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Effects of ozone and sulfur dioxide on superoxide dismutase levels in bean and radish leaves Chanway, Christopher Peter 1983

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EFFECTS OF OZONE AND SULFUR DIOXIDE ON SUPEROXIDE DISMUTASE LEVELS IN BEAN AND RADISH LEAVES Christopher Peter Chanway B.Sc, The University of Winnipeg, 1978 B.S.Agric, The University of Manitoba, 1980 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Plant Science) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA (c) Christopher Peter Chanway, 1983 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Plant Science The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date May 27. 1983 DE-6 (3/81) ABSTRACT The primary leaves of bush bean plants pretreated daily with non-injurious, low levels of ozone ( 0 . 0 2 or 0 . 0 5 ppm) pass through stages of varying susceptibility to a subsequent acute dose. Previous work showed that this variation in response to acute dose could only partly be accounted for by stomatal behaviour. Present studies i n -dicate that the oxy-radical scavenger superoxide dismutase (SOD) appears to play no role in the phenomenon. No observed changes in SOD levels following various low ozone pretreatment regimes were related to susceptibility to acute injury in compaEisons with plants maintained M ' f i l t e r e d a i r as controls. The only significant change in SOD levels which appeared to be related to ozone occurred simultaneously with the appearance of vis i b l e symptoms of injury following exposure to 0^ concentrations greater than 0.1 ppm. The nature of the effect on SOD levels and the degree of visible injury was related to plant age and temperature. Younger plants or plants fumigated at a lower temperature (23°C) showed less injury after acute exposure to ozone than older plants or ones exposed at a higher temperature (33°C). SOD activity was higher than controls when a lower temperature growth regime ( 2 3 ° / l 6°C) was used, but dropped to below control levels when higher temperatures ( 3 3 ° / 2 3°C) were used. The anti-ozone agent "ethylene diurea", EDU, provided protection against acute injury, but had no effect on SOD levels in either primary or t r i f o l i a t e leaves prior to exposure to an acute dose. SOD levels in the f i r s t and second leaves of radish plants pre-treated daily with low ozone ( 0 . 0 2 ppm) were not substantially different i i i from c o n t r o l s before or dftex an acute exposure to ozone. In c o n t r a s t to bean l e a v e s , pretreatment of r a d i s h w i t h low ozone a l s o had no e f f e c t on the amount of i n j u r y induced by an acute dose. Low SGv, pretreatment (0.1 ppm) of the primary leaves of bush bean or the f i r s t two leaves of r a d i s h d i d not a f f e c t SOD a c t i v i t y . V i s i b l e i n j u r y a f t e r an acute S0^ exposure (2.0 ppm) was a l s o not a f f e c t e d by subacute pretreatment i n bean. However, p r e t r e a t e d r a d i s h leaves were predisposed to acute i n j u r y . In c o n t r a s t w i t h the e f f e c t of ozone, acute SO^  exposure of the primary leaves of bean r e s u l t e d i n a decrease i n SOD a c t i v i t y com-pared w i t h p l a n t s grown i n f i l t e r e d a i r , r e g a r d l e s s of pretreatment regime. A s i m i l a r t r e n d was observed i n t K e r f i r s t two leaves of r a d i s h only i f p r e t r e a t e d w i t h subacute S02» SOD a c t i v i t y i n leaves p r e t r e a t e d w i t h f i l t e r e d a i r was not a f f e c t e d by acute S0 o fumigation. iv TABLE OP CONTENTS Abstract i i Introduction 1 Review of Literature 3 Pretreatment Effects 3 Effects of Free Radicals 8 Superoxide Dismutase 15 Materials and Methods 23 Fumigation with Ozone 23 Fumigation with SO^  • 25 Assessment of Injury 25 Analytical 26 Results 31 Beans Treated with Ozone 37 Relation of SOD to Injury Symptoms 39 Effect of EDU on Bean Leaves 42 SOD and Acute Injury 46 Low Temperature Effects ( 2 3 ° / l 6 ° C) 48 High Temperature Effects (32° / 2 3 ° C) 52 Radish Treated with Ozone 52 Effects of Subacute Sulfur Dioxide 56 Effects of Acute Sulfur Dioxide 56 Discussion 62 Summary 73 Literature Cited 75 V LIST OF TABLES 1 . Reactivity of chloroplast components with superoxide: their reaction rate constants and concentration in chloroplasts .... 19 2 . SOD activity before acute dose and chlorophyll content after acute dose in beans treated with 0 . 1 ppm ozone 6 hours per day 44 3 . Effect of EDU on SOD level of bean leaves before acute ex-posure, and on %LAN after acute exposure 45 4 . %LAN in plants receiving acute ozone or S O 2 treatment after pretreatment with either f i l t e r e d a i r or subacute doses of 0 ^ or S 0 2 58 5 . SOD activity in plants receiving acute 0 ^ or S 0 | treatment compared with plants maintained in f i l t e r e d a i r or sub-acute concentrations of 0 ? or S 0 O 59 v i LIST OF FIGURES 1. Relationship between l o g AC^Q and volume of bovine erythrocyte SOD added 28 2 . SOD a c t i v i t y and protein content of primary bean leaves with PVP i n extraction medium expressed as a % of values obtained without using PVP 32 3 . Diagrammatic representation of SDS-PAGE columns containing proteins of f i r s t t r i f o l i a t e leaves of bush bean 33 4 . SOD a c t i v i t y i n the primary leaves of bean ex-pressed on a l e a f area and dry weight basis 34 5 . SOD a c t i v i t y i n the f i r s t two leaves of radish expressed on a l e a f area or dry weight basis 35 6. Comparison of i n j u r y assays using primary leaves of bean 36 7. E f f e c t of d a i l y 6-h pretreatment with ozone on %LAN of primary leaves of bean r e s u l t i n g from treatment with 0 .4 ppm ozone 38 8 . E f f e c t of d a i l y 6-h ozone pretreatment on SOD a c t i v i t y i n primary leaves of bean p r i o r to t r e a t -ment with 0 .4 ppm ozone 40 9 . Percentage i n h i b i t i o n of SOD i n primary leaves of bean by add i t i o n of 1mM KCN to the assay 41 10. SOD a c t i v i t y i n primary leaves of bean treated d a i l y with 0.1 ppm ozone ( 6 h ) , expressed as a percent of controls maintained i n charcoal-f i l t e r e d - a i r 43 11. E f f e c t of ozone treatment on s p e c i f i c l e a f area of primary bean leaves 47 12. E f f e c t of four successive exposures ot 0 .4 ppm ozone on SOD a c t i v i t y and cumulative %LAN i n primary leaves of bean 49 13* Conditions as i n Figure 12, but exposures com-menced 6 days from emergence 50 v i i 14. Conditions as i n Figure 12, but exposures com-menced 9 days from emergence 51 15. E f f e c t of four successive exposures to 0.4 ppm ozone of SOD a c t i v i t y and cumulative %LAN i n primary leaves of bean at 3 2 ° / 2 3 ° C 53 16. Conditions as i n Figure 15, but exposures com-menced 6 days from emergence 54 17. Conditions as i n Figure 15» but exposures com-menced 9 days from emergence 55 18. SOD a c t i v i t y i n the f i r s t two leaves of radish exposed to 0.02 ppm ozone 6 hours d a i l y 57 19. SOD a c t i v i t y i n the f i r s t two leaves of radish and the primary leaves of bean exposed to 0.1 ppm S09 continuously 60 v i i i ACKNOWLEDGEMENTS I would l i k e to express sincere thanks to Dr.V.C.Runeckles f o r h i s guidance and support throughout t h i s program. I would also l i k e to express my appreciation to Peter Garnett . f o r h i s advice and expertise i n technical matters. F i n a l l y , I am g r a t e f u l to Dr.F.B.Holl and Dr.M.Shaw f o r generously allowing me use of t h e i r labratory equipment and instruments during my work, and D r . P . A . J o l l i f f e f o r introducing me to computers. i x LIST OF ABBREVIATIONS EDU - N-(2-(2 - 0 x 0 - 1-imidazolidinyl)-ethyl)-N'-phenyl urea ("ethylene diurea") d - day h - hour LAN - l e a f area n e c r o t i c Or? - superoxide r a d i c a l 0, - ozone OH* - hydroxyl r a d i c a l PAGE - polyaery1amide gel electrophoresis ppm - parts per m i l l i o n , by volume, m i c r o l i t e r s per l i t e r PUFA - polyunsaturated f a t t y a c i d SO2 - s u l f u r dioxide SOD - superoxide dismutase TCA - t r i c h l o r o a c e t i c a c i d 1 INTRODUCTION The phenomenon by which previous exposure to low ambient levels of a pollutant may confer a protective effect to foliage exposed to a subsequent acute dose may in part be attributable to biochemical changes in the plant tissue (Zahn, 1970; Runeckles and Rosen, 1974)• Specifically, the protective effect observed by pretreating bush bean Phaseolus vulgaris L. cv. Pure Gold wax with 0.02 ppm ozone (0 )^ daily can be correlated partly with a dampening of stomatal action, effectively reducing the amount of the oxidant entering the pretreated plant during subsequent acute fumigation (Runeckles and Rosen, 1977). How-ever, this phenomenon holds true only for pretreatment concentrations below 0.04 ppm 0^  administered for six hours daily. Higher pretreat-ment concentrations predispose plants to injury from a subsequent acute dose, which suggests that other factors may be involved in the response. The idea that free radicals may contribute to the mechanism by which a i r pollutants cause tissue damage is not new (Pryor, 1976; Leshem, 1981). Recently, evidence has been put forth supporting the view that injury caused by sulfur dioxide (S02)(Tanaka and Sugahara, 1980) and 0^  (Lee and Bennett, 1982) involves the superoxide radical, 0£. Both studies followed the levels of superoxide dismutase (SOD) as an indicator of the internal steady state concentrations of O^*. Treatment with the experimental chemical, N-(2-(2-oxo-1-imidazol-idinyl)-ethyl)-N-phenyl urea ("ethylene diurea", EDU) has been reported to confer ozone-tolerance to sensitive species (Carnahan et a l . , 1978? Legassicke and Ormrod, 1981). Lee and Bennett (1982) reported that 2 EDU-induced tolerance to ozone in snap beans, Phaseolus vulgaris L. cv. Bush Blue Lake 290, involves induction of SOD as an oxy-radical scavenger. The f i r s t objective of this study was to determine the role of SOD, i f any, in the protective effect conferred by ozone pretreatment on the primary leaves of bush bean and to extend these studies to radish; the second objective was to investigate the proposed role of SOD in EDU-conferred ozone-tolerance in bush bean. The third objective was to verify the reported protective effect of SO^-induced SOD on foliage subsequently treated with acutely injurious SO^  levels. 