged periods of anoxic stress.
MATERIALS AND METHODS Species and Growth Conditions. The three species in this series of experiments were: Iris germanica L. (var Quechei, supplied by the University Botanic Garden), Iris pseudacorus L. and Glyceria maxima (Hartm.) Holmberg, collected locally. G. maxima is the least tolerant of anoxia (less than 1 week (1), while L germanica is of intermediate tolerance and L pseudacorus is very tolerant (2 and 8 weeks, respectively) (14). Plants were allowed to grow on in the greenhouse, and prior to experimentation were bathed for 1 min with 0.16 mM chloramphenicol, and incubated aerobically for 48 to 72 h wrapped in moist filter paper at 20°C. ' Supported by research grants from the Natural Environment Re- The main series of experiments concerning anoxic versus postansearch Council (to L. S. M.) and from the Academy of Finland (to K. V. oxic SOD activity were conducted within the three spring months and the protein synthesis inhibitor experiment was performed F.). 2 Present address: Scottish Crop Research Institute, Invergowrie, Dun- the following spring. Anoxic incubation of rhizome material was accomplished by dee DD2 5DA, Scotland, United Kingdom. 3 Abbreviations: SOD, superoxide dismutase; XOD, xanlthine oxidase; filling anaerobe jars (Gaspak, Becton Dickinson and Co.) containing a palladium catalyst to remove traces of oxygen, in the NBT, nitro blue tetrazolium. 1016
1017
SUPEROXIDE DISMUTASE AS AN ANAEROBIC POLYPEPTIDE anaerobic workbench (Forma Scientific, Ohio) and subsequently placing them in incubators at 20°C. The anoxic atmosphere thus consisted of 90% N2 and 10% H2. The jars also contained methylene blue indicator strips which remain colorless in the absence of oxygen. During the postanoxic phase in air rhizomes were kept on moist filter paper at 20°C. SOD Assay: Xanthine-Xanthine Oxidase Method. Superoxide dismutase was assayed spectrophotometrically, based on the method by McCord and Fridovich (20). A Unicam SP 1800 Ultraviolet Spectrophotometer was used attached to a waterbath at 25 ± 1 C, which kept the temperature of the cuvettes constant. The reaction mixture (total volume, 3 ml) contained: 50 mm Kphosphate buffer (pH 7.8), 1.0 mm NaN3, 0.10 mM EDTA, 0.01 mM ferricyt c, and 1.0 mM xanthine. The reaction was started by adding XOD, which was diluted approximately 10 times in order to produce an increase in A at 550 nm of 0.025 unit/min. The substrate for SOD is the unstable superoxide free radical, which in this indirect assay is continuously generated at a controlled rate by XOD. Cyt c in the assay mixture is reduced by superoxide at a known rate, which on the addition of SOD will decrease. Under these conditions, the amount of SOD required to inhibit the rate of reduction of Cyt c by 50% is defined as one unit of activity. Calibration curves (0.1-0.5 ,ug SOD/ml) were made daily with bovine liver SOD (Sigma). The curves were linear up to 30 to 35% inhibition. Buffer containing Cyt c and xanthine was made up weekly and stored at -20C. Each commercial preparation of horse heart Cyt c (Sigma) was first checked spectrophotometrically to ascertain the true concentration of ferricyt c. Dilutions of buttermilk XOD (Sigma) were made daily, and the rate of reduction of Cyt c was checked regularly throughout the assay. Peroxidases and Cyt oxidase are known to interfere with this assay, by using Cyt c as a substrate (5), so 1.0 mM azide was added to the buffer to block these possible reactions (16). A low rate of Cyt c reduction was noted on addition of I. pseudacorus (7 d anoxic incubation onward) extract before XOD was added to start the assay reaction. This interfering reaction, probably due to a peroxidase, was eliminated by diluting the extract, a step anyway necessary because of the high activity in this species. Higher concentrations of azide could not be used since it has been shown to inhibit the Cu,Zn-SOD isozyme (I17). Tissue Preparation. Rhizome extracts were prepared first by grinding with a pestle and mortar 0.5 g slices in 3 ml 0.05 M Kphosphate (pH 7.8), containing 0.1 mM EDTA and 1.0 mM NaN3. After 15 min centrifugation at 8730g at 5°C, the supernatant was passed through a filter, pore size 1.2 ,um (Millipore). The extract was then desalted by running through a Sephadex G-25 column (Pharmacia), which had been equilibrated with the extraction buffer. Some dilution of the extract occurred at this step, and in the case of G. maxima and L germanica the extracts were then concentrated in Centricon centrifugal microconcentrators (Amicon). Due to the large amounts of SOD found in I. pseudacorus, however, the extract was diluted 2- to 4-fold. The extraction procedure was carried out as far as possible at temperatures below +5°C. Soluble PVP, 3% (w/v) was added to the extraction buffer in the case of I. germanica and I. pseudacorus in order to give maximal catalytic activities. Addition of PVP did not enhance SOD activity in G. maxima and so it was omitted from the extraction procedure. Protein Assay. Soluble protein content of the extracts was taken as a reference value for SOD activity and measured by the binding of bromophenol blue to proteins under acidic conditions, the bound form absorbing light at 610 nm wavelength (6). BSA was used as a standard. It was noted that PVP interferes with this protein assay, causing some overestimation of protein content so that the specific activity of SOD in the Iris species may be consistently slightly underestimated.
