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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 p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  requirements f o r an advanced degree at the  the  University  of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make it  f r e e l y a v a i l a b l e f o r reference  and  study.  I further  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may department or by h i s or her  be granted by  the head o f  representatives.  my  It i s  understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain  s h a l l not be allowed without my  permission.  Department of  Plant  Science  The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date  DE-6  (3/81)  May 27.  1983  written  ABSTRACT The primary leaves of bush bean plants pretreated d a i l y with non-injurious, low l e v e l s of ozone ( 0 . 0 2  or 0 . 0 5 ppm) pass through  stages of varying s u s c e p t i b i l i t y to a subsequent acute dose.  Previous  work showed that t h i s v a r i a t i o n i n response to acute dose could only p a r t l y be accounted f o r by stomatal behaviour.  Present studies i n -  dicate that the oxy-radical scavenger superoxide dismutase appears to play no role i n the phenomenon.  (SOD)  No observed changes i n  SOD l e v e l s following various low ozone pretreatment regimes were related to s u s c e p t i b i l i t y to acute injury i n compaEisons with plants maintained M ' f i l t e r e d a i r as controls. The only s i g n i f i c a n t change i n SOD l e v e l s which appeared to be related to ozone occurred simultaneously with the appearance of v i s i b l e symptoms of injury following exposure to 0^ concentrations greater than 0.1 ppm.  The nature of the e f f e c t on SOD levels and  the degree of v i s i b l e injury was related to plant age and temperature. Younger plants or plants fumigated at a lower temperature (23°C) showed less injury a f t e r acute exposure to ozone than older plants or ones exposed at a higher temperature (33°C).  SOD a c t i v i t y was  higher than controls when a lower temperature growth regime  (23°/l6°C)  was used, but dropped to below control l e v e l s 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 e f f e c t on SOD l e v e l s i n either primary or t r i f o l i a t e leaves p r i o r to exposure to an acute dose. SOD l e v e l s i n the f i r s t and second leaves of radish plants pretreated d a i l y with low ozone (0.02  ppm) were not substantially  different  iii  from c o n t r o l s b e f o r e o r dftex an a c u t e exposure t o ozone.  In contrast  t o bean l e a v e s , p r e t r e a t m e n t o f 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 o f i n j u r y i n d u c e d by an a c u t e dose. Low  SGv, p r e t r e a t m e n t (0.1  ppm)  o f the p r i m a r y l e a v e s o f bush  bean o r t h e f i r s t two l e a v e s o f r a d i s h d i d n o t a f f e c t SOD V i s i b l e i n j u r y a f t e r an a c u t e S0^ exposure a f f e c t e d by subacute p r e t r e a t m e n t i n bean.  (2.0  ppm)  activity.  was a l s o n o t  However, p r e t r e a t e d r a d i s h  l e a v e s were p r e d i s p o s e d t o a c u t e i n j u r y . I n c o n t r a s t w i t h the e f f e c t o f ozone, a c u t e SO^ p r i m a r y l e a v e s o f bean r e s u l t e d i n a d e c r e a s e i n SOD  exposure o f t h e a c t i v i t y com-  p a r e d 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 o f p r e t r e a t m e n t regime.  A s i m i l a r t r e n d was o b s e r v e d i n t K e r f i r s t two l e a v e s o f r a d i s h  o n l y 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 pretreated  w i t h f i l t e r e d a i r was n o t a f f e c t e d by a c u t e S0  o  fumigation.  iv  TABLE OP CONTENTS  Abstract  i i  Introduction  1  Review of Literature  3  Pretreatment E f f e c t s  3  E f f e c t s of Free Radicals  8 15  Superoxide Dismutase  23  Materials and Methods  23  Fumigation with Ozone Fumigation with SO^  25  •  Assessment of Injury  25  Analytical  26 31  Results Beans Treated with Ozone  37  Relation of SOD to Injury Symptoms  39  E f f e c t of EDU on Bean Leaves  42  SOD and Acute Injury  46  Low Temperature E f f e c t s ( 2 3 ° / l 6 ° C)  48  High Temperature E f f e c t s (32°/23°  52  C)  Radish Treated with Ozone  52  E f f e c t s of Subacute Sulfur Dioxide  56  E f f e c t s of Acute Sulfur Dioxide  56  Discussion  62  Summary  73  Literature Cited  75  V  LIST OF TABLES  1. 2.  3. 4.  Reactivity of chloroplast components with superoxide: t h e i r reaction rate constants and concentration i n chloroplasts ....  19  SOD a c t i v i t y before acute dose and chlorophyll content a f t e r acute dose i n beans treated with 0 . 1 ppm ozone 6 hours per day  44  E f f e c t of EDU on SOD l e v e l of bean leaves before acute exposure, and on %LAN a f t e r acute exposure  45  %LAN i n plants receiving acute ozone or S O 2 treatment a f t e r pretreatment with either f i l t e r e d a i r or subacute doses of 0 ^ or S 0  58  SOD a c t i v i t y i n plants receiving acute 0 ^ or S 0 | treatment compared with plants maintained i n f i l t e r e d a i r or subacute concentrations of 0 ? or S 0  59  2  5.  O  vi  LIST OF FIGURES  1.  2.  3.  4.  5.  6.  7.  8.  9.  10.  11.  12.  13*  R e l a t i o n s h i p between l o g AC^Q and volume o f bovine e r y t h r o c y t e SOD added  28  SOD a c t i v i t y and p r o t e i n c o n t e n t o f p r i m a r y bean l e a v e s w i t h PVP i n e x t r a c t i o n medium expressed as a % o f v a l u e s o b t a i n e d w i t h o u t u s i n g PVP  32  Diagrammatic r e p r e s e n t a t i o n o f 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  SOD a c t i v i t y i n the p r i m a r y l e a v e s o f bean exp r e s s e d on a l e a f a r e a and d r y weight b a s i s  34  SOD a c t i v i t y i n the f i r s t two l e a v e s o f r a d i s h e x p r e s s e d on a l e a f a r e a o r d r y weight b a s i s  35  Comparison 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 bean  36  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 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 from treatment w i t h 0 . 4 ppm ozone  38  E f f e c t o f d a i l y 6-h ozone p r e t r e a t m e n t on SOD a c t i v i t y i n p r i m a r y l e a v e s o f bean p r i o r t o t r e a t ment w i t h 0 . 4 ppm ozone  40  Percentage 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 1mM KCN t o the assay  41  SOD a c t i v i t y i n p r i m a r y l e a v e s o f bean t r e a t e d d a i l y w i t h 0.1 ppm ozone ( 6 h ) , e x p r e s s e d as a percent of c o n t r o l s maintained i n c h a r c o a l filtered-air  43  E f f e c t o f ozone treatment on s p e c i f i c l e a f a r e a o f p r i m a r y bean l e a v e s  47  E f f e c t o f f o u r s u c c e s s i v e exposures o t 0 . 4 ppm ozone on SOD a c t i v i t y and c u m u l a t i v e %LAN i n p r i m a r y l e a v e s o f bean  49  C o n d i t i o n s as i n F i g u r e 12, b u t exposures menced 6 days from emergence  50  com-  vii  14.  15.  16.  17.  18.  19.  C o n d i t i o n s as i n F i g u r e 12, b u t exposures commenced 9 days from emergence  51  E f f e c t o f f o u r s u c c e s s i v e exposures to 0.4 ppm ozone o f SOD a c t i v i t y and c u m u l a t i v e %LAN i n p r i m a r y l e a v e s o f bean a t 3 2 ° / 2 3 ° C  53  C o n d i t i o n s as i n F i g u r e 15, b u t exposures commenced 6 days from emergence  54  C o n d i t i o n s as i n F i g u r e 15» b u t exposures commenced 9 days from emergence  55  SOD a c t i v i t y i n the f i r s t two l e a v e s o f r a d i s h exposed t o 0.02 ppm ozone 6 hours d a i l y  57  SOD a c t i v i t y i n the f i r s t two l e a v e s o f r a d i s h and the p r i m a r y l e a v e s o f bean exposed t o 0.1 ppm S0 9 c o n t i n u o u s l y  60  viii  ACKNOWLEDGEMENTS  I would l i k e t o express s i n c e r e thanks f o r h i s guidance  and s u p p o r t throughout  I would a l s o l i k e  t o Dr.V.C.Runeckles  t h i s program.  t o express my a p p r e c i a t i o n t o P e t e r G a r n e t t .  f o r h i s a d v i c e and e x p e r t i s e i n t e c h n i c a l m a t t e r s . Finally,  I am g r a t e f u l t o D r . F . B . H o l l and Dr.M.Shaw f o r  g e n e r o u s l y a l l o w i n g me use o f t h e i r l a b r a t o r y equipment and i n s t r u m e n t s d u r i n g my work, and D r . P . A . J o l l i f f e f o r i n t r o d u c i n g me t o computers.  ix  LIST OF ABBREVIATIONS  EDU  - N-(2-(2-0x0-1-imidazolidinyl)-ethyl)-N'-phenyl diurea")  d  - day  h  - hour  LAN  - leaf area necrotic  Or?  - superoxide  0,  - ozone  OH*  - hydroxyl r a d i c a l  radical  PAGE - polyaery1amide ppm  urea  gel electrophoresis  - p a r t s p e r m i l l i o n , by volume, m i c r o l i t e r s  PUFA - p o l y u n s a t u r a t e d f a t t y SO2  - sulfur dioxide  SOD  - superoxide  TCA  - trichloroacetic  dismutase acid  acid  per l i t e r  ("ethylene  1 INTRODUCTION  The phenomenon by which previous exposure to low ambient l e v e l s of a pollutant may  confer a protective effect to f o l i a g e exposed to  a subsequent acute dose may  i n part be attributable to biochemical  changes i n the plant tissue (Zahn, 1970;  Runeckles and Rosen, 1974)•  S p e c i f i c a l l y , the protective e f f e c t observed by pretreating bush bean Phaseolus vulgaris L. cv. Pure Gold wax with 0.02  ppm ozone (0^)  daily  can be correlated p a r t l y with a dampening of stomatal action, e f f e c t i v e l y 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 f o r pretreatment concentrations below 0.04  ppm  0^ administered f o r s i x hours d a i l y .  Higher pretreat-  ment concentrations predispose plants to injury from a subsequent acute dose, which suggests that other factors may be involved i n the response. The idea that free r a d i c a l s may contribute to the mechanism by which a i r pollutants cause tissue damage i s not new Leshem, 1981).  (Pryor,  1976;  Recently, evidence has been put f o r t h supporting the  view that injury caused by s u l f u r dioxide (S02)(Tanaka and Sugahara, 1980) 0£.  and 0^ (Lee and Bennett, 1982)  involves the superoxide r a d i c a l ,  Both studies followed the l e v e l s of superoxide dismutase  (SOD)  as an indicator of the i n t e r n a l steady state concentrations of O^*. Treatment with the experimental chemical, N-(2-(2-oxo-1-imidazolidinyl)-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 i n snap beans, Phaseolus v u l g a r i s L. cv. Bush Blue Lake 290, involves induction of SOD as an oxy-radical scavenger. The f i r s t objective of t h i s study was to determine the r o l e of SOD,  i f any, i n the protective e f f e c t 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 r o l e of SOD i n EDU-conferred ozone-tolerance i n bush bean.  The t h i r d objective  was to v e r i f y the reported protective e f f e c t of SO^-induced  SOD on  f o l i a g e subsequently treated with acutely injurious SO^ l e v e l s .  3  REVIEW OF LITERATURE  Pretreatment E f f e c t s  The movement of a gaseous pollutant i s passive (Heath, 1980) thus t o t a l flux of such a gas into a plant can be approximated by Fick's law f o r d i f f u s i o n :  (1)  j = g C  where J = mass flow or f l u x , g = conductance of the gas, and C = the concentration gradient, which i s generally assumed to be l i n e a r across the conductance region.  Since resistance i s the r e c i p r o c a l of con-  ductance, the equation can be rewritten as: j ,  -S  (2)  total Total resistance (r, , ,) = r + r + r (3) total a s m ' = the resistance from bulk a i r to the boundary layer near v  where r  7  v  the stomate; r = stomatal resistance; and r = resistance encountered ' s ' m from the substomatal chamber to the c e l l u l a r i n t e r i o r , including c e l l walls and membranes. Two s i g n i f i c a n t factors should be noted from equation 2.  First,  under f i e l d conditions where an appreciable wind speed at the canopy layer often e x i s t s , 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 i n determining f l u x .  Second, the difference between p o l -  lutant exposure and e f f e c t i v e dose absorbed by the plant should be emphasized (Runeckles, 1974)*  C l a s s i c a l l y , pollutant exposure has  4  been described by the term "dose", which i s the product of concentration and time.  From equation 2 i t can be seen that even a high con-  centration y i e l d i n g a large "C" component can lead to a small e f f e c t i v e dose ( i . e . the amount of pollutant a c t u a l l y entering the plant) when any or a l l components of ^- ^ ^ r  0  a  r  e  a  ample, i f stomates are closed, r  g  proportionately l a r g e .  For ex-  w i l l be very large and a high "C"  w i l l s t i l l r e s u l t i n a small e f f e c t i v e dose. Previous  exposure to an a i r pollutant can a l t e r s u s c e p t i b i l i t y  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, environmental conditions, and pretreatment dosage of the pollutant. Considering the importance of stomata i n determining pollutant f l u x , many pretreatment studies have focussed on t h e i r r o l e as an explanation 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 f l e c k i n g from sub-  sequent s i m i l i a r exposures, while plants exposed i n i t i a l l y to 0.08 ppm f o r 7 hours were l e s s injured by t h i s greater dose administered the next day.  