3 REVIEW OF LITERATURE Pretreatment Effects The movement of a gaseous pollutant i s passive (Heath, 1980) thus total flux of such a gas into a plant can be approximated by Fick's law for diffusion: j = g C (1) where J = mass flow or flux, g = conductance of the gas, and C = the concentration gradient, which i s generally assumed to be linear across the conductance region. Since resistance i s the reciprocal of con-ductance, the equation can be rewritten as: j , -S (2) total Total resistance (r, , ,) = r + r + r (3) v t o t a l 7 a s m v ' where r = the resistance from bulk a i r to the boundary layer near the stomate; r = stomatal resistance; and r = resistance encountered ' s ' m from the substomatal chamber to the cellular interior, including c e l l walls and membranes. Two significant factors should be noted from equation 2. Fi r s t , under f i e l d conditions where an appreciable wind speed at the canopy layer often exists, r w i l l be very small; thus r , as determined by 3« S .. the size and degree of opening of the stomates, becomes extremely important in determining flux. Second, the difference between pol-lutant exposure and effective dose absorbed by the plant should be emphasized (Runeckles, 1974)* Classically, pollutant exposure has 4 been described by the term "dose", which is the product of concentra-tion and time. From equation 2 i t can be seen that even a high con-centration yielding a large "C" component can lead to a small effective dose (i.e. the amount of pollutant actually entering the plant) when any or a l l components of r^- 0^ a^ a r e proportionately large. For ex-ample, i f stomates are closed, r g w i l l be very large and a high "C" w i l l s t i l l result in a small effective dose. Previous exposure to an a i r pollutant can alter susceptibility of a plant to a subsequent acute dose (Macdowell, 1965; Heck and Dunning, 1967; Runeckles and Rosen, 1974» 1977). The type of response i s dependent on several factors including plant species and age, en-vironmental conditions, and pretreatment dosage of the pollutant. Considering the importance of stomata in determining pollutant flux, many pretreatment studies have focussed on their role as an explan-ation of the phenomena. For example, Macdowell (1965) observed that tobacco (Nicotiana tabacum L. cv. White Gold) exposed to a low dose of 0^  (about 0.04 ppm, 7 h per day) was predisposed to flecking from sub-sequent similiar exposures, while plants exposed i n i t i a l l y to 0.08 ppm for 7 hours were less injured by this greater dose administered the next day. He speculated that reduced stomatal opening in plants ex-posed to the higher concentration may have contributed to the effect. Heck and Dunning (1967) verified the antagonistic effect 6£ two successive exposures i n causing v i s i b l e leaf injury. Using tobacco (Nicotiana tabacum L. cv. Bel W3) and bean (Phaseolus vulgaris L. cv. Pinto), they found that two 30-minute exposures to 0.3 ppm 0^  separated by one, two, or three hours resulted in less injury when compared with 5 plants exposed to the same concentration for one hour. The length of the interexposure period did not significantly affect the response. These authors speculated that stomatal closure after the f i r s t fum-igation may have contributed to the response, but also that the plants may have partially "recovered" during the "rest" period. Wilton et a l . (1972) suggested that stomatal mechanisms are not central to observed pretreatment effects of 0^  in various cultivars of bluegrass (Poa pratensis L. cvC's Merion, Kenblue, Windsor, Belturf, and 117-27-6). In their experiments, plants were exposed to 0.3 ppm 0j for 2 to 4 h and injury was recorded. The grass was then clipped to a length of 2.5 cm and allowed to regrow to a height of 20 cm. Further exposure of the regrown grass to 0.3 ppm 0^  resulted in less injury than had been previously recorded. In these results, stomatal acclimation in plants fumigated once seems to be ruled out as a pos-sible explanation of the protective effect of pretreatment. Bicak (1978)» working with bean (Phaseolus vulgaris L. cv. Pure Gold wax) and radish (Raphanus sativus L. cv. Cherry Belle), admin-istered the same total dose of 0^  in several different ways by varying the concentration over a 7 h period. In a l l cases, concentration was manipulated such that a peak was reached either at the beginning, middle, or end of the treatment. In both species, most severe visible injury was correlated with early peak concentrations. If stomatal mech-anisms were involved in the response, a similiar correlation between r and visible injury would be expected. However, no obvious relation-s ship could be detected when mean r , or values of r at the time of s s maximum concentration were compared with injury. These data clearly 6 suggest that factors not related to stomatal mechanisms influence the injury response to 0^. Runeckles and Rosen (1974) showed that the primary leaves of bean (Phaseolus vulgaris L. cv. Pure Gold wax) and the top pairs of leaves in mint cuttings (Mentha arvensis L.) pretreated with daily low 0^  (0.02 ppm 6 h per day) behave differently, in terms of visible f o l i a r injury, when subsequently exposed to an acute dose. Mint pretreated for seven days became predisposed to injury by an acute dose, while pretreated bean was substantially less injured by a subsequent acute exposure than controls pretreated in f i l t e r e d a i r . Stomatal measure-ments indicated that partially dampened stomatal opening in ozone-treated beans could be correlated to the lower f o l i a r injury after one acute dose. However, stomatal conductance in filtered-air-pre-treated plants dropped below that of the ozone-pretreated plants after the acute treatment, yet these plants were more susceptible to injury by a second acute exposure. Hence stomatal action only partially ex-plains these results. Further work by these authors (Runeckles and Rosen, 1977) showed that 0^  pretreatment effects in bean (same cultivar) were dependent on both the number and concentration of pretreatment doses administered. Plants were shown to pass with age through varying stages of increased or decreased susceptibility to an acute dose depending on pretreatment concentration. Plants treated with 0.02 ppm 0^  for 6 h daily were i n i t i a l l y more susceptible to a subsequent acute dose, but after 3 to 4 days became more resistant to acute injury when compared with filtered-air-treated controls. A somewhat higher pretreatment con-7 centration of 0.05 ppm resulted in the plants being less susceptible i n i t i a l l y to an acute dose, during the f i r s t 4 days of pretreatment, but further exposure at this concentration resulted in predisposition to injury by a subsequent acute dose. S t i l l higher pretreatment con-centrations had previously been shown to increase susceptibility to acute injury compared with filtered-air-treated controls (Runeckles and Rosen, 1974)• These results suggest that the plant Response i s simply speeded up by increasing pretreatment concentration, i f the assumption is made that plants pretreated at 0.02 ppm 0^  would even-tually become predisposed to acute injury. Thus, plants pretreated at the lower level of ozone show a relatively long period of reduced susceptibility (following an i n i t i a l period of increased susceptib-i l i t y ) ; at 0.05 ppm 0^  this phase is reduced to 3 to 4 days, while at 0.1 ppm no phase of reduced susceptibility occurs. Transpiration measurements before and during acute fumigation suggested stomatal involvement in the differential susceptibility observed by Runeckles and Rosen (1977)• For example, the primary leaves of bean plants pretreated with 0.02 ppm 0^  daily un t i l they were in the susceptible phase were shown to transpire at rates similiar to controls, but upon exposure to 0.4 ppm 0 ,^ were unable to close their stomata as rapidly as filtered-air-treated controls. However, when such ozone-pretreated plants entered the less susceptible phase, trans-piration rates before and during acute fumigation were shown to be lower than those of plants pretreated in f i l t e r e d - a i r . Thus, when ozone-pretreated plants are more susceptible than controls, dampened guard c e l l .activity may contribute to the phenomenon. The resulting 8 slower stomatal closure would cause exposure to the oxidant for a longer time, compared with filtered-air-treated controls. When the plants had entered the less susceptible phase, stomatal aperture was apparently decreased relative to controls, which would r e s t r i c t the amount of ozone that could be absorbed even when stomates are open. Varying susceptibility to an acute dose following pretreatment also occurs with SGv,. Zahn (1970) found that wheat and barley pretreated with 0 .4 ppm SO^ were significantly less injured than non-pretreated controls after acute exposure, and that this positive effect was en-hanced by increasing the pretreatment duration from 77 to 133 hours. Further work with a l f a l f a (Medicago sativa L.) and larch (Larix decidua L.) revealed no effect of subacute SO2 pretreatment on an acute dose response, inspiring Zahn's (1970) conclusion that monocots react positively to such pretreatment while dicots are not affected or show a slight negative effect. Effects of Free Radicals It appears that some sort of biochemical acclimation or hardening within the pretreated plant may also be partly responsible for the ob-served effects. A review of the reported effects that pollutants have on biochemical processes in plants reveals the limited state of our understanding of the injury mechanisms at the present time. For example, depending on species and dose, SO^ has been reported to either stimulate, — —2 inhibit, or not affect respiration (Black, 1982). Using HSOj and S 0 ^ (hydration products of S 0 _ ) photosynthetic electron transport and 9 ribulose-T ,5 bisphosphate carboxylase are both inhibited (Heath, 1980). After SO^  exposure, quantitative and qualitative changes in pool sizes of amino acids and reducing sugars have also been reported (Malhotra and Sarkar, 1979)• Ozone is thought to stimulate respiration, but this may be a generalized response to injury (Levitt, 1980). Photosynthesis, on the other hand, i s generally reported to be inhibited by 0^  (Heath, 1980). Reactions of cellular components with the oxidant include cleavage of the nicotinamide ring of NADP(H) (Mudd, 1974)» oxidation of aromatic amino acids (Mudd, 19^9) and oxidation of free sulfhydryls to disulfides (Heath, 1980). Ozone also reacts with l i p i d s directly by ozonolysis or indirectly by in i t i a t i o n of l i p i d peroxidation (Mudd, 1982). The mechanism(s) of these responses have not been determined, but Pryor's (1976) volume discusses mounting evidence for the participation of free radicals i n biological systems. A free radical i s an atom or molecule which contains an odd number of electrons and which may be pos-i t i v e l y charged, negatively charged, or neutral. Owing to their unstable electronic configuration, most free radicals are highly reactive. Thus, in any biological system, one can speculate upon a number of effects that may be caused by this sort of random reactivity, such as inactiv-ation of key metabolic enzymes and loss of membrane l i p i d f l u i d i t y . While many species of free redicals are known to exist, two which are l i k e l y to have significant effects on biological systems are the super-oxide radical, 0^ , and the hydroxyl radical, 0H» . Fluxes of 0^' have been shown to inactivate viruses (Lavelle et a l . , 1973)f to induce l i p i d peroxidation (Kellogg and Fridovich, 1975)» to damage membranes (Gold-berg and Stern, 1976; Kellogg and Fridovich, 1977)» and to k i l l c e l ls 10 (Michelson and Buckingham, 1974). Target molecules which are attacked by superoxide include proteins, nucleic acids, polyunsaturated fatty acids (PUFA), and carbohydrates (Leshem, 1981). There has been a suggestion that molecules of DNA may be "nicked" by free radicals, but conclusive proof is lacking (Pryor, 1976; Leshem, 1981). In the plant kingdom, phenomena such as post harvest f r u i t deterioration, leaf senescence, accelerated spoilage of cut flowers (Leshem, 1981) and sunscald (Rabinowitch and Sklan, 1980) have been reported to involve free radicals. Superoxide may be formed in several ways, including photooxidative processes. These reactions are effected by photosensitizers, chemicals which pass on energy of excitation from absorbed light to oxygen. The result is the formation of high energy singlet oxygen (ground state molecular oxygen is in the t r i p l e t state), or 0g via univalent electronic reduction. Such photosensitizers include riboflavin, por-phyrins, and anthraquinones. Superoxide may also be generated as a result of normal electron flow in photosynthesis. Mehler (1951) showed that oxygen can be reduced to HgO^ by illuminated chloroplasts. Indirect evidence using superoxide dismutase (SOD), an 0£ scavenger, suggests that 0~* i s produced in illuminated chloroplasts (Asada et a l . , 1976). For ex-ample, the superoxide mediated photoreduction of cytochrome "c" by spinach (Spinacia oleracea L.) chloroplasts can be inhibited by SOD (Nelson et a l . , 1972). Electron paramagnetic resonance evidence 18 (Harbour and Bolton, 1975) as well as 0 work (Glidewell and Raven, 1975) point to the univalent pathway of oxygen reduction in chloro-11 