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Effects of treatment with low ozone concentrations on stomatal behavior, growth, and susceptibility to… Rosen, Peter Mark 1979

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EFFECTS OF TREATMENT WITH LOW OZONE CONCENTRATIONS ON STOMATAL BEHAVIOR, GROWTH, AND SUSCEPTIBILITY TO ACUTE OZONE INJURY by Pe te r Mark Rosen B . A . , U n i v e r s i t y o f C h i c a g o , 1970 M . S c . , Utah S t a t e U n i v e r s i t y , 1972 T h e s i s Submit ted In P a r t i a l F u l f i l l m e n t Of The Requirements For The Degree of Doctor o f P h i l o s o p h y i n the Department o f P l a n t S c i e n c e i n THE PACQMT OF GRADUATE ST UDIES . We accept t h i s t h e s i s as conforming to the r e q u i r e d s t a n d a r d The U n i v e r s i t y o f B r i t i s h Columbia O^Wrr)., 197 9 © Petes' Mark Rosen, f 979 In presenting this thesis in partial fulfilment of the requirements fo an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. n ,. P l a n t Science Department or ^ _ _ _ _ _ The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 October 31, 1979 i A b s t r a c t Sub-acu te ozone exposures (doses below the t h r e s h o l d f o r v i s i b l e i n j u r y ) have been found to a f f e c t p l a n t growth, s tomata l b e h a v i o r , and the extent o f v i s i b l e i n j u r y i n response to sub-sequent acute exposures (doses above the t h r e s h o l d f o r v i s i b l e i n j u r y ) . The i n t e r r e l a t i o n s h i p s o f these e f f e c t s v a r i e d a c c o r d i n g to the magnitude and t i m i n g of the p re t rea tment dose and the p a r t i c u l a r s p e c i e s and c u l t i v a r t e s t e d . When s e e d l i n g s of bush bean (Phaseolus v u l g a r i s L . cv Pure Go ld Wax) were r a i s e d from seed i n f i l t e r e d a i r and then p're-t r e a t e d w i th 0.02 p a r t s per m i l l i o n (ppm) ozone f o r s i x days ( s i x hours per d a y ) , b e g i n n i n g at e i g h t days a f t e r sowing , s u s c e p t i b i l i t y to acute i n j u r y (the extent o f v i s i b l e i n j u r y a f t e r an acute exposure) was g r e a t e r than tha t o f c o n t r o l s m a i n t a i n e d throughout i n f i l t e r e d a i r . S e e d l i n g s exposed to t h i s low ozone t reatment f o r an a d d i t i o n a l seven days became l e s s s u s c e p t i b l e to acute i n j u r y than c o n t r o l s ma in ta ined i n f i l t e r e d a i r . Treatment d i f f e r e n c e s i n acute s u s c e p t i b i l i t y were found to c o r r e s p o n d to d i f f e r e n c e s i n s tomata l b e h a v i o r . A t the e a r l y s tage o f i n c r e a s e d s u s c e p t i b i l i t y , ozone-p 're-t r e a t e d p l a n t s and c o n t r o l s m a i n t a i n e d i n f i l t e r e d a i r had the same s tomata l r e s i s t a n c e when measured i n f i l t e r e d a i r , but o z o n e - p r e t r e a t e d stomata had a reduced tendency, to c l o s e i n response to an acute ozone exposure (0.4 ppm). At the l a t e r s tage of decreased acute s u s c e p t i b i l i t y , o z o n e - p r e t r e a t e d i i stomata s t i l l had t h i s reduced s e n s i t i v i t y to o z o n e - i n d u c e d c l o s u r e , but ozone p re t rea tment a l s o r e s u l t e d i n an o v e r a l l i n c r e a s e i n s tomata l r e s i s t a n c e i n f i l t e r e d a i r , p r i o r to acute e x p o s u r e s . S tomata l en t ry o f ozone may thus p r o v i d e , i n p a r t , an e x p l a n a t i o n f o r the d i f f e r e n c e s i n s u s c e p t i b i l i t y r e s u l t i n g from these t reatments i n b e a n . Exposure of mint c u t t i n g s (Mentha a r v e n s i s L . ) to 0.02 ppm ozone i n c r e a s e d t h e i r acute s u s c e p t i b i l i t y s l i g h t l y . The ozone t o l e r a n c e of mint was approx imate ly s i x t imes g r e a t e r than tha t of bean s e e d l i n g s r a i s e d i n f i l t e r e d a i r (based on the dosage r e q u i r e d f o r e q u i v a l e n t degrees o f v i s i b l e i n j u r y ) . In exper iments w i th p o t t e d g rapev ines ( V i t i s l a b r u s c a n a , B a i l e y ) i n open- top f i e l d chambers exposed to ambient a i r ( c o n t a i n i n g " n a t u r a l l y o c u r r i n g " ozone) or c h a r c o a l - f i l t e r e d a i r ( c o n t a i n i n g reduced ozone l e v e l s ) f o r an e n t i r e growing s e a s o n , growth was found to be g r e a t e r i n ambient a i r i n two o f th ree c u l t i v a r s t e s t e d . T h i s growth s t i m u l a t i o n was g r e a t e s t i n l e a v e s , but o c c u r r e d a l s o i n t runks and roo ts (both dry weight and f r e s h w e i g h t ) . W i t h i n the th ree c u l t i v a r s ( ' I v e s ' , ' C o n c o r d ' , and 'De laware ' ) the growth s t i m u l a t i o n was d i r e c t l y r e l a t e d to the s e n s i t i v i t y o f f i e l d - g r o w n v i n e s to ozone i n j u r y ; the most s e n s i t i v e c u l t i v a r , ' I v e s ' , showed the g r e a t e s t growth i n c r e a s e i n ambient a i r as compared to f i l t e r e d a i r . Only one o f the g rapev ine c u l t i v a r s . showed any s t a t i s t i c a l l y s i g n i f i c a n t e f f e c t o f c h r o n i c exposure to ambient ozone on acute s u s c e p t i b i l i t y . In tha t c a s e , ' D e l a w a r e ' , the c u l t i v a r i i i most r e s i s t a n t to ozone i n j u r y i n the f i e l d , was p r e d i s p o s e d to acute i n j u r y by c h r o n i c p re t rea tment w i t h low ozone. With g r a p e v i n e s , there were no s t a t i s t i c a l l y s i g n i f i c a n t e f f e c t s o f low ozone pre t rea tment on s tomata l b e h a v i o r , but t rends i n the data i n d i c a t e d tha t ozone p re t rea tment tended to decrease both s tomata l r e s i s t a n c e when measured i n f i l t e r e d a i r and c l o s u r e i n response to acute ozone exposures (0.75 ppm). In exper iments w i t h g r a p e v i n e s , u n l i k e those w i t h b e a n s , decreased s u s c e p t i b i l i t y appeared to be more c l o s e l y r e l a t e d to an i n c r e a s e i n o v e r a l l v i g o r , a s s o c i a t e d w i th a lower s tomata l r e s i s t a n c e i n f i l t e r e d a i r , r a t h e r than to a s tomata l mechanism r e g u l a t i n g ozone e n t r y i n t o the l e a f . A l t h o u g h there were d i f f e r e n c e s between s p e c i e s i n the r o l e tha t stomata p l a y i n the response to p re t rea tment w i t h s u b - a c u t e ozone d o s e s , there was the s u g g e s t i o n o f a g e n e r a l p a t t e r n o f i n c r e a s e d tendenc ies toward a p r o t e c t i v e e f f e c t o f ozone pre t rea tment i n more o z o n e - s e n s i t i v e s p e c i e s (bean versus mint) or c u l t i v a ' r s ( ' I v e s ' and ' C o n c o r d ' ve rsus ' D e l a w a r e ' ) . i v TABLE OF CONTENTS Abstract. • • • • • • • » . . , . , . ) . , . . i I n t r o d u c t i o n 1 Review of L i t e r a t u r e 5 Dose- In j u r y - B a c k g r o u n d 5 P r e t r e a t m e n t - S u s c e p t i b i l i t y 10 Stomata . 1 6 Growth 31 M a t e r i a l s and Methods 36 Bean and Mint . 3 6 Grapev ines 39 R e s u l t s . . 44 S u s c e p t i b i l i t y 44 Stomata 58 Growth 6 7 D i s c u s s i o n . - 80 Summary. - 98 L i t e r a t u r e C i t e d .102 V LIST OF TABLES 1. Ozone m o n i t o r i n g d a t a , August 1 to S e p t . 9, 1977, f o r open- top chamber exper iment 42 2. Acute i n j u r y s u s c e p t i b i l i t y o f greenhouse r a i s e d bean and mint a f t e r s u b - a c u t e p re t rea tment . .45 3. Acute i n j u r y s u s c e p t i b i l i t y o f p o t t e d grapev ines grown i n ambient a i r or c h a r c o a l -f i l t e r e d a i r . 52 4. S tomata l b e h a v i o r o f p o t t e d g rapev ines grown i n ambient a i r or c h a r c o a l - f i l t e r e d a i r .66 5. Growth of p o t t e d ' I v e s ' v i n e s i n ambient a i r or c h a r c o a l - f i l t e r e d a i r . . . . . . . . 71 6. Growth of p o t t e d • ' C o n c o r d ' V v i n e s i n ambient a i r or c h a r c o a l - f i l t e r e d a i r 72 7. Growth o f p o t t e d 'De laware ' v i n e s i n ambient a i r or c h a r c o a l - f i l t e r e d a i r 73 8. ' D e l a w a r e ' : s i m p l e c o r r e l a t i o n s o f growth , i n j u r y , ' and s t o m a t a l b e h a v i o r , September 75 9. ' I v e s ' : s i m p l e c o r r e l a t i o n s o f growth, i n j u r y , and s tomata l b e h a v i o r , September 76 10. ' C o n c o r d ' : s i m p l e c o r r e l a t i o n s o f growth , i n j u r y , and s t o m a t a l b e h a v i o r , September 78 11. Summary of i n d i c a t i o n s from v i n e - t o - v i n e c o r r e l a t i o n s of growth and ozone s u s c e p t i b i l i t y i n g r a p e v i n e s , 79 v i . LIST OF"FIGURES 1. Mint bed i n greenhouse. . . 37 2. Acute ozone injury to bean. . : . .37 3. Acute fumigation chamber for transpiration measurement 37 4. Open-top chamber i n vineyard. 40 5. Acute ozone fumigation chamber, grapevine experiments 40 6. Acute ozone injury to bean plants pretreated with various sub-acute doses 47 7. Eff e c t of increasing pretreatment dose on acute ozone s u s c e p t i b i l i t y of beans . 49 8. Acute ozone s u s c e p t i b i l i t y of beans afte r growth i n continuous f i l t e r e d a i r or various patterns of daily exposure to 0.0 2 ppm ozone 55 9. Stomatal conductance of beans during sub-acute pretreatment and subsequent acute treatment with ozone . .60} 10. Transpiration rates of attached primary bean leaves during acute ozone exposure, after pretreatment with either f i l t e r e d a i r or 0.02 ppm ozone .62 11. Height of bean plants grown in f i l t e r e d a i r or 0.02 ppm ozone 68 12. Model of ozone pretreatment mechanism. .84 13. Growth of potted 'Ives' vines i n ambient a i r or c h a r c o a l - f i l t e r e d a i r 93 14. Growth of potted 'Concord' vines in ambient a i r or c h a r c o a l - f i l t e r e d a i r 94 v i i Acknowledgements I would l i k e to express my thanks to Dr . V . C . R u n e c k l e s , f o r h i s p a t i e n t and p e r c e p t i v e s u p e r v i s i o n through the course of t h i s s t u d y , and to Dr . P e t e r A . J o l l i f f e , f o r h i s exper t a d v i c e on numerous q u e s t i o n s of exper imenta l method and w r i t t e n p r e s e n t a t i o n . I am e s p e c i a l l y g r a t e f u l to Dr . Wal te r J . Kender , and the e n t i r e s t a f f o f the Department of Pomology and V i t i c u l t u r e , New York S ta te A g r i c u l t u r a l Exper iment S t a t i o n , Geneva, f o r t h e i r suppor t and i n s p i r a t i o n . I would a l s o l i k e to express my a p p r e c i a t i o n to f a m i l y and f r i e n d s , f o r t h e i r q u i e t f a i t h . v i i i L i s t o f A b b r e v i a t i o n s ABA - a b s c i s i c a c i d h - hours 3 kPa - 10 p a s c a l s , 1 p a s c a l = 1 newton per square meter K - s tomata l conductance ; i n v e r s e o f s tomata l r e s i s t a n c e s (see R ) LAN - p e r c e n t l e a f a rea n e c r o t i c min - minutes NAR - net a s s i m i l a t i o n r a t e , change i n p l a n t dry weight per u n i t l e a f a rea PAN - p e r o x y a c e t y l n i t r a t e PAR - p h o t s y n t h e t i c a l l y a c t i v e r a d i a t i o n , -ppm - p a r t s per m i l l i o n , by volume, m i c r o l i t e r s per l i t e r RH - r e l a t i v e h u m i d i t y R-^  - l e a f r e s i s t a n c e to water vapor f l u x R g - s tomata l r e s i s t a n c e to water vapor f l u x SE - s t a n d a r d e r r o r o f the mean sec - seconds wt - weight ^ E - 10" 6 E i n s t e i n s ; 1 E i n s t e i n = 6.02 x 1 0 2 3 t imes the energy of 1 quantum of l i g h t o f a g i v e n wavelength 1 I n t r o d u c t i o n The p o s s i b i l i t y o f a d e l e t e r i o u s e f f e c t ' o f ozone on a g r i c u l t u r a l p r o d u c t i o n was f i r s t noted i n the 1 9 5 0 ' s , on the b a s i s of v i s i b l e symptoms of i n j u r y ( l e a f f l e c k ) observed on grape (R ichards et a l . , 1958) and t o b a c c o . ( H e g g e s t a d and M i d d l e t o n , 1959) . A grea t dea l o f work s i n c e tha t t ime has been concerned w i t h the s p e c i f i c c h a r a c t e r i z a t i o n of t h i s type of i n j u r y , the i n v e s t i g a t i o n o f env i ronmenta l f a c t o r s which l e a d to a g r e a t e r or l e s s e r degree o f s u s c e p t i b i l i t y , and o ther aspec ts o f the s i t u a t i o n which might a i d a g r i c u l t u r a l s c i e n t i s t s i n recommending c u l t u r a l p r a c t i c e s to a l l e v i a t e the p rob lem. In a d d i t i o n to t h i s p o s s i b i l i t y o f p r o d u c t i o n l o s s e s a r i s i n g from exposures r e s u l t i n g i n v i s i b l e i n j u r y , and the consequent l o s s of a c t i v e p h o t o s y n t h e t i c a r e a , more r e c e n t l y , i n v e s t i g a t i o n s i n t o the e f f e c t o f ozone on a g r i c u l t u r e have c o n s i d e r e d the e f f e c t s o f exposure to doses below the t h r e s h o l d f o r v i s i b l e i n j u r y . Whi le t h i s a re a l o g i c a l l y i n c l u d e s a number of s t u d i e s i n t o what have r e c e n t l y been c a l l e d " s u b t l e e f f e c t s , " (Heck et a l . , 1977) i . e . , p h y s i o l o g i c a l and b i o c h e m i c a l e f f e c t s which may occur p r i o r to v i s i b l e i n j u r y , i t s major focus has been the c h r o n i c e f f e c t s , p a r t i c u l a r l y the e f f e c t of l o n g - t e r m exposure to s u b - a c u t e doses on p l a n t growth. By way of d e f i n i t i o n , " a c u t e " here r e f e r s to an ozone 2 exposure tha t r e s u l t s i n the c h a r a c t e r i s t i c symptoms of v i s i b l e i n j u r y i n the s p e c i e s c o n s i d e r e d , u s u a l l y some form of n e c r o t i c f l e c k . " S u b - a c u t e " r e f e r s to any exposure below t h i s t h r e s h o l d , whereas " c h r o n i c " r e f e r s s p e c i f i c a l l y to a l o n g - t e r m s u b - a c u t e exposure tha t r e s u l t s i n another r e c o g n i z a b l e form o f i n j u r y , u s u a l l y c h l o r o s i s , premature s e n e s c e n c e , and growth r e d u c t i o n (see W e i n s t e i n , 1975) . Work i n t h i s t h e s i s i n v o l v e s one p a r t i c u l a r aspec t o f s u b - a c u t e e f f e c t s , the e f f e c t o f s u b - a c u t e exposure on the extent o f i n j u r y i n response to subsequent acute e x p o s u r e . Because acute ozone exposure i n the f i e l d i s l i k e l y to be accompanied by s u b - a c u t e exposure and an i n c r e a s e d background l e v e l o f ozone , knowledge of the i n t e r a c t i o n between these two types of exposure i s n e c e s s a r y to improve our a b i l i t y to e x p l a i n the t o t a l e f f e c t o f f i e l d e x p o s u r e s . P r e v i o u s workers have shown tha t an acute ozone exposure may e i t h e r p r e d i s p o s e a p l a n t to g r e a t e r i n j u r y from a sub-sequent acute exposure or p r o t e c t i t from the i n j u r i o u s e f f e c t o f the second exposure ( e . g . Macdowa l l , 1965) . S h i f t i n g our i n t e r e s t to s u b - a c u t e p re t rea tment exposures r a i s e s the p o s s i b i l i t y o f a more e f f e c t i v e form of a c c l i m a t i o n , based on a p o s s i b l e p r o t e c t i v e e f f e c t , and i n t r o d u c e s the q u e s t i o n of a c c l i m a t i o n w i t h i n an e v o l u t i o n a r y framework, based on the n a t u r a l background l e v e l s o f ozone . The mechanism most o f t e n c o n s i d e r e d as a b a s i s f o r the p r o t e c t i v e i n t e r a c t i o n o f two acute ozone exposures i n v o l v e s 3 the e f f e c t o f the p r i o r exposure on s tomata l r e s i s t a n c e (R-s) • A l though the e x i s t e n c e o f t h i s mechanism has never been demonstrated e x p e r i m e n t a l l y , i t f o l l o w s n a t u r a l l y from the known tendency of ozone to induce s t o m a t a l c l o s u r e (Macdowal l , 1965), and the importance o f stomata i n " r e g u l a t i n g s u s c e p t i b i l i t y to ozone i n s o f a r . a s they c o n t r o l the e n t r y o f the t o x i c gas i n t o the l e a f (Rich et a l . , 1970) . Stomata l c l o s u r e such as occurs i n response to acute p re t rea tment doses may a l s o occur i n response to s u b - a c u t e p re t rea tment d o s e s , thereby r e d u c i n g s u s c e p t i b i l i t y to subsequent acute e x p o s u r e s , b u t , i n the l o n g e r d u r a t i o n o f s u b - a c u t e p r e t r e a t m e n t , the a s s o c i a t e d r e d u c t i o n i n the exchange o f water vapor and carbon d i o x i d e (CC^) c o u l d a l s o be expected to have a s i g n i f i c a n t e f f e c t on p l a n t growth. The c u r r e n t b a t t e r y o f t echn iques used to assess ozone t o l e r a n c e , v i s i b l e i n j u r y i n response to acute exposure and growth r e d u c t i o n s i n response to c h r o n i c e x p o s u r e , w i t h the use of c h a r c o a l - f i l t e r e d a i r as a c o n t r o l t r e a t m e n t , s e r i o u s l y underes t imates the c o m p l e x i t y o f the s i t u a t i o n . The p o s s i b i l i t i e s of a c c l i m a t i o n to background l e v e l s of ozone e i t h e r d u r i n g a s i n g l e ep isode i n the f i e l d or over the e v o l u t i o n a r y h i s t o r y of the s p e c i e s must be c o n s i d e r e d i n o rder to f u r t h e r our u n d e r s t a n d i n g o f p l a n t response to c u r r e n t a n t h r o p o g e n i c emiss ions of ozone p r e c u r s o r s . The f i r s t o b j e c t i v e o f the work d e s c r i b e d i n t h i s t h e s i s was to p r o v i d e i n f o r m a t i o n about the e f f e c t of p re t rea tment 4 w i t h s u b - a c u t e ozone doses on s u s c e p t i b i l i t y to acute ozone i n j u r y , and p a r t i c u l a r l y to i n v e s t i g a t e the p o s s i b i l i t y o f a c c l i m a t i o n to ozone. Whi le any i n t e r a c t i o n between s u b - a c u t e and acute exposure w i l l a f f e c t the i n t e r p r e t a t i o n of p l a n t responses to ozone observed i n the f i e l d , and the p r e d i c t i o n of d o s e - i n j u r y r e l a t i o n s h i p s based on c o n t r o l l e d e x p o s u r e s , knowledge about the p o s s i b i l i t y o f ozone a c c l i m a t i o n i n p a r t i c u l a r i s v i t a l l y n e c e s s a r y to improve our a b i l i t y to adapt a g r i c u l t u r a l p r a c t i c e s to areas o f ozone s t r e s s . In b r e e d i n g f o r ozone t o l e r a n c e , f o r example, or i n any a g r i c u l t u r a l s i t u a t i o n where ozone exposure may be r e s p o n s i b l e f o r y i e l d r e d u c t i o n s , the o c c u r r e n c e of a c c l i m a t i o n c o u l d d r a s t i c a l l y a f f e c t the e f f i c i e n c y of c u l t u r a l p r a c t i c e s d e s i g n e d to c o u n t e r r a c t tha t s t r e s s . The second o b j e c t i v e o f t h i s s tudy was to i n v e s t i g a t e the r e l a t i o n s h i p o f the s tomata l response to s u b - a c u t e and acute ozone exposure w i t h the observed v i s i b l e i n j u r y r e s p o n s e s , and p a r t i c u l a r l y to examine the r o l e o f avo idance mechanisms, e x c l u s i o n o f ozone and r e d u c t i o n i n the e f f e c t i v e d o s e , i n any p o s s i b l e a c c l i m a t i o n . The immediate p o s s i b i l i t y o f e x p l o i t i n g ozone a c c l i m a t i o n i n a g r i c u l t u r a l s i t u a t i o n s depends to some extent on the r o l e of s tomata l e x c l u s i o n mechanisms, and the r e l a t i o n of p r o t e c t i v e s tomata l c l o s u r e to p l a n t growth. C o n s e q u e n t l y , the t h i r d o b j e c t i v e of t h i s s tudy was to i n v e s t i g a t e the r e l a t i o n s h i p of p r o t e c t i v e s tomata l c l o s u r e to p l a n t growth over an e n t i r e growing s e a s o n . 