He speculated that reduced stomatal opening i n plants ex-  posed to the higher concentration may have contributed to the e f f e c t . Heck and Dunning (1967) v e r i f i e d the antagonistic e f f e c t 6£ two successive exposures i n causing v i s i b l e l e a f i n j u r y .  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 i n l e s s injury when compared with  5  plants exposed to the same concentration f o r one hour.  The length of  the interexposure period d i d not s i g n i f i c a n t l y a f f e c t the response. These authors speculated that stomatal closure a f t e r the f i r s t fumi g a t i o n may have contributed to the response, but also that the plants may have p a r t i a l l y "recovered" during the " r e s t " period. Wilton et a l . (1972) suggested that stomatal mechanisms are not central to observed pretreatment effects of 0^ i n various c u l t i v a r s of bluegrass (Poa pratensis L. cvC's Merion, Kenblue, Windsor, Belturf, and 117-27-6).  In t h e i r experiments, plants were exposed to 0.3 ppm  0j f o r 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 i n less injury than had been previously recorded.  In these r e s u l t s , stomatal  acclimation i n plants fumigated once seems to be ruled out as a poss i b l e explanation of the protective e f f e c t of pretreatment. Bicak (1978)» working with bean (Phaseolus vulgaris L. cv. Pure Gold wax)  and radish (Raphanus sativus L. cv. Cherry B e l l e ) , admin-  i s t e r e d the same t o t a l dose of 0^ i n several d i f f e r e n t 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 v i s i b l e  i n j u r y was correlated with early peak concentrations.  I f stomatal mech-  anisms were involved i n the response, a s i m i l i a r c o r r e l a t i o n between r  and v i s i b l e injury would be expected. However, no obvious r e l a t i o n s ship could be detected when mean r , or values of r at the time of s s maximum concentration were compared with i n j u r y . These data c l e a r l y  6  suggest that factors not related to stomatal mechanisms influence the i n j u r y response to  0^.  Runeckles and Rosen (1974) showed that the primary leaves of bean (Phaseolus v u l g a r i s L. cv. Pure Gold wax) and the top pairs of leaves 0^  i n mint cuttings (Mentha arvensis L.) pretreated with d a i l y low (0.02  ppm 6 h per day) behave d i f f e r e n t l y , i n terms of v i s i b l e f o l i a r  injury, when subsequently exposed to an acute dose.  Mint pretreated  f o r seven days became predisposed to injury by an acute dose, while pretreated bean was s u b s t a n t i a l l y less injured by a subsequent exposure than controls pretreated i n f i l t e r e d a i r .  acute  Stomatal measure-  ments indicated that p a r t i a l l y dampened stomatal opening i n ozonetreated beans could be correlated to the lower f o l i a r injury a f t e r one acute dose.  However, stomatal conductance  in filtered-air-pre-  treated plants dropped below that of the ozone-pretreated plants a f t e r the acute treatment, yet these plants were more susceptible to injury by a second acute exposure.  Hence stomatal action only p a r t i a l l y ex-  p l a i n s these r e s u l t s . Further work by these authors (Runeckles and Rosen, 1977)  showed  that 0^ pretreatment e f f e c t s i n bean (same c u l t i v a r ) 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 s u s c e p t i b i l i t y to an acute dose depending on pretreatment concentration.  Plants treated with 0.02  ppm 0^ f o r 6 h daily were  i n i t i a l l y more susceptible to a subsequent acute dose, but a f t e r 3 to 4 days became more r e s i s t a n t to acute injury when compared with f i l t e r e d - a i r - t r e a t e d controls.  A somewhat higher pretreatment con-  7  centration of 0.05 ppm resulted i n the plants being l e s s 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 t h i s concentration resulted i n predisposition to injury by a subsequent acute dose.  S t i l l higher pretreatment con-  centrations had previously been shown to increase s u s c e p t i b i l i t y to acute injury compared with f i l t e r e d - a i r - t r e a t e d controls (Runeckles and Rosen, 1974)•  These r e s u l t s suggest that the plant Response i s  simply speeded up by increasing pretreatment concentration,  i f the  assumption i s made that plants pretreated at 0.02 ppm 0^ would event u a l l y become predisposed  to acute i n j u r y .  Thus, plants pretreated  at the lower l e v e l of ozone show a r e l a t i v e l y long period of reduced s u s c e p t i b i l i t y (following an i n i t i a l period of increased  susceptib-  i l i t y ) ; at 0.05 ppm 0^ t h i s phase i s reduced to 3 to 4 days, while at 0.1 ppm no phase of reduced s u s c e p t i b i l i t y  occurs.  Transpiration measurements before and during acute  fumigation  suggested stomatal involvement i n the d i f f e r e n t i a l s u s c e p t i b i l i t y observed by Runeckles and Rosen (1977)•  For example, the primary  leaves of bean plants pretreated with 0.02 ppm 0^ d a i l y u n t i l they were i n the susceptible phase were shown to transpire at rates s i m i l i a r to controls, but upon exposure to 0.4 ppm 0^, were unable to close t h e i r stomata as r a p i d l y as f i l t e r e d - a i r - t r e a t e d controls.  However, when  such ozone-pretreated plants entered the l e s s susceptible phase, transp i r a t i o n rates before and during acute fumigation were shown to be lower than those of plants pretreated i n 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 r e s u l t i n g  8 slower stomatal closure would cause exposure to the oxidant f o r a longer time, compared with f i l t e r e d - a i r - t r e a t e d controls.  When the  plants had entered the l e s s susceptible phase, stomatal aperture was apparently decreased r e l a t i v e 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 s u s c e p t i b i l i t y to an acute dose following pretreatment also occurs with SGv,.  Zahn (1970) found that wheat and barley pretreated  with 0 . 4 ppm SO^ were s i g n i f i c a n t l y less injured than non-pretreated controls a f t e r acute exposure, and that t h i s p o s i t i v e effect was enhanced by increasing the pretreatment duration from 77 to 133 hours. Further work with a l f a l f a (Medicago s a t i v a L.) and l a r c h (Larix decidua L.) revealed no e f f e c t of subacute SO2 pretreatment on an acute dose response, i n s p i r i n g Zahn's (1970) conclusion that monocots react p o s i t i v e l y to such pretreatment while dicots are not affected or show a s l i g h t negative e f f e c t .  E f f e c t s of Free Radicals  I t appears that some sort of biochemical acclimation or hardening within the pretreated plant may also be p a r t l y responsible f o r the observed e f f e c t s .  A review of the reported effects that pollutants have  on biochemical processes i n plants reveals the l i m i t e d 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,  — i n h i b i t , or not a f f e c t r e s p i r a t i o n (Black, 1982).  — 2  Using HSOj and S 0 ^  (hydration products of S 0 _ ) photosynthetic electron transport and  9  ribulose-T,5 bisphosphate carboxylase are both i n h i b i t e d (Heath, 1980). After SO^ exposure, quantitative and q u a l i t a t i v e changes i n pool sizes of amino acids and reducing sugars have also been reported (Malhotra and Sarkar, 1979)•  Ozone i s thought to stimulate r e s p i r a t i o n , but t h i s  may be a generalized response to injury (Levitt, 1980).  Photosynthesis,  on the other hand, i s generally reported to be i n h i b i t e d by 0^ (Heath, 1980).  Reactions of c e l l u l a r components with the oxidant include  cleavage of the nicotinamide r i n g of NADP(H) (Mudd, 1974)» oxidation of aromatic amino acids (Mudd, 19^9) and oxidation of free sulfhydryls to d i s u l f i d e s (Heath, 1980).  Ozone also reacts with l i p i d s d i r e c t l y by  ozonolysis or i n d i r e c t l y by i n 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 f o r the p a r t i c i p a t i o n of free r a d i c a l s i n b i o l o g i c a l systems.  A free r a d i c a l i s an atom or  molecule which contains an odd number of electrons and which may be posi t i v e l y charged, negatively charged, or n e u t r a l .  Owing to t h e i r unstable  electronic configuration, most free r a d i c a l s are highly r e a c t i v e .  Thus,  i n any b i o l o g i c a l system, one can speculate upon a number of effects that may be caused by this sort of random r e a c t i v i t y , such as i n a c t i v ation of key metabolic enzymes and loss of membrane l i p i d  fluidity.  While many species of free r e d i c a l s are known to exist, two which are l i k e l y to have s i g n i f i c a n t effects on b i o l o g i c a l systems are the superoxide r a d i c a l , 0^ , and the hydroxyl r a d i c a l , 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 (Goldberg and Stern, 1976; Kellogg and Fridovich, 1977)» and to k i l l  cells  10 (Michelson and Buckingham, 1974).  Target molecules which are  attacked  by superoxide include proteins, nucleic acids, polyunsaturated acids (PUFA), and carbohydrates (Leshem, 1981). suggestion  fatty  There has been a  that molecules of DNA may be "nicked" by free r a d i c a l s , but  conclusive proof i s lacking (Pryor, 1976; Leshem, 1981).  In the plant  kingdom, phenomena such as post harvest f r u i t deterioration, l e a f senescence, accelerated spoilage of cut flowers (Leshem, 1981) and sunscald (Rabinowitch and Sklan, 1980) have been reported to involve free r a d i c a l s . Superoxide may be formed i n several ways, including processes.  photooxidative  These reactions are effected by photosensitizers, chemicals  which pass on energy of e x c i t a t i o n from absorbed l i g h t to oxygen. The r e s u l t i s the formation of high energy s i n g l e t oxygen (ground state molecular oxygen i s i n the t r i p l e t state), or 0g electronic reduction.  v i a univalent  Such photosensitizers include r i b o f l a v i n , por-  phyrins, and anthraquinones. Superoxide may also be generated as a r e s u l t of normal electron flow i n photosynthesis. reduced to HgO^  Mehler (1951) showed that oxygen can be  by illuminated chloroplasts.  Indirect evidence using  superoxide dismutase (SOD), an 0£ scavenger, suggests that 0~* i s produced i n 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 i n h i b i t e d 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 i n chloro-  11  p l a s t s , suggesting that the H ° 2 reported by Mehler (1951) 2  i s more  l i k e l y a r e s u l t of the reaction: 0~. + 0~- + 2 H — »  H 0  +  2  2  +0  2  rather than the divalent reduction of molecular oxygen as was  ini-  t i a l l y thought. Various enzymatic processes such as the breakdown of xanthine to u r i c a c i d i n purine catabolism  also produce 0~»  . The  oxidative  nature of r e s p i r a t i o n i t s e l f r e s u l t s i n superoxide production even though the major oxygen u t i l i z i n g enzyme, cytochrome oxidase, accomplishes 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 f a t t y acids cont a i n i n g the cis,cis-1,5-pentadiene system, including l i n o l e i c l i n o l e n i c acids) can produce Cv,* may  (Leshem, 1981).  and  In addition, PUFA  become "free r a d i c a l i z e d " v i a E - abstraction during t h i s process +  (Leshem, 1981). Ozone has also been implicated i n i n i t i a t i n g c e r t a i n free r a d i c a l producing reactions, e s p e c i a l l y i n membrane f a t t y acids (Pryor, 1976). The highly reactive ozone molecule i s capable of reacting with a wide v a r i e t y of organic molecules, including alkanes, alkenes, aldehydes, and amines (Pryor, 1976), often with resultant free r a d i c a l production. Various l i n e s of experimental evidence support the idea that ozone has r e l a t i v e l y profound effects on c e l l membranes.  The water-  logged spots t y p i c a l of 0^ injury to leaves are a r e f l e c t i o n of the loss of i n t r a c e l l u l a r compartmentation (Heath, 1980)  and  increased  membrane leakiness (Perchorowicz and Ting, 1974)*  Several obser-  vations suggest that ozone damage i s at least i n part a t t r i b u t a b l e to membrane l i p i d oxidation.  For example, malondialdehyde (MDA),  a product of l i p i d peroxidation, accumulates a f t e r ozone treatment (Tomlinson and Rich, 1970; Mudd et a l . , 1971; Frederick and Heath, 1974)•  Work by the l a s t authors showed that ozone exposure also  resulted i n decreased l e v e l s of PUFA and s t e r o l s i n plant t i s s u e s . Protection against ozone i n j u r y i s afforded by c e r t a i n antioxidants such as <<-tocopherol (Pryor, 1976), while studies with liposomes have indicated that l i p i d peroxidation does indeed r e s u l t i n leakiness (Goldstein and Weissman, 1977; Hicks and G a l b i c k i , 1978). The most l i k e l y source of 0~«  i n injured tissue i s l i p i d per-  oxidation of membrane PUFA (Pauls and Thompson, 1981). normal metabolic circumstances,  Even under  the danger of i n i t i a t i n g  peroxida-  t i o n of the c l o s e l y associated hydrophobic t a i l s v i a H -abstraction +  and free r a d i c a l production i s always present.  