plasts, suggesting that the H 2°2 reported by Mehler (1951) is more l i k e l y a result of the reaction: 0~. + 0~- + 2 H + — » H 20 2 + 0 2 rather than the divalent reduction of molecular oxygen as was i n i -t i a l l y thought. Various enzymatic processes such as the breakdown of xanthine to uric acid in purine catabolism also produce 0~» . The oxidative nature of respiration i t s e l f results in superoxide production even though the major oxygen u t i l i z i n g enzyme, cytochrome oxidase, accom-plishes tetravalent oxygen reduction to water '-without the release of any toxic intermediates (Fridovich, 1978)• The action of lipoxygen-ase (which catalyzes the oxidation of unsaturated fatty acids con-taining the cis,cis-1,5-pentadiene system, including l i n o l e i c and linolenic acids) can produce Cv,* (Leshem, 1981). In addition, PUFA may become "free radicalized" via E+- abstraction during this process (Leshem, 1981). Ozone has also been implicated in init i a t i n g certain free radical producing reactions, especially in membrane fatty acids (Pryor, 1976). The highly reactive ozone molecule is capable of reacting with a wide variety of organic molecules, including alkanes, alkenes, alde-hydes, and amines (Pryor, 1976), often with resultant free radical production. Various lines of experimental evidence support the idea that ozone has relatively profound effects on c e l l membranes. The water-logged spots typical of 0^  injury to leaves are a reflection of the loss of intracellular compartmentation (Heath, 1980) and increased membrane leakiness (Perchorowicz and Ting, 1974)* Several obser-vations suggest that ozone damage is at least in part attributable to membrane l i p i d oxidation. For example, malondialdehyde (MDA), a product of l i p i d peroxidation, accumulates after ozone treatment (Tomlinson and Rich, 1970; Mudd et a l . , 1971; Frederick and Heath, 1974)• Work by the last authors showed that ozone exposure also resulted in decreased levels of PUFA and sterols in plant tissues. Protection against ozone injury is afforded by certain antioxidants such as <<-tocopherol (Pryor, 1976), while studies with liposomes have indicated that l i p i d peroxidation does indeed result in leak-iness (Goldstein and Weissman, 1977; Hicks and Galbicki, 1978). The most l i k e l y source of 0~« in injured tissue is l i p i d per-oxidation of membrane PUFA (Pauls and Thompson, 1981). Even under normal metabolic circumstances, the danger of i n i t i a t i n g peroxida-tion of the closely associated hydrophobic t a i l s via H +-abstraction and free radical production is always present. Due to the reactiv-i t y of ozone, only a few molecules exposed to a portion of the hydro-phobic membrane interior could i n i t i a t e peroxidative processes. Pauls and Thompson (1980) showed that the free radical scaven-gers benzoate and n-propyl-gallate inhibit ozone-induced phase trans-itions in bean (Phaseolus vulgaris L. cv. Kinghorn) cotyledon li p o -somes. Similiarities were shown to exist between ozone-treated and senescing tissue i n that both underwent membrane l i p i d transitions from the liquid-crystalline phase to a "leaky" mixture of.the li q u i d -crystalline and gel phases; but addition of free radical scavengers, in both cases, inhibited MDA formation and prevented the gel phase transition. 13 Once c e l l membranes are damaged, ozone molecules may gain access to the c e l l in relatively large numbers. It has been established that ozone is very reactive towards free sulfhydryls, which are ox-idized to disulfides, or in extreme cases, sulfoxides (Mudd, 1973; Heath et a l . , 1974)* Consequently, exposure to 0^  could subject proteins to deleterious structural changes. With membranes disrupted and enzymes inactivated, photosynthetic electron transport would be blocked (Coulson and Heath, 1974)* The resulting autoxidations of various electron transport components could result in 0~» production. Also of significance to the production of superoxide is the potential cleavage of the nicotinamide ring of NADP(H) (Mudd, 1974). Radmer and Ollinger (1980) concluded that photoreduction of oxygen occurs at the expense of reducing equivalents generated in photo-system 1, in Scenedesmus obliquus (Turpin) Kuetzing. Speculation that molecular oxygen may act as the terminal electron acceptor of photosystem 1 in higher plants has been put forth (Leshem, 1981; Foster and Hess, 1982). This univalent reduction would result in the production of superoxide, an .idea that helps explain the obser-vation that most SOD activity in plants i s localized i n the chloro-plast (Asada et a l . , 1973; Jackson et a l . , 1978; Kono et a l . , 1979; Foster and Edwards, 1980). Thus ozone may act indirectly to enhance intracellular 0~* production by inactivation of N A D P ( H ) . Respiratory activity increases significantly with the onset of vi s i b l e symptoms (Levitt, 1980). Also, C02 output from tobacco c a l -lus cultures was increased by 65% after ozone fumigation (Anderson and Taylor, 1973)• Stimulated respiration could also mean stimulated superoxide production i f normal respiration contributes to production of the free radical. Finally, the potential for the formation of various radicals exists when ozone simply dissolves in water. Molecular oxygen and various other products result when ozone decomposes spontaneously in aqueous solution (Heath, 1980). While i t i s thought that large amounts of 0H~ are produced by this process (Weiss, 1935)» extensive qual-itative and quantitative analysis of the products with respect to biological systems has not yet been undertaken (Heath, 1975)* There are indications that damage attributed to 02* may really be due to the action of 0H« (Leshem, 1981). For example, methional (CH^- S-CILy- CH^ CHO), when exposed to an enzymatic source of both 0^* and H2O2, i s oxidatively attacked resulting in the production of ethylene (Fridovich, 1978). Ethanol or benzoate (compounds known to scavenge 0H~ selectively while remaining unreactive towards Q>£ or ^2^2^ itthi^it ethylene production in these systems. The addition of SOD or catalase also inhibited ethylene production, further supporting the hypothesis that 0H» is the active agent since 02* and ^ 2^2 can interact to form 0H~ . Under the catalysis of iron ions, the Haber-Weiss reaction i s thought to occur, where: (Leshem, 1981). The Fenton reaction may also occur where hydrogen peroxide reacts with divalent ferrous iron to form the hydroxyl rad-i c a l , a hydroxyl ion, and a trivalent f e r r i c ion: Since most iron in biological systems i s chelated in f e r r i c form, H2°2 + °2 ~~* 0 H " + 0 H * °2 + Fe 3+ 15 Cohen (1978) suggests that superoxide may react with the chelated iron forming ferrous iron and molecular oxygen. Thus, i t can be seen that 0H« production i s closely tied to 0~» metabolism, which empha-sizes the need for an adequate defence mechanism against this toxic oxygen species. Superoxide Dismutase Superoxide dismutase, which catalyzes the disproportionation of superoxide to hydrogen peroxide and molecular oxygen via the reaction: 0~. + 0~» + 2H +—> H 20 2 + 0 2 has been detected in virtua l l y a l l organisms surveyed which respire aerobically. This is somewhat surprising considering that 02« w i l l spontaneously dismutate to the same products with a relatively large rate constant (Fridovich, 1978; Elstner, 1979). The reason for this apparently redundant but ubiquitous enzymatic defence i s at least two-fold. Khan's (1970) observation that spon-taneous dismutation of superoxide produces molecular oxygen, but in the highly energetic singlet state helps to explain the f i r s t ad-vantage. Dissipation of this extra energy can result in direct oxid-ation of membrane l i p i d s to peroxides (Halliwell, 1981), which en-courages free radical production via R+- abstraction from the l i p i d peroxide (Pryor, 1976). Thus, without SOD, one toxic form of oxygen becomes another, and complete detoxification has not occurred. Secondly, while superoxide i s a transient species with a lifetime in the nano-second range due to spontaneous disproportionation, SOD-catalyzed 16 dismutation i s 10 -fold faster at physiological pH (Fridovich, 1978)* SOD i s a family of enzymes, the members being distinguishable on the basis of their metal cofactor. Iron-containing (FeSOD) and man-ganese-containing (MnSOD) enzymes are characteristic of prokaryotes and are closely related in their amino acid sequences (Fridovich, 1976, 1978)* Enzymes containing both copper and zinc (CuZnSOD) are char-acteristic of eukaryotes and seem to have evolved independently as they have no sequences homologous to those of FeSOD or MnSOD. The cytosols of eukaryotes generally contain both CuZnSOD and MnSOD, which are distinguishable by their differential sensitivity to cyanide. CuZnSOD activity i s inhibited by cyanide while MnSOD i s not affected (Fridovich, 1975). Cupro-zinc SOD is a homodimer of molecular weight 32,000. Each 2+ 2+ subunit contains one atom of Cu and Zn (Fridovich, 1976). The cupro-zinc enzyme isolated from yeast, Neurospora crassa, spinach, chicken, or cow are a l l strikingly similar, showing only minor d i f -ferences in amino acid composition (Fridovich, 1976). Structurally, the subunits resemble a cylinder whose wall i s made up of eight strands of the peptide chain arranged i n antiparallel fashion (Richardson et a l . , 1975). Two coils protrude from one side of the beta-barrel, which together enclose and constitute the active s i t e . The atoms of Cu and Zn are in close proximity at the active site and are joined by a common ligand. However, the Cu is relatively exposed while the Zn is more hidden within the molecular structure. Somewhat less i s known about the structure of MnSOD. It has a subunit weight of 22,900 (Fridovich, 1976). Analysis of i t s amino acid sequence has led to predictions that i t does not have as extensive a fi-17 structure as i t s CuZn counterpart, but has a compact globular con-formation devoid of protruding loops (Steinman and Naik, 1978)* Without complete s t r u c t u r a l a n a l y s i s , i t i s not c l e a r exactly how many metal binding s i t e s are present i n the enzyme. Generally, i t s metal content has been reported to vary between one and four atoms per molecule (F r i d o v i c h , 1976). The number of subunits also v a r i e s from two to four depending on the source of enzyme (Bridges and S a l i n , 1981). Kono et a l . (1979) found the molecular weight of MnSOD from kidney bean to be 44»000, suggesting that i t i s a dimer. I t has been established that at l e a s t three isozymes of SOD e x i s t i n Phaseolus v u l g a r i s L. (Asada et a l . , 1977} Bridges and S a l i n , 1981). Two of these use copper and zi n c as t h e i r cofactors and the t h i r d man-ganese. The CuZnS0D*is l o c a l i z e d mainly i n the chloro p l a s t stroma (Asada et a l . , 1973; Asada et a l . , 1977)» while some a c t i v i t y i s as-sociated with the thylakoid membranes (Jackson et a l . , 1978; Foyer and H a l l , 1981). I t i s not c l e a r at present whether or not the l a t t e r a c t i v i t y i s a r t i f a c t u a l . MnSOD a c t i v i t y , which i s responsible f o r 25% to 37% of the t o t a l a c t i v i t y i n kidney bean leaves (Kono et a l . , 1979) i s l a r g e l y associated with the mitochondrial f r a c t i o n . The reason that the spontaneous dismutation of 0^ * i s r e l a t i v e l y slow compared with the SOD-catalyzed reaction i s , at l e a s t i n part, thought to be due to the s i m i l a r charge on the superoxide substrate molecules. E l e c t r o s t a t i c repulsion between the anions prevents the close approach that would allow the reac t i o n to occur. To circumvent t h i s problem, alternate reduction and reoxidation of a c a t a l y s t during successive encounters with 0^ * could occur, allowing f o r the tra n s f e r of one electron to