5 Review of L i t e r a t u r e D o s e - I n j u r y - B a c k g r o u n d An environment where ozone l e v e l s are c o n s i s t e n t l y e l e v a t e d due to an th ropogen ic p r o d u c t i o n of ozone p r e c u r s o r s r e c e i v e s a combina t ion of i n c r e a s e d b a c k g r o u n d , i n c r e a s e d mean, and o c c a s i o n a l peak c o n c e n t r a t i o n s . G iven a p o i n t source o f p o l l u t a n t s , o f known e m i s s i o n s , the d i s t r i b u t i o n of exposure c o n c e n t r a t i o n s can be e s t i m a t e d by lognormal e q u a t i o n s . With s u l f u r d i o x i d e (SC^) , such equat ions can be used to e s t i m a t e the exposure f u n c t i o n ( L a r s e n , 1971) and can be u s e f u l l y a p p l i e d to the d e s i g n o f l o n g - t e r m exper iments which would s i m u l a t e f i e l d e x p o s u r e s . By t a k i n g the f r e q u e n c i e s o f exposure to v a r i o u s c o n c e n t r a t i o n s d e r i v e d from these e q u a t i o n s , and combining them randomly , the exper imenter c o u l d a v o i d any unwarranted assumptions about the exposure p a t t e r n . Because ozone i s a secondary p o l l u t a n t , produced i n the atmosphere i n d i r e c t l y from p r e c u r s o r s , u n l i k e p r imary p o l l u t a n t s such as which are emi t ted d i r e c t l y by p o i n t s o u r c e s , L a r s e n ' s t echn ique i s not a p p l i c a b l e to ozone. Models of f i e l d ozone exposures are dependent on the p o o r l y unders tood p r o c e s s e s o f p r e c u r s o r c o n v e r s i o n , and are most o f t e n c o n s t r u c t e d s o l e l y on the b a s i s o f e m p i r i c a l m o n i t o r i n g data (Johnson et a l . , 1976; E s c h e n r o e d e r , 1977; R e v l e t t , 1978) . A g e n e r a l 6 mathemat ica l d e s c r i p t i o n of the exposure f u n c t i o n i s t h e r e f o r e not p o s s i b l e w i t h ozone , and the b e s t tha t c o u l d be done a l o n g such l i n e s would n e c e s s a r i l y be l o c a l i t y s p e c i f i c . .In exper iments w i t h S 0 ? , exposure to the mean v a l u e s d e r i v e d from the combina t ion of peaks p r e d i c t e d by L a r s e n ' s techn ique was shown not to r e s u l t i n i n j u r y e q u i v a l e n t to tha t caused by exposure to randomized combinat ions o f peaks w i th the same o v e r a l l mean (Male , L . , p e r s o n a l communica t ion ) . T h i s n o n - l i n e a r i t y between dose and i n j u r y has been shown w i th ozone a l s o , though i t has never been demonstrated i n the same manner. U s i n g exposure t imes o f up to f o u r hours (h ) , and c o n c e n t r a t i o n s of ozone up to 0.6 p a r t s per m i l l i o n (ppm), w i th p i n t o bean and t o b a c c o , Heck, Dunning , and Hindawi (1966) showed tha t a " d e f i n i t e t h r e s h o l d c o n c e n t r a t i o n and p r e s e n t a t i o n time are r e q u i r e d b e f o r e i n j u r y i s i n i t i a t e d . " For both s p e c i e s , the t h r e e - d i m e n s i o n a l response s u r f a c e of acute i n j u r y x c o n c e n t r a t i o n x t ime was complex, but the g e n e r a l t r e n d was f o r l o n g e r exposures at lower c o n c e n t r a t i o n s to r e s u l t i n l e s s i n j u r y than e q u i v a l e n t doses p r e s e n t e d as h i g h e r c o n c e n t r a t i o n s f o r s h o r t e r p e r i o d s of t ime . Q u a l i t a t i v e d i f f e r e n c e s i n the i n j u r y response a t d i f f e r e n t c o n c e n t r a t i o n s or exposure t i m e s , w i t h i n a s h o r t - t e r m r a n g e , may be a t t r i b u t e d to a v a r i e t y o f p h y s i o l o g i c a l t h r e s h o l d e f f e c t s , but there i s as y e t no t h e o r e t i c a l framework f o r s p e c u l a t i o n i n t h i s a r e a . In the con tex t o f f i e l d - t y p e exposures of longer d u r a t i o n , however, at l e a s t one major 7 q u a l i t a t i v e d i s t i n c t i o n has been r e c o g n i z e d , the d i s t i n c t i o n between acute and c h r o n i c r e s p o n s e s . The Amer ican P h y t o p a t h o l o g i c a l S o c i e t y g l o s s a r y (see W e i n s t e i n , 1975) d e f i n e s two types o f a i r p o l l u t i o n i n j u r y as f o l l o w s : • "Acute i n j u r y - - i n j u r y u s u a l l y i n v o l v i n g n e c r o s i s which deve lops w i t h i n s e v e r a l hours to a few days a f t e r s h o r t -term exposure to a p o l l u t a n t , and e x p r e s s e d as f l e c k , s c o r c h , or b i f a c i a l n e c r o s i s . C h r o n i c i n j u r y - - i n j u r y which deve lops o n l y a f t e r l o n g -term or r e p e a t e d exposure to an a i r p o l l u t a n t , and expressed as c h l o r o s i s , b r o n z i n g , premature s e n e s c e n c e , reduced growth r a t e ; can i n c l u d e n e c r o s i s . " By c i r c u l a r d e f i n i t i o n , these two types o f i n j u r y can be l o o s e l y i d e n t i f i e d w i th two types o f e x p o s u r e , acute exposure and c h r o n i c exposure ; whereby an acute exposure (or acute dose) i s one which leads to acute i n j u r y and a c h r o n i c exposure (or c h r o n i c dose) i s one which leads to c h r o n i c i n j u r y . T h i s s imp le i d e n t i f i c a t i o n masks a number o f a m b i g u i t i e s , however, such as the d e s c r i p t i o n of exposures which l e a d to i n j u r i o u s p h y s i o l o g i c a l changes which are not m a n i f e s t as v i s i b l e symptoms. The terms " h i d d e n i n j u r y " or " i n v i s i b l e i n j u r y " were i n t r o d u c e d e a r l y i n the 19th c e n t u r y (see McCune et a l . , 1967 f o r a h i s t o r y o f t h i s u s a g e ) , but the re i s a g r e a t d e a l o f c o n t r o v e r s y about t h e i r usage (as i t i s i n h e r e n t l y a p p e a l i n g to " c o n s e r v a t i o n i s t s " and i n h e r e n t l y u n a p p e a l i n g to " i n d u s -t r i a l i s t s " ) . McCune et a l . (1967) suggested tha t the terms be d i s c a r d e d as a redundant f o r m u l a t i o n f o r c h r o n i c i n j u r y . 8 Heck et a l . (1977) have p roposed the term " s u b t l e e f f e c t s " to d e s c r i b e b i o c h e m i c a l and p h y s i o l o g i c a l changes induced by p o l l u t a n t exposure tha t are not m a n i f e s t as v i s i b l e symptoms. The c o n s i d e r a t i o n , o f l o n g - t e r m exposures to low l e v e l s of p o l l u t a n t s i n t r o d u c e s the q u e s t i o n o f d e t e r m i n i n g the normal background c o n c e n t r a t i o n s to which p l a n t s are. exposed , and r a i s e s the problem o f p a r t i t i o n i n g these background l e v e l s i n t o a p o r t i o n r e s u l t i n g f r o m ' n a t u r a l causes and a p o r t i o n r e s u l t i n g from a n t h r o p o g e n i c s o u r c e s . B e l l and Clough (1973) , f o r example, t e n t a t i v e l y d e s i g n a t e d a y i e l d r e d u c t i o n i n r y e -g r a s s , i n response to c h r o n i c exposure to S C ^ , as i n v i s i b l e i n j u r y , a l though conced ing tha t the p l a n t s were s l i g h t l y c h l o r o t i c as compared to p l a n t s grown i n f i l t e r e d a i r . They contended tha t t h i s s l i g h t c h l o r o s i s would not be e v i d e n t i n the f i e l d because any crop of r y e g r a s s grown outdoors i n Eng land w o u l d ' m a n i f e s t the same symptoms. For ozone , the absence o f h i s t o r i c a l da ta on a tmospher ic c o n c e n t r a t i o n s make t h i s problem i n a c c e s s i b l e to d i r e c t s o l u t i o n . The r e c e n t d i s c o v e r y o f r e l a t i v e l y h i g h ozone l e v e l s i n r u r a l a r e a s , f a r removed from the t y p i c a l urban sources of ozone p r e c u r s o r s , i n cases where the long d i s t a n c e t r a n s p o r t o f ozone or ozone p r e c u r s o r s does not appear p r o b a b l e ( e . g . S t a s i u k and C o f f e y , 1974) , has c o m p l i c a t e d the i s s u e s t i l l f u r t h e r . Chambers (1976). argues tha t s i g n i f i c a n t accumula t ions of ozone i n the atmosphere would not have o c c u r r e d p r i o r to the w idespread use o f pe t ro leum f u e l s i n 9 the 20th c e n t u r y . S t a s i u k and C o f f e y (1974) , however , c a l c u l a t e d that c u r r e n t h i g h ozone l e v e l s i n r u r a l areas c o u l d not be accounted f o r by t r a n s p o r t o f p r e c u r s o r s from urban a r e a s , and sugges ted tha t t r a n s p o r t from the s t r a t o s p h e r e down to the t roposphere might be the c a u s e . By e x t e n s i o n , t h i s same mechanisms) c o u l d have accounted f o r h i g h background l e v e l s o f ozone throughout the e v o l u t i o n a r y h i s t o r y of c u r r e n t p l a n t s p e c i e s . I n t e n s i v e r e c e n t s tudy of t h i s p o s s i b i l i t y , and o f the long range t r a n s p o r t of ozone and ozone p r e c u r s o r s , has not c l a r i f i e d these q u e s t i o n s (see Ludwig et a l . , 1977; C o f f e y et a l . , 1977; Chang and We ins tock , 1977; D i m i t r i a d e s and A l t s h u l l e r , 1978) . These workers c o n s i d e r the p o s s i b i l i t y tha t h i g h r u r a l l e v e l s may be the r e s u l t o f m u l t i p l e c a u s e s , i n c l u d i n g , i n a d d i t i o n to t r a n s p o r t down from the s t r a t o s p h e r e , l o c a l s o u r c e s , long range t r a n s p o r t , and the d e s t r u c t i o n o f ozone by n i t r i c ox ide i n the urban env i ronment . Bennet t et a l . , (1974) argue tha t n a t u r a l sources o f ozone may account f o r s i g n i f i c a n t e v o l u t i o n a r y a d a p t a t i o n to ozone i n p l a n t s , on the b a s i s of p l a n t growth s t i m u l a t i o n s observed i n response to low ozone l e v e l s added to f i l t e r e d a i r . The i n t e r p r e t a t i o n o f exper iments i n v o l v i n g c h r o n i c exposure to ozone must take i n t o account t h i s p o s s i b i l i t y tha t f i l t e r e d a i r t reatments do not r e p r e s e n t v a l i d c o n t r o l s . In s t u d y i n g the i n t e r a c t i o n between s u b - a c u t e and acute e x p o s u r e s , i t must a l s o be determined how pre t rea tment w i t h f i l t e r e d a i r p r i o r 10 to acute exposure compares to p re t rea tment w i t h low l e v e l s of ozone. P r e t r e a t m e n t - S u s c e p t i b i l i t y The a l t e r a t i o n of p l a n t response to ozone by p r e v i o u s ozone exposure can m a n i f e s t i t s e l f e i t h e r as an i n c r e a s e i n s u s c e p t i b i l i t y t o ^ i n j u r y ( p r e d i s p o s i t i o n ) or a r e d u c t i o n i n s u s c e p t i b i l i t y to i n j u r y ( p r o t e c t i o n ) . V i s i b l e i n j u r y i s o n l y one form of i n j u r y e x p r e s s i o n , and i t i s not unreasonab le to suppose tha t exposure to doses below the t h r e s h o l d f o r v i s i b l e i n j u r y w i l l n o n e t h e l e s s i n j u r e the p l a n t i n some way which may combine .w i th i n j u r y from subsequent exposures which are above that t h r e s h o l d . T h i s phenomenon w i l l then appear as p r e d i s p o s i t i o n , or synerg ism between the two d o s e s . P o s s i b l e mechanisms o f p r o t e c t i o n by p r i o r exposures are l e s s s t r a i g h t f o r w a r d , but such phenomena are known to o c c u r i n response to a wide v a r i e t y o f o t h e r p l a n t s t r e s s e s as a r e s u l t o f a c c l i m a t i o n . L e v i t t (1972) ment ions c o l d , h e a t , d r o u g h t , low l i g h t , u l t r a v i o l e t l i g h t , sodium c h l o r i d e , heavy m e t a l s , o ther t o x i c ions (copper , s e l e n i u m , manganese, z i n c , chromium, b o r a t e ) , and h i g h p r e s s u r e , i n t h i s c a t e g o r y . P r e v i o u s work w i t h ozone p r e t r e a t m e n t s has i n d i c a t e d t h a t , under d i f f e r i n g c o n d i t i o n s , e i t h e r o f these phenomena, p r e d i s p o s i t i o n or p r o t e c t i o n , may o c c u r . Macdowal l (1965) i n c l u d e d ozone i t s e l f among the c h e m i c a l f a c t o r s he s t u d i e d w i t h r e s p e c t to the p r e d i s p o s i t i o n o f 11 tobacco to ozone i n j u r y , and c o n c l u d e d tha t "a low dose of p o l l u t i n g ozone o f t e n appeared to act s y n e r g i s t i c a l l y w i t h a second c o n s e c u t i v e low d o s e , " whereas " h i g h doses o f p o l l u t a n t a c t e d a n t a g o n i s t i c a l l y a g a i n s t h i g h doses t h a t immedia te ly f o l l o w e d . " These c o n c l u s i o n s were based on f l e c k i n d i c e s i n f i e l d p l o t s a f t e r exposure to ambient ep isodes on a s e r i e s o f d a y s . By M a c d o w a l l ' s t e r m i n o l o g y , r o u g h l y c a l c u l a t e d on the b a s i s of a c o n s t a n t c o n c e n t r a t i o n d u r i n g seven h of exposure per day , the "low" dose of 0.26 ppm-h y i e l d s an average c o n c e n t r a t i o n of about 0.04 ppm, w h i l e the " h i g h " dose o f 0.54 ppm-h works out to a seven h mean of a p p r o x i m a t e l y 0.08 ppm. Macdowal l goes on to say that " i n h i b i t i o n of s t o m a t a l opening may have been a c o n t r i b u t i n g f a c t o r " (to the antagonism) based on the s o l v e n t i n f i l t r a t i o n method o f d e t e r m i n i n g s tomata l o p e n i n g . The on ly ev idence p r e s e n t e d f o r t h i s c o n c l u s i o n , however, was the o b s e r v a t i o n o f c l o s e d stomata on some newly f l e c k e d tobacco l e a v e s . U s i n g a s i m i l a r system of watch ing symptom development on tobacco exposed to ambient ozone , i n an a rea where ambient l e v e l s were i n the range of 0.03 to 0.10 ppm, Heagle and Heck (1974) observed the same phenomenon of s y n e r g i s t i c i n t e r a c t i o n between c o n s e c u t i v e low d o s e s . They h e l d some p l a n t s i n f i l t e r e d a i r w h i l e expos ing o thers c o n t i n u o u s l y to u n f i l t e r e d a i r , and found tha t new symptom development was g r e a t e r on p l a n t s which had been c o n t i n u o u s l y exposed to u n f i l t e r e d a i r than on p l a n t s which were s w i t c h e d from f i l t e r e d 12 a i r to u n f i l t e r e d a i r . T h i s sugges ted tha t one a s p e c t o f c h r o n i c i n j u r y which was not e x p r e s s e d i n v i s i b l e symptoms was a p r e d i s p o s i t i o n , a case o f h idden or i n v i s i b l e i n j u r y which combined w i t h and a m p l i f i e d v i s i b l e symptoms i n response to l a t e r e x p o s u r e s . Heck and Dunning (1967) , i n l a b o r a t o r y e x p e r i m e n t s , found the phenomenon of an tagon ism, whereby h i g h doses may p r o t e c t p l a n t s from i n j u r y i n response to subsequent h i g h d o s e s . Working w i t h tobacco and p i n t o b e a n , they compared i n j u r y from two, 30 minute (min) f u m i g a t i o n s w i t h 0.30 ppm ozone s e p a r a t e d by one, two, or th ree h , w i th the e f f e c t o f a s i n g l e 60 min f u m i g a t i o n . They d i d not f i n d any s i g n i f i c a n t d i f f e r e n c e s between the v a r i o u s lengths o£ r e c o v e r y t ime between e x p o s u r e s , b u t , i n a l l c a s e s , i n j u r y from a 60 min exposure was g r e a t e r than i n j u r y from two 30 min e x p o s u r e s . In a d d i t i o n to the p o s s i b i l i t y o f s tomata l c l o s u r e i n response to the f i r s t e x p o s u r e , Heck and Dunning a l s o sugges ted the p o s s i b i l i t y o f p a r t i a l r e c o v e r y d u r i n g the i n t e r f u m i g a t i o n i n t e r v a l to e x p l a i n these r e s u l t s . Heck and Dunning a l s o exposed tobacco to i n t e r m i t t e n t d a i l y doses o f ozone , two exposures per day , r a n g i n g from one h a l f to f o u r h , o f c o n c e n t r a t i o n s r a n g i n g from 0.02 to 0.10 ppm, i . e . , t o t a l d a i l y doses r a n g i n g from 0.0 5 to 0.20 ppm-h. They c o n t i n u e d t h i s f o r f i v e d a y s , and assayed i n j u r y each day. For t reatments w i t h e q u i v a l e n t d a i l y d o s e s , i n d i f f e r e n t p a t t e r n s , e . g . , two exposures per day of one h a l f h 13 each to 0.10 ppm versus one exposure per day o f one h to to 0.0 5 ppm, they found tha t the s h o r t e r exposures to h i g h e r c o n c e n t r a t i o n s l e d to g r e a t e r i n j u r y . The e x p l a n a t i o n f o r t h i s may l i e e i t h e r i n the b a l a n c e between a n t a g o n i s t i c and s y n e r g i s t i c c o m b i n a t i o n , depending on c o n c e n t r a t i o n , or i t may be another e x p r e s s i o n of the t h r e s h o l d f u n c t i o n noted by Heck, Dunning , and Hindawi (1966) . Due to the l e n g t h o f t ime r e q u i r e d f o r p e n e t r a t i o n of ozone through the stomata and i n t o the i n j u r y - s e n s i t i v e p a l i s a d e t i s s u e s , these d i f f e r e n c e s due to exposure d u r a t i o n may a l s o r e f l e c t d i f f e r e n c e s i n e f f e c -t i v e d o s e , i . e . , the d u r a t i o n o f a p a r t i c u l a r i n t e r n a l c o n c e n t r a t i o n - o f ozone^see R u n e c k l e s , 1974). W i l t o n et a l . (19 72) , work ing w i t h v a r i o u s c u l t i v a r s of b l u e g r a s s a l s o found p r o t e c t i o n from subsequent acute exposures by p r i o r o n e s . They fumigated w i t h 0.30 ppm ozone f o r two or f o u r h , assayed i n j u r y , c l i p p e d t h e . g r a s s to a l e n g t h of 2.5 c e n t i m e t e r s (cm), a l l o w e d regrowth to 20 cm i n the greenhouse , and repea ted the f u m i g a t i o n . They found tha t there was l e s s i n j u r y i n response to the second f u m i g a t i o n than there had been i n response to the f i r s t . T h i s p r o t o c o l goes a long way toward r u l i n g out s tomata l mechanisms (which would in- t h i s case be at l e a s t i n d i r e c t ) . T h e i r r e s u l t s a l s o i n d i c a t e d that the extent o f the p r o t e c t i o n rendered by the f i r s t exposure was c o r r e l a t e d p o s i t i v e l y w i t h the ozone s e n s i t i v i t y o f a p a r t i c u l a r c u l t i v a r . None of these p r e v i o u s exper iments on the e f f e c t o f ozone 14 p re t rea tment on acute ozone s u s c e p t i b i l i t y examined the e f f e c t o f a s u b - a c u t e p re t rea tment dose . The i n t e r a c t i o n o f s u b - a c u t e exposure w i th subsequent acute exposure has been s t u d i e d , however, w i th S C ^ . Zahn (1963) showed a p r o t e c t i v e e f f e c t of s u b - a c u t e SC^ pre t rea tment i n wheat and l a r c h , and s u g g e s t e d , on the b a s i s o f measurements o f s u l f u r c o n t e n t , tha t the mechanism was not s t o m a t a l . The s u l f u r conten t a f t e r acute exposure was e q u i v a l e n t i n t o l e r a n t , p r e t r e a t e d p l a n t s and i n s u s c e p t i b l e , u n p r e t r e a t e d p l a n t s . P i e r r e (1977) a l s o showed a p r o t e c t i v e e f f e c t o f s u b - a c u t e SC>2 p r e t r e a t m e n t , i n P e l a r g o n i u m , w i t h data based not on v i s i b l e i n j u r y symptoms but on enzyme a c t i v i t i e s . Ke assayed f o r g lutamate dehydro-genase , a s p a r t a t e a m i n o t r a n s f e r a s e , p h o s p h o f r u c t o k i n a s e , g l u -c o s e - 6 -phosphate dehydrogenase , and m a l i c enzyme, a l l o f which were d e c r e a s e d by acute SO^ ^t reatment . P re t rea tment w i t h low l e v e l s o f SO^, he f o u n d , negated t h i s e f f e c t . Horsman and We l lburn (1977) a l s o i n v e s t i g a t e d the e f f e c t o f acute SC^ fumi -g a t i o n on the a c t i v i t y o f r i b u l o s e d iphosphate c a r b o x y l a s e , g lutamate o x a l o a c e t a t e t r a n s a m i n a s e , and p e r o x i d a s e , i n dock (Rumex o b t u s i f o l i u s ) , c o l l e c t e d from an area o f h i g h background SC^ and from an area o f low background S C ^ . They found tha t i n response to acute f u m i g a t i o n w i t h S C ^ , r i b u l o s e d iphosphate c a r b o x y l a s e a c t i v i t y was decreased and p e r o x i d a s e a c t i v i t y was i n c r e a s e d i n p l a n t s from low background a r e a s , w h i l e the re was no e f f e c t i n p l a n t s from h i g h background a r e a s . G e n e r a l l y , i n the exper iments o f P i e r r e , and those of 15 Horsman and Wellburn, the e f f e c t s of treatment with low l e v e l s of SC>2 were not q u a l i t a t i v e l y d i f f e r e n t from the e f f e c t s of acute fumigation, although they o c c u r r e d to a l e s s e r extent. The e f f e c t s o f S0 ? pretreatment on the response to acute fumigation were a l s o c o n f i n e d to l e v e l s o f enzyme'activity r a t h e r than to the spectrum of enzymes a f f e c t e d or the d i r e c t i o n of the e f f e c t . U n l i k e the type of q u a l i t a t i v e d i s t i n c t i o n between acute and c h r o n i c , the e f f e c t of SC>2 exposure i n the experiments of P i e r r e , and those of Horsman and Wellburn, represented more of a continuum between vari o u s exposure l e v e l s . Although there was no.evidence given i n t h i s work about the e f f e c t o f low l e v e l exposure on growth, there was some i m p l i c a t i o n , t h e r e f o r e , that the e f f e c t was e i t h e r i n s i g n i f i c a n t , or a s l i g h t r e d u c t i o n . I f p r o t e c t i o n from acute exposures was a s s o c i a t e d with the absence of any growth e f f e c t , or with a growth s t i m u l a t i o n , two aspects of a c c l i m a t i o n to low p o l l u t a n t l e v e l s c o u l d be s a i d to have been a c t i n g i n co n c e r t . I f a p r o t e c t i v e e f f e c t of. low l e v e l exposure was a s s o c i a t e d with a r e d u c t i o n i n growth, however, the two processes might be s a i d to have been working i n o p p o s i t i o n with r e s p e c t to optimum p l a n t p r o d u c t i v i t y i n a p o l l u t e d environment. In an area where low background l e v e l s were not continuous, however, and where the occurrence of low background l e v e l s was c l o s e l y a s s o c i a t e d with subsequent acute episodes, the e x i s t e n c e of a mechanism that induced a p r o t e c t i v e e f f e c t even at the expense o f some 16 growth r e d u c t i o n might s t i l l be advantageous . P r o t e c t i v e e f f e c t s based on s t o m a t a l c l o s u r e are one example i n which p r o t e c t i o n might be expected to be a s s o c i a t e d w i th a growth r e d u c t i o n . To c l a r i f y the i n t e r a c t i o n between s u b - a c u t e and acute e x p o s u r e s , i t i s n e c e s s a r y to unders tand t h e i r e f f e c t s , bo th i n d e p e n d e n t l y and i n c o m b i n a t i o n , on s tomata l b e h a v i o r . Stomata Study o f the r e l a t i o n s h i p between ozone and p l a n t stomata f a l l s rough ly i n t o two a r e a s . The f i r s t a r e a , which encompasses a m a j o r i t y o f the work done, concerns the e f f e c t o f s tomata l a p e r t u r e d u r i n g ozone exposure on the ex ten t o f i n j u r y to the p l a n t . In t h i s work, an attempt i s made to r e l a t e the e f f e c t o f v a r i a t i o n s i n the env i ronment , e i t h e r d u r i n g ozone exposure or d u r i n g the growth o f the p l a n t p r i o r to exposure , to the ex tent o f i n j u r y , by i n v o k i n g the e f f e c t o f those env i ronmenta l v a r i a t i o n s on s t o m a t a l a p e r t u r e . Exper imenters i n t h i s a rea have c o n c l u d e d e i t h e r ( a ) . t h a t s tomata l changes induced by changing env i ronmenta l c o n d i t i o n s are p r i m a r i l y r e s p o n s i b l e f o r the c o r r e s p o n d i n g changes i n ozone s u s c e p t i b i l i t y o f the i n t e r n a l l e a f t i s s u e , (b) tha t changes i n s u s c e p t i b i l i t y are independent o f changes i n s t o m a t a l a p e r t u r e , or (c) tha t changes i n stomata are a p a r t i a l but not complete e x p l a n a t i o n o f changes i n s u s c e p t i b i l i t y . Most c o n c l u s i o n s have f a l l e n i n t o c a t e g o r y ( c ) , a l though o c c a s i o n a l l y 17 (a) ( e . g . , F l e t c h e r et a l . , 1972) or .