Due to the r e a c t i v -  i t y of ozone, only a few molecules exposed to a portion of the hydrophobic membrane i n t e r i o r could i n i t i a t e peroxidative  processes.  Pauls and Thompson (1980) showed that the free r a d i c a l scavengers benzoate and n-propyl-gallate i n h i b i t ozone-induced phase transi t i o n s i n bean (Phaseolus vulgaris L. cv. Kinghorn) cotyledon somes.  S i m i l i a r i t i e s were shown to exist between ozone-treated  lipoand  senescing tissue i n that both underwent membrane l i p i d t r a n s i t i o n s from the l i q u i d - c r y s t a l l i n e phase to a "leaky" mixture of.the l i q u i d c r y s t a l l i n e and gel phases; but addition of free r a d i c a l scavengers, i n both cases, i n h i b i t e d 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 i n r e l a t i v e l y large numbers.  I t has been established  that ozone i s very reactive towards free sulfhydryls, which are oxi d i z e d to d i s u l f i d e s , or i n extreme cases, sulfoxides (Mudd, 1973; Heath et a l . , 1974)*  Consequently, exposure to 0^ could subject  proteins to deleterious s t r u c t u r a l changes.  With membranes disrupted  and enzymes inactivated, photosynthetic electron transport would be blocked (Coulson and Heath, 1974)*  The r e s u l t i n g autoxidations of  various electron transport components could r e s u l t i n 0~» production. Also of s i g n i f i c a n c e to the production of superoxide  i s the  potential cleavage of the nicotinamide r i n g of NADP(H) (Mudd, 1974). Radmer and O l l i n g e r (1980) concluded  that photoreduction  of oxygen  occurs at the expense of reducing equivalents generated i n photosystem 1, i n Scenedesmus obliquus (Turpin) Kuetzing. that molecular oxygen may  Speculation  act as the terminal electron acceptor of  photosystem 1 i n higher plants has been put f o r t h (Leshem, 1981; Foster and Hess, 1982).  This univalent reduction would r e s u l t i n  the production of superoxide,  an .idea that helps explain the obser-  vation that most SOD a c t i v i t y i n plants i s l o c a l i z e d i n the chlorop l a s t (Asada et a l . , 1973; Jackson et a l . , 1978; Kono et a l . , 1979; Foster and Edwards, 1980).  Thus ozone may act i n d i r e c t l y to enhance  i n t r a c e l l u l a r 0~* production by i n a c t i v a t i o n of  NADP(H).  Respiratory a c t i v i t y increases s i g n i f i c a n t l y with the onset of v i s i b l e symptoms (Levitt, 1980). lus cultures was  Also, C02 output from tobacco c a l -  increased by 65% a f t e r ozone fumigation (Anderson  and Taylor, 1973)•  Stimulated r e s p i r a t i o n could also mean stimulated  superoxide production i f normal r e s p i r a t i o n contributes to production of the free r a d i c a l . F i n a l l y , the potential f o r the formation of various r a d i c a l s exists when ozone simply dissolves i n water.  Molecular oxygen and  various other products r e s u l t when ozone decomposes spontaneously i n aqueous solution (Heath, 1980).  While i t i s thought that large amounts  of 0H~ are produced by t h i s process (Weiss, 1935)» extensive quali t a t i v e and quantitative analysis of the products with respect to b i o l o g i c a l systems has not yet been undertaken (Heath, 1975)* There are indications that damage attributed to 02* may r e a l l y 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 r e s u l t i n g i n the production of ethylene (Fridovich, 1978).  Ethanol or benzoate (compounds known to  scavenge 0H~ s e l e c t i v e l y while remaining unreactive towards Q>£ or ^2^2^  i t t h i ^ i t ethylene production i n these systems.  The addition of  SOD or catalase also i n h i b i t e d ethylene production, further supporting the hypothesis that 0H» i s the active agent since 02* and ^2^2 can interact to form 0H~ . Under the c a t a l y s i s of iron ions, the HaberWeiss reaction i s thought to occur, where: H  (Leshem, 1981).  2°2  +  °2 ~~*  0 H  "  +  0  H  * °2  The Fenton reaction may also occur where hydrogen  peroxide reacts with divalent ferrous iron to form the hydroxyl radi c a l , a hydroxyl ion, and a t r i v a l e n t f e r r i c ion: + Fe3+ Since most i r o n i n b i o l o g i c a l systems i s chelated i n f e r r i c form,  15  Cohen (1978) suggests that superoxide may react with the chelated i r o n forming ferrous iron and molecular oxygen.  Thus, i t can be seen  that 0H« production i s c l o s e l y t i e d to 0~» metabolism, which emphasizes the need f o r 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 v i a the reaction: 0~. + 0~» + 2 H — > H 0 +  2  2  +0  2  has been detected i n v i r t u a l l y a l l organisms surveyed which respire aerobically.  This i s somewhat s u r p r i s i n g considering that 0 « w i l l 2  spontaneously dismutate to the same products with a r e l a t i v e l y large rate constant (Fridovich, 1978; Elstner, 1979). The reason f o r t h i s apparently redundant but ubiquitous enzymatic defence i s at l e a s t two-fold.  Khan's (1970) observation that spon-  taneous dismutation of superoxide produces molecular oxygen, but i n the highly energetic s i n g l e t state helps to explain the f i r s t advantage.  Dissipation of this extra energy can r e s u l t i n d i r e c t oxid-  ation of membrane l i p i d s to peroxides ( H a l l i w e l l , 1981), which encourages free r a d i c a l production v i a R - abstraction from the l i p i d +  peroxide (Pryor, 1976).  Thus, without SOD, one toxic form of oxygen  becomes another, and complete d e t o x i f i c a t i o n has not occurred.  Secondly,  while superoxide i s a transient species with a l i f e t i m e i n the nanosecond range due to spontaneous disproportionation,  SOD-catalyzed  16  dismutation i s 10 - f o l d f a s t e r at physiological pH (Fridovich, 1978)* SOD i s a family of enzymes, the members being distinguishable on the basis of t h e i r metal cofactor.  Iron-containing  (FeSOD) and man-  ganese-containing (MnSOD) enzymes are c h a r a c t e r i s t i c of prokaryotes and are c l o s e l y related i n t h e i r amino acid sequences (Fridovich, 1976, 1978)*  Enzymes containing both copper and zinc (CuZnSOD) are char-  a c t e r i s t i c 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 t h e i r d i f f e r e n t i a l s e n s i t i v i t y to cyanide. CuZnSOD a c t i v i t y i s i n h i b i t e d by cyanide while MnSOD i s not affected (Fridovich, 1975). Cupro-zinc SOD i s a homodimer of molecular weight 32,000.  2+ subunit contains one atom of Cu  Each  2+ and Zn  (Fridovich, 1976). The  cupro-zinc enzyme i s o l a t e d from yeast, Neurospora crassa,  spinach,  chicken, or cow are a l l s t r i k i n g l y s i m i l a r , showing only minor d i f ferences i n amino a c i d 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 a n t i p a r a l l e l fashion 1975).  (Richardson et a l . ,  Two c o i l s 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 i n close proximity at the active s i t e and are joined by a common ligand.  However, the Cu i s r e l a t i v e l y exposed while the Zn  i s more hidden within the molecular structure. Somewhat less i s known about the structure of MnSOD. subunit weight of 22,900 (Fridovich, 1976).  I t has a  Analysis of i t s amino acid  sequence has l e d to predictions that i t does not have as extensive a fi-  17  s t r u c t u r e as i t s CuZn c o u n t e r p a r t , b u t has a compact g l o b u l a r conf o r m a t i o n d e v o i d o f p r o t r u d i n g l o o p s (Steinman Without  complete  and Naik, 1978)*  s t r u c t u r a l a n a l y s i s , i t i s n o t c l e a r e x a c t l y how  many metal b i n d i n g s i t e s a r e p r e s e n t i n t h e enzyme.  Generally, i t s  metal c o n t e n t has been r e p o r t e d t o v a r y between one and f o u r atoms p e r molecule  ( F r i d o v i c h , 1976).  from two t o f o u r depending  1981).  The number o f s u b u n i t s a l s o v a r i e s  on t h e s o u r c e o f enzyme ( B r i d g e s and S a l i n ,  Kono e t a l . (1979) found the m o l e c u l a r weight  o f MnSOD from  k i d n e y bean t o be 44»000, s u g g e s t i n g t h a t i t i s a dimer. I t has been e s t a b l i s h e d t h a t a t l e a s t t h r e e isozymes i n Phaseolus v u l g a r i s L. (Asada e t a l . , Two  o f SOD e x i s t  1977} B r i d g e s and S a l i n , 1981).  o f these use copper and z i n c as t h e i r c o f a c t o r s and t h e t h i r d man-  ganese.  The CuZnS0D*is l o c a l i z e d m a i n l y i n t h e c h l o r o p l a s t  (Asada e t a l . ,  1973;  Asada e t a l . ,  1977)» w h i l e some a c t i v i t y i s a s -  s o c i a t e d w i t h t h e t h y l a k o i d membranes (Jackson e t a l . , Hall,  1981).  1978;  I t i s n o t c l e a r a t p r e s e n t whether o r n o t the  activity is artifactual.  stroma  F o y e r and latter  MnSOD a c t i v i t y , which i s r e s p o n s i b l e f o r 25%  t o 37% o f t h e t o t a l a c t i v i t y i n k i d n e y bean l e a v e s (Kono e t a l . ,  1979)  i s l a r g e l y a s s o c i a t e d w i t h the m i t o c h o n d r i a l f r a c t i o n . The r e a s o n t h a t t h e spontaneous d i s m u t a t i o n o f 0^* i s r e l a t i v e l y slow compared w i t h t h e SOD-catalyzed  reaction i s , a t least i n part,  thought t o be due t o t h e s i m i l a r charge on t h e s u p e r o x i d e s u b s t r a t e molecules.  E l e c t r o s t a t i c r e p u l s i o n between t h e a n i o n s p r e v e n t s t h e  c l o s e approach  t h a t would a l l o w t h e r e a c t i o n t o o c c u r .  To circumvent  t h i s problem, a l t e r n a t e r e d u c t i o n and r e o x i d a t i o n o f a c a t a l y s t d u r i n g s u c c e s s i v e encounters w i t h 0^* c o u l d o c c u r , a l l o w i n g f o r t h e t r a n s f e r o f one e l e c t r o n t o ansuperoxide m o l e c u l e w i t h o u t r e q u i r i n g c l o s e  prox-  18  i m i t y o f the second s u b s t r a t e m o l e c u l e .  F r i d o v i c h (1978) p o i n t s  out  t h a t t h i s type o f mechanism seems t o be employed by every SOD examined to date.  I f "E" denotes the enzyme and  MMe" the metal c o f a c t o r , t h e  mechanism can be w r i t t e n : E-Me  11  + 0~» —> E-Me " 11  E-Me " 11  If  + 02  (1 )  + 0~» + 2 H + — > E-Me  11  + R" 2 0 2  (2)  CuZnSOD i s the c a t a l y s t , i n d i c a t i o n s are t h a t copper o s c i l l a t e s  between c u p r i c and ing  1  1  cuprous s t a t e s d u r i n g the r e a c t i o n , w i t h Zn p l a y -  mainly a s t r u c t u r a l r o l e .  v a l e n t and  I n MnSOD-catalyzed r e a c t i o n s , t r i -  d i v a l e n t s t a t e s o f manganese are  involved.  While o t h e r s u p e r o x i d e d e t o x i f y i n g mechanisms e x i s t , the u b i q u i t o u s n a t u r e o f SOD i n r e s p i r i n g systems s t r o n g l y suggests t h a t i t p l a y s a major r o l e i n c e l l u l a r o x y - r a d i c a l s c a v e n g i n g .  Asada e t a l .  (1977) l i s t s e v e r a l p o s s i b l e c h l o r o p l a s t components c a p a b l e o f scavenging  0~» It  ( T a b l e 1 ). can be seen t h a t none o f the components p r o v i d e s  whose e f f i c i e n c y o f s c a v e n g i n g 02» that provided in  a list  by CuZnSOD.  i s greater  than a few  a system percent o f  Other a n t i o x i d a n t s which c o u l d be i n c l u d e d  o f c e l l u l a r defenses i n c l u d e <*•-tocopherol a n d ^ - c a r o t e n e .  However, t h e i r q u a n t i t a t i v e c o n t r i b u t i o n as s c a v e n g i n g agents i n v i v o has  not been a s s e s s e d .  T h i s i s a l s o the case f o r c y t o k i n i n s and  process o f photorespiration.  the  C y t o k i n i n s may serve a d u a l r o l e i n f r e e  r a d i c a l metabolism: a s scavengers u s i n g the <<-carbon (Leshem, 1981), and by p r e v e n t i n g  the f o r m a t i o n  o f s u p e r o x i d e by b i n d i n g x a n t h i n e  o x i d a s e under c e r t a i n c i r c u m s t a n c e s (Leshem, 1981). respiration i s s t i l l  l a r g e l y s p e c u l a t i v e , but  The  r o l e o f photo-  some workers have hypoth-  e s i z e d t h a t , w i t h o u t i t s oxygen consumption c a p a b i l i t y , the l e v e l s o f  T a b l e 1. R e a c t i v i t y o f c h l o r o p l a s t components with superoxide t h e i r r e a c t i o n r a t e c o n s t a n t s and c o n c e n t r a t i o n i n c h l o r o p l a s t s (Asada et a l . , 1977).  R e a c t i o n with CL*  cytochrome f •  (Fe  Reaction Rate Constants (pH=7.8)  Concentration in C h l o r o p l a s t (M)  (Fe  3 +  )  6.1 x 1 0  6  6.2 x 1 0 ~  5  (Cu  2 +  )  1.1 x 1 0  6  6.2 x 1 0 "  5  )  2 +  plastocyanin • (Cu ) +  ferredoxin •  Mn  (Fe  (Fe 3 +  )  6.2 x 10~^  )  6.0 x 1 0  6  4.0 x 1 0  2.7 x 1 0  5  2.5 x 1 0 ~  3  GSH — • GSSG  6.7 x 1 0  5  3.5 x 1 0 ~  3  CuZn SOD  2.0 x 1 0  9  8.0 x 1 0 ~  6  2 +  —• Mn  2 +  3 +  ascorbate  •  - 4  dehydroascorbate  20  various high energy species may overwhelm c e l l u l a r defence mechanisms, r e s u l t i n g i n i n j u r y or death (Foyer and H a l l , 1980; Leshem, 1981). Consequently, while SOD seems to be a major part of the defence mechanism against active oxygen, i t should be r e a l i z e d that a l t e r native scavenging systems e x i s t . The f i r s t report of SOD being involved i n the response of a plant to a i r pollutant stress came from Tanaka and Sugahara (1980). worked with poplar, Populus euramericana (Spinacea oleracea L. cv. New A s i a ) .  