ansuperoxide molecule without r e q u i r i n g close prox-18 imity of the second substrate molecule. F r i d o v i c h (1978) points out that t h i s type of mechanism seems to be employed by every SOD examined to date. I f "E" denotes the enzyme and MMe" the metal cofactor, the mechanism can be wri t t e n : E-Me11 + 0~» —> E-Me11"1 + 02 (1 ) E-Me11"1 + 0~» + 2H +—> E-Me11 + R"202 (2) I f CuZnSOD i s the c a t a l y s t , i n d i c a t i o n s are that copper o s c i l l a t e s between cupric and cuprous states during the reaction, with Zn play-in g mainly a s t r u c t u r a l r o l e . In MnSOD-catalyzed reactions, t r i -valent and d i v a l e n t states of manganese are involved. While other superoxide d e t o x i f y i n g mechanisms e x i s t , the u b i -quitous nature of SOD i n r e s p i r i n g systems strongly suggests that i t plays a major r o l e i n c e l l u l a r oxy-radical scavenging. Asada et a l . (1977) l i s t several p o s s i b l e c h l o r o p l a s t components capable of scaveng-in g 0~» (Table 1 ). I t can be seen that none of the components provides a system whose e f f i c i e n c y of scavenging 02» i s greater than a few percent of that provided by CuZnSOD. Other antioxidants which could be included i n a l i s t of c e l l u l a r defenses include <*•-tocopherol and^ -carotene. However, t h e i r q u a n t i t a t i v e contribution as scavenging agents i n vivo has not been assessed. This i s also the case f o r cytokinins and the process of photorespiration. Cytokinins may serve a dual r o l e i n free r a d i c a l metabolism: as scavengers using the <<-carbon (Leshem, 1981), and by preventing the formation of superoxide by binding xanthine oxidase under c e r t a i n circumstances (Leshem, 1981). The r o l e of photo-r e s p i r a t i o n i s s t i l l l a r g e l y speculative, but some workers have hypoth-esized that, without i t s oxygen consumption c a p a b i l i t y , the l e v e l s of Table 1. Reac t i v i t y of ch lo rop las t components with superoxide t h e i r react ion rate constants and concentrat ion in ch lo rop las ts (Asada et a l . , 1977). Reaction with CL* Reaction Rate Concentrat ion in Constants (pH=7.8) Ch lorop las t (M) cytochrome f ( Fe 3 + ) 6.1 x 10 6 6.2 x 10~ 5 • ( Fe 2 + ) p lastocyanin (Cu 2 + ) 1.1 x 10 6 6.2 x 1 0 " 5 • (Cu+) fe r redox in ( Fe 2 + ) 6.2 x 10~^ • ( Fe 3 + ) M n 2 + — • M n 3 + 6.0 x 10 6 4.0 x 1 0 - 4 ascorbate • 2.7 x 10 5 2.5 x 10~ 3 dehydroascorbate GSH — • GSSG 6.7 x 10 5 3.5 x 10~ 3 CuZn SOD 2.0 x 10 9 8.0 x 10~ 6 20 various high energy species may overwhelm cellular defence mechanisms, resulting in injury or death (Foyer and Hall, 1980; Leshem, 1981). Consequently, while SOD seems to be a major part of the defence mechanism against active oxygen, i t should be realized that alter-native scavenging systems exist. The f i r s t report of SOD being involved in the response of a plant to a i r pollutant stress came from Tanaka and Sugahara (1980). They worked with poplar, Populus euramericana (Dode) Guinier, and spinach (Spinacea oleracea L. cv. New Asia). In one set of experiments, poplar trees were subjected to continuous fumigation with 0.1 ppm SO^  while the levels of CuZnSOD and catalase were monitored. SOD activity was shown to increase significantly in trees pretreated with SO,, after four days of fumigation, to a maximum level 4.4 times the control values after 20 days of pretreatment. Plants were then subjected to an acute fumigation of 2.0 ppm SO^  for two hours. Chlorophyll con-tent was measured and used as an index of injury. Plants pretreated with daily low SO2 were found to be less injured after acute fumigation, in accord with Zahn's (1970) work. Thus, Tanaka and Sugahara.(1980) postulated a cause and effect relationship in which subacute SO^-pretreatment elevated CuZnSOD levels and resulted in protection against acute SO^  injury. A second, less direct line of evidence was obtained using the copper chelating agent, diethyldithiocarbamate (DDTC). Spinach leaves sprayed with 2% DDTC showed a 65% to 77% decrease in SOD activity. Treated and control plants were then exposed to 0.5 ppm §0^ to test the relative injury response. Chlorophyll analysis showed that plants treated with DDTC were more injured than the untreated controls. These 21 results supported their hypothesis that SOD plays a central role i n protection against SO2 stress. The experimental chemical N-(2-(2-roxo-1-imidazolidinyl)-ethyl)-N-phenylurea "ethylene diurea" (EDIl) has been shown to confer toler-ance to ozone-sensitive species (Carnahan et a l . , 1978). However, these authors offered no suggestion concerning the mechanism involved. Recently, Lee and Bennett (1982) have correlated increased SOD activ-i t y with EDU-induced ozone tolerance in snap beans. Phaseolus vul- garis L. cv. Bush Blue Lake 290 plants were watered with an EDU solution at three to four weeks of age. Visible t r i f o l i a t e leaf injury after acute ozone exposure was reduced by 55% when 25 mg EDU per 15-cm pot was added while no visible injury was detected when 50 mg per pot was added. SOD activity was shown to be only slightly stimulated by the application of 25 mg EDU per pot when compared with untreated controls, but was more than doubled in plants receiving 50 mg EDU per pot. Polyacrylamide gel electrophoresis showed quantitative differences in proteins of molecular weight 32,000 and 16,000 daltons in extracts of EDU-treated plants compared with controls. These proteins were thought to be the undissociated CuZnSOD dimer and dis-sociated subunits, respectively. Lee and Bennett (1982) concluded that EDU acts by inducing certain oxidant scavenging enzymes including SOD and that this added activity then enables plants to withstand stress from oxy-radicals generated within the plant as a result of normal metabolic activity or of external stresses such as ozone. Recently, McKersie et a l . (1982) examined SOD activity i n suscept-ible and tolerant cultivars of Phaseolus vulgaris L.. They found no difference in the int r i n s i c SOD levels of the cultivars that could be related to ozone sensitivity, but observed a tendency for SOD activity to increase after acute ozone exposure. These researchers concluded that SOD plays only a minor role, i f any, in the differen-t i a l sensitivity of bean cultivars to ozone. 23 MATERIALS AMD METHODS Bush bean (Phaseolus vulgaris L. cv. Pure Gold wax) seeds (ob-tained from Buckerfield's Ltd.), were planted 2 cm deep, two seeds per 15-cm diameter pot in ste r i l i z e d potting s o i l containing 2% peat by volume. Radish (Raphanus sativus L. cv. Cherry Belle) was planted 1 cm deep, several seeds per 15-cm diameter pot. Pots with bean seeds were kept in the greenhouse and transferred to charcoal-^filtered-air upon seedling emergence. Pots with radish seeds were kept in the greenhouse, thinned to three plants per pot, and transferred to char-coal-filtered-air eleven days after seedling emergence. Fumigation with Ozone Two types of exposure chambers were used for ozone fumigation. Pretreatment with subacute ozone concentrations (0.02 to 0.1 ppm) was done in modified growth cabinets (Conviron, Model E F 7 ) . Charcoal-f i l t e r e d - a i r was supplied to provide one complete a i r change every nine minutes. Internal fans provided complete and rapid mixing and distribution. An 11-h photoperiod was used in a l l exposure chambers. Day and night temperatures were 20°C and 15°C respectively. Acute fumigations were administered in smaller Plexiglas chambers (120 x 35 x 45 cm). In these, the charcoal-filtered-air supply resulted in one complete a i r change every thirteen minutes. Daytime temperature in these was maintained at 23+2°C by. passing f i l t e r e d a i r through 24 an a i r conditioner. Night temperature was 16±2°C. Experiments re-quiring elevated temperatures were performed by placing a thermo-sta t i c a l l y controlled 750 watt car interior heater (Allstate Model 86043) in each chamber. These experiments had day and night temp-eratures of 32±2°C and 23-2°C respectively. In both chamber types photosyhthetically active radiation at plant level was 0.2 m s , and relative humidity varied between 55% and 75%» Plants were watered to f i e l d capacity on alternate days in the larger exposure chambers and daily in the smaller ones. To com-pare differences between the two chamber systems, two experiments at a pretreatment concentration of 0.1 ppm and one at 0.05 ppm employed the smaller chambers for both subacute and acute fumigation. No significant differences between the systems were detected using Stud-ent's t test. Subacute ozone concentrations (0,02 to 0.1 ppm) were generated by passing a stream of charcoal-filtered-air over a bank of twelve germicidal ultraviolet lamps (Sylvania type B-4W Model 4511) con-tained in an air-tight f o i l - l i n e d Plexiglas chamber (30 x 10 x 10 cm). Acute ozone concentrations (0.275 to 0.8 ppm) were achieved by placing an ozone generator (Welsbach Model RB-4) directly in front of the f i l t e r e d a i r stream supplying the chamber. Ozone was monitored continuously (Dasibi Environmental Corp. Ozone Monitor Model 1003AH). Monitors were calibrated regularly at the Environmental Laboratory, British Columbia Ministry of Environment on the University of British Columbia campus. Both UV photometry and gas-phase titration with n i t r i c oxide were used to calibrate the ozone 25 source used as a primary standard (McQuaker, 1981). A l l exposures started three hours after the beginning of the photo-period and lasted six hours daily unless otherwise specified. Fumigation with SCy, A l l subacute and acute exposures were administered in the smaller Plexiglas chambers. SCv, was introduced at the desired flow rate from a tank (1% SO^  in a i r ) . The concentration was monitored either con-tinuously or at 25 minute internals. The SOg monitor used (Thermo Electron Pulsed Fluorescent SCv, Analyzer, Series 43) was also cal-ibrated regularly using U.S. National Bureau of Standards primary stan-dards of S02. Assessment of Injury For acute injury assessment in pretreatment experiments using 0^  or SGv,* acute doses were administered for one to four days, unt i l the i n i t i a t i o n of acute injury symptoms. After acute exposures, the plants were l e f t in f i l t e r e d a i r for one to three days to develop symptoms f u l l y . Injury was assessed by visual rating of percent leaf area nec-rotic (%LAN) for each leaf, estimated to the nearest 5%. Values for leaves were then averaged to yi e l d an estimate of plant injury. Injury was assessed using the primary leaves of bean in a l l experiments except those testing EDU, where the f i r s t t r i f o l i a t e s were also measured. In a l l experiments with radish, the f i r s t two leaves appearing after emergence were assayed. Chlorophyll and protein content of the prim-ary leaves were also measured and used to compare injury between treat-26 merits. (Details of the procedures used in these assays are presented in the "Analytical" section). Since adaxial leaf surfaces sustain most of the injury after acute ozone fumigation, measurement of the relative chlorophyll content in the upper c e l l layers of the leaf using reflectance spectrophotometry (Runeckles and Resh, 1974).also provided, a measure of ozone injury. Reflectance measurements were made using a Perkin-Elmer, Coleman Model 124 spectrophotometer equipped with a diffuse reflectance integrating sphere. The instrument was adjusted to 100% reflectance at 550 nm using a gypsum standard. Disks from the basal portion of the leaf were placed directly in the sample holder for reflectance measurement. Analytical Assays for SOD were performed two to three hours after the start of the photoperiod. Tissue for analysis was obtained by using a cork borer to cut discs of known area from the leaves. Samples of approx-2 imately 7 to 15 cm were used for dry weight