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EPR investigation of free radicals in excised and attached leaves subjected to ozone and sulphur dioxide.. 1988

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EPR INVESTIGATION OF FREE RADICALS IN EXCISED AND ATTACHED LEAVES SUBJECTED TO OZONE AND SULPHUR DIOXIDE AIR POLLUTION by MANIVALDE VAARTNOU B.A., The U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1972 M . S c , The U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1980 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department o f P l a n t S c i e n c e ) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA ©Manivalde V a a r t n o u , 1988 In presenting this thesis in partial fulfilment of the requirements for 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 or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of P l a n t S c i e n c e The University of British Columbia Vancouver, Canada Date O c t o b e r 3 , 1988 DE-6 (2/88) ABSTRACT The X-band EPR s p e c t r o m e t r y system was m o d i f i e d t o a l l o w f o r t h e i n s i t u m o n i t o r i n g o f f r e e r a d i c a l changes i n a t t a c h e d , i n t a c t p l a n t l e a v e s , w h i c h were caused by s t r e s s f a c t o r s such as exposure t o e x c e s s i v e photon f l u x d e n s i t y , ozone o r s u l p h u r d i o x i d e . T h i s was done t h r o u g h use o f t h e dewar i n s e r t o f t h e v a r i a b l e t e m p e r a t u r e a c c e s s o r y as a g u i d e , t h e c o n s t r u c t i o n o f 'T' shaped c e l l u l o s e a c e t a t e h o l d e r s t o which l e a v e s c o u l d be a t t a c h e d w i t h a d h e s i v e t a p e , and m o d i f i c a t i o n o f t h e gas f l o w system used f o r c o n t r o l l e d t e m p e r a t u r e s t u d i e s . K i n e t i c s t u d i e s o f f r e e r a d i c a l f o r m a t i o n were p o s s i b l e w i t h l e a v e s w h i c h had m i n i m a l u n d e r l y i n g F e + + and M n + + s i g n a l s . I n l e a v e s w i t h l a r g e u n d e r l y i n g s i g n a l s a V a r i a n s o f t w a r e program was used t o s u b t r a c t o v e r l a p p i n g s i g n a l s from each o t h e r , t h e r e b y r e v e a l i n g t h e f r e e - r a d i c a l s i g n a l changes w h i c h o c c u r r e d under d i f f e r e n t l i g h t regimes and s t r e s s c o n d i t i o n s . P r e l i m i n a r y i n v e s t i g a t i o n d i s c l o s e d t h e f o r m a t i o n o f a new s i g n a l upon p r o l o n g e d exposure t o f a r - r e d l i g h t and t h e e f f e c t o f oxygen d e p l e t i o n upon p h o t o s y n t h e t i c S i g n a l s I and I I . Leaves s u b j e c t t o h i g h photon f l u x d e n s i t y r e v e a l an u n r e p o r t e d f r e e - r a d i c a l s i g n a l , w h i c h decays upon exposure t o microwave r a d i a t i o n ; and c o n c o m i t a n t damage t o Photosystems I and I I . Upon e l i m i n a t i o n o f t h i s s i g n a l l e a v e s r e t u r n t o t h e undamaged s t a t e o r r e v e a l permanent damage t o e i t h e r p h o t o - system, depending upon t h e degree of damage. Kentucky b l u e g r a s s and p e r e n n i a l r y e g r a s s l e a v e s s u b j e c t t o low l e v e l s o f ozone (up t o 80ppb) f o r p e r i o d s o f 8 h o u r s show no changes i n f r e e - r a d i c a l s i g n a l f o r m a t i o n . A t i n t e r m e d i a t e l e v e l s o f ozone (80-250ppb) a new f r e e - r a d i c a l s i g n a l was formed w i t h i n 3 h o u r s o f f u m i g a t i o n , S i g n a l I I was d e c r e a s e d and S i g n a l I decayed. These changes were r e v e r s i b l e i f f u m i g a t i o n was t e r m i n a t e d . A t f u m i g a t i o n l e v e l s e x c e e d i n g 250ppb a d i f f e r e n t new i r r e v e r s i b l e f r e e - r a d i c a l s i g n a l was formed i n d a r k n e s s w i t h i n 1.5 h o u r s of f u m i g a t i o n . R a d i s h , Kentucky b l u e g r a s s and p e r e n n i a l r y e g r a s s l e a v e s s u b j e c t t o h i g h l e v e l s o f s u l p h u r d i o x i d e (10-500ppm) r e v e a l t h e f o r m a t i o n o f S i g n a l I upon i r r a d i a t i o n w i t h broad-band w h i t e o r 650nm l i g h t , t h e r e b y i n d i c a t i n g an i n t e r r u p t i o n o f normal e l e c t r o n f l o w from P S I I t o P S I . Damage t o t h e o x y g e n - e v o l v i n g complex and r e a c t i o n c e n t r e o f P S I I i s a l s o r e v e a l e d t h r o u g h changes i n S i g n a l I I and t h e M n + + s i g n a l . These changes i n t h e normal EPR s i g n a l s a r e dose-dependent. Leaves s u b j e c t t o low l e v e l s o f s u l p h u r d i o x i d e (600-2000ppb) r e v e a l t h e d i s a p p e a r a n c e o f S i g n a l I a f t e r 3 h o u r s o f f u m i g a t i o n and t h e f o r m a t i o n o f a i v new f r e e - r a d i c a l s i g n a l w i t h p a r a m e t e r s s i m i l a r t o t h e s u l p h u r t r i o x i d e f r e e - r a d i c a l s i g n a l . These l a t t e r changes a r e p a r t i a l l y r e v e r s i b l e upon t e r m i n a t i o n o f f u m i g a t i o n . A f t e r p r o l o n g e d exposure t o e i t h e r ozone o r s u l p h u r d i o x i d a f r e e - r a d i c a l s i g n a l w i t h p arameters s i m i l a r t o t h e s u p e r o x i d a n i o n f r e e - r a d i c a l s i g n a l i s formed i n p l a n t l e a v e s . V TABLE OF CONTENTS Page No. ABSTRACT i i TABLE OF CONTENTS V LIST OF TABLES v i i i LIST OF FIGURES i x ABBREVIATIONS X V ACKNOWLEDGEMENTS xv i 1.0 INTRODUCTION 1 2.0 LITERATURE REVIEW 6 2.1 FREE RADICALS AND OXYGEN TOXICITY 6 2.2 PHOTOINHIBITION 14 2.3 PHOTOSYNTHETIC ELECTRON TRANSPORT 2 0 2.3.1 Photosystem I I 2 3 2.3.2 Cytochrome bgf Complex 27 2.3.3 Photosystem I 28 2.4 EPR STUDIES WITH CHLOROPLAST, SUBCHLOROPLAST AND ENZYME PREPARATIONS 3 0 3.0 DEVELOPMENT OF METHODOLOGY 4 2 3.1 OBJECTIVES 42 3.2 METHODS 42 3.2.1 S p e c t r o m e t e r O p e r a t i o n 42 3.2.2 L e a f H o l d e r 46 3.2.3 Atmosphere M o d i f i c a t i o n 53 v i TABLE OF CONTENTS (Continued) Page No. 3.2.4 S p i n T r a p p i n g 54 3.2.5 P l a n t M a t e r i a l s 54 3.3 PHENOMENA ASSOCIATED WITH EPR SPECTROMETRY OF LEAF SEGMENTS 55 3.3.1 N a t u r a l V a r i a t i o n i n P h o t o s y n t h e t i c EPR S i g n a l s i n E x c i s e d P i e c e s o f Leaves 55 3.3.2 V a r i a b i l i t y i n P h o t o s y n t h e t i c EPR S i g n a l s i n Whole, A t t a c h e d G r a s s Leaves 59 3.3.3 P o s s i b i l i t i e s and L i m i t a t i o n s o f K i n e t i c S t u d i e s 63 3.3.4 S i g n a l S u b t r a c t i o n 77 3.3.4.1 A New L i g h t - i n d u c e d F r e e - R a d i c a l S i g n a l 77 3.3.4.2 Oxygen E f f e c t s on S i g n a l s I and I I 80 3.3.5 E f f e c t s o f P h o t o i n h i b i t i o n 89 3.4 DISCUSSION 104 4.0 OZONE STUDIES 110 4.1 INTRODUCTION 110 4.2 METHODS 111 4.3 RESULTS 111 4.3.1 E f f e c t s o f Low L e v e l s o f 0 3 112 4.3.2 E f f e c t s o f I n t e r m e d i a t e L e v e l s o f 0-, 112 v i i TABLE OF CONTENTS (Continued) Page No. 4.3.3. E f f e c t s o f Hig h L e v e l s o f 0 3 121 4.4 DISCUSSION 129 5.0 SULPHUR DIOXIDE STUDIES 13 3 5.1 INTRODUCTION 13 3 5.2 METHODS 13 4 5.3 RESULTS 13 5 5.3.1 Detached R a d i s h L e a f Segments 135 5.3.1.1 S i g n a l I S t u d i e s 135 5.3.1.2 M n + + S i g n a l 147 5.3.1.3 S i g n a l I I U + S 148 5.3.2 A t t a c h e d , I n t a c t G r a s s Leaves 150 5.3.2.1 H i g h S 0 2 L e v e l s 150 5.3.2.2 Low S 0 2 L e v e l s 152 5.4 DISCUSSION 160 6.0 GENERAL DISCUSSION 172 7.0 SUMMARY AND CONCLUSIONS 193 8.0 LITERATURE CITED 198 APPENDIX A 224 APPENDIX B 2 30 APPENDIX C 2 32 v i i i LIST OF TABLES Page No. 1. I r r a d i a t i o n i n t e n s i t i e s r e l e v a n t t o t h e s t u d i e s o f t h i s i n v e s t i g a t i o n . 45 2. R e l a t i o n s h i p o f t h e u n c h a r a c t e r i z e d EPR f r e e - r a d i c a l s i g n a l ( S i g n a l N 7 1 0 ) i n i n t a c t r y e g r a s s l e a v e s w i t h t i m e o f exposure t o 710nm l i g h t . 8 0 i x LIST OF FIGURES Page No. 1. C u r r e n t c o n c e p t o f t h e Z scheme f i r s t p r o p osed by H i l l and B e n d a l l (1960). 22 2. P r i m a r y p h o t o s y n t h e t i c s i g n a l s found i n r e a c t i o n c e n t r e and c h l o r o p l a s t p r e p a r a t i o n s and i n i n t a c t l e a f t i s s u e . 31 3. C r o s s s e c t i o n a l v i e w o f t h e equipment arrangement. 44 4. C o n v e n t i o n a l f a c t o r y (Wang L a b o r a t o r i e s I n c . ) b i o l o g i c a l t i s s u e h o l d e r . 48 5. E 2 3 i c a v i t y and dewar i n s e r t . 50 6. 'T'-shaped c e l l u l o s e a c e t a t e l e a f h o l d e r used i n t h e EPR s t u d i e s . 52 7. Equipment s e t u p f o r s t u d i e s w i t h a t t a c h e d , i n t a c t g r a s s l e a v e s . 56 8. EPR s i g n a l s o b t a i n e d from h e a l t h y r a d i s h l e a f p i e c e s i n d i f f e r e n t l i g h t r e gimes, r e c o r d e d i m m e d i a t e l y a f t e r e x c i s i o n o f t h e p i e c e s . 57 9. EPR s i g n a l s o b t a i n e d from t h e p r e v i o u s l y h e a l t h y r a d i s h l e a f p i e c e s d e p i c t e d i n F i g u r e 8, i n d i f f e r e n t l i g h t r e g i m e s , r e c o r d e d 2 h o u r s a f t e r e x c i s i o n , w i t h t h e p i e c e s h e l d i n d a r k n e s s i n t h e c a v i t y d u r i n g t h e i n t e r v a l . 60 10. Changes i n t h e M n + + , da r k , and w h i t e l i g h t - i n d u c e d s i g n a l s i n i n t a c t , a t t a c h e d Kentucky b l u e - g r a s s l e a v e s a f t e r 5 days i n t h e s p e c t r o m e t e r . 62 X LIST OF FIGURES (Continued) Page No. 11. K i n e t i c s o f S i g n a l I f o r m a t i o n and decay i n un f u m i g a t e d r a d i s h l e a f p i e c e s w i t h d i f f e r e n t l i g h t t r e a t m e n t s . 64 12. K i n e t i c s o f S i g n a l I f o r m a t i o n and decay i n d i f f e r e n t l i g h t t r e a t m e n t s i n r a d i s h l e a f p i e c e s p r e v i o u s l y f u m i g a t e d w i t h a p p r o x i m a t e l y 4 00ppm S 0 2 f o r 10 m i n u t e s . 66 13. EPR spectrum from a deta c h e d p i n e n e e d l e . 69 14. EPR spectrum from an e x c i s e d maple l e a f p i e c e . 70 15. EPR spectrum from an e x c i s e d oak l e a f p i e c e . 71 16. EPR spectrum from an e x c i s e d i v y l e a f p i e c e . 72 17. EPR spectrum from an e x c i s e d c h e r r y l e a f p i e c e . 7 3 18. White l i g h t - i n d u c e d s p e c t r a from an i n t a c t , a t t a c h e d p e r e n n i a l r y e g r a s s l e a f . 7 6 19. Comparison o f o r i g i n a l S i g n a l I and S i g n a l N 7 1 Q i n a t t a c h e d , i n t a c t Kentucky b l u e g r a s s l e a v e s . 79 20. Combined S i g n a l s I and H u + S from an a t t a c h e d , i n t a c t K entucky b l u e g r a s s l e a f . 82 21. S i g n a l H u + S from an a t t a c h e d , i n t a c t K entucky b l u e g r a s s l e a f h e l d under N 2. 83 22. White l i g h t - i n d u c e d s i g n a l from an a t t a c h e d , i n t a c t K entucky b l u e g r a s s l e a f . 84 X I LIST OF FIGURES (Continued) Page No. 23. True 710nm l i g h t - i n d u c e d s i g n a l ( S i g n a l I) from an i n t a c t , a t t a c h e d Kentucky b l u e g r a s s l e a f . 86 24. Changes i n t h e d a r k and w h i t e l i g h t - i n d u c e d s i g n a l s i n an i n t a c t , a t t a c h e d Kentucky b l u e g r a s s l e a f caused by removal o f 0 2• 87 25. Examples o f t h e f r e e - r a d i c a l s i g n a l found i n K entucky b l u e g r a s s l e a v e s o f p l a n t s grown under h i g h photon f l u x d e n s i t y . 90 26. Examples o f t h e f r e e - r a d i c a l s i g n a l found i n l e a v e s o f p e r e n n i a l r y e g r a s s p l a n t s grown under h i g h photon f l u x d e n s i t y . 91 27. Examples o f t h e f r e e - r a d i c a l s i g n a l found i n l e a v e s o f b a r n y a r d g r a s s grown i n f u l l summer s u n l i g h t . 93 28. Examples o f t h e f r e e - r a d i c a l s i g n a l found i n l e a v e s o f Kentucky b l u e g r a s s p l a n t s grown under h i g h photon f l u x d e n s i t y 8 days a f t e r t h e p l a n t s were moved t o t h e s p e c t r o m e t r y l a b o r a t o r y . 95 29. Decay o f t h e f r e e - r a d i c a l s i g n a l found i n K entucky b l u e g r a s s l e a v e s grown i n c o n d i t i o n s o f h i g h photon f l u x d e n s i t y , upon exposure t o microwave r a d i a t i o n i n t h e s p e c t r o m e t e r c a v i t y . 96 30. S p e c t r a o f t h e f r e e - r a d i c a l s i g n a l found i n K entucky b l u e g r a s s l e a v e s grown under h i g h photon f l u x d e n s i t y a f t e r exposure t o microwave r a d i a t i o n i n t h e s p e c t r o m e t e r c a v i t y f o r d i f f e r e n t t i m e p e r i o d s i n d a r k n e s s . 97 31. S p e c t r a o f p e r e n n i a l r y e g r a s s l e a v e s grown under h i g h photon f l u x d e n s i t y . 99 x i i LIST OF FIGURES (Continued) Page No. 32. S i g n a l Npj o f Kentucky b l u e g r a s s l e a v e s exposed t o h i g h photon f l u x d e n s i t y . 100 33. A l t e r n a t i v e r e s p o n s e s o f Kentucky b l u e g r a s s l e a v e s c o n t a i n i n g S i g n a l Npj a f t e r e xposure t o microwave r a d i a t i o n i n t h e s p e c t r o m e t e r c a v i t y . 102 34. A l t e r n a t i v e r e s p o n s e s o f Kentucky b l u e g r a s s l e a v e s c o n t a i n i n g S i g n a l Npj a f t e r e xposure t o microwave r a d i a t i o n i n t h e s p e c t r o m e t e r c a v i t y . 103 35. EPR s i g n a l s from an i n t a c t , a t t a c h e d Kentucky b l u e g r a s s l e a f p r i o r t o f u m i g a t i o n . 113 36. EPR s i g n a l s from an i n t a c t , a t t a c h e d Kentucky b l u e g r a s s l e a f a f t e r exposure t o lOOppb ozone f o r 2 h o u r s . 115 37. EPR s i g n a l s from an i n t a c t , a t t a c h e d Kentucky b l u e g r a s s l e a f a f t e r exposure t o lOOppb ozone f o r 3 h o u r s . 116 38. EPR s i g n a l s from an i n t a c t , a t t a c h e d Kentucky b l u e g r a s s l e a f 20 minutes a f t e r t e r m i n a t i o n o f a 3 hour exposure t o lOOppb ozone. 118 39. EPR s i g n a l s from an i n t a c t , a t t a c h e d Kentucky b l u e g r a s s l e a f 16 hou r s and 20 m i n u t e s a f t e r a 3 hour exposure t o lOOppb ozone. 120 40. 710nm l i g h t - i n d u c e d s i g n a l s from an i n t a c t , a t t a c h e d Kentucky b l u e g r a s s l e a f p r i o r t o f u m i g a t i o n . 122 X l l l LIST OF FIGURES (Continued) Page No. 41. D i f f e r e n c e s i n f r e e - r a d i c a l s i g n a l s i n i n t a c t , a t t a c h e d Kentucky b l u e g r a s s l e a v e s a f t e r e x p o s ure t o 0.5ppm ozone f o r 1.5 h o u r s . 123 42. L i g h t - d e p e n d e n t f r e e - r a d i c a l s i g n a l s i n i n t a c t , a t t a c h e d Kentucky b l u e g r a s s l e a v e s a f t e r 1.5 ho u r s o f f u m i g a t i o n w i t h 0.5ppm ozone. 126 43. F r e e - r a d i c a l s i g n a l d i f f e r e n c e s i n i n t a c t , a t t a c h e d p e r e n n i a l r y e g r a s s l e a v e s f u m i g a t e d w i t h lppm ozone f o r 30 minutes a f t e r s u b t r a c t i o n o f i n i t i a l S i g n a l I I U + S . 128 44. K i n e t i c s o f S i g n a l I f o r m a t i o n i n e x c i s e d u n f u m i g a t e d r a d i s h l e a f p i e c e s i n d i f f e r e n t l i g h t r e g i m e s . 137 45. K i n e t i c s o f S i g n a l I f o r m a t i o n i n S 0 2 ~ f u m i g a t e d (400ppm) r a d i s h l e a f p i e c e s i n d i f f e r e n t l i g h t r e g imes. 139 46. EPR s i g n a l s i n d i f f e r e n t l i g h t regimes from a r a d i s h l e a f p i e c e a f t e r 50 minutes o f f u m i g a t i o n w i t h 400ppm S 0 2 • 141 47. The e f f e c t o f 400ppm S 0 2 f u m i g a t i o n d u r a t i o n on t h e i n t e n s i t y o f t h e w h i t e l i g h t - i n d u c e d S i g n a l I i n e x c i s e d r a d i s h l e a f p i e c e s . 142 48. The e f f e c t o f i n t e r r u p t i o n o f 400ppm S 0 2 f u m i g a t i o n on t h e k i n e t i c s o f t h e w h i t e l i g h t - i n d u c e d f o r m a t i o n o f S i g n a l I i n e x c i s e d r a d i s h l e a f p i e c e s . 144 49. The l a g t i m e p r i o r t o t h e i n d u c t i o n o f t h e w h i t e l i g h t - i n d u c e d S i g n a l I i n r e l a t i o n t o S 0 o c o n c e n t r a t i o n . 145 x i v LIST OF FIGURES (Continued) Page No, 50. The M n + + s i g n a l i n h e a l t h y and f u m i g a t e d r a d i s h l e a f p i e c e s . 148 51. Comparative k i n e t i c s o f t h e i n c r e a s e i n t h e Mn s i g n a l and t h e d e c r e a s e i n S i g n a l I I U + S i n e x c i s e d r a d i s h l e a f p i e c e s d u r i n g f u m i g a t i o n w i t h 400ppm S 0 2 . 149 52. EPR s i g n a l s from h e a l t h y and S 0 2 ~ f u m i g a t e d a t t a c h e d , i n t a c t p e r e n n i a l r y e g r a s s l e a v e s . 151 53. L i g h t - i n d u c e d EPR s i g n a l changes i n a t t a c h e d , i n t a c t K entucky b l u e g r a s s l e a v e s f u m i g a t e d w i t h 600ppb S 0 2 . 153 54. L i g h t - i n d u c e d EPR s i g n a l changes i n a t t a c h e d , i n t a c t K entucky b l u e g r a s s l e a v e s f u m i g a t e d w i t h 600ppb S 0 2 . 156 55. EPR 710nm l i g h t - i n d u c e d and w h i t e l i g h t - i n d u c e d s i g n a l s i n i n t a c t , a t t a c h e d Kentucky b l u e g r a s s l e a v e s 16 hou r s a f t e r t e r m i n a t i o n o f f u m i g a t i o n w i t h 600ppb S 0 2 . 157 56. S i g n a l N S o x , found i n a t t a c h e d , i n t a c t K e ntucky b l u e g r a s s l e a v e s a f t e r f u m i g a t i o n w i t h 600ppb S 0 2 f o r 4 h o u r s . 159 X V ABBREVIATIONS BPN N - t - b u t y l - a - p h e n y l n i t r o n e DCMU 3 - ( 3 , 4 - d i c h l o r o p h e n y l ) - 1 , 1 - d i m e t h y l u r e a DMPO 5 , 5 - d i m e t h y l - p y r r o l i n e - N - o x i d e DPPH 1 , l - d i p h e n y l - 2 - p i c r y l - h y d r a z y l EDU N - ( 2 - ( 2 - o x o - l - i m i d a z o l i d i n y l ) - e t h y l ) - N ' - p h e n y l u r e a ("ethylene d i u r e a " ) FAD F l a v i n a d enine d i n u c l e o t i d e kDa k i l o D a l t o n MDA M a l o n d i a l d e h y d e NADP N i c o t i n a m i d e adenine d i n u c l e o t i d e phosphate OEC O x y g e n - e v o l v i n g complex PBN 4 - p y r i d y l - l - o x i d e - N - t e r t - b u t y l - n i t r o n e PFD Photon f l u x d e n s i t y PS I Photosystem I P S I I Photosystem I I PUFA P o l y u n s a t u r a t e d f a t t y a c i d SOD S u p e r o x i d e d i s m u t a s e TEMPO 2 , 2 , 6 , 6 - t e t r a m e t h y l - l - p i p e r i d i n e - N - o x y l TIRON 1 , 2 - d i h y d r o x y b e n z e n e - 3 , 5 - d i s u l p h o n a t e xv i ACKNOWLEDGEMENTS T h i s t h e s i s i s d e d i c a t e d t o : K i t t y F u r b a l l V a a r t n o u because she i s a l o t o f f u n , has a mind o f h e r own and c o u l d e a s i l y p l a y b o t h r o l e s i n "A T a l e o f Two K i t t i e s " . I would l i k e t o thank my w i f e V a l o r i e and d a u g h t e r C h r i s t i e f o r b o t h s u p p o r t and t o l e r a n c e o v e r an u n u s u a l l y extended p e r i o d o f t i m e , and thank V a l f o r p r i n t i n g t h e many d r a f t s o f t h i s t h e s i s . I would a l s o l i k e t o acknowledge t h e c o n t r i b u t i o n o f my r e s e a r c h s u p e r v i s o r , Dr. V.C. R u n e c k l e s : f i n a n c i a l s u p p o r t t h r o u g h h i s N.R.C. g r a d u a t e a s s i s t a n t s h i p s ; e x t e n s i v e u s e f u l e d i t o r i a l h e l p w i t h t h e m a n u s c r i p t ; and a d v i c e and encouragement t h r o u g h o u t t h e c o u r s e o f t h e r e s e a r c h . I n a d d i t i o n , t h e s u p p o r t o f t h e o t h e r s i n my immediate f a m i l y was a p p r e c i a t e d : P a r e n t s Dr. Herman and Mrs. H e l l a V a a r t n o u , b r o t h e r s P e t e r and E r i k , and s i s t e r s - i n - l a w C i n d y and L i n d a . L a s t , b u t n o t l e a s t , t h e C e c i l crowd o f 1967-74 - s c a t t e r e d from Kamloops t o M o n t r e a l t o Tucson t o K u a l a Lumpur - t h e memories rem a i n : Ken, Gord, E r i c , Susan, Bob, R o b i n H., R o b i n E., Barb, B r u c e , Maureen, Joanne, Russ, W i l l i e , B r ock, Dune and S t e v e . 1.0 INTRODUCTION P l a n t s , i n t h e i r n a t u r a l environment, a r e s u b j e c t t o numerous s t r e s s e s which may e i t h e r r e t a r d o r t e r m i n a t e t h e i r growth and development. Many s t r e s s f u l c o n d i t i o n s , such as exp o s u r e t o e x c e s s i v e r a d i a t i o n o r a s h o r t a g e o f w a t e r , may o c c u r n a t u r a l l y , b u t some s t r e s s e s , such as exposure t o e l e v a t e d l e v e l s o f gaseous a i r p o l l u t a n t s , a r e l a r g e l y due t o t h e a c t i v i t y o f man ( L e v i t t , 1980) . P l a n t s r e s pond t o such s t r e s s e s i n v a r i o u s ways. O v e r a l l growth may be a f f e c t e d , and c h a r a c t e r i s t i c symptoms may d e v e l o p i n r e s p o n s e t o d i f f e r e n t s t r e s s e s . I n many i n s t a n c e s , n e c r o s i s o f c e l l s and t i s s u e s may o c c u r . However, such r e s p o n s e s a r e t h e u l t i m a t e m a n i f e s t a t i o n o f v a r i o u s a c t i o n s o c c u r r i n g a t t h e b i o c h e m i c a l l e v e l w i t h i n t h e c e l l s o f t h e p l a n t , w h i c h l e a d t o d i s r u p t i o n s o f normal m e t a b o l i c p r o c e s s e s . I n t h e case o f a i r p o l l u t a n t s , such as ozone and s u l p h u r d i o x i d e , t h e r e d u c t i o n s i n p l a n t growth which t h e y cause have been, a t l e a s t i n p a r t , a t t r i b u t e d t o r e d u c t i o n s i n p h o t o - s y n t h e s i s ( B l a c k and Unsworth, 1979; K o z i o l and J o r d a n , 1978; S i s s o n e t a l . 1981). One s u g g e s t e d cause o f t h i s r e d u c t i o n i s t h e impairment o f t h e p h o t o s y n t h e t i c e l e c t r o n t r a n s p o r t c h a i n t h r o u g h t h e f o r m a t i o n o f d e t r i m e n t a l f r e e r a d i c a l s (Tanaka and Sugahara, 1980; S a k a k i e t a l . 1983). - 1 - E l e c t r o n p a r a m a g n e t i c resonance (EPR) s p e c t r o s c o p y , a b r i e f d e s c r i p t i o n o f w h i c h i s p r e s e n t e d i n Appendix A, a l l o w s t h e s t u d y o f f r e e r a d i c a l s , i . e . c h e m i c a l s p e c i e s w h i c h c o n t a i n u n p a i r e d e l e c t r o n s . EPR has been found t o be u s e f u l f o r t h e s t u d y o f m e t a b o l i c e l e c t r o n t r a n s p o r t c h a i n s because some o f t h e components o f such c h a i n s have u n p a i r e d e l e c t r o n s i n e i t h e r t h e i r o x i d i z e d o r r e d u c e d s t a t e . The n a t u r a l f o r m a t i o n o f f r e e r a d i c a l s i n p h o t o s y n t h e s i z i n g c h l o r o p l a s t s has been known s i n c e t h e p i o n e e r i n g EPR s t u d i e s o f Commoner e t a_l. (1956, 1957). Subsequent i n v e s t i g a t i o n s by numerous w o r k e r s have shown t h a t one f r e e - r a d i c a l s i g n a l ( S i g n a l I) o b s e r v e d i n i s o l a t e d c h l o r o p l a s t s i l l u m i n a t e d by f a r - r e d l i g h t (700-730nm) i s a s s o c i a t e d w i t h Photosystem I (PSI) ( B e i n a r t e t al. 1962; M a l k i n , 1982). A second s i g n a l ( S i g n a l I I ) i s i n c r e a s e d by i l l u m i n a t i o n o v e r a wide range o f wave l e n g t h s l e s s t h a n 7 00nm and has been been a s s o c i a t e d w i t h o x i d a t i o n - r e d u c t i o n r e a c t i o n s i n t h e w a t e r - s p l i t t i n g p r o c e s s o f Photosystem I I ( P S I I ) (Babcock and Sauer, 1973a). P l a n t l e a v e s a l s o r e v e a l a s i x peak M n + + s i g n a l , t h e i n t e n s i t y o f w h i c h v a r i e s g r e a t l y among s p e c i e s and w i t h p l a n t l e a f age ( M i s h r a e t a l . 