(b) ( e . g . , Evans and T i n g 1974a) have a f f o r d e d a b e t t e r e x p l a n a t i o n of e x p e r i m e n t a l r e s u l t s . The second area of s tudy on the r e l a t i o n s h i p between ozone and stomata concerns the e f f e c t of ozone exposure on s tomata l a p e r t u r e i t s e l f , i . e . , d i r e c t e f f e c t s on guard c e l l f u n c t i o n . E x p e r i m e n t a l r e s u l t s i n t h i s area can be c l a s s i f i e d as showing tha t e i t h e r (a) ozone induces s t o m a t a l c l o s u r e , (b) ozone induces s tomata l o p e n i n g , or (c) ozone has no e f f e c t on s tomata . Under d i f f e r i n g env i ronmenta l c o n d i t i o n s , and w i th d i f f e r e n t s p e c i e s , each o f these c a t e g o r i e s of response has been f o u n d . These two areas may be i n t e r r e l a t e d , p a r t i c u l a r l y i n work on ozone p re t rea tment e f f e c t s , because o z o n e - i n d u c e d s tomata l c l o s u r e may a l s o p r o t e c t p l a n t s from ozone i n j u r y . M a n s f i e l d (1973) used a s i m i l a r two-par t d i s t i n c t i o n i n c h a r a c t e r i z i n g work on ozone and p l a n t s t o m a t a , i n h i s r e v i e w , "The r o l e o f stomata i n d e t e r m i n i n g the responses o f p l a n t s to a i r p o l l u t a n t s , " where he b e g i n s , " S t u d i e s of the p a r t p l a y e d by stomata i n d e t e r m i n i n g the s u s c e p t i b i l i t y o f p l a n t s to a i r p o l l u t a n t s can be c o n v e n i e n t l y c o n s i d e r e d under two main h e a d i n g s . F i r s t , the re are i n v e s t i g a t i o n s of the r o l e o f stomata as the p o r t s of en t ry of p o l l u t a n t s , which have u s u a l l y taken i n t o account s tomata l a p e r t u r e s at d i f f e r e n t t imes o f day and under d i f f e r e n t c o n d i t i o n s . S e c o n d l y , the re are s t u d i e s whose main concern has been the r e a c t i o n of the stomata to the p r e s e n c e of a i r p o l l u t a n t s , and the consequent e f f e c t on water r e l a t i o n s and the p h y s i o l o g y of the p l a n t g e n e r a l l y . " Much of the e a r l i e s t work on p l a n t response to ozone 18 r e c o g n i z e d the p o s s i b l e importance o f s t o ma t a , and ga thered i n d i r e c t ev idence f o r t h e i r invo lvement i n d e t e r m i n i n g the ex tent o f i n j u r y . Macdowal l et a l . (1964) used e v a p o t r a n s p i -r a t i o n r a t i o s i n t h e i r equa t ions f o r the p r e d i c t i o n o f i n j u r y . Bobrov (1952) and Juhren et a l . (1957) no ted tha t the banding p a t t e r n of smog i n j u r y to g rasses c o r r e s p o n d e d to s t o m a t a l bands on the l e a f . G e n e r a l l y , however , the r o l e o f stomata has been a c c e p t e d w i thou t r i g o r o u s e x a m i n a t i o n . Heck (1968) i n a rev iew o f the env i ronmenta l f a c t o r s i n f l u e n c i n g ozone i n j u r y , c o n c l u d e d t h a t stomata were both the on ly r o u t e , and o n l y the r o u t e , o f ozone e n t r y i n t o the p l a n t . The second p a r t o f t h i s c o n c l u s i o n i s i n d i s p u t a b l e , w h i l e the f i r s t i s s u p p o r t e d by remarkably l i t t l e e v i d e n c e . Heggestad and M i d d l e t o n (1959) showed tha t c o a t i n g the upper s u r f a c e o f p i n t o bean l e a v e s w i t h l a n o l i n had no e f f e c t on i n j u r y , whereas c o a t i n g both the upper and the lower s u r f a c e s p r e v e n t e d i n j u r y (the stomata o f p i n t o beans are l o c a t e d on the lower s u r f a c e ) . V a l l i (1971) c o a t e d the u n d e r s i d e of tobacco l e a v e s and p r o t e c t e d them c o m p l e t e l y from exposures to up to seven ppm ozone , but t h i s ev idence o f complete p r o t e c t i o n i s u n i q u e , and i n v o l v e s c o a t i n g the e n t i r e e p i d e r m i s , not j u s t the s tomata . I am unaware o f any exper iments u s i n g s i m i l a r mechan ica l means to s e p a r a t e s tomata l pathways from o ther p o s s i b l e pathways of d i f f u s i o n i n t o the l e a f , or any a t a l l to examine the f e a t u r e s o f o t h e r p o s s i b l e pathways. I n d i r e c t ev idence tha t stomata are the o n l y rou te o f 19 ozone e n t r y comes from exper iments where env i ronmenta l v a r i a t i o n a f f o r d s p a r t i a l p r o t e c t i o n , c o r r e s p o n d i n g to p a r t i a l s tomata l c l o s u r e . The o n l y t e s t o f the r e l a t i v e importance of the s tomata l pathway i n such exper iments comes from the na ture o f the c o r r e l a t i o n between a p e r t u r e and i n j u r y , a , t y p e of a n a l y s i s which a l lows f o r a l a r g e degree of u n c e r t a i n t y . Under changing c o n d i t i o n s , the importance o f stomata may not be c o n s t a n t , as i n d i c a t e d by the work of Lee (1965) on the invo lvement o f s tomata l b e h a v i o r and l e a f sugar conten t i n the response o f tobacco to ozone. Lee s a y s , "The r e l a t i v e importance o f s t o m a t a l w id th to l e a f s u s c e p t i b i l i t y i s determined by o ther c o n d i t i o n s o f the l e a f and perhaps a l s o by (the) c o n c e n t r a t i o n o f ozone to which the l e a v e s are exposed . . . l eaves w i t h open stomata i n the i n i t i a l s tage o f ozone t reatment b u i l d up a t o x i c l e v e l o f ozone i n the t i s s u e sooner than those w i th the stomata c l o s e d ; but the c o n c e n t r a t i o n o f ozone i n the l e a f n e c e s s a r y to produce v i s i b l e symptoms depends on o ther c o n d i t i o n s of the l e a f . . . l eaves w i t h open stomata would have more damage than those w i t h c l o s e d s tomata . T h i s might be the case u s u a l l y observed i n the f i e l d . However, s tomata l c l o s u r e cannot p revent damage from ozone when the c o n d i t i o n s w i t h i n p l a n t t i s s u e s are f a v o r a b l e f o r ozone i n j u r y a n d the c o n c e n t r a t i o n o f ozone i s h i g h . " The r e l a t i v e importance of stomata may a l s o be a f f e c t e d by env i ronmenta l v a r i a b l e s which a f f e c t boundary l a y e r r e s i s t a n c e , such as wind v e l o c i t y , and thus the percen tage o f t o t a l l e a f r e s i s t a n c e (R^) which i s c o n t r o l l e d by s t o m a t a l r e s i s t a n c e (R-s) (see T i n g and H e a t h , 1975; Ashenden and M a n s f i e l d , 1977) . The e n t i r e area of env i ronmenta l v a r i a t i o n and i t s e f f e c t 20 on ozone s u s c e p t i b i l i t y was summarized by Treshow (1970) who noted tha t the env i ronmenta l v a r i a b l e s which i n f l u e n c e stomata "appeared to exer t the g r e a t e s t i n f l u e n c e on p l a n t response to p o l l u t a n t s , " and tha t " v i r t u a l l y every m e t e o r o l o g i c a l and edaphic f a c t o r i n the environment i n f l u e n c e s the p r e d i s p o s i t i o n and s e n s i t i v i t y o f p l a n t s to o z o n e . " Heck 's (1968) rev iew l i s t e d some o f the f a c t o r s which were presumed to have an impor tant e f f e c t on p o l l u t a n t r e s p o n s e : l i g h t ( q u a l i t y , d u r a t i o n , and i n t e n s i t y ) , t empera tu re , CC^ c o n c e n t r a t i o n , h u m i d i t y , w i n d , s o i l m o i s t u r e , s o i l a e r a t i o n , n u t r i e n t l e v e l s , and s o i l t e x t u r e . A l l these c o n d i t i o n s were f u r t h e r s u b d i v i d e d a c c o r d i n g to t h e i r e f f e c t s e i t h e r p r i o r to or d u r i n g exposure . Dunning and Heck (1977) examined the e f f e c t o f l i g h t i n t e n s i t y , t empera tu re , and r e l a t i v e h u m i d i t y on the response of bean and tobacco to ozone . They c o n c l u d e d ; "we b e l i e v e tha t the v a r i o u s i n t e r a c t i o n s a s s o c i a t e d w i t h exposure environments are t r i g g e r e d p r i m a r i l y by a change i n s tomata l a p e r t u r e a s s o c i a t e d w i t h changes i n l e a f water p o t e n t i a l , " and that the 'Var ious i n t e r a c t i o n s a s s o c i a t e d w i t h growth environments ( p r i o r to exposure) are t r i g g e r e d p r i m a r i l y by changes i n l i p i d c o m p o s i t i o n and membrane s t r u c t u r e . " T h e i r p a p e r , however, c o n t a i n e d no measurements o f s t o m a t a l a p e r t u r e , l e a f water p o t e n t i a l , l i p i d c o m p o s i t i o n , or membrane s t r u c t u r e . There i s a good dea l o f e v i d e n c e , however , which i n c l u d e s measurements o f s tomata l a p e r t u r e , which shows t h a t 21 env i ronmenta l changes may induce both s tomata l c l o s u r e and p r o t e c t i o n from ozone i n j u r y c o n c u r r e n t l y . F l e t c h e r et a l . (1972) p a i n t e d the pr imary l eaves of bean p l a n t s w i t h ten m i l l i g r a m s per l i t e r (mgli^) a b s c i s i c a c i d (ABA) and exposed them to 0.20 to 0.40 ppm ozone f o r th ree h . Stomata l a p e r t u r e decreased from 10.7 microns (yuJ) i n c o n t r o l s to 4. 2 /A i n A B A - t r e a t e d l e a v e s . I n j u r y , as measured by c h l o r o p h y l l e x t r a c t i o n six days a f t e r e x p o s u r e , was g r e a t e r i n u n t r e a t e d p l a n t s (751 more c h l o r o p h y l l i n A B A - t r e a t e d p l a n t s ) . Ozone exposure induced s tomata l c l o s u r e i n both A B A - t r e a t e d l eaves and u n t r e a t e d l e a v e s , e q u i v a l e n t l y , from 10.7 to 4.9 i n u n t r e a t e d l e a v e s , and from 4.2 to 2 . 9 ^ i n A B A - t r e a t e d l e a v e s . A p a r t from the q u e s t i o n of whether o t h e r p h y s i o l o g i c a l changes induced by ABA i n a d d i t i o n to the s tomata l e f f e c t s were p a r t i a l l y r e s p o n s i b l e f o r ozone p r o t e c t i o n (the authors t e s t e d f o r t h i s w i t h measurements of p r o t e i n and RNA i n a d d i t i o n to c h l o r o p h y l l ) , one c o u l d o b j e c t to such an exper iment on the grounds tha t a c o a t i n g of ten mg 1 ^ ABA d i d not r e p r e s e n t an env i ronmenta l v a r i a t i o n w i t h i n the r e g i o n o f .expected changes . The. exper iment d i d serve to show, however, tha t s tomata l c l o s u r e may reduce ozone i n j u r y . The use o f a s imp le f a c t o r l i k e added ABA (or such t h i n g s as temperature or humid i ty ) as an independent v a r i a b l e i s more l i k e l y to r e v e a l a c l o s e r e l a t i o n between c l o s u r e and p r o t e c t i o n . The use of a complex f a c t o r such as age or c u l t i v a r would be more l i k e l y to show tha t s t o m a t a l c l o s u r e , 22 when and i f i t d i d o c c u r , i s o n l y a p a r t i a l e x p l a n a t i o n o f p r o t e c t i o n . O.ther exper iments have used s imp le f a c t o r s which are more l i k e l y than a c o a t i n g o f ten mg 1 1 ABA to r e p r e s e n t p o s s i b l e f i e l d s i t u a t i o n s , and n a t u r a l p r o t e c t i v e mechanisms. • . Q u i t e a few workers have shown tha t ozone s e n s i t i v i t y i n c r e a s e s w i t h i n c r e a s i n g h u m i d i t y (Menser, 1962; H u l l and Went, 1952; Thomas and H e n d r i c k s , 1956; Ot to and D a i n e s , 1969; Davis and Wood, 1973; Leone and Brennan , 1969), and i n some cases ( e . g . , Ot to and D a i n e s , 1969) t h i s has been shown to be c l o s e l y a s s o c i a t e d w i t h d e c r e a s i n g R . The s t o m a t a l mechanism i n v o l v e d i n t h i s h u m i d i t y response i s not e n t i r e l y c l e a r . I t may i n v o l v e d i r e c t e f f e c t s on the l o s s o f water from guard c e l l s , " p e r i s t o m a t a l t r a n s p i r a t i o n , " (Lange et a l . , 1971; S c h u l z e et a l . , 1972) , o r , i f the h u m i d i t y exposure i s o f long enough d u r a t i o n , i t c o u l d be media ted by changes i n l e a f water p o t e n t i a l . A d d i t i o n a l s i n g l e f a c t o r s s t u d i e d , w i t h the i n c l u s i o n of c o n c u r r e n t measurements o f R , which show a c l o s e i n v e r s e r e l a t i o n between R g and ozone i n j u r y , i n c l u d e CO2 c o n c e n t r a t i o n ( M a n s f i e l d and M a j e r n i k , 1970) and po tass ium n u t r i t i o n (Leone, 1976) . In some c a s e s , the use o f more complex f a c t o r s as independent v a r i a b l e s has a l s o demonstrated a c l o s e c o r r e l a t i o n of R s and ozone s e n s i t i v i t y . Siedman and Riggan (1968) noted that p l a n t s grown i n c o n t r o l l e d environments had a 23 reduced s e n s i t i v i t y to a i r p o l l u t a n t s d u r i n g the w i n t e r , and showed tha t t h i s cor responded to an endogenous rhythm whereby s tomata l a p e r t u r e was r e d u c e d , i n d e p e n d e n t l y o f env i ronmenta l c o n d i t i o n s . Dean (1972) looked at s i x c u l t i v a r s o f t o b a c c o , o f v a r y i n g ozone s e n s i t i v i t i e s , and found a good cor respondence between i n c r e a s i n g ozone s e n s i t i v i t y and i n c r e a s e s i n both s tomata l s i z e and s t o m a t a l d e n s i t y . T ingey et a l . (1973a) s t u d i e d the v a r i a t i o n o f i n j u r y w i t h age i n the f i r s t t r i f o l i a t e l e a f o f soybean and found a good' cor respondence between a decrease i n i n j u r y a f t e r 20 days from s e e d i n g and an i n c r e a s e i n R . Other s t u d i e s based on the use of complex e x t e r n a l f a c t o r s show c o n t r a s t i n g r e s u l t s . Evans and T i n g (1974a) s t u d i e d changes i n the s e n s i t i v i t y o f the pr imary l eaves of bean w i t h age and found tha t "bean l e a f s e n s i t i v i t y was a f u n c t i o n o f more i n t e r n a l c i r c u m s t a n c e s , and gas exchange was never the l i m i t i n g f a c t o r . " S i m i l a r r e s u l t s , on the v a r i a t i o n of c o t t o n s e n s i t i v i t y to ozone w i t h p l a n t age , where s e n s i t i v i t y was not c o r r e l a t e d w i th R , were found by T i n g and Dugger (1968) . In c o n t r a s t , G l a t e r et a l . (1962) found tha t the s tage o f ozone s u s c e p t i b i l i t y i n tobacco cor responded to the onset o f f u n c t i o n a l stomata and the expans ion o f i n t r a c e l l u l a r s p a c e s . Dugger et a l . (1962) s t u d i e d the e f f e c t o f p r e - e x p o s u r e dark p e r i o d s on ozone s e n s i t i v i t y i n bean and showed tha t changes i n ozone s u s c e p t i b i l i t y induced by these t reatments 24 had no d i r e c t r e l a t i o n s h i p to the degree of stomatal opening d u r i n g exposure. They suggested that changes i n i n j u r y were a f u n c t i o n of carbohydrate r e s e r v e s i n the p l a n t at the time of exposure, whereby d e p l e t e d r e s e r v e s i n c r e a s e d s u s c e p t i b i l i t y . T h i s was e x p l a i n e d by the a c t i o n of carbohydrates as a source of reducing compounds, pre v e n t i n g o x i d a t i o n of such compounds as p r o t e i n and ItCpid i n the c e l l membranes. The authors concluded, based on a review of the l i t e r a t u r e , t h a t stomata must be open f o r i n j u r y to occur, but that beyond t h a t , "stomatal opening i s not the primary f a c t o r i n p r e d i s p o s i n g p l a n t s to i n j u r y from ozone." In c o n t r a s t to the work of Dean (1972) mentioned above, Tin g and Dugger (1971) a l s o s t u d i e d the d i f f e r e n c e i n ozone s e n s i t i v i t y between two tobacco c u l t i v a r s , Bel B and B e l W3, but d i d not f i n d a s i m i l a r r e l a t i o n s h i p between s u s c e p t i b i l i t y and stomatal s i z e or density'. Dean's s e r i e s of c u l t i v a r s , i t should be noted, contained Bel W3 but not Bel B. T i n g and Dugger d i s c u s s e d a more general change i n water balance, whereby t h e i r t o l e r a n t c u l t i v a r , Bel B, had s m a l l e r roots and a g e n e r a l l y lower t r a n s p i r a t i o n r a t e . In the l i g h t , R^ was higher i n Bel B and l e a f water p o t e n t i a l s were lower, which suggested to them that d i f f e r e n c e s might be a s c r i b a b l e to stem conductance. Turner et a l . (1972) s t u d i e d the r e l a t i o n s h i p of R^ and ozone s e n s i t i v i t y i n f o u r tobacco c u l t i v a r s , Bel W3, 6524 (a 25 t o l e r a n t ( c u l t i v a r ) , and two c u l t i v a r s o£ i n t e r m e d i a t e t o l e r a n c e (Conn 49 and 6 590) . They found tha t R^ was g e n e r a l l y r e l a t e d to ozone s e n s i t i v i t y , i n comparing the extremes of s e n s i t i v i t y , but the i n t e r m e d i a t e c u l t i v a r s used i n t h e i r s tudy demonstrated some anomal ies i n t h i s r e l a t i o n s h i p which l e d them to conc lude tha t the " d i f f e r e n c e s ( in stem conductance) are not s u f f i c i e n t to account f o r d i f f e r e n c e s i n v i s i b l e damage." M a n s f i e l d (1973) c o n c l u d e d , w i t h r e s p e c t to the e f f e c t o f s t o m a t a l a p e r t u r e on ozone i n j u r y , that " the b a l a n c e o f the ev idence suggests tha t s t o m a t a l a p e r t u r e s are o f importance i n d e t e r m i n i n g the s e n s i t i v i t y o f p l a n t s d u r i n g exposure to a i r p o l l u t a n t s . " D e s p i t e some i n d i c a t i o n s to the c o n t r a r y (see Dugger et a l . , 1962; Evans and T i n g , 1974a) , M a n s f i e l d ' s c o n c l u s i o n w i t h r e g a r d to the second a r e a , the e f f e c t o f ozone exposure on s t o m a t a , was l e s s e q u i v o c a l . He s a i d , "s tomata c l o s e i n response to o z o n e . " E v i d e n c e f o r t h i s c l o s u r e has come from some o f the e a r l i e s t work on the e f f e c t s o f a i r p o l l u t a n t s on p l a n t s . K o r i t z and Went (1952) showed tha t t r a n s p i r a t i o n was reduced by about 80% i n tomato p l a n t s a f t e r exposure to a mix tu re o f 0.1 ppm ozone and 1-n-hexene v a p o r s . With d a i l y doses o f two h per day of tha t m i x t u r e , over f o u r d a y s , they found tha t t h i s e f f e c t was not r e v e r s i b l e upon removal o f the a r t i f i c i a l smog. T h i s i r r e v e r s i b l e c l o s u r e , t h e r e f o r e , was somewhat 26 d i f f e r e n t from the r e v e r s i b l e c l o s u r e induced by ozone found by H i l l and L i t t l e f i e l d (1969) . H i l l and L i t t l e f i e l d exposed a l a r g e number o f s p e c i e s , o a t s , b a r l e y , wheat , t o b a c c o , p i n t o bean , l ima b e a n , bush bean , c h a r d , c o r n , c a u l i f l o w e r , sugar b e e t , p o t a t o , and tomato, and found tha t c o n c e n t r a t i o n s as low as 0.1 ppm c o u l d cause p a r t i a l s tomata l c l o s u r e (as measured by p l a s t i c l e a f i m p r e s s i o n s ) and complementary decreases i n p h o t o s y n t h e s i s and t r a n s p i r a t i o n . They used s h o r t - t e r m e x p o s u r e s , one to two h , and found these ' e f f e c t s to be l a r g e l y r e v e r s i b l e upon removal o f the ozone, but d i d not g i v e r e p e a t e d d o s e s . These two types o f o z o n e - i n d u c e d s tomata l c l o s u r e , the s h o r t - t e r m r e v e r s i b l e " type found by H i l l , and L i t t l e f i e l d (1969) and the l o n g e r term dampening found by K o r i t z and Went (1952) encompass the b a s i c forms o f d i r e c t ozone e f f e c t s on s tomata . Another type ( e . g . , M a c k n i g h t , 1968) o f ozone-induced c l o s u r e may occur as an i n d i r e c t r e s u l t o f damage to ep idermal t i s s u e . The s h o r t - t e r m r e v e r s i b l e type o f c l o s u r e was f u r t h e r c h a r a c t e r i z e d by R i c h and Turner (1972) , i n two c u l t i v a r s o f t o b a c c o , t o l e r a n t C o n s o l i d a t e d L and s e n s i t i v e B e l W3. They s t u d i e d the v a r i a t i o n s i n the c l o s u r e response under d i f f e r e n t m o i s t u r e c o n d i t i o n s . U s i n g 60 min exposures to 0 .20-0.25 ppm they found c l o s u r e w i t h i n ten min i n the t o l e r a n t c u l t i v a r , w h i l e c l o s u r e i n the s u s c e p t i b l e c u l t i v a r o c c u r r e d 27 much more s l o w l y . C l o s u r e was r e v e r s i b l e upon' removal o f the ozone. T h i s c l o s u r e , however, was l i m i t e d to dry a tmospheres , 371 r e l a t i v e h u m i d i t y (RH). In a mois t atmosphere (73I ;RH) , stomata o f both c u l t i v a r s c l o s e d more s l o w l y , and the r a t e was equal f o r the two. c u l t i v a r s . In exper iments w i t h b e a n s , R i c h and Turner found tha t stomata o f w e l l watered p l a n t s , w i t h a l e a f water p o t e n t i a l o f - 3 . 