and chlorophyll deter-2 mination. Samples of 15 to 60 cm were used for the SOD extract. The tissue was homogenized for 25 seconds in 40 ml 50mM -/potassium phos-phate buffer, pH 7*8, at 4°C. The homogenate was f i l t e r e d through four layers of cheesecloth and centrifuged at 10,000 g for 15 minutes. The supernatant was then immediately tested for SOD activity and protein content. SOD was assayed photochemically according to Beauchamp and Fridovich (1971) with the modifications of Dhindsa et a l . (1981). The assay is based on the a b i l i t y of SOD to scavenge 0* produced photochemically 27 from riboflavin before i t reduces the dye, nitroblue tetrazolium (NBT). Reduction of NBT results in a significant increase in absorbance at 560 nm which is measured after several minutes of illumination. The 3 ml reaction mixture contained 50 mmoles potassium phosphate buffer pH 7.8» 13 mmoles methionine, 75 mmoles NBT, 2 mmoles riboflavin, 0.1 mmoles EDTA, and 0 - 150 4I of the enzyme extract. After addition of the enzyme, the tubes were shaken and placed approximately 30 cm below a single 20 watt "Cool White" fluorescent lamp in a f o i l - l i n e d box (45 x 20 x 30 cm). The reaction was started by turning on the light and ran for 12 minutes, at which time the absorbance in tubes without enzyme had reached 0.18 ± 0.01 0D units at 56O nm. For each enzyme ex-tract, assays were carried out using at least three different volumes of extract in order to determine the relationship between absorption and volume of extract. A plot of log A - ^ Q versus volume was found to be linear as shown in Figure 1 (r = 0.99 p<0.00l). One unit of enzyme activity was defined as the volume of extract required to inhibit color development in the tubes by 50% (Beauchamp and Fridovich, 1971)• A l l assay runs contained an internal standard of bovine erythrocyte SOD. Protein content in the extract was determined by the method of Bradford (1976). One hundred M! of the supernatant was added to 5 ml of a reaction mixture containing 0.01% Coomassie B r i l l i a n t Blue G-250, 4.7% ethanol, and 8.5% phosphoric acid. The tubes were shaken and allowed to develop color for 20 minutes. Protein content (mg ml~^ ) was determined by comparing A - Q ^ to a standard curve obtained using bovine serum albumin. Extracted chlorophyll was estimated by the method of Bruinsma (1963)» Leaf tissue was stored in the freezer (-9°C) un t i l i t was 2 8 1.4 h 1.3 L o L O CU o c ro Xi S-o in CD o 1.2 •1.1 r-1.0 h "0.9 h '0.8 L 25 50 75 Microliters of Extract Added 100 Figure 1. Relationship between log A 5 6 Q and volume of bovine erythrocyte SOD extract added: Extract contained 5 mg/ml SOD as lyophilized powder. Due to the nature of the assay, absorbance decreases as SOD is added, hence, values of Log Abs 5 6 Q decrease as volume increases. 29 assayed. The disks were homogenized in 50 ml of cold 80% acetone for 45 seconds and the resultant extract was passed through a sintered glass f i l t e r under suction. Absorbances at 645» 652, and 663 nm were measured and the use of Bruinsma's equation: 20.2A645 + 8A 6 6 3 + 2 7.8A 6 5 2 2 yielded estimates of chlorophyll concentration (mg ml""^  ). PAGE was done according to Davis (1964) using 7«5% acrylamide containing 0.1% SDS. The proteins were completely dissociated by immersing samples for 1 minute in boiling water. Twenty nanogram protein samples were loaded in each well. Electrophoresis was carried out in 0.1M Tris-glycine buffer (pH 8.3) at room temperature at 5 niA per tube for about 6 hours. Gels were stained for 1 hour in a solution con-taining 0.1% Coomassie Blue and 50% TCA, and then destained i n a 7% acetic acid-20% methanol mixture. Duplicate samples were prepared for the localization of both enz-yme protein and enzyme activity. SOD was located on gels according to the negative staining technique of Beauchamp and Fridovich (1971)• After electrophoresis, gels were immersed in ice-cold 50mM potassium phosphate buffer for 10 to 15 minutes. The gels were rinsed with dis-t i l l e d water and put in tubes containing 2.45mM NBT at room temperature for 15 to 20 minutes. Subsequently, they were transferred to a 50mM -5 potassium phosphate solution containing 2.8 x 10 M riboflavin, also at room temperature, for 15 minutes. The gels were then washed with and suspended in 0.1mM EDTA prior to illumination. After irradiation the gels were stained blue except at bands with SOD activity. 30 A l l control and treatment means were tested for significance using Students t test for comparing two means on each day on which assays were done. To condense the presentation of results, treatment data are often presented as a percentage of control data. Standard errors were computed according to the equation: r = 1 Z f < (r ) 2 + (z-r ) 2 where z £ r = x - r z x y ± r y with r = associated standard error of the mean (Jeffreys, 1932). A l l data in tables and graphs are means and standard errors. When standard error bars of treatment and control means do not overlap, or where the error bar of the ratio of treatment to control does not cross the 100% value, means are significantly different (p<0.l). 31 RESULTS Recently, Lee and Bennett (1982) have stressed the importance of including PVP in the extraction medium when isolating SOD. They ar-gue that phenolic compounds naturally present in bean leaf tissues form complexes with proteins resulting in the formation of qiiinones. Quinones in turn oxidize essential protein functional groups, or form covalent bonds with proteins, reducing apparent SOD activity in the assay. To investigate this possibility, tissue samples were taken from opposite sides of the midrib of primary bean leaves ranging in age from 4 to 15 days past emergence. The samples were extracted in buffer, with or without the addition of 3% PVP. As shown in Figure 2, SOD activity was found to be slightly higher (7-10%) in non-PVP ex-tracts when assayed photochemically. PAGE extracts prepared with or without PVP were indistinguishable (Figure 3). While protein content i s slightly higher when the phenol scavenger i s used i t apparently does not protect SOD (Figure 2). Consequently, the presence of PVP i s not c r i t i c a l to obtaining accurate results when assaying for SOD. The temporal patterns of SOD activity in the developing primary leaves of bean and radish are shown in Figures 4 and 5 respectively. Younger leaves were found to contain more activity than older leaves whether expressed on an area or dry weight basis. Central to the work in this thesis i s the r e l i a b i l i t y of assays used to estimate plant injury after exposure to 0^  or SO^. Figure 6 shows a comparison of three different assays used to determine injury to the primary leaves of bean at different stages of susceptibility 32 120 h 110 h Q O 100 90 80 120 no 100 90 80 o o 10 12 14 16 18 Days from Emergence Figure 2. SOD activity and protein content of primary bean leaves with PVP in extraction medium expressed as a % of values obtained without using PVP. (•) SOD activ-ity. (•) Protein content. n=6. • A F i g u r e 3. d H H B D D i a g r a m m a t i c r e p r e s e n t a t i o n o f SDS-PAGE co lumns c o n -t a i n i n g p r o t e i n s o f f i r s t t r i f o l i a t e l e a v e s o f bush b e a n . 20 ng samp les were l o a d e d on 7 .5% a c r y l a m i d e c o n t a i n i n g 0 . 1 % SDS. A , B, C , and E s t a i n e d f o r p r o t e i n w i t h Coom-a s s i e b r i l l i a n t b l u e . D and F s t a i n e d f o r SOD a c t i v i t y . (A) l e a f p r o t e i n s i n c r u d e e x t r a c t ; (B) l e a f p r o t e i n s e x t r a c t e d w i t h PVP ; (C) p l a n t s t r e a t e d w i t h 75 mg EDU; (D) l e a f p r o t e i n s i n c r u d e e x t r a c t s t a i n e d f o r SOD a c -t i v i t y ; (E j c o m m e r c i a l l y a v a i l a b l e b o v i n e e r y t h r o c y t e SOD s t a i n e d f o r p r o t e i n ; (F) c o m m e r c i a l l y a v a i l a b l e b o v i n e e r y t h r o c y t e SOD s t a i n e d f o r SOD a c t i v i t y . (D) and (F ) a r e n e g a t i v e r e p r e s e n t a t i o n s , o f g e l s s t a i n e d f o r SOD a c t i v i t y . 35 11 12 13 14 15 16 17 18 19 20 21 22 23 Days from Emergence Figure 5. SOD activity in the f i r s t two leaves of radish ex-pressed on a leaf area (•) or dry weight (•) basis. n=6. 36 D 1 . . . . 1 1 1 1 ) I 1 i 0 2 4 6 8 10 12 14 Days f rom Emergence F i g u r e 6 . C o m p a r i s i o n o f i n j u r y a s s a y s u s i n g p r i m a r y l e a v e s o f b e a n : %LAN ( v ) , % r e f l e c t a n c e (•), p r o t e i n c o n t e n t based on d r y w e i g h t (O ) . P l a n t s were p r e t r e a t e d w i t h e i t h e r 0 . 0 5 ppm ozone o r f i l t e r e d - a i r b e f o r e a c u t e ozone d o s e . n=10-12. 37 to an acute dose of ozone. Conventionally, %LM has been used for this purpose (Runeckles and Rosen, 1974* 1977) and was shown to be a satis-factory method of injury assessment (Figure 6). Protein content in the SOD extract was found to be inversely well correlated (r = 0.91 p<0.001) with values of. percentage leaf area necrotic and was consistently used as a check on injury rating by visual estimation of LAN. Chloro-phyll estimation of adaxial leaf surfaces using reflectance spectro-photometry also showed similiar trends, but this technique is ideally suited to the assessment of chronic injury and was used sparingly throughout the research. Chlorophyll extracts in acetone ^ proved to be far too insensitive to detect differences between treatments in the various subacute pretreatment experiments, and therefore were not used as an assay for injury. Beans Treated with Ozone The results persented in Figure 7 confirm work reported earlier by Runeckles and Rosen (1977)» showing the effects of 0^  pretreatment on subsequent acute injury i n the primary leaves of bean. Plants exposed to 0.02 ppm 0^  for six hours daily from emergence are i n i t i a l l y more susceptible to a subsequent acute dose of ozone than f i l t e r e d - a i r -grown controls. Plants receiving an additional four or ten days of pretreatment are progressively less injured than controls (r = 0.44 P<0.001). In contrast, plants exposed to 0.05 ppm 0^  for six hours daily are less susceptible to a subsequent acute dose after two pretreat-ments. However, additional days of pretreatment increase susceptibility 38 200 -175 -150 h 25 h 0 I I I I I I I I I I I I L _ _ 0 1 2 3 4 5 6 7 ' 8 9 10 11 12 Days f r om Emergence F i g u r e 7. E f f e c t o f d a i l y 6-h p r e t r e a t m e n t w i t h ozone (•, 0 . 0 2 ppm and D , . 0 . 0 5 ppm) on %LAN o f p r i m a r y l e a v e s o f bean r e s u l t i n g f rom t r e a t m e n t w i t h 0 . 4 ppm ozone on day i n -d i c a t e d . n = l 2 - 1 4 . 39 and predispose the leaves to acute ozone injury (r^= 0.16 p<0.021). The pattern of extractable SOD in these leaves, relative to con-tro l s , i s shown i n Figure 8. It can be seen that SOD activity in pre-treated plants was similiar regardless of pretreatment dosage and did not vary with time ( 0.02 ppm - r 2= 0.12 p<0.82; 0.05 ppm - r 2= 0.01 p<0.18). Significant differences from controls were observed only on days 4 and 6 at pretreatment concentrations of 0.02 ppm and 0.05 ppm, respectively. However, at these times, pretreated and control plants exhibited a similiar degree of injury after acute 0^  treatment (Figure 7). When differences from the controls were observed in suscept-i b i l i t y to acute injury i.e. days 2 and 12 at 0.02 ppm and days 2 and 9 at 0.05 ppm (Figure 7), no corresponding significant differences in SOD activity were observed (Figure 8). While no apparent changes in overall SOD level occurred as a result of pretreatment with subacute ozone, i t was possible that a shif t in the ratio of cupro-zinc to manganese SOD results from low 0^  exposure. To test this possibility, 1mM KCN was included in SOD assays, but, on days 2 and 12 of pretreatment with 0.02 ppm 0^, when dif f e r -ential susceptibility to acute 0^  occurs, there was no difference in the proportion of SOD activity inhibited by cyanide (Figure 9). However, changes in CN-sensitive SOD activity did occur as both pre-treated (r 2= 0.78 p<0.019) and control (r 2= 0.82 p<0.013) plants aged. Relation of SOD to Injury Symptoms Plants exposed to 0.1 ppm 0^  for six hours daily began to show signs of visible injury after 2 to 3 days of treatment. The symptoms 40 140 ^ 120 h oj_ +-> o o +-> Q O I/O 100 80 h 60 10 12 14 16 Days from Emergence Figure 8 . Effect of daily 6-h ozone pretreatment (•, 0.02 ppm and •, 0.05 ppm) on SOD activity in primary leaves of bean prior to treatment with 0.4 ppm ozone on day indicated. n=12-14. 