1971; McCain e t a l . 1984). S i g n a l I I i s superimposed on t h e f o u r t h peak from t h e low f i e l d end o f t h i s M n + + s i g n a l , w h i l e S i g n a l I i s superimposed on S i g n a l I I . I n a d d i t i o n , a b r o a d s l o p i n g s i g n a l a t t r i b u t e d t o F e + + (Treharne and E y s t e r , 1962; T r e h a r n e e t a l . 1964) u n d e r l i e s t h e M n + + s i g n a l (Appendix C ) . -2- Independent o f p o s s i b l e d i r e c t e f f e c t s on p h o t o s y n t h e t i c p r o c e s s e s , a i r p o l l u t a n t s have been h y p o t h e s i z e d as g i v i n g r i s e t o d e l e t e r i o u s f r e e r a d i c a l s w h i c h a r e g e n e r a l l y d i s r u p t i v e t o t h e normal f u n c t i o n i n g o f c e l l membranes (Mudd, 1973,1982). F r e e r a d i c a l s a r e a l s o h y p o t h e s i z e d as p r o v i d i n g t h e b a s i s f o r s enescence i n p l a n t t i s s u e s (Leshem, 1981). I n p a r t i c u l a r , f o r m a t i o n o f t o x i c oxygen-based s p e c i e s such as t h e s u p e r o x i d e ( 0 2 ~ ) and h y d r o x y l (OH*) f r e e r a d i c a l s has been proposed as t h e p r i m a r y cause o f i n j u r y . However, t h i s has n o t been dem o n s t r a t e d i n i n t a c t l e a v e s , nor has i t been c o n c l u s i v e l y e s t a b l i s h e d i n s t u d i e s u s i n g c h l o r o p l a s t o r s u b c h l o r o p l a s t p r e p a r a t i o n s under ambient c o n d i t i o n s . N e v e r t h e l e s s , f r e e r a d i c a l s have been c a u s a l l y i m p l i c a t e d i n p l a n t senescence ( C h i a e t a l . 1981, 1982; McRae and Thompson, 1983; Thompson e t a l . 1987), and premature senescence i s a symptom f r e q u e n t l y a s s o c i a t e d w i t h t h e exposure o f p l a n t s t o a i r p o l l u t a n t s . A l t h o u g h t h e p o l l u t a n t - i n d u c e d p r o d u c t i o n o f t h e s u p e r o x i d e and o t h e r f r e e r a d i c a l s has been proposed as t h e p r i m a r y cause o f t h e g e n e r a l impairment o f membrane f u n c t i o n s t h r o u g h o u t t h e c e l l f o l l o w i n g p o l l u t a n t u p t a k e , Lee and B e n n e t t (1982) s u g g e s t e d t h a t such a d v e r s e a f f e c t s were m i n i m i z e d i n t h e p r e s e n c e o f t h e s u p e r o x i d e d i s p r o p o r t i o n a t i n g enzyme, s u p e r o x i d e d i s m u t a s e (SOD). However, Chanway and R u n e c k l e s (1984a, 1984b) and M c K e r s i e e t a l . (1982) were u n a b l e t o c o n f i r m t h e proposed r o l e o f SOD i n r e d u c i n g t h e h a r m f u l e f f e c t s o f ozone. -3- The s u p e r o x i d e a n i o n r a d i c a l has been d e t e c t e d i n r a p i d f l o w enzyme systems (Palmer e t a l . 1964; Bray e t a_l. 1964 ; Knowles e t a l . 1969). D e t e c t i o n o f t h e s u p e r o x i d e (Harbour and B o l t o n , 1975; McRae and Thompson, 1983) and h y d r o x y l (Harbour and B o l t o n , 1978) f r e e r a d i c a l s has a l s o been d e m o n s t r a t e d by t h e use o f s p i n - t r a p p i n g EPR t e c h n i q u e s w i t h c h l o r o p l a s t and o t h e r s u b c e l l u l a r p r e p a r a t i o n s . However, few s t u d i e s have been c o n d u c t e d on t h e changes i n f o r m a t i o n o f EPR f r e e - r a d i c a l s i g n a l s r e s u l t i n g from exposure t o ozone o r s u l p h u r d i o x i d e (Rowlands e t a l . 1970; S h i m a z a k i e t a l . 1984b). F u r t h e r m o r e , t h e y a l l used e x c i s e d t i s s u e s o r c h l o r o p l a s t p r e p a r a t i o n s a f t e r p l a n t s had been exposed t o t h e a i r p o l l u t a n t s f o r v a r i o u s t i m e p e r i o d s r a t h e r t h a n o b s e r v i n g t h e s i g n a l s d u r i n g f u m i g a t i o n . Such approaches p r e c l u d e making o b s e r v a t i o n s on t h e dynamics of f r e e r a d i c a l f o r m a t i o n d u r i n g exposure t o gaseous p o l l u t a n t s . A l s o , e f f e c t s o b s e r v e d i n e x c i s e d l e a v e s o r c h l o r o p l a s t p r e p a r a t i o n s a r e n o t n e c e s s a r i l y i n d i c a t i v e o f e f f e c t s o c c u r r i n g i n i n t a c t , n o r m a l l y f u n c t i o n i n g , a t t a c h e d l e a v e s . The f i r s t o b j e c t i v e o f t h e p r e s e n t s t u d y was t h e r e f o r e t o d e v e l o p a method p e r m i t t i n g t h e o b s e r v a t i o n o f EPR s i g n a l s i n whole, a t t a c h e d l e a v e s , a t room t e m p e r a t u r e . The second o b j e c t i v e was t o o b s e r v e t h e changes i n t h e p h o t o s y n t h e t i c s i g n a l s o c c u r r i n g when l e a v e s were f u m i g a t e d w i t h ozone o r s u l p h u r d i o x i d e . A t h i r d o b j e c t i v e was t o o b s e r v e t h e -4- appearance o f new s i g n a l s d u r i n g o r subsequent t o f u m i g a t i o n , and t o d e t e r m i n e how t h e s e changes r e f l e c t e d t h e i n i t i a l s t a g e s o f p l a n t r e s p o n s e . A l t h o u g h t h e o v e r a l l o b j e c t i v e was t o i n v e s t i g a t e f r e e r a d i c a l changes i n d u c e d by exposure t o gaseous a i r p o l l u t a n t s , i t was a l s o n e c e s s a r y t o i n v e s t i g a t e t h e v a r i o u s f a c t o r s w h i c h i n f l u e n c e EPR s p e c t r o s c o p y o f i n t a c t t i s s u e s , such as p h o t o i n h i b i t i o n and oxygen t o x i c i t y e f f e c t s , i n o r d e r t o o b t a i n a c l e a r u n d e r s t a n d i n g o f t h e p o t e n t i a l and l i m i t a t i o n s o f s t u d y i n g i n t a c t t i s s u e . -5- 2.0 LITERATURE REVIEW 2.1 F r e e R a d i c a l s and Oxygen T o x i c i t y F r e e r a d i c a l s a r e m o l e c u l e s o r atoms wh i c h c o n t a i n u n p a i r e d e l e c t r o n s . They may be p o s i t i v e l y o r n e g a t i v e l y c h a r g e d , o r may be n e u t r a l . Most f r e e r a d i c a l s a r e h i g h l y r e a c t i v e because o f t h e i r u n s t a b l e e l e c t r o n i c c o n f i g u r a t i o n . T h i s r e a c t i v i t y may cause d e l e t e r i o u s e f f e c t s i n b i o l o g i c a l systems, such as a l o s s o f membrane f l u i d i t y o r i n a c t i v a t i o n o f numerous enzymes. A l s o , because f r e e r a d i c a l s c o n t a i n an u n p a i r e d e l e c t r o n t h e y c r e a t e a n o t h e r f r e e r a d i c a l when t h e y r e a c t w i t h a n o n - r a d i c a l . Thus, t h e y have t h e p o t e n t i a l t o i n i t i a t e a c h a i n r e a c t i o n and g r e a t l y i n c r e a s e t h e damage caused by t h e i n i t i a t i n g r a d i c a l . I n r e c e n t y e a r s , t h e i n v o l v e m e n t o f oxygen-based f r e e r a d i c a l s i n b i o l o g i c a l systems has been proposed as a l i k e l y f a c t o r c o n t r i b u t i n g t o s t r e s s - r e l a t e d i n j u r y s u f f e r e d by p l a n t s ( P r y o r , 1976) . Oxygen t o x i c i t y i s u s u a l l y n o t a s c r i b e d t o m o l e c u l a r oxygen, b u t t o t h e r e a c t i v e s p e c i e s d e r i v e d from i t . M o l e c u l a r oxygen (dioxygen) i s r e l a t i v e l y u n r e a c t i v e because o f t h e s p i n r e s t r i c t i o n o f i t s two u n p a i r e d e l e c t r o n s . I n i t s ground s t a t e , m o l e c u l a r oxygen has two u n p a i r e d e l e c t r o n s o f t h e same s p i n w h i c h occupy d i s t i n c t o r b i t a l s o f e q u a l energy. These u n p a i r e d e l e c t r o n s c o n s t i t u t e a s p i n r e s t r i c t i o n t o c o v a l e n t b o n d i n g -6- s i n c e one o f t h e s p i n s must be i n v e r t e d b e f o r e 0 2 can r e a c t w i t h a r e d u c t a n t ( A l l e n and H i l l , 1978). T h i s i n v e r s i o n r e q u i r e s energy as w e l l as t i m e , and as t h e t i m e f o r a s p i n i n v e r s i o n (10 s) i s l o n g i n comparison t o t h e l i f e t i m e o f a — i o . m o l e c u l a r c o l l i s i o n (10 s ) , t h e p r o b a b i l i t y o f oxygen b e i n g i n v o l v e d i n many r e a c t i o n s i s low ( A l l e n and H i l l , 1978). The s p i n r e s t r i c t i o n can be overcome i f s u f f i c i e n t energy i s s u p p l i e d t o t h e ground s t a t e 0 2 t o form e i t h e r o f two s p e c i e s o f t h e e x c i t e d s i n g l e t s t a t e oxygen i n whi c h t h e s p i n s a r e o c c u p y i n g e i t h e r s e p a r a t e o r b i t a l s (^g) o r t h e same o r b i t a l ("*Ag) • These can r e t u r n t o t h e ground s t a t e e i t h e r t h r o u g h r a d i a t i o n o f e x c e s s energy o r by energy t r a n s f e r t o a n o t h e r m o l e c u l e . Of t h e s e s p e c i e s , ( E g ) i s r a p i d l y quenched and does not engage i n s i g n i f i c a n t c h e m i s t r y ( R a b i n o w i t c h and F r i d o v i c h , 1983) . ("^Ag) i s more s t a b l e and i s i n v o l v e d i n d y e - s e n s i t i z e d o x i d a t i o n s c a l l e d photodynamic e f f e c t s . Photodynamic e f f e c t s can be an i m p o r t a n t a s p e c t o f oxygen t o x i c i t y ( N i l s s o n and Kearns, 1973). Green p l a n t s , w h i c h a r e n a t u r a l l y exposed t o i n t e n s e l i g h t and oxygen, c o n t a i n t h e p h o t o s e n s i t i z e r , c h l o r o p h y l l . The s p i n r e s t r i c t i o n can a l s o be overcome by a one e l e c t r o n t r a n s f e r t o 0 2 . T h i s e n e r g e t i c a l l y f a v o u r a b l e r e a c t i o n r e s u l t s i n f o r m a t i o n o f t h e s u p e r o x i d e r a d i c a l ( 0 2 ~ ) (Samuel and S t e c k e l , 1974). The u n i v a l e n t r e d u c t i o n o c c u r s i n b o t h a b i o t i c and b i o l o g i c a l o x i d a t i o n s . -7- The s u p e r o x i d e r a d i c a l ( 0 2 ~ ) and a n o t h e r oxygen-based f r e e r a d i c a l , t h e h y d r o x y l r a d i c a l (OH'), a r e most f r e q u e n t l y c i t e d as h a v i n g d e t r i m e n t a l e f f e c t s on b i o l o g i c a l systems. S u p e r o x i d e has been known t o i n d u c e l i p i d p e r o x i d a t i o n ( K e l l o g g and F r i d o v i c h , 1975), i n j u r e membranes ( G o l d b e r g and S t e r n , 1976; K e l l o g g and F r i d o v i c h , 1977), i n a c t i v a t e v i r u s e s ( L a v e l l e e t a l . 1973) and cause t h e d e a t h o f c e l l s ( M i c h e l s o n and Buckingham, 1974). M o l e c u l e s s u s c e p t i b l e t o s u p e r o x i d e a t t a c k i n c l u d e p r o t e i n s , n u c l e i c a c i d s , p o l y u n s a t u r a t e d f a t t y a c i d s (PUFA) and c a r b o h y d r a t e s (Leshem, 1981). P l a n t senescence and f r u i t r i p e n i n g a r e t h o u g h t t o be p r o c e s s e s a s s o c i a t e d w i t h i n c r e a s e d f r e e r a d i c a l f o r m a t i o n , i n p a r t i c u l a r , t h e f o r m a t i o n o f f r e e r a d i c a l s d e r i v e d from oxygen ( F r e n k e l , 1978; H a l l i w e l l , 1981; R a b i n o w i t c h and S k l a n , 1981; Buchvarov and G a n t c h e f f , 1984) . A c c e l e r a t e d s p o i l a g e o f c u t f l o w e r s (Leshem, 1981), wounding (Thompson e t a l . 1987), a g i n g ( P r i e s t l e y e t a l . 1980) and s u n s c a l d ( R a b i n o w i t c h and S k l a n , 1980) may a l s o i n v o l v e f r e e r a d i c a l a c t i v i t y . Numerous o x i d a t i v e enzymes a r e known t o g e n e r a t e t h e s u p e r o x i d e r a d i c a l i n b i o l o g i c a l systems. T h i s o c c u r s i n r e a c t i o n s i n v o l v i n g oxygen w h i c h a r e m ediated by c h l o r o p l a s t and m i t o c h o n d r i a l membranes. Some examples o f such enzymes i n c l u d e x a n t h i n e o x i d a s e , a l d e h y d e o x i d a s e , d i h y d r o o r o t i c d i o x y g e n a s e , i n d o l e a m i n e d i o x y g e n a s e and 2 - n i t r o p r o p a n e d i o x y g e n a s e (Palmer e t a l . 1964; Bray e t a l . 1964; Knowles e t a l . 1969; F r i d o v i c h , -8- 1981). Superoxide may also be formed through photooxidative processes. These react ions are mediated by p h o t o s e n s i t i z e r s such as r i b o f l a v i n , porphyrins and anthroquinones. These chemicals pass e x c i t a t i o n energy absorbed from l i g h t to oxygen with the r e s u l t a n t formation of high energy s i n g l e t oxygen or superoxide v i a univalent oxygen reduction (Ballou et a l . 1969). EPR s p i n t rapping was used to demonstrate the presence of the superoxide r a d i c a l i n young c h l o r o p l a s t suspensions exposed to elevated l e v e l s of oxygen (Harbour and Bolton, 1975) and i n aged c h l o r o p l a s t suspensions (McRae and Thompson, 1983). Super- oxide production has also been shown i n mitochondria (Rich and Bonner, 1978), microsomes (Kuthan et a l . 1982) and n u c l e i (Patton et a l . 1980). Photosynthetic e lec t ron flow may also cause the formation of the superoxide r a d i c a l . Asada et a l . (1976), presented evidence using superoxide dismutase (SOD), a scavenger of superoxide, which suggested that superoxide i s produced i n i l l u m i n a t e d c h l o r o p l a s t s . SOD was also shown to i n h i b i t the photoreduction of cytochrome c by spinach c h l o r o p l a s t suspensions, thereby i m p l i c a t i n g the a c t i v i t y of the superoxide r a d i c a l i n t h i s photoreduction (Nelson et a l . 1972). Oxygen can be reduced to H 2 0 2 by i l l u m i n a t e d c h l o r o p l a s t s (Mehler, 1951). The r e a c t i o n probably involves the superoxide r a d i c a l as an intermediate : - 9 - °2~ + °2~ + 2 H y i e l d s H 2 0 2 + 0 2 (Harbour and B o l t o n , 1975; G l i d e w e l l and Raven, 1975) E n z y m a t i c p r o c e s s e s such as t h e breakdown o f x a n t h i n e t o u r i c a c i d i n p u r i n e c a t a b o l i s m a l s o produce s u p e r o x i d e . The o x i d a t i v e n a t u r e o f r e s p i r a t i o n i t s e l f r e s u l t s i n s u p e r o x i d e p r o d u c t i o n even though t h e major enzyme wh i c h u t i l i z e s oxygen, cytochrome o x i d a s e , r educes t e t r a v a l e n t oxygen t o w a t e r w i t h o u t t h e r e l e a s e o f any t o x i c i n t e r m e d i a t e ( F r i d o v i c h , 1978) . L i p o x y g e n a s e a c t i v i t y can a l s o produce s u p e r o x i d e and PUFA can become c o n v e r t e d t o f r e e r a d i c a l s v i a H + a b s t r a c t i o n d u r i n g t h i s p r o c e s s (Leshem, 1981) . Ozone has been i m p l i c a t e d i n i n i t i a t i n g f r e e r a d i c a l p r o d u c i n g r e a c t i o n s , e s p e c i a l l y i n membrane f a t t y a c i d s ( P r y o r , 1976). The r e a c t i v e ozone m o l e c u l e i s c a p a b l e o f r e a c t i n g w i t h numerous o r g a n i c m o l e c u l e s . These i n c l u d e a l k a n e s , a l k e n e s , a l d e h y d e s and amines ( P r y o r , 1976). F r e q u e n t l y t h i s i n t e r a c t i o n can r e s u l t i n f r e e r a d i c a l f o r m a t i o n . There i s a l s o p o t e n t i a l f o r t h e f o r m a t i o n o f v a r i o u s f r e e r a d i c a l s when ozone d i s s o l v e s i n w a t e r . The p rominent p r o d u c t o f t h i s r e a c t i o n i s p r o b a b l y t h e h y d r o x y l r a d i c a l (Weiss, 1935), but w i t h i n b i o l o g i c a l systems t h e r e has been no d e t a i l e d a n a l y s i s o f t h e p r o d u c t s as y e t (Heath, 1975). The most l i k e l y s o u r c e o f s u p e r o x i d e i n i n j u r e d t i s s u e s i s -10- l i p i d p e r o x i d a t i o n o f membrane PUFA (Tomlinson and R i c h , 1970; P a u l s and Thompson, 1981a, 1981b). Even under normal m e t a b o l i c c i r c u m s t a n c e s , t h e danger o f i n i t i a t i n g p e r o x i d a t i o n o f t h e c l o s e l y a s s o c i a t e d h y d r o p h o b i c t a i l s v i a H + a b s t r a c t i o n and f r e e r a d i c a l p r o d u c t i o n i s always p r e s e n t . Due t o t h e r e a c t i v i t y o f ozone, o n l y a few m o l e c u l e s exposed t o a p o r t i o n o f t h e h y d r o p h o b i c membrane c o u l d i n i t i a t e p e r o x i d a t i v e p r o c e s s e s . However, Mudd (1982) c a u t i o n s t h a t t h e p e r o x i d a t i o n may, i n some i n s t a n c e s , be c o n f u s e d w i t h o z o n e - i n d u c e d o z o n o l y s i s , as t h e most r e a d i l y measured p r o d u c t , m a l o n d i a l d e h y d e (MDA), i s a p r o d u c t o f b o t h r e a c t i o n s . T o x i c f r e e r a d i c a l f o r m a t i o n has been proposed as a p r i m a r y e v e n t i n t h e sequence t h r o u g h w h i c h ozone may cause d e t r i m e n t a l changes t o t h e p h o t o s y n t h e t i c p r o c e s s ( S a k a k i e t a l . 1983). F r e e r a d i c a l - i n d u c e d membrane impairment i s o f t e n c i t e d as t h e r e a s o n f o r p h o t o s y n t h e t i c r e d u c t i o n (Mudd, 1973, 1982). The degree o f impairment has been c o r r e l a t e d t o t h e l e v e l o f s u p e r o x i d e d i s m u t a s e (SOD) p r e s e n t i n t h e l e a v e s (Lee and B e n n e t t , 1982), but t h i s was not c o n f i r m e d by subsequent i n v e s t i g a t o r s ( M c K e r s i e e t a l . 1982; Chanway and R u n e c k l e s , 1984a, 1984b). These d i v e r s e r e s u l t s a r e p r o b a b l y a t t r i b u t a b l e t o i n h e r e n t d i f f e r e n c e s i n p l a n t m a t e r i a l and e x p e r i m e n t a l c o n d i t i o n s . S a k a k i e t a l . (1983) found t h a t l e v e l s o f SOD i n s p i n a c h c h l o r o p l a s t s u s p e n s i o n s from l e a v e s p r e v i o u s l y f u m i g a t e d w i t h 0.5ppm 0 3 were d e c r e a s e d 50 p e r c e n t by 4 h o u r s o f f u m i g a t i o n . However, t h e r e was no change i n SOD l e v e l s when s p i n a c h l e a v e s were f u m i g a t e d w i t h 0.1 o r 0.2ppm 0 3 f o r 24 h o u r s , bu t t h e r e was a 50 p e r c e n t r e d u c t i o n when l e a v e s were f u m i g a t e d w i t h 0.3ppm 0 3 f o r 24 h o u r s . Thus, t h e e f f e c t s on SOD seem t o be exposure dependent. A t t h e l o w e r f u m i g a t i o n l e v e l s , MDA l e v e l s i n c r e a s e d even though SOD, c h l o r o p h y l l a, c h l o r o p h y l l b, L - a s c o r b a t e and d e h y d r o - L - a s c o r b a t e l e v e l s remained unchanged ( S a k a k i e t a l . 1983). T h i s s u p p o r t s t h e c o n t e n t i o n t h a t i n c r e a s e s i n MDA l e v e l s a r e a f u n c t i o n o f o z o n o l y s i s , n o t l i p i d p e r o x i d a t i o n (Mudd, 1982). I t has been s u g g e s t e d t h a t m o l e c u l a r oxygen may a l s o a c t as t h e t e r m i n a l a c c e p t o r o f Photosystem I i n h i g h e r p l a n t s (Leshem e t a l . 1981; F o s t e r and Hess, 1982). W h i l e t h i s i s s t r i c t l y a h y p o t h e s i s a t t h e p r e s e n t t i m e , t h e u n i v a l e n t r e d u c t i o n would r e s u l t i n t h e p r o d u c t i o n o f s u p e r o x i d e . The p r e s e n c e o f t h e s u p e r o x i d e t h u s c r e a t e d would p a r t i a l l y e x p l a i n t h e p r e s e n c e o f t h e m a j o r i t y o f SOD a c t i v i t y i n t h e c h l o r o p l a s t s o f p l a n t s (Asada e t a l . 1973; Kono e t a l . 1979; F o s t e r and Edwards, 1980). Mudd e t a l . (1974) a l s o p o i n t out t h a t p o t e n t i a l s u p e r o x i d e p r o d u c t i o n c o u l d o c c u r t h r o u g h c l e a v a g e o f t h e n i c o t i n a m i d e r i n g o f NADP(H). S u p e r o x i d e g e n e r a t e d i n aqueous media i s u n s t a b l e , p a r t i c u l a r l y w i t h d e c r e a s i n g pH, whereas i n an a p r o t i c medium such as t h e i n t e r i o r o f t h e h y d r o c a r b o n b i l a y e r i n biomembranes, -12- i t s l i f e t i m e i s l o n g e r ( A l l e n and H i l l , 1978). S u p e r o x i d e can s p o n t a n e o u s l y d i s m u t a t e t o form H 2 0 2 as shown below: 2 0 2 ~ + 2H + y i e l d s H 2 0 2 + 0 2 T h i s i s t h e r e a c t i o n t h a t i s a l s o c a t a l y z e d by t h e enzyme, s u p e r o x i d e d i s m u t a s e . D e s p i t e t h e p r o l i f e r a t i o n o f r e p o r t s w h i c h a t t r i b u t e damage t o b i o l o g i c a l systems t o t h e a c t i v i t y o f t h e s u p e r o x i d e r a d i c a l , o t h e r s t u d i e s s u g g e s t t h a t a t l e a s t some o f t h i s damage may be caused by t h e more t o x i c h y d r o x y l r a d i c a l (Leshem, 1981). F r i d o v i c h (1978) showed t h a t a d d i t i o n o f s u p e r o x i d e d i s m u t a s e (SOD) o r c a t a l a s e i n h i b i t e d t h e p r o d u c t i o n o f e t h y l e n e from m e t h i o n a l (CH 3-S-CH 2-CH 2-CH0) exposed t o an e n z y m a t i c s o u r c e o f b o t h s u p e r o x i d e and H 2 0 2 . However, t h e a d d i t i o n o f e t h a n o l o r b e n z o a t e , b o t h o f wh i c h scavenge t h e h y d r o x y l r a d i c a l s e l e c t i v e l y , w h i l e r e m a i n i n g u n r e a c t i v e t o s u p e r o x i d e o r H 2 0 2 , a l s o i n h i b i t e d t h e p r o d u c t i o n o f e t h y l e n e . T h i s s u p p o r t s t h e c o n t e n t i o n t h a t t h e h y d r o x y l r a d i c a l i s t h e a c t i v e agent s i n c e s u p e r o x i d e and H 2 0 2 can combine t o form t h e h y d r o x y l r a d i c a l , t h r o u g h i r o n c a t a l y s i s o f t h e Haber-Weiss r e a c t i o n shown below: H2°2 + °2~ Y i e l d s 0 H * + 0 H " + °2 (Leshem, 1981) -13- A n o t h e r a l t e r n a t i v e f o r t h e f o r m a t i o n o f t h e h y d r o x y l r a d i c a l i s t h e Fenton r e a c t i o n i n w h i c h H 2 0 2 r e a c t s w i t h d i v a l e n t f e r r o u s i r o n t o form t h e h y d r o x y l r a d i c a l , a h y d r o x y l i o n and a t r i v a l e n t f e r r i c i o n : H 2 0 2 + F e + 2 y i e l d s 0H~ + OH' + Fe 4" 3 Once formed, t h e h y d r o x y l r a d i c a l i s e x t r e m e l y r e a c t i v e and a t t a c k s numerous t a r g e t m o l e c u l e s i n d i s c r i m i n a t e l y . Thus, t h e r e a r e no s p e c i f i c s c a v e n g e r s f o r t h i s r a d i c a l w i t h i n p l a n t s as t h e y would be r e q u i r e d a t u n r e a l i s t i c a l l y h i g h c o n c e n t r a t i o n s . I t appears t h a t organisms defend t h e m s e l v e s a g a i n s t t h e e f f e c t s o f t h e h y d r o x y l r a d i c a l t h r o u g h v a r i o u s a n t i o x i d a n t s w h i c h p r e v e n t t h e f o r m a t i o n o f t h e p r e c u r s o r s t o t h e h y d r o x y l r a d i c a l . S p e c i f i c a l l y , c a t a l a s e s and p e r o x i d a s e s scavenge H 2 0 2 (Asada e t a l . 1975), w h i l e s u p e r o x i d e d i s m u t a s e s scavenge 0 2 ~ (Kono and F r i d o v i c h , 1982). 