They  (Dode) Guinier, and spinach  In one set of experiments,  poplar trees were subjected to continuous fumigation with 0.1 ppm SO^ while the l e v e l s of CuZnSOD and catalase were monitored.  SOD a c t i v i t y  was shown to increase s i g n i f i c a n t l y i n trees pretreated with SO,, a f t e r four days of fumigation, to a maximum l e v e l 4.4 times the control values a f t e r 20 days of pretreatment.  Plants were then subjected to  an acute fumigation of 2.0 ppm SO^ f o r two hours. tent was measured and used as an index of i n j u r y .  Chlorophyll conPlants pretreated  with d a i l y low SO2 were found to be less injured a f t e r acute fumigation, i n accord with Zahn's (1970) work.  Thus, Tanaka and Sugahara.(1980)  postulated a cause and e f f e c t relationship i n which subacute SO^pretreatment elevated CuZnSOD l e v e l s and resulted i n protection against acute SO^ i n j u r y . A second, less d i r e c t l i n e of evidence was obtained using the copper chelating agent, diethyldithiocarbamate (DDTC).  Spinach leaves  sprayed with 2% DDTC showed a 65% to 77% decrease i n SOD a c t i v i t y . Treated and control plants were then exposed to 0.5 ppm §0^ to test the r e l a t i v e injury response.  Chlorophyll analysis showed that plants  treated with DDTC were more injured than the untreated controls.  These  21  r e s u l t s supported t h e i r hypothesis that SOD plays a central r o l e i n protection against SO2 s t r e s s . The experimental chemical  N-(2-(2-roxo-1-imidazolidinyl)-ethyl)-N-  phenylurea "ethylene diurea" (EDIl) has been shown to confer t o l e r 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 a c t i v i t y with EDU-induced ozone tolerance i n snap beans.  Phaseolus v u l -  g a r i s 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  i n j u r y a f t e r acute ozone exposure was reduced by 55% when 25 mg EDU per 15-cm pot was added while no v i s i b l e injury was detected when 50 mg per pot was added.  SOD a c t i v i t y was shown to be only s l i g h t l y  stimulated by the application of 25 mg EDU per pot when compared with untreated controls, but was more than doubled i n plants r e c e i v i n g 50 mg EDU per pot.  Polyacrylamide gel electrophoresis showed quantitative  differences i n proteins of molecular weight 32,000 and 16,000 daltons i n extracts of EDU-treated plants compared with controls.  These  proteins were thought to be the undissociated CuZnSOD dimer and d i s sociated subunits, respectively.  Lee and Bennett (1982) concluded  that EDU acts by inducing certain oxidant scavenging enzymes including SOD and that this added a c t i v i t y then enables plants to withstand stress from oxy-radicals generated within the plant as a r e s u l t of normal metabolic a c t i v i t y or of external stresses such as ozone. Recently, McKersie et a l . (1982) examined SOD a c t i v i t y i n suscepti b l e and tolerant c u l t i v a r s of Phaseolus vulgaris L..  They found  no difference i n the i n t r i n s i c SOD l e v e l s of the c u l t i v a r s that could  be related  to ozone s e n s i t i v i t y , but observed a tendency f o r SOD  a c t i v i t y to increase a f t e r acute ozone exposure.  These researchers  concluded that SOD plays only a minor r o l e , i f any, i n the d i f f e r e n t i a l s e n s i t i v i t y of bean c u l t i v a r s to ozone.  23  MATERIALS AMD METHODS  Bush bean (Phaseolus vulgaris L. cv. Pure Gold wax) seeds (obtained from Buckerfield's Ltd.), were planted 2 cm deep, two seeds per 15-cm diameter pot i n s t e r i l i z e d potting s o i l containing 2% peat by volume.  Radish (Raphanus sativus L. cv. Cherry B e l l e ) was planted  1 cm deep, several seeds per 15-cm diameter pot.  Pots with bean seeds  were kept i n the greenhouse and transferred to charcoal-^filtered-air upon seedling emergence.  Pots with radish seeds were kept i n the  greenhouse, thinned to three plants per pot, and transferred to charc o a l - f i l t e r e d - a i r eleven days a f t e r seedling emergence.  Fumigation with Ozone  Two types of exposure chambers were used f o r ozone fumigation. Pretreatment with subacute ozone concentrations (0.02  to 0.1 ppm) was  done i n 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 i n a l l exposure chambers.  Day and night temperatures  were 20°C and 15°C respectively.  Acute  fumigations were administered i n smaller Plexiglas chambers (120 x 35 x 45 cm). In these, the c h a r c o a l - f i l t e r e d - a i r supply resulted i n one complete a i r change every thirteen minutes.  Daytime  temperature  i n 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 r e -  q u i r i n g elevated temperatures were performed by p l a c i n g a thermos t a t i c a l l y controlled 750 watt car i n t e r i o r heater ( A l l s t a t e Model 86043) i n each chamber.  These experiments had day and night temp-  eratures of 32±2°C and 23-2°C r e s p e c t i v e l y . In both chamber types photosyhthetically active r a d i a t i o n at plant l e v e l was 0.2 and 75%»  m s  , and r e l a t i v e humidity varied between  55%  Plants were watered to f i e l d capacity on alternate days i n  the larger exposure chambers and d a i l y i n 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  the smaller chambers f o r both subacute and acute fumigation.  employed No  s i g n i f i c a n t differences between the systems were detected using Student's t t e s t . Subacute ozone concentrations (0,02  to 0.1  ppm) were generated  by passing a stream of c h a r c o a l - f i l t e r e d - a i r over a bank of twelve germicidal u l t r a v i o l e t lamps (Sylvania type B-4W Model 4511)  con-  tained i n an a i r - t i g h t f o i l - l i n e d P l e x i g l a s 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)  d i r e c t l y i n 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 c a l i b r a t e d regularly at  the Environmental Laboratory, B r i t i s h Columbia Ministry of Environment on the University of B r i t i s h Columbia campus.  Both UV photometry and  gas-phase t i t r a t i o n with n i t r i c oxide were used to c a l i b r a t e the ozone  25  source used as a primary standard (McQuaker, 1981). A l l exposures started three hours a f t e r the beginning of the photoperiod and lasted s i x hours d a i l y unless otherwise s p e c i f i e d .  Fumigation with SCy,  A l l subacute and acute exposures were administered i n the smaller Plexiglas chambers. a tank (1%  SCv, was introduced at the desired flow rate from  SO^ i n a i r ) .  The concentration was monitored either con-  tinuously or at 25 minute i n t e r n a l s .  The SOg monitor used (Thermo  Electron Pulsed Fluorescent SCv, Analyzer, Series 43)  was also c a l -  ibrated r e g u l a r l y using U.S. National Bureau of Standards primary standards of  S0 2 .  Assessment of Injury  For  acute injury assessment  i n pretreatment experiments using 0^  or SGv,* acute doses were administered f o r one to four days, u n t 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 i n f i l t e r e d a i r f o r one to three days to develop symptoms fully. rotic  Injury was assessed by v i s u a l r a t i n g of percent l e a f area nec(%LAN) f o r each l e a f , estimated to the nearest 5%.  Values f o r  leaves were then averaged to y i e l d an estimate of plant i n j u r y .  Injury  was assessed using the primary leaves of bean i n a l l experiments except those t e s t i n g 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 a f t e r emergence were assayed. ary  Chlorophyll and protein content of the prim-  leaves were also measured and used to compare injury between t r e a t -  26  merits.  (Details of the procedures used i n these assays are presented  i n the " A n a l y t i c a l " section).  Since adaxial l e a f surfaces sustain  most of the injury a f t e r acute ozone fumigation, measurement of the r e l a t i v e chlorophyll content i n the upper c e l l layers of the l e a f using reflectance spectrophotometry a measure of ozone i n j u r y .  (Runeckles and Resh, 1974).also provided,  Reflectance measurements were made using  a Perkin-Elmer, Coleman Model 124 spectrophotometer d i f f u s e reflectance integrating sphere.  equipped with a  The instrument was adjusted  to 100% reflectance at 550 nm using a gypsum standard.  Disks from the  basal portion of the l e a f were placed d i r e c t l y i n the sample holder f o r reflectance measurement.  Analytical  Assays f o r SOD were performed two to three hours a f t e r the s t a r t of the photoperiod.  Tissue f o r 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 f o r dry weight and chlorophyll deter-  2 mination.  Samples of 15 to 60 cm were used f o r the SOD extract.  The tissue was homogenized f o r 25 seconds i n 40 ml 50mM -/potassium phosphate 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 f o r SOD a c t i v i t y and protein  content. SOD was assayed photochemically according to Beauchamp and Fridovich (1971) with the modifications of Dhindsa et a l . (1981). based on the a b i l i t y of SOD to scavenge 0*  produced  The assay i s  photochemically  27  from r i b o f l a v i n before i t reduces the dye, nitroblue tetrazolium (NBT). Reduction of NBT results i n a s i g n i f i c a n t increase i n absorbance at 560 nm which i s measured a f t e r several minutes of i l l u m i n a t i o n .  The 3  ml reaction mixture contained 50 mmoles potassium phosphate buffer pH 7.8» 13 mmoles methionine, 75 mmoles NBT, 2 mmoles r i b o f l a v i n , 0.1 mmoles EDTA, and 0 - 150 4 I of the enzyme extract.  A f t e r addition  of the enzyme, the tubes were shaken and placed approximately 30 cm below a single 20 watt "Cool White" fluorescent lamp i n 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 l i g h t  and ran f o r 12 minutes, at which time the absorbance i n tubes without enzyme had reached 0.18 ± 0.01 0D units at 56O nm.  For each enzyme ex-  t r a c t , assays were c a r r i e d out using at l e a s t three d i f f e r e n t volumes of extract i n order to determine the relationship between absorption and volume of extract.  A plot of l o g A - ^ Q versus volume was found to  be l i n e a r as shown i n Figure 1 (r = 0.99  p<0.00l).  One unit of enzyme  a c t i v i t y was defined as the volume of extract required to i n h i b i t color development  i n the tubes by 50% (Beauchamp and Fridovich, 1971)•  A l l assay runs contained an i n t e r n a l standard of bovine erythrocyte SOD. Protein content i n 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 a c i d . allowed to develop color f o r 20 minutes.  The tubes were shaken and 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 i n the freezer (-9°C) u n t i l i t was  28  1.4  h  1.3  L  1.2 o LO CU  o  •1.1  r-  1.0  h  "0.9  h  '0.8  L  c  ro Xi S-  o  in CD  o  25  50  75  100  Microliters of Extract Added Figure 1.  Relationship between log A 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 A b s decrease as volume increases. 5 6 Q  56Q  29  assayed.  The disks were homogenized i n 50 ml of cold 80% acetone f o r  45 seconds and the resultant extract was passed through a sintered Absorbances a t 645» 652, and 663 nm were  glass f i l t e r under suction.  measured and the use of Bruinsma's equation: 20.2A  645  + 8A  6 6 3  + 2 .8A 7  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 f o r 1 minute i n b o i l i n g water. protein samples were loaded i n each w e l l .  Twenty nanogram  Electrophoresis was c a r r i e d  out i n 0.1M T r i s - g l y c i n e buffer (pH 8.3) at room temperature at 5 niA per tube f o r about 6 hours.  Gels were stained f o r 1 hour i n 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 f o r the l o c a l i z a t i o n of both enzyme protein and enzyme a c t i v i t y .  SOD was located on gels according to  the negative s t a i n i n g technique of Beauchamp and Fridovich (1971)• After electrophoresis, gels were immersed i n i c e - c o l d 50mM potassium phosphate buffer f o r 10 to 15 minutes.  The gels were rinsed with d i s -  t i l l e d water and put i n 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 at room temperature, f o r 15 minutes.  M r i b o f l a v i n , also  The gels were then washed with  and suspended i n 0.1mM EDTA p r i o r to i l l u m i n a t i o n .  After irradiation  the gels were stained blue except at bands with SOD a c t i v i t y .  30  A l l control and treatment means were tested f o r s i g n i f i c a n c e using Students t test f o r comparing two means on each day on which assays were done.  To condense the presentation of r e s u l t s , treatment data  are often presented as a percentage of control data.  Standard errors  were computed according to the equation: = 1  r Z  (r ) + (z-r ) 2  2  f <  where z £ r = x - r z x y  ±  r  y  with r = associated standard error of the mean (Jeffreys, 1932). A l l data i n 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 r a t i o of treatment to control does not cross the 100% value, means are s i g n i f i c a n t l y d i f f e r e n t (p<0.l).  