41 0 2 4 6 8 10 12 Days f r om Emergence F i g u r e 9 . P e r c e n t a g e i n h i b i t i o n o f SOD i n p r i m a r y l e a v e s o f bean by a d d i t i o n o f 1 mM KCN t o t h e a s s a y . P l a n t s were r a i s e d e i t h e r i n f i l t e r e d - a i r (•), o r 0 . 0 2 ppm ozone 6 h d a i l y (•). n=12. 42 were typified as minute areas of localized chlorosis and necrosis resulting in a spotted or flecked appearance to the adaxial leaf sur-face. With the onset of such visible symptoms, the SOD level in these leaves was found to increase significantly above control values, as shown in Figure 10. Continued pretreatments were found by Runeckles and Resh (1974) to result in an accumulation of chronic symptoms, and this observation can be correlated with the increased SOD ac-t i v i t y of the injured plants depicted in Figure 10. The effect on SOD persisted through the course of the experiment and reached a level of 57% more enzyme activity than the controls by day 18. In order to investigate the relative sensitivity of pretreated plants to acute treatment, nine-day old bean plants exposed daily to 0.1 ppm 0^  from emergence and filtered-air-treated controls were simultaneously exposed to an acute dose of ozone. As shown in Table 2, parallel samples of plants from each treatment revealed that pre-treated plants had 21% more SOD activity than the controls. However, pretreated plants were also approximately 30% more susceptible to the acute dose (Table 2, as revealed by reflectance measurements), as was observed earlier by Runeckles and Rosen (1977)• Effect of EDU on Bean Leaves Experiments with EDU confirmed i t s protective effect on primary leaves of bean exposed to ozone (Table 3). On the average, over 70% of the control leaf area was necrotic one day after the administration of an acute 0^  dose, while EDU-treated plants were essentially unin-jured. However, the effect of the chemical on SOD activity was insig-43 175 F 0 2 4 6 8 10 12 14 16 18 20 22 Days from Emergence Figure 10. SOD activity in primary leaves of bean treated daily with 0.1 ppm ozone (6h), expressed as percent of controls maintained in charcoal-filtered air. Treatment commenced on day 5. n=12-14. 44 T a b l e 2. SOD a c t i v i t y b e f o r e a c u t e dose and r e l a t i v e c h l o r o p h y l l c o n t e n t (as % r e f l e c t a n c e ) a f t e r a c u t e dose i n 9-day o l d beans p r e -t r e a t e d f r o m emergence w i t h e i t h e r f i l t e r e d a i r ( C o n t r o l ) o r 0.1 ppm ozone ( P r e t r e a t e d ) . A c u t e dose was a d m i n i s t e r e d f o r 2 days (n = 1 2 ) . Enzyme a c t i v i t y e x p r e s s e d on l e a f a r e a b a s i s . T r e a t m e n t SOD _ 2 P r e t r e a t e d A c t i v i t y P r e t r e a t e d R e f l e c t a n c e ( u n i t s cm" ) (as % o f C o n t r o l ) (as % o f C o n t r o l ) C o n t r o l 8 . 9 8 \ 0 . 2 3 P r e t r e a t e d 1 0 . 9 0 - 0 .16 100 121 - 4% 100 131 - 7% 45 Table 3. Effect of EDU on SOD level of bean leaves before acute exposure, and on %LAN after acute exposure. Primary leaves were on plants 6-8 days old. 200 mg EDU added per pot; acute dose 0.8 ppm for 2 h (n = 16). First trifoliate leaves were on plants 4-5 weeks old. 300 mg EDU added per pot; acute dose 0.8 ppm, 6h per day for 4 days (n = 12). Injury estimated 1 day after acute dose. _2 SOD activity (units cm ) %LAN Leaves Control EDU-treated Control EDU-treated Primary 10.30 - 0.68 10.97 - 0.68 7 2 - 6 0 Trifoliate 10.31 - 0.12 10.54 - 0.20 7 3 - 7 3 - 1 *EDU dissolved in water and added to pots 24 h before SOD assay and acute exposure. 46 nificant. To test the effect of EDU on t r i f o l i a t e leaves, plants were grown for 4 to 5 weeks past emergence, u n t i l the f i r s t t r i f o l i a t e s were f u l l y expanded. While these aged plants were more resistant to high 0^  con-centrations than the one-week old plants previously tested, the same trend was observed when injury symptoms developed. Control plants were substantially injured or k i l l e d by the acute treatment while EDU-treated plants showed l i t t l e or no injury. When assayed photochem-i c a l l y , the effect of EDU on SOD was again insignificant (Table 3). PAGE also showed no effect of EDU on bands exhibiting SOD activity (Figure 3 C ) . SOD and Acute Injury Analysis of primary bean leaves before and after receiving acute ozone doses suggested that SOD was more active in injured leaves when compared to filtered-air-grown plants. A detailed study which mon-itored this apparent induction of SOD as leaves were subjected to acute doses of 0^  was carried out. The acute injury response to the oxidant generally involves tissue collapse, effectively decreasing specific leaf area (SLA) by thickening the leaf (Bennett and Runeckles, 1977)* Increasing the dose of ozone results in a progressive shift to an early decrease in SLA such that leaves treated with 0.4 ppm are substantially thickened after 4 days of treatment compared with con-trols (r = 0.43 p<0.15).(Figure 11). For this reason, i t i s prefer-able that enzyme activity be expressed on a dry weight basis when treat-ments involve 0^  doses greater than 0.1 ppm 6 hours daily. Plants were grown in an atmosphere of f i l t e r e d a i r for 3»6, or 9 47 120 h-110 L_ o (_> ro CD =1 rO 01 <_> (/) 90 U 80 L_ 70 8 9 10 11 12 13 Days o f T r e a t m e n t Figure 11. Effect of ozone treatment on specific leaf area ( # of square centimeters per gram dry weight ) of primary bean leaves at 0.05 ppm (•), 0.1 ppm (•), and 0.4 ppm(A) 6 h daily. n=12. 48 days past emergence at which point four successive treatments of 0.4 ppm 0^  for 6 hours daily were administered. %LAN and SOD activity were recorded after each daily 0^  exposure and these values were compared with ones from controls maintained throughout in f i l t e r e d a i r . The two temperature regimes used were day/night temperatures of 2 3 7 l 6 ° C and 32 ° /23 ° C. Lower Temperature Effects ( 2 5 ° / l 6 ° C) When fumigated at 23° C, the youngest plants sustained the least injury. Symptoms f i r s t appeared midway through the third acute dose (Figure 12). A relatively large increase in SOD activity (37%) occurred simultaneously (Figure 12). While %LAN in these plants increased due to the fourth and f i n a l acute dose, there was no corresponding significant change in SOD activity, which remained about 30% above the control level. A somewhat different situation was noticed when 5-day old plants were treated in the same way. In these, SOD activity was again significantly higher than the controls after one acute dose, but vi s i b l e injury was also present (Figure 13) and %LAN progressively increased as subsequent acute doses were administered, to a f i n a l value of 54%. SOD activity, relative to controls, also increased with subsequent acute doses throughout the experiment, unt i l treated plants had 60% more SOD activity than controls. However, i t should be noted that the f i n a l increase was observed following a day on which no acute dose was administered. Plants receiving their f i r s t acute dose at day 9 (Figure 14) 0 1 2 3 4 5 Days Past First Exposure Figure 12. Effects of four successive exposures to 0,4 ppm ozone on SOD activity (•) and cumulative %LAN (•) in primary leaves of bean. Day/Night temperature was 23°/16°C. Ex-posures commenced 3 days from emergence. n=12. 80 0 1 2 3 4 5 Days Past First Exposure Figure 13. Conditions as in figure 12. Exposures commenced 6 days from emergence. 51 Figure 14. Conditions as in figure 12. Exposures commenced 9 days from emergence. 52 showed similar trends to the 5-day old plants with regard to SOD levels, hut the fi n a l %LAN was higher. High Temperature Effects ( 3 2 0 / 2 5 ° C) Two-day old plants receiving acute doses at 52° C were sub-stantially more injured than plants treated at 23° C (compare Figures 12 and 15). Symptoms were initiated on day 2 rather than day 3 of the 0., treatment and fi n a l %LAN was almost double that 3 observed at 23° C. SOD activity in the plants treated at 32° C was only slightly affected by the fumigations, increasing to. a level about 15% greater than controls at the end of the experiment. Plants receiving treatments beginning at either 6 or 9 days past emergence were also more injured than those treated at the lower temperature (compare Figures 13 and 16, and 14 and 17, resp-ectively). The patterns of SOD activity in these plants relative to the controls were dramatically different from those treated at the lower temperature. Here, a slight stimulation of SOD was noticed after one and two days of fumigation (Figures 16 and 17)» but subsequent treatment with 0^  resulted in significant declines in SOD activity to levels 13% and 19% lower than controls in plants when fumigation started at 5 and 8 days of age respectively (Figures 16 and 17). Radish Treated with Ozone SOD activity in the f i r s t two leaves of radish subjected to low 0, treatments of 0.02 ppm six hours daily tended to remain at 53 o u > O O 100 H 75 —I 50 25 Days Past First Exposure Figure 15. Effects of four successive exposures to 0.4 ppm ozone on SOD activity (•) and cumulative %LAN (•) in primary leaves of bean. Day/Night temperature was 32°/23°C. Ex-posures commenced 3 days from emergence. n=12. 54 100 1 2 3 4 5 Days Past First Exposure Figure 16. Conditions as in figure 15. Exposures commenced 6 days from emergence. 55 Days Past First Exposure Figure 17. Conditions as in figure 15. Exposures commenced 9 days from emergence. 56 or slightly above that of controls, u n t i l two weeks of exposure, at which time i t decreased significantly, as shown i n Figure 18. Contrary to the response observed in bean, this pretreatment had no effect on %LAN after an acute dose of 0^  was administered (Table 4). SOD activity in plants which had been exposed to an acute dose was slightly but significantly greater than in filtered-air-treated controls (Table 5 ) t even though the pretreatment alone tended to reduce SOD ac t i v i t y . Effects of Subacute Sulfur Dioxide Considerable fluctuation was observed in the SOD levels of primary leaves of bean pretreated with 0.1 ppm continuously from emer-gence, as shown in Figure 19. However, the differences were small and barely significant. Visible leaf injury following acute exposure was also unaffected by low S0£ pretreatment (Table 4)» SOD activity in the f i r s t two leaves of radish showed no signi-ficant change when treated with low SO^  (Table 5)» but, such treat-ment predisposed the leaves to visible injury after acute SO^  exposure (Table 4-)* Effects of Acute Sulfur Dioxide Unlike ozone, acute SO^  exposure tended to decrease SOD activity in the primary leaves of bean, compared with filtered-air-grown controls, regardless of pretreatment regime (Table 5)» Ln radish, a similar trend was observed only in those plants pretreated with 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Days o f P r e t r e a t m e n t SOD a c t i v i t y in the f i r s t two leaves of radish ex-posed to 0.02 ppm ozone 6 h da i l y . n=12. T a b l e 4 . %LAN i n p l a n t s r e c e i v i n g a c u t e ozone o r s u l f u r d i o x i d e t r e a t m e n t a f t e r p r e t r e a t m e n t w i t h e i t h e r f i l t e r e d a i r o r s u b - a c u t e doses o f 0- o r S 0 o . A . ) R a d i s h and O z o n e . P l a n t s 15 days p a s t f i r s t p r e t r e a t m e n t . A c u t e t r e a t m e n t ( 0 . 