2.2 P h o t o i n h i b i t i o n I n j u r y t o t h e p h o t o s y n t h e t i c a p p a r a t u s o f p l a n t s i n d u c e d by exposure t o e x c e s s i v e l i g h t has been documented f o r many y e a r s (Emerson, 1936; R a b i n o w i t c h , 1945; Kok, 1956). The i n t e n s i t y o f l i g h t needed t o i n d u c e p h o t o i n h i b i t i o n v a r i e s from f u l l s u n l i g h t (2000/imol m - 2 s - 1 ) t o a l i t t l e as 50/imol m ~ 2 s _ 1 f o r a l g a e grown a t low photon f l u x d e n s i t y (PFD) (Whitelam and Codd, 1983). Damage t o t h e p h o t o s y n t h e t i c p r o c e s s can be caused by -14- exposure t o l i g h t i n t h e u l t r a v i o l e t w a v e l e n g t h ( C a l d w e l l , 1981), by e x c e s s i v e l i g h t i n t h e v i s i b l e p a r t o f t h e spectrum (Kok, 1956; C r i t c h l e y , 1981) o r by i n t e r a c t i o n o f l i g h t i n t h e s e s p e c t r a (Warner and C a l d w e l l , 1983). H e r e i n , o n l y damage whi c h o c c u r s t h r o u g h e x c e s s i v e exposure t o v i s i b l e l i g h t i s d i s c u s s e d . Thus, p h o t o i n h i b i t i o n i s d e f i n e d as t h e r e d u c t i o n i n p h o t o - s y n t h e t i c c a p a c i t y , w h i c h i s not dependent upon changes i n pigment c o n c e n t r a t i o n , b u t w h i c h i s i n d u c e d by exposure t o e x c e s s i v e i n t e n s i t i e s o f v i s i b l e l i g h t i n t h e 400-700nm r e g i o n . T h i s i n h i b i t i o n i s not caused by C 0 2 l i m i t a t i o n s . The terms p h o t o o x i d a t i o n , p h o t o i n a c t i v a t i o n , p h o t o l a b i l i t y , s o l a r i z a t i o n and photodynamic r e a c t i o n s have a l s o been a p p l i e d t o e v e n t s t h a t l e a d t o t h e r e d u c t i o n o f p h o t o s y n t h e t i c c a p a c i t y w h i c h o c c u r s t h r o u g h exposure t o s t r o n g l i g h t (Powles, 1984). P h o t o x i d a t i o n i s u s u a l l y used t o d e s c r i b e t h e p h o t o - d e s t r u c t i o n o f p h o t o s y n t h e t i c pigments by s t r o n g l i g h t . T h i s i s u s u a l l y e v i d e n t as t h e b l e a c h i n g o f pigments and i s b o t h l i g h t and oxygen dependent. I n t h i s c a s e p r o l o n g e d t r e a t m e n t r e s u l t s i n t h e d e a t h o f t h e c e l l o r organism. P h o t o o x i d a t i o n o f pigments o c c c u r s a f t e r i n i t i a l p h o t o i n h i b i t i o n (Myers and B u r r , 1940; Kok e t a l . 1965; S a t o h , 1970a), w i t h a d i s t i n c t t i m e l a g between t h e two p r o c e s s e s . The s t a r t o f p h o t o o x i d a t i o n u s u a l l y o c c u r s a f t e r p h o t o i n h i b i t i o n has r e a c h e d a maximum ( S i r o n v a l and K a n d l e r , 1958). -15- E v i d e n c e o f p h o t o i n h i b i t i o n has been shown i n b o t h a q u a t i c and l a n d p l a n t s . Land p l a n t s can be c l a s s i f i e d as e i t h e r shade o r sun p l a n t s . I n a d d i t i o n t o growth i n t h e i r n a t u r a l e n v i r o n m e n t , sun p l a n t s can g e n e r a l l y grow i n c o n d i t i o n s o f l o w e r photon f l u x d e n s i t y (Bjorkman, 1973,1981). The r e v e r s e o f t e n does n ot h o l d t r u e f o r shade p l a n t s , because o f s u s c e p t i b i l i t y t o p h o t o i n h i b i t i o n . F o r i n s t a n c e , Bjorkman and Holmgren (1963) and Bjorkman (1968) showed t h a t p o p u l a t i o n s o f S o l i d a g o v i r q a u r e a from open h a b i t a t s were a b l e t o grow i n t h e sun b u t were a l s o a b l e t o s u r v i v e and grow, though a t s l o w e r r a t e s , when h e l d under c o n d i t i o n s o f lo w e r l i g h t i n t e n s i t y . I n c o n t r a s t , p o p u l a t i o n s o r i g i n a t i n g from low l i g h t i n t e n s i t y h a b i t a t s grew w e l l under low PFD b u t had m i n i m a l growth a t h i g h e r l i g h t i n t e n s i t i e s . I n t h e s e s t u d i e s some p h o t o o x i d a t i v e b l e a c h i n g o c c u r r e d on t h e exposed p a r t s o f l e a v e s b u t was not r e s p o n s i b l e f o r t h e i n i t i a l i n h i b i t i o n o f p h o t o s y n t h e s i s . The p h o t o i n h i b i t i o n was e v i d e n t as a d e c l i n e i n l i g h t - s a t u r a t e d p h o t o s y n t h e s i s and photon y i e l d . P l a n t s have d i f f e r e n t mechanisms f o r d i s s i p a t i n g t h e e x c e s s i v e l i g h t energy. A t t h e b i o p h y s i c a l l e v e l , p l a n t s have t h e p o t e n t i a l f o r t h e s p i l l o v e r o f e x c i t a t i o n energy from P S I I t o P S I . A t t h e m e t a b o l i c l e v e l , t h e maintenance o f m i n i m a l p h o t o s y n t h e t i c c a r b o n m e t a b o l i s m can m i n i m i z e p h o t o i n h i b i t i o n , w h i l e a t t h e m o r p h o l o g i c a l l e v e l , l e a f movement i n re s p o n s e t o h i g h PFD can p r e v e n t p h o t o i n h i b i t i o n (Powles, 1984). D i f f e r e n t s p e c i e s a d j u s t i n d i f f e r i n g ways t o exposure t o i n t e n s e l i g h t . Some s p e c i e s , such as F r a q a r i a v i r q i n i a n a . which i s n o t e x c l u s i v e l y a shade p l a n t , a l s o show l o w e r l i g h t - s a t u r a t e d p h o t o s y n t h e t i c c a p a c i t y and photon y i e l d when grown i n c o n d i t i o n s o f f u l l s u n l i g h t . Sun p l a n t s , grown o r i g i n a l l y a t low PFD, e x h i b i t t h e same c h a r a c t e r i s t i c s i f moved s u d d e n l y t o a h i g h e r PFD environment (Bunce e t a l . 1977; Powles and C r i t c h l e y , 1980; Powles and Thorne, 1981; Powles and Bjorkman, 1983). P l a n t s w h i c h e x p e r i e n c e t h i s i n i t i a l p h o t o i n h i b i t i o n may respond by i n c r e a s i n g t h e i r p h o t o s y n t h e t i c c a p a c i t y a t t h e h i g h e r PFD ( P r e n z e l and L i c h t e n t h a l e r , 1982) o r may s u f f e r p h o t o o x i d a t i v e b l e a c h i n g and c e l l d e a t h (Louwerse and Zweerde, 1977). The mechanism t h r o u g h which exposure t o e x c e s s i v e l i g h t c a u ses a d e c l i n e and d i s r u p t i o n i n t h e p h o t o s y n t h e t i c p r o c e s s i s p o o r l y u n d e r s t o o d . E a r l y e x p e r i m e n t s w i t h d i l u t e s u s p e n s i o n s o f a l g a l c e l l s l e d t o t h e c o n c l u s i o n t h a t i n t e n s e l i g h t " i n h i b i t e d t h e p r i m a r y p h o t o c h e m i c a l p r o c e s s , t h e r e b y c a u s i n g a r e d u c t i o n i n t h e c a p a c i t y f o r e f f i c i e n t l y p a s s i n g on l i g h t quanta and a c o n c o m i t a n t d i m i n u a t i o n o f l i g h t - s a t u r a t e d p h o t o s y n t h e s i s " (Kok, 1956). Subsequent e x p e r i m e n t a t i o n w i t h shade p l a n t s i n d i c a t e d t h a t P S I I was i m p a i r e d when shade p l a n t s were grown a t a h i g h e r PFD. PSI was a l s o damaged, but t o a l e s s e r e x t e n t (Bjorkman, 1968). S i m i l a r e f f e c t s were shown i n s t u d i e s where sun p l a n t s were i n i t i a l l y grown i n shade and t h e n exposed t o a PFD comparable t o f u l l s u n l i g h t . C h l o r o p l a s t t h y l a k o i d s i s o l a t e d from t h e s e l e a v e s showed some degree o f i n h i b i t i o n o f l i g h t - s a t u r a t e d , u n c o u p l e d e l e c t r o n t r a n s p o r t c a p a c i t y (Powles and C r i t c h l e y , 1980; Powles and Bjorkman, 1983), depending upon l e n g t h o f exposure t i m e ( C r i t c h l e y , 1981). A g a i n , t h e r e was l e s s i n h i b i t i o n o f PSI a c t i v i t y and p h o t o p h o s p h o r y l a t i o n was not i n h i b i t e d by t h e h i g h e r PFD ( C r i t c h l e y , 1981). S t u d i e s o f P S I I f l u o r e s c e n c e a t 77K a l s o i n d i c a t e d damage t o P S I I . P h o t o i n h i b i t i o n was shown as an i n h i b i t i o n o f l i g h t - s a t u r a t e d p h o t o s y n t h e s i s , photon y i e l d and P S I I e l e c t r o n t r a n s p o r t . F l u o r e s c e n c e k i n e t i c s o f t h e exposed upper l e a f s u r f a c e a t 77K r e v e a l e d t i m e and PFD-dependent i n h i b i t i o n o f P S I I maximal f l u o r e s c e n c e ( F m a x ) w i t h m i n i m a l changes i n t h e i n i t i a l f l u o r e s c e n c e ( F Q ) . L i t t l e change i n F m a x o c c u r r e d on t h e s e l f - s h a d e d l o w e r s u r f a c e s o f l e a v e s . F m a x c o u l d n o t be r e s t o r e d by t h e a d d i t i o n o f donors which bypass t h e w a t e r - s p l i t t i n g r e a c t i o n ; t h e r e b y s u g g e s t i n g t h a t t h e i n a c t i v a t i o n o f P S I I o c c u r r e d t h r o u g h an e f f e c t on t h e r e a c t i o n c e n t r e complex (Powles and Bjorkman, 1983). P h o t o i n h i b i t i o n i s not oxygen-dependent, as s i m i l a r r e s u l t s were o b t a i n e d when t h e oxygen p a r t i a l p r e s s u r e was a t a t m o s p h e r i c l e v e l s o r c l o s e t o z e r o ( B e l a y and Fogg, 1978; B e l a y , 1981). However, p h o t o i n h i b i t i o n a t moderate PFD l e v e l s a l s o o c c u r s i n t h e absence o f C 0 2 . R e s u l t s from e x p e r i m e n t s c o n d u c t e d under a h i g h PFD w i t h o u t C 0 2 were s i m i l a r t o t h o s e -18- o b t a i n e d i n t h e p r e s e n c e o f C 0 2 and a h i g h PFD (Powles e t a l . 1979; Powles and C r i t c h l e y , 1980). P h o t o i n h i b i t i o n t h r o u g h exposure t o h i g h PFD i s i n c r e a s e d t h r o u g h t h e s y n e r g i s t i c a c t i o n o f s t r e s s from low t e m p e r a t u r e (Fork e t a l . 1980; Baker e t a l . 1983), h i g h t e m p e r a t u r e (Ludlow and Borkman, 1983) and w a t e r d e f i c i t ( G a uhl, 1979). The p r e c i s e mechanisms t h r o u g h which e x c e s s i v e PFD causes damage t o Photosystems I and I I , r e s p e c t i v e l y , a r e unknown a t t h e p r e s e n t t i m e but c l e a r l y d i f f e r . P S I I e l e c t r o n t r a n s p o r t and p r i m a r y p h o t o c h e m i s t r y i n h i b i t i o n o c c u r i d e n t i c a l l y i n t h e p r e s e n c e o r absence o f oxygen. T h i s s u g g e s t s t h a t t h e g e n e r a t i o n o f oxygen r a d i c a l s i s u n l i k e l y t o be t h e cause o f t h e p h o t o i n h i b i t i o n . K y l e e t a l . (1984) s u g g e s t e d t h a t p h o t o - i n h i b i t o r y damage i n Chlamydomonas r e i n h a r d i i was due t o t h e l o s s o f t h e 32 k i l o D a l t o n (kDa) h e r b i c i d e - b i n d i n g p o l y p e p t i d e , w h i c h i s a l s o t h e p o l y p e p t i d e c a r r y i n g t h e t h e s e c o n d a r y e l e c t r o n a c c e p t o r quinone (QB). However, C l e l a n d and C r i t c h l e y (1985) found t h a t p h o t o i n h i b i t i o n under s i m i l a r c o n d i t i o n s was n o t accompanied by t h e l o s s o f t h e 32 kDa p r o t e i n . They c o n c l u d e t h a t t h e r e d u c t i o n i n F m a x o c c u r s because o f t h e g e n e r a t i o n o f a v e r y e f f i c i e n t f l u o r e s c e n c e quencher d u r i n g p h o t o i n h i b i t i o n . They s u g g e s t t h a t t h e quencher may be a m o d i f i e d form o f QA, caused e i t h e r by a t t a c k by a h y d r o x y l r a d i c a l w i t h subsequent h y d r o x y l a t i o n o r by i n t e r a c t i o n o f t h e quinone w i t h t h i o l groups on t h e p o l y p e p t i d e . They s u g g e s t t h a t -19- t h e l o s s o f t h e 32 kDa p r o t e i n i s a r e s u l t , n o t a cause o f p h o t o i n h i b i t i o n . Some s u p p o r t i s a v a i l a b l e f o r t h e i r h y p o t h e s i s as Den Haan e t a l . (1973) found an u n i d e n t i f i e d quencher (T) w h i c h was g e n e r a t e d i n a l i g h t f l a s h and d i s a p p e a r e d i n a d a r k r e a c t i o n o f t h e o r d e r o f lO/zs. Such a quencher may have a l o n g e r l i f e d u r i n g a p h o t o i n h i b i t o r y e p i s o d e . I n c o n t r a s t , t h e p h o t o i n h i b i t i o n o f PSI e l e c t r o n t r a n s p o r t r e q u i r e s t h e p r e s e n c e o f oxygen (Powles and Bjorkman, 1983) and e l e c t r o n t r a n s p o r t from P S I I (Satoh, 1970b). The s i t e o f PSI i n h i b i t i o n has been shown t o be v e r y c l o s e t o t h e PSI r e a c t i o n c e n t r e ( S a t o h , 1970c) and t h e oxygen r e q u i r e m e n t s u g g e s t s t h a t t o x i c oxygen compounds may be i n v o l v e d i n PSI p h o t o i n h i b i t i o n . R ecovery from p h o t o i n h i b i t i o n has r e c e i v e d l i t t l e a t t e n t i o n t o d a t e b u t i s a v e r y slow p r o c e s s i n r e l a t i o n t o t h e speed o f o n s e t o f p h o t o i n h i b i t i o n (Powles, 1984). 2.3 P h o t o s y n t h e t i c E l e c t r o n T r a n s p o r t P h o t o s y n t h e t i c e l e c t r o n t r a n s p o r t i n green p l a n t s e n t a i l s t h e a b s o r p t i o n o f l i g h t energy by a l a r g e a r r a y o f l i g h t - h a r v e s t i n g p i g m e n t s , and subsequent t r a n s f e r o f t h i s energy by re s o n a n c e t o t h e r e a c t i o n c e n t r e s o f two d i f f e r e n t p h otosystems, Photosystems I and I I (PSI and P S I I ) . There i t i s c o n v e r t e d i n t o c h e m i c a l energy. The photosystems a c t t h r o u g h a s e r i e s o f -20- o x i d a t i o n - r e d u c t i o n r e a c t i o n s i n t h e n o n - c y c l i c t r a n s f e r o f e l e c t r o n s from H 20 t o NADP +. I n t h e l i g h t , P S I I g e n e r a t e s a s t r o n g o x i d a n t c a p a b l e o f o x i d i z i n g w a t e r , and, c o n c o m i t a n t l y , a weak r e d u c t a n t , w h i l e PSI g e n e r a t e s a s t r o n g r e d u c t a n t c a p a b l e o f r e d u c i n g NADP +, and, c o n c o m i t a n t l y , a weak o x i d a n t ( M a l k i n , 1982). H i l l and B e n d a l l (1960) proposed t h e Z scheme ( F i g u r e 1) w h i c h p l a c e s t h e components o f t h e e l e c t r o n t r a n s p o r t c h a i n a c c o r d i n g t o t h e i r redox p o t e n t i a l s and e s t a b l i s h e s a b a s i s f o r e n e r g e t i c c o n s i d e r a t i o n s . The v e c t o r i a l charge s e p a r a t i o n i n t h e r e a c t i o n c e n t r e s and t h e v e c t o r i a l t r a n s p o r t o f p r o t o n s p r o v i d e t h e e l e c t r o c h e m i c a l g r a d i e n t a c r o s s t h e t h y l a k o i d membrane w h i c h d r i v e s t h e s y n t h e s i s o f ATP, as f i r s t s u g g e s t e d by M i t c h e l l (1961). L a t e r a l m o b i l i t y o f t h e i n t e g r a l p r o t e i n complexes i n t h e t h y l a k o i d membrane f i r s t p roposed i n t h e f l u i d - m o s a i c model o f S i n g e r and N i c h o l s o n (1972) has been c o n f i r m e d i n t h e 1980's. There i s an extreme l a t e r a l h e t e r o g e n e i t y i n t h e d i s t r i b u t i o n o f P S I I and PSI i n a p p r e s s e d grana and stroma-exposed t h y l a k o i d membranes, r e s p e c t i v e l y (Andersson and Anderson, 1980). The m o b i l e e l e c t r o n c a r r i e r s , p l a s t o q u i n o n e and p l a s t o c y a n i n , which r e s p e c t i v e l y r e c e i v e e l e c t r o n s from P S I I and donate e l e c t r o n s t o P S I , a r e t h o u g h t t o l i n k t h e photosystems by moving l a t e r a l l y t h r o u g h t h e p l a n e o f t h e t h y l a k o i d membranes between t h e g r a n a l -21- To FAD, NADP & d a r k r e a c t i o n s • P700 2H 20 PS I PS I I F i g u r e 1. C u r r e n t c o n c e p t o f t h e Z scheme f i r s t p r o p o s e d by H i l l and B e n d a l l (1960). OEC=Oxygen-evolving complex; Z=the immediate e l e c t r o n donor t o P680; D=an a u x i l i a r y e l e c t r o n donor t o P680; P680=photosystem I I r e a c t i o n c e n t r e c h l o r o p h y l l a; Pheo=pheophytin; Q L = u n i d e n t i f i e d component wh i c h may be an e l e c t r o n a c c e p t o r ; Q A = i r o n - p l a s t o q u i n o n e e l e c t r o n a c c e p t o r ; QB=second i r o n - p l a s t o q u i n o n e e l e c t r o n a c c e p t o r ; P Q = p l a s t o q u i n o n e ; cyt=cytochrome b g - f complex; P C = p l a s t o c y a n i n ; P700=photosystem I r e a c t i o n c e n t r e c h l o r o p h y l l a; a.Q and a ^ u n i d e n t i f i e d e a r l y e l e c t r o n a c c e p t o r s ; X=a p o s s i b l e i r o n - s u l p h u r e l e c t r o n a c c e p t o r ; B and A = i r o n - s u l p h u r c e n t r e s ; F d = F e r r e d o x i n ; FAD=Flavin a d e n i n e d i n u c l e o t i d e ; NADP=Nicotinamide a d e n i n e d i n u c l e o t i d e phosphate. Combined from G o v i n d j e e e t a l . (1985), Haehnel (1984) and M a l k i n (1982). -22- and s t r o m a l r e g i o n s ( F o r d and B a r b e r , 1983). The g r e a t e r p a r t o f t h e e l e c t r o n t r a n s p o r t c h a i n from H 20 t o NADP+ i s o r g a n i z e d i n t o t h r e e membrane-spanning complexes. These a r e t h e P S I I complex, t h e cytochrome b 6 - f complex, and t h e PSI complex. Each c o n t a i n s s e v e r a l e x t r i n s i c and i n t r i n s i c p o l y p e p t i d e s . These complexes do not i n t e r a c t d i r e c t l y b u t a r e l i n k e d by s m a l l e l e c t r o n c a r r i e r s . P l a s t o q u i n o n e l i n k s t h e P S I I complex w i t h t h e cytochrome b_ 6-f complex and p l a s t o c y a n i n l i n k s t h e cytochrome b_ 6-f complex w i t h t h e PSI complex. F e r r e d o x i n m e d i a t e s t h e e l e c t r o n t r a n s f e r from PSI t o t h e membrane-bound f e r r e d o x i n - N A D P + r e d u c t a s e and p o s s i b l y t o t h e cytochrome b ^ - f complex i n c y c l i c e l e c t r o n t r a n s p o r t (Haehnel, 1984) . 2.3.1 Photosystem I I Photosystem I I i s a c h l o r o p h y l l - c o n t a i n i n g p r o t e i n complex i n v o l v e d i n l i g h t - i n d u c e d oxygen e v o l u t i o n and p l a s t o q u i n o n e r e d u c t i o n . The scheme i s o u t l i n e d below: D QL 2H 20 °2 OEC — Z P680 — • Pheo QA-^QB-^-PQ where OEC i s t h e oxygen e v o l v i n g complex; P680 i s a c h l o r o p h y l l a m o l e c u l e w h i c h i s t h e p r i m a r y e l e c t r o n donor, D and Z a r e -23- e l e c t r o n d o n o r s ; Pheo i s p h e o p h y t i n a; QA and QB a r e i r o n - p l a s t o s e m i q u i n o n e e l e c t r o n a c c e p t o r s ; QL i s p o s t u l a t e d t o be a n o t h e r e l e c t r o n a c c e p t o r ; and PQ i s p l a s t o q u i n o n e . A quantum absorbed i n t h e antenna pigments i s t r a n s f e r r e d t o t h e r e a c t i o n c e n t r e and i n d u c e s an e l e c t r o n t r a n s f e r from t h e p r i m a r y donor P680 t o t h e i n t e r m e d i a t e a c c e p t o r , p h e o p h y t i n a, (Pheo) w i t h i n a few hundred p i c o s e c o n d s . The f o l l o w i n g e l e c t r o n t r a n s f e r from Pheo t o QA i s l i k e l y t o be t h e e l e c t r o g e n i c s t e p (van Gorkum e t a l . 1983) i n t h e charge s e p a r a t i o n o f P S I I from t h e i n s i d e t o t h e o u t s i d e o f t h e t h y l a k o i d membrane. E l e c t r o n t r a n s f e r from QA~, whi c h has been i d e n t i f i e d as a p l a s t o - s e m i q u i n o n e - F e + 2 complex (Nugent e t a l . 1981a), i n v o l v e s a two e l e c t r o n g a t e mechanism (Bouges-Bocquet, 1977). The se c o n d a r y a c c e p t o r (QB) i s a l s o a p l a s t o q u i n o n e m o l e c u l e w h i c h i s n o t exchanged w i t h a n o t h e r p l a s t o q u i n o n e m o l e c u l e o f t h e p o o l (PQ) u n t i l i t i s f u l l y r e d u c e d . The mechanism i s as f o l l o w s : 1 s t f l a s h : QAQB —+~ QA"QB QAQB~(stable) 2nd f l a s h : Q A Q B " Q A ~ Q B ~ — — QAQBH2 — Q A Q B 2H + PQ PQH2 One r e l e v a n t a s p e c t o f P S I I i s t h e l o c a t i o n o f t h e i n h i b i t i o n s i t e o f 3 - ( 3 , 4 - d i c h l o r o p h e n y l ) - 1 , 1 - d i m e t h y l u r e a (DCMU) and a t r a z i n e - t y p e i n h i b i t o r s o f P S I I . B i n d i n g by t h e s e i n h i b i t o r h e r b i c i d e s i s c o m p e t i t i v e w i t h t h e b i n d i n g o f p l a s t o q u i n o n e t o t h e QB s i t e ( V e l t h u y s and Amesz, 1974; C r o f t s -24- and W r a i g h t , 1983) and r e s u l t s i n a b l o c k i n t h e e l e c t r o n t r a n s p o r t c h a i n . The f u n c t i o n o f t h e p o s t u l a t e d a c c e p t o r QL i s n o t known a t p r e s e n t ( E c k e r t and Renger, 1980). E l e c t r o n t r a n s f e r from t h e donor Z t o P68 0 + can be f o l l o w e d by EPR measurements o f t h e r a p i d l y r e v e r s i b l e S i g n a l II V£, a t t r i b u t e d t o Z +. S i m u l t a n e o u s measurements a t room t e m p e r a t u r e o f t h e EPR s i g n a l o f Z + and t h e decay o f P 6 8 0 + i n T r i s - t r e a t e d t h y l a k o i d s p r o v i d e d e v i d e n c e t h a t Z i s t h e immediate donor t o P 6 8 0 + (Babcock and Sauer, 1983b). The donor D i s r e p r e s e n t e d by EPR S i g n a l I I + s . T h i s s p e c i e s i s o n l y m a r g i n a l l y u n d e r s t o o d as y e t , b u t appears t o be a secondary donor on a s i d e c h a i n (Boussac and E t i e n n e , 1982a, 1982b). Once o s c i l l a t i o n s were found i n t h e oxygen y i e l d a f t e r s h o r t s a t u r a t i n g f l a s h e s ( J o l i o t e t a l . 1969), Kok e t a l . (1970) f o r m u l a t e d t h e k i n e t i c S-state-scheme f o r oxygen e v o l u t i o n . On e x c i t a t i o n o f i t s a s s o c i a t e d P S I I r e a c t i o n c e n t r e complex, each i n d i v i d u a l o x y g e n - e v o l v i n g complex goes t h r o u g h a sequence o f i n c r e a s i n g o x i d a t i o n s t a t e s from S Q t o S 4. A f t e r t h e enzyme r e a c h e s t h e S 4 s t a t e , i t r e l e a s e s oxygen and r e t u r n s t o t h e S 0 s t a t e . The l i g h t - i n d u c e d t r a n s i t i o n s from S Q t o S j and from S 2 t o S 3 r e s u l t i n t h e r e l e a s e o f one p r o t o n , and t h e t r a n s i t i o n from S 3 t o S Q v i a S 4 r e s u l t s i n t h e r e l e a s e o f two p r o t o n s (Bowes and C r o f t s , 1978; F o r s t e r e t a l . 1981). P r o t o n r e l e a s e from S Q t o S j b u t not from S j t o S 2 was c o n f i r m e d by R u t h e r f o r d e t a l . (1984b). Manganese i s a l s o i n v o l v e d i n t h e mechanism o f oxygen e v o l u t i o n (Cheniae and M a r t i n , 1970). T r e a t m e n t - i n d u c e d r e l e a s e o f Mn from t h e membrane shows t h e p r e s e n c e o f a t l e a s t t h r e e d i f f e r e n t p o o l s o f Mn i n c h l o r o p l a s t s . Weakly bound Mn, not i n v o l v e d i n oxygen e v o l u t i o n , can be removed by i n c u b a t i o n w i t h d i v a l e n t i o n s o r EDTA (Yocum e t a l . 1981). A p p r o x i m a t e l y t w o - t h i r d s o f t h e r e m a i n i n g s t r o n g l y bound Mn can be removed w i t h h e a t , h y d r o x y l a m i n e o r T r i s - w a s h i n g t r e a t m e n t (Amesz, 1983; Dismukes, 1986). I f t h e r e m a i n i n g v e r y s t r o n g l y bound Mn i s e x t r a c t e d from a P S I I r e a c t i o n c e n t r e , r e a d d i t i o n o f f o u r Mn atoms p e r r e a c t i o n c e n t r e g i v e s t h e maximal r e a c t i v a t i o n o f e l e c t r o n t r a n s p o r t (Klimov e t a l . 1982). However, o f t h e s e , o n l y two Mn atoms p e r r e a c t i o n c e n t r e a r e v i t a l , as t h e o t h e r two can be r e p l a c e d by o t h e r d i v a l e n t c a t i o n s w i t h o u t l o s s o f a c t i v i t y . C h l o r i d e has a l s o been shown t o be a component o f t h e o x y g e n - e v o l v i n g complex. I t s e x a c t f u n c t i o n i s unknown bu t i t may a c t as a b r i d g i n g l i g a n d between Mn atoms ( C r i t c h l e y , 1985). O t h e r components o f P S I I i n c l u d e a number o f p o l y p e p t i d e s . Three o f t h e s e , w h i c h have m o l e c u l a r w e i g h t s o f 17, 2 3 and 3 3 kDa, r e s p e c t i v e l y , a r e found i n t h e o x y g e n - e v o l v i n g complex (OEC) (Kuwabara and Mu r a t a , 1982, 1983; Yamamoto e t a l . 1981, 1983) . These p r o t e i n s have absorbance peaks a t 275-276nm, bu t none i n t h e v i s i b l e r e g i o n and a r e monomeric i n s o l u t i o n . They -26- do n o t c o n t a i n Mn, o t h e r m e t a l s , o r c h l o r o p h y l l ( J a n s s o n e t a l . 