2 b a r s , -320 k i l o p a s c a l s (kPa ) , c l o s e d from 2.5 to 5.2 seconds per cent imer (sec cm )^ a f t e r ten min exposure to 0 .20 -0 .25 ppm, which was more s l o w l y than the s tomata l c l o s u r e i n w a t e r - s t r e s s e d p l a n t s , w i t h a l e a f water p o t e n t i a l o f - 5 . 4 bars (-540 k P a ) , which c l o s e d from 2.9 to 8.4 sec cm ^ i n the same p e r i o d . Exper iments w i t h w e l l - w a t e r e d bean p l a n t s showed tha t a l t h o u g h c l o s u r e o c c u r r e d i n a dry atmosphere , i n a mois t atmosphere stomata d i d not respond at a l l to ozone. Bean p l a n t s a l s o had a h i g h e r i n i t i a l R s i n the dry atmosphere , l e a d i n g the authors to conc lude tha t lower amounts o f i n j u r y i n dry atmospheres might be a t t r i b u t a b l e to both h i g h e r i n i t i a l R g and g r e a t e r degrees o f c l o s u r e i n response to f u m i g a t i o n . T h i s complementary r e l a t i o n s h i p between i n i t i a l R g and c l o s u r e i n response to ozone need not be c o n s t a n t , however. In the exper iments o f Leone (1976) , p o t a s s i u m - d e f i c i e n t p l a n t s had a h i g h e r i n i t i a l R g and a l e s s e r degree o f c l o s u r e i n response to ozone f u m i g a t i o n . The f a c t tha t s tomata l c l o s u r e i n response to ozone may p r o v i d e the b a s i s f o r p r o t e c t i o n from acute ozone exposure 28 was p o i n t e d out by Eng le and Gableman (1966). They compared the response of d i f f e r e n t i a l l y s e n s i t i v e i n b r e d l i n e s o f on ion to 0.3 ppm ozone f o r 30-60 min . Working w i t h the ozone t o l e r a n t l i n e s W4 and SW52, and the s u s c e p t i b l e l i n e s TDYG and SW34, they found tha t d i f f e r e n c e s i n s u s c e p t i b i l i t y were c l o s e l y r e l a t e d to d i f f e r e n c e s i n s t o m a t a l response to / ozone . O b s e r v a t i o n s o f t i p b u r n i n j u r y i n f i e l d - p l a n t e d and p o t t e d bu lbs of these i n b r e d l i n e s and t h e i r F l h y b r i d s i n d i c a t e d tha t ozone t o l e r a n c e was a dominant g e n e t i c f a c t o r , perhaps c o n t r o l l e d by a s i n g l e gene p a i r . The l i n e s W4 and SW34, and t h e i r F l h y b r i d s were a l s o used to s tudy s t o m a t a l ' r e s p o n s e to ozone , i n the l a b o r a t o r y , by o b s e r v a t i o n o f water l o s s . T r a n s p i r a t i o n i n W4 and the W4 x SW34 h y b r i d was found to decrease a f t e r 30. min of exposure to 0.4 ppm ozone , w h i l e there was no change i n t h e • t r a n s p i r a t i o n r a t e of SW34 g i v e n the same exposure . I f ozone was removed a f t e r 90 min o f f u m i g a t i o n i n W4 or SW34, t r a n s p i r a t i o n - was found to r e c o v e r . Stomata were a l s o observed under a low power mic roscope d u r i n g f u m i g a t i o n w i t h 0.3 ppm o z o n e , and i t was found tha t s tomata l c l o s u r e i n ozone t o l e r a n t l i n e s o c c u r r e d i n i t i a l l y at a r a t e three to f i v e t imes s lower than c l o s u r e i n response to d a r k n e s s . A f t e r one c y c l e of c l o s u r e and reopen ing i n o z o n e - f r e e a i r , however, c l o s u r e i n response to a second ozone exposure o c c u r r e d at a r a t e a p p r o x i m a t e l y equal to the darkness r e s p o n s e . An average c y c l e f o r the ozone t o l e r a n t l i n e s was 40 min to c l o s e upon the f i r s t 29 e x p o s u r e , ten min to reopen upon removal o f the ozone , ten min to r e c l o s e upon r e f u m i g a t i o n , and 20 min to r e o p e n . Stomata of s u s c e p t i b l e SW34 p l a n t s d i d not c l o s e at a l l w i t h i n the two h p e r i o d p r i o r to the appearance of v i s i b l e i n j u r y symptoms. T h i s type o f response may be o f importance i n the i n t e r a c t i o n o f s u b - a c u t e and acute e x p o s u r e s , and i t i s e v i d e n t tha t repea ted exposures may have a s i g n i f i c a n t e f f e c t on the c l o s u r e response i t s e l f . T h i s f a c t o r must be c o n s i d e r e d , however, i n combina t ion w i t h changes i n i n i t i a l s tomata l a p e r t u r e . A l t h o u g h the p o l l u t a n t used i n the exper iments of Bonte' et a l . ; (1977) was SO^, tha t work i s i n s t r u c t i v e i n terms of the k inds o f c o n s i d e r a t i o n s which may be impor tant i n a compar ison o f the e f f e c t s o f i n i t i a l a p e r t u r e and c l o s u r e i n response to f u m i g a t i o n . A survey of 11 c u l t i v a r s of Pe la rgon ium w i t h v a r y i n g SC^ s u s c e p t i b i l i t i e s r e v e a l e d no s i g n i f i c a n t r e l a t i o n s h i p between s t o m a t a l s i z e or f requency and SC^ s e n s i t i v i t y . With c o n t r o l l e d exposures o f two c u l t i v a r s , one t o l e r a n t and one s e n s i t i v e , however, Bonte' et a l . found s i g n i f i c a n t d i f f e r e n c e s i n s t o m a t a l b e h a v i o r which c o u l d have accounted f o r the d i f f e r e n t SG^ s e n s i t i v i t i e s . Speed o f s tomata l c l o s u r e i n response to S C ^ , and t o t a l volume of gas exchanged d u r i n g the f u m i g a t i o n were s i g n i f i c a n t l y d i f f e r e n t between c u l t i v a r s , i n both wet and dry a tmospheres . In dry a i r , the t o t a l volume exchanged (as a p e r c e n t o f the p o s s i b l e , volume w i thout any 30 s tomata l c l o s u r e ) d i f f e r e d between the c o n t r o l and SG^ t rea tment , whereas i t d i d not i n wet a i r . In wet a i r , the f i n a l mean a p e r t u r e s of stomata d i f f e r e d , whereas i n dry a i r they d i d n o t . T h i s i n d i c a t e d tha t the dry a i r d i f f e r e n c e was due to both a d i f f e r e n t i n i t i a l a p e r t u r e and the f a c t tha t c l o s u r e o c c u r r e d to the same end p o i n t . In wet a i r , a l though the i n i t i a l a p e r t u r e s d i f f e r e d , the end p o i n t d i d a l s o . Two r a t i o s (aper ture i n dry a i r / a p e r t u r e i n wet a i r i ' , and t o t a l volume exchanged i n dry a i r / t o t a l volume exchanged i n wet a i r ) were s i g n i f i c a n t l y h i g h e r i n the S C ^ - s e n s i t i v e c u l t i v a r , i n d i c a t i n g tha t the d i f f e r e n t i a l s e n s i t i v i t y to SC^ was accompanied by a d i f f e r e n t i a l s e n s i t i v i t y to a tmospher ic h u m i d i t y . M a n s f i e l d and M a j e r n i k (19 70) found that exposure to SC^ c o u l d cause an i n c r e a s e i n s tomata l a p e r t u r e i n atmospheres of r e l a t i v e humid i ty g r e a t e r than 40%, and a decrease i n s t o m a t a l a p e r t u r e s at lower r e l a t i v e h u m i d i t i e s . Whi le the i n t e r a c t i o n between s t o m a t a l response to humid i ty and s tomata l response to ozone does not appear to p r e s e n t the same c o m p l e x i t i e s as the i n t e r a c t i o n o f humid i ty response and SO^ r e s p o n s e , both the r a t e of c l o s u r e i n response to ozone and the f i n a l a p e r t u r e d u r i n g f u m i g a t i o n have impor tant i m p l i c a t i o n s f o r the r e l a t i o n s h i p between the e f f e c t s o f ozone on s tomata l b e h a v i o r and i t s e f f e c t on acute s u s c e p t i b i l i t y and growth. i 31 Growth Changes i n s tomata l b e h a v i o r induced by l o n g - t e r m exposure to low l e v e l s of ozone may be accompanied by changes i n p l a n t p r o d u c t i v i t y , e i t h e r as a d i r e c t r e s u l t o f s t o m a t a l e f f e c t s or as a r e s u l t o f o ther p h y s i o l o g i c a l changes induced by ozone exposure . Work w i t h both c o n t r o l l e d exposures to ozone and w i t h ambient a i r v e r s u s f i l t e r e d a i r t reatments has demonstrated the o c c u r r e n c e of such growth e f f e c t s . On p e r e n n i a l p l a n t s , Thompson et a l . (1969) r e p o r t e d , on the b a s i s of f i l t e r e d a i r v e r s u s u n f i l t e r e d open- top chamber t r e a t m e n t s , tha t ambient o x i d a n t l e v e l s i n C a l i f o r n i a reduced both the f r u i t y i e l d and v e g e t a t i v e growth (p run ings) o f g r a p e v i n e s . The ambient o x i d a n t s i n tha t environment were a mix ture of ozone and PAN, and Thompson et a l . r e p o r t e d peaks o f up to 0.25 ppm d u r i n g the growing season i n t h e i r s t u d y . They a l s o r e p o r t e d r e d u c t i o n s i n the sugar conten t o f b e r r i e s and i n the c h l o r o p h y l l content o f l eaves as a r e s u l t o f o x i d a n t e x p o s u r e . Thompson and T a y l o r (1969) showed tha t t h i s same t reatment reduced y i e l d i n lemon and o r a n g e , and Thompson et a l . (1967) showed tha t ambient o x i d a n t s reduced both water use and apparent p h o t o s y n t h e s i s i n those same s p e c i e s . Barnes (1972a) r e p o r t e d decreases i n p h o t o s y n t h e t i c r a t e 32 a f t e r exposure of p i n e s e e d l i n g s to 0 .05-0 .10 ppm ozone f o r f i v e to e i g h t e e n weeks. He a l s o showed c o r o l l a r y -i n c r e a s e s i n r e s p i r a t i o n r a t e . Long- te rm exposure of p i n e s e e d l i n g s to 0.05 ppm ozone d e c r e a s e d the c a r b o h y d r a t e content w h i l e i n c r e a s i n g the a s c o r b i c a c i d content (Barnes , 1972b). Brewer and F e r r y (1974) showed that ambient o x i d a n t s i n C a l i f o r n i a reduced the l i n t and seed y i e l d o f c o t t o n by about 151. In soybeans , l o n g - t e r m exposure to loxv ozone l e v e l s was shown to reduce seed y i e l d s (Heagle et a l . , 1974) . Feder (1970) showed tha t exposure to 0.10 ppm ozone , f i v e to seven h per day, f o r one to th ree months, reduced f l o w e r i n g , s i d e b r a n c h i n g , and l e a f s i z e i n geranium and c a r n a t i o n , w h i l e i n c r e a s i n g i n t e r n o d e l e n g t h . Exposure to 0.10 ppm ozone f o r t> f i v e h per day , f o r 14 d a y s , t o t a l l y i n h i b i t e d f l o w e r i n g i n duckweed (Feder and S u l l i v a n , 1969) w h i l e r e d u c i n g the f r o n d d o u b l i n g r a t e by 50%. Craker (1972) showed an o z o n e - i n d u c e d r e d u c t i o n of f l o w e r i n g i n p e t u n i a , and Heggestad (1973) demonstrated a r e d u c t i o n i n y i e l d w i t h p o t a t o exposed to ambient ozone on the A t l a n t i c C o a s t . The overwhelming m a j o r i t y of the work on the e f f e c t s of ozone exposure on p l a n t growth, l i k e those papers d e s c r i b e d above, has found decreases i n both v e g e t a t i v e and r e p r o d u c t i v e growth i n response to ozone. At tempts to s u b d i v i d e the e f f e c t on v e g e t a t i v e growth i n t o the be low-ground and above-" ground p o r t i o n s o f the p l a n t have g e n e r a l l y shown a g r e a t e r 33 e f f e c t on roo t growth. Heagle et a l . (1973) found an equal r e d u c t i o n i n the top and r o o t f r e s h weights o f B e l W3 t o b a c c o , but p o i n t e d out tha t top f r e s h weight a l s o i n c l u d e d water l o s s due to i n j u r y l e s i o n s . T ingey et a l . (1973b) exposed soybean to 0.10 ppm ozone f o r e i g h t h per day , f i v e days per week, f o r the f i r s t th ree weeks o f growth, and found top f r e s h weight reduced by 21% and roo t f r e s h weight reduced by 25%, w h i l e roo t dry weight was reduced by 21% and top dry weight by o n l y n i n e p e r c e n t . Treatment w i t h low ozone l e v e l s a l s o had' a l a r g e r e f f e c t on r o o t growth i n r a d i s h (T ingey et a l . , 1971) , 50% r e d u c t i o n i n r o o t s as compared to 20% f o r l e a v e s . Bennet t and Runeckles (1977) showed tha t r o o t growth was more s e n s i t i v e than top growth, i n c l o v e r and r y e g r a s s , to growth r e d u c t i o n s induced by exposure to 0.03 or 0.09 ppm ozone, and suggested tha t r e d u c t i o n s i n top growth were accompanied by a compensatory i n c r e a s e i n net a s s i m i l a t i o n r a t e (NAR). Blum and T ingey (1977) , work ing w i th s o y b e a n , determined tha t r e d u c t i o n i n r o o t growth was a r e s u l t o f f o l i a r e x p o s u r e , not r o o t e x p o s u r e , by expos ing l eaves and r o o t s to ozone (on whole p l a n t s ) both t o g e t h e r and s e p a r a t e l y . The r e p e r c u s s i o n s o f f o l i a r i n j u r y on f o l i a r growth or r o o t growth may e v i d e n t l y va ry w i d e l y , even i n c l o s e l y r e l a t e d s p e c i e s . Wi lhour and Nee ley (1977) showed tha t 19% f o l i a r i n j u r y i n western whi te p i n e l e d to s i g n i f i c a n t growth r e d u c t i o n s o n l y i n f o l i a g e dry w e i g h t , w h i l e 20% f o l i a r i n j u r y i n Ponderosa p i n e l e d to s i g n i f i c a n t growth r e d u c t i o n s o n l y i n r o o t dry w e i g h t . 34 Bennett and Oshima (1976), work ing w i t h c a r r o t , found tha t r o o t s were more s e n s i t i v e than l eaves to growth-r e d u c t i o n s r e s u l t i n g from o z o n e - i n d u c e d f o l i a r i n j u r y . In tha t exper iment , i n the p resence of f o l i a r i n j u r y , l e a f number and l e n g t h were i n c r e a s e d , a l t h o u g h l e a f dry weight was n o t . Bennet t et a l . (1974) rev iewed the l i t e r a t u r e on ozone e f f e c t s on growth and p o i n t e d out tha t apparent s t i m u l a t i o n s o f growth by ozone c o u l d r e s u l t from compar ison w i th c h a r c o a l -f i l t e r e d c o n t r o l s . In a i r p o l l u t i o n e x p e r i m e n t s , the a d d i t i o n o f a c h a r c o a l f i l t e r to p r o v i d e " c l e a n " a i r i s o f t e n c o n s i d e r e d an ozone or o x i d a n t e f f e c t when i t s h o u l d more a c c u r a t e l y be c o n s i d e r e d a c h a r c o a l f i l t e r e f f e c t . In compar ison o f ambient a i r t reatments w i t h c h a r c o a l - f i l t e r e d c o n t r o l s t h e r e i s always the p o s s i b i l i t y t h a t the . c h a r c o a l f i l t e r i s removing a tmospher ic c o n s t i t u e n t s which are advantageous to the p l a n t . I f the e x p e r ime n t a l p l a n t s have adapted to the p resence of these a tmospher ic c o n s t i t u e n t s , p l a n t s grown i n c h a r c o a l - f i l t e r e d a i r would not r e p r e s e n t t rue examples o f normal growth. In comparing c h a r c o a l - f i l t e r e d c o n t r o l s to p l a n t s r e c e i v i n g f i l t e r e d a i r p l u s a s m a l l amount o f ozone, there remains the p o s s i b i l i t y tha t c h a r c o a l f i l t r a t i o n does not r e p r e s e n t a c o n t r o l , i f p l a n t s , have adapted to a low a tmospher ic ozone l e v e l over the 35 course of e v o l u t i o n or d e l i b e r a t e s e l e c t i o n due to n a t u r a l sources of ozone. The data o f Bennet t et a l . (1974) suggested, t h i s p o s s i b i l i t y . Comparing f i l t e r e d a i r w i th f i l t e r e d a i r p l u s ozone , exposure to 0.03 ppm ozone i n c r e a s e d t o t a l p l a n t dry weight i n b a r l e y and smartweed, and top dry weight i n bean . T h e ' p o s s i b i l i t y o f a growth s t i m u l a t i o n induced by s u b - a c u t e exposure suggests one a s s o c i a t i o n between growth e f f e c t s and e f f e c t s on s u s c e p t i b i l i t y whereby both a growth s t i m u l a t i o n and a p r o t e c t i v e e f f e c t are two aspec ts of a g e n e r a l l y b e n e f i c i a l e f f e c t o f low ozone t r e a t m e n t , r e f l e c t i n g two forms of a c c l i m a t i o n , over two d i f f e r e n t time s c a l e s . The g e n e r a l r e l a t i o n s h i p between the more commonly observed growth r e t a r d i n g e f f e c t o f s u b - a c u t e exposure and a l o n g - t e r m s t o m a t a l dampening r a i s e s the q u e s t i o n of whether a growth s t i m u l a t i o n may a l s o be a s s o c i a t e d w i t h some s t o m a t a l e f f e c t , perhaps s tomata l o p e n i n g , a l though t h i s would tend to decrease the p r o t e c t i v e e f f e c t . None o f the e x i s t i n g work i n t h i s area speaks d i r e c t l y to the concerns of t h i s t h e s i s , the r e l a t i o n s h i p o f the growth e f f e c t s o f s u b - a c u t e exposure to the e f f e c t s on acute s u s c e p t i b i l i t y and s tomata l b e h a v i o r . 36 M a t e r i a l s and Methods Bean and Mint Bush bean (Phaseolus v u l g a r i s L. cv Pure Go ld Wax) was p l a n t e d two cm deep, two to f i v e seeds per p o t , i n p a s t e u r i z e d s o i l . A f t e r emergence, one or two u n i f o r m s e e d l i n g s were r e t a i n e d per pot f o r e x p e r i m e n t a l u s e . Treatments began e i t h e r immedia te ly a f t e r sowing or a f t e r an e i g h t to f o u r t e e n day i n t e r v a l . Mint (Mentha a r v e n s i s L.) came from a g e n e t i c a l l y u n i f o r m s t o c k m a i n t a i n e d i n the greenhouse ( F i g . 1 ) . Stem c u t t i n g s were r o o t e d i n sand f o r two weeks and p o t t e d i n p a s t e u r i z e d s o i l i n ten cm p l a s t i c p o t s . A x i l l a r y shoots., were removed f o u r days p r i o r to the s t a r t o f experiments, . Exper iments were done i n c o n t r o l l e d environment chambers m a i n t a i n e d at 20/15 degrees C e l s i u s (C) d a y / n i g h t , w i t h an 11 h p h o t o p e r i o d (8 AM to 7 PM). A l l ozone exposures were f o r s i x h per day (11 AM to 5 PM). Ozone was genera ted by p a s s i n g a stream of compressed a i r or oxygen through a corona d i s c h a r g e t u b e , and was mon i to red w i t h Mast (Model 724) ozone meters p l a c e d w i t h i n the chambers. Meters were c a l i b r a t e d p e r i o d i c a l l y d u r i n g exper iments a g a i n s t 1% NBKI a c c o r d i n g to the method of the F e d e r a l R e g i s t e r , November 25 , 1971. In exper iments u s i n g compressed a i r i n ozone g e n e r a t i o n , no n i t r o g e n d i o x i d e was d e t e c t a b l e , by the method of Sal tzman 37 F i g u r e 3- Acute f u m i g a t i o n chamber f o r t r a n s p i r a t i o n measure-ment 38 (1954), at any of the ozone c o n c e n t r a t i o n s used i n the exper iments . No ozone was d e t e c t e d i n the chambers s u p p l i e d w i th f i l t e r e d a i r . R e l a t i v e humid i ty was found to v a r y , between 55 and 801, l a r g e l y depending on the number o f p l a n t s i n the chamber. P l a n t numbers per chamber were t h e r e f o r e kept the same i n f i l t e r e d a i r and ozone t r e a t m e n t s . Pots were watered to c a p a c i t y on a l t e r n a t e d a y s . Photosyn-t h e t i c a l l y a c t i v e r a d i a t i o n (PAR) i n the chambers, at p l a n t l e v e l , was 0.4 m i c r o e i n s t e i n s per square c e n t i m e t e r per - 2 -1 minute ( ^ E cm min ) , as measured w i t h an Isco (Model SR) s p e c t r o r a d i o m e t e r . A i r velocity . at p l a n t l e v e l was 0.5 meters per second (m sec "*") . Acute i n j u r y ( F i g . 2) was assayed v i s u a l l y , as p e r c e n t pr imary l e a f a rea n e c r o t i c (LAN) a f t e r the p l a n t s had been i n an ambient environment f o r at l e a s t one day f o l l o w i n g acute t rea tment . Stomata l r e s i s t a n c e (P g ) was measured on the lower s u r f a c e o f p r imary bean l eaves w i t h a Lambda (Model L i 60) d i f f u s i o n porometer . T r a n s p i r a t i o n was measured by d i f f e r e n t i a l thermocouple psychrometry i n a system c o n t a i n i n g one a t t a c h e d pr imary l e a f s e a l e d i n a p l e x i g l a s s chamber, 15 x 11 x 5 cm ( F i g . 