31  RESULTS  Recently, Lee and Bennett  (1982) have stressed the importance  including PVP i n the extraction medium when i s o l a t i n g SOD.  of  They ar-  gue that phenolic compounds n a t u r a l l y present i n bean l e a f tissues form complexes with proteins r e s u l t i n g i n the formation of qiiinones. Quinones i n turn oxidize essential protein functional groups, or form covalent bonds with proteins, reducing apparent SOD a c t i v i t y i n the assay.  To investigate t h i s p o s s i b i l i t y , tissue samples were taken  from opposite sides of the midrib of primary bean leaves ranging i n age from 4 to 15 days past emergence.  The samples were extracted i n  buffer, with or without the addition of 3% PVP.  As shown i n Figure 2,  SOD a c t i v i t y was found to be s l i g h t l y higher (7-10%) i n non-PVP ext r a c t s when assayed photochemically.  PAGE extracts prepared with or  without PVP were indistinguishable (Figure 3).  While protein content  i s s l i g h t l y 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 r e s u l t s when assaying f o r SOD. The temporal patterns of SOD a c t i v i t y i n the developing primary leaves of bean and radish are shown i n Figures 4 and 5 r e s p e c t i v e l y . Younger leaves were found to contain more a c t i v i t y than older leaves whether expressed on an area or dry weight basis. Central to the work i n this thesis i s the r e l i a b i l i t y  of assays  used to estimate plant injury a f t e r exposure to 0^ or SO^.  Figure 6  shows a comparison of three d i f f e r e n t assays used to determine  injury  to the primary leaves of bean at d i f f e r e n t stages of s u s c e p t i b i l i t y  32  120  h  120  110  h  no  100  100 o o  Q O  90  90  80  80 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 activi t y . (•) Protein content. n=6.  • d A  Figure  B  3.  H H 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 c o l u m n s 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 bean. 20 ng s a m p l e s w e r e 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 Cooma 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 P V P ; (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 ; (Ej 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 expressed on a leaf area (•) or dry weight (•) basis. n=6.  36  D1  .... )  1 0  2  1  1 4  1 6 Days f r o m  F i g u r e 6.  I  8  10  1 12  i  14  Emergence  Comparision of i n j u r y assays using primary leaves of 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 o z o n e o r f i l t e r e d - a i r b e f o r e a c u t e o z o n e d o s e . n=10-12.  37  to an acute dose of ozone.  Conventionally, %LM  purpose (Runeckles and Rosen, 1974* 1977) and was factory method of i n j u r y assessment (Figure 6).  has been used f o r t h i s shown to be a s a t i s Protein content i n  the SOD extract was found to be inversely well correlated (r = 0.91 p<0.001) with values of. percentage l e a f area necrotic and was consistently used as a check on injury r a t i n g by v i s u a l estimation of LAN.  Chloro-  p h y l l estimation of adaxial l e a f surfaces using reflectance spectrophotometry also showed s i m i l i a r trends, but t h i s technique i s i d e a l l y suited to the assessment of chronic i n j u r y and was used sparingly throughout the research.  Chlorophyll extracts i n acetone ^proved to  be f a r too i n s e n s i t i v e to detect differences between treatments i n the various subacute pretreatment experiments, and therefore were not used as an assay f o r i n j u r y .  Beans Treated with Ozone  The r e s u l t s persented i n Figure 7 confirm work reported  earlier  by Runeckles and Rosen (1977)» showing the effects of 0^ pretreatment on subsequent acute i n j u r y i n the primary leaves of bean.  Plants exposed  to 0.02 ppm 0^ f o r s i x hours d a i l y 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 l e s s injured than controls (r = 0.44  P<0.001). In contrast, plants exposed to 0.05 ppm 0^ f o r s i x hours d a i l y are l e s s susceptible to a subsequent acute dose a f t e r two pretreatments.  However, additional days of pretreatment increase s u s c e p t i b i l i t y  38  200  -  175  -  150  h  25  h  0  I  I  0  1  I  I  I  I  I  I  2  3  4  5  6  7  Days f r o m F i g u r e 7.  I  I  I  I  ' 8  9  10  11  L__  12  Emergence  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 o z o n e (•, 0.02 ppm a n d D , . 0 . 0 5 ppm) on %LAN o f p r i m a r y l e a v e s o f b e a n r e s u l t i n g f r o m t r e a t m e n t w i t h 0 . 4 ppm o z o n e on day i n dicated. n=l2-14.  39  and predispose the leaves to acute ozone injury (r^= 0.16  p<0.021).  The pattern of extractable SOD i n these leaves, r e l a t i v e to cont r o l s , i s shown i n Figure 8.  I t can be seen that SOD a c t i v i t y i n pre-  treated plants was s i m i l i a r regardless of pretreatment dosage and d i d not vary with time ( 0.02 ppm - r = 0.12 2  p<0.18).  p<0.82; 0.05 ppm - r = 0.01 2  S i g n i f i c a n t differences from controls were observed only  on days 4 and 6 a t pretreatment concentrations of 0.02 ppm and 0.05 ppm, respectively.  However, a t these times, pretreated and control  plants exhibited a s i m i l i a r degree of injury a f t e r acute 0^ treatment (Figure 7).  When differences from the controls were observed i n 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 s i g n i f i c a n t differences i n SOD a c t i v i t y were observed (Figure 8). While no apparent changes i n o v e r a l l SOD l e v e l occurred as a r e s u l t of pretreatment with subacute ozone, i t was possible that a s h i f t i n the r a t i o of cupro-zinc to manganese SOD results from low 0^ exposure.  To test t h i s p o s s i b i l i t y , 1mM KCN was included i n SOD assays,  but, on days 2 and 12 of pretreatment with 0.02 ppm 0^, when d i f f e r e n t i a l s u s c e p t i b i l i t y to acute 0^ occurs, there was no difference i n the proportion of SOD a c t i v i t y i n h i b i t e d by cyanide (Figure 9). However, changes i n CN-sensitive SOD a c t i v i t y did occur as both pretreated ( r = 0.78 2  p<0.019) and control ( r = 0.82 2  p<0.013) plants aged.  Relation of SOD to Injury Symptoms  Plants exposed to 0.1 ppm 0^ f o r s i x hours d a i l y began to show signs of v i s i b l e i n j u r y a f t e r 2 to 3 days of treatment.  The symptoms  40  140  ^o  j_ +->  120  h  o o  100 +-> Q O I/O  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 Days  Figure  9.  8 from  10  12  Emergence  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 o z o n e 6 h d a i l y (•). n=12.  42  were t y p i f i e d as minute areas of l o c a l i z e d chlorosis and necrosis r e s u l t i n g i n a spotted or flecked appearance face.  to the adaxial l e a f sur-  With the onset of such v i s i b l e symptoms, the SOD l e v e l i n these  leaves was found to increase s i g n i f i c a n t l y above control values, as shown i n Figure 10.  Continued pretreatments were found by Runeckles  and Resh (1974) to r e s u l t i n an accumulation of chronic symptoms, and t h i s observation can be correlated with the increased SOD act i v i t y of the injured plants depicted i n Figure 10.  The e f f e c t on  SOD persisted through the course of the experiment and reached a l e v e l of 57% more enzyme a c t i v i t y than the controls by day 18. In order to investigate the r e l a t i v e s e n s i t i v i t y of pretreated plants to acute treatment, nine-day o l d bean plants exposed d a i l y to 0.1 ppm 0^ from emergence and f i l t e r e d - a i r - t r e a t e d controls were simultaneously exposed to an acute dose of ozone.  As shown i n Table 2,  p a r a l l e l samples of plants from each treatment revealed that pretreated plants had 21% more SOD a c t i v i t y 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 e a r l i e r by Runeckles and Rosen (1977)•  E f f e c t of EDU on Bean Leaves  Experiments with EDU confirmed i t s protective e f f e c t on primary leaves of bean exposed to ozone (Table 3).  On the average, over 70%  of the control l e a f area was necrotic one day a f t e r the administration of an acute 0^ dose, while EDU-treated plants were e s s e n t i a l l y uninjured.  However, the e f f e c t of the chemical on SOD a c t i v i t y 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 d o s e a n d 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 ( a s % r e f l e c t a n c e ) a f t e r a c u t e d o s e i n 9-day o l d b e a n s p r e t r e a t e d f r o m e m e r g e n c e 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 d o s e was a d m i n i s t e r e d f o r 2 d a y s (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 . Treatment  SOD _ ( u n i t s cm" )  Pretreated A c t i v i t y (as % o f C o n t r o l )  Control Pretreated  8.98 \ 0.23 10.90 - 0.16  100  2  121 - 4%  Pretreated (as % o f  Reflectance Control)  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  10.30 - 0.68  10.97 - 0.68  72-6  Trifoliate 10.31 - 0.12  10.54 - 0.20  73-7  Primary  EDU-treated 0 3-1  *EDU dissolved in water and added to pots 24 h before SOD assay and acute exposure.  46  nificant. To test the e f f e c t of EDU on t r i f o l i a t e leaves, plants were grown f o r 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 r e s i s t a n t to high 0^ con-  centrations than the one-week o l d plants previously tested, the same trend was observed when i n j u r y symptoms developed.  Control plants  were s u b s t a n t i a l l y injured or k i l l e d by the acute treatment while EDUtreated plants showed l i t t l e or no i n j u r y . When assayed photochemi c a l l y , the e f f e c t of EDU on SOD was again i n s i g n i f i c a n t (Table  3).  PAGE also showed no e f f e c t of EDU on bands exhibiting SOD a c t i v i t y (Figure 3 C ) .  SOD and Acute Injury  Analysis of primary bean leaves before and a f t e r receiving acute ozone doses suggested that SOD was more active i n injured leaves when compared to filtered-air-grown plants.  A detailed study which mon-  i t o r e d t h i s apparent induction of SOD as leaves were subjected to acute doses of 0^ was c a r r i e d out. The acute injury response to the oxidant generally involves tissue collapse, e f f e c t i v e l y  decreasing  s p e c i f i c l e a f area (SLA) by thickening the l e a f (Bennett and Runeckles, 1977)*  Increasing the dose of ozone r e s u l t s i n a progressive s h i f t to  an early decrease i n SLA such that leaves treated with 0.4 ppm are substantially thickened a f t e r 4 days of treatment compared with cont r o l s (r = 0.43  p<0.15).(Figure  11).  For t h i s reason, i t i s prefer-  able that enzyme a c t i v i t y be expressed on a dry weight basis when t r e a t ments involve 0^ doses greater than 0.1 ppm 6 hours d a i l y . Plants were grown i n an atmosphere of f i l t e r e d a i r f o r 3»6, or 9  47  120  h-  110  L_  90  U  80  L_  o (_> ro CD  =1 rO  01  <_> (/)  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^ f o r 6 hours d a i l y were administered.  %LAN and SOD a c t i v i t y  were recorded a f t e r each d a i l y 0^ exposure and these values were compared with ones from controls maintained throughout i n f i l t e r e d air.  The two temperature regimes used were day/night temperatures  of 2 3 7 l 6 ° C and 3 2 ° / 2 3 ° C.  Lower Temperature E f f e c t s ( 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 t h i r d  acute dose (Figure 12). a c t i v i t y (37%)  A r e l a t i v e l y large increase i n SOD  occurred simultaneously (Figure 12).  While %LAN i n  these plants increased due to the fourth and f i n a l acute dose, there was no corresponding s i g n i f i c a n t change i n SOD a c t i v i t y , which remained about 30% above the control l e v e l . A somewhat d i f f e r e n t s i t u a t i o n was noticed when 5-day o l d plants were treated i n the same way.  In these, SOD a c t i v i t y was again  s i g n i f i c a n t l y higher than the controls a f t e r one acute dose, but v i 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 a c t i v i t y , r e l a t i v e to controls, also increased  with subsequent acute doses throughout the experiment, u n t i l treated plants had 60% more SOD a c t i v i t y 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 t h e i r 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. Exposures commenced 3 days from emergence. n=12.  80  0  1  2  3  4  5  Days Past F i r s t 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 s i m i l a r trends to the 5-day o l d plants with regard to SOD l e v e l s , hut the f i n a l %LAN was higher.  High Temperature E f f e c t s ( 3 2 0 / 2 5 °  C)  Two-day o l d plants receiving acute doses at 52° C were subs t a n t i a l l y more injured than plants treated at 23° C (compare Figures 12 and 15).  Symptoms were i n i t i a t e d on day 2 rather than  day 3 of the 0., treatment and f i n a l %LAN was almost double that  3  observed at 2 3 ° C.  SOD a c t i v i t y i n the plants treated at 32° C  was only s l i g h t l y affected by the fumigations,  increasing to. a l e v e l  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, ectively).  and 14 and 17,  resp-  The patterns of SOD a c t i v i t y i n these plants r e l a t i v e  to the controls were dramatically d i f f e r e n t from those treated at the lower temperature.  