1983; Kuwabara and M u r a t a , 1982, 1983; Yamamoto e t a l . 1983). T h e i r f u n c t i o n i s unknown but may be c a t a l y t i c , r e g u l a t o r y o r s t r u c t u r a l . R e c e n t l y , H u n z i k e r e t a l , . (1987) p r e s e n t e d e v i d e n c e f o r an a s s o c i a t i o n between t h e 33kDa p r o t e i n and oxygen e v o l u t i o n , b u t S t y r i n g e t a l . (1987) found no r e q u i r e m e n t f o r t h i s p r o t e i n i f s u f f i c i e n t C l ~ was p r e s e n t . Other p o l y p e p t i d e s i n c l u d e two c h l o r o p h y l l a - p r o t e i n complexes w i t h a p o p r o t e i n s o f 47 and 50 kDa, r e s p e c t i v e l y ( D e l e p e l a i r e and Chua, 1979). The f i r s t o f t h e s e i s a s s o c i a t e d w i t h t h e s e condary e l e c t r o n a c c e p t o r QB w h i l e t h e l a t t e r may be a s s o c i a t e d w i t h t h e r e a c t i o n c e n t r e o f P S I I . A l s o p r e s e n t a r e t h e 32 kDa p o l y p e p t i d e i n v o l v e d i n t h e b i n d i n g o f QB, and cytochrome , w h i c h has a m o l e c u l a r w e i g h t o f 10 kDa. 2.3.2 Cytochrome b_ 6-f Complex T h i s s e c t i o n o f t h e e l e c t r o n t r a n s p o r t c h a i n i s an i n t e g r a l p r o t e i n complex w h i c h i n c l u d e s two cytochrome p o l y p e p t i d e s . These a r e t h e p o l y p e p t i d e o f cytochrome f o f 34 kDa, and a p o l y p e p t i d e w i t h two cytochrome b_6 hemes o f 23 kDa. O t h e r components a r e t h e h i g h p o t e n t i a l F e 2 S 2 " R i e s k e " p r o t e i n o f 20 kDa and a bound p l a s t o q u i n o l . The complex s t r u c t u r e , as known t o d a y , i s c o mpleted w i t h a s t r u c t u r a l p o l y p e p t i d e o f 17 kDa and a s m a l l p e p t i d e o f 5 kDa ( B e n d a l l , 1982; Cramer and Whitmarsh, 1977; Hauska e t a l . 1983). The p r e c i s e mechanism o f -27- e l e c t r o n t r a n s f e r w i t h i n t h i s complex has y e t t o be e l u c i d a t e d . 2.3.3 P hotosystem I The PSI complex c o n s i s t s o f t h e r e a c t i o n c e n t r e c h l o r o p h y l l P700, w h i c h f u n c t i o n s as t h e p r i m a r y e l e c t r o n donor o f P S I , and f i v e e l e c t r o n a c c e p t o r s , as shown below: P700 * a Q m a-L m X m ( c e n t r e B - c e n t r e A) , where P700 i s r e c o g n i z e d as a form o f c h l o r o p h y l l a, b u t t h e p r e c i s e s t r u c t u r e i s unknown; d i f f e r e n t i n v e s t i g a t o r s have p r o v i d e d e v i d e n c e s u p p o r t i n g a dimer ( N o r r i s e t a_l. 1971) , a monomer (O'M a l l e y and Babcock, 1984b), and an e n o l i z e d monomer ( W a s i e l e w s k i e t a l . 1981). The c h a r g e s s e p a r a t e d i n t h e r e a c t i o n c e n t r e a r e s t a b i l i z e d by f a s t c o n s e c u t i v e e l e c t r o n t r a n s f e r t h r o u g h t h e c h a i n o f e l e c t r o n a c c e p t o r s shown above. The r e c e n t l y d e t e c t e d i n i t i a l a c c e p t o r s aQ and a-̂  may be monomeric c h l o r o p h y l l a a n i o n s (Gast e t a l . 1983; M a l k i n , 1982). A c c e p t o r a-^ i s c l o s e l y a s s o c i a t e d w i t h X, and i f X i s d e s t r o y e d , does n ot p a r t i c i p a t e i n PSI e l e c t r o n t r a n s f e r (Warden and Golbeck, 1987). The t h i r d e l e c t r o n a c c e p t o r , X, has p r o p e r t i e s s i m i l a r t o an i r o n - s u l p h u r c e n t r e ( K o i k e and Katoh, 1982; M c i n t o s h and B o l t o n , 1976) but t h e e x a c t s t r u c t u r e i s unknown. The f i n a l e l e c t r o n a c c e p t o r s a r e t h e i r o n - s u l p h u r c e n t r e s A and B, which i n t e r a c t c l o s e l y w i t h each o t h e r ( M a l k i n , 1982). I t has been shown t h a t t h e s e -28- a r e bound f e r r e d o x i n s h a v i n g F e 4 S 4 c e n t r e s (Cammack and Evans, 1975). T h e i r r e d u c t i o n can be ob s e r v e d as absorbance changes a t 430nm (Ke, 1972). E l e c t r o n t r a n s f e r between X, and c e n t r e s A and B has y e t t o be d e f i n i t i v e l y e l u c i d a t e d . I n i t i a l r e p o r t s o f t h e redox p o t e n t i a l s o f t h e s e a c c e p t o r s i n s p i n a c h c h l o r o p l a s t s s u g g e s t l i n e a r t r a n s f e r from X t o B t o A (Ke e t a l . 1973; Evans e t a l . 1974). However, redox p o t e n t i a l s a r e d i f f e r e n t i n some systems ( M a l k i n , 1982). The s u c c e s s f u l r e d u c t i o n o f A a f t e r i n a c t i v a t i o n o f B (Golbeck and Warden, 1982) and t h e c h e m i c a l r e d u c t i o n o f B p r i o r t o A (Nugent e t a l . 1981b) has l e d t o a h y p o t h e s i s t h a t X i s t h e b r a n c h p o i n t l e a d i n g t o p a r a l l e l f u n c t i o n o f c e n t r e s A and B (Nugent e t a l . 1981b). The p r i m a r y e l e c t r o n donor, P700, and a c c e p t o r s a Q and a j , a r e c o n t a i n e d i n t h e l a r g e s t s u b u n i t o f t h e PSI r e a c t i o n c e n t r e complex w h i c h has a m o l e c u l a r w e i g h t o f 7 0 kDa ( B e n g i s and N e l s o n , 1977). The t h r e e l a t e r a c c e p t o r s , X, A and B, a r e a p p a r e n t l y l o c a t e d i n two s m a l l e r s u b u n i t s h a v i n g m o l e c u l a r w e i g h t s o f a p p r o x i m a t e l y 15-18 kDa ( M o l l e r e t a l . 1981). -29- 2.4 EPR S t u d i e s EPR s p e c t r o s c o p y has been used e x t e n s i v e l y t o s t u d y t h e components o f t h e p h o t o s y n t h e t i c e l e c t r o n t r a n s p o r t c h a i n . I n f a c t , many o f t h e components were d i s c o v e r e d t h r o u g h t h e use o f EPR. W i t h t h e e x c e p t i o n o f i n i t i a l o b s e r v a t i o n s made r e g a r d i n g S i g n a l s I and I I , t h e m a j o r i t y o f t h e work has been done a t l i q u i d h e l i u m o r l i q u i d n i t r o g e n t e m p e r a t u r e s u s i n g c h l o r o p l a s t and r e a c t i o n c e n t r e p r e p a r a t i o n s . I n i t i a l l y , Commoner e t a l . (1956, 1957), w o r k i n g a t 35C, d i s c o v e r e d t h e p r e s e n c e o f EPR s i g n a l s i n t o b a c c o c h l o r o p l a s t s i l l u m i n a t e d w i t h l i g h t . One s i g n a l , now known as S i g n a l I ( F i g u r e 2 c ) , i s a s s o c i a t e d w i t h P S I . I t c o n s i s t s o f a s i n g l e peak, has a g - v a l u e o f 2.0024-2.0025, and a peak-to-peak l i n e w i d t h o f 7.5-9 gauss. I n c h l o r o p l a s t p r e p a r a t i o n s from h e a l t h y p l a n t s t h i s s i g n a l i s o n l y i n d u c e d by i l l u m i n a t i o n w i t h f a r - r e d (700-730nm) l i g h t . I t i s t r a n s i e n t and d i s a p p e a r s w i t h i n m i l l i s e c o n d s a f t e r t e r m i n a t i o n o f i l l u m i n a t i o n (Warden and B o l t o n , 1974a). T h i s s i g n a l i s a s c r i b e d t o t h e o x i d i z e d form o f t h e c h l o r o p h y l l a o f t h e r e a c t i o n c e n t r e o f PSI ( B e i n e r t e t a l . 1962). S i g n a l I i s a l s o formed upon i r r a d i a t i o n o f c h l o r o p l a s t s u s p e n s i o n s w i t h r e d ( B l u m e n f e l d e t al. 1974; Babcock and Sauer, 1975a) o r broad-band w h i t e l i g h t (Babcock and Sauer, 1975b) when e l e c t r o n t r a n s p o r t from P S I I t o PSI i s b l o c k e d by an i n h i b i t o r s uch as DCMU ( V e l t h u y s and Amesz, 1974). -30- a 3236 3256 3276 M a g n e t i c f i e l d (Gauss) F i g u r e 2. P r i m a r y p h o t o s y n t h e t i c s i g n a l s found i n r e a c t i o n c e n t r e and c h l o r o p l a s t p r e p a r a t i o n s and i n i n t a c t l e a f t i s s u e . These s i g n a l s were o b t a i n e d i n t h e p r e s e n t s t u d y from Kentucky b l u e g r a s s l e a v e s . a. Combined S i g n a l I and I I + ; b. S i g n a l I I U + S ; c. S i g n a l I ( o b t a i n e d by s u b t r a c t i o n o f B from a ) . -31- The second major s i g n a l d i s c o v e r e d by Commoner e t a l . (1956,57), now known as S i g n a l I I ( F i g u r e 2 b ) , i s a s s o c i a t e d w i t h Photosystem I I , i s a s y m m e t r i c a l , has f i v e l i n e s o f unequal i n t e n s i t y and s p l i t t i n g , a g - v a l u e around 2.0046 and a peak-to-peak l i n e w i d t h o f 18-20 gauss ( E s s e r , 1974a). T h i s s i g n a l was f i r s t known as a d a r k s i g n a l , b u t subsequent i n v e s t i g a t i o n s have shown t h a t i t , t o o , has components w h i c h a r e dependent upon l i g h t , as o x i d a t i o n i n d a r k n e s s r e s u l t s i n e l i m i n a t i o n o f 50% o f t h e s i g n a l (Weaver, 1968; Babcock and Sauer, 1975a). The two components o f t h i s s i g n a l a r e now known as S i g n a l I I U and S i g n a l I I S , w i t h t h e s u b s c r i p t s , u and s, s t a n d i n g f o r unchanging and slow, r e s p e c t i v e l y . The com p o s i t e s i g n a l i s known as S i g n a l I I U + S . T h i s s i g n a l i s i n d u c e d by broad-band w h i t e l i g h t and has s e v e r a l decay phases (Warden and B o l t o n , 1974b). The s l o w e s t o f t h e s e phases has a h a l f - l i f e e x c e e d i n g 2 hours (Weaver, 1968). Recent s t u d i e s by R u t h e r f o r d (1985) have shown t h a t S i g n a l H u + S i s composed o f an o v e r l a p o f two d i s t i n c t f o u r l i n e s i g n a l s . Which o f t h e s e emanates from any g i v e n membrane i s dependent upon t h e o r i e n t a t i o n o f t h e membrane t o t h e magnetic f i e l d ( p a r a l l e l o r p e r p e n d i c u l a r ) . I n whole p l a n t t i s s u e s t h e s i g n a l s o v e r l a p t o form S i g n a l I I U + S - DCMU and a t r a z i n e - t y p e h e r b i c i d e s which i n h i b i t P S I I a t QB ( V e l t h u y s and Amesz, 1974), do not p r e v e n t t h e f o r m a t i o n o f S i g n a l H u + S ( L o z i e r and B u t l e r , 1973; E s s e r , 1974b). The e x a c t s t r u c t u r e o f t h e s p e c i e s g i v i n g r i s e t o S i g n a l I I U + S i s not known b u t i t was f i r s t a t t r i b u t e d t o a p l a s t o s e m i q u i n o n e (Weaver, 1962; K o h l and Wood, 1969) o r p l a s t o c h r o m a n o x y l r a d i c a l ( K o h l e t a l . 1969). S u b s e q u e n t l y , i t was p r o p o s e d t h a t S i g n a l H u + S o r i g i n a t e s from a p l a s t o s e m i q u i n o n e a n i o n r a d i c a l p e r t u r b e d by a m e t a l c a t i o n ( H a l e s and Case, 1981; H a l e s and Das Gupta, 1981). More r e c e n t l y , i t was su g g e s t e d t h a t S i g n a l I I U + S r e p r e s e n t s a p l a s t o q u i n o n e c a t i o n r a d i c a l i n whi c h t h e e l e c t r o n - d o n a t i n g a b i l i t y o f t h e quin o l - O H groups has been d e c r e a s e d (O'Malley and Babcock, 1984a). The f u n c t i o n a l r o l e o f t h e s p e c i e s g i v i n g r i s e t o S i g n a l I I U + S has not been a s c e r t a i n e d but e a r l y h y p o t h e s e s s u g g e s t e d t h a t S i g n a l H u + S r e f l e c t e d t h e b e h a v i o u r o f some q u i n o i d a l compound on t h e r e d u c i n g s i d e o f P S I I w h i c h was l o c a t e d near t h e e l e c t r o n t r a n s p o r t c h a i n (Weaver 1962; Warden and B o l t o n , 1974b). More r e c e n t l y , S i g n a l I I U + S has been a s s i g n e d t o D, a donor on an a u x i l i a r y p a t h t o P680 (Boussac and E t i e n n e , 1982a, 1982b) (see F i g u r e 1 ) . S i g n a l I I U + S i s d i s t i n g u i s h e d from two o t h e r s i g n a l s , S i g n a l I I f (Babcock and Sauer, 1975a, 1975b) and S i g n a l I I v f ( B l a n k e n s h i p e t a l . 1975a; Warden e t a l . 1976). The f and v f s t a n d f o r f a s t and v e r y f a s t , r e s p e c t i v e l y . The l a t t e r s i g n a l s , w h i c h r e p r e s e n t l i g h t - g e n e r a t e d f r e e r a d i c a l s d e t e c t e d i n c h l o r o p l a s t s u s p e n s i o n s by EPR, a r e t h e EPR m a n i f e s t a t i o n s o f Z +, t h e p r i m a r y donor t o P680 (Yocum and Babcock, 1981). The d i f f e r e n c e i n nomenc l a t u r e r e p r e s e n t s t h e f a c t t h a t t h e - 3 3 - l i g h t - g e n e r a t e d r e d u c t i o n o f Z can be f a s t o r v e r y f a s t , d e pending upon t h e s t a g e o f t h e o x y g e n - e v o l v i n g complex. I n o x y g e n - e v o l v i n g c h l o r o p l a s t s , Z + r e d u c t i o n o c c u r s i n t h e s u b - m i l l i s e c o n d t i m e range; t h u s i t s EPR s i g n a l i s d e s i g n a t e d S i g n a l H V f - When o x y g e n - e v o l v i n g a b i l i t y has been i n h i b i t e d , Z + i s s t a b l e w e l l i n t o t h e m i l l i s e c o n d - r a n g e ; t h u s i t s s i g n a l i s d e s i g n a t e d S i g n a l I I f (Babcock and Sauer, 1975b; Babcock e t a l . 1976). The v a r i o u s components of S i g n a l I I a l l have t h e same shape, g - v a l u e and l i n e w i d t h (see F i g u r e 2 b ) . M a l k i n and Bearden (1971) used EPR s p e c t r o s c o p y t o d e m o n s t r a t e t h e p r e s e n c e of an i r o n - s u l p h u r p r o t e i n i n c h l o r o p l a s t membranes m a i n t a i n e d a t l i q u i d h e l i u m t e m p e r a t u r e s . I l l u m i n a t i o n o f c h l o r o p l a s t s m a i n t a i n e d a t 10K produced a p a r a m a g n e t i c s p e c i e s w i t h a spectrum h a v i n g g - v a l u e s o f g x=1.86, gy=1.94 and g z=2.05. T h i s i s now known as i r o n - s u l p h u r c e n t r e A. The a s s o c i a t i o n o f t h i s c e n t r e w i t h PSI was shown by Bearden and M a l k i n (1972a), s i n c e f a r - r e d l i g h t , w h i c h a c t i v a t e s P S I , a l s o r e d u c e s t h e i r o n - s u l p h u r c e n t r e . F u r t h e r a s s o c i a t i o n w i t h P700 was i n d i c a t e d t h r o u g h t h e o b s e r v e d 1:1 s t o i c h i o m e t r y between p h o t o - o x i d i z e d P7 00 and t h e p h o t o - r e d u c e d i r o n - s u l p h u r c e n t r e A (Bearden and M a l k i n , 1972b). A t c r y o g e n i c t e m p e r a t u r e s t h e o x i d i z e d P700 and reduced i r o n - s u l p h u r c e n t r e A a r e s t a b l e i n t h e d a r k a f t e r c e s s a t i o n o f i l l u m i n a t i o n . However, when t h e t e m p e r a t u r e i s r a i s e d from 77K t o 150K t h e r e i s a back r e a c t i o n a r i s i n g from a c h a rge -34- r e c o m b i n a t i o n between t h e s e s p e c i e s (Bearden and M a l k i n , 1972b; M a l k i n and Bearden, 1974; V i s s e r e t a l . 1974). A t 150K t h e r e i s an a p p r o x i m a t e 4 0% decay i n b o t h t h e P7 00 and i r o n - s u l p h u r c e n t r e A (Bearden and M a l k i n , 1972b). T h i s decay i s b i p h a s i c (Bearden and M a l k i n , 1972b), b u t t h e r e a s o n f o r t h e b i p h a s i c decay has y e t t o be e x p l a i n e d e x c e p t f o r t h e t r i v i a l a n a l y s i s t h a t t h e r e i s h e t e r o g e n e i t y i n t h e PSI complex. T h i s was a t t r i b u t e d t o consequences a r i s i n g from f r e e z i n g o f t h e samples ( L o z i e r and B u t l e r , 1974). A second i r o n - s u l p h u r c e n t r e , B, was d i s c o v e r e d c o n c o m i t a n t l y ( M a l k i n and Bearden, 1971; Evans e t a l . 1972). The spectrum o f t h i s c e n t r e , which has g - v a l u e s o f g x=1.89, gy=1.94, g z=2.05, o v e r l a p s t h a t o f C e n t r e A, e x c e p t when C e n t r e A has been p r e v i o u s l y reduced c h e m i c a l l y . A 1:1 s t o i c h i o m e t r i c r e l a t i o n s h i p has been shown between c e n t r e s A and B ( M a l k i n and Bearden, 1978) and t h e d i s a p p e a r a n c e o f t h e 1.86 component o f t h e EPR spectrum o f C e n t r e A c o r r e l a t e s w e l l w i t h t h e appearance o f t h e 1.89 component of C e n t r e B (Ke e t a l . 1973; Evans e t a l . 1974). These c e n t r e s appear t o be bound f e r r e d o x i n s h a v i n g F e 4 S 4 c e n t r e s (Evans e t a l . 1975). EPR s i g n a l s from t h e s e c e n t r e s have y e t t o be found a t ambient t e m p e r a t u r e s . The p r e s e n c e o f t h e e l e c t r o n a c c e p t o r X was f i r s t shown by M c i n t o s h and B o l t o n (1976) i n s t u d i e s u s i n g p u r i f i e d s p i n a c h -35- Photosystem I p a r t i c l e s m a i n t a i n e d a t 10K. T h i s a c c e p t o r , w h i c h has an EPR s i g n a l h a v i n g g - v a l u e s o f g x=1.75, gy=1.86, g z=2.07, i s d i s c e r n i b l e i f b o t h C e n t r e s A and B a r e p r e v i o u s l y r e d u c e d . T h i s c e n t r e may be a n o t h e r i r o n - s u l p h u r p r o t e i n ( M c i n t o s h and B o l t o n , 1976) o r i t may be a c h l o r o p h y l l a n i o n r a d i c a l i n t e r a c t i n g w i t h a n o t h e r p a r a m a g e n t i c c e n t r e such as an i r o n atom (Rupp e t al. 1979). S h u v a l o v e t a l . (1979) used Photosystem I p a r t i c l e s m a i n t a i n e d a t 5K t o show t h e p r e s e n c e o f an e l e c t r o n a c c e p t o r p r i o r t o X. T h i s e a r l y a c c e p t o r was shown t o have a f r e e - r a d i c a l s i g n a l a t a g - v a l u e o f 2.004, w i t h a l i n e w i d t h o f a p p r o x i m a t e l y 10 gauss. H e a t h c o t e and Evans (1980), and B a l t i m o r e and M a l k i n (1980) a t t r i b u t e d a f r e e - r a d i c a l s i g n a l w h i c h had a g - v a l u e o f 2.003 and a l i n e w i d t h o f 12-14 gauss t o a-j^. T h i s was a s s i g n e d t o a c h l o r o p h y l l a monomer. S u b s e q u e n t l y , B o n n e r j e a and Evans (1982) showed t h a t t h e 12-14 gauss s i g n a l o r i g i n a t e d from t h e o v e r l a p o f two f r e e r a d i c a l s ; one h a v i n g a g - v a l u e o f 2.0051, and t h e second a g - v a l u e o f 2.0024. When b o t h s i g n a l s were formed a t c r y o g e n i c t e m p e r a t u r e s a f t e r 50min i l l u m i n a t i o n , t h e c o m p o s i t e EPR s i g n a l had a g - v a l u e o f 2.0033 and a l i n e w i d t h o f 13 gauss. C o n c u r r e n t l y , (Gast e t a l . 1983) w o r k i n g under s t r o n g l y r e d u c i n g c o n d i t i o n s , found two f r e e r a d i c a l s , h a v i n g g - v a l u e s o f 2.0017 and 2.0054, and r e s p e c t i v e l i n e w i d t h s o f a p p r o x i m a t e l y 11.5 and 10.8 gauss. The i n d i v i d u a l s i g n a l s a r e now a s c r i b e d t o t h e e l e c t r o n -36- a c c e p t o r s a Q and alf r e s p e c t i v e l y . The i d e n t i t y o f t h e s p e c i e s g i v i n g r i s e t o t h e s e s i g n a l s i s not y e t c l e a r , b u t t h e a Q s i g n a l v a l u e i s c o m p a t i b l e w i t h t h a t o f a c h l o r o p h y l l a n i o n monomer ( B o n n e r j e a and Evans, 1982). B l a n k e n s h i p e t a l . (1975b) o b s e r v e d t h e f i r s t p o l a r i z e d EPR s i g n a l i n p h o t o s y n t h e s i s . T h i s s i g n a l r esembled an i n v e r t e d s t e a d y - s t a t e P 7 0 0 + spectrum and was f i r s t a t t r i b u t e d t o a t r i p l e t mechanism ( M a c i n t o s h and B o l t o n , 1979). S u b s e q u e n t l y , upon t h e d i s c o v e r y o f ag and a^, i t was shown t h a t t h e r a d i c a l p a i r mechanism was r e s p o n s i b l e . The s i g n a l , w h i c h has d e f i n i t e o r i e n t a t i o n e f f e c t s (Dismukes e t a l . 1978), i s now a t t r i b u t e d t o a r a d i c a l p a i r i n t e r a c t i o n between P7 0 0 + and a Q (Gast and H o f f , 1979) , o r between P 7 0 0 + and a-^ p o l a r i z e d t h r o u g h exchange i n t e r a c t i o n w i t h a Q ( B r o a d h u r s t e t a l . 1986; H o f f , 1984). T h i s was c o n f i r m e d when s i g n a l s a t t r i b u t a b l e t o t h e p o l a r i z e d t r i p l e t s t a t e (P ) were d i s c o v e r e d (Frank e t a l . 1979a, 1979b; R u t h e r f o r d and M u l l e t , 1981; R u t h e r f o r d e t a l . 1981). O t h e r components o f t h e p h o t o s y n t h e t i c e l e c t r o n t r a n s p o r t c h a i n have been m i n i m a l l y s t u d i e d b u t Nugent e t a l . (1981c) showed t h a t t h e p r i m a r y a c c e p t o r o f P S I I , QA, was a q u i n o n e - i r o n complex g i v i n g r i s e t o an EPR s i g n a l h a v i n g a g - v a l u e o f 1.82. T h i s s i g n a l can be i n d u c e d by i l l u m i n a t i o n o f r e a c t i o n c e n t r e p a r t i c l e s a t low t e m p e r a t u r e s o r by c h e m i c a l r e d u c t i o n . A r e c e n t s t u d y i n d i c a t e s t h a t QB i s a l s o an i r o n - q u i n o n e complex + 3 w h i c h can be f o l l o w e d t h r o u g h t h e appearance o f an Fe s i g n a l a t a g - v a l u e o f 7.9 (Zimmermann and R u t h e r f o r d , 1986). Other s i g n a l s c o n n e c t e d t o t h e e l e c t r o n t r a n s p o r t c h a i n i n c l u d e a m u l t i l i n e Mn s i g n a l a s s o c i a t e d w i t h t h e S 2 s t a t e o f Mn i n t h e o x y g e n - e v o l v i n g complex (Dismukes and S i d e r e r , 1981; Zimmermann and R u t h e r f o r d , 1984), a newly d i s c o v e r e d s i g n a l w i t h a g - v a l u e o f 4.1, a l s o s e e m i n g l y a s s o c i a t e d w i t h t h e S 2 s t a t e (Casey and Sauer, 1984a, 1984b; De P a u l a and Brudwig, 1985) and v a r i o u s s i g n a l s a s s o c i a t e d w i t h cytochrome b 5 5 g ( R u t h e r f o r d e t a l . 1984a), cytochrome b 5 6 3 (Bergstrom, 1985) and t h e cytochrome b 6 - f complex ( M a l k i n and Vanngard, 1980; Nugent and Evans, 1980). P l a n t l e a v e s a l s o show a manganous i o n (Mn + +) EPR spectrum (Theg and S a y r e , 1979; McCain e t a l . 1984). The e x a c t r o l e o f t h i s E P R - d e t e c t a b l e M n + + has y e t t o be e l u c i d a t e d , a s i t u a t i o n w h i c h i s c o m p l i c a t e d by t h e o c c u r r e n c e o f s e v e r a l p o o l s o f bound M n + + i n c h l o r o p l a s t s (Amesz, 1983). The M n + + d e t e c t a b l e by EPR s p e c t r o m e t r y w i t h o u t p r i o r t r e a t m e n t i s n e i t h e r t h e M n + + i n v o l v e d i n w a t e r o x i d a t i o n nor t h e hexaaquomanganese ( I I ) i o n (McCain e t a l . 