3 ) . A i r f low through the system was 1.2 l i t e r s per minute (1 min ^) . A i r was pumped from o u t d o o r s , f i l t e r e d through c h a r c o a l , and kept at a r e l a t i v e humid i ty of 50% when e n t e r i n g the f u m i g a t i o n chamber by means o f a s e r i e s of temperature c o n t r o l l e d b u b b l e r s . Mean a i r temperature i n the chamber was 39 32 C, mean l e a f temperature was 30 C. PAR at the l e a f was - 2 -1 4 . 5 ^ E cm min . Ozone was genera ted w i t h a Mon i to r Labs (Model 8250) ozone source and moni tored w i t h a Mast (Model 724) ozone meter . Grapev ines O n e - y e a r - o l d r o o t e d c u t t i n g s of th ree grape c u l t i v a r s ( V i t i s l a b r u s c a n a , B a i l e y ) were p l a n t e d i n 12 cm c l a y pots i n a mix ture of 1:1:1 (v /v ) s o i l : p e a t : p e r l i t e , i n e a r l y May. They were reduced to a s i n g l e shoot and a l l o w e d to l e a f out o u t s i d e at the V i n e y a r d L a b o r a t o r y i n F r e d o n i a , New Y o r k . On June 15 they were p l a c e d w i t h i n open- top chambers b u i l t to the d e s i g n of Heagle et a l . (1973) ( F i g . 4) except tha t chamber h e i g h t was i n c r e a s e d to n ine f e e t (2.7 m e t e r s ) . Pots were p l a c e d on the ground around the p e r i m e t e r of a chamber c o n t a i n i n g a s i n g l e 2 0 - y e a r - o l d ' C o n c o r d ' v i n e t r a i n e d a c c o r d i n g to the Umbre l l a K n i f f i n system i n the c e n t e r . Pots were watered to c a p a c i t y week ly , and each pot r e c e i v e d one ounce (28.4g) of 50-50-50 N-P-K f e r t i l i z e r on August 19. With two t r e a t m e n t s , c h a r c o a l - f i l t e r e d a i r and ambient a i r , , the exper iment was done a c c o r d i n g to a randomized b l o c k d e s i g n , w i th two d u p l i c a t e . chambers t r e a t e d as b l o c k s . Samples were randomized a c c o r d i n g to chamber p o s i t i o n . Ozone was moni to red f o r f i v e m in , once each 65 m i n , F i g u r e 5- Acute ozone f u m i g a t i o n chamber 41 i n each chamber, u s i n g both a D a s i b i ( M o d e l 1003AH) and a M o n i t o r Labs (Model 8410E) ozone m o n i t o r . Over the p e r i o d August f i r s t to September n i n t h , the o v e r a l l mean ozone c o n c e n t r a t i o n s were 0.04 ppm i n the ambient a i r , 0.03 ppm i n the ambient a i r chambers, and 0.01 ppm i n the f i l t e r e d a i r chambers. The e f f i c i e n c y of f i l t r a t i o n i n open- top f i e l d chambers i s not as g rea t as i n growth c a b i e n t s , and t h i s i s an impor tant l i m i t a t i o n to t h e i r e f f e c t i v e n e s s i n exper iments w i t h low ozone c o n c e n t r a t i o n s . S i x v i n e s of each c u l t i v a r were h a r v e s t e d from each t reatment ( three per chamber) on J u l y t w e l f t h , August n i n t h , and September t w e l f t h , and d i v i d e d i n t o l e a f ( i n c l u d i n g p e t i o l e ) , s h o o t , t r u n k , and r o o t p o r t i o n s . Those p o r t i o n s were we ighed, d r i e d o v e r n i g h t ( l eaves and shoots ) or f o r 48 h ( t runk and r o o t s ) at 70 C, and reweighed . Lea f a rea was e s t i m a t e d by comparing the dry weight o f 2 th ree l e a f s e c t i o n s of th ree cm each chosen at random from the e n t i r e canopy , w i th t o t a l l e a f dry w e i g h t . An a d d i t i o n a l s i x v i n e s per c u l t i v a r , per t r e a t m e n t , were fumigated w i t h 0.75 ppm ozone , f o r s i x h , i n a c l e a r p l a s t i c chamber ( F i g . 5) 121: x 30 x 84 cm, p l a c e d w i t h i n a w a l k - i n growth chamber. Temperature d u r i n g f u m i g a t i o n was 25 C and r e l a t i v e humid i ty was 80%. Ozone was genera ted by p a s s i n g f i l t e r e d a i r over a T a b l e 1. Ozone m o n i t o r i n g d a t a , Aug. 1 to Sept . 9, 1977 i n open- top chamber experiment Treatment O v e r a l l mean Maximum # Readings g r e a t e r  than .08 ppm  M L 7 DAS Z ML DAS ML DAS Ambient a i r chamber (1) .03 .03 .12 .10 19 7 Ambient a i r chamber (2) .03 .03 .13 .10 17 5 C h a r c o a l f i l t e r e d chamber (1) • .01 .01 .09 .06 1 0 C h a r c o a l f i l t e r e d chamber (2) .01 .01 .11 .10 • 6 4 Ambient a i r .04 .04 .13 .13 53 41 samples for the two monitors were removed simultaneously monitors were calibrated every two weeks against a Monitor Labs ozone source x o n e r e a d i n g = maximum d u r i n g 5 minute p e r i o d , once per 65 minutes ^Moni to r Labs (Model 8410 E) ozone moni tor z D a s i b i (Model 1003 AH) ozone moni tor 4^ 43 u l t r a v i o l e t b u l b , and moni tored w i t h a Mast (Model 724) - 2 -1 ozone meter . PAR i n the chamber was 9.8 E cm min a s . measured w i t h a Lambda (Model L i 185) r a d i o m e t e r , at p l a n t l e v e l , and a i r f low through the chamber was 350 1 min 1 . S tomata l r e s i s t a n c e (R s ) was measured on the lower s u r f a c e of ."leaf. f ^ J - f r ^ ^ a s ; a l ) ) on each v i n e , immedia te ly p r i o r to the i n t r o d u c t i o n of ozone , and two h a f t e r the i n t r o d u c t i o n o f ozone , w i t h a Lambda (Model L i 65) d i f f u s i o n porometer . Symptoms o f acute ozone i n j u r y s i m i l a r to those d e s c r i b e d p r e v i o u s l y ( S h a u l i s et a l . , 1972) were assayed the f o l l o w i n g day on two l e a f d i s c s taken from t h i s same l e a f , one from each s i d e of the m i d r i b , and assayed v i s u a l l y , f o r p e r c e n t a rea n e c r o t i c a f t e r c l e a r i n g o f the c h l o r o p h y l l by b o i l i n g f o r 15-20 min i n 70% e t h a n o l and r i n s i n g i n acetone f o r an a d d i t i o n a l 15-20 min . P h o t o s y n t h e t i c r a t e was a l s o measured, on v i n e s from the September h a r v e s t , i n ambient a i r , i n the greenhouse , w i t h supp lementa l l i g h t p r o v i d e d w i t h f l o o d l a m p s , to p r o v i d e - 2 -1 from 3.6 to 6.6 /A. E cm min PAR. A 5 x 3 x 1/2 cm c u v e t t e a t t a c h e d to the u n d e r s i d e of the l e a f was connected to a Beckman (Model 215A) i n f r a r e d gas a n a l y z e r and a Genera l E a s t e r n (Model 1100AP) dew p o i n t hygrometer . For the September h a r v e s t , the same v i n e s were used f o r both the growth measurements, and f u m i g a t i o n assays so tha t v i n e - t o -v i n e c o r r e l a t i o n s o f i n j u r y , s tomata l b e h a v i o r , and growth parameters c o u l d be a s s e s s e d . 44 R e s u l t s S u s c e p t i b i l i t y To determine the e f f e c t o f s u b - a c u t e p re t rea tment doses o f ozone on p l a n t response to a subsequent acute d o s e , bean s e e d l i n g s and mint c u t t i n g s r a i s e d i n the greenhouse were exposed to 0.02 ppm ozone , s i x h per day , f o r seven d a y s , p r i o r to an exposure to an acute dose o f 0.15 ppm f o r s i x h (bean) or 0.3 ppm f o r 18 h (m in t ) . These r e s p e c t i v e acute doses l e d to e q u i v a l e n t n u m e r i c a l r a t i n g s o f acute i n j u r y , i n the r e s p e c t i v e s p e c i e s , i n p l a n t s p r e t r e a t e d w i t h f i l t e r e d a i r . V i s i b l e i n j u r y , the percentage o f the area o f the pr imary l eaves tha t was n e c r o t i c (LAN), a f t e r acute t reatment i n p l a n t s p r e t r e a t e d w i th a s u b - a c u t e ozone expo-sure was compared to v i s i b l e i n j u r y a f t e r the same acute ex-posure on p l a n t s p r e t r e a t e d f o r the same p e r i o d i n f i l t e r e d a i r (Table 2 ) . I n j u r y i n bean p l a n t s was reduced from 331 LAN i n the p l a n t s p r e t r e a t e d w i t h f i l t e r e d a i r to 14% LAN i n the o z o n e - p r e t r e a t e d p l a n t s . T h i s p r o t e c t i o n o f bean p l a n t s from an acute dose by s u b - a c u t e p re t rea tment i n d i c a t e d the p resence o f some form of a c c l i m a t i o n . T h i s may t e n t a t i v e l y be c a l l e d h a r d e n i n g , by ana logy to the ha rden ing o f p l a n t s to c o l d , drought , - and o t h e r p l a n t s t r e s s e s . ' In the more o z o n e - t o l e r a n t s p e c i e s , m i n t , the same ozone pre t rea tment had l i t t l e e f f e c t on s u s c e p t i b i l i t y to 45 T a b l e 2. Acute i n j u r y s u s c e p t i b i l i t y o f g r e e n h o u s e - r a i s e d bean and mint a f t e r s u b - a c u t e p r e t r e a t m e n t . . . W Acute i n j u r y % l e a f a rea n e c r o t i c Y X Pre t rea tment Bean Mint F i l t e r e d a i r . 33 * 3.2 32 * 1. 0.02 ppm ozone 14 ± 2.5 3 9 - 2 The r e s u l t s d e s c r i b e d i n t h i s t a b l e and i n F i g u r e s 6 to 10 have p r e v i o u s l y appeared i n : R u n e c k l e s , V . C . , and P.M. Rosen. 1974. C a n . J . B o t . 52: 2607- 2610 R u n e c k l e s , V . C . , and P.M. Rosen. 1977. C a n . J . B o t . 55:193-197 W+ S E , n = 24 - 38 v Acute dose : 0.3 ppm per 6 h per day , 3 days Y 1 A c u t e dose: 0.15 ppm f o r 6 h values of n refer to numbers of plants values for a single plant represent the mean of two primary leaves in bean within experiments, single plants are treated as replicates experiments were replicated 2-3 times 46 acute i n j u r y , i n c r e a s i n g i t s l i g h t l y . Comparing the two s p e c i e s , t h e r e f o r e , the tendency toward harden ing appears to be d i r e c t l y r e l a t e d to s u s c e p t i b i l i t y to acute i n j u r y , i . e . , the more s e n s i t i v e s p e c i e s , b e a n , shows a g r e a t e r tendency toward h a r d e n i n g . To i n v e s t i g a t e the range of c o n d i t i o n s under which t h i s harden ing e f f e c t o c c u r r e d i n b e a n s , s e e d l i n g s of two a g e s , e i g h t and f o u r t e e n days from sowing , and t h r e e .dura t ions of p re t rea tment w i t h low ozone were u s e d ' >, f i v e , s e v e n , and ten d a y s . W i t h i n t h i s range of v a r i a t i o n i n age and l e n g t h o f p r e t r e a t m e n t , s e n s i t i v i t y was c o n s i s t e n t l y d i m i n i s h e d by ozone pre t rea tment ( F i g . 6 ) . The e f f e c t o f i n c r e a s i n g the p re t rea tment dose was a l s o examined by i n c r e a s i n g pre t rea tment c o n c e n t r a t i o n i n seven-day p re t rea tments b e g i n n i n g e i g h t days a f t e r sowing . I n c r e a s i n g the p re t rea tment c o n c e n t r a t i o n i n c r e a s e d the s u s c e p t i b i l i t y to acute i n j u r y (as shown i n F i g . 6 ) . Response to the p re t rea tment thus appeared to c o n s i s t o f both a p r o t e c t i v e e f f e c t o c c u r r i n g below a t h r e s h o l d dosage and a p r e d i s p o s i t i o n o c c u r r i n g above that dosage . To look more e x t e n s i v e l y at the e f f e c t o f i n c r e a s i n g pre t rea tment dosage on acute s u s c e p t i b i l i t y , i t was more conven ien t to vary the d u r a t i o n of the p re t rea tment dose r a t h e r than the c o n c e n t r a t i o n . T h i s was done w i th pre t rea tment c o n c e n t r a t i o n s o f both 0.02 ppm and 0.05 ppm 47 1 \ 1 1 0 0 .02 0.04 0.06 0 .08 0.10 Pre t rea tment Ozone C o n c e n t r a t i o n (ppm) F i g u r e 6. Acute ozone i n j u r y to bean p l a n t s p r e t r e a t e d w i th v a r i o u s s u b - a c u t e d o s e s . Numbers on curve r e f e r to age (days from sowing) at s t a r t o f p r e -t r e a t m e n t ; • , 7 d a y s , acu te dose 0.15 ppm, 6 h ; •., 7 d a y s , acute d o s e , 0.4 ppm, 6h^»5 d a y s , -A-7. days • 10 d a y s , acute d o s e , 0.4 ppm for 2x6h; +_ SE of r e p r e s e n t a t i v e t r e a t m e n t s , n=6-10. See note to Tab le 2. ,48-. ozone ( F i g . 7 ) . U s i n g a c o n s t a n t p re t rea tment c o n c e n t r a t i o n o f 0.02 ppm, and v a r y i n g the d u r a t i o n of p re t rea tment from one to n ine d a y s , i t appeared tha t four days of p re t rea tment were r e q u i r e d f o r the onset o f p r o t e c t i o n ( F i g . 7 ) . T h i s p r o t e c t i v e e f f e c t remained r e l a t i v e l y c o n s t a n t f o r at l e a s t a f u r t h e r f i v e d a y s . The range o f p re t rea tment dosage e f f e c t i v e f o r p r o t e c t i o n was thus between 0.48 (i.e..', 4 days of 0.02 ppm; 6 h per day) and 1.08- ppm-h. T h i s may be compared to i n f o r m a t i o n from pre t rea tments w i t h i n c r e a s i n g c o n c e n t r a t i o n s ( F i g . 6 ) , i n which approx imate ly 0.06 ppm was r e q u i r e d f o r p r e d i s p o s i t i o n ; f o r seven d a y s , s i x h per day , or a t o t a l dose of 2.52 ppm-h. The approximate l i n e a r i t y o f t h i s response was a l s o t e s t e d by p re t rea tment at a c o n s t a n t c o n c e n t r a t i o n o f 0.05 ppm f o r one to n i n e d a y s . In tha t e x p e r i m e n t , the range of e f f e c t i v e p r o t e c t i o n was from one to th ree days ( F i g . 7 ) , a range i n dosage from 0.30 to 0.90 ppm-h. S i g n i f i c a n t p r e d i s p o s i t i o n o c c u r r e d a f t e r s i x d a y s , at t o t a l dosages of 1.80 ppm-h and g r e a t e r . At tempts to determine whether , . i n d a i l y exposure to 0.02 ppm, s u s c e p t i b i l i t y would u l t i m a t e l y have i n c r e a s e d to a s tage o f p r e d i s p o s i t i o n , were not p o s s i b l e w i t h t h i s system because o f the senescence o f the pr imary l eaves w i t h i n the time p e r i o d of the r e q u i r e d exper iment . Hence, i t was not p o s s i b l e to determine more p r e c i s e l y whether p r e d i s p o s i t i o n was a s t r i c t f u n c t i o n o f cumula t i ve dosage , i . e . , whether p r e d i s p o s i t i o n would have , , I I I Days from Sowing Figure 7 . E f f e c t of increasing pretreatment dose on acute ozone s u s c e p t i b i l i t y of bean. Daily pretreatments, 6 h per day, with either 0 . 0 2 ppm (») or 0 . 0 5 ppm (H) ozone, a f t e r transfer from f i l t e r e d a i r at day 8 ; + SE of representative treatments, n=6. See. note to Table 2 . 50 o c c u r r e d a f t e r approx imate ly ten days of exposure to 0.02 ppm. The i n t e r r e l a t i o n s h i p between 0.02 ppm and 0.05 ppm treatments at lower d o s a g e s , however, sugges ted tha t t h i s might be s o . The range o f e f f e c t i v e p r o t e c t i o n appeared to be from about 0.30 to 1.00 ppm-h, f o l l o w e d by a t r a n s i t i o n to p r e d i s p o s i t i o n at about 1.00 to 1.80 ppm-h. Above t h a t , the ex tent o f p r e d i s p o s i t i o n i n c r e a s e d w i t h d o s e , at l e a s t up to about 2.7 ppm-h (nine d a y s , 0.05 ppm). E f f e c t i v e dosage appeared to be s i m p l y the p r o d u c t o f c o n c e n t r a t i o n and t i m e , w i t h no extreme n o n - l i n e a r i t y i n the ranges t e s t e d (0.0 2 ppm to 0.0 5 ppm, one to n i n e d a y s ) . Whi le these ranges p r o v i d e a good assessment of the k i n d s o f s u b - a c u t e exposures which may occur immedia te ly p r i o r to an acute exposure d u r i n g f i e l d e p i s o d e s , s tudy o f the i n t e r a c t i o n s between the e f f e c t s o f s u b - a c u t e exposure on acute s u s c e p t i b i l i t y and on p l a n t growth r e q u i r e d a l o n g e r p re t rea tment exposure . E f f e c t s o f s e a s o n - l o n g s u b - a c u t e exposure on acu te s u s c e p t i b i l i t y were examined i n t h r e e c u l t i v a r s of g rape . ' I v e s ' i s the most s e n s i t i v e grape c u l t i v a r to acute exposures i n the l a b o r a t o r y , and shows the most i n j u r y i n the f i e l d , i n areas of h i g h ambient ozone . 'De laware ' i s the most t o l e r a n t , and ' C o n c o r d ' i s o f i n t e r m e d i a t e s u s c e p t i b i l i t y (Kender and C a r p e n t e r , 1974) , A f t e r p re t rea tment f o r one to th ree months w i t h e i t h e r f i l t e r e d a i r 51 or ambient a i r ( r e p r e s e n t i n g averages of 0.01 or 0.03 ppm ozone , r e s p e c t i v e l y - - s e e T a b l e 1 ) , v i n e s were exposed to an acute dose of 0.75 ppm ozone f o r s i x h . The most ozone t o l e r a n t o f the three c u l t i v a r s , ' D e l a w a r e ' , was the on ly one / i i i which there • was a s t a t i s t i c - a l l y - s i g n i f i c a n t e f f e c t of p r e t r e a t m e n t . In that c a s e , a f t e r th ree months o f p r e t r e a t m e n t , v i n e s p r e t r e a t e d w i t h ambient a i r were p r e d i s p o s e d to acute i n j u r y as compared to v i n e s p r e t r e a t e d w i th f i l t e r e d a i r (Table 3 ) . T h i s f i n d i n g suppor ted the compar ison between bean and m i n t , which suggested tha t more o z o n e - t o l e r a n t p l a n t s have a reduced tendency to h a r d e n i n g , o r , i n t h i s c a s e , an i n c r e a s e d tendency to p r e d i s p o s i t i o n . The range o f p r e t r e a t -ment doses encountered i n s e a s o n - l o n g exposures i s l i k e l y to be above the r e g i o n o f p r o t e c t i o n , so tha t w i t h i n t h i s a r e a , the phenomenon of p r e d i s p o s i t i o n i n t o l e r a n t p l a n t s w i l l p r o b a b l y be the phenomenon of g r e a t e s t c o n c e r n . Trends i n the data from Tab le 3, however, a l though below the l e v e l of s t a t i s t i c a l s i g n i f i c a n c e , suggested tha t i n ' C o n c o r d ' and ' I v e s ' an e a r l y season p e r i o d of p r o t e c t i o n was f o l l o w e d by a l a t e season tendency toward p r e d i s p o s i t i o n . A l t h o u g h t h i s e f f e c t o f p re t rea tment was super imposed on a d i f f e r e n t curve of changing acute s u s c e p t i b i l i t y w i th t i m e , the p a t t e r n o f change i n acute s u s c e p t i b i l i t y w i th changing p re t rea tment dosage here was s i m i l a r to the r e s u l t s o b t a i n e d w i th bean . 52 T a b l e 5. Acute i n j u r y s u s c e p t i b i l i t y o f p o t t e d g r apev ines grown i n ambient a i r (AA) or c h a r c o a l - f i l t e r e d a i r *(CF) . V INJURY Date JULY AUGUST SEPTEMBER Treatment CF AA CF AA CF AA Ives 20.0 20.0 39. 5 2 2.0 1.3 2.5 C u l t i v a r  Concord 8.1 4.8 39 . 8 21.3 1.1 1. 8 Delaware 0.0 0.0 1.0 3.2 S i g n i f i c a n c e of t reatment e f f e c t L e a f a rea n e c r o t i c a f t e r 6 h , 0.75 ppm ozone * = 51 l e v e l : Two way a n a l y s i s o f v a r i a n c e , t reatment x t ime 53 The i n t e r p r e t a t i o n of exper iments w i t h greenhouse-r a i s e d bean p l a n t s , and compar ison w i t h f i e l d e x p o s u r e s , depends on assumptions about normal background l e v e l s o f ozone . Use o f the word " h a r d e n i n g " to d e s c r i b e the p r o t e c t i v e e f f e c t o f s u b - a c u t e p re t rea tment assumes tha t a c c l i m a t i o n to low ozone o c c u r r e d over the course of these e x p e r i m e n t s . I f a low background c o n c e n t r a t i o n o f ozone i n the atmosphere r e p r e s e n t s a norm, and radaptation^"- has o c c u r r e d over a l o n g e r t ime s c a l e , the d i f f e r e n c e i n acute s u s c e p t i b i l i t y which has been a s c r i b e d to h a r d e n i n g might be more a c c u r a t e l y d e s c r i b e d as d e h a r d e n i n g , i . e . , a harmfu l e f f e c t o f f i l t e r e d a i r p re t rea tment as compared to p re t rea tment w i t h normal low ozone c o n c e n t r a t i o n s . In e i t h e r c a s e , o f c o u r s e , the a d a p t a t i o n to ozone would .have deve loped over the same time s c a l e , but i n the one case i t would e x i s t as a c a p a c i t y f o r h a r d e n i n g , w h i l e i n the o t h e r i t would e x i s t as a " p r e f e r e n c e " f o r growth i n low ozone c o n c e n t r a t i o n s . To t e s t these a l t e r n a t i v e s , beans were grown from seed i n e i t h e r con t inuous f i l t e r e d a i r or f i l t e r e d a i r to which 0.02 ppm ozone was added f o r s i x h per day. Some o f each type of p l a n t s were then t r a n s f e r r e d to the a l t e r n a t e regime b e f o r e acute f u m i g a t i o n . I f h a r d e n i n g o c c u r r e d as a r e s u l t o f exposure to 0.02 ppm we-would expect to see a decrease i n the s u s c e p t i b i l i t y o f those p l a n t s s t a r t e d i n f i l t e r e d a i r 54 and then swi tched to d a i l y ozone exposures as compared to those p l a n t s remain ing i n f i l t e r e d a i r th roughout . I f f i l t e r e d a i r i t s e l f was harmfu l we would expect to see an i n c r e a s e i n the s u s c e p t i b i l i t y o f those p l a n t s s t a r t e d i n d a i l y exposures to 0.02 ppm ozone and then sw i tched to f i l t e r e d a i r , as compared to those p l a n t s exposed to d a i l y low ozone th roughout . Comparing f i r s t the e f f e c t o f con t inuous t reatment i n f i l t e r e d a i r w i th con t inuous d a i l y ozone pre t rea tments ( F i g . 8 A , C , s o l i d l i n e s ) , there was a s m a l l but s i g n i f i c a n t l y g r e a t e r s u s c e p t i b i l i t y i n the p l a n t s grown i n f i l t e r e d a i r i n the e a r l y s tages o f growth (day 13 ) , which p e r s i s t e d through day 17 but d i s a p p e a r e d by day 21, d u r i n g which time the p l a n t s s u b j e c t e d to e i t h e r regime became s i g -n i f i c a n t l y more s u s c e p t i b l e to acute i n j u r y . T h i s e a r l y decrease i n s u s c e p t i b i l i t y was comparable to tha t observed i n g r e e n h o u s e - r a i s e d beans ( F i g . 