Recovery Experiments. Enzyme recovery experiments with mixtures of samples and standards were carried out for each species. A recovery value of 88.1 ± 3.0% was found for G. maxima (n = 3); 104.0 + 10.6%, for I. germanica (n = 3); 94.2 + 2.8%, for L pseudacorus(n = 3). SOD Assay: Photochemical Method. To test the accuracy of SOD activity measurements by xanthine-XOD spectrophotometric assay, a second method was used for the further investigation into the high activities found in L pseudacorus rhizomes. This photochemical determination of SOD activity is based on the photoreduction of NBT by light in the presence of riboflavin and methionine. NBT is reduced to blue formazan, which has a strong absorbance at 560 nm wavelength. Under aerobic assay conditions SOD inhibits the formation of blue formazan. The reaction mixture (3 ml) contained 1.3 AM riboflavin, 13 mM methionine, 63 AM NBT, and 0.05 M K-phosphate with 0.10 mm EDTA. This assay has been stated as being most reliable and reproducible (9). One SOD unit was defined as 50% inhibition of the basic rate of reaction according to McCord and Fridovich (20). However, to establish a linear relationship between SOD activity and inhibition, V/v transformation was used. Linear correlation gave an equation: SOD U/ml = (0.953V/v - 0.907) x dilution factor The correlation coefficient for this line was 0.990 (Fig. 1). Since plant tissues contain naturally occurring small molecular antioxidants (1 1, 18), their presence was tested for by passing the extract through molecular filters with pore sizes equivalent to mol wt of 10,000 and 30,000. The small molecular fraction was estimated to account for only 3 to 8% of the total SOD activity measured in L pseudacorus and this activity did not increase during anoxic stress. Experiments with pure bovine liver SOD added to plant samples produced a recovery percentage of 98.5 ± 2.7%. Protein Synthesis Inhibitor Experiment. To observe whether the induced SOD activity is synthesized de novo or activated during anaerobiosis, an experiment with the protein synthesis 9
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0 1 3 SOD units 2 FIG. 1. Inhibition of the photoreduction of nitro blue tetrazolium by SOD of raw extract of I. pseudacorus rhizome tissue. Since inhibition is not linear with SOD concentration, a V/v transformation was used to obtain linearity (V = basic reaction rate without rhizome extract, v = reaction rate with extract). When 50% inhibition of basic reaction rate was used as one unit of SOD activity, equation for the line was SOD U = 0.953V/v - 0.907. Correlation coefficient for this line was 0.99.
Plant Physiol. Vol. 85, 1987
MONK ET AL. 1018 inhibitors cycloheximide and actinomycin D was carried out. The photochemical method for SOD activity measurements was used. Transverse slices of rhizome tissue (2 mm thick) were incubated anaerobically in 100 ml flasks on a shaker at 20°C in
the dark. The flasks contained 68 mM K-phosphate, 0.1 mM EDTA, 100 gg/ml cycloheximide, and/or 50 ,ug/ml actinomycin D. To exclude bacterial contamination during the incubation 0. 16 mm chloramphenicol was added to all samples. SOD activity in the tissue was determined at 0, 4, 7, and 11 d anoxia. Control material was incubated under the same conditions but without the protein synthesis inhibitors. Total protein content of the rhizome extracts from tissue slices was estimated using a kit supplied by Sigma based on Peterson's modification (27) of the micro-Lowry method with precipitation of proteins. Inhibition of SOD by KCN. In order to find out what proportion of Cu,Zn-SOD vs Mn-SOD was present in the activities measured in aerobic and anoxic rhizome material, an inhibition experiment with KCN was performed (20) using the photochemical method for estimations of SOD activity. KCN (3 mM) was added to the cuvette before illumination.