1984). I t appears t o be t h e weakly bound o r s t o r a g e p o o l M n + + ( G o l d f e l d and B l u m e n f e l d , 1979: Amesz, 1983) but i t s f u n c t i o n i s not known t o d a t e . Most p l a n t l e a v e s a l s o r e v e a l a b r o a d F e + + s i g n a l w h i c h -38- s l o p e s down from low t o h i g h f i e l d , and u n d e r l i e s t h e s i x - p e a k M n + + s i g n a l (Treharne and E y s t e r , 1962). The f u n c t i o n o f t h e i r o n r e s p o n s i b l e f o r t h i s s i g n a l i s not known a t p r e s e n t . The e f f e c t o f S 0 2 upon f r e e - r a d i c a l S i g n a l s I and I I U + S , and t h e M n + + s i g n a l , has not been e x t e n s i v e l y i n v e s t i g a t e d t o d a t e . Rowlands e t a l . (1970) f u m i g a t e d r a d i s h , p i n t o bean and soybean p l a n t s w i t h S 0 2 c o n c e n t r a t i o n s r a n g i n g from 0.05 t o 300ppm. D a i l y exposure p e r i o d s v a r i e d from 1 t o 20 hours f o r spans o f 18 t o 27 days, depending upon t h e e x p e r i m e n t . S u b s e q u e n t l y , l e a v e s were e x c i s e d and s p e c t r a o b t a i n e d were compared t o s p e c t r a from c o n t r o l l e a v e s a t ambient (3 00 K) and l i q u i d n i t r o g e n (77 K) t e m p e r a t u r e s . A f t e r f u m i g a t i o n w i t h low l e v e l s o f S 0 2 (0.05 and 0.5ppm) t h e y found no d i f f e r e n c e s i n th e s p e c t r a e x c e p t f o r a change i n t h e h y p e r f i n e s p l i t t i n g o f t h e second and t h i r d l i n e s o f t h e M n + + spectrum when t h e s p e c t r a were r e c o r d e d a t 77 K. However, when p i n t o bean l e a v e s were f u m i g a t e d w i t h h i g h c o n c e n t r a t i o n s (100-300ppm) o f S 0 2 f o r 1 hour f o r 18 days, s p e c t r a r e c o r d e d a t 300 K showed a l a r g e i n c r e a s e i n t h e M n + + s i g n a l and a d e c r e a s e i n a ' f r e e - r a d i c a l ' s i g n a l h a v i n g a g - v a l u e around 2.002 3. They a l s o s t a t e , ( w i t h o u t p r o v i d i n g d a t a ) , t h a t a s i m i l a r e f f e c t o c c u r r e d when a c h l o r o p l a s t s u s p e n s i o n was exposed t o a h i g h b u t u n s p e c i f i e d l e v e l o f S 0 2 . The i n v e s t i g a t o r s o f f e r o n l y v e r y t e n t a t i v e c o n c l u s i o n s b u t s u g g e s t t h a t t h e i n c r e a s e i n t h e M n + + spectrum may be a s s o c i a t e d w i t h S 0 2 - c a u s e d changes i n Photosystem I I . I n a r e l a t e d e x p e r i m e n t t h e y compared t h e forms o f s i g n a l s , d e s c r i b e d as S i g n a l I and S i g n a l I I U + S , o b t a i n e d w i t h c h l o r o - p l a s t s u s p e n s i o n s p r e p a r e d from s p i n a c h l e a v e s , w h i c h were i l l u m i n a t e d w i t h w h i t e l i g h t , b e f o r e and a f t e r e x p o sure t o 0.5ppm S 0 2 f o r 4 hours and su g g e s t e d t h a t S 0 2 had caused a change i n t h e r e l a t i o n s h i p o f PSI t o P S I I t o ac c o u n t f o r t h e d i f f e r e n c e s . S h i m a z a k i e t a l . (1984b) f u m i g a t e d s p i n a c h l e a v e s w i t h 2ppm S 0 2 f o r 45 mi n u t e s and t h e n t r a c e d t h e k i n e t i c s o f S i g n a l I i n e x c i s e d s e c t i o n s o f t h e s e l e a v e s . A f t e r t h e p l a n t s were removed from t h e f u m i g a t i o n chamber t h e l e a f segments were i l l u m i n a t e d w i t h f a r - r e d l i g h t (>700nm). T h i s i r r a d i a t i o n , w h i c h i n d u c e d t h e f o r m a t i o n o f S i g n a l I , was t h e n f o l l o w e d by e i t h e r i l l u m i n a t i o n w i t h broad-band w h i t e l i g h t o r by e l i m i n a t i o n o f a l l l i g h t f o c u s e d upon t h e s p e c t r o m e t e r c a v i t y window. They found t h a t n e i t h e r t h e f o r m a t i o n o f S i g n a l I i n f a r - r e d l i g h t n o r i t s decay i n d a r k n e s s was a f f e c t e d by t h e S 0 2 f u m i g a t i o n . However, t h e r a t e o f decay o f S i g n a l I i n w h i t e l i g h t was red u c e d by t h e f u m i g a t i o n . They c o n c l u d e d , from t h e s e d a t a and o t h e r s ( S h i m a z a k i and Sugahara, 1979, 1980; S h i m a z a k i e t a l . 1984a), t h a t t h e S 0 2 f u m i g a t i o n r e v e r s i b l y i n h i b i t s t h e p h o t o s y n t h e t i c w a t e r - s p l i t t i n g enzyme system and i n j u r e s t h e r e a c t i o n c e n t r e o f P S I I when f u m i g a t i o n i s p r o l o n g e d . EPR i n v e s t i g a t i o n o f t h e e f f e c t s o f exposure t o 0 3 on t h e -40- f o r m a t i o n o f f r e e r a d i c a l s i n p l a n t l e a v e s , c h l o r o p l a s t o r s u b c h l o r o p l s t p r e p a r a t i o n s , i s even more l i m i t e d . Rowlands e t a l . (1970) f u m i g a t e d soybean and p i n t o bean p l a n t s w i t h 0.5ppm 0 3 f o r 1 hour o r w i t h 0.05ppm 0 3 f o r 4 h o u r s , and compared t h e s p e c t r a o b t a i n e d from f u m i g a t e d s e c t i o n s o f l e a v e s w i t h t h o s e o f s p e c t r a p r e v i o u s l y o b t a i n e d from u n f u m i g a t e d s e c t i o n s o f t h e same l e a f . A t t h e l o w e r c o n c e n t r a t i o n o f ozone, no d i f f e r e n c e s i n t h e p h o t o s y n t h e t i c s i g n a l s were found, n o r was t h e r e e v i d e n c e f o r t h e f o r m a t i o n o f new f r e e r a d i c a l s . A t t h e h i g h e r c o n c e n t r a t i o n , t h e r e was a l a r g e i n c r e a s e i n f r e e r a d i c a l f o r m a t i o n around t h e g - v a l u e o f "2.000". U n f o r t u n a t e l y , t h e i r s p e c t r a were r e c o r d e d , and a r e p r e s e n t e d , o v e r a scan range o f 1000 gauss, t h u s p r o h i b i t i n g p r e c i s e c h a r a c t e r i z a t i o n o f t h i s s i g n a l . The i n c r e a s e appears t o be independent o f l i g h t as t h e y do n o t mention i r r a d i a t i o n o f t h e s p e c t r o m e t e r c a v i t y . However, t h e i m p r e c i s e g - v a l u e l e a d s o n l y t o s p e c u l a t i o n r e g a r d i n g whether t h i s i n c r e a s e d s i g n a l i s an i n c r e a s e d S i g n a l I , an i n c r e a s e d S i g n a l I I U + S , o r i s r e p r e s e n t a t i v e o f a new f r e e r a d i c a l . The a u t h o r s do not attempt t o e x p l a i n f u r t h e r t h e s i g n a l i n c r e a s e (Rowlands e t a l . 1970). As y e t , no EPR s t u d i e s have been r e p o r t e d i n w h i c h t h e i n v i v o E P R - d e t e c t a b l e changes i n S i g n a l I and H u + S w h i c h o c c u r d u r i n g f u m i g a t i o n w i t h a gaseous p o l l u t a n t were i n v e s t i g a t e d . -41- 3.0 DEVELOPMENT OF METHODOLOGY 3.1 O b j e c t i v e s The o b j e c t i v e s o f t h i s s t u d y were: 1) t o d e v e l o p a system c a p a b l e o f d e t e c t i n g f r e e r a d i c a l s i n i n t a c t p l a n t l e a v e s , r a t h e r t h a n c e l l u l a r o r s u b c e l l u l a r p r e p a r a t i o n s o r e x c i s e d l e a f t i s s u e , i n o r d e r t o d e t e c t s i g n a l f o r m a t i o n o r decay o c c u r r i n g i n s i t u ; 2) t o make t h e system c a p a b l e o f d e t e c t i n g changes i n f r e e - r a d i c a l s i g n a l s r e s u l t i n g from exposure o f t h e l e a f t o changes i n a t m o s p h e r i c c o m p o s i t i o n ; 3) t h r o u g h i d e n t i f i c a t i o n , i f p o s s i b l e , o f any new f r e e r a d i c a l s and, t h r o u g h a n a l y s i s o f t h e changes w h i c h o c c u r i n t h e p h o t o s y n t h e t i c f r e e - r a d i c a l s i g n a l s , t o i n c r e a s e o ur u n d e r s t a n d i n g o f t h e mechanisms by which gaseous a i r p o l l u t a n t s i n j u r e p l a n t c e l l s and d i s r u p t t h e p h o t o s y n t h e t i c p r o c e s s . 3.2 Methods 3.2.1 S p e c t r o m e t e r O p e r a t i o n S p e c t r o m e t r y was c a r r i e d out a t room t e m p e r a t u r e i n an -42- X-band (9GHz) s p e c t r o m e t e r ( C e n t u r y S e r i e s , Model E-109 V a r i a n I n s t r u m e n t D i v i s i o n ) w i t h an a t t a c h e d computer (Model 9835B, H e w l e t t - P a c k a r d Co.)- The T M 1 1 Q c a v i t y was used i n t h e s t u d i e s w i t h e x c i s e d l e a f p i e c e s p l a c e d i n t h e s t a n d a r d b i o l o g i c a l t i s s u e h o l d e r (Wang L a b o r a t o r i e s I n c . ) . I n subsequent s t u d i e s u s i n g t h e c e l l u l o s e a c e t a t e l e a f h o l d e r ( d e s c r i b e d below i n S e c t i o n 3.2.2), t h e E 2 3 1 c a v i t y and v a r i a b l e t e m p e r a t u r e a c c e s s o r y equipment were used. Both c a v i t i e s have an a c c e s s p o r t f o r i l l u m i n a t i o n , w h i c h was p r o v i d e d by a c o l l i m a t e d beam from a 400W t u n g s t e n lamp ( s l i d e p r o j e c t o r ) , d i r e c t e d towards t h e c a v i t y p o r t ( F i g u r e 3 ) . O p t i c a l bandpass f i l t e r s (PTR O p t i c s L t d . ) i n s e r t e d i n t h e l i g h t p a t h were used t o o b t a i n 650 and 710nm s p e c t r a l bands. Broad-band w h i t e l i g h t was o b t a i n e d by o m i t t i n g f i l t e r s from t h e p a t h o f t h e l i g h t beam. The r e l e v a n t i r r a d i a t i o n i n t e n s i t i e s a c h i e v e d a r e p r e s e n t e d i n T a b l e 1. I n s t r u m e n t s e t t i n g s were v a r i e d t o o b t a i n maximum c l a r i t y o f s i g n a l s . I n most c a s e s , optimum p h o t o s y n t h e t i c s i g n a l s were a c h i e v e d w i t h t h e f o l l o w i n g s e t t i n g s : t i m e c o n s t a n t 0.5sec; scan r a t e lOOG/min; m o d u l a t i o n 1.0x10 G; r e c e i v e r g a i n 5 . 0 x l 0 4 ; power lOmW. Fo r b e t t e r r e s o l u t i o n , i n some c a s e s t h e o r d i n a t e was s u b s e q u e n t l y i n c r e a s e d t h r o u g h use o f t h e V a r i a n f a c t o r y s o f t w a r e package and a t t a c h e d computer. Where t h e i n s t r u m e n t s e t t i n g s v a r i e d from t h e above t h e y a r e p r e s e n t e d w i t h t h e s p e c t r a i n t h e a p p r o p r i a t e f i g u r e s . -43- B P r o j e c t o r Figure 3 . Cross s e c t i o n a l view of the equipment arrangement. - 4 4 - T a b l e 1. I r r a d i a t i o n i n t e n s i t i e s r e l e v a n t t o t h e s t u d i e s o f t h i s i n v e s t i g a t i o n . E xposure c o n d i t i o n MEm s~L Wm W i t h i n * s p e c t r o m e t e r c a v i t y 650nm l i g h t 710nm l i g h t broad-band w h i t e l i g h t 1.50 .75 72 . 00 . 68 .42 46. 00 Room l i g h t * * w i t h i n s p e c t r o m e t e r c a v i t y 3 . 60 .90 F u l l s u n l i g h t I n greenhouse ( t o p o f benches) I n greenhouse (below benches) 1. 50x10- 4 . 65x10' 40. 00 8 .20x10' 2 . 35x10' 24 . 00 * R a d i a t i o n i n t e n s i t i e s w i t h i n t h e s p e c t r o m e t e r c a v i t y were based upon h a l v i n g t h e i n t e n s i t i e s a t t h e s u r f a c e o f t h e c a v i t y , by t h e r a d i a t i o n p o r t , as p e r m a n u f a c t u r e r ' s s p e c i f i c a t i o n s . ** These f i g u r e s a r e o n l y i n c l u d e d f o r purposes o f comparison as a l l s p e c t r o m e t r y was done w i t h t h e room l i g h t o f f , t h u s t h e o n l y i l l u m i n a t i o n was p r o v i d e d by t h e p r o j e c t o r beam. K i n e t i c t r a c e s were r e c o r d e d d i r e c t l y onto EPR d a t a paper. F r e e r a d i c a l s i g n a l s were r e c o r d e d , u s i n g t h e V a r i a n s o f t w a r e package, o n t o t h e computer s c r e e n and t h e n s t o r e d on t a p e s t o p r o v i d e t h e c a p a b i l i t y o f s u b t r a c t i n g s i g n a l s r e c o r d e d under d i f f e r e n t l i g h t o r f u m i g a t i o n regimes from each o t h e r . The t i m e r e q u i r e d t o r e c o r d a s i g n a l , s t o r e i t onto a t a p e and r e t u r n t o t h e s c a n module f o r a new scan was a p p r o x i m a t e l y 6 m i n u t e s . S i g n a l s w h i c h were found t o decay r a p i d l y , such as t h o s e s u b s e q u e n t l y d e s c r i b e d i n S e c t i o n 3.3.5, E f f e c t s o f Photo- i n h i b i t i o n , were r e c o r d e d d i r e c t l y onto paper, so t h a t s e v e r a l s i g n a l s c o u l d be r e c o r d e d w i t h i n a 6 minute span, t h u s a l l o w i n g f o r c o m p a r i s o n o f s i g n a l s i z e a t v a r i o u s p o i n t s i n t i m e . F r e e r a d i c a l g - v a l u e s (see Appendix A) were d e t e r m i n e d by c o m p a r i s o n w i t h t h e known s t a n d a r d DPPH (1,1 d i p h e n y l - 2 - p i c r y l - h y d r a z y l ) , w h i c h has a g - v a l u e o f 2.0037 (Appendix B ) . 3.2.2 L e a f H o l d e r The r e q u i r e m e n t f o r c h a n g i n g t h e c o m p o s i t i o n o f t h e s u r r o u n d i n g a i r w h i l e t h e l e a f was i n t h e s p e c t r o m e t e r c a v i t y n e c e s s i t a t e d t h e development o f a t e c h n i q u e f o r k e e p i n g t h e l e a f immobile i n a f l o w i n g gas stream. C o n v e n t i o n a l use o f t h e s t a n d a r d f a c t o r y b i o l o g i c a l t i s s u e h o l d e r i s n o t s u i t a b l e because t h e c o v e r o f t h i s h o l d e r p r e v e n t s gas exchange from o c c u r r i n g . Thus, f o r gaseous a i r p o l l u t a n t s t u d i e s , f l u x t o t h e l e a f would be p r e v e n t e d . The use o f l e a v e s o r l e a f p i e c e s w i t h o u t any s u p p o r t i n g m a t e r i a l i s not p o s s i b l e because t h e gas f l o w r e q u i r e d f o r i n s i t u s t u d i e s causes movement o f t h e l e a f i n t h e c a v i t y , w h i c h i n t u r n p r e v e n t s t h e e s t a b l i s h m e n t o f r e s o n a n c e . I n i t i a l l y , t h e f o l l o w i n g t e c h n i q u e was used. The c o v e r p l a t e o f t h e h o l d e r was removed and t h e l e a f t i s s u e was h e l d i n p l a c e w i t h a d h e s i v e t a p e . T h i s a l l o w e d ready a c c e s s o f t h e components o f t h e gas system t o one l e a f s u r f a c e . S i n c e t h e f a c t o r y q u a r t z t i s s u e h o l d e r ( F i g u r e 4) can be s u p p o r t e d from t h e t o p o f t h e c a v i t y , t h i s a l l o w e d atmosphere m o d i f i c a t i o n by passage o f a i r t h r o u g h t h e bottom o p e n i n g o f t h e c a v i t y v i a t h e t e f l o n t u b i n g used t o t r a n s p o r t n i t r o g e n i n c o n t r o l l e d o r low t e m p e r a t u r e s t u d i e s . C o n s e q u e n t l y , t h e l e a f t i s s u e c o u l d be f u m i g a t e d w h i l e i t was i n t h e c a v i t y w i t h t h e s p e c t r o m e t e r f u n c t i o n i n g . T h i s i n i t i a l p r o c e d u r e a l l o w e d i n s i t u f u m i g a t i o n o f e x c i s e d p i e c e s o f l e a v e s w i t h some degree o f s u c c e s s , b u t a b s o l u t e h o l d e r s t a b i l i t y was not always a c h i e v e d . However, a major drawback was t h a t t h e f a c t o r y h o l d e r gave r i s e t o a br o a d EPR s i g n a l w h i c h , w h i l e i t d i d not i n t e r f e r e w i t h d e t e c t i o n o f t h e r e l e v a n t s i g n a l s a t r e c e i v e r g a i n l e v e l s o f up t o 5 . 0 x l 0 4 , began t o i n t e r f e r e a t t h e h i g h e r g a i n s e t t i n g s needed f o r some l e a f samples. W h i l e t h i s e x t r a n e o u s s i g n a l c o u l d be c o r r e c t e d -47- F i g u r e 4. C o n v e n t i o n a l f a c t o r y (Wang L a b o r a t o r i e s I n c . ) b i o l o g i c a l t i s s u e h o l d e r . -48- f o r by s p e c t r a l s u b t r a c t i o n , a more s u i t a b l e h o l d e r was sought. I n o r d e r t o m a i n t a i n t h e l e a f sample m o t i o n l e s s i n t h e c a v i t y , i t was d e c i d e d t o use t h e c a v i t y dewar i n s e r t o f t h e V a r i a n v a r i a b l e t e m p e r a t u r e a c c e s s o r y as a g u i d e . T h i s i n s e r t has an i n t e r n a l d i a m e t e r o f 5mm. I t s use n e c e s s i t a t e d c h a n g i n g t o t h e E 2 31 c a v i t y , w h i c h i s adapted f o r v a r i a b l e t e m p e r a t u r e s t u d i e s ( F i g u r e 5 ) . T r i a l s were co n d u c t e d w i t h h o l d e r s made o f s e v e r a l d i f f e r e n t m a t e r i a l s , o f v a r i o u s d i m e n s i o n s , as f o l l o w s . 1) a g l a s s m i c r o s c o p e s l i d e h o l d e r H o l d e r s made from g l a s s m i c r o s c o p e s l i d e s were found t o g e n e r a t e s t r o n g EPR s i g n a l s i n c l u d i n g t h o s e a t t r i b u t a b l e t o Fe, Mn and Cu, and an u n i d e n t i f i e d f r e e r a d i c a l . A l t h o u g h t h e s e s i g n a l s c o u l d be c o r r e c t e d by s u b t r a c t i o n , t h i s was an u n d e s i r a b l e f e a t u r e . 2) a m i c r o s c o p e c o v e r s l i p s e c t i o n The use o f t h i s m a t e r i a l caused much l e s s n o i s e t h a n t h e c o n v e n t i o n a l f a c t o r y h o l d e r when t h e s p e c t r o m e t e r was s e t a t a h i g h r e c e i v e r g a i n . However, t h e m a t e r i a l was v e r y d i f f i c u l t t o work w i t h because o f t h e f r a g i l i t y o f t h e narrow p i e c e s w h i c h were r e q u i r e d t o f i t i n t o t h e dewar i n s e r t . - 4 9 - g r a s s l e a f EPR c a v i t y dewar i n s e r t gas s a m p l i n g l i n e l e a f h o l d e r E 2 3 1 w a v e g u i d e t r a n s f e r t u b e c a b l e F i g u r e 5. E 2 3 1 c a v i t y and dewar i n s e r t , -50- 3) T e f l o n and f i l m h o l d e r s . V a r i o u s f i l m s o f T e f l o n and o t h e r p o l ymers were found t o have s t r o n g e r background s i g n a l s t h a n t h e m i c r o s c o p e c o v e r s l i p , b u t l e s s t h a n t h e m i c r o s c o p e s l i d e . They were u s a b l e b u t not o p t i m a l . d) C e l l u l o s e a c e t a t e C e l l u l o s e a c e t a t e f i l m , 0.1mm t h i c k , t h e m a t e r i a l commonly used f o r overhead p r o j e c t i o n , was found t o be i d e a l . T h i s m a t e r i a l was chosen because o f i t s d e s i r e d p h y s i c a l s t r e n g t h and r o b u s t n e s s , and t h e f a c t t h a t i t d i d not r e v e a l any EPR s i g n a l s i n t h e r e g i o n s o f i n t e r e s t . The d i m e n s i o n s o f t h e 'T' shaped c e l l u l o s e a c e t a t e h o l d e r f i n a l l y adopted f o r use ( F i g u r e 6) were chosen so t h a t t h e t a i l f i t t e d s n u g l y l e n g t h w i s e i n t o t h e c a v i t y dewar, l e a v i n g ample room f o r unimpeded gas f l o w and f o r t h e i n s e r t i o n o f a sample l i n e t h r o u g h w h i c h gas samples c o u l d be w ithdrawn f o r a n a l y s i s . I n t a c t l e a v e s o r e x c i s e d l e a f p i e c e s (3x15mm) were a t t a c h e d t o t h e t a i l o f t h e h o l d e r w i t h a d h e s i v e t a p e . The l o c a t i o n s o f t h e h o l d e r and t h e t a p e a t t a c h m e n t s a r e a l s o shown i n F i g u r e 6. I n a l l c a s e s t h e a b a x i a l s u r f a c e o f t h e l e a f was p l a c e d a g a i n s t t h e h o l d e r . -51- b F i g u r e 6. "I"-shaped c e l l u l o s e a c e t a t e l e a f h o l d e r used i n t h e EPR s t u d i e s , a. L e a f h o l d e r ; b. C r o s s s e c t i o n o f h o l d e r i n t h e v a r i a b l e t e m p e r a t u r e a c c e s s o r y dewar i n s e r t . -52- 3.2.3 Atmosphere M o d i f i c a t i o n S i n c e t h e c r o s s - s e c t i o n a l a r e a o f t h e c a v i t y dewar i n s e r t was 16.5mm2, a v e l o c i t y o f 2 m/s was a c h i e v e d w i t h a f l o w r a t e o f a p p r o x i m a t e l y 2 1/min, t h e c o n d i t i o n s used i n subsequent s t u d i e s w i t h ozone and s u l p h u r d i o x i d e . P r e l i m i n a r y r e s u l t s , o b t a i n e d by i l l u m i n a t i n g r a d i s h l e a f p i e c e s w i t h 710nm o r 650nm l i g h t , had i n d i c a t e d t h a t t h e magnitude o f E P R S i g n a l s I and I I U + S o b t a i n e d was u n a f f e c t e d by a i r f l o w r a t e . E n r i c h e d l e v e l s o f d i f f e r e n t gases were a c h i e v e d by i n j e c t i n g a g a s - i n - a i r m i x t u r e i n t o t h e a i r stream, f l o w i n g f i r s t t h r o u g h a m i x i n g chamber, and t h e n i n t o t h e v a r i a b l e t e m p e r a t u r e a c c e s s o r y system v i a t h e t u b i n g n o r m a l l y used t o s u p p l y gases i n c o n t r o l l e d t e m p e r a t u r e s t u d i e s . Ozone was g e n e r a t e d by p a s s i n g m e d i c a l grade a i r t h r o u g h a c h a r c o a l f i l t e r and t h e n t h r o u g h an ozone g e n e r a t o r ( D a s i b i I n s t r u m e n t C o . ) . Fo r t h e s u l p h u r d i o x i d e s t u d i e s , a s u p p l y o f s t a n d a r d 1 p e r c e n t s u l p h u r d i o x i d e i n a i r was i n j e c t e d t h r o u g h a f l o w m e t e r / n e e d l e v a l v e i n t o t h e m i x i n g chamber. P o l l u t a n t c o n c e n t r a t i o n s w i t h i n t h e dewar i n s e r t were m o n i t o r e d t h r o u g h a 2mm e x t e r n a l d i a m e t e r t e f l o n s a m p l i n g l i n e i n s e r t e d i n t o t h e t o p o f t h e dewar i n s e r t as shown i n F i g u r e 6. The s a m p l i n g l i n e was con n e c t e d t o e i t h e r an ozone m o n i t o r (Model 1003AX, D a s i b i I n s t r u m e n t Co.) o r a s u l p h u r d i o x i d e m o n i t o r (Model 43, T h e r m o e l e c t r o n C o r p . ) / depending upon t h e p o l l u t a n t under i n v e s t i g a t i o n . 3.2.4 S p i n T r a p p i n g Numerous a t t e m p t s were made t o i n c o r p o r a t e t h e f r e e r a d i c a l - s p e c i f i c s p i n t r a p s DMPO ( 5 , 5 - d i m e t h y l - p y r r o l i n e - N - o x i d e ) and T i r o n ( 1 , 2 - d i h y d r o x y b e n z e n e - 3 , 5 - d i s u l p h o n a t e ) , and t h e non- s p e c i f i c s p i n t r a p s PBN ( 4 - p y r i d y l - l - o x i d e - N - t e r t - b u t y l - n i t r o n e ) , TEMPO ( 2 , 2 , 6 , 6 - t e t r a m e t h y l - l - p i p e r i d i n e - N - o x y l ) and BPN ( N - t - b u t y l - a - p h e n y l n i t r o n e ) , i n t o p l a n t l e a v e s . V a r i o u s approaches were t r i e d . F i r s t , 20mM s o l u t i o n s o f t h e s p i n t r a p s were s e p a r a t e l y p a i n t e d onto t h e e x t e r i o r o f t h e t i s s u e h o l d e r . The a p p r o p r i a t e p o l l u t a n t was t h e n r e l e a s e d and a sc a n was r e c o r d e d t o a c t as a c o n t r o l . S u b s e q u e n t l y t h e s p i n t r a p s were p a i n t e d o n t o t h e e x t e r i o r o f t h e l e a f t i s s u e , i n c o r p o r a t i o n was att e m p t e d by uptake t h r o u g h t h e p e t i o l e f o r p e r i o d s o f 2, 24 and 4 8 h o u r s and i n c o r p o r a t i o n was at t e m p t e d by vacuum i n f i l t r a t i o n . S p i n t r a p c o n c e n t r a t i o n s were a l s o d e c r e a s e d t o lOmM and i n c r e a s e d t o lOOmM. No s p i n a dducts were formed, r e g a r d l e s s o f p o l l u t a n t o r method o f at t e m p t e d i n c o r p o r a t i o n . 3.2.5 P l a n t M a t e r i a l s P l a n t s o f r a d i s h (Raphanus s a t i v u s L. cv C h e r r y B e l l e ) , p e r e n n i a l r y e g r a s s ( L o l i u m perenne L.) and Kentucky b l u e g r a s s -54- (Poa p r a t e n s i s L.) were grown from seed i n p o t s c o n t a i n i n g s t a n d a r d greenhouse p o t t i n g s o i l mix (85% loam; 15% peat) f e r t i l i z e d w i t h 14-14-14 (Osmocote) f e r t i l i z e r ( S i e r r a C h e m i c a l Company; M i l p i t a s , C a l i f o r n i a , USA). F o r s t u d i e s i n v o l v i n g a t t a c h e d , i n t a c t l e a v e s , t h e g r a s s p l a n t s were grown i n 5cm p o t s , w h i c h c o u l d be p l a c e d on a p l a t f o r m i n f r o n t o f t h e s p e c t r o m e t e r magnet, t o one s i d e o f t h e l i g h t b e a m . d i r e c t e d a t t h e c a v i t y window ( F i g u r e 7 ) . 