6 , 7 ) , a l l o w i n g f o r d i f f e r e n c e s i n the environment d u r i n g e a r l y growth. The t r a n s f e r o f p l a n t s from d a i l y 0.02 ppm ozone to cont inuous f i l t e r e d a i r on day e i g h t ( F i g . 8A) or on day ten ( F i g . 8C) d i d not r e s u l t i n any s i g n i f i c a n t change i n the p a t t e r n o f i n c r e a s i n g s u s c e p t i b i l i t y w i t h t i m e . - . 55 F i g u r e 8. Acute ozone s u s c e p t i b i l i t y o f beans a f t e r growth i n cont inuous f i l t e r e d a i r o r v a r i o u s p a t t e r n s o f d a i l y exposure to 0.02 ppm ozone. S u s c e p t i b i l i t y c a l c u l a t e d as acute i n j u r y per t o t a l acute dose (about 3 ppm-h) . V e r t i c a l bars are s t a n d a r d e r r o r s (n=4). Arrows i n d i c a t e day on which t r a n s f e r between pre t rea tment regimes' o c c u r r e d . P re t rea tments : o , c o n t -ihous f i l t e r e d a i r ; 4*', d a i l y 0.02 ppm o z o n e ; . © ' , . . t rans-f e r r e d from d a i l y ozone to cont inuous f i l t e r e ' d a i r ; © , t r a n s f e r r e d from cont inuous f i l t e r e d a i r to d a i l y ozone . A , B , C , and D re fe r , to s e p a r a t e e x p e r i m e n t s . See note to T a b l e 2. 57 Hence i t i s apparent that the p r e v i o u s l y noted d i f f e r e n c e s between f i l t e r e d a i r and ozone pre t rea tment o f greenhouse r a i s e d p l a n t s cannot be e x p l a i n e d i n terms of the harmfu l e f f e c t o f f i l t e r e d a i r . In c o n t r a s t , the r e v e r s e t r a n s f e r (from cont inuous f i l t e r e d a i r to d a i l y 0.02 ppm ozone) r e s u l t e d i n a r a p i d i n c r e a s e i n s u s c e p t i b i l i t y , which appeared to r e q u i r e at l e a s t two d a i l y exposures to m a n i f e s t i t s e l f . Thus the e f f e c t i s c l e a r a f t e r t h r e e , f o u r , or f i v e days o f ozone pre t rea tment ( F i g . 8 A , C, D ) , but had not appeared a f t e r two days ( F i g . 8 B ) . A f t e r t h i s e a r l y r i s e , the s u s c e p t i b i l i t y o f those t r a n s f e r r e d p l a n t s remained c o n s t a n t over the next e i g h t d a y s , at a l e v e l which was s i g n i f i c a n t l y lower than that o f p l a n t s s u b j e c t e d to any o ther p r e t r e a t m e n t . The magnitude o f the d i f f e r e n c e i n s u s c e p t i b i l i t y 21 days a f t e r sowing , between p l a n t s grown i n f i l t e r e d a i r throughout and those t r a n s f e r r e d to low ozone , was about the same as the d i f f e r e n c e between the s u s c e p t i b i l i t y o f p l a n t s grown i n f i l t e r e d a i r and those grown i n low ozone , at day 13 ( F i g . 8 A , C ) . As a consequence i t appears tha t there was a decrease caused by low ozone pretreatment in the pattern'of increasing suscept ibi l i ty with age of plants from sowing. T h i s d e c r e a s e , however, appeared o n l y a f t e r the i n i t i a l i n c r e a s e i n s u s c e p t i b i l i t y i n p l a n t s t r a n s f e r r e d from f i l t e r e d a i r to low ozone. In the case o f p l a n t s r a i s e d from seed i n low 58 ozone, the e f f e c t d i s a p p e a r e d by 21 days ( F i g - 8 A , C ) . T h i s l a t t e r i n c r e a s e i n s u s c e p t i b i l i t y seems to be analogous to the t r e n d toward an i n c r e a s e d p r e d i s p o s i n g e f f e c t o f p re t rea tment w i th i n c r e a s i n g p re t rea tment dosage , as noted w i th g r e e n h o u s e - r a i s e d p l a n t s . The exper iments w i th f i l t e r e d a i r or 0.02 ppm ozone from seed thus r e v e a l e d an e a r l y s tage i n the pre t rea tment e f f e c t on s u s c e p t i b i l i t y , a s tage o f p r e d i s p o s i t i o n , when compared to exper iments w i th g r e e n h o u s e - r a i s e d p l a n t s . They a l s o demonstrated that the major e f f e c t i n those exper iments w i th g r e e n h o u s e - r a i s e d p l a n t s was d e s c r i b a b l e as a ha rden ing r a t h e r than a d e h a r d e n i n g . Stomata In exper iments w i th g r e e n h o u s e - r a i s e d bean s e e d l i n g s , the p o s s i b i l i t y that s tomata l b e h a v i o r was i n v o l v e d i n the observed e f f e c t o f s u b - a c u t e p re t rea tment on acute s u s c e p t i b i l i t y was t e s t e d by measur ing the R g of those p l a n t s d u r i n g the o z o n e - f r e e p a r t o f the d a i l y c y c l e , d u r i n g both s u b - a c u t e p re t rea tment and the subsequent acute t rea tment . I t appeared tha t s tomata l c l o s u r e induced by the pre t rea tment exposure might be at l e a s t a p a r t i a l e x p l a n a t i o n of the e f f e c t on i n j u r y . The s tomata l conductance (K ) o f g r e e n h o u s e - r a i s e d bean p l a n t s p r e t r e a t e d w i t h th ree d a i l y doses ( s i x h) o f 0.02 ppm ozone was reduced 35% as compared to p l a n t s p r e t r e a t e d w i t h f i l t e r e d a i r ( F i g . 9 ) . A f t e r a f u r t h e r two days of p r e t r e a t m e n t , the p l a n t s were s u b j e c t e d to two acute doses of ozone. V i s i b l e i n j u r y i n these p l a n t s a f t e r the acute exposures was 53% l e s s i n o z o n e - p r e t r e a t e d p l a n t s than i n p l a n t s p r e t r e a t e d w i th f i l t e r e d a i r . A t l e a s t p a r t o f t h i s p r o t e c t i v e e f f e c t would appear to have been due to reduced en t ry o f ozone i n t o the l e a f d u r i n g the acute exposure as a r e s u l t o f p r e t r e a t m e n t -induced s tomata l c l o s u r e . Two types o f s tomata l c l o s u r e are apparent i n F i g u r e 9. In a d d i t i o n to the p r e t r e a t m e n t - i n d u c e d c l o s u r e observed on day f o u r , f u r t h e r c l o s u r e i s e v i d e n t i n both t reatments a f t e r the f i r s t acute dose on day f i v e . The s tomata l c l o s u r e i n response to acute exposure may have been due to the p h y s i c a l damage to ep iderma l and m e s o p h y l l i c t i s s u e s induced by tha t e x p o s u r e , and the r e l a t i v e e f f e c t on the two t reatments was not a f f e c t e d by an a d d i t i o n a l acute e x p o s u r e , i n d i c a t i n g that at t h i s s tage s tomata l a p e r t u r e was not a p r imary determinant o f f u r t h e r i n j u r y . S i n c e these s tomata l e f f e c t s were measured d u r i n g the o z o n e - f r e e p e r i o d s , n e i t h e r o f the types of s tomata l c l o s u r e observed here was r e v e r s i b l e by removal o f the ozone. In a d d i t i o n to t h i s i r r e v e r s i b l e form o f s tomata l r e s p o n s e , there may a l s o be a component o f the s tomata l e f f e c t o f ozone which occurs o n l y d u r i n g the p resence of ozone 6-0 4 5 6 7 Days A f t e r T r a n s f e r from F i l t e r e d A i r F i g u r e 9- Stomatal conductance of beans d u r i n g sub-acute pretreatment and subsequent acute treatment with ozone. Fourteen-day-old seed-l i n g s t r a n s f e r r e d to continuous f i l t e r e d a i r (O) or d a i l y 6 h exposure to 0.02 ppm ozone (») f o r 5 days p r i o r to acute doses (*) of 0.40 ppm, ± SE, n=6. Stomatal measurements made 1.5 h b e f o r e ozone exposure and 1.5 h a f t e r b e g i n n i n g of l i g h t p e r i o d . See note to Table 2. 61 i n the atmosphere. S p e c i f i c a l l y , there i s the p o s s i b i l i t y that s u b - a c u t e p re t rea tment may a f f e c t acute s u s c e p t i b i l i t y by a f f e c t i n g s tomata l response to acute ozone i n a manner that can on ly be measured d u r i n g the presence of the acute ozone dose and not d u r i n g the o z o n e - f r e e p e r i o d . To i n v e s t i g a t e t h i s p o s s i b i l i t y , t r a n s p i r a t i o n was measured d u r i n g acute f u m i g a t i o n f o r p l a n t s p r e t r e a t e d w i th f i l t e r e d a i r f o l l o w e d by 0.02 ppm ozone a c c o r d i n g to the schedu le d e s c r i b e d i n F i g u r e 8 A . Stomata l response to acute f u m i g a t i o n was observed at two s tages i n the s u s c e p t i b i l i t y r e s p o n s e , the e a r l y s tage o f i n c r e a s e d s u s c e p t i b i l i t y (14 days) and the l a t e r s tage of decreased s u s c e p t i b i l i t y (21 d a y s ) . A t 14 d a y s , when the s u s c e p t i b i l i t y o f o z o n e - p r e t r e a t e d p l a n t s was g r e a t e r than tha t o f p l a n t s p r e t r e a t e d w i th f i l t e r e d a i r , stomata had a decreased a b i l i t y to c l o s e i n response to acute ozone exposure which may e x p l a i n t h e i r i n c r e a s e d s u s c e p t i b i l i t y ( F i g . 10A)-. A l t h o u g h there was no d i f f e r e n c e i n t r a n s p i r a t i o n r a t e s between the two t reatments p r i o r to the b e g i n n i n g of acute ozone f u m i g a t i o n , stomata o f the p l a n t s p r e t r e a t e d w i t h f i l t e r e d a i r began c l o s u r e immediate ly upon the i n t r o d u c t i o n of 0.40 ppm ozone, whereas t r a n s p i r a t i o n i n the o z o n e - p r e t r e a t e d p l a n t s c o n t i n u e d to i n c r e a s e f o r two h b e f o r e c l o s u r e began . T h i s reduced s e n s i t i v i t y o f stomata to c l o s u r e i n the p resence o f h i g h ozone c o n c e n t r a t i o n s , i n o z o n e - p r e t r e a t e d p l a n t s , 6 2 F i g u r e 10. . T r a n s p i r a t i o n r a t e s o f a t t a c h e d pr imary bean l eaves d u r i n g acute ozone e x p o s u r e , a f t e r p r e -t reatment w i th e i t h e r f i l t e r e d a i r or 0.02 ppm ozone . A , 14 days from sowing; B , 21 days from sowing . Pre t rea tments :. © , con t inuous f i l t e r e d air - ; © , t r a n s f e r r e d from cont inuous f i l t e r e d a i r to d a i l y 0.0 2 ppm ozone .(6 h per day) 7 days from sowing . Ozone (0.40 ppm) i n t r o d u c e d at arrow. Res u l t s shown here a re from a r e p r e s e n t a t i v e r u n , J: t rea tments were r e p l i c a t e d 2-4 t i m e s . See note to T a b l e 2. 63 o 1100 1200 1300 1400 1500 1600 Hour of Day 64 c r e a t e d a p e r i o d d u r i n g which the stomata were open wider and the atmosphere c o n t a i n e d p o t e n t i a l l y t o x i c c o n c e n t r a t i o n s of ozone. The i n c r e a s e d en t ry of ozone i n t o the l e a f at that t ime c o u l d have accounted f o r the i n c r e a s e d s u s c e p t i b i l i t y o f o z o n e - p r e t r e a t e d p l a n t s to acute i n j u r y at t h i s s t a g e . At 21 d a y s , the absence o f any decrease i n t r a n s p i -r a t i o n i n response to acute f u m i g a t i o n , i n o z o n e - p r e t r e a t e d p l a n t s , i n d i c a t e d a c o n t i n u a t i o n o f the reduced s e n s i t i v i t y o f o z o n e - p r e t r e a t e d stomata to h i g h ozone c o n c e n t r a t i o n s t^'ig. 10B). At 21 daysj the i n i t i a l t r a n s p i r a t i o n r a t e s were lower i n o z o n e - p r e t r e a t e d p l a n t s than they were i n p l a n t s p r e t r e a t e d w i th f i l t e r e d a i r . The t reatment d i f f e r e n c e r e s u l t i n g i n decreased s u s c e p t i b i l i t y to acute i n j u r y may thus a l s o be p a r t i a l l y e x p l a i n e d by s tomata l b e h a v i o r , i n t h i s c a s e , by the decrease i n i n i t i a l a p e r t u r e , caused by l o n g - t e r m low l e v e l p re t rea tment r a t h e r than by the e f f e c t o f low ozone pre t rea tment on s tomata l response to h i g h ozone c o n c e n t r a t i o n s such as was r e s p o n s i b l e f o r the i n c r e a s e d s u s c e p t i b i l i t y at 14 d a y s . I t appeared then that the component o f s tomata l b e h a v i o r which a f f e c t e d response to acute exposure on ly d u r i n g the presence o f h i g h ozone c o n c e n t r a t i o n s was a l s o i n v o l v e d i n the response to low ozone pre t rea tment d u r i n g the e a r l y stage of i n c r e a s e d s u s c e p t i b i l i t y . In c o n t r a s t , the l a t e r p r o t e c t i v e e f f e c t of low ozone pre t rea tment was a s s o c i a t e d 65 w i th an i r r e v e r s i b l e e f f e c t on stomata as a r e s u l t o f which they showed ho response to acute exposure . F u r t h e r i n v e s t i g a t i o n o f these p o s s i b i l i t i e s was conducted i n exper iments w i t h g r a p e v i n e s , to determine the e x i s t e n c e and r e l a t i v e importance o f these types o f s tomata l response d u r i n g a s e a s o n - l o n g exposure to low l e v e l s of ozone, and to determine t h e i r r e l a t i o n s h i p i n tha t i n s t a n c e to changes i n acute s u s c e p t i b i l i t y and growth. V i n e s f o r these exper iments were removed from the f i l t e r e d a i r or ambient a i r p re t rea tment chambers p e r i o d i c a l l y through the growing season and R was measured i n f i l t e r e d a i r and a f t e r two h o f f u m i g a t i o n w i t h 0.75 ppm ozone . As shown i n T a b l e 4, there were no s i g n i f i c a n t t reatment e f f e c t s on e i t h e r i n i t i a l R i n f i l t e r e d a i r or the change s & i n R g i n response to acute f u m i g a t i o n , i n any o f the c u l t i v a r s t e s t e d . Exposure to s u b - a c u t e p re t rea tment c o n c e n t r a t i o n s approx imate ly e q u i v a l e n t to those used i n exper iments w i th b e a n , f o r an e n t i r e growing s e a s o n , had no measurable e f f e c t on s t o m a t a l b e h a v i o r i n . g r a p e . The p r e d i s p o s i t i o n of 'De laware ' v i n e s to acute i n j u r y d i d not appear to be r e l a t e d to an e f f e c t on p o l l u t a n t e n t r y , a l though the data t rends f o r 'De laware ' a f t e r th ree months o f p re t rea tment d i d not c o n t r a d i c t a p o s s i b l e mechanism o f tha t t y p e . Thus at t h i s t ime , o z o n e - p r e t r e a t e d 'De laware ' v i n e s tended to have both a lower i n i t i a l R g and a l e s s e r degree o f c l o s u r e i n response to acute f u m i g a t i o n . Tab1e 4. S tomata l b e h a v i o r o f p o t t e d grapev ines grown i n ambient a i r (AA) or c h a r c o a l - f i l t e r e d a i r ( C F ) . Date Treatment V Ives i n i t i a l R change i n R s C u l t i v a r i n i t i a l R S • W Concord cTTange i n R Delaware i n i t i a l change R i n R JULY AUGUST SEPTEMBER CF AA CF AA CF AA 12 . 5 7 . 9 2.1 2.. 9 7. 7 5.8 0 . 8 2.1 1.3 , 1.4 - 0 . 3 -0 .2 4.1 4.0 3,4 3.1 5.1 4.4 0.4 •0.1 0 . 4 0 . 2 1.7 1.0 7.7 9 .1 5.1 4 . 2 3.5 2 . 2 3.9 3.4 V D a t a show no s i g n i f i c a n t t reatment e f f e c t s , S% l e v e l , two way a n a l y s i s of v a r i a n c e , t reatment x t ime W -1 Stomata l r e s i s t a n c e (sec cm ) at s t a r t o f ozone f u m i g a t i o n X -1 Change in s tomata l r e s i s t a n c e (s ec cm ) a f t e r 2 h of 0.75 ppm ozone ON 67 Growth The invo lvement o f an i r r e v e r s i b l e dampening o f s tomata l f u n c t i o n w i t h the p r o t e c t i v e e f f e c t o f s u b - a c u t e ozone p r e t r e a t m e n t , i n b e a n , suggested the p o s s i b i l i t y o f an accompanying r e d u c t i o n i n p l a n t growth. Both the growth and s u s c e p t i b i l i t y responses c o u l d r e s u l t from a s i n g l e , p r i m a r i l y s t o m a t a l , mechanism. I t was a l s o p o s s i b l e , however, tha t the e f f e c t o f s u b - a c u t e p re t rea tment on acute s u s c e p t i b i l i t y was independent o f any e f f e c t on growth, or t h a t i t c o u l d be accompanied by an i n c r e a s e d r a t e o f p l a n t growth. Exposure of bean s e e d l i n g s to s u b - a c u t e ozone t reatments was found to i n h i b i t stem growth by 21 days from sowing (P ig - 11) . S e e d l i n g s grown from seed i n 0.02 ppm ozone were found to be s h o r t e r than those grown i n f i l t e r e d a i r . T r a n s f e r from 0.02 ppm ozone to f i l t e r e d a i r , at day e i g h t , had no e f f e c t on stem h e i g h t by day 25, i n d i c a t i n g the importance of the f i r s t e i g h t days o f growth f o r the e f f e c t o f ozone on stem growth. T r a n s f e r frOm f i l t e r e d a i r to low ozone at day e i g h t a l s o had no e f f e c t i n r e l a t i o n to growth i n f i l t e r e d a i r on stem h e i g h t by day 25, a l though a d i f f e r e n c e may have been b e g i n n i n g to appear at tha t t i m e . A f t e r b e i n g swi tched from f i l t e r e d a i r to 0.02 ppm ozone at day e i g h t , the acute s u s c e p t i b i l i t y o f bean s e e d l i n g s was s i g n i f i c a n t l y reduced by day 21, so tha t even i f t h i s 68 Figure 11. Days from Sowing Height of bean plants grown i n f i l t -ered a i r or 0 . 0 2 ppm ozone-. Treatments: O, f i l t e r e d a i r from seed; <• , switched from f i l t e r e d a i r to low ozone ( 0 . 0 2 ppm, 6 h per day) at day 8 ; • , low ozone from seed; o, switched from low ozone to f i l t -ered a i r at day 8 , ± SE of representative treatments, n=4. 69 switch d i d l e a d to a s i g n i f i c a n t h e i g h t r e d u c t i o n at some po i n t beyond 25 days, the two responses (height and s u s c e p t i b i l i t y ) would not be synchronous. This would be expected, hox^ever, i f both of these responses were mediated by the same stomatal mechanism, s i n c e the l a g i n the growth response c o u l d be expected to be g r e a t e r . The p o s s i b i l i t y of a common stomatal mechanism l i n k i n g a p r o t e c t i v e e f f e c t of sub-acute treatment and a r e d u c t i o n i n growth was not c o n t r a d i c t e d by the r e s u l t s of these experiments on bean stem growth, but the data showed no e f f e c t on stem h e i g h t o f exposure to 0.02 ppm ozone a f t e r the f i r s t e i g h t days of growth. Data on the growth of beans during the f i r s t 25 days a f t e r sowing-were not adequate f o r an e v a l u a t i o n of the r e l a t i o n s h i p between the growth and s u s c e p t i b i l i t y e f f e c t s of low ozone pretreatment. To answer questions about that r e l a t i o n s h i p i t was necessary to examine the data from experiments on the exposure of grapevines to low ozone f o r an e n t i r e growing season. For these experiments, p o t t e d grapevines were grown i n open-top f i e l d chambers r e c e i v i n g e i t h e r ambient a i r or c h a r c o a l - f i l t e r e d a i r . Vines were removed from the chambers p e r i o d i c a l l y f o r an assessment of s u s c e p t i b i l i t y to acute ozone i n j u r y (Table 3). At those same i n t e r v a l s , p a r a l l e l samples were removed f o r assessment of vine growth, by measurement of f r e s h weight and dry weight of leaves, stems, trunk, and r o o t s . 7Q In v ines o f the ' I v e s ' c u l t i v a r , growth was g r e a t e r i n ambient a i r than i n f i l t e r e d a i r (Table 5 ) . Both top weight and r o o t w e i g h t , and both dry weight and f r e s h w e i g h t , were a f f e c t e d approx imate ly e q u a l l y by these t r e a t m e n t s . In v i n e s of the ' C o n c o r d ' c u l t i v a r a l s o , growth was s i g n i f i c a n t l y g r e a t e r i n ambient a i r (Table 6 ) . However, i n t h i s c u l t i v a r o n l y l e a f we ights (both dry and f r e s h ) were s i g n i f i c a n t l y a f f e c t e d . In ' D e l a w a r e ' , there were no s i g n i f i c a n t d i f f e r e n c e s i n v i n e growth between f i l t e r e d a i r or ambient a i r t reatment (Table 7 ) . The r a n k i n g o f these c u l t i v a r s w i t h r e s p e c t to the e f f e c t o f ambient a i r ve rsus f i l t e r e d a i r t reatment appeared to be d i r e c t l y r e l a t e d to t h e i r i n j u r y s e n s i t i v i t y (Kender and C a r p e n t e r , 1974). The c u l t i v a r most s e n s i t i v e to ozone i n j u r y , ' I v e s ' , showed the g r e a t e s t e f f e c t s on growth. The most ozone t o l e r a n t c u l t i v a r , ' D e l a w a r e ' , showed the l e a s t e f f e c t on growth. ' D e l a w a r e ' , however, showed the g r e a t e s t e f f e c t o f t h i s same t reatment on acute s u s c e p t i b i l i t y (Table 3 ) . Comparing c u l t i v a r s , the growth e f f e c t o f low ozone t rea tment , t h e r e f o r e , appeared to be i n v e r s e l y r e l a t e d to the pretreatment effect ' on s u s c e p t i b i l i t y . B a s e l i n e c u l t i v a r s e n s i t i v i t y to ozone i n j u r y and the s t i m u l a t o r y e f f e c t s o f low ozone t reatment on growth on the o ther hand were p o s i t i v e l y c o r r e l a t e d , w h i l e both o f these were a s s o c i a t e d w i th the absence of a tendency toward a p r e d i s p o s i n g e f f e c t T a b l e 5. Growth o f p o t t e d ' I v e s ' v i n e s i n ambient a i r (AA) or c h a r c o a l - f i l t e r e d a i r (CF ) . l e a f roo t a rea top dry w t / f r e s h wt F resh xveight C R ) Dry weight (cm ) Lwt J D a t e v Leaves shoot t runk r o o t s l eaves" shoot t runk roo ts r o o t shoot l eaves JULY CF 6.9 4.1 11.3 26.6 1. 