Here, a s l i g h t stimulation of SOD was noticed  a f t e r one and two days of fumigation  (Figures 16 and 17)» but  subsequent treatment with 0^ resulted i n s i g n i f i c a n t declines i n SOD a c t i v i t y to l e v e l s 13% and 19% lower than controls i n plants when fumigation  started at 5 and 8 days of age respectively (Figures  16 and 17).  Radish Treated with Ozone  SOD a c t i v i t y i n the f i r s t two leaves of radish subjected to low 0,  treatments of 0.02  ppm s i x hours d a i l y tended to remain at  53  100  H  75  —I  50  o u  25  >  O O  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. Exposures commenced 3 days from emergence. n=12.  54  100  1  2  3  4  5  Days Past First Exposure Figure 16.  Conditions as in figure days from emergence.  15. Exposures commenced 6  55  Days Past First Exposure Figure 17.  Conditions as in figure 15. Exposures commenced 9 days from emergence.  56 or s l i g h t l y above that of controls, u n t i l two weeks of exposure, at which time i t decreased s i g n i f i c a n t l y , as shown i n Figure 18. Contrary to the response observed i n bean, this pretreatment had no e f f e c t on %LAN a f t e r an acute dose of 0^ was administered (Table 4).  SOD a c t i v i t y i n plants which had been exposed to an acute dose  was s l i g h t l y but s i g n i f i c a n t l y greater than i n f i l t e r e d - a i r - t r e a t e d controls (Table 5 ) t reduce SOD  even though the pretreatment alone tended to  activity.  E f f e c t s of Subacute Sulfur Dioxide  Considerable f l u c t u a t i o n was observed i n the SOD l e v e l s of primary leaves of bean pretreated with 0.1 ppm gence, as shown i n Figure 19. and barely s i g n i f i c a n t .  continuously from emer-  However, the differences were small  V i s i b l e l e a f injury following acute exposure  was also unaffected by low S0£ pretreatment (Table 4)» SOD a c t i v i t y i n the f i r s t two leaves of radish showed no s i g n i f i c a n t change when treated with low SO^  (Table 5)» but, such treat-  ment predisposed the leaves to v i s i b l e injury a f t e r acute SO^ exposure (Table 4-)*  E f f e c t s of Acute Sulfur Dioxide  Unlike ozone, acute SO^ exposure tended to decrease SOD  activity  i n the primary leaves of bean, compared with filtered-air-grown controls, regardless of pretreatment regime (Table 5)»  Ln radish,  a s i m i l a r trend was observed only i n those plants pretreated with  3  4  5  6  7  Days o f  8  9  10  11  12  13  14  15  16  Pretreatment  SOD a c t i v i t y in the f i r s t two leaves of radish exposed to 0.02 ppm ozone 6 h d a i l y . n=12.  Table 4. %LAN i n p l a n t s r e c e i v i n g a c u t e o z o n e o r s u l f u r d i o x i d e treatment a f t e r pretreatment with e i t h e r f i l t e r e d a i r or sub-acute d o s e s 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 d a y s 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 . Injury est 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 d a y a f t e r a c u t e d o s e , (n = 1 2 ) . Filtered air  pretreated  0 . 0 2 ppm 0 , 6 h d a y  4 6 - 5  BJ  4 5 - 6  B e a n and S 0 . P l a n t s 12 d a y s p a s t e m e r g e n c e . Acute t r e a t ment ( 2 . 0 ppm f o r 2 h) on d a y 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 ) . ?  Filtered air  pretreated  0.1  ppm SO,, c o n t i n u o u s l y  5 3 - 9  C.)  1  5 3 - 8  R a d i s h and S 0 . P l a n t s 12 d a y s p a s t f i r s t d a y o f p r e t r e a t ment. Acute t r e a t m e n t ( 2 . 0 ppm f o r 2 h) on d a y 1 1 . Injury 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 d a y a f t e r a c u t e d o s e , (n = 1 2 ) . ?  Filtered air  pretreated  2 2 - 7 ***  0.1  ppm S 0  ?  continuously  46 -  p < 0.01 w h e r e " p " i s t h e l e v e l o f s i g i f i c a n t from f i l t e r e d a i r c o n t r o l .  8*** difference  59  T a b l e 5. SOD a c t i v i t y i n p l a n t s r e c e i v i n g acute ozone or SO,, treatment compared with p l a n t s maintained in f i l t e r e d a i r o r subacute c o n c e n t r a t i o n s o f ozone o r SO,,. A c t i v i t y expressed as u n i t s per gram dry w e i g h t . (n=12). A.)  B.)  C.)  Radish and Ozone. 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 . Acute treatment (0.27 ppm 6 h d a i l y ) f o r 3 days. Filtered air maintained  0.02 ppm 6 h daily  Filtered air then acute  5700 - 60  5200 - 2 0 * * *  5570 - 60*  Bean and S O . P l a n t s 12 days p a s t emergence. ment (2.0 ppm f o r 2 h) on day 11. ?  Filtered air maintained  0.10 ppm continuously  Filtered air then acute  4950 - 220  4890 - 110  3060 - 250'  0.02 ppm 6 h day" then acute  6000 - 8 0 * * *  Acute  treat-  0.10 ppm c o n t i n u o u s l y then acute 3260 - 250***  P l a n t s 12 days past f i r s t day o f p r e t r e a t Radish and SO, ment. Acute treatment (2.0 ppm f o r 2 h) on day 11. Filtered air maintained  0.10 ppm continuously  Filtered air then acute  5160 - 70  4920 - 240  5110 - 210  0.10 ppm c o n t i n u o u s l y then acute 4090 - 4 6 0 * * *  * p < 0.10 ** p < 0.05 * * * p < 0.01 "p"  i s the l e v e l  o f s i g n i f i c a n t d i f f e r e n c e from f i l t e r e d a i r  control  60  120 L_  ft  /  110  /  -  100  /  /  /  /  /  i\  \ \ \  A-  1  90  80 0  1  J  I  I  I  L  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  Days of Pretreatment Figure 19.  SOD activity in the f i r s t two leaves of radish (•), and the primary leaves of bean (•) exposed to 0.1 ppm S0~ continuously. n=12.  c o n t i n u o u s subacute SO,,. to a c u t e SO^  P l a n t s grown i n f i l t e r e d a i r and  showed no change i n SOD  activity  exposed  r e l a t i v e t o those main-  t a i n e d i n f i l t e r e d a i r f o r the whole experiment.  DISCUSSION  One o f t h e m a j o r what r o l e ,  (1977)  Rosen  subacute acute in  i f any,  air-grown  4 o r d a y 6).  difference 7,  (Figures  enzyme  bean  was t o determine that  leaves  stages  Runeckles and pretreated  apparent  that  SOD i s n o t i n v o l v e d  i n ozone-pretreated and  (Figures  7 and 8 ) .  i s onedayon which  SOD a c t i v i t y controls  However,  days  that  i t i s on these  i n s u s c e p t i b i l i t y t o the acute Significant  SOD a c t i v i t y  filtered-  A t each  than  o n days  with  o f s u s c e p t i b i l i t y t o s*  i s substantially higher  SOD a c t i v i t y  possibility existed  insensitive  SOD w a s  specifically  insensitive ratio  levels  a r e compared there  work  pretreat-  i nt h e  (Figure  no significant  dose was observed  differences  was i n d i f f e r e n t  8,  i n acute  (Figure  injury  t o pretreatment  7 and 8 ) .  Total  not  I t becomes  corresponding days).  were noted  the  plants  primary  through varying  when  concentration  of this  i n t h eresponse  where  treatment.  controls  pretreated day  observed,  the response  ment  SOD p l a y s  ozone pass  ozone  objectives  wasunaffected that  b y ozone p r e t r e a t m e n t , b u t  theproportion of CN-sensitive:CN-  responding t o ozone.  designed  activity,  to include  spot  was o c c u r r i n g on days  checks  While  regular  indicated  a treatment  experiments  were  m o n i t o r i n g o f t h e CNthat  effect  no shift  i nt h e  was observed  (Figure  9). SOD a c t i v i t y after p p m 0^  visible  injury  has  occurred  daily  had  l i t t l e  6 hours  occurred.  c a nbe enhanced  More  dramatically,  by treatment (Figures  effect  two-day  with  ozone,  10, 12-14).  o n SOD a c t i v i t y old plants  but only  Thus, until  0.1 flecking  showed n o n e c r o t i c  63 tissue or change i n SOD a c t i v i t y a f t e r two daily exposures to 0.4 ppm ozone (Figure 12, days 1 and 2), but as soon as a s l i g h t amount of necrotic tissue could be detected (Figure 12, day 3)» the SOD l e v e l 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 l e v e l increased as v i s i b l e symptoms accumulated due to repeated fumigations, which supports the idea that SOD i s r e l a t e d to injury and not 0^ i t s e l f .  Consequently, SOD can be thought of as secondary  i n the defense against ozone i n j u r y . U n t i l 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  i n expanding primary bean leaves (Figures 12-14).  In addition, SOD  l e v e l s are highest i n young primary l e a f tissue, i n 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 a c t i v i t y and p a r a l l e l increase i n s u s c e p t i b i l i t y to acute injury i s of causal significance i n bean.  This i s a questionable conclusion to reach,  though, as SOD l e v e l i s only one of many physiological and biochemical factors that change with age. For example, expansion of the l e a f means that l e a f area, l e a f 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 a f t e r reaching maximum l e v e l s early i n development.  Contrary to Lee  and Bennett's (1982) suggestion, i t would be hasty to conclude that d e c l i n i n g SOD l e v e l s i n plants tissue are responsible f o r increased  64 susceptibility to ozone as plants age. The response of EDU-treated beans to acute ozone fumigation was clearly demonstrated.  Primary and t r i f o l i a t e 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 i n plants treated with EDU, but results in Table 3 do not support this finding. It i s d i f f i c u l t 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 i s 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 i s the fact that different varieties 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 a l . (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 interpretation of data. When expressed on a percentage of control basis, SOD activity in EDU-treated primary leaves i s 107% while trifoliates  had 102% of control activity (Table 3).  Neither result i s statis-  t i c a l l y significant, while associated differences significant.  in %LAN were highly  Lee and Bennett (1982) show a strikingly similar corr-  elation in their work, yet, they conclude SOD i s 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 satisfactory 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^,  i n i t i a l l y impart a water-logged or flecked appearance to the adaxial leaf surface and result i n areas of adaxial or bifacial chlorbtic and necrotic tissue (Tomlinson arid Rich, 1974)• A significant consequence of such injury i s a decrease in specific leaf area, SLA, resulting from ozone-induced tissue collapse and cessation of f o l i a r 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 i s a suitable baseline for enzyme  a c t i v i t y when t e s t i n g the effects of 0.1 ppm ozone, or l e s s , but tissue showing acute injury symptoms would appear to have greatly increased SOD a c t i v i t y compared to controls, simply because more tissue per u n i t l e a f area i s present. Both protein and chorophyll content substantially decrease following acute ozone exposure (Olszyk and T i b b i t t s , 1982), again r e s u l t i n g i n spuriously high estimates of SOD a c t i v i t y i n fumigated plants i f either of the parameters were used as a basis f o r enzyme activity.  Total plant dry weight i s also affected by acute ozone  fumigation (Tingey, 1974; Olszyk and T i b b i t t s , 1982) but s t i l l represents the basis with the l e a s t i n t r i n s i c error f o r expression of SOD  activity. When experiments comparing %LAN  out at a higher temperature, apparent  (Figures 15-17).  and SOD a c t i v i t y were c a r r i e d  some i n t e r e s t i n g tendencies became  The same general trend between %LAN  and  plant age was observed at 3 2 ° / 2 3 ° C, where youngest plants showed the l e a s t i n j u r y .  At a l l ages, %LAN  3 2 ° / 2 3 ° C compared with 2 3 % 6° C.  was- s i g n i f i c a n t l y higher at 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 d a i l y fumigation.  This p o s s i b i l i t y  was  not investigated further i n the present studies. P l o t t i n g r e l a t i v e SOD a c t i v i t i e s a f t e r fumigation (Figures 1517) revealed a substantial temperature-ozone i n t e r a c t i o n .  Two-day  o l d plants subjected to successive acute doses at 3 2 ° / 2 3 ° C again showed stimulation of the enzyme, while v i s i b l e injury covered about  67 60% of the l e a f area.  When five-day o l d plants were studied, SOD  a c t i v i t y i n the fumigated beans increased i n i t i a l l y , but dropped below that of the controls a f t e r the t h i r d dose had been administered. t h i s time, 80% of the l e a f area was i n j u r e d .  At  The f i n a l acute exposure  resulted i n a further depression of SOD a c t i v i t y to 19% below the control l e v e l with a corresponding LAN greater than 90% (Figure 16). The trends wer s i m i l a r when eight-day old plants were used (Figure 17). These observations are i n sharp contrast to the responses observed at 2 3 ° / l 6 ° c.  A f t e r three acute doses, five-day o l d plants  treated under this temperature regime had 25% more SOD a c t i v i t y than controls, but only 50% LAN. The f i n a l acute exposure further increased SOD a c t i v i t y to 35% above the control l e v e l but LAN increased only marginally to 55% (Figure 13).  Similar trends were observed using  eight-day o l d plants (Figure 14). The observed depression of SOD a c t i v i t y i s only an i n d i r e c t © e f f e c t of temperature (Figures 12-17).  Under high temperature  regime, v i s i b l e injury to the leaves occurs e a r l i e r and i s more extensive by the end of the experiment compared with treatments 1. administered at 2 3 ° / l 6 ° C.  