3.3 Phenomena A s s o c i a t e d w i t h EPR S p e c t r o m e t r y o f L e a f T i s s u e 3.3.1 N a t u r a l V a r i a t i o n i n P h o t o s y n t h e t i c EPR S i g n a l s i n E x c i s e d P i e c e s o f Leaves The t e m p o r a l v a r i a t i o n i n EPR s i g n a l s from e x c i s e d p i e c e s o f r a d i s h l e a v e s i s shown i n F i g u r e s 8 and 9. F i g u r e 8 shows t h e s i g n a l s o b t a i n e d i m m e d i a t e l y f o l l o w i n g e x c i s i o n and mounting i n t h e c a v i t y . R e s i d u a l S i g n a l I I U + S , a t t r i b u t a b l e t o a component o f Photosystem I I , i s ob s e r v e d i n d a r k n e s s and i s enhanced i n 650nm l i g h t ; S i g n a l I , a s s o c i a t e d w i t h Photosystem I , i s superimposed when t h e s i g n a l s a r e r e c o r d e d i n 710nm l i g h t . I n h e a l t h y l e a v e s , under broad-band w h i t e l i g h t o f much h i g h e r i n t e n s i t y ( T a b l e 1 ) , o n l y a s m a l l S i g n a l I i s superimposed upon S i g n a l H u + S when s i g n a l s a r e r e c o r d e d i m m e d i a t e l y a f t e r e x c i s i o n ( F i g u r e s 8a,8b). A f t e r s u b t r a c t i o n (see F i g u r e 2) o f S i g n a l H u + S from t h e combined s i g n a l 0, -55- F i g u r e 7. Equipment s e t u p f o r s t u d i e s w i t h a t t a c h e d , i n t a c t g r a s s l e a v e s . -56- a 10 G D Low f i e l d maximum ( S i g n a l I I ) Hi g h f i e l d minimum ( S i g n a l I I ) Low f i e l d maximum ( S i g n a l I) Hi g h f i e l d minimum) ( S i g n a l I) 650 710 W F i g u r e 8. EPR s i g n a l s o b t a i n e d from h e a l t h y r a d i s h l e a f p i e c e s i n d i f f e r e n t l i g h t r e g i m e s , r e c o r d e d i m m e d i a t e l y a f t e r e x c i s i o n o f t h e p i e c e s . a. 4-week o l d p a l e green l e a f ; b. 6-week o l d d a r k g r e e n l e a f ; c. c h l o r o t i c , t u r g i d l e a f . Recorded i n : D = d a r k ; 650 = 650nm l i g h t ; 710 = 710nnm l i g h t ; W = broad-band w h i t e l i g h t . -57- o b t a i n e d i n 7l0nm l i g h t , S i g n a l I was found t o have a p e a k - t o - peak w i d t h o f 8-9 gauss and a g - v a l u e o f 2.0025. S i g n a l H u + S was found t o have a peak-to-peak w i d t h o f a p p r o x i m a t e l y 19 gauss and a g - v a l u e o f 2.0046. These c h a r a c t e r i s t i c s a r e s i m i l a r t o t h o s e r e p o r t e d i n t h e r e l e v a n t l i t e r a t u r e (Appendix B ) . P i e c e s o f 6-week o l d r a d i s h l e a v e s w h i c h were d a r k green t y p i c a l l y y i e l d e d l a r g e r s i g n a l s i n s e m i - d a r k n e s s , under 650nm l i g h t , under 710nm l i g h t and under broad-band w h i t e l i g h t ( F i g u r e 8a) t h a n younger, p a l e green l e a v e s (4 weeks o l d ) ( F i g u r e 8 b ) . F o r each t y p e o f l e a f , t h e s p e c t r a were r e c o r d e d i n t h e sequence i n d i c a t e d , on t h e same p i e c e o f l e a f , a f t e r e x posure t o 3 m i n u t e s o f t h e a p p r o p r i a t e l i g h t regime. The shade o f greenness o f t h e l e a f was found t o be a more i m p o r t a n t d e t e r m i n a n t o f s i g n a l i n t e n s i t y t h a n l e a f age. The dependence o f t h e s i g n a l s upon t h e p r e s e n c e o f c h l o r o p h y l l was i n d i c a t e d by t h e l a c k o f s i g n a l s from c o m p l e t e l y c h l o r o t i c , b u t s t i l l f u l l y t u r g i d o l d e r l e a v e s , such as t h o s e shown i n F i g u r e 8c. The dependence o f t h e s i g n a l s on f u n c t i o n i n g c h l o r o p l a s t s was v e r i f i e d u s i n g v a r i e g a t e d C o l e u s sp. l e a v e s . I r r a d i a t i o n o f p i e c e s t a k e n from t h e green p a r t s o f such l e a v e s r e v e a l e d s i m i l a r s i g n a l s t o t h o s e o b t a i n e d from green r a d i s h l e a v e s , w h i l e i l l u m i n a t i o n o f p i e c e s t a k e n from c h l o r o p h y l l - f r e e p i n k o r w h i t e a r e a s r e s u l t e d i n t r a c e s comparable t o t h o s e o b t a i n e d from t h e c h l o r o t i c r a d i s h l e a v e s ( C o l e u s sp. s p e c t r a n o t shown). -58- A f t e r two hour s w i t h i n t h e c a v i t y , w i t h o u t i l l u m i n a t i o n from t h e p r o j e c t o r beam, t h e s i g n a l s i n F i g u r e 9 were r e c o r d e d f o r t h e same l e a f p i e c e s as t h o s e used f o r F i g u r e 8. A l l s i g n a l s can be seen t o have changed, w i t h t h e l a r g e s t change o c c u r r i n g i n t h e d a r k g r e e n , 6-week o l d l e a v e s i l l u m i n a t e d w i t h 650nm o r broad-band w h i t e l i g h t ( F i g u r e 9 b ). These changes i n t h e s i g n a l s were not d i r e c t l y a t t r i b u t a b l e t o d e s i c c a t i o n , as t r i a l s w i t h d e t a c h e d l e a f p i e c e s m a i n t a i n e d i n c l o s e t o 100 p e r c e n t r e l a t i v e h u m i d i t y showed comparable changes i n t h e s i g n a l s w i t h t i m e ( s p e c t r a n o t shown). The major p a r t o f t h e changes i n t h e s i g n a l s o b s e r v e d under any l i g h t t r e a t m e n t o c c u r r e d i n t h e second hour a f t e r detachment from t h e p l a n t . S i m i l a r changes i n s i g n a l s were a l s o o b s e r v e d i n d e t a c h e d l e a f p i e c e s o f p e r e n n i a l r y e g r a s s and Ken t u c k y b l u e g r a s s ( s p e c t r a n o t shown). 3.3.2 V a r i a b i l i t y i n P h o t o s y n t h e t i c EPR S i g n a l s i n Whole, A t t a c h e d G r a s s Leaves I n c o n t r a s t t o t h e s i t u a t i o n d e s c r i b e d f o r e x c i s e d l e a f p i e c e s , when h e a l t h y , i n t a c t . a t t a c h e d g r a s s l e a v e s were m a i n t a i n e d i n t h e s p e c t r o m e t e r c a v i t y , no changes i n t h e s i g n a l s o c c u r r e d f o r a t l e a s t 72 h o u r s . I n t h e s e t r i a l s , u s i n g b o t h p e r e n n i a l r y e g r a s s and Kentucky b l u e g r a s s l e a v e s , t h e t e s t l e a f remained mounted i n t h e c a v i t y f o r 5 days. The p l a n t s were a D 650 710 W F i g u r e 9. EPR s i g n a l s o b t a i n e d from t h e p r e v i o u s l y h e a l t h y r a d i s h l e a f p i e c e s d e p i c t e d i n F i g u r e 8, i n d i f f e r e n t l i g h t r e g i m e s , r e c o r d e d 2 h o u r s a f t e r e x c i s i o n , w i t h t h e p i e c e s h e l d i n d a r k n e s s i n t h e c a v i t y d u r i n g t h e i n t e r v a l . a. 4-week o l d p a l e g r e e n l e a f ; b. 6-week o l d d a r k g r e e n l e a f ; c. c h l o r o t i c , t u r g i d l e a f . Recorded i n : D = d a r k ; 650 = 650nm l i g h t ; c = 710nm l i g h t ; W = broad-band w h i t e l i g h t . -60- w a t e r e d d a i l y and were exposed t o 14hr room l i g h t and l O h r d a r k n e s s . D u r i n g t h e i n t e r v a l s between t h e r e c o r d i n g o f t h e s i g n a l s o n l y i n d i r e c t room l i g h t e n t e r e d t h e c a v i t y d u r i n g t h e p h o t o p e r i o d and t h e l e a f was i l l u m i n a t e d by t h e p r o j e c t o r beam o n l y d u r i n g s i g n a l measurement. EPR s i g n a l s were r e c o r d e d a f t e r t h e p l a n t s had been h e l d i n room l i g h t f o r 2 h o u r s each day. The s p e c t r o m e t e r was t u r n e d o f f a f t e r each day's measurements. A f t e r f i v e days i n t h e s p e c t r o m e t e r c a v i t y , w i t h t h e m a g n e t i c f i e l d o f f , s e v e r a l changes were o b s e r v e d t o have o c c u r r e d i n t h e EPR s i g n a l s . A r e p r e s e n t a t i v e s e r i e s o f s p e c t r a o b t a i n e d w i t h Kentucky b l u e g r a s s l e a v e s i s shown i n F i g u r e 10. The changes were: 1) t h e near e l i m i n a t i o n o f t h e m u l t i p l e band M n + + s i g n a l t y p i c a l l y a s s o c i a t e d w i t h h e a l t h y b l u e g r a s s l e a v e s ( F i g u r e 10a,b); 2) t h e c o n c o m i t a n t e l i m i n a t i o n o f t y p i c a l S i g n a l I I U + S ( F i g u r e 1 0 a ) ; and 3) t h e appearance o f a new s i g n a l under broad-band w h i t e l i g h t ( F i g u r e 10b,d). These changes o n l y o c c u r r e d i n t h e p a r t o f t h e s p e c i f i c l e a f m a i n t a i n e d i n t h e c a v i t y ; s i g n a l s from o t h e r p a r t s o f t h a t l e a f o r from o t h e r l e a v e s o f t h e same p l a n t , w h i c h were not i n t h e c a v i t y f o r t h e f i v e day p e r i o d , were not a f f e c t e d i n t h i s i n t e r v a l as shown i n F i g u r e 10c. The w h i t e l i g h t - i n d u c e d s i g n a l o b s e r v e d a f t e r 5 days i s shown more c l e a r l y i n F i g u r e lOd, f o l l o w i n g s u b t r a c t i o n o f t h e o r i g i n a l s i g n a l s . T h i s s i g n a l , w hich has a g - v a l u e o f 2.0046 and a peak-to-peak w i d t h o f a p p r o x i m a t e l y 10 gauss, i s c l e a r l y d i f f e r e n t from S i g n a l I , which has a g - v a l u e o f 2.0025 -61- F i g u r e 10. Changes i n t h e Mn, d a r k , and w h i t e - l i g h t i n d u c e d s i g n a l s i n i n t a c t , a t t a c h e d Kentucky b l u e g r a s s l e a v e s a f t e r 5 days i n t h e s p e c t r o m e t e r . a. Top - d a r k s i g n a l a t s t a r t ; Bottom - d a r k s i g n a l a f t e r l e a f t a p e d t o h o l d e r w i t h i n c a v i t y f o r 5 d a y s ; b. Top - w h i t e l i g h t s i g n a l a t s t a r t ; Bottom - w h i t e l i g h t s i g n a l a f t e r l e a f t a p e d t o h o l d e r w i t h i n c a v i t y f o r 5 d a y s ; c. Top - w h i t e l i g h t s i g n a l a t s t a r t from a d i f f e r e n t l e a f on same p l a n t as l e a f used f o r F i g u r e s 10a and 10b; Bottom - w h i t e l i g h t s i g n a l from l e a f used f o r lOc-Top a f t e r 5 days w i t h t h e p l a n t on t h e s p e c t r o m e t e r p l a t f o r m b u t t h e l e a f n o t t a p e d t o t h e h o l d e r ; d. Top - (10b Bottom - 10b Top) - (10a Bottom - 10a T o p ) ; Bottom - DPPH (g=2.0037). -62- and a w i d t h o f 7.5-9 gauss. The l e a f - t o - l e a f d i f f e r e n c e s which o c c u r on t h e same p l a n t a r e shown by t h e c o m p a r i s o n o f t h e upper s p e c t r a i n F i g u r e s 10b and 10c, which were o b t a i n e d i n w h i t e l i g h t from d i f f e r e n t l e a v e s o f t h e same p l a n t . 3.3.3 P o s s i b i l i t i e s and L i m i t a t i o n s o f K i n e t i c S t u d i e s I t i s p o s s i b l e t o f o l l o w t h e r i s e and decay of f r e e - r a d i c a l s i g n a l s o v e r t i m e , by l o c k i n g t h e s p e c t r o m e t e r onto t h e low o r h i g h f i e l d peak of t h e a p p r o p r i a t e s i g n a l . P r i o r t o k i n e t i c t r a c k i n g i t i s n e c e s s a r y t o f i r s t r e c o r d t h e s i g n a l i n o r d e r t o a s c e r t a i n i t s p r e c i s e l o c a t i o n i n t h e magnetic f i e l d . Once i t s l o c a t i o n i n t h e m agnetic f i e l d has been e s t a b l i s h e d , subsequent t r i a l s w i t h d i f f e r e n t l e a f p i e c e s can be done t o f o l l o w t h e k i n e t i c s o f s i g n a l f o r m a t i o n and decay. F o r example, t h e t r a c e d e p i c t e d i n F i g u r e 11 shows t h e t y p i c a l changes w h i c h o c c u r i n S i g n a l I i n h e a l t h y r a d i s h l e a f p i e c e s exposed i n sequence t o t h e d i f f e r e n t l i g h t r e g i m e s . To o b t a i n t h e d a t a t h e s p e c t r o - meter was l o c k e d onto t h e low f i e l d maximum (see F i g u r e 8 ) . The t r a c e shows t h e appearance o f a l a r g e S i g n a l I upon i r r a d i a t i o n w i t h 710nm l i g h t , m i n i m a l o c c u r r e n c e o f t h i s s i g n a l on exposure t o 650nm o r broad-band w h i t e l i g h t , and t h e r a p i d decay o f t h e s i g n a l when t h e q u a l i t y o f l i g h t i s changed o r i l l u m i n a t i o n c e a s e s . These o b s e r v a t i o n s a r e s i m i l a r t o t h o s e o f B l u m e n f e l d e t a l . (1974), who worked w i t h p i e c e s o f l e a v e s from s e v e r a l p e r e n n i a l s p e c i e s . 0 I I I 16 t i m e (min) W 710 D 710 W 650 t 16 | | | 32 t i m e (min) F i g u r e 11. K i n e t i c s o f S i g n a l I f o r m a t i o n and decay i n u n f u m i g a t e d r a d i s h l e a f p i e c e s w i t h d i f f e r e n t l i g h t t r e a t m e n t s Arrows (A ) i n d i c a t e t i m e s a t w h i c h l i g h t was changed. D = d a r k , 650 = 650nm l i g h t ; 710 = 710nm l i g h t ; W = broad-band w h i t e l i g h t . I n c o n t r a s t , F i g u r e 12 shows t h e changes i n t h e low f i e l d maximum of S i g n a l I d u r i n g t h e same sequence o f l i g h t r e g i m e s , w h i c h o c c u r i n p i e c e s o f r a d i s h l e a v e s p r e v i o u s l y f u m i g a t e d f o r 10 m i n u t e s w i t h a p p r o x i m a t e l y 400ppm S 0 2 , a m a s s i v e dose o f t h i s gaseous a i r p o l l u t a n t . A g a i n , i n d a r k n e s s , S i g n a l I i s b a r e l y d e t e c t a b l e , and i s markedly i n c r e a s e d upon i r r a d i a t i o n w i t h 7l0nm l i g h t , a l t h o u g h t h e r a t e o f i n c r e a s e i s l e s s t h a n i n u n t r e a t e d t i s s u e ( F i g u r e 1 1 ) . However, f o l l o w i n g S 0 2 t r e a t m e n t , s i g n a l s o f a p p r o x i m a t e l y comparable s i z e r e s u l t from i r r a d i a t i o n w i t h e i t h e r 650nm o r 710nm l i g h t and a s t i l l l a r g e r S i g n a l I r e s u l t s from i r r a d i a t i o n w i t h t h e broad-band w h i t e l i g h t . These d a t a , and o t h e r changes caused by exposure t o S 0 2 a r e d i s c u s s e d i n d e t a i l i n S e c t i o n 5.0, b u t i t i s a p p a r e n t t h a t t h e m a s s i v e i n f l u x o f S 0 2 i n t o t h e l e a f has a f f e c t e d t h e normal p h o t o s y n t h e t i c e l e c t r o n t r a n s f e r p r o c e s s found i n h e a l t h y l e a v e s . I n r a d i s h l e a v e s , u s i n g e x c i s e d l e a f t i s s u e , i t i s t h u s p o s s i b l e t o f o l l o w t h e EPR s i g n a l s i n d u c e d by t r e a t m e n t w i t h d i f f e r e n t l i g h t q u a l i t i e s , and t o d e t e r m i n e how t h e s e s i g n a l s may be i n f l u e n c e d by gas t r e a t m e n t , w i t h i n t h e l i m i t e d 60 minute p e r i o d f o l l o w i n g e x c i s i o n , b e f o r e t h e changes i n d u c e d by e x c i s i o n b e g i n t o be a p p r e c i a b l e . -65- F i g u r e 12. K i n e t i c s o f S i g n a l I f o r m a t i o n and decay i n d i f f e r e n t l i g h t t r e a t m e n t s i n r a d i s h l e a f p i e c e s p r e v i o u s l y f u m i g a t e d w i t h a p p r o x i m a t e l y 400ppm S 0 2 f o r 10 m i n u t e s . Arrows (4 ) i n d i c a t e t i m e s a t whi c h l i g h t was changed. D = d a r k , 650 = 650nm l i g h t ; 710 = 710nm l i g h t ; W = broad-band w h i t e l i g h t . -66- However, p l a n t l e a v e s a r e c a p a b l e o f a d a p t a t i o n ( B l u m e n f e l d e t a l . 1974; Ruuge and Tihkonov, 1972; Tihkonov and Ruuge 1975a, 1975b; Andreyeva e t a l . 1979). P r o l o n g e d exposure t o e i t h e r d a r k n e s s o r i n t e n s e broad-band w h i t e l i g h t c auses a d e l a y i n t h e f o r m a t i o n o f S i g n a l I when t h e l e a f i s s u b s e q u e n t l y exposed t o 710nm l i g h t . S e v e r a l h o u r s o f d a r k n e s s a r e r e q u i r e d t o d e l a y i t s appearance, b u t as l i t t l e as 3 minutes exposure t o i n t e n s e w h i t e l i g h t i s s u f f i c i e n t t o r e t a r d i t s f o r m a t i o n upon exposure t o 710nm l i g h t , as shown i n t h e lo w e r p a r t o f t h e t r a c e i n F i g u r e 11. Thus, i f one w i s h e s t o r e c o r d t h e s i g n a l s a f t e r a l e n g t h y k i n e t i c t r a c e i n e i t h e r d a r k n e s s o r broad-band w h i t e l i g h t , t h e 710nm l i g h t - i n d u c e d S i g n a l I w i l l n o t be formed i m m e d i a t e l y upon i l l u m i n a t i o n w i t h 710nm l i g h t . T h i s can be o f im p o r t a n c e when t h e t e c h n i q u e o f s i g n a l s u b t r a c t i o n i s used t o c l a r i f y t h e changes i n s p e c t r a r e s u l t i n g from d i f f e r e n t t r e a t m e n t s o f t h e l e a f as d i s c u s s e d below i n S e c t i o n 3.3.4. The p e r i o d o f t i m e r e q u i r e d t o c o u n t e r t h e e f f e c t s o f d a r k n e s s o r broad-band w h i t e l i g h t i n c r e a s e s w i t h i n c r e a s i n g t i m e o f exposure t o t h e s e l i g h t regimes ( d a t a n o t p r e s e n t e d ) and v a r i e s somewhat from l e a f t o l e a f . I n o r d e r t o m i n i m i z e t h i s problem i n l o n g - t e r m s t u d i e s i t i s t h e r e f o r e n e c e s s a r y t o i l l u m i n a t e t h e l e a f w i t h l i g h t o f t h e a p p r o p r i a t e w a v e l e n g t h (710nm o r 650nm) f o r s e v e r a l m i n u t e s p r i o r t o r e c o r d i n g t h e EPR s p e c t r a . A more s e v e r e problem i n a t t e m p t i n g t o t r a c k t h e p h o t o s y n t h e t i c S i g n a l s I and H u + S o c c u r s when t h e s i g n a l s o f -67- i n t e r e s t a r e superimposed upon o t h e r s i g n a l s o f comparable o r l a r g e r magnitude, i f t h e s e , t o o , change w i t h e x p e r i m e n t a l t r e a t m e n t . I n l e a v e s o f many s p e c i e s , EPR s p e c t r o m e t r y r e v e a l s t h e b r o a d F e + + and s i x - p e a k M n + + s i g n a l s d i s c u s s e d e a r l i e r i n S e c t i o n 2.4. S i g n a l s I and I I U + S , and a l l f r e e - r a d i c a l s i g n a l s , a r e u n d e r l a i n f i r s t by t h e F e + + s i g n a l and a l s o by t h e f o u r t h peak from t h e low f i e l d end o f t h e M n + + s i g n a l . Thus, i f t h e s e s i g n a l s change as a r e s u l t o f any t r e a t m e n t , t h e ap p a r e n t r i s e o r decay o f t h e p h o t o s y n t h e t i c o r o t h e r f r e e - r a d i c a l s i g n a l s m o n i t o r e d by t h e k i n e t i c s c a n may be c o m p l e t e l y i n a c c u r a t e , e s p e c i a l l y i n t i s s u e s i n w h i c h t h e i r o n and manganese s i g n a l s a r e much l a r g e r t h a n t h e f r e e - r a d i c a l s i g n a l s o f i n t e r e s t . U n f o r t u n a t e l y , t h i s appears t o be t h e case f o r many, i f n o t most, p l a n t s p e c i e s . F i g u r e s 13-17 i n d i c a t e t h e EPR s p e c t r a o b t a i n e d from p i e c e s o f t h e l e a v e s o f f i v e s p e c i e s s e l e c t e d a r b i t r a r i l y . These s p e c t r a were r e c o r d e d o v e r 5000 gauss w i t h t h e l e a f p i e c e s h e l d i n d a r k n e s s i n t h e c a v i t y . I n o r d e r t o a v o i d t r u n c a t i n g t h e s p e c t r a , t h e s p e c t r o m e t e r r e c e i v e r g a i n was h a l v e d t o 2.5x10000. The s i x - p e a k M n + + s i g n a l and t h e s l o p i n g F e + + s i g n a l w h i c h u n d e r l i e s i t a r e p r e s e n t i n each spectrum and t h e i r i n t e n s i t i e s v a r y from s p e c i e s t o s p e c i e s . A t t h i s l o w e r e d r e c e i v e r g a i n , S i g n a l I I U + S i s not a p p a r e n t i n t h e s p e c t r a from t h e p i n e n e e d l e ( F i g u r e 13) and maple l e a f ( F i g u r e 1 4 ) , and i s o n l y p r e s e n t as a v e r y s m a l l p e r t u r b a t i o n i n t h e -68- 701 3251 M a g n e t i c f i e l d (Gauss) 5701 F i g u r e 13. EPR spectrum from a d e t a c h e d p i n e n e e d l e . Recorded i n d a r k n e s s . R e c e i v e r g a i n - 2.5 x 10000; Microwave f r e q u e n c y - 9.188 701 3251 5701 M a g n e t i c f i e l d (Gauss) F i g u r e 14. EPR spectrum from an e x c i s e d maple l e a f p i e c e . Recorded i n d a r k n e s s . R e c e i v e r g a i n - 2.5 x 10000; Microwave f r e q u e n c y - 9.188 -70- S i g n a l I I 701 3251 5701 M a g n e t i c f i e l d (Gauss) F i g u r e 15. EPR spectrum from an e x c i s e d oak l e a f p i e c e . Recorded i n d a r k n e s s . R e c e i v e r g a i n - 2.5 x 10000; Microwave f r e q u e n c y - 9.188. -71- M a g n e t i c f i e l d (Gauss) F i g u r e 16. EPR spectrum from an e x c i s e d i v y l e a f p i e c e . Recorded i n d a r k n e s s . R e c e i v e r g a i n - 2.5 x 10000; Microwave f r e q u e n c y - 9.188 -72- 701 3251 5701 M a g n e t i c f i e l d (Gauss) F i g u r e 17. EPR spectrum from an e x c i s e d c h e r r y l e a f p i e c e . R ecorded i n d a r k n e s s . R e c e i v e r g a i n - 2.5 x 10000; Microwave f r e q u e n c y - 9.188 f o u r t h Mn peak i n t h e s p e c t r a from t h e oak ( F i g u r e 15) and i v y ( F i g u r e 16) l e a v e s . The s p e c t r a from t h e p i n e n e e d l e ( F i g u r e 13) and c h e r r y l e a f ( F i g u r e 17) show a d d i t i o n a l s i g n a l s r i d i n g on t h e second and f o u r t h M n + + peak, r e s p e c t i v e l y . A t t h i s s c a l e i t i s i m p o s s i b l e t o d i s c e r n t h e e x a c t g - v a l u e s o f t h e s e s i g n a l s . However, t h e c h e r r y l e a f s i g n a l w i t h a g - v a l u e c l o s e t o 2.000 cannot be S i g n a l I , which r e q u i r e s l i g h t f o r i n d u c t i o n , and i s p o s s i b l y t h e u n i d e n t i f i e d o r g a n i c f r e e r a d i c a l s i g n a l ( M i s h r a e t a l . 1971; P r i e s t l e y e t a l . 1980) g e n e r a l l y a s s o c i a t e d w i t h aged p l a n t m a t e r i a l . As i s r e a d i l y a p p a r e n t , t h e manganese s i g n a l s and t h e broad s l o p i n g i r o n s i g n a l upon which t h e manganese s i g n a l s t h e m s e l v e s r i d e a r e much l a r g e r t h a n S i g n a l I I U + S . Thus, any changes i n t h e s e s i g n a l s w i l l d i s t o r t t h e k i n e t i c t r a c k i n g o f S i g n a l I : ru+s- A l t h o u g h t h e use o f g r a s s l e a v e s a t t a c h e d t o t h e p l a n t o f f e r s t h e advantage o f p r o v i d i n g s t a b l e p h o t o s y n t h e t i c EPR s p e c t r a o v e r a much l o n g e r t i m e p e r i o d t h a n e x c i s e d l e a f p i e c e s , a l l o f t h e g r a s s e s s u r v e y e d r e v e a l e d d i s t i n c t F e + + and M n + + s p e c t r a , as shown t y p i c a l l y by t h e Kentucky b l u e g r a s s s p e c t r a i n F i g u r e 10. J u s t as t h e s e s i g n a l s can change o v e r t i m e w i t h p r o l o n g e d r e s i d e n c e i n t h e chamber, so t o o can t h e y be m o d i f i e d as a r e s u l t o f p o l l u t a n t gas t r e a t m e n t . Such changes i n an a t t a c h e d p e r e n n i a l r y e g r a s s l e a f t r e a t e d w i t h ozone a r e shown i n -74- F i g u r e 18. The spectrum i n F i g u r e 18a, r e c o r d e d o v e r 1600 gauss, under broad-band w h i t e l i g h t , shows t y p i c a l c o m p o s i t e F e + + / M n + + s i g n a l s p r i o r t o f u m i g a t i o n w i t h ozone. A f t e r 1 hour o f f u m i g a t i o n w i t h lppm ozone i n t h e c a v i t y , b o t h t h e M n + + and F e + + s i g n a l s have i n c r e a s e d s l i g h t l y ( F i g u r e 18b) bu t a f t e r 14 hours o f exposure t o t h e ozone and w h i t e l i g h t , t h e M n + + s i g n a l has g r e a t l y d i m i n i s h e d w h i l e t h e F e + + s i g n a l has g r e a t l y i n c r e a s e d ( F i g u r e 1 8 c ) , as e v i d e n c e d by t h e i n c r e a s e i n o v e r a l l s l o p e i n t h e c e n t r e o f t h e spectrum. The imminent demise o f t h e f u m i g a t e d p a r t o f t h e l e a f i s i n d i c a t e d by t h e l a r g e f r e e - r a d i c a l s i g n a l found near t h e 2.000 g - v a l u e . However, even t h e t r a c k i n g o f t h i s l a r g e f r e e - r a d i c a l s i g n a l would be confounded by t h e s h i f t s i n t h e u n d e r l y i n g M n + + and F e + + s i g n a l s . These c o m p l i c a t i o n s t h e r e f o r e l i m i t t h e t r a c k i n g o f t h e changes i n t h e p h o t o s y n t h e t i c f r e e - r a d i c a l s i g n a l s i n d u c e d by gaseous a i r p o l l u t a n t s t o s p e c i e s such as r a d i s h , w h i c h have v e r y m i n i m a l M n + + and F e + + EPR s i g n a l s i n r e l a t i o n t o t h e s i z e o f t h e i r p h o t o s y n t h e t i c f r e e - r a d i c a l s i g n a l s . I n p a r t i c u l a r , i t i s u n f o r t u n a t e t h a t such t r a c k i n g does n ot appear t o be u s a b l e w i t h any o f t h e g r a s s s p e c i e s i n v e s t i g a t e d , i n s p i t e o f t h e a t t r a c t i v e n e s s t h a t t h e y can be s t u d i e d i n t a c t w i t h o u t t h e appearance o f t h e a r t i f i c i a l a g i n g e f f e c t s d i s c u s s e d e a r l i e r , and a r e t h u s p o t e n t i a l l y more u s e f u l f o r a s s e s s i n g t h e l o n g e r - t e r m e f f e c t s o f low c o n c e n t r a t i o n s o f gaseous p o l l u t a n t s . -75- 2450 3250 4050 M a g n e t i c f i e l d (Gauss) F i g u r e 18. White l i g h t - i n d u c e d s p e c t r a from an i n t a c t , a t t a c h e d p e r e n n i a l r y e g r a s s l e a f . R e c e i v e r g a i n - 10.0x10000; Microwave f r e q u e n c y - 9.184. a. p r i o r t o f u m i g a t i o n ; b. a f t e r 30 minutes o f lppm ozone; c. a f t e r 14 h o u r s o f lppm ozone. The l e a f was m a i n t a i n e d i n w h i t e l i g h t c o n t i n u o u s l y . -76- The o n l y a l t e r n a t i v e appears t o be t o r e c o r d t h e a b s o l u t e s i g n a l s w i t h t r e a t e d and c o n t r o l l e a v e s under d i f f e r e n t l i g h t r e gimes a t a p p r o p r i a t e t i m e s , and t h e n compare t h e s p e c i f i c s p e c t r a , f o r example by measuring peak-to-peak h e i g h t s , o r by s i g n a l s u b t r a c t i o n s c o nducted t o a d j u s t f o r t h e s e o t h e r s i g n a l changes, as d e s c r i b e d below. 