7 0 . 8 5.1 7.0 324. 3 •3.0 0. 26 0 . 21 0 . 24 AA 9.7 6.0 12.0 33. 6 2.3 1.1 5.3 7.9 438.0 2. ,6-' 0 . 24 0 .18 0.23 AUG CF 6.4 3.2 15. 5 27 .6 . 1.8 0.8 7.3 7.2 367.8 2.8 0. 26 0 . 26 0 . 29 AA 10 . 3 7.1 14.6 38. 2 2 . 7 1.8 6.8 8.9 .561.9 2.0 0.24 0 . 26 0 .27 SEPT CF 5. 3 4.4 13.5 30 . 5 1.8 1.3 7.1 8. 5 314.1 3.1 0.28 0 . 29 0 . 35 AA 7.5 5.6 13.4 42.6 2.4 1.9 7.1 10.4 372.9 2 . 5 0 . 26 0.34 0 . 33 S i g n i f i c a n c e w of t reatment * * ft ft ft ft ft ftft e f f e c t v T r e a t m e n t begun June 15 w * = 5%, * * = 1%; two way a n a l y s i s o f v a r i a n c e , t reatment x t ime , n=6 T a b l e 6. Growth o f p o t t e d ' C o n c o r d ' v ines i n ambient a i r (AA) or c h a r c o a l - f i l t e r e d a i r (CF ) . F resh weight (S) Dry weight (g) l e a f a rea (cm ) roo t top Lwt J dry w t / f r e s h wt D a t e V l eaves shoot t runk r o o t s leaves shoot t runk roo ts r o o t shoot l eaves JULY CF 8.3 6.2 16.1 29.1 2.0 1.1 .7 .5 7.8 426.3 2.5 0 .27 0 .19 0 . 24 AA 11. 3 7.1 : 17.5 33..5' 2.9 1.4 8.0. 7.7 465. 3 1.9 0 .23 0 .19 0 .25 AUG ,CF 11. 5 7.7 20 .4 38.9 3.3 1.9 10.0 8.5 634. 5 1.6 0 .22 0 .25 0. 29 AA 13.8 8.8 15. 6 60.0 4.1 2.3 7.7 10.1 760 . 8 1.7 0 . 20 0 . 26 0.29 SEPT CF 7.7 5.9 21. 3 4 3.9 2.9 ' 2 . 2 1-2.0 11.6 553. 5 2.3 0 .27 0 . 38 0 . 39 AA 9 . 3 5.9 19 .6 47.0 3.5 2 . 2 11.2 12.2 555.9 2.2 0 . 26 0 . 36 0.37 w S i g n i f i c a n c e o f t reatment * e f f e c t v Treatment begun June 15 w. 5%; two way a n a l y s i s o f v a r i a n c e , t reatment x t ime, n-6 T a b l e 7. Growth o f p o t t e d 'De laware ' v ines i n ambient a i r ' ( A A ) or c h a r c o a l - f i l t e r e d a i r (CF ) . Date v F r e s h weight (g)  l eaves shoot t runk r o o t s l e a f root area top Dry weight (g) , l eaves shoot t runk roo ts dry wt / - f resh wt r o o t . s h o o t l e a v e s JULY CF 8. 2 4.0 17. 2 18.2 1.7 0,7 8.1 5.5 449 .4 2.4 0 . 30 0.17 0 . 21 AA 6. 8 3.1 17. 5 15.1 1.6 0.5 8.3 4.6 384 .0 2.3 0 . 29 0.17 0 .23 SEPT CF 5. 1 2.9 14. 8 24.4 1.6 0 . 8 8.1 6.6 429 .0 2.8 0 . 27 0 . 29 0 .31 AA 6. 8 3.7 18. 5 30.4 2.1 1.0 10.0 7.8 537 . 6 2.6 0 . 26 0 . 29 0 . 30 S i g n i f i c a n c e _ . _ - a n NS - - - -o f t reatment e f f e c t v T r e a t m e n t begun June 15 w Two way a n a l y s i s o f v a r i a n c e , t reatment x t i m e , n=6 74 of low ozone t rea tment . However, i t was found that c o r r e l a t i o n s between b a s e l i n e s e n s i t i v i t y to ozone ( r e g a r d l e s s of the e f f e c t o f low ozone t r e a t m e n t ) , the e f f e c t o f low ozone t r e a t -ment on growth, and the e f f e c t o f low ozone t reatment on acute s u s c e p t i b i l i t y , based on c u l t i v a r - t o - c u l t i v a r v a r i a t i o n , were not n e c e s s a r i l y i n d i c a t i v e of c o r r e -l a t i o n s between these same q u a n t i t i e s based on v i n e - t o -v i n e v a r i a t i o n w i t h i n c u l t i v a r s . To t e s t t h i s l a t t e r p o i n t , v i n e s h a r v e s t e d at the end o f the growing season were s u b j e c t e d to the v a r i o u s ana lyses s e r i a l l y , r a t h e r than i n p a r a l l e l samples . Acute i n j u r y to 'De laware ' v i n e s was p o s i t i v e l y c o r r e l a t e d w i th f r e s h and dry weights o f l eaves and shoots (Table 8 ) . There was a l s o a p o s i t i v e c o r r e l a t i o n i n tha t c u l t i v a r between acute i n j u r y and i n i t i a l R g at the time o f acute f u m i g a t i o n . V i n e s w i t h a h i g h e r R g ( s m a l l e r s tomata l aper tu re ) were more s u s c e p t i b l e to acute i n j u r y , i n d i c a t i n g that the r o l e o f stomata i n p o l l u t a n t en t ry was o f secondary importance to an i n d i r e c t a s s o c i a t i o n between i n c r e a s e d top growth and increased--suscept ib i l i ty - . . • ' In ' I v e s ' , there were no s i g n i f i c a n t c o r r e l a t i o n s between any of the growth parameters measured and acute i n j u r y (Table 9 ) . C o r r e l a t i o n s between s tomata l v a r i a b l e s and i n j u r y were a l s o not s t a t i s t i c a l l y s i g n i f i c a n t , but T a b l e 8. "De laware ' : s imple c o r r e l a t i o n s of growth, i n j u r y , and s tomata l b e h a v i o r , September. C o r r e l a t i o n c o e f f i c i e n t v F resh weight Dry weight i n j u r y i n i t i a l R leaves shoot t runk r o o t s leaves shoot t runk roots r a t e .65* .83* - . 0 5 .53 P h o t o s y n t h e t i c w Change x I n i t i a l ^ .64* .76* .21 .29 07 .44. 60*- .11 p h o t o s y n t h e t i c r a t e .26 - . 1 9 26 .59 25 .31 55 .02 change i n R - . 2 9 .07 - . 3 0 - . 2 4 - . 30 - . 24 - . 30 - .36 22 - .06 .20 .41 06 59 .11 i n R s_ 04 39 R .63* v n = 10, * = s i g n i f i c a n t at 5% l e v e l - 2 ~ 1 w i n ambient a i r , 3.5 to 6.6 / * E cm min PAR •^Change i n s tomata l r e s i s t a n c e a f t e r 2 h o u r s , 0.75 ppm ozone z L e a f i n j u r y (LAN) 24 hours a f t e r a 6 hour f u m i g a t i o n w i th 0.75 ppm ozone ^Stomata l r e s i s t a n c e at s t a r t o f ozone f u m i g a t i o n T a b l e 9. ' I v e s ' : s imple c o r r e l a t i o n s of growth, i n j u r y , and s tomata l b e h a v i o r , September. C o r r e l a t i o n c o e f f i c i e n t F r e s h weight . Dry weight P h o t o s y n t h e t i c * Change x I n i t i a l ^ leaves shoot t runk r o o t s leaves shoot t runk roo ts r a t e i n R g R g m j ury i n i t i a l R s change i n R 33 - .07 - . 4 8 .23 78* - .42 - . 1 9 - . 4 4 19 - .01 - . 2 2 .61 29 - . 0 5 - .46 .12 p h o t o s y n t h e t i c r a t e . 53 .26'" - . 23 .74* .76* - . 4 6 - . 19 .25 .10 - .21 52 34 • - . 27 15 68* 41 41 59 58 33 39 - .51 v n = 9, * = s i g n i f i c a n t at 5% l e v e l 2 1 w i n ambient a i r , 3.5 to 6.6 f j i cm min PAR x Change i n s tomata l r e s i s t a n c e a f t e r 2 h o u r s , 0.75 ppm ozone ^Stomata l r e s i s t a n c e at s t a r t o f ozone f u m i g a t i o n z L e a f i n j u r y (LAN) 24 hours a f t e r a 6 hour f u m i g a t i o n w i th 0.75 ppm ozone. as 77 there was some i n d i c a t i o n o f a n e g a t i v e c o r r e l a t i o n between i n j u r y and i n i t i a l R g . S i n c e i n i t i a l R s was n e g a t i v e l y c o r r e l a t e d w i th l e a f w e i g h t , t h i s may have i n d i c a t e d an i n d i r e c t a s s o c i a t i o n between top growth and s u s c e p t i b i l i t y , mediated by stomata i n t h e i r r o l e as the s i t e o f p o l l u t a n t e n t r y , whereby i n c r e a s e d top growth would i n c r e a s e s u s c e p t i b i l i t y , c o n t r a r y to the e f f e c t seen i n 1 D e l a w a r e ' . In ' C o n c o r d ' v i n e s a l s o , there .were no s i g n i f i c a n t c o r r e l a t i o n s between i n j u r y and growth (Table 10). R e l a t i o n s h i p s between p h o t o s y n t h e t i c r a t e and v a r i o u s growth p a r a m e t e r s , however, sugges ted the p o s s i b i l i t y o f a complex i n t e r a c t i o n between growth and i n j u r y w i t h a g r e a t e r tendency toward i n j u r y i n v i n e s w i t h h i g h e r p h o t o s y n t h e t i c r a t e s , l a r g e r t r u n k s , and reduced top growth. There was no i n d i c a t i o n t h a t t h i s i n d i r e c t i n t e r a c t i o n between growth and i n j u r y was mediated by s tomata l mechanisms. V i n e - t o - v i n e c o r r e l a t i o n s , w i t h i n c u l t i v a r s , thus r e v e a l e d a v a r i e t y , o f systems of a s s o c i a t i o n between i n j u r y s u s c e p t i b i l i t y , v i n e growth, and s t o m a t a l b e h a v i o r . In a l l c a s e s , i n c r e a s e d ozone s u s c e p t i b i l i t y c o u l d be l o o s e l y a s s o c i a t e d w i t h some form of growth i n c r e a s e , though the b a l a n c e between growth dependent on c u r r e n t p h o t o s y n t h e s i s o r on r e s e r v e s s t o r e d i n the t runk v a r i e d from c u l t i v a r to c u l t i v a r (see T a b l e 11). T a b l e 1 0 / ' C o n c o r d ' : s imple c o r r e l a t i o n s between growth, i n j u r y , and s tomata l b e h a v i o r , September. C o r r e l a t i o n c o e f f i c i e n t v m j ury i n i t i a l R s change i n R Fresh weight Dry weight l eaves shoot t runk r o o t s leaves shoot t runk roo ts - . 0 4 - . 0 2 .51 .02 P h o t o s y n t h e t i c w Change x I n i t i a l ^ 24 .11 .35 - . 4 6 - .03 15 - . 22 - . 1 8 p h o t o s y n t h e t i c r a t e - . 59* - . 72* - . 28 - . 4 9 .01 - . 0 5 .48 .01 .28 .16 .30 - .49 .07 .02 - . 22 - . 19 .69* .72* - . 22 r a t e 55 28 19 i n R R - . 40 - .01 - .19 v n = 11, * = s i g n i f i c a n t at 51 l e v e l 2 1 w i n ambient a i r , 3.5 to 6. 6 f*E cm min PAR x Change i n s tomata l r e s i s t a n c e a f t e r 2 h o u r s , 0.75 ppm ozone ^Stomata l r e s i s t a n c e at s t a r t of ozone f u m i g a t i o n z L e a f i n j u r y (LAN) 24 hours a f t e r a 6 hour f u m i g a t i o n w i t h 0.75 ppm ozone T a b l e 11. Summary o f i n d i c a t i o n s from v i n e - t o - v i n e c o r r e l a t i o n s , w i t h i n c u l t i v a r s C o r r e l a t i o n w i t h ozone i n j u r y s u s c e p t i b i l i t y V C u l t i v a r Growth Parameter S tomata l Behav io r Top Trunk P h o t o s y n t h e t i c w I n i t i a l x Change 7 wt wt r a t e R i n R_ s Ives + • + 0 Concord - + +. 0 0 Delaware + - 0 + . 0 v + , p o s i t i v e ; - , n e g a t i v e ; 0, no c o r r e l a t i o n w i n ambient a i r , 3.6 to 6.6 yW.E cm ^min PAR s tomata l r e s i s t a n c e at s t a r t o f ozone f u m i g a t i o n ^change i n s t o m a t a l r e s i s t a n c e a f t e r 2 h , 0.75 ppm ozone 80 DISCUSSION The p r o t e c t i v e e f f e c t o f a p re t rea tment ozone exposure a g a i n s t a subsequent acute exposure has been p r e v i o u s l y observed by Macdowal l (1965), Heck and Dunning (1967) , and W i l t o n et a l . (1969). A p r e d i s p o s i n g e f f e c t o f p r i o r exposure has been observed by Macdowal l (1965) and Heagle and Heck (1974). In each o f these p r e v i o u s c a s e s , however, the p re t rea tment dose was i n i t s e l f an acute d o s e . The r e s u l t s d e s c r i b e d here f o r bean and mint r e p r e s e n t the f i r s t r e p o r t e d case o f a s i g n i f i c a n t i n t e r a c t i o n between s u b - a c u t e p re t rea tment and subsequent acute exposure . The e x i s t e n c e o f t h i s type of response means tha t the assessment o f an acute ozone ep isode i n the f i e l d cannot be based s imp ly on the magnitude o f the dose above the t h r e s h o l d f o r v i s i b l e i n j u r y , nor can the t o t a l e f f e c t i v e dose f o r a s e r i e s o f ep isodes be c a l c u l a t e d s imp ly by summation o f exposures above tha t t h r e s h o l d . The e f f e c t o f every acute exposure must be q u a l i f i e d by c o n s i d e r a t i o n o f the ozone env i ronment , even i f exposure l e v e l s are below the v i s i b l e i n j u r y t h r e s h o l d , f o r a t l e a s t s e v e r a l days p r i o r to the acute exposure . L i k e w i s e , i n l a b o r a t o r y exper iments d e s i g n e d to examine the response to acute e x p o s u r e , r e s e a r c h e r s must s p e c i f y the ozone environment i n which p l a n t s were grown 81 p r i o r to acute exposure . The d i f f e r e n c e between c h a r c o a l -f i l t e r e d a i r or ambient a i r , i n t h i s growth env i ronment , may e v i d e n t l y have a l a r g e e f f e c t on p l a n t response to the acute exposure . The extent and f r e q u e n c y , i n n a t u r e , o f ozone c o n c e n t r a t i o n s as low as those found i n f i l t e r e d a i r , i s q u e s t i o n a b l e (Bennett et a l . , 1974) . I t was, t h e r e f o r e , not obvious from r e s u l t s on the s u b - a c u t e p re t rea tment o f g r e e n h o u s e - r a i s e d beans whether the d i f f e r e n c e between f i l t e r e d a i r or s u b - a c u t e ozone pre t rea tment r e p r e s e n t e d an a c c l i m a t i o n to low ozone l e v e l s d u r i n g the course o f the exper iments (hardening) or a d e b i l i t a t i n g e f f e c t o f f i l -t e r e d a i r t reatment d u r i n g the course o f the exper iments (dehardening) on p l a n t s a c c l i m a t e d to or n a t u r a l l y s e l e c t e d f o r t o l e r a n c e to low ozone l e v e l s i n the atmosphere. Exper iments i n which bean p l a n t s were r a i s e d from seed i n the f i l t e r e d a i r o r low ozone environments showed tha t the s w i t c h from f i l t e r e d a i r or low ozone environments was the major component of the suscept ibi l i ty change and . ' had a l a r g e r e f f e c t on s u s c e p t i b i l i t y than the s w i t c h from low ozone to f i l t e r e d a i r . T h i s i n d i c a t e d tha t h a r d e n i n g , not d e h a r d e n i n g , was the major component o f the observed p r o t e c t i v e e f f e c t . I t a p p e a r e d , however, that there was some a d d i t i o n a l i n t e r a c t i o n of p l a n t age w i t h the response to low ozone p r e t r e a t m e n t . The e f f e c t s of p re t rea tment w i t h low ozone on the pr imary l eaves of bean can be d i v i d e d i n t o th ree s t a g e s . 82 F i r s t , a s tage of p r e d i s p o s i t i o n , f o l l o w e d by a s tage of p r o t e c t i o n , and a t h i r d s t a g e , a l s o c h a r a c t e r i z e d by p r e d i s p o s i t i o n . These th ree s tages may be r e l a t e d to o ther f e a t u r e s o f development i n p r imary l e a v e s of bean , s i n c e the f i r s t s tage o c c u r s at a time when the l e a v e s are s t i l l e x p a n d i n g , the second stage o c c u r s at a t ime when expans ion i s r e a c h i n g i t s maximum,' and the t h i r d stage o c c u r s at the time when expans ion i s c o m p l e t e . Under the e x p e r i m e n t a l c o n d i t i o n s i n t h i s t h e s i s , the s u s c e p t i b i l i t y of the p r i m a r y l e a v e s c o n t i n u e d to i n c r e a s e throughout the 21-day p e r i o d a f t e r sowing. U l t i m a t e l y , as the l e a v e s matured , we can expect tha t l e a f s u s c e p t i b i l i t y would have begun to d e c r e a s e . The lower s u s c e p t i b i l i t y o f ozone p r e t r e a t e d p l a n t s at 21 d a y s , t h e r e f o r e , may be viewed e i t h e r as a p r o l o n g a t i o n o f the s u s c e p t i b i l i t y a s s o c i a t e d w i th more j u v e n i l e t i s s u e , or an a c c e l e r a t e d senescence and more Cr)apid achievement of the s u s c e p t i b i l i t y a s s o c i a t e d w i t h more mature t i s s u e . P l a n t s r a i s e d from seed i n low ozone , and rema in ing t h e r e , d i d not show an e f f e c t on s u s c e p t i b i l i t y p a r a l l e l to those r a i s e d i n f i l t e r e d a i r and l a t e r sw i tched to low ozone. A s imple e x p l a n a t i o n of t h i s d i f f e r e n c e would d e s c r i b e a p r o t e c t i v e p r o c e s s o c c u r r i n g i n response to low ozone exposure a f t e r 14 days from sowing ( F i g . 12 Z) and an a n t a g o n i s t i c p r o c e s s o c c u r r i n g i n response to low ozone exposure e a r l i e r ( F i g . 12 Y ) . 83 Process Z , which p r o t e c t s p l a n t s from acute ozone i n j u r y , would be induced by growth i n low ozone a f t e r the 8 - 1 4 day growth s tage and would be m a i n t a i n e d through m a t u r i t y . The a n t a g o n i s t i c p r o c e s s , Y , which a c t s c o u n t e r to the e f f e c t o f Z , would be produced i n response to growth i n low ozone p r i o r to tha t s t a g e . P rocess Y has i t s e l f a s l i g h t l y p r o t e c t i v e p r o p e r t y , a l t h o u g h i t s major r o l e i s to suppress the e f f e c t o f Z . T h u s , i f a p l a n t had been exposed to low ozone c o n c e n t r a t i o n s e a r l y i n development , the p r o t e c t i v e e f f e c t o f low ozone exposures l a t e r i n development would be n e g a t e d . T h i s t ime-dependence i n the response to sub-acute p re t rea tment g i v e s some suppor t to the i n d i c a t i o n that ha rden ing i s the o p e r a t i v e p r o c e s s h e r e . Bean p l a n t s must germinate i n an e n v i r o n -ment where the ozone c o n c e n t r a t i o n i s below 0.02 ppm f o r the p r o t e c t i v e p r o c e s s to be o p t i m a l l y e f f e c t i v e . T h i s sugges ts a s e a s o n a l d imens ion to the harden ing p r o c e s s , whereby p l a n t s i n areas of background ozone c o n c e n t r a t i o n s below 0.02 ppm e a r l y i n the season would be more r e s i s t a n t to combined s u b - a c u t e and acute exposures l a t e r i n the 84 o co CO Juvenile 8-14 Days Mature Leaf Age Figure 12. Model of ozone pretreatment mechanism. Effe c t of exposure.to ozone pretreatment at juvenile stage (Y), mature stage (Z), and both juvenile and mature stages (YZ) on s u s c e p t i b i l i t y to acute ozone injury. 85 season than p l a n t s i n areas where h i g h background l e v e l s of ozone were more c o n t i n u o u s . T h i s phenomenon may account f o r some o f the d i f f e r e n c e between exper iments w i t h g-reenhouse-r a i s e d and f i l t e r e d a i r - r a i s e d bean p l a n t s . I t may a l s o be added to the l i s t o f c a u t i o n s i n comparing the grape exper iments to those w i t h b e a n . . The p e r i o d from e a r l y May to m i d - J u n e , when grapev ines were a l lowed to l e a f out under ambient c o n d i t i o n s , may have reduced the tendency toward p r o t e c t i o n by sub-acute p re t rea tment l a t e r i n the s e a s o n . I f grapes are s i m i l a r to bean , i n tha t i n c r e a s ' i n g ^ p r e t r e a t m e n t c o n c e n t r a t i o n s above 0.02 ppm would i n c r e a s e s u s c e p -t i b i l i t y to acute i n j u r y , then the h i g h e r p re t rea tment c o n c e n t r a t i o n s i n the open- top chambers would a l s o reduce the p r o b a b i l i t y o f h a r d e n i n g . The p r e d i s p o s i t i o n of 'De laware ' to acute ozone i n j u r y by s e a s o n - l o n g s u b - a c u t e p re t rea tment i n d i c a t e d a form of i n v i s i b l e , or c h r o n i c i n j u r y s i m i l a r to tha t d i s c u s s e d by Heagle and Heck (1974) and Macdowal l (1965) . T h i s response can be e x p l a i n e d by a g r e a t e r than a d d i t i v e i n t e r a c t i o n o f i n j u r y below the v i s i b l e t h r e s h o l d and i n j u r y above tha t t h r e s h o l d , g i v i n g the appearance of a form o f s y n e r g i s m . The g r e a t e r tendency toward t h i s response i n o z o n e - t o l e r a n t p l a n t s 86 may be e x p l a i n e d as an i n c r e a s e d p o s s i b i l i t y o f a c c l i m a t i o n i n o z o n e - s e n s i t i v e p l a n t s , under the assumpt ion tha t i n c r e a s e d s e n s i t i v i t y to ozone i n j u r y may be a s s o c i a t e d w i th some p h y s i o l o g i c a l b a s i s f o r a p r o t e c t i v e r e s p o n s e . In p a r t i c u l a r , t h i s concept o f h y p e r s e n s i t i v i t y as a b a s i s f o r ozone p r o t e c t i o n has been the f o u n d a t i o n f o r p a s t s p e c u l a t i o n about the p o s s i b l e r o l e o f stomata i n a c c l i m a t i o n . Based on the observed e f f e c t o f a s e r i e s o f acute ep isodes i n the f i e l d , w i t h t o b a c c o , Macdowal l (1965) c o n c l u d e d tha t lower p r i o r doses had a p r e d i s p o s i n g e f f e c t toward l a t e r d o s e s , w h i l e h i g h e r p r i o r doses had a p r o t e c t i v e e f f e c t . W. J o h n s t o n ( p e r s o n a l communicat ion) ' , work ing w i t h s o y b e a n , has observed t h i s same t r e n d i n l a b o r a t o r y exper iments w i t h p re t rea tment c o n c e n t r a t i o n s , as low as 0.03 ppm. Both o f these r e s e a r c h e r s have i n t e r p r e t e d t h e i r r e s u l t s as s u p p o r t i n g the h y p o t h e s i s tha t the p r o t e c t i v e e f f e c t o f p re t rea tment exposures i s p a r t l y a s tomata l e f f e c t . R e s u l t s d e s c r i b e d i n t h i s t h e s i s a l s o suggest tha t the p r o t e c t i v e e f f e c t o f p re t rea tment i s due to i t s e f f e c t on s tomata . The s tomata l e f f e c t o f low ozone exposure has been d i v i d e d here i n t o a g e n e r a l e f f e c t on s tomata l r e s i s t a n c e , and an e f f e c t on the speed o f response to acute ozone exposure . The p r o -t e c t i v e aspec t o f the s tomata l e f f e c t o f p re t rea tment 87 appears to b e , the g e n e r a l e f f e c t , n o t ' t h e e f f e c t on the speed o f response to acute ozone. Whi le i t i s not r e a s o n a b l e to assume tha t t h i s g e n e r a l e f f e c t on stomata i s r e v e r s e d as p re t rea tment c o n c e n t r a t i o n 1 i n c r e a s e s , i t does appear tha t the s tomata l e f f e c t may be o v e r r i d d e n by the p r e d i s p o s i n g e f f e c t o f p re t rea tment as the p re t rea tment c o n c e n t r a t i o n i n c r e a s e s . The most commonly noted e f f e c t o f ozone exposure on s tomata l b e h a v i o r i n v o l v e s an i n d u c t i o n o f s tomata l c l o s u r e by acute l e v e l s o f ozone tha t i s r a p i d l y r e v e r s i b l e upon removal o f the ozone ( e . g . , H i l l and L i t t l e f i e l d , 1969).. The a s p e c t o f s tomata l b e h a v i o r which i s i n v o l v e d w i t h the p r o t e c t i v e e f f e c t o f low ozone p r e t r e a t m e n t , demon-s t r a t e d i n the exper iments i n t h i s t h e s i s , appears to be a c h r o n i c dampening of s tomata l a p e r t u r e tha t i s not r e v e r s i b l e upon ozone r e m o v a l . T h i s i r r e v e r s i b l e type o f response has o c c a s i o n a l l y been noted i n p r e v i o u s exper iments (Macknight , 1968; K o r i t z and Went, 1952). The p o s s i b l e combina t ion o f these two types o f ozone-induced c l o s u r e i n exper iments w i t h g r e e n h o u s e - r a i s e d p l a n t s i s u n c l e a r . In exper iments w i t h g r e e n h o u s e - r a i s e d bean , the lower i n i t i a l R g o f f i l t e r e d a i r - p r e t r e a t e d p l a n t s l e d to a g r e a t e r s u s c e p t i b i l i t y to acute inj iury and to a g r e a t e r degree o f s tomata l c l o s u r e i n response to the acute exposure . Both of these e f f e c t s are o f the i r r e v e r -s i b l e t y p e , however, because a l l measurements were made i n f i l t e r e d a i r . 