2 7 ppm 6 h d a i l y ) f o r 3 d a y s . I n j u r y e s -t i m a t e d on t h e f i r s t two l e a v e s o f p l a n t s 1 day a f t e r a c u t e d o s e , (n = 1 2 ) . F i l t e r e d a i r p r e t r e a t e d 0 . 0 2 ppm 0 , 6 h day 1 4 6 - 5 4 5 - 6 B J Bean and S 0 ? . P l a n t s 12 days p a s t emergence . A c u t e t r e a t -ment ( 2 . 0 ppm f o r 2 h) on day 1 1 . I n j u r y e s t i m a t e d on p r i m a r y l e a v e s 1 day a f t e r a c u t e d o s e , (n = 1 2 ) . F i l t e r e d a i r p r e t r e a t e d 0.1 ppm SO,, c o n t i n u o u s l y 5 3 - 9 5 3 - 8 C . ) R a d i s h and S 0 ? . P l a n t s 12 days p a s t f i r s t day o f p r e t r e a t -ment . A c u t e t r e a t m e n t ( 2 . 0 ppm f o r 2 h) on day 1 1 . I n j u r y e s t i m a t e d on t h e f i r s t two l e a v e s o f p l a n t s 1 day a f t e r a c u t e d o s e , (n = 1 2 ) . F i l t e r e d a i r p r e t r e a t e d 0.1 ppm S 0 ? c o n t i n u o u s l y 2 2 - 7 46 - 8 * * * * * * p < 0.01 where " p " i s t h e l e v e l o f s i g i f i c a n t d i f f e r e n c e f rom f i l t e r e d a i r c o n t r o l . 59 Table 5. SOD a c t i v i t y in plants rece iv ing acute ozone or SO,, treatment compared with plants maintained in f i l t e r e d a i r or subacute concentrat ions of ozone or SO,,. A c t i v i t y expressed as uni ts per gram dry weight. (n=12). A.) Radish and Ozone. Plants 15 days past f i r s t pretreatment. Acute treatment (0.27 ppm 6 h da i l y ) f o r 3 days. F i l t e r e d a i r maintained 0.02 ppm 6 h da i l y F i l t e r e d a i r then acute 0.02 ppm 6 h day" then acute 5700 - 60 5200 - 20*** 5570 - 60* 6000 - 80*** B.) Bean and SO ? . Plants 12 days past emergence. Acute t reat-ment (2.0 ppm fo r 2 h) on day 11. F i l t e r e d a i r maintained 0.10 ppm cont inuously F i l t e r e d a i r then acute 0.10 ppm cont inuously then acute 4950 - 220 4890 - 110 3060 - 250' 3260 - 250*** C.) Radish and SO, Plants 12 days past f i r s t day of pretreat-ment. Acute treatment (2.0 ppm fo r 2 h) on day 11. F i l t e r e d a i r maintained 0.10 ppm cont inuously F i l t e r e d a i r then acute 0.10 ppm cont inuously then acute 5160 - 70 4920 - 240 5110 - 210 4090 - 460*** * p < 0.10 * * p < 0.05 * * * p < 0.01 "p " i s the leve l of s i g n i f i c a n t d i f f e rence from f i l t e r e d a i r control 60 120 L_ 110 - 100 90 80 / ft / / i \ / \ / \ / \ / A-1 J I I I L 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Days of Pretreatment Figure 19. SOD activity in the first two leaves of radish (•), and the primary leaves of bean (•) exposed to 0.1 ppm S0~ continuously. n=12. continuous subacute SO,,. Plants grown i n f i l t e r e d a i r and exposed to acute SO^ showed no change i n SOD a c t i v i t y r e l a t i v e to those main-tained i n f i l t e r e d a i r f o r the whole experiment. D I S C U S S I O N One o f t h e m a j o r o b j e c t i v e s o f t h i s w o r k w a s t o d e t e r m i n e w h a t r o l e , i f a n y , SOD p l a y s i n t h e r e s p o n s e t h a t R u n e c k l e s a n d R o s e n (1977) o b s e r v e d , w h e r e p r i m a r y b e a n l e a v e s p r e t r e a t e d w i t h s u b a c u t e o z o n e p a s s t h r o u g h v a r y i n g s t a g e s o f s u s c e p t i b i l i t y t o s* a c u t e o z o n e t r e a t m e n t . I t b e c o m e s a p p a r e n t t h a t SOD i s n o t i n v o l v e d i n t h e r e s p o n s e w h e n e n z y m e l e v e l s i n o z o n e - p r e t r e a t e d a n d f i l t e r e d -a i r - g r o w n c o n t r o l s a r e c o m p a r e d ( F i g u r e s 7 a n d 8 ) . A t e a c h p r e t r e a t -m e n t c o n c e n t r a t i o n t h e r e i s o n e d a y o n w h i c h SOD a c t i v i t y i n t h e p r e t r e a t e d p l a n t s i s s u b s t a n t i a l l y h i g h e r t h a n c o n t r o l s ( F i g u r e 8 , d a y 4 o r d a y 6). H o w e v e r , i t i s o n t h e s e d a y s t h a t n o s i g n i f i c a n t d i f f e r e n c e i n s u s c e p t i b i l i t y t o t h e a c u t e d o s e w a s o b s e r v e d ( F i g u r e 7, c o r r e s p o n d i n g d a y s ) . S i g n i f i c a n t d i f f e r e n c e s i n a c u t e i n j u r y w e r e n o t e d o n d a y s SOD a c t i v i t y w a s i n d i f f e r e n t t o p r e t r e a t m e n t ( F i g u r e s 7 a n d 8 ) . T o t a l SOD a c t i v i t y w a s u n a f f e c t e d b y o z o n e p r e t r e a t m e n t , b u t t h e p o s s i b i l i t y e x i s t e d t h a t t h e p r o p o r t i o n o f C N - s e n s i t i v e : C N -i n s e n s i t i v e SOD w a s r e s p o n d i n g t o o z o n e . W h i l e e x p e r i m e n t s w e r e n o t s p e c i f i c a l l y d e s i g n e d t o i n c l u d e r e g u l a r m o n i t o r i n g o f t h e C N -i n s e n s i t i v e a c t i v i t y , s p o t c h e c k s i n d i c a t e d t h a t n o s h i f t i n t h e r a t i o w a s o c c u r r i n g o n d a y s a t r e a t m e n t e f f e c t w a s o b s e r v e d ( F i g u r e 9). SOD a c t i v i t y c a n b e e n h a n c e d b y t r e a t m e n t w i t h o z o n e , b u t o n l y a f t e r v i s i b l e i n j u r y h a s o c c u r r e d ( F i g u r e s 10, 12-14). T h u s , 0.1 ppm 0^  6 h o u r s d a i l y h a d l i t t l e e f f e c t o n SOD a c t i v i t y u n t i l f l e c k i n g o c c u r r e d . M o r e d r a m a t i c a l l y , t w o - d a y o l d p l a n t s s h o w e d n o n e c r o t i c 63 tissue or change in SOD activity after two daily exposures to 0.4 ppm ozone (Figure 12, days 1 and 2), but as soon as a slight amount of necrotic tissue could be detected (Figure 12, day 3)» the SOD level jumped to 37% above the control value. Certainly i f ozone i t s e l f was inducing SOD, two days at 0.4 ppm should have e l i c i t e d some response. At both concentrations (0.1 ppm and 0.4 ppm), the SOD level increased as visible symptoms accumulated due to repeated fumigations, which supports the idea that SOD i s related to injury and not 0^  i t s e l f . Consequently, SOD can be thought of as secondary in the defense against ozone injury. Until f u l l expansion i s reached, younger leaves are generally less susceptible to ozone damage than older ones, at which point the trend can reverse i t s e l f (Ting, 1974)• This tendency was observed in expanding primary bean leaves (Figures 12-14). In addition, SOD levels are highest i n young primary leaf tissue, in both bean and radish, and decline with age (Figures 4 and 5)« Lee and Bennett (1982) have suggested that this age-related drop i n SOD activity and parallel increase in susceptibility to acute injury i s of causal significance in bean. This i s a questionable conclusion to reach, though, as SOD level i s only one of many physiological and biochemical factors that change with age. For example, expansion of the leaf means that leaf area, leaf thickness and density of stomates-all change with time. Chlorophyll, protein, and starch content are age-related as well; the former two parameters .also .decline with age after reaching maximum levels early in development. Contrary to Lee and Bennett's (1982) suggestion, i t would be hasty to conclude that declining SOD levels in plants tissue are responsible for increased 64 susceptibility to ozone as plants age. The response of EDU-treated beans to acute ozone fumigation was clearly demonstrated. Primary and trifoliate leaves showed l i t t l e or no injury after acute exposure (Table 3) while severe injury was observed on controls. Lee and Bennett (1982) correlated this ozone tolerance to enhanced SOD activity in plants treated with EDU, but results in Table 3 do not support this finding. It is difficult to determine why there are differences in data with respect to the effect of EDU on SOD activity, but some basic differences between the studies exist. For example, Lee and Bennett (1982) used an assay system which relies on a biological source of Op • ., while the one employed in this work utilized 0^* generated photochemically. A documented problem characteristic of the former system is the large variation in results due to poor control of superoxide generation (Beauchamp and Fridovich, 1971)• Experiences of this nature prompted the adoption of the photochemical method in the present studies. Perhaps 6£ greater significance is the fact that different var-ieties of Phaseolus vulgaris L. were used in the two studies. Diff-erential behaviour between cultivars with respect to enzyme activity would not be surprising. While no comparisons have been made studying the effect of ozone on SOD activity, McKersie et al . (1982) found a 3-fold range in the inherent levels of the enzyme in ten cultivars of white bean. A final difference between the studies may l i e in the inter-pretation of data. When expressed on a percentage of control basis, SOD activity in EDU-treated primary leaves is 107% while trifoliates had 102% of control activity (Table 3). Neither result is statis-tically significant, while associated differences in %LAN were highly significant. Lee and Bennett (1982) show a strikingly similar corr-elation in their work, yet, they conclude SOD is of imagor importance to ozone tolerance. When they treated each plant with 25 mg EDU, SOD activity was effectively unchanged, increasing to 107% of the control level, while %LAN was less than half the control value. Such a finding does not suggest a central role for SOD in the response to acute ozone. Problems are encountered when one attempts to find a satisfac-tory basis on which enzyme activity can be expressed after visible injury has occurred. Chronic injury symptoms, typically occurring after repeated exposure to a slightly injurious concentration of ozone (0.1 ppm 6 hours daily for bean) consist of highly localized areas of chlorosis or necrosis, mainly on the adaxial leaf surface, which result in a flecked appearance to the leaf. Acute injury symptoms, noticable after one or two daily exposures to 0.4 ppm 0^, initially impart a water-logged or flecked appearance to the adaxial leaf surface and result in areas of adaxial or bifacial chlorbtic and necrotic tissue (Tomlinson arid Rich, 1974)• A significant consequence of such injury is a decrease in specific leaf area, SLA, resulting from ozone-induced tissue collapse and cessation of foliar growth and expansion. The effects of subacute, chronic, and acute concentrations of ozone on SLA were compared (Figure 11). Subacute ozone treatment has no significariit effect; exposure to 0.1 ppm, which causes chronic symptoms to occur, tends to increase leaf thickness but not substantially, while repeated acute ozone exposure decreases SLA by over 25%. Thus, leaf area is a suitable baseline for enzyme a c t i v i t y when testing the effects of 0.1 ppm ozone, or less, but tissue showing acute injury symptoms would appear to have greatly increased SOD activity compared to controls, simply because more tissue per unit leaf area is present. Both protein and chorophyll content substantially decrease following acute ozone exposure (Olszyk and Tibbitts, 1982), again resulting in spuriously high estimates of SOD activity in fumigated plants i f either of the parameters were used as a basis for enzyme acti v i t y . Total plant dry weight is also affected by acute ozone fumigation (Tingey, 1974; Olszyk and Tibbitts, 1982) but s t i l l rep-resents the basis with the least i n t r i n s i c error for expression of SOD activity. When experiments comparing %LAN and SOD activity were carried out at a higher temperature, some interesting tendencies became apparent (Figures 15-17). The same general trend between %LAN and plant age was observed at 32 ° /23° C, where youngest plants showed the least injury. At a l l ages, %LAN was- significantly higher at 32° /23 ° C compared with 23% 6° C. Macdowell (1963) suggests the higher night temperature i s more important than the day temperature. He implies that photosynthetic reserves w i l l be consumed at a greater rate during higher night temperatures affording the plant less protection during the next daily fumigation. This possibility was not investigated further in the present studies. Plotting relative SOD activities after fumigation (Figures 15-17) revealed a substantial temperature-ozone interaction. Two-day old plants subjected to successive acute doses at 32 ° /23 ° C again showed stimulation of the enzyme, while visible injury covered about 67 60% of the leaf area. When five-day old plants were studied, SOD activity in the fumigated beans increased i n i t i a l l y , but dropped below that of the controls after the third dose had been administered. At this time, 80% of the leaf area was injured. The f i n a l acute exposure resulted in a further depression of SOD activity to 19% below the control level with a corresponding LAN greater than 90% (Figure 16). The trends wer similar when eight-day old plants were used (Figure 17). These observations are in sharp contrast to the responses ob-served at 2 3 ° / l 6 ° c. After three acute doses, five-day old plants treated under this temperature regime had 25% more SOD activity than controls, but only 50% LAN. The f i n a l acute exposure further increased SOD activity to 35% above the control level but LAN increased only marginally to 55% (Figure 13). Similar trends were observed using eight-day old plants (Figure 14). The observed depression of SOD activity i s only an indirect © effect of temperature (Figures 12-17). Under high temperature regime, vi s i b l e injury to the leaves occurs earlier and is more -extensive by the end of the experiment compared with treatments 1. administered at 2 3 ° / l 6 ° C. Five and eight-day old plants grown at 32 ° /23 ° C show the greatest v i s i b l e injury and lowest SOD activity of a l l treatments examined. Hence, i t appears that when LAN reaches a c r i t i c a l threshold value, around 75-80% (Figures 16 and 17» day 3) the leaf loses i t s ' a b i l i t y to synthesize or activate SOD, resulting in a loss of enzyme activity relative to controls. Once this c r i t i c a l threshold i s reached, ozone molecules may actually penetrate c e l l envelopes and directly react with the protein moieties of SOD causing inactiviation or denaturation and contribute to the 68 observed decreased in ac t i v i t y . Since plants treated at 23°/l6° C or two-day old plants treated at 32°/23° C sustain less than 75% LAN, they are able to maintain elevated levels of SOD (Figures 13-15)* PVP i s often included in experimental protocols as an insurance against loss of protein or enzyme activity during the extraction procedure. Lee and Bennett's (1982) work is the f i r s t to suggest that this phenol scavanger i s essential to an accurate SOD assay when using plant tissue. Several authors (Asada et a l . , 1973; Giannopolitis and Ries, 1977; Jackson et a l . , 1978; Rabinowitch and Sklan, 1980 and 1981; Bridges and Salin, 1981) have used crude extracts, without PVP in their extraction medium, when isolating SOD from plants. This latter observation alone does not preclude the possibility that, u n t i l now, the importance of PVP i n SOD extrac-tion has been overlooked. However, in conjunction with results in this thesis (Figure 2), there is no evidence to uphold Lee and Bennett's (1982) claim. A possible explanation l i e s i n the fact that two different SOD assays were used. Lee and Bennett colori-metrically measured the reduction in cytochrome "c" by 02* , generated biologically from xanthine and xanthine oxidase. Superoxide generated in the photochemical assay comes from light-stimulated riboflavin, and the color change of the dye, NBT, i s followed. It i s possible that PVP removes compounds which specifically interfere with the former assay while having l i t t l e effect on the photochemical method. It i s interesting to note that while PVP does apparently protect protein (Figure 2), SOD does not seem to benefit. In fact, the inverse relationship between protein content and SOD activity, in the presence of PVP, suggests that either PVP is removing an inter-69 fering substance from the extract which mimics SOD activity, or con-versely, that PVP directly or indirectly causes an inhibition of SOD activity while exerting a protective effect over protein in general. It i s not possible to distinguish between these po s s i b i l i t i e s from the data in this thesis. Following exposure to a pollutant, assays for plant injury usually consist of a visual estimate of %LAS or some measure of chlorophyll content. Both methods have been c r i t i c i z e d for their lack of precision (Olszyk and Tibbitts, 1982). Visual estimates are l i k e l y to vary between observers, while chlorophyll content i s not necessarily sensitive enough to detect subtle differences between treatments. Protein content would be expected to decline as injury occurs i f one regards the damage process as an increase in the rate of senescence (Pauls and Thompson, 1980, 1981). During this process, protein i s broken down into glut-amine and asparagine and a net export of these amino acids occurs from the dying tissue (Dr. W. Woodbury, personal communication). Consequently, measurement:of the protein content in crude extracts i n the present study always provided an unambiguous measure of injury which detected very small differences between treatments (Figure 6). While this method of estimating injury seems to have been ignored to date, i t i s widely applicable to the problem at hand, especially i f further biochemical analysis of the plant material i s required. Data from experiments with radish and ozone tend to support the conclusions reached with bean and ozone, though the experiments were not as extensive as in the latt e r case. Pretreatment of radish with low ozone had no substantial effect of SOD activity (Figure 18), but unlike bean, after 11 days of low ozone, no difference in response to an acute dose could be observed between treatments. This may simply 70 mean that the plants were at an "indifferent" stage with respect to acute ozone tolerance or that stages of varying susceptibility do not occur i n pretreated radish. SOD activity also seemed to increase after acute symptoms developed, although the response was not as consistent as in bean (Table 4)» The main objective i n working with SO^  was to attempt a veri-fication of Tanaka and Sugahara's (1980) work with S02 and poplar. Pretreatment of primary leaves in bean and radish with subacute SO^  continuously had l i t t l e or no effect on SOD activity (Table 5). Administration of acute S02 revealed no effect of pretreatment, with respect to vi s i b l e injury, in bean (Table 4), while SO^-pre-treated radish plants were twice as susceptible to injury compared with filtered-air-grown controls. In both cases, i t i s apparent that subacute fumigation does not affect SOD activity, and hence the conclusions of Tanaka arid Sugahara (1980) could not be substan-tiated in these species. It should be emphasized that plants exposed to acute S02 respond very differently from those fumigated with acute ozone. Under similar temperature regimes, injurious ozone treatment tends to induce or activate SOD in primary bean leaves (Figures 12-14), while leaves injured by acute SO^  show less SOD activity than controls maintained in f i l t e r e d a i r (Table 5)» SOD activity in radish tended to not respond to injurious S02 or 0^  treatment compared with controls maintained i n f i l t e r e d a i r . However, trends i n SOD activity similar to those observed in bean were seen in radish when plants were pre-treated with subacute levels of the pollutants instead of f i l t e r e d a i r (Table 5). The f i r s t two leaves of radish pretreated with low ozone (0.02 ppm) showed an increase i n SOD activity after acute 0^  treatment while leaves pretreated with subacute SO^  had less SOD activity after the acute SO^  dose. The general failure to induce SOD under any but the most extreme conditions leads one to question the extent to which i t is possible for SOD to respond to i t s environment. In plants, the majority of SOD activity i s CuZnSOD, and is associated with the chloroplast (Kono et a l . , 1979: Asada et a l . , 1979). It i s this form of the enzyme that has been affected in the only two reports claiming induction of the enzyme in leaf tissue (Tanaka and Sugahara, 1980; Lee and Bennett, 1982). The only other unambiguous reports of CuZnSOD i n -duction are in the prokaryotes Saccharomyces cerevisiae and Photo- bacterium leignathi when exposed to hyperoxic conditions (Gregory et a l . , 1974; Puget and Michelson, 1974). Rat lung SOD, presumably the CuZn form, has also been induced by high oxygen tension (Pridovich, 1980). MnSOD in Ej_ c o l i i s quite responsive to high oxygen and paraquat treatments (Pridovich, 1978). Foster and Hess (1980; 1982) have studied the response of several enzymes in cotton and maize to an atmosphere containing 75% oxygen. Most enzymes, including SOD, were unaffected, leading them to conclude that the greater proportion (Foster and Hess, 1980) and higher specific activity (Groden and Beck, 1979) of CuZnSOD in land plants may permit tolerance to large fluctuations in atmospheric oxygen and presumably superoxide. Since CuZnSOD does not seem to be particularly responsive to potentially inductive conditions, the "indifferent" behavior of the enzyme in atmospheres containing 0^  or 0^  does not necessarily rule out superoxide as a key product of exposure. SOD may simply respond 72 to the stress by becoming activated rather than by increasing i t s overall pool size. This possibility suggests that following SOD levels to estimate the degree of 0^ stress in plants may be too indirect to be of use. The same hyperoxic studies (Poster and Hess, 1980, 1982) showed a two- to three-fold increase in the activity of glutatione reductase under high 0^. The enzyme generates NADP+ in the process of gluta-thione reduction. Increasing the pool size of this oxidized cofactor would reduce the chance of oxygen accepting photosystem 1 electrons and producing superoxide. It may be f r u i t f u l then, to conduct an exhaustive study of enzyme responses in plants subjected to ozone, with emphasis on those l i k e l y to relieve oxidative stress. 73 SUMMARY 1 . PVP has no significant effect on the photochemical assay for SOD. 2 . Changes in SOD of primary bean leaves pretreated with daily low ozone (0 .02 or 0.05 ppm) are not related to the protective effect from an acute dose that this pretreatment confers, relative to filtered-air-grown controls. 3 . EDU confers ozone tolerance to primary and t r i f o l i a t e bush bean leaves but does not affect SOD activity. 4 . SOD activity in the primary leaves of bean increases only as chronic or acute symptoms of ozone injury appear when fumigation i s carried out at 23°C. 5 . Primary leaves of bean are more injured by an acute dose of ozone when administered at 33°C compared with treatment at 23°C. 6 . A threshold LAN exists (approximately 75%) above which SOD can not be enhanced in the primary leaves of bean due to extensive cellular disruption. Consequently, SOD activity in bean exposed to ozone at 33°C decreases below control levels because the injury threshold i s surpassed. 7. SOD in the f i r s t two leaves of radish is not affected by daily low (0.02 ppm) ozone pretreatment. The acute injury response is also 74 not a f f e c t e d by t h i s pretreatment w h i l e SOD i s s l i g h t l y s t i m u l a t e d by the acute dose. 8. Continuous pretreatment w i t h subacute SO^ (0.1 ppm) does not a f f e c t v i s i b l e i n j u r y a f t e r acute SO^ exposure or SOD a c t i v i t y i n the primary leaves of bean. 9. 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