3.3.4 S i g n a l S u b t r a c t i o n The H e w l e t t - P a c k a r d computer a t t a c h e d t o t h e s p e c t r o m e t e r p e r m i t s t h e s u b t r a c t i o n o f s p e c t r a from each o t h e r . C o n t r o l s p e c t r a can be s t o r e d on t a p e and s u b s e q u e n t l y s u b t r a c t e d from s p e c t r a o b t a i n e d a f t e r t r e a t m e n t . T h i s a l l o w s f o r t h e c h a r a c t e r i z a t i o n o f s m a l l , new f r e e r a d i c a l s w h i c h o v e r l a p e s t a b l i s h e d f r e e r a d i c a l s . A s i m p l e example o f r e s u l t s a t t a i n a b l e w i t h t h i s t e c h n i q u e has been p r e s e n t e d i n F i g u r e 2. The t i m e r e q u i r e d t o r e c o r d t h e s i g n a l , s t o r e i t on t a p e and r e t u r n t o t h e sc a n module t o r e c o r d t h e n e x t s i g n a l i s 6 mi n u t e s . 3.3.4.1. A New L i g h t - i n d u c e d F r e e R a d i c a l S i g n a l When a t t a c h e d p e r e n n i a l r y e g r a s s l e a v e s were m a i n t a i n e d i n c h a r c o a l - f i l t e r e d a i r i n 710nm l i g h t i n t h e s p e c t r o m e t e r c a v i t y f o r 7 h o u r s , a d o w n f i e l d s h i f t i n t h e peak o f t h e s i g n a l i n d u c e d by 710nm l i g h t became a p p a r e n t , w i t h a c o n c o m i t a n t s h i f t i n g - v a l u e . T h i s s u g g e s t e d t h e f o r m a t i o n o f a new s i g n a l w i t h a h i g h e r g - v a l u e t h a n S i g n a l I . To c l a r i f y t h i s new s i g n a l , t h e spectrum o b t a i n e d i n 710nm l i g h t a t t h e s t a r t o f t h e ex p e r i m e n t was s u b t r a c t e d from t h e 710nm l i g h t - i n d u c e d spectrum a f t e r 7 h o u r s . T h i s s u b t r a c t i o n r e v e a l e d t h e s m a l l f r e e - r a d i c a l s i g n a l ( S i g n a l N 7 1 Q ) shown i n F i g u r e 19b. Subsequent t r i a l s w i t h b o t h a t t a c h e d Kentucky b l u e g r a s s and a t t a c h e d p e r e n n i a l r y e g r a s s l e a v e s d i s c l o s e d t h a t S i g n a l N 7 1 0 was formed a f t e r p r o l o n g e d exposure t o 710nm l i g h t . Depending upon t h e l e a f , t h e s i g n a l appeared anywhere from 3 0 t o 60 minutes a f t e r t h e o n s e t o f i l l u m i n a t i o n , r e g a r d l e s s o f whether o r n o t a i r was p a s s i n g t h r o u g h t h e c a v i t y . S i g n a l N 7 1 0 , which i s r e v e a l e d by s u b t r a c t i o n o f t h e l i g h t - i n d u c e d s i g n a l a t t h e o n s e t o f i l l u m i n a t i o n , from t h e l i g h t - i n d u c e d s i g n a l a f t e r 30 t o 60 minutes o f i l l u m i n a t i o n , has a peak-to-peak w i d t h o f 8.5 gauss, a g - v a l u e o f a p p r o x i m a t e l y 2.0054 and i s i n d u c e d by 710nm l i g h t ( T a b l e 2 ) . I t r e a c h e s a maximum w i t h i n 120 m i n u t e s o f i l l u m i n a t i o n . The maximum h e i g h t o f S i g n a l N 7 1 Q i s a p p r o x i m a t e l y 45 p e r c e n t o f t h e 710nm l i g h t - i n d u c e d S i g n a l I . I t i s c l e a r l y n o t an i n c r e a s e i n S i g n a l I as i t d i f f e r s i n g - v a l u e ( S i g n a l I = 2.0025). The i d e n t i t y o f S i g n a l N 7 1 0 i s unknown; t h e g - v a l u e i s i d e n t i c a l t o t h a t o f t h e e a r l y e l e c t r o n a c c e p t o r , a ^ d e s c r i b e d by Gast e t a l . (1983), b u t t h e l i n e w i d t h i s l e s s (Appendix B ) . J I I I L 3236 3276 M a g n e t i c f i e l d (Gauss) b M a g n e t i c f i e l d (Gauss) F i g u r e 19. Comparison o f o r i g i n a l S i g n a l I and S i g n a l N 7 1 0 i n a t t a c h e d , i n t a c t Kentucky b l u e g r a s s l e a v e s . a. O r i g i n a l S i g n a l I (710nm l i g h t - i n d u c e d s i g n a l - d a r k s i g n a l ) ; b. S i g n a l N 7 1 0 a f t e r l e a f h e l d i n 710nm l i g h t f o r 1 hour ((710nm l i g h t s i g n a l a f t e r 1 hour - d a r k s i g n a l a f t e r 1 hour) - o r i g i n a l S i g n a l I ) . Microwave f r e q u e n c y - 9.190. -79- T a b l e 2. R e l a t i o n s h i p o f appearance o f t h e u n c h a r a c t e r i z e d EPR f r e e - r a d i c a l s i g n a l ( S i g n a l N 7 1 Q ) i n i n t a c t r y e g r a s s l e a v e s w i t h t i m e o f exposure t o 710nm l i g h t . Time o f exposure (min) 30 120 240 320 420 New 710nm l i g h t - i n d u c e d s i g n a l ( S i g n a l N 7 1 0 ) * * Peak-to-peak w i d t h 8 8.5 8.5 8.5 8.5 H e i g h t * 29 42 37 39 43 g - v a l u e 2.0055 2.0055 2.0054 2.0054 2.0054 • R e l a t i v e u n i t s * * 0 b t a i n e d by s u b t r a c t i o n o f t h e o r i g i n a l 710nm l i g h t - i n d u c e d s i g n a l from t h e 710nm l i g h t - i n d u c e d s i g n a l a t t h e i n d i c a t e d t i m e s . I n each c a s e , t h e t r u e l i g h t - i n d u c e d s i g n a l s were o b t a i n e d by p r i o r s u b t r a c t i o n o f t h e a p p r o p r i a t e d a r k s i g n a l s . 3.3.4.2 Oxygen E f f e c t s on S i g n a l s I and I I U + S Changes i n t h e oxygen c o n t e n t o f t h e a i r p a s s i n g o v e r t h e l e a f t i s s u e s u p p o r t e d i n t h e c a v i t y have a p r o f o u n d i n f l u e n c e on t h e p h o t o s y n t h e t i c S i g n a l s I and I I U + S . F i g u r e 20 d e p i c t s t h e combined s i g n a l ( S i g n a l H u + S + S i g n a l I) when Kentucky -80- b l u e g r a s s l e a v e s a r e i l l u m i n a t e d w i t h 710nm l i g h t . F i g u r e 20a shows t h e t y p i c a l s i g n a l when a i r i s passe d t h r o u g h t h e c a v i t y a t t h e s t a n d a r d r a t e o f 2 1/min. F i g u r e 2 0b shows t h e d e c r e a s e d s i g n a l r e c o r d e d a f t e r t h e l e a f was h e l d i n 710nm l i g h t f o r 30 mi n u t e s d u r i n g which 0 2 was removed by f l u s h i n g t h e system w i t h N 2• F i g u r e 20c shows t h e absence o f S i g n a l I i n d u c t i o n by 710nm l i g h t f o l l o w i n g l e a f exposure t o 4 mi n u t e s each o f d a r k n e s s and w h i t e l i g h t . T h i s s i g n a l was r e c o r d e d a f t e r t h e l e a f had been re e x p o s e d t o 4 minutes o f 710nm l i g h t i n o r d e r t o e l i m i n a t e any p o s s i b l e l i g h t a d a p t a t i o n e f f e c t s caused by t h e exposure t o w h i t e l i g h t (see S e c t i o n 3.3.3). F i g u r e 20d shows t h e s i g n a l o b t a i n e d a f t e r an a d d i t i o n a l hour i n N 2 w i t h t h e l e a f h e l d i n 710nm l i g h t f o r t h a t t i m e , and s u g g e s t s a composi t e reduced S i g n a l I I U + S and reduced S i g n a l I . F i g u r e 21 shows t h e s i g n a l s o b t a i n e d w i t h no d i r e c t c a v i t y i l l u m i n a t i o n when t h e l e a f was exposed t o a i r ( F i g u r e 2 1 a ) , and a f t e r 36 and 96 minutes exposure t o N 2 ( F i g u r e s 21b and 2 1 c ) , and s u g g e s t s a p r o g r e s s i v e d e c r e a s e i n S i g n a l I I U + S . F i g u r e 22 shows t h e s i g n a l s o b t a i n e d when t h e l e a f was i l l u m i n a t e d w i t h broad-band w h i t e l i g h t d u r i n g t h e same sequence o f a t m o s p h e r i c c o n d i t i o n s and s u g g e s t s a d e c r e a s e i n b o t h S i g n a l I I U + S and t h e m i n i m a l w h i t e l i g h t - i n d u c e d S i g n a l I . I n o r d e r t o r e v e a l t h e changes i n S i g n a l I more c l e a r l y , t h e d a r k s i g n a l s r e c o r d e d a t t h e a p p r o p r i a t e t i m e s were s u b t r a c t e d -81- J I I I L J I I I L 3236 3276 3236 3276 M a g n e t i c f i e l d (Gauss) M a g n e t i c f i e l d (Gauss) F i g u r e 20. Combined S i g n a l s I and H u + S from an a t t a c h e d , i n t a c t K entucky b l u e g r a s s l e a f . a. i n a i r ; b. i n N 2 f o r 30 m i n u t e s ; c. i n N 2 , 12 minutes a f t e r 20b; t h e l e a f was i n d a r k n e s s f o r 4 m i n u t e s and w h i t e l i g h t f o r 4 m i n u t e s i n t h e 12 minute i n t e r v a l ; d. i n N,, 60 m i n u t e s a f t e r 2 0c. The l e a f was h e l d i n 710nm l i g h t t h r o u g h o u t e x c e p t f o r t h e r e c o r d i n g o f t h e d a r k and w h i t e l i g h t - i n d u c e d s i g n a l s shown i n F i g u r e s 21b and 22b, r e s p e c t i v e l y . These were r e c o r d e d i n t h e i n t e r v a l between b and c o f t h i s f i g u r e . Microwave f r e q u e n c y - 9.190. -82- 10 G b 3236 3276 M a g n e t i c f i e l d (Gauss) F i g u r e 21. S i g n a l H u + S from an i n t a c t , a t t a c h e d Kentucky b l u e g r a s s l e a f . a. i n a i r ; b. i n N, f o r 36 m i n u t e s ; c. i n N 2 f o r 96 m i n u t e s . The l e a f was h e l d i n 710nm l i g h t d u r i n g t h e e x p e r i m e n t e x c e p t f o r t h e t i m e s when t h e t r a c e s shown i n b (above) and F i g u r e 22b were r e c o r d e d . I n t h i s f i g u r e , a and c were r e c o r d e d p r i o r t o , and a f t e r , exposure t o t h e 7l0nm l i g h t , r e s p e c t i v e l y . Microwave f r e q u e n c y - 9.190. -83- 10 G 3236 3276 M a g n e t i c f i e l d (Gauss) F i g u r e 22. White l i g h t - i n d u c e d s i g n a l from an a t t a c h e d , i n t a c t K e ntucky b l u e g r a s s l e a f . a. i n a i r ; b. i n N 2 f o r 42 m i n u t e s ; c. i n N 2 f o r 102 m i n u t e s . The l e a f was h e l d i n 710 l i g h t t h r o u g h o u t , e x c e p t f o r t h e t i m e s when t h e t r a c e s shown i n b (above) and F i g u r e 21b were r e c o r d e d . I n t h i s f i g u r e , a and c were r e c o r d e d p r i o r t o , and a f t e r , e x p o sure t o 90 m i n u t e s o f 7l0nm l i g h t . Microwave f r e q u e n c y - 9.190. -84- from t h e c o m p o s i t e s i g n a l as shown i n F i g u r e 23. S p e c i f i c a l l y , F i g u r e 23a was o b t a i n e d by s u b t r a c t i n g 21a from 20a, F i g u r e 23b by t h e s u b t r a c t i o n o f 21b from 20b and F i g u r e 23c by t h e s u b t r a c t i o n o f 21c from 2 0d. The s i g n a l s shown i n F i g u r e s 23a-23c a l l have t h e peak-to-peak l i n e w i d t h o f 7.5 gauss and g - v a l u e o f 2.0025 which i s a s s o c i a t e d w i t h S i g n a l I (see A p pendix B ) . Thus, i t i s a p p a r e n t t h a t once S i g n a l I has been formed under c o n d i t i o n s o f c l e a n a i r and 710nm l i g h t , m i n i m a l decay o c c u r s when t h e oxygen i s r e p l a c e d by n i t r o g e n ( F i g u r e 23b) f o r 30 m i n u t e s . However, i f t h e l i g h t i s t h e n b r i e f l y t u r n e d o f f , t h e n t h e s i g n a l i s not r e s t o r e d w i t h i n 4 m i n u tes o f e xposure t o 710nm l i g h t as p r e v i o u s l y shown. Hence t h e r e i s e v i d e n c e f o r some oxygen-dependence o f t h e f o r m a t i o n o f t h e s p e c i e s r e p r e s e n t e d by t h e s i g n a l s i n c e a f t e r 1 a d d i t i o n a l hour o f e x p osure t o 7l0nm l i g h t under n i t r o g e n o n l y a p p r o x i m a t e l y 4 0 p e r c e n t o f S i g n a l I i s r e s t o r e d ( F i g u r e 2 3 c ) . F i g u r e 24a shows t h e p o r t i o n o f S i g n a l I I U + S w h i c h i s l o s t as a r e s u l t o f t h e s u b s t i t u t i o n o f t h e N 2 f o r a i r . T h i s was o b t a i n e d by s u b t r a c t i n g F i g u r e 21c from F i g u r e 21a. F i g u r e 24b shows t h e d i f f e r e n c e s i g n a l o b t a i n e d under broad-band w h i t e l i g h t . T h i s was o b t a i n e d by s u b t r a c t i n g F i g u r e 22c from F i g u r e 22a. However, t h i s change i s a c o m p o s i t e o f t h e d i f f e r e n c e w h i c h o c c u r s i n d a r k n e s s and t h e d i f f e r e n c e i n t h e w h i t e l i g h t - i n d u c e d component. T h e r e f o r e , t o o b t a i n t h e r e a l d i f f e r e n c e i n t h e w h i t e l i g h t component i t i s n e c e s s a r y t o -85- 10 G M a g n e t i c f i e l d (Gauss) F i g u r e 23. True 710nm l i g h t - i n d u c e d s i g n a l ( S i g n a l I ) from an i n t a c t , a t t a c h e d Kentucky b l u e g r a s s l e a f . a. i n a i r ; b. i n N 2 f o r 30 m i n u t e s ; c. i n N 2 f o r 90 m i n u t e s . A l l f i g u r e s were o b t a i n e d by s u b t r a c t i n g t h e dark s i g n a l from t h e combined s i g n a l o b t a i n e d i n 710nm l i g h t . Microwave f r e q u e n c y - 9.190. -86- g=2.0025 3236 3276 M a g n e t i c f i e l d (Gauss) F i g u r e 24. Changes i n t h e d a r k and w h i t e l i g h t - i n d u c e d s i g n a l s i n an i n t a c t , a t t a c h e d Kentucky b l u e g r a s s l e a f caused by removal o f 0 2 . a. Loss o f S i g n a l H u + S caused by 0 2 removal ( O r i g i n a l d a r k s i g n a l - d a r k s i g n a l a f t e r 90 m i n u t e s N 2 ) ; b. d i f f e r e n c e i n t h e w h i t e l i g h t - i n d u c e d s i g n a l a f t e r removal o f 0 2 ( O r i g i n a l w h i t e l i g h t i n d u c e d s i g n a l a f t e r 90 m i n u t e s N 2 ; c. True d i f f e r e n c e i n w h i t e l i g h t - i n d u c e d s i g n a l caused by removal o f 0 2 ( F i g u r e 24b-24a); d. True w h i t e l i g h t - i n d u c e d s i g n a l p r i o r t o removal o f 0 2 ( O r i g i n a l w h i t e l i g h t - i n d u c e d s i g n a l - o r i g i n a l d a r k s i g n a l ) . Microwave f r e q u e n c y - 9.190. -87- s u b t r a c t F i g u r e 24a from F i g u r e 24b. The r e s u l t o f t h i s s u b t r a c t i o n i s shown i n F i g u r e 24c. T h i s d i f f e r e n c e s i g n a l i s n e a r l y i d e n t i c a l t o t h e d i f f e r e n c e s i g n a l o b t a i n e d when t h e o r i g i n a l d a r k s i g n a l ( F i g u r e 21a) i s s u b t r a c t e d from t h e o r i g i n a l w h i t e l i g h t - i n d u c e d s i g n a l ( F i g u r e 22a) as shown i n F i g u r e 24d. The s m a l l s i g n a l s shown i n F i g u r e s 24c and 24d have g - v a l u e s o f 2.0025 and peak-to-peak l i n e w i d t h s o f 7.5 gauss, i d e n t i c a l t o S i g n a l I o b t a i n e d upon i r r a d i a t i o n w i t h 710nm l i g h t . Thus, t h e use o f t h i s s u b t r a c t i o n t e c h n i q u e i n d i c a t e s t h a t t h e s u b s t i t u t i o n o f N 2 f o r 0 2 causes a 60 p e r c e n t r e d u c t i o n i n S i g n a l I i n d u c e d by 710nm l i g h t , e l i m i n a t e s a l l o f t h e m i n i m a l S i g n a l I i n d u c e d by broad-band w h i t e l i g h t and causes a decay o f a p p r o x i m a t e l y 30 p e r c e n t o f S i g n a l I I U + S . S i g n a l I I U + S decays w h i l e t h e l e a f i s h e l d under 710nm l i g h t b u t S i g n a l I i s o n l y m i n i m a l l y a f f e c t e d d u r i n g t h i s p e r i o d . The i n i t i a l l o s s o f S i g n a l I under 710nm l i g h t i s comparable i n s i z e t o t h e l o s s o f t h e S i g n a l I l o s t under broad-band w h i t e l i g h t . However, once t h e 710nm l i g h t - i n d u c e d S i g n a l I formed under a i r decays i n d a r k n e s s , o n l y 40 p e r c e n t o f i t can be r e s t o r e d i n 710nm l i g h t under N 2 i n t h e n e x t hour. These d a t a s u p p o r t t h e e a r l i e r o b s e r v a t i o n s t h a t S i g n a l I i s heter o g e n e o u s (Bearden and M a l k i n , 1972b) b u t a l s o s u g g e s t t h a t S i g n a l I I U + S i s heterogeneous and has some oxygen dependence. -88- 3.3.5 E f f e c t s o f P h o t o i n h i b i t i o n I n t h e p r e s e n t s t u d y , p r e l i m i n a r y i n v e s t i g a t i o n s showed t h a t l e a v e s o f p l a n t s grown under c o n d i t i o n s o f h i g h photon f l u x d e n s i t y (PFD) d u r i n g t h e summers o f 1986 and 1987 d i d not d i s p l a y t h e t y p i c a l p h o t o s y n t h e t i c s i g n a l s found i n c h l o r o p l a s t s u s p e n s i o n s and l e a v e s grown under l e s s i n t e n s e l i g h t . L i g h t i n t e n s i t y d u r i n g t h i s p e r i o d was c l o s e t o 1500/iE m s i n f u l l s u n l i g h t and j u s t under 465/^tE m s i n t h e greenhouse. T h i s c o n f o u n d i n g f a c t o r was e l i m i n a t e d by s u b s e q u e n t l y g r o w i n g new e x p e r i m e n t a l p l a n t s d u r i n g t h e summer under t h e greenhouse benches a t a reduced l i g h t i n t e n s i t y o f a p p r o x i m a t e l y 40JZE m ~ 2 s - 1 . However, l e a v e s o f p l a n t s grown i n h i g h PFD were s t u d i e d i n o r d e r t o i n c r e a s e t h e c u r r e n t body o f i n f o r m a t i o n p e r t a i n i n g t o p h o t o i n h i b i t o r y e f f e c t s o f i n t e n s e s u n l i g h t . When C-3 p l a n t s grown i n f u l l summer s u n l i g h t o r i n t h e greenhouse a t a l i g h t i n t e n s i t y r e a c h i n g 4 65JIE m " s x were used, e x c i s e d p i e c e s o f r a d i s h , p e r e n n i a l r y e g r a s s o r Kentucky b l u e g r a s s l e a v e s and i n t a c t , a t t a c h e d l e a v e s o f t h e g r a s s e s were found t o y i e l d a l a r g e EPR s i g n a l i n d a r k n e s s and when i r r a d i a t e d w i t h 710nm o r w h i t e l i g h t . Examples o f t h i s s i g n a l a r e p r e s e n t e d i n F i g u r e s 25 and 26, f o r Kentucky b l u e g r a s s and p e r e n n i a l r y e g r a s s , r e s p e c t i v e l y . These f i g u r e s i n d i c a t e two t y p e s o f r e s p o n s e , depending upon t h e l e a f , r e g a r d l e s s o f s p e c i e s . The t o p s p e c t r a i n each f i g u r e i n d i c a t e t h e p r e s e n c e -89- 10 G F i g u r e 25. Examples o f t h e f r e e - r a d i c a l s i g n a l found i n Kentucky b l u e g r a s s l e a v e s o f p l a n t s grown under h i g h photon f l u x d e n s i t y . a. d a r k n e s s ; b. i n 710nm l i g h t ; c. i n w h i t e l i g h t . T o p = t y p i c a l r e s p o n s e o f l e a v e s from p l a n t s grown i n greenhouse; B o t t o m = t y p i c a l r e s p o n s e o f l e a v e s from p l a n t s grown i n f u l l s u n l i g h t . -90- F i g u r e 26. Examples o f t h e f r e e - r a d i c a l s i g n a l found i n l e a v e s o f p e r e n n i a l r y e g r a s s p l a n t s grown under h i g h photon f l u x d e n s i t y . a. d a r k n e s s ; b. i n 710nm l i g h t ; c. i n w h i t e l i g h t . T o p = t y p i c a l r e s p o n s e o f l e a v e s from p l a n t s grown i n greenhouse; B o t t o m = t y p i c a l r e s p o n s e o f l e a v e s from p l a n t s grown i n f u l l s u n l i g h t . -91- o f a new s i g n a l ( S i g n a l Npj) i n a d d i t i o n t o t h e p r e s e n c e o f t h e e s t a b l i s h e d S i g n a l I and S i g n a l I I U + S . T h i s was t h e t y p i c a l r e s p o n s e i n l e a v e s grown on t h e greenhouse benches. The bottom s p e c t r a o f each f i g u r e i n d i c a t e t h e p r e s e n c e o f S i g n a l I I U + S and t h e new S i g n a l N p i b u t a l s o i n d i c a t e t h a t S i g n a l I , formed i n 710nm l i g h t , has not been formed, s i n c e s i g n a l s i n a l l l i g h t regimes a r e i d e n t i c a l . T h i s was t h e t y p i c a l r e s p o n s e i n l e a v e s grown o u t s i d e i n f u l l s u n l i g h t . W h i l e t h e s e were t h e most f r e q u e n t r e s p o n s e s o f p l a n t s grown i n t h e greenhouse and o u t s i d e i n f u l l s u n l i g h t , r e s p e c t i v e l y , some l e a v e s o f b o t h s p e c i e s showed t h e o p p o s i t e r e s p o n s e . A p p r o x i m a t e l y 5% o f l e a v e s grown o u t s i d e s t i l l r e v e a l e d t h e p r e s e n c e o f S i g n a l I and a s i m i l a r p e r c e n t a g e o f l e a v e s grown i n t h e greenhouse d i d not r e v e a l t h e f o r m a t i o n o f S i g n a l I i n 710nm l i g h t . The g - v a l u e s o f t h e s e combined s i g n a l s a r e 2.0049 f o r F i g u r e s 25b and 26b (Top s p e c t r a ) and 2.0051 f o r a l l o t h e r s p e c t r a i n t h e s e f i g u r e s . I n a l l l e a v e s o f a C-4 p l a n t , b a r n y a r d g r a s s ( E c h i n o c h l o a c r u s g a l l i (L.) Beauv.), w h i c h was grown i n f u l l s u n l i g h t , S i g n a l N p I was r e v e a l e d and S i g n a l I was not formed i n 710nm l i g h t ( F i g u r e 2 7 ) . The g - v a l u e o f t h e combined s i g n a l i n b a r n y a r d g r a s s l e a v e s was found t o be 2.0051. F u r t h e r m o r e , once i n d u c e d , t h i s new s i g n a l i s l o n g - l i v e d , as l e a v e s o f p l a n t s o r i g i n a l l y grown under h i g h PFD, w h i c h were moved under greenhouse benches o r t o t h e s p e c t r o m e t r y l a b o r a t o r y f o r p e r i o d s o f up t o e i g h t days, s t i l l r e v e a l e d t h i s s i g n a l , as 10 G F i g u r e 27. Examples o f t h e f r e e - r a d i c a l s i g n a l found i n l e a v o f b a r n y a r d g r a s s grown i n f u l l summer s u n l i g h t . a. d a r k n e s s ; b. i n 710nm l i g h t ; c. i n w h i t e l i g h t . -93- shown i n F i g u r e 28. S i m i l a r l y , i f l e a f p i e c e s from p l a n t s grown under h i g h PFD were e x c i s e d and s t o r e d i n w a t e r f o r e i g h t days, S i g n a l N p I was s t i l l p r e s e n t ( d a t a n o t p r e s e n t e d ) . I n most l e a v e s , t h e s i g n a l was r a p i d l y e l i m i n a t e d upon ex p o s u r e o f t h e l e a f t o microwave r a d i a t i o n i n t h e s p e c t r o m e t e r c a v i t y . The k i n e t i c s o f t h e s i g n a l decay were found t o v a r y somewhat, from l e a f t o l e a f , b u t a t y p i c a l example i s shown i n F i g u r e 29. These k i n e t i c s , w hich a r e independent o f c a v i t y l i g h t , were o b t a i n e d by l o c k i n g t h e s p e c t r o m e t e r onto t h e low f i e l d maximum o f t h e s i g n a l shown i n F i g u r e s 25-28. I n i t i a l l y , t h e r e i s a s m a l l i n c r e a s e i n S i g n a l N p I , b u t w i t h i n two min u t e s a maximum h e i g h t i s a c h i e v e d and decay s e t s i n . I n n e a r l y a l l l e a v e s grown i n t h e greenhouse (see below) t h e s i g n a l was found t o d i s a p p e a r w i t h i n 15 mi n u t e s o f exposure t o microwave r a d i a t i o n . The k i n e t i c t r a c e shown i n F i g u r e 29 was o b t a i n e d w i t h a Kentucky b l u e g r a s s l e a f i r r a d i a t e d w i t h 710nm l i g h t b u t t h e k i n e t i c s o f t h e r i s e and decay o f S i g n a l N p i were n o t a f f e c t e d by t h e i r r a d i a t i o n o f t h e l e a f t h r o u g h t h e c a v i t y p o r t as s i m i l a r r e s u l t s were o b t a i n e d i n 7l0nm l i g h t , i n 650nm l i g h t , i n broad-band w h i t e l i g h t and i n d a r k n e s s . A r e p r e s e n t a t i v e example o f t h e a b s o l u t e s i z e o f t h e s i g n a l i n Kentucky b l u e g r a s s l e a v e s h e l d i n d a r k n e s s d u r i n g exposure t o microwave r a d i a t i o n i n t h e c a v i t y f o r v a r i o u s t i m e p e r i o d s i s shown i n F i g u r e 30. -94- 10 G F i g u r e 28. Examples o f t h e f r e e - r a d i c a l s i g n a l found i n l e a v e s o f K e n t u c k y b l u e g r a s s p l a n t s grown under h i g h p h o t o n f l u x d e n s i t y 8 days a f t e r t h e p l a n t s were moved t o t h e s p e c t r o m e t r y l a b o r a t o r y , a. i n d a r k n e s s ; b. i n 710nm l i g h t . -95- 15 I I I I 3 0 t i m e (min) F i g u r e 29. Decay o f t h e f r e e - r a d i c a l s i g n a l found i n Kentucky b l u e g r a s s l e a v e s grown i n c o n d i t i o n s o f h i g h photon f l u x d e n s i t y , upon exposure t o microwave r a d i a t i o n i n t h e s p e c t r o m e t e r c a v i t y . -96- 10 G F i g u r e 30. S p e c t r a o f t h e f r e e - r a d i c a l s i g n a l found i n Kentucky b l u e g r a s s l e a v e s grown under h i g h photon f l u x d e n s i t y a f t e r e x p o s u r e t o microwave r a d i a t i o n i n t h e s p e c t r o m e t e r c a v i t y f o r d i f f e r e n t t i m e p e r i o d s i n d a r k n e s s , a. 15 s e c ; b. 2 min; c. 6 min; d. 10 min; e. 15 min; f . 