Five and eight-day o l d plants grown at  3 2 ° / 2 3 ° C show the greatest v i s i b l e injury and lowest SOD a c t i v i t y 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 l e a f loses i t s ' a b i l i t y to synthesize or activate SOD, r e s u l t i n g i n a loss of enzyme a c t i v i t y r e l a t i v e to controls.  Once  this c r i t i c a l threshold i s reached, ozone molecules may a c t u a l l y penetrate c e l l envelopes and d i r e c t l y react with the protein moieties of SOD causing i n a c t i v i a t i o n or denaturation and contribute to the  68  observed decreased i n a c t i v i t y .  Since plants treated at 23°/l6° C  or two-day o l d plants treated at 32°/23° C sustain less than 75% LAN, they are able to maintain elevated l e v e l s of SOD  (Figures 13-15)*  PVP i s often included i n experimental protocols as an insurance against loss of protein or enzyme a c t i v i t y during the extraction procedure.  Lee and Bennett's (1982) work i s the f i r s t to suggest  that this phenol scavanger i s essential to an accurate SOD assay when using plant t i s s u e .  Several authors (Asada et a l . ,  Giannopolitis and Ries, 1977;  1973;  Jackson et a l . , 1978; Rabinowitch and  Sklan, 1980 and 1981; Bridges and Salin, 1981) have used crude extracts, without PVP i n t h e i r extraction medium, when i s o l a t i n g SOD from plants.  This l a t t e r observation alone does not preclude  the p o s s i b i l i t y that, u n t i l now, tion has been overlooked.  the importance of PVP i n SOD extrac-  However, i n conjunction with r e s u l t s i n  t h i s thesis (Figure 2), there i s no evidence to uphold Lee and Bennett's (1982) claim.  A possible explanation l i e s i n the f a c t  that two d i f f e r e n t SOD assays were used.  Lee and Bennett c o l o r i -  m e t r i c a l l y measured the reduction i n cytochrome "c" by 02*  ,  generated b i o l o g i c a l l y from xanthine and xanthine oxidase.  Superoxide  generated i n the photochemical assay comes from light-stimulated r i b o f l a v i n , and the c o l o r change of the dye, NBT,  i s followed. I t  i s possible that PVP removes compounds which s p e c i f i c a l l y i n t e r f e r e with the former assay while having l i t t l e e f f e c t on the photochemical method. I t i s i n t e r e s t i n g to note that while PVP does apparently protect protein (Figure 2), SOD does not seem to b e n e f i t .  In f a c t , the  inverse r e l a t i o n s h i p between protein content and SOD a c t i v i t y , i n the presence of PVP,  suggests that either PVP i s removing an i n t e r -  69  f e r i n g substance from the extract which mimics SOD a c t i v i t y , or conversely, that PVP d i r e c t l y or i n d i r e c t l y causes an i n h i b i t i o n of  SOD  a c t i v i t y while exerting a protective e f f e c t over protein i n general. I t i s not possible to distinguish between these p o s s i b i l i t i e s from the data i n t h i s t h e s i s . Following exposure to a pollutant, assays f o r plant injury usually consist of a v i s u a l estimate of %LAS or some measure of chlorophyll content.  Both methods have been c r i t i c i z e d f o r t h e i r lack of precision  (Olszyk and T i b b i t t s , 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 i n the rate of senescence 1980, 1981).  (Pauls and Thompson,  During this process, protein i s broken down into g l u t -  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 i n 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, e s p e c i a l l y 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 i n the l a t t e r case.  Pretreatment of radish  with low ozone had no substantial e f f e c t of SOD a c t i v i t y (Figure 18), but unlike bean, a f t e r 11 days of low ozone, no difference i n response to an acute dose could be observed between treatments.  This may  simply  70  mean that the plants were at an " i n d i f f e r e n t " stage with respect to acute ozone tolerance or that stages of varying s u s c e p t i b i l i t y do not occur i n pretreated radish.  SOD a c t i v i t y also seemed to  increase a f t e r acute symptoms developed, although the response was not as consistent as i n bean (Table 4)» The main objective i n working with SO^ was to attempt a v e r i f i c a t i o n of Tanaka and Sugahara's (1980) work with S0  2  and poplar.  Pretreatment of primary leaves i n bean and radish with subacute SO^ continuously had l i t t l e or no e f f e c t on SOD a c t i v i t y (Table 5). Administration of acute S0  2  revealed no e f f e c t of pretreatment,  with respect to v i s i b l e injury, i n 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 a f f e c t SOD a c t i v i t y , and hence the conclusions of Tanaka arid Sugahara (1980) could not be substant i a t e d i n these species. I t should be emphasized  that plants exposed to acute S0  2  respond very d i f f e r e n t l y from those fumigated with acute ozone. Under similar temperature regimes, injurious ozone treatment tends to induce or activate SOD i n primary bean leaves (Figures 12-14), while leaves injured by acute SO^ show less SOD a c t i v i t y than controls maintained i n f i l t e r e d a i r (Table 5)» to not respond to injurious S0 maintained i n f i l t e r e d a i r .  2  SOD a c t i v i t y i n radish tended  or 0^ treatment compared with controls  However, trends i n SOD a c t i v i t y s i m i l a r  to those observed i n bean were seen i n radish when plants were pretreated  with subacute l e v e l s 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 a c t i v i t y a f t e r acute 0^ treatment while leaves pretreated with subacute SO^ had l e s s SOD a c t i v i t y a f t e r the acute SO^ dose. The general f a i l u r e to induce SOD under any but the most extreme conditions leads one to question the extent to which i t i s possible f o r SOD to respond to i t s environment.  In plants, the majority of  SOD a c t i v i t y i s CuZnSOD, and i s associated with the chloroplast (Kono et a l . , 1979: Asada et a l . , 1979).  I t i s t h i s form of the  enzyme that has been affected i n the only two reports claiming induction of the enzyme i n l e a f tissue (Tanaka and Sugahara, 1980; Lee and Bennett, 1982).  The only other unambiguous reports of CuZnSOD i n -  duction are i n the prokaryotes  Saccharomyces cerevisiae and Photo-  bacterium l e i g n a t h i when exposed to hyperoxic 1974;  Puget and Michelson,  1974).  conditions (Gregory et a l . ,  Rat lung SOD, presumably the CuZn  form, has also been induced by high oxygen tension (Pridovich, 1980). MnSOD i n 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 i n cotton and maize to an atmosphere containing 75% oxygen. Most enzymes, i n c l u d i n g SOD, were unaffected, leading them to conclude that the greater proportion (Foster and Hess, 1980) and higher s p e c i f i c a c t i v i t y (Groden and Beck, 1979) of CuZnSOD i n land plants may permit tolerance to large fluctuations i n atmospheric oxygen and presumably superoxide.  Since CuZnSOD does not seem to be p a r t i c u l a r l y  responsive  to p o t e n t i a l l y inductive conditions, the " i n d i f f e r e n t " behavior of the enzyme i n 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 o v e r a l l pool s i z e .  This p o s s i b i l i t y suggests that following SOD  l e v e l s to estimate the degree of 0^  stress i n plants may be too  i n d i r e c t to be of use. The same hyperoxic studies (Poster and Hess, 1980, 1982) showed a two- to three-fold increase i n the a c t i v i t y of glutatione reductase under high 0^.  The enzyme generates NADP i n the process of gluta+  thione reduction.  Increasing the pool size of t h i s oxidized cofactor  would reduce the chance of oxygen accepting photosystem 1 electrons and producing exhaustive  superoxide.  I t may be f r u i t f u l then, to conduct an  study of enzyme responses i n plants subjected to ozone,  with emphasis on those l i k e l y to r e l i e v e oxidative s t r e s s .  73  SUMMARY  1.  PVP has no s i g n i f i c a n t e f f e c t on the photochemical assay f o r SOD.  2.  Changes i n SOD of primary bean leaves pretreated with d a i l y low ozone ( 0 . 0 2 or 0 . 0 5 ppm) are not related to the protective e f f e c t from an acute dose that this pretreatment confers, r e l a t i v e 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 a f f e c t SOD a c t i v i t y .  4.  SOD a c t i v i t y i n 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 i n the primary leaves of bean due to extensive c e l l u l a r disruption.  Consequently, SOD a c t i v i t y i n bean exposed to ozone at  33°C decreases below control l e v e l s because the injury threshold i s surpassed.  7.  SOD i n the f i r s t two leaves of radish i s not affected by d a i l y low ( 0 . 0 2 ppm) ozone pretreatment.  The acute injury response i s also  74  n o t a f f e c t e d by t h i s p r e t r e a t m e n t w h i l e SOD  i s slightly  stimulated  by the a c u t e dose.  8.  Continuous p r e t r e a t m e n t w i t h subacute SO^ v i s i b l e i n j u r y a f t e r a c u t e SO^  (0.1 ppm)  exposure o r SOD  does n o t  affect  a c t i v i t y i n the  p r i m a r y l e a v e s o f bean.  9.  P r e t r e a t m e n t o f the f i r s t two l e a v e s o f r a d i s h w i t h subacute  SO^  p r e d i s p o s e s l e a v e s t o v i s i b l e i n j u r y f o l l o w i n g an a c u t e dose o f SO^ b u t does n o t a f f e c t SOD  10.  Acute SO^  activity.  exposure causes a decrease i n SOD  a c t i v i t y o f the p r i m a r y  l e a v e s o f bean r e g a r d l e s s o f p r e t r e a t m e n t regime w h i l e a  similiar  t r e n d i s o b s e r v e d i n the f i r s t two l e a v e s o f r a d i s h o n l y a f t e r p r e t r e a t m e n t w i t h subacute  S0 . o  75  LITERATURE CITED  Anderson, W.C, and O.C. Taylor. 1973. Ozone induced carbon dioxide evolution i n tobacco c a l l u s cultures. P h y s i o l . Plant. 28:419-423. Asada, K., M. Urano, and M. Takahashi. 1973. Subcellular location of superoxide dismutase i n spinach leaves and preparation and prope r t i e s o f c r y s t a l l i n e spinach superoxide dismutase. Eur. J . Biochem. 36:257-266. Asada, K., S. Takahashi, and Y. Kona. 1976. Superoxide dismutases i n photosynthetic organisms, pp. 551-564 In Advances i n Experimental Medicine and Biology. V o l . 74. Iron and Copper Proteins. K. Yasunobu, H.F. Mower, and 0. Hayaishi (Eds.) Plenum Press, New York. Asada, K., M. Takshashi, K. Tanaka, and Y. Nakano. 1977. Formation of active oxygen and i t s fate i n chloroplasts. pp. 45-63 In Biochemical and Medical Aspects of Active Oxygen. 0. Hayaishi and K. Asada (Eds.) University Park Press, Baltimore. Baum, J.A., and J.G. Scandalios. 1981. I s o l a t i o n and characterisation of the c y t o s o l i c and mitochondrial superoxide dismutases of maize. Arch. Biochem. Biophys. 206:249-264. Beauchamp, CO., and I. Fridovich. 1971. Superoxide dismutase: Improved assays and an assay applicable to acrylamide g e l s . Anal. Biochem. 44:276-287. Bennett, J.P., and V.C. Runeckles. 1977. Effects of low. ozone on growth of Crimson Clover and Annual Ryegrass. Crop Science.  17:443-445.  Bicak, C.J. 1978. Plant response to variable ozone regimes of constant dosage. M.Sc. Thesis. University of B r i t i s h Columbia. Black, V.J. 1982. E f f e c t s o f s u l f u r dioxide on physiological processes i n plants, pp. 67-91 l£. Effects of Gaseous A i r P o l l u t i o n i n Agriculture and H o r t i c u l t u r e . M.H. Unsworth and D.P. Ormrod (Eds.) Butterworth S c i e n t i f i c , Toronto. Bradford, M. 1976. A rapid and sensitive method f o r the quant i t a t i o n of microgram quantities of protein u t i l i z i n g the p r i n c i p l e o f protein-dye binding. Anal. Biochem. 72:248-254* Bridges, S.M., and M.L. S a l i n . 1981. D i s t r i b u t i o n of iron-containing superoxide dismutase i n vascular plants. Plant P h y s i o l . 68:  275-278.  76  Bruinsma, J . 1963* The q u a n t i t a t i v e a n a l y s i s o f c h l o r o p h y l l a and b i n p l a n t e x t r a c t s . Photochem. P h o t o b i o l . 2:241-249* Carnahan, J . E . , E . L . Jenner, and E.K.W. Wat. 1978. P r e v e n t i o n o f ozone i n j u r y by a new p r o t e c t a n t c h e m i c a l . P h y t o p a t h . 68:  1225-1229. Cohen, G. 1 9 7 8 . The g e n e r a t i o n o f h y d r o x y l r a d i c a l s i n b i o l o g i c systems: t o x i c o l o g i c a l a s p e c t s . Photochem. P h o t o b i o l . 28:  669-675. C o u l s o n , C.L., and R.L. Heath. 1974. I n h i b i t i o n o f t h e photosynt h e t i c c a p a c i t y o f i s o l a t e d c h l o r o p l a s t s by ozone. P l a n t P h y s i o l .  53:32-38. D a v i s , B . J . 1 9 6 4 . D i s c g e l e l e c t r o p h o r e s i s . 11. Method and a p p l i c a t i o n t o human serum p r o t e i n s . Ann, N.Y. Acad. S c i . 121:404-427» Dhindsa, R.S., P.P. Dhindsa, and T.A. Thorpe. 1981. L e a f senescence: c o r r e l a t e d w i t h i n c r e a s e d l e v e l s o f membrane p e r m e a b i l i t y and l i p i d p e r o x i d a t i o n , and d e c r e a s e d l e v e l s o f s u p e r o x i d e dismutase and catalase. J . Exp. B o t . 32:93-101. E l s t n e r , E . F . 