RESULTS SOD Activity in Anoxic and Postanoxic Rhizomes. Superoxide dismutase activity was determined in rhizomes of three species after 3 (G. maxima only), 7, 14, 21, and 28 d anoxia, and during a 48 h postanoxic recovery phase. Each of the three species examined showed a different pattern of development of SOD as a result of anoxic stress (Fig. 2). Remarkable rises in levels of SOD were found in I. pseudacorus rhizomes during the course of the anoxic treatments. Seven d anoxia resulted in a 3½/2-fold increase in activity and at the end of 14 d anoxia an 8-fold rise was apparent, while 21 d anaerobiosis produced an 1 -fold increase; 28 d anoxia brought about a 13-fold increase in levels of SOD to 1576 U/mg protein. The longer the period of imposed oxygen deprivation, the greater the increase in SOD activity seen. These high activities were maintained in every case during the postanoxic recovery phase. In L germanica rhizomes initial levels of activity were similar to that measured in the related I. pseudacorus. No significant change in level was found at the end of 7 d anoxia, but 14 d oxygen deprivation resulted in a 170% increase; 21 d anoxia brought about a 120% increase. However, 28 d produced a slight drop in activity compared to initial levels. During the 48 h recovery phase after 7 d anoxic stress, increases in SOD up to 197 U/mg protein were noted. During recovery phases after 14 and 21 d anaerobiosis, the relatively high levels found during anoxia are approximately maintained. In contrast, there is a postanoxic increase in SOD activity to 159 U/mg protein in rhizomes from the 28 d treatment, which falls sharply again at 48 h. In G. maxima rhizomes over twice as high SOD levels as in the Iris species were found at the beginning of the experiment. After 3 d anoxia, however, a drop of about 50% was recorded. Decreases in activity of similar proportion were also seen in the longer periods of anoxia. In rhizomes exposed to 3 and 7 d anaerobiosis, SOD activities increased during the 48 h postanoxic phase to levels approaching those at the beginning of the experiment. After the 14, 21, and 28 d anoxic treatments SOD activity fell away during the postanoxic phase to levels only 30% of the initial measurements. Protein Synthesis Inhibitor Experiment. Results of the protein synthesis inhibitor experiment are shown in Figure 3. During 11 d incubation under anoxic conditions SOD activity in I. pseudacorus rhizome slices increased by 44%. Slices incubated with actinomycin D behaved in a similar manner, whereas tissue with cycloheximide decreases in SOD activity by 47%. Incubation with both actinomycin D and cycloheximide resulted in an even
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FIG. 2. The development of superoxide dismutase activity, as determined by the xanthine-xanthine oxidase system, on return to air in (A) I. pseudacorus, (B) I. germanica and G. maxima rhizomes after anoxic incubation of up to 28 d. The column sets represent 0 to 2 d postanoxic treatment, and the different columns within the sets represent 0 (0), 3 (E), 7 (S), 14 (O), 21 (E), and 28 (U) d of previous incubation under anoxia. (n = 3-6, bars indicate standard error of mean).
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the tissue dying under these severe conditions. KCN inhibited 84.0% and 75.5% of the SOD activity present in aerobic and anaerobic tissues, respectively, indicating that Cu,Zn-SOD is the prevalent form of the enzyme in both envionments (Table I). Boiling the extract for 20 min resulted in about 64% decrease in activity showing that SOD in L pseudacorus is relatively heat stable in raw extract, as is the case with many other higher plant SODs (1 1).
DISCUSSION The level of SOD found in anaerobic rhizomes of the three species in this series of experiments, was closely associated with the ability of the particular species to survive anoxia (Fig. 2). I. pseudacorus showed considerable increases in SOD activity during anoxia, and these high levels (up to 13-fold rise, compared to initial levels) were maintained during the recovery phase in air. This species has been observed to survive 8 weeks oxygen deprivation (14). High SOD activities may contribute to the tolerance of anoxia by providing adequate defences against oxygen toxicity on restoration of oxygen supply. It is possible that the modest increases seen in the related I. germanica are not large enough, and hence oxidative damage could be a determin-
SUPEROXIDE DISMUTASE AS AN ANAEROBIC POLYPEPTIDE 14
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