20 min; g. 30 min. -97- However, i f t h e s i g n a l i s r e c o r d e d and t h e microwave r a d i a t i o n i s i m m e d i a t e l y t u r n e d o f f , t h e i d e n t i c a l decay o c c u r s and no s i g n a l i s found when t h e k l y s t r o n i s t u r n e d on a g a i n 15 mi n u t e s l a t e r , as shown i n t h e s p e c t r a i n F i g u r e 31. On t h e o t h e r hand, i f t h e l e a f i s m a i n t a i n e d i n t h e s p e c t r o m e t e r c a v i t y f o r p e r i o d s up t o 2 h o u r s , w i t h o u t t u r n i n g on t h e microwave r a d i a t i o n t o r e c o r d a s i g n a l , t h e s i g n a l shown i n F i g u r e s 25-29 i s s t i l l p r e s e n t . T h i s i s t h e case w i t h o r w i t h o u t t h e magnetic f i e l d t u r n e d on, i n d i c a t i n g t h a t t h e s i g n a l i s not a f f e c t e d by t h e p r e s e n c e o f t h e magnetic f i e l d ( d a t a n o t shown). P h o t o s y n t h e t i c S i g n a l I I U + S , as shown i n F i g u r e s 3Of and 30g, appears t o be u n a f f e c t e d , b u t S i g n a l I may, o r may n o t , be p r e s e n t i n l e a v e s which e x h i b i t t h e new EPR s i g n a l , depending upon t h e l e a f ( F i g u r e s 25,26). The new s i g n a l ( S i g n a l N p i ) can be c h a r a c t e r i z e d as b e i n g s y m m e t r i c a l , w i t h an ap p r o x i m a t e peak-to-peak w i d t h o f 9 gauss and a g - v a l u e o f 2.0056 ( F i g u r e 3 2 c ) . T h i s f i g u r e was o b t a i n e d a f t e r r e d u c t i o n o f S i g n a l N p i by exposure t o microwave r a d i a t i o n i n t h e s p e c t r o m e t e r c a v i t y and s u b t r a c t i o n o f S i g n a l I and S i g n a l H u + S ( F i g u r e 32b) from t h e o r i g i n a l c o m p o s i t e s i g n a l shown i n F i g u r e 32a, w h i c h was r e c o r d e d i n 710nm l i g h t . These parameters do not match t h o s e of any s i g n a l i n t h e r e l e v a n t l i t e r a t u r e . An i d e n t i c a l s i g n a l i s r e v e a l e d i f S i g n a l H u + S i s s u b t r a c t e d from t h e o r i g i n a l c o m p o s i t e s i g n a l r e c o r d e d i n d a r k n e s s o r broad-band w h i t e l i g h t , o r i f S i g n a l I I U + S i s s u b t r a c t e d from s p e c t r a r e c o r d e d i n -98- F i g u r e 31. S p e c t r a o f p e r e n n i a l r y e g r a s s l e a v e s grown under h i g h photon f l u x d e n s i t y . Top - a t s t a r t o f e x p e r i m e n t ; Bottom - 15 m i n u t e s a f t e r t h e t o p s i g n a l s were r e c o r d e d , w i t h t h e k l y s t r o n o f f i n t h e i n t e r v a l , a. i n d a r k n e s s ; b. i n 710nm l i g h t ; c. i n w h i t e l i g h t . 3236 3276 M a g n e t i c f i e l d (Gauss) F i g u r e 32. S i g n a l N p i o f Kentucky b l u e g r a s s l e a v e s exposed t o h i g h p h o t o n f l u x d e n s i t y , a. Composite s i g n a l r e c o r d e d i n 7l0nm l i g h t a t s t a r t o f e x p e r i m e n t ( S i g n a l N p j , S i g n a l I and S i g n a l I I U + S ) ; b. S i g n a l I + S i g n a l H u + S a f t e r e l i m i n a t i o n o f S i g n a l N p i by e x p o s u r e t o 15 m i n u t e s microwave r a d i a t i o n ; c. S i g n a l N p i , o b t a i n e d by s u b t r a c t i n g b from a. Microwave f r e q u e n c y - 9.190. -100- 710nm l i g h t w h i c h do not r e v e a l S i g n a l I ( d a t a n o t shown). A f t e r e x p o sure t o microwave r a d i a t i o n , l e a v e s w h i c h a r e a f f e c t e d by exposure t o h i g h PFD d i f f e r i n t h e i r c a p a c i t y t o r e v e r t t o t h e e s t a b l i s h e d r e s p o n s e s a s s o c i a t e d w i t h t h e r e v e l a t i o n o f p h o t o s y n t h e t i c S i g n a l s I and H u + S under v a r i o u s l i g h t r e g i m e s . The re s p o n s e o f each s p e c i e s v a r i e s , depending upon t h e degree o f damage and i s i l l u s t r a t e d i n F i g u r e s 3 3 and 34 f o r Kentucky b l u e g r a s s . I n extreme c a s e s , S i g n a l N p j cannot be removed, even a f t e r 1 hour o f exposure t o microwave r a d i a t i o n i n t h e s p e c t r o m e t e r c a v i t y , r e g a r d l e s s o f l i g h t regime ( F i g u r e 33a). T h i s o n l y o c c u r r e d w i t h a few l e a v e s o f p l a n t s grown i n f u l l s u n l i g h t o u t s i d e t h e greenhouse, n e v e r w i t h l e a v e s o f p l a n t s grown i n t h e greenhouse. An a l t e r n a t i v e r e s p o n s e from l e a v e s grown i n f u l l s u n l i g h t i s t h e e l i m i n a t i o n o f S i g n a l N p j , b u t no appearance o f S i g n a l I , e i t h e r under i r r a d i a t i o n w i t h 710nm o r broad-band w h i t e l i g h t ( F i g u r e 3 3b). I n such l e a v e s , S i g n a l I cannot be i n d u c e d even by c o n t i n u o u s i r r a d i a t i o n w i t h 710nm l i g h t f o r p e r i o d s o f up t o 2 h o u r s . T h i s was t h e most p r e v a l e n t r e sponse o f l e a v e s o f b a r n y a r d g r a s s . A t h i r d a l t e r n a t i v e i s shown i n F i g u r e 34a. I n t h i s i n s t a n c e S i g n a l N p i has been e l i m i n a t e d and S i g n a l I i s formed upon i r r a d i a t i o n w i t h 710nm l i g h t . However, S i g n a l I , as i n d i c a t e d by i t s g - v a l u e and peak-to-peak l i n e w i d t h , i s a l s o formed upon i r r a d i a t i o n w i t h broad-band w h i t e l i g h t . The f o u r t h -101- D 710 W a b F i g u r e 33. A l t e r n a t i v e r e s p o n s e s o f Ken t u c k y b l u e g r a s s l e a v e s c o n t a i n i n g S i g n a l N p i a f t e r exposure t o microwave r a d i a t i o n i n t h e s p e c t r o m e t e r c a v i t y . a. r e t e n t i o n o f S i g n a l N p i ; b. e l i m i n a t i o n o f S i g n a l N p j , and a l a c k o f g e n e r a t i o n o f S i g n a l I i n 710nm l i g h t . D=darkness; 710=in 710nm l i g h t ; W=in broad-band w h i t e l i g h t . -102- D 710 W a b ' F i g u r e 34. A l t e r n a t i v e r e s p o n s e s o f Ken t u c k y b l u e g r a s s l e a v e s c o n t a i n i n g S i g n a l N p j a f t e r e xposure t o microwave r a d i a t i o n i n t h e s p e c t r o m e t e r c a v i t y . a. e l i m i n a t i o n o f S i g n a l N p i and f o r m a t i o n o f S i g n a l I i n w h i t e l i g h t ; b. t y p i c a l S i g n a l I and I I U + S a s s o c i a t e d w i t h l e a v e s , c h l o r o p l a s t p r e p a r a t i o n s and r e a c t i o n c e n t r e p r e p a r a t i o n s n o t exposed t o h i g h photon f l u x d e n s i t y . D=darkness; 710=in 710nm l i g h t ; W=in broad-band w h i t e l i g h t . -103- p o s s i b l e r e s p o n s e i s shown i n F i g u r e 34b as a r e t u r n t o t h e s i g n a l s found i n h e a l t h y l e a v e s w i t h t h e f o r m a t i o n o f S i g n a l I upon i r r a d i a t i o n w i t h 710nm l i g h t , b u t not upon i r r a d i a t i o n w i t h broad-band w h i t e l i g h t . T h i s l a t t e r r e s p o n s e was t h e most p r e v a l e n t i n Kentucky b l u e g r a s s l e a v e s o f p l a n t s grown i n t h e greenhouse, w h i l e t h e a d d i t i o n a l f o r m a t i o n o f S i g n a l I under broad-band w h i t e l i g h t was t h e most common r e s p o n s e o f p e r e n n i a l r y e g r a s s p l a n t s grown i n t h e greenhouse. 3.4 D i s c u s s i o n The r e s u l t s p r e s e n t e d i l l u s t r a t e t h e c a p a b i l i t y o f t h e t e c h n i q u e d e v e l o p e d t o d e t e r m i n e f r e e r a d i c a l s i n s i t u i n l e a f t i s s u e s . The methodology e n a b l e s t h e i n v e s t i g a t o r t o f o l l o w changes i n EPR s i g n a l s r e s u l t i n g from exposure t o p o l l u t a n t g a s e s , from o t h e r changes i n gas c o m p o s i t i o n o r from o t h e r t r e a t m e n t s . The t e c h n i q u e i l l u s t r a t e d i n t r a c k i n g changes i n S i g n a l I , a s s o c i a t e d w i t h Photosystem I , can a l s o be used t o f o l l o w changes i n S i g n a l I I U + S , a s s o c i a t e d w i t h Photosystem I I , and t h e u b i q u i t o u s M n + + s i g n a l i f t h e u n d e r l y i n g F e + + s i g n a l i s u n a f f e c t e d by t h e t r e a t m e n t . The appearance o f new f r e e - r a d i c a l s i g n a l s can be m o n i t o r e d by f o l l o w i n g t h e k i n e t i c s o f s i g n a l appearance d i r e c t l y , i f p r e l i m i n a r y s t u d i e s have shown t h a t o n l y one s i g n a l i s a f f e c t e d . I f two s i g n a l s , w h i c h respond t o d i f f e r e n t l i g h t r e g i m e s , a r e found t o o v e r l a p , t h e n b o t h s i g n a l s can be r e c o r d e d s e q u e n t i a l l y under t h e i r r e s p e c t i v e -104- l i g h t r egimes a f t e r v a r i o u s f u m i g a t i o n p e r i o d s . Subsequent s u b t r a c t i o n o f t h e s i g n a l s o b t a i n e d a t d i f f e r e n t t i m e s w i l l r e v e a l new EPR s i g n a l s a t t r i b u t a b l e t o t h e e f f e c t s o f t r e a t m e n t . The problems a s s o c i a t e d w i t h t h e d i f f e r e n c e s i n i n h e r e n t s i g n a l i n t e n s i t y from l e a f t o l e a f a r e m i n i m i z e d by s u b t r a c t i n g o r i g i n a l s i g n a l s found i n l e a v e s p r i o r t o f u m i g a t i o n from t h o s e found i n t h e same l e a v e s a f t e r f u m i g a t i o n , i n o r d e r t o o b s e r v e t h e r e l a t i v e changes i n t h e s i g n a l s . F u r t h e r m o r e , i f d i f f e r e n t changes i n f r e e - r a d i c a l s i g n a l appearance o c c u r i n d a r k n e s s t h a n i n l i g h t , t h e t r u e l i g h t - i n d u c e d f r e e - r a d i c a l s i g n a l can be d e t e r m i n e d by m u l t i p l e s u b t r a c t i o n s . The s m a l l d i f f e r e n c e s i n g - v a l u e s and peak-to-peak w i d t h s o f t h e v a r i o u s s i g n a l s r e q u i r e i n s t r u m e n t p r e c i s i o n and a c c u r a t e r e c o r d i n g because t h e d i f f e r e n c e s i n t h e s e parameters a r e t h e b a s i s upon w h i c h s i g n a l s a r e d i f f e r e n t i a t e d . The s p e c t r o m e t e r p r e c i s i o n i s a t t e s t e d t o by t h e f a c t t h a t t h a t s e v e r a l hundred 710nm l i g h t - i n d u c e d s i g n a l s ( S i g n a l I) were a l l found t o have a g - v a l u e o f 2.0025, w i t h an e r r o r range o f 0.0002. T h i s e r r o r i s i d e n t i c a l t o t h e measurement e r r o r a s s o c i a t e d w i t h t h e s t a n d a r d used f o r co m p a r i s o n , DPPH. O p e r a t o r e r r o r i s l i m i t e d t o f a u l t y p lacement o f t h e r e c o r d i n g paper on t h e r e c o r d e r . A l l s i g n a l s d i s c u s s e d i n t h i s t h e s i s were r e c o r d e d a t l e a s t 8 t i m e s , and y i e l d e d v i r t u a l l y i d e n t i c a l g - v a l u e s and l i n e w i d t h s . -105- S t u d i e s i n v o l v i n g f u m i g a t i o n o f e x c i s e d l e a v e s have t o be l i m i t e d t o p e r i o d s o f between 1 and 1.5 h o u r s because o f t h e e f f e c t s o f e x c i s i o n (wounding), w h i c h r e s u l t i n changes i n f r e e - r a d i c a l s i g n a l s i n t h e v i c i n i t y o f t h e 2.000 g - v a l u e a s s o c i a t e d w i t h p h o t o s y n t h e t i c s i g n a l s . However, t h i s problem i s l a r g e l y overcome t h r o u g h t h e use o f i n t a c t , a t t a c h e d g r a s s l e a v e s w h i c h can be p l a c e d i n t h e c a v i t y w i t h o u t wounding. The g r a s s l e a v e s can be h e l d i n a i r i n t h e s p e c t r o m e t e r c a v i t y f o r 72 h o u r s w i t h o u t t h e f o r m a t i o n o f new f r e e - r a d i c a l s i g n a l s . T h i s a l l o w s t h e use o f r e a l i s t i c c o n c e n t r a t i o n s o f p o l l u t a n t s w h i l e m o n i t o r i n g f r e e r a d i c a l changes o v e r l o n g e r p e r i o d s o f t i m e . A l t h o u g h d e v e l o p e d s p e c i f i c a l l y t o p e r m i t i n v e s t i g a t i o n o f t h e e f f e c t o f gaseous a i r p o l l u t a n t s on f r e e r a d i c a l f o r m a t i o n i n i n t a c t l e a v e s , t h e method c o u l d be used t o i n v e s t i g a t e changes r e s u l t i n g from o t h e r p h y s i c a l o r c h e m i c a l t r e a t m e n t s . The permanence o f any changes i n d u c e d by t r e a t m e n t s can a l s o be i n v e s t i g a t e d because t h e s i g n a l s can be o b s e r v e d a t v a r i o u s t i m e p e r i o d s a f t e r c e s s a t i o n o f t r e a t m e n t . The method c o u l d a l s o p e r m i t t h e d e t e c t i o n o f t r a n s i e n t f r e e r a d i c a l s , such t h e s u p e r o x i d e and h y d r o x y l f r e e r a d i c a l s , i f a p p r o p r i a t e s p i n t r a p s c o u l d be i n c o r p o r a t e d i n t o e x c i s e d p l a n t l e a f t i s s u e . The d a t a p r e s e n t e d a l s o s u p p o r t t h e c o n t e n t i o n t h a t f r e e r a d i c a l f o r m a t i o n i s a c o n t r i b u t o r y f a c t o r i n p h o t o i n h i b i t i o n . -106- U n f o r t u n a t e l y , t h e f r e e r a d i c a l ( S i g n a l N p i ) formed by t h e exp o s u r e t o i n t e n s i v e l i g h t cannot be i d e n t i f i e d by EPR c h a r a c t e r i s t i c s a l o n e . The d i f f e r e n t i a l r e s p o n s e a f t e r l e a v e s have been exposed t o microwave r a d i a t i o n i n t h e c a v i t y i s i n d i c a t i v e o f t h e degree o f damage s u f f e r e d by t h e p l a n t . P l a n t l e a v e s w h i c h e i t h e r r e t a i n S i g n a l N p i , o r show no S i g n a l I a f t e r e l i m i n a t i o n o f S i g n a l N p i , have been s e v e r e l y damaged and a r e not f u n c t i o n a l i n t h e p h o t o s y n t h e t i c p r o c e s s a t t h a t t i m e . D e f i n i t e damage t o Photosystem I i s i n d i c a t e d by t h e l a c k o f S i g n a l I f o r m a t i o n upon exposure t o 710nm l i g h t . Photosystem I I may, o r may not be, damaged a l s o , b u t t h i s can not be a s c e r t a i n e d i n t h e absence o f S i g n a l I f o r m a t i o n . S i g n a l I I U + S i s u n a f f e c t e d by t h e f o r m a t i o n o f S i g n a l N p i , b u t o t h e r components o f Photosystem I I may be damaged by t h e p r e s e n c e o f t h e f r e e r a d i c a l r e v e a l e d by t h e p r e s e n c e o f S i g n a l N p i . However, i t i s n e c e s s a r y t o a s c e r t a i n t h e r e s p o n s e o f S i g n a l I t o d i f f e r e n t i r r a d i a t i o n r e g imes b e f o r e damage t o Photosystem I I can be p o s t u l a t e d . As t h e r e i s no f o r m a t i o n o f S i g n a l I , even i n 710nm l i g h t , i n t h e above c a s e s , c o n s i d e r a t i o n o f i n t e r r u p t i o n o f e l e c t r o n t r a n s f e r from P S I I t o PSI would o n l y be s p e c u l a t i o n . Damage t o t h e s e l e a v e s has a p p a r e n t l y p a s s e d t h e r e v e r s i b l e p h o t o i n h i b i t o r y s t a g e and may have r e a c h e d t h e p h o t o o x i d a t i v e s t a g e l e a d i n g t o d e a t h . -107- Damage t o Photosystem I I , o r t h e e l e c t r o n t r a n s p o r t c h a i n , i s i n d i c a t e d i n l e a v e s i n w h i c h S i g n a l N p i decays upon ex p o s u r e t o microwave r a d i a t i o n , b u t which a l s o r e v e a l t h e f o r m a t i o n o f S i g n a l I upon i r r a d i a t i o n w i t h broad-band w h i t e l i g h t . The f o r m a t i o n o f t h e w h i t e l i g h t - i n d u c e d S i g n a l I i n d i c a t e s t h a t t h e e l e c t r o n t r a n s f e r from P S I I t o PSI n o r m a l l y a s s o c i a t e d w i t h exposure t o w h i t e l i g h t has been i n t e r r u p t e d and t h e r e d u c t i o n o f t h e l i g h t - o x i d i z e d c h l o r o p y l l a f r e e r a d i c a l g i v i n g r i s e t o S i g n a l I i s no l o n g e r o c c u r r i n g . T h e r e f o r e , e i t h e r P S I I has been damaged o r t h e e l e c t r o n t r a n s p o r t c h a i n i s b l o c k e d . The f i n d i n g s from t h e s e s t u d i e s a r e c o n s i s t e n t w i t h t h e s u g g e s t i o n s t h a t p h o t o i n h i b i t i o n p r e c e d e s p h o t o o x i d a t i o n , w i t h t h e l a t t e r p r o c e s s r e s u l t i n g i n d e a t h o f t h e l e a f (Kok e t a l . 1965). The f l u o r e s c e n c e s t u d i e s which i n d i c a t e d t h a t P S I I i s c o n s i s t e n t l y damaged by p h o t o i n h i b i t i o n (Bjorkman, 1968; Powles and Bjorkman, 198 3) cannot be c o n f i r m e d by EPR s p e c t r o m e t r y a l o n e , but t h e s t u d i e s which i n d i c a t e d t h a t PSI may a l s o be damaged (Bjorkman, 1968; C r i t c h l e y , 1981) have been c o n f i r m e d . The s i t e o f P S I I damage can not be a s c e r t a i n e d from EPR d a t a but d a t a showing e l i m i n a t i o n o f S i g n a l I a r e h i g h l y s u p p o r t i v e of e a r l i e r d a t a w h i c h i n d i c a t e d t h a t t h e s i t e o f PSI i n h i b i t i o n i s v e r y c l o s e t o t h e r e a c t i o n c e n t r e o f PSI ( S atoh, 1970b). The new u n i d e n t i f i e d f r e e r a d i c a l , d e p i c t e d as S i g n a l N p i , -108- may be t h e ' f l u o r e s c e n c e quencher' d i s c o v e r e d by Den Haan e t a l . (1973) and s u b s e q u e n t l y p o s t u l a t e d t o be t h e major cause o f p h o t o i n h i b i t i o n ( C l e l a n d and C r i t c h l e y , 1985). However, t h e r o l e o f t h e f r e e r a d i c a l g i v i n g r i s e t o S i g n a l N p I i s c u r r e n t l y unknown and b e f o r e t h i s can be c l a r i f i e d i t i s f i r s t n e c e s s a r y t o i d e n t i f y t h e f r e e r a d i c a l shown i n F i g u r e 32c as S i g n a l N p i . The g r e a t e r and sometimes permanent damage s u f f e r e d by l e a v e s on p l a n t s grown i n f u l l s u n l i g h t c o n f i r m s t h a t p h o t o i n h i b i t o r y damage i s dependent upon exposure ( C r i t c h l e y , 1981). The d i f f e r i n g r e s p o n s e s i n f r e e r a d i c a l f o r m a t i o n i n Kentucky b l u e g r a s s and p e r e n n i a l r y e g r a s s l e a v e s upon exposure t o microwave r a d i a t i o n s u g g e s t t h a t Kentucky b l u e g r a s s i s more t o l e r a n t t o i n t e n s e l i g h t t h a n p e r e n n i a l r y e g r a s s . -109- 4.0 OZONE STUDIES 4.1 I n t r o d u c t i o n The e f f e c t o f ozone on v e g e t a t i o n ranges from v i s i b l e i n j u r y w h i c h can l e a d t o n e c r o s i s ( C o s t o n i s , 1970), t o l e s s o b v i o u s changes i n t h e m e t a b o l i c p r o c e s s e s o f p l a n t s (Mudd e t a l . 1969,1971; F r e d e r i c k and Heath, 1974; T i n g e y , 1974). P l a n t p h y s i o l o g y and b i o c h e m i s t r y can be a f f e c t e d w i t h c o n c o m i t a n t r e d u c t i o n s i n p l a n t growth ( F l a g l e r and Youngner, 1982a, 1982b). Exposure t o ozone causes r e d u c t i o n s i n p h o t o s y n t h e s i s ( C o u l s o n and Heath, 1974) and a l t e r s p h o t o s y n t h a t e p o o l s ( M c L a u g h l i n and McConathy, 1983) and p a r t i t i o n i n g among p l a n t organs (Oshima e t a l . 1978). The degree o f damage i s a f u n c t i o n o f g e n e t i c ( W i l t o n e t a l . 1972) and e n v i r o n m e n t a l f a c t o r s (Heck and Dunning, 1967). F r e e r a d i c a l s have been i m p l i c a t e d i n ozone t o x i c i t y i n p l a n t systems (Rowlands e t a l . 1970; Asada e t a l . 1977). These may be formed by ozone d e c o m p o s i t i o n i n aqueous systems o r by oxygen r e d u c t i o n i n t h e c h l o r o p l a s t s (Weiss, 1935; M e h l e r , 1951). The p r e s e n t s t u d y was u n d e r t a k e n t o i n v e s t i g a t e t h e i n s i t u changes i n f r e e r a d i c a l appearance and decay w h i c h o c c u r when p l a n t l e a v e s a r e f u m i g a t e d w i t h d i f f e r e n t l e v e l s o f ozone. -110- 4.2 Methods P l a n t m a t e r i a l s , EPR s p e c t r o m e t r y and t h e l e a f h o l d e r were as d e s c r i b e d e a r l i e r ( S e c t i o n 3.2). P r e l i m i n a r y i n v e s t i g a t i o n s w i t h e x c i s e d p i e c e s o f l e a v e s i n d i c a t e d t h a t no changes i n f r e e r a d i c a l f o r m a t i o n o c c u r r e d upon exposure t o low and i n t e r m e d i a t e l e v e l s o f ozone (up t o 2 50ppb) d u r i n g t i m e p e r i o d s o f up t o one hour. T h e r e f o r e , a l l f u r t h e r s t u d i e s i n v o l v e d t h e use o f 10-12 week o l d i n t a c t , a t t a c h e d Kentucky b l u e g r a s s and p e r e n n i a l r y e g r a s s l e a v e s . Ozone was g e n e r a t e d by p a s s i n g m e d i c a l grade a i r a t a r a t e o f 2 1/min t h r o u g h a c h a r c o a l f i l t e r , and t h e n t h r o u g h a D a s i b i ozone g e n e r a t o r . Ozone l e v e l s were m o n i t o r e d w i t h a D a s i b i Model 1003AX m o n i t o r . P l a n t s were moved i n t o t h e s p e c t r o m e t r y l a b o r a t o r y 2 ho u r s p r i o r t o t h e s t a r t o f any t r e a t m e n t . The room l i g h t was on d u r i n g t h i s p e r i o d . P r e v i o u s l i g h t a d a p t a t i o n e f f e c t s (Tikhonov and Ruuge, 1975a) were t h e n s t a n d a r d i z e d by e x p o s i n g t h e l e a v e s t o 30 seconds o f 710nm l i g h t f o l l o w e d by 3 minutes o f d a r k n e s s i n t h e c a v i t y . 4.3 R e s u l t s R e s u l t s were s i m i l a r w i t h b o t h g r a s s e s . Hence, t h e m a j o r i t y o f d a t a p r e s e n t e d h e r e a r e from e x p e r i m e n t s w i t h K e n t u c k y b l u e g r a s s l e a v e s , s i n c e t h e y e x h i b i t l e s s i n t e r f e r e n c e from t h e Mn and Fe s i g n a l s d i s c u s s e d e a r l i e r ( S e c t i o n 2.4). 4.3.1 E f f e c t s o f Low L e v e l s o f 0 3 When l e a v e s were f u m i g a t e d a t low l e v e l s o f 0 3 , (up t o 80ppb) f o r p e r i o d s o f t i m e up t o 8 h o u r s , no changes o c c u r r e d i n t h e p h o t o s y n t h e t i c EPR s i g n a l s , and no new EPR f r e e - r a d i c a l s i g n a l s were e v i d e n t . The s m a l l f r e e r a d i c a l ( S i g n a l N 7 1 0 ) whi c h i s formed under 710nm l i g h t i s i n d u c e d by t h e c o n t i n u a l e xposure t o 710nm l i g h t , and i s independent o f ozone t r e a t m e n t . D e t a i l s o f t h i s f r e e r a d i c a l a r e p r e s e n t e d i n S e c t i o n 3.3.4.1. Knowledge o f S i g n a l N 7 1 0 i s c r i t i c a l