1 9 7 9 . Oxygen a c t i v a t i o n and s u p e r o x i d e dismutase i n chloroplasts. pp. 411-415 In Encyclopedia o f Plant Physiology. V o l . 6 . P h o t o s y n t h e s i s 1 1 : Carbon Metabolism and R e l a t e d P r o cesses. M. Gibbs and E . L a t z k o ( E d s . ) S p r i n g e r - V e r l a g , New York. P o s t e r , J.G., and G.E. Edwards. 1980. L o c a l i z a t i o n o f s u p e r o x i d e dismutase i n l e a v e s o f C 3 and CA p l a n t s . P l a n t and C e l l P h y s i o l .  21:895-906. F o s t e r , J.G., and J . L . Hess. 1980. Responses o f s u p e r o x i d e dismutase and g l u t a t h i o n e r e d u c t a s e a c t i v i t i e s i n c o t t o n l e a f t i s s u e exposed t o an atmosphere e n r i c h e d i n oxygen. P l a n t P h y s i o l . 6 6 :  482-487. F o s t e r , J.G., and J . L . Hess. 1982. Oxygen e f f e c t s on maize l e a f s u p e r o x i d e dismutase and g l u t a t h i o n e r e d u c t a s e . Phytochem.  21:1527-1532. F o y e r , C.H., and D. H a l l . 1980. Superoxide dismutase a c t i v i t y i n the f u n c t i o n i n g c h l o r o p l a s t . pp. 380-389 I n C l i n i c a l and B i o chemical A s p e c t s o f Superoxide and Superoxide Dismutase. J.V. B a n n i s t e r and H.A.O. H i l l (Eds.) E l s e v i e r / N o r t h H o l l a n d , New Y o r k . F r e d e r i c k , P.E., and R.L. Heath. 1 9 7 4 . Ozone-induced f a t t y a c i d and v i a b i l i t y changes i n C h l o r e l l a . Plant Physiol. 55'15-19.  77  Fridovich, I. 147-159.  1975*  Superoxide dismutase.  Arih. Rev. Biochem.  44:  F r i d o v i c h , I . 1 9 7 6 . Superoxide d i s m u t a s e s : s t u d i e s i n s t r u c t u r e and function, pp. 5 3 0 - 5 3 9 I n Advances i n E x p e r i m e n t a l M e d i c i n e and B i o l o g y . V o l . 7 4 . I r o n and Copper P r o t e i n s . K. Yasunobu, H.F. Mower, and 0 . Hayaishe (Eds.) Plenum P r e s s , New Y o r k . Fridovich, I. 875-880.  1978.  The b i o l o g y o f oxygen r e d i c a l s .  Science. 2 0 1 :  F r i d o v i c h , I . 1 9 7 9 . Superoxide and s u p e r o x i d e d i s m u t a s e s . pp. 6 7 - 9 0 In Advances i n I n o r g a n i c B i o c h e m i s t r y . V o l . 1 . G.L. E i c h o r n and L.G. M a r z i l l i (Eds.) E l s e v i e r / N o r t h H o l l a n d , New York. G i a n n o p o l i t i s , C.N., and S.K. R i e s . 1977* Superoxide d i s m u t a s e s . Occurence i n h i g h e r p l a n t s . P l a n t P h y s i o l . 59:309-314*  1.  G l i d e w e l l , S., and J.A. Raven. 1 9 7 5 * Measurement o f simultaneous oxygen e v o l u t i o n and uptake i n H y d r o d i c t y o n a f r i c a n u m . J . Exp. Bot. 26:479-488. Goldberg, B., and A. S t e r n . 1 9 7 6 . Superoxide a n i o n as a m e d i a t o r o f drug-induced o x i d a t i v e h e m o l y s i s . J . B i o l . Chem. 251:6468-6470* G o l d s t e i n j I.M., and G. Weissman. 1 9 7 7 * Effects of generation o f s u p e r o x i d e a n i o n on t h e p e r m e a b i l i t y o f l i p o s o m e s . Biochem. B i o p h y s . Res. Commun. 75:604-609* Gregory, E.M., and I . F r i d o v i c h . 1974* b a c i l l u s plantarum. J . Bacteriol.  Oxygen metabolism 117:166-169.  i n Lacto-  Groden, D., and E . Beck. 1 9 7 9 * H2O2 d e s t r u c t i o n by ascorbate-dependent systems from c h l o r o p l a s t s . Biochem. B i o p h y s . A c t a . 5 4 6 : 426-435* H a l l i w e l l , B. 1 9 8 2 . Superoxide and superoxide-dependent h y d r o x y l r a d i c a l s a r e important i n oxygen t o x i c i t y . 272.  formation of TIBS. 7:270-  Harbour, J.R., and J.R. B o u l t o n . 1 9 7 5 * Superoxide f o r m a t i o n i n s p i n a c h c h l o r o p l a s t s : ESR d e t e c t i o n by s p i n t r a p p i n g . Biochem. B i o p h y s . Res. Commun. 64:803-807. Heath, R.L., P. C h i m i k l i s , and P. F r e d e r i c k . 1 9 7 4 . Role o f potassium and l i p i d s i n ozone i n j u r y t o p l a n t membranes, pp. 5 8 - 7 5 ^ n A i r P o l l u t i o n R e l a t e d t o P l a n t Growth. W.M. Dugger (Ed.) Am. Chem. Soc. Symp. S e r . Washington D.C. Heath, R.L. 1 9 7 5 * Ozone, p p . 2 3 - 5 5 In Responses o f P l a n t s t o A i r Pollutants. J.B. Mudd and T.T. K o z l o w s k i (Eds.) Academic P r e s s , New York.  78  Heath, R.L. 1980. I n i t i a l events i n injury to plants by a i r p o l lutants. Ann. Rev. Plant P h y s i o l . 31:395-431. Heck, W.W., and J.A. Dunning. 19&7* T h effects of ozone on tobacco and pinto bean as conditioned by several ecological f a c t o r s . J . A i r P o l l u t . Control Assoc. 17:112-114. e  Hicks, M., and J.M. G a l b i c k i . 1978. A quantitative relationship between permeability and the degree of peroxidation i n l i p o some membranes. Biochem. Biophys. Res. Commun. 80:704-708. Jackson, C , J . Dench, A.L. Moore, B. H a l l i w e l l , C.H. Foyer, and D.O. H a l l . 1978. Subcellular l o c a l i z a t i o n and i d e n t i f i c a t i o n of superoxide dismutase i n the leaves of higher plants. Eur. J . Biochem. 91:339-344. Jeffreys, H. 1932. On the theory of errors and least squares. Proc. Roy. Soc. (London), A. 138:48-55. Kellogg I I I , E.W., and I. Fridovich. 1975* Superoxide, hydrogen peroxide, and s i n g l e t oxygen i n l i p i d peroxidation by a xanthine oxidase system. J . B i o l . Chem. 250:8812-8817. Kellogg I I I , E.W., and I. Fridovich. 1977. Liposome oxidation and erythrocyte l y s i s by enzymatically generated superoxide and hydrogen peroxide. J . B i o l . Chem. 252:6721-6728. Khan, A.D". 1970. Singlet molecular oxygen from superoxide anion and sensitized flourescence of organic molecules. Science. 168:476-477. Kono, Y., M. Takahashi, and K. Asada. 1979. Superoxide dismutases from kidney bean leaves. Plant and C e l l Physiol. 20:1229-1235. Kruiper, P.J.C. 1972. Water transport across membranes. Plant P h y s i o l . 23:158-172.  Ann. Rev.  Lavelle, F., A.M. Michelson, and L. Dimitrejevic. 1973. B i o l o g i c a l protection by superoxide dismutase. Biochem. Biophys. Res. Commun. 55:350-357. Lee, E.H., and J.H. Bennett. 1982. Superoxide dismutase. A poss i b l e protective enzyme against ozone injury i n snap beans (Phaseolus v u l g a r i s L . ) . Plant Physiol. 69:1444-1449. Legassicke, B.C., and D.P. Ormrod. 1981. Suppression of ozoneinjury on tomatoes by ethylene diurea i n controlled environments and i n the f i e l d . HortSci. 16:183-184. Lehninger, A.L.  1976.  Biochemistry.  Worth Publishers, New York.  Leshem, Y.Y. 1981. Oxy free radicals and plant senescence. New i n Plant P h y s i o l . 12:1-4.  What's  79  L e v i t t , J . 1980. Man-made s t r e s s e s , pp. 507-530 I n Responses o f P l a n t s t o Environmental S t r e s s e s . V o l . I I . Academic P r e s s , New York. Macdowall, F.D.H. 1965* P r e d i s p o s i t i o n o f tobacco t o ozone damage. Can. J . P l a n t S c i e n c e . 45:1-12. M a l h o t r a , S.S., and S.K. S a r k a r . 1979. Effects o f sulfur dioxide on sugar and f r e e amino a c i d c o n t e n t o f p i n e s e e d l i n g s . P l a n t  Physiol.  47:223-228.  McCord, J.M., and I . F r i d o v i c h . 1969* Superoxide dismutase. An enzymatic f u n c t i o n f o r e r y t h r o c u p r e i n . J . B i o l . Chem. 244:  6049-6055.  McKersie, B.D., W.D. B e v e r s d o r f , and P. H u c l . 1982. The r e l a t i o n s h i p between ozone i n s e n s i t i v i t y , l i p i d - s o l u b l e a n t i o x i d a n t s , and s u p e r o x i d e dismutase i n Phaseolus v u l g a r i s . Can. J . B o t .  60:2686-2691.  McQuaker, N.R., H. Haboosheh, and W. B e s t . 1981. Standards used i n the t r a c e a n a l y s i s o f s e l e c t e d g a s e s . I n t e r n a t i o n a l Lab. Jan./Feb. 1981. Mehler, A.H. 1951. S t u d i e s on r e a c t i o n s o f i l l u m i n a t e d c h l o r o p l a s t s . I . Mechanism o f the r e d u c t i o n o f oxygen and o t h e r H i l l r e a g e n t s . A r c h . Biochem. B i o p h y s . 33:65-77* M i c h e l s o n , A.M., and M.E. Buckingham. 1974* E f f e c t s o f superoxide r a d i c a l s on myoblast growth and d i f f e r e n t i a t i o n . Biochem. B i o p h y s . Res. Commun. 58:1079-1086. Mudd, J.B., R. L e a v i t t , A. Ongun, and T.T. McManus. o f ozone w i t h amino a c i d s and p r o t e i n .  19^9-  Atmos. E n v i r o n .  Reaction 3:669-682.  Mudd, J.B., T.T. McManus, A. Ongun, and T.E. McCullogh. 1971 * I n h i b i t i o n o f g l y c o l i p i d b i o s y n t h e s i s i n c h l o r o p l a s t s by ozone and s u l f h y d r y l r e a g e n t s . P l a n t P h y s i o l . 48:335-339* Mudd, J.B. 1973* B i o c h e m i c a l e f f e c t s o f some a i r p o l l u t a n t s on plants, pp. 31-47 I n A i r P o l l u t i o n Damage t o P l a n t s . Adv. Chem. S e r . V o l . 122. _ J.A. Naegele (Ed.) ;Am. Chem. S o c , Washington D.C. Mudd, J.B., F . Leh, and T.T. McManus. 1974* R e a c t i o n o f ozone w i t h n i c o t i n a m i d e and i t s d e r i v a t i v e s . A r c h . Biochem. B i o p h y s . 161s  408-419.  Mudd, J.B. 1982. E f f e c t s o f o x i d a n t s on m e t a b o l i c f u n c t i o n , pp.189203 I n E f f e c t s o f Gaseous A i r P o l l u t i o n i n A g r i c u l t u r e and H o r t i c u l t u r e . M.H. Unsworth and D.P. Ormrod (Eds.) Butterworth S c i e n t i f i c , Toronto.  80  Nelson, N., H. Nelson, and E. Racker. 1972. Photoreaction of FMNT r i c i n e and i t s p a r t i c i p a t i o n i n photophosphorylation. Photochem. Photobiol. 16:481-489. Olszyk, D.M., and T.W. T i b b i t t s . 1982. Evaluation of injury to expanding leaves of peas exposed to s u l f u r dioxide and ozone. J . Amer. Soc. Hort. 107:266-271. Pauls, K.P., and J.E. Thompson. 1980. In v i t r o simulation of senescence-related membrane damage by ozone-induced l i p i d peroxidation. Nature. 283:504-506. Pauls, K.P., and J.E. Thompson. 1981. Effects of i n v i t r o treatment with ozone on the physical and chemical properties of membranes. P h y s i o l . Plant. 53:255-262. Perchorowicz, J.T., and I.P. Ting. 1974. Ozone effects on plant c e l l permeability. Am. J . Bo't. 61 :787-793. Pryor, W.A. 1976. The r o l e of free r a d i c a l reactions i n b i o l o g i c a l systems, pp. 1-49 In Free Radicals i n Biology. Vol 1. W.A. Pryor (Ed.) Academic Press, New York. Puget, K., and A.M. Michelson. 1974* Isolation of a new coppercontaining superoxide dismutase: bacteriocuprein. Biochem. Biophys. Res. Commun. 58:830-838. Rabinowitch, H.D., and D. Sklan. 1980. A possible protective agent against sunscald i n tomatoes (Lycopersicon esculentum M i l l . ) . Planta 148:162-167. Rabinowitch, H.D., and D. Sklan. 1981. Superoxide dismutase a c t i v i t y i n ripening cucumber and pepper f r u i t . Physiol. Plant. 52:380-  384.  Radmer, R.J., and 0. O l l i n g e r . 1980. Light-driven uptake of oxygen, carbon dioxide, and bicarbonate by the green algae Scenedesmus. Plant P h y s i o l . 65:723-729. Rich, S., and H. Tomlinson. 1974* Mechanisms of ozone injury to plants, pp. 76-82 In A i r P o l l u t i o n Effects on Plant Growth. M. Dugger (Ed.) Am. Chem. S o c , Washington D.C. Richardson, J.S., K.A. Thomas, and D.C. Richardson. 1975* Alphacarbon coordinates f o r bovine CuZn superoxide dismutase. Biochem. Biophys. Res. Commun. 63:986-992. Rosen, P. 1979. Plant response to low ozone treatments. University of B r i t i s h Columbia.  Ph.D. Thesis.  81  Runeckles, V.C. 1974* Dosage of a i r pollutants and damage to vegetation. Environ. Conserv. 1:305-308. Runeckles, V . C , and H.M. Resh. 1974* The assessment of chronic ozone injury to leaves by reflectance spectrophotometry. Atmos. Environ. 9:447-452. Runeckles, V . C , and P. Rosen. 1974* Effects of pretreatment with low ozone concentrations on ozone Injury to bean and mint. Can.  J . Bot.  52:2607-2610.  Runeckles, V . C , and P. Rosen. 1977* Effects of ambient ozone pretreatment on transpiration and s u s c e p t i b i l i t y to ozone i n j u r y .  Can. J . Bot.  55:193-197.  Steinman, H.M., V.R. Naik, J.L, Abernathy, and R.L. H i l l . 1974. Bovine erythrocyte superoxide dismutase: complete amino acid sequence. J . B i o l . Chem. 249:7326-7338. Tanaka, K., and K. Sugahara. 1980. Role of superoxide dismutase i n the defence against SO2 t o x i c i t y and induction of superoxide d i s mutase with SO2 fumigation. Res. Rep. N a t l . Inst. Env. Stud. 11:  155-164.  Ting, I.P., J . Perchorowicz, and L. Evans. 1974. E f f e c t of ozone on plant c e l l permeability, pp. 8-21 In A i r P o l l u t i o n Effects on Plant Growth. M. Dugger (Ed.) Am. Chem. S o c , Washington D.C Tingey, D.T. 1974. Ozone-induced a l t e r a t i o n s i n the metabolite pools and enzyme a c t i v i t i e s of plants, pp. 40-57 In. A i r P o l l u t i o n Effects on Plant Growth. M. Dugger (Ed.) Am. Chem. S o c , Washinton D.C. Tomlinson, H., and S. Rich. 1970. L i p i d peroxidation as a r e s u l t of injury i n bean leaves exposed to ozone. Phytopath. 60:1531-1532. Weiss, J . 1935. Investigation on the r a d i c a l HO2 i n solution. Faraday Soc. 31:668-681.  Trans.  Wilton, A.C., J . J . Murray, H.E. Heggestad, and F.V. Juska. 1972. T o l erance and s u s c e p t i b i l i t y of Kentucky bluegrass (Poa pratensis L.) c u l t i v a r s to a i r p o l l u t i o n , i n the f i e l d and i n an ozone chamber. J . Environ. Qual. 1:112-114. Zahn, R. 1970. The e f f e c t on plants of a combination of sub-acute and toxic s u l f u r dioxide doses. Staub. Reinhalt. Luft. 30:20-23.  

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