88 T h e o r e t i c a l l y , s t o m a t a l c l o s u r e i n response to ozone may be d i v i d e d i n t o th ree t y p e s : (1) a r e v e r s i b l e type of c l o s u r e i n response to acute exposure (due to changes i n p h y s i o l o g i c a l p r o p e r t i e s such as membrane s t r u c t u r e or a c t i v i t y , c a r b o h y d r a t e c o n t e n t , e t c . ; (2) an i r r e v e r s i b l e type o f c l o s u r e i n response to l o n g - t e r m s u b - a c u t e e x p o s u r e s , perhaps due to the a c c u m u l a t i o n of changes i n s i m i l a r types of p h y s i o l o g i c a l f a c t o r s as i n (1 ) ; (3) an i r r e v e r s i b l e type o f c l o s u r e i n response to acute exposures which i s a s s o c i a t e d w i t h the appearance of n e c r o t i c l e s i o n s and d i r e c t p h y s i c a l damage to ep iderma l and m e s o p h y l l i c t i s s u e s s u r r o u n d i n g the s tomata . The types o f c l o s u r e observed i n exper iments w i t h g r e e n h o u s e - r a i s e d beans are p r o b a b l y (2) and (3 ) . E x a m i n a t i o n o f type (1) c l o s u r e r e q u i r e s measurement o f s tomata l b e h a v i o r i n the p r e s e n c e o f h i g h ozone c o n c e n t r a t i o n s such as was done i n exper iments w i t h beans r a i s e d i n f i l t e r e d a i r arid then sw i tched to low ozone a f t e r seven d a y s . Those exper iments sugges ted tha t s tomata l c l o s u r e types (1) and (2) may be p r o g r e s s i v e s tages i n a s i n g l e type o f s tomata l response to ozone. The e f f e c t s o f low ozone p re t rea tment on s u s c e p t i b i l i t y i n those exper iments p a r a l l e l e d the p r o g r e s s i n t h i s s t o m a t a l r e s p o n s e . In the e a r l y s t a g e , where s u s c e p t i b i l i t y to acute exposure was i n c r e a s e d by s u b - a c u t e p r e t r e a t m e n t , the magnitude o f the r e v e r s i b l e c l o s u r e induced by acute ozone , type (1 ) , was d e c r e a s e d . In the second s t a g e , when s u s c e p t i b i l i t y was d e c r e a s e d , the i r r e v e r s i b l e type of 89 c l o s u r e induced by s u b - a c u t e e x p o s u r e , type (2 ) , had appeared . T h i s p r o g r e s s i o n from a r e d u c t i o n i n type (1) c l o s u r e to the appearance o f type (2) c l o s u r e does not h e l p to e l u c i d a t e whether the same system o f p h y s i o l o g i c a l changes i s a c t i v e i n both c a s e s . The two s tages may be e x p l a i n e d by a s i n g l e mechanism, however, whereby ozone p r o g r e s s i v e l y reduces the a b i l i t y o f stomata to respond to env i ronmenta l changes . The d e s e n s i t i z a t i o n would move from an e a r l y s tage i n which s t o m a t a l s e n s i t i v i t y to ozone i t s e l f was decreased to a l a t e r s tage i n which there was a more g e n e r a l dampening of s tomata l f u n c t i o n . In g r a p e v i n e e x p e r i m e n t s , no s i g n i f i c a n t e f f e c t s o f low ozone t reatment on s tomata l b e h a v i o r were o b s e r v e d . In the on ly case o f a s i g n i f i c a n t t reatment e f f e c t on s u s c e p t i b i l i t y , the p r e d i s p o s i t i o n o f ' D e l a w a r e ' , the t o l e r a n t c u l t i v a r , t rends i n the data on s tomata l b e h a v i o r at the t ime o f p r e d i s p o s i t i o n , September, showed tha t o z o n e - p r e t r e a t e d p l a n t s (ambient a i r t reatment) tended to have a lower i n i t i a l R g than f i l t e r e d a i r - p r e t r e a t e d p l a n t s (type (2) c l o s u r e ) , w i t h e s s e n t i a l l y no change, or a s l i g h t r e d u c t i o n i n the ex ten t o f c l o s u r e i n response to h i g h ozone c o n c e n t r a t i o n s ( type (1)). In p a r t , t h i s compares w i t h the r e s u l t s on b e a n , i n that the e f f e c t o f low ozone p re t rea tment on s u s c e p t i b i l i t y i n the l a t e r s tages appeared to be a s s o c i a t e d w i t h changes i n type (2) c l o s u r e . In 'De laware ' g r a p e v i n e s , however, there was l e s s type (2) c l o s u r e i n o z o n e - p r e t r e a t e d p l a n t s and a g r e a t e r 9 0 s u s c e p t i b i l i t y to acute ozone i n j u r y , w h i l e i n bean there was more type (2) c l o s u r e i n o z o n e - p r e t r e a t e d p l a n t s and a l e s s e r s u s c e p t i b i l i t y to acute ozone i n j u r y . I g n o r i n g the e f f e c t s o f low ozone p r e t r e a t m e n t , and c o n c e n t r a t i n g on the g e n e r a l r a n k i n g o f the th ree grape c u l t i v a r s i n the f i e l d , the ozone t o l e r a n c e o f 'De laware ' appears to be a s s o c i a t e d w i t h a g r e a t e r degree o f type (1) c l o s u r e ( c f . T a b l e 4 ) . The s tomata l b a s i s , i f any, o f the p re t rea tment r e s p o n s e , though r e l a t e d to the g e n e r a l c u l t i v a r s e n s i t i v i t y to ozone i n j u r y i n the f i e l d , does not seem to be based on the same aspec ts o f s tomata l b e h a v i o r . C o n c l u s i o n s about the r e l a t i o n o f s t o m a t a l response and s u s c e p t i b i l i t y response to low ozone p re t rea tment based on the r e l a t i v e responses i n the ' C o n c o r d ' and ' I v e s ' c u l t i v a r s are. a good d e a l more t e n u o u s , due to the absence o f any s t a t i s t i c a l l y s i g n i f i c a n t e f f e c t s . Based on t rends i n the acute i n j u r y data i n August (Table 3) there appeared to be an e q u i v a l e n t tendency toward p r o t e c t i o n i n both c u l t i v a r s . Based on the r a n k i n g o f s tomata l r e s p o n s e s , however, and suppor ted by the r a n k i n g o f c u l t i v a r s a c c o r d i n g to i n j u r y s e n s i t i v i t y i n the f i e l d , ' C o n c o r d ' might be expec ted to be i n t e r m e d i a t e between ' I v e s ' and ' D e l a w a r e ' . In ' C o n c o r d ' , the t r e n d was f o r a s l i g h t d e c r e a s e , or no change, i n both type (1) and type (2) c l o s u r e . In ' I v e s ' the tendency seen i n ' C o n c o r d ' and 'De laware ' f o r a s l i g h t decrease i n type (1) has d i s a p p e a r e d , and , more i m p o r t a n t l y , the r e d u c t i o n i n type 91 (2) c l o s u r e ( ' I v e s ' ) and absence o f type (2) c l o s u r e ( ' C o n c o r d ' ) , r e v e a l s a t r e n d toward an i n c r e a s e i n type (2) c l o s u r e ( ' D e l a w a r e ' ) . The t r e n d toward i n c r e a s i n g p r o t e c t i v e e f f e c t would thus be accompanied by a t r e n d toward i n c r e a s e s i n i r r e v e r s i b l e s tomata l c l o s u r e induced by l o n g - t e r m low l e v e l e x p o s u r e s , s i m i l a r to t h a t observed at the p r o t e c t i v e s tage o f the response i n beans . Whi le t h i s c o n c l u s i o n i s s p e c u l a t i v e , i t i s c l e a r tha t o f the two types of s tomata l b e h a v i o r which may be a s s o c i a t e d w i th changes i n s u s c e p t i b i l i t y ( the h y p e r s e n s i t i v e mechanism, i . e . , changes i n type (1) c l o s u r e , or l o n g - t e r m i r r e v e r s i b l e e f f e c t s , i . e . , type (2) c l o s u r e ) , the l a t t e r appears more l i k e l y to be i n v o l v e d . The paradox h e r e , however, i s tha t the h y p e r s e n s i t i v e mechanism seems to a f f o r d the b e s t p o s s i b i l i f o r c o u p l i n g a c c l i m a t i o n to the c o m b i n a t i o n o f h i g h and low ozone c o n c e n t r a t i o n s encountered i n the f i e l d , w i thou t an accompanying growth r e d u c t i o n . The a s s o c i a t i o n o f p r o t e c t i o n w i t h i r r e v e r s i b l e dampening of s t o m a t a l , a p e r t u r e would appear to suppor t an a s s o c i a t i o n o f p r o t e c t i o n w i t h r e d u c t i o n s i n p l a n t growth. Whi le bean exper iments were not adequate to address t h i s p o i n t , the exper iments w i t h g r a p e v i n e s i n d i c a t e d that t h i s was not the c a s e . On a c u l t i v a r - t o - c u l t i v a r b a s i s , i t appears o n l y tha t the absence of a p r e d i s p o s i t i o n e f f e c t i s a s s o c i a t e d w i t h the p r e s e n c e o f a growth s t i m u l a t i o n . S i n c e there were a l s o no s i g n i f i c a n t s u s c e p t i b i l i t y e f f e c t s i n the two c u l t i v a r s 92 which showed s i g n i f i c a n t e f f e c t s on growth, there can be no f i r m c o n c l u s i o n s about a p o s i t i v e a s s o c i a t i o n between growth s t i m u l a t i o n and p r o t e c t i o n . The s e a s o n a l t rends i n ozone e f f e c t s on v i n e growth and acute s u s c e p t i b i l i t y , however, do suggest the e x i s t e n c e o f t h i s r e l a t i o n s h i p . The g r e a t e s t s t i m u l a t i o n of growth i n ' I v e s ' tops appeared at the August h a r v e s t ( F i g . 13 ) , w h i l e the ozone-induced s t i m u l a t i o n of ' I ves ' ; r o o t growth c o n t i n u e d to i n c r e a s e throughout the growing s e a s o n . The o z o n e - i n d u c e d s t i m u l a t i o n o f ' C o n c o r d ' l e a f growth was g r e a t e s t at the J u l y h a r v e s t , and the extent o f tha t e f f e c t d e c r e a s e d as the season p r o g r e s s e d ( F i g . 14 ) . In bo th c u l t i v a r s , the s e a s o n a l p r o f i l e o f the s t i m u l a t o r y e f f e c t o f low ozone on l e a f growth p a r a l l e l e d the development o f the e f f e c t on s u s c e p t i b i l i t y to acute ozone i n j u r y (Tab le 3 ) , i n a manner which suggested a p o s i t i v e a s s o c i a t i o n between s t i m u l a t i o n o f l e a f growth and p r o t e c t i o n from acute ozone i n j u r y . S i n c e i t was p o i n t e d out by Bennet t et a l . i n 1974, so l i t t l e work has been done on the s t i m u l a t i o n o f p l a n t growth by exposure to low ozone c o n c e n t r a t i o n s tha t the re i s r e a l l y no b a s i s f o r s p e c u l a t i o n about the p o s s i b l e p h y s i o l o g i c a l mechanisms o f t h i s r e s p o n s e . One p o s s i b i l i t y t e s t e d i n t h i s t h e s i s was based on work i n v o l v i n g growth r e d u c t i o n s induced by exposure to low ozone c o n c e n t r a t i o n s . Bennet t and Runeckles (1977) e x p l a i n e d the f a c t tha t 9 3 or-O O 6 o- -O' Aug Sept l x J J u l y Time of Year ' Aug - P - P o o CO •o C\J E o ccj u < <H cd Sept Growth o f p o t t e d 'Ives' v i n e s i n ambient a i r (•) o r c h a r c o a l - f i l t e r e d a i r ( o ) . Fresh we igh tC—) and dry - w e i g h t (--•> o f l eaves (A), shoots (B), and r o o t s (C); and l e a f a re a (D). Data from Table 5-94 o - J I Aug Sept J u l y Aug Sept Time o f Year Growth o f p o t t e d ' C o n c o r d ' v i n e s i n ambient a i r (•) o r c h a r c o a l - f i l t e r e d a i r (o). F r e s h weight (—) and dry weight (—) o f l e a v e s ( A ) , and r o o t dry weight r a t i o (B ) . Data from T a b l e k. 95 reduced l e a f areas i n c l o v e r and r y e g r a s s i n response to low ozone exposures were accompanied by e q u i v a l e n t or s l i g h t l y h i g h e r NAR's by c o n c l u d i n g tha t a compensat ion o c c u r r e d i n NAR to c o u n t e r a c t the e f f e c t o f ozone on l e a f a r e a . To e x p l a i n the i n c r e a s e d l e a f a rea i n exper iments w i t h p o t t e d g r a p e v i n e s , the complementary h y p o t h e s i s was deve loped tha t t h i s i n c r e a s e d l e a f area was a compensatory r e a c t i o n to a p r imary e f f e c t of ozone i n d e c r e a s i n g NAR i n t h i s s p e c i e s . T h i s p o s s i b i l i t y i s not unreasonab le i n c u t t i n g s of the type used f o r the g r a p e v i n e e x p e r i m e n t s , because growth i n the season observed was h i g h l y dependent on e x i s t i n g c a r b o h y d r a t e r e s e r v e s . I f m o b i l i z a t i o n o f these r e s e r v e s was c o n t r o l l e d by some feedback mechanism r e g u l a t e d by p r o d u c t i o n o f p h o t o s y n t h a t e i n the l e a v e s , the c a p a c i t y might e x i s t t o . p r o d u c e more new growth under c o n d i t i o n s more u n f a v o r a b l e to c u r r e n t p h o t o s y n t h e s i s . Measurements o f p h o t o s y n t h e t i c r a t e s on v i n e s from the September h a r v e s t , however, r e v e a l e d no d i f f e r e n c e s between v i n e s t r e a t e d w i t h ambient or f i l t e r e d a i r . Whi le i t i s p o s s i b l e tha t measurements made e a r l i e r i n the growing season might have been a b e t t e r i n d i c a t i o n o f a p o s s i b l e compensat ion e f f e c t , the r e s u l t s do not suppor t t h i s assumpt ion o f a l e a f a rea i n c r e a s e based on r e d u c t i o n s i n p h o t o s y n t h e t i c r a t e . There i s no ev idence tha t t h i s type o f l o n g - t e r m 96 a d a p t a t i o n of g rapev ines to low ozone c o n c e n t r a t i o n s , r e f l e c t e d i n i n c r e a s e d growth r a t e s , i s accompanied by any immediate r e d u c t i o n s i n acute ozone t o l e r a n c e , s t o m a t a l a p e r t u r e , or p h o t o s y n t h e s i s . T h i s phenomenon i s d i s t i n c t from the i n d u c t i o n of acute o z o n e - t o l e r a n c e by s u b - a c u t e exposure which i s accompanied by a dampening of s tomata l f u n c t i o n . Whi le t h i s s h o r t e r term a c c l i m a t i o n c a l l s i n t o q u e s t i o n the use o f f i l t e r e d a i r i n exper iments on acute ozone s u s c e p t i b i l i t y , the o c c u r r e n c e o f such growth s t i m u l a t i o n s must n e c e s s a r i l y c a l l i n t o q u e s t i o n w idespread assumptions about the f o r m a t i v e e v o l u t i o n a r y i n f l u e n c e of o z o n e - f r e e env i ronments . The i n d i c a t i o n s i n t h i s t h e s i s tha t ozone a c c l i m a t i o n may o c c u r suggest a number of areas where a d d i t i o n a l r e s e a r c h i s n e c e s s a r y . Perhaps the most immedia te ly urgent o f these i s the need to r e - e v a l u a t e . , i n the l i g h t o f p o s s i b l e a c c l i m a t i o n , the r e s u l t s o f pas t at tempts at b r e e d i n g ozone t o l e r a n c e i n t o crop p l a n t s . New surveys and s e l e c t i o n exper iments d e s i g n e d to e x p l o i t the p o s s i b i l i t y o f a c c l i m a t i o n must a l s o be u n d e r t a k e n . Whi le s t o m a t a l e x c l u s i o n has been shown here to be an impor tant f a c t o r i n some i n s t a n c e s of ozone a c c l i m a t i o n , there i s a l s o a need f o r a d d i t i o n a l exper iments to e x p l o r e the q u e s t i o n o f p o s s i b l e n o n - s t o m a t a l mechanisms o f ozone p r o t e c t i o n which may be induced by exposure to low ozone c o n c e n t r a t i o n s . 9 7 A d d i t i o n a l work i s a l s o necessary to e l u c i d a t e the r e l a t i o n s h i p between ozone a c c l i m a t i o n and growth. The evident a s s o c i a t i o n between p r o t e c t i o n from acute i n j u r y and.long-term i r r e v e r s i b l e c l o s u r e of stomata, i n bean, i l l u s t r a t e s the p o s s i b i l i t y of a necessary t r a d e o f f between acute ozone t o l e r a n c e and o v e r a l l p o t e n t i a l p r o d u c t i v i t y , w h ile the i m p l i e d r e l a t i o n s h i p of i n c r e a s e d growth and gre a t e r ozone t o l e r a n c e , i n grapevines, independent of stomatal e f f e c t s , suggests the e x i s t e n c e of other forms of a c c l i m a t i o n which may be p o t e n t i a l l y more u s e f u l . 98 SUMMARY 1. Sub-acu te ozone exposures had a s i g n i f i c a n t e f f e c t on the development o f v i s i b l e i n j u r y i n response to subsequent acute e x p o s u r e s . 2. T h i s e f f e c t may take the form o f e i t h e r p r o t e c t i o n from i n j u r y or p r e d i s p o s i t i o n to g r e a t e r i n j u r y . In bean , lower p re t rea tment doses l e d to p r o t e c t i o n w h i l e h i g h e r doses l e d to p r e d i s p o s i t i o n . 3. The p r o t e c t i v e aspect o f t h i s e f f e c t may be d e s c r i b e d as h a r d e n i n g , observed as an o z o n e - i n d u c e d change o c c u r r i n g d u r i n g the course o f the e x p e r i m e n t s , r a t h e r than as a p r e d i s p o s i n g e f f e c t o f f i l t e r e d a i r . 4. As the time o f exposure to s u b - a c u t e ozone i n c r e a s e d , i n b e a n , the e f f e c t on s u s c e p t i b i l i t y to acute i n j u r y passed through three s t a g e s : an e a r l y s tage of i n c r e a s e d s u s c e p t i b i l i t y , a middle s tage of decreased s u s c e p t i b i l i t y , and a l a t e r s tage o f i n c r e a s e d s u s c e p t i b i l i t y . 5. The f i r s t two s tages i n tha t response were a s s o c i a t e d w i t h the e f f e c t o f p re t rea tment on s tomata . The e a r l y p r e d i s p o s i t i o n was a s s o c i a t e d w i t h a reduced tendency f o r s tomata l c l o s u r e i n response to acute ozone e x p o s u r e , while the protective stage was associated with a general stomatal closure, independent of acute exposure. Pretreatment doses which had a s i g n i f i c a n t protective effect in bean increased the acute ozone suscep-t i b i l i t y of mint cuttings s l i g h t l y . In grape, season-long sub-acute exposures to ozone had a stimulatory e f f e c t on plant growth in 2 of 3 cul t i v a r s tested. In the t h i r d c u l t i v a r , which showed no effect of ozone on growth, vines were predisposed to acute injury by sub-acute pretreatment. Comparing c u l t i v a r s , there was some indication that an increasing trend toward protection by sub-acute pretreatment may have been associated with the same type of stomatal closure observed in bean. Within c u l t i v a r s , there was some indication that growth stimulation may have been associated with increased acute s u s c e p t i b i l i t y at the end of the growing season, and decreased acute suscep-t i b i l i t y at mid-season. Exper iment Summary P l a n t Treatment C o n d i t i o n s Ozone D u r a t i o n C o n c e n t r a t i o n (days) (PP m )  Bean .02 7 ( t reatments b e g i n n i n g 8 days a f t e r . 02 - . 10 7 sowing) .02 7 , .02 14 .02 7 .02 14 Environment P r i o r to Treatment E f f e c t s o f Treatment ( c o n t r o l s exposed to c h a r c o a l -f i l t e r e d a i r ) greenhouse greenhouse f i l t e r e d a i r f i l t e r e d a i r f i l t e r e d a i r f i l t e r e d a i r decrease i n acute s u s c e p t i b i l i t y i n c r e a s e i n acute s u s c e p t i b i l i t y w i t h i n c r e a s i n g dose i n c r e a s e i n acute s u s c e p t i b i l i t y . decrease i n s tomata l c l o s u r e induced by acute ozone exposure decrease i n acute s u s c e p t i b i l i t y . i n c r e a s e d s tomata l r e s i s t a n c e i n f i l t e r e d a i r p r i o r to acute exposure and through-out acute exposure no e f f e c t on stem h e i g h t no e f f e c t on stem he igh t Exper iment Summary (cont inued) P l a n t Treatment C o n d i t i o n s Ozone D u r a t i o n C o n c e n t r a t i o n (days) M in t .02 Grape see T a b l e 1 1-3 months cv ' I v e s ' Grape " 1-3 months cv ' C o n c o r d ' Grape " 1-3 months cv 'De laware ' Envi ronment P r i o r to Treatment E f f e c t s o f Treatment ( c o n t r o l s exposed to c h a r c o a l - f i l t e r e d a i r )  greenhouse s m a l l i n c r e a s e i n acute s u s c e p t i b i l i t y ambient i n c r e a s e d growth , no e f f e c t on acu te suscep t i b i l i t y o r s t o m a t a l b e h a v i o r ambient i n c r e a s e d growth , no e f f e c t on acu te suscep t i b i l i t y or s t o m a t a l b e h a v i o r ambient no e f f e c t on g rowth , i n c r e a s e d acu te suscep t i b i l i t y , no e f f e c t on s tomata l b e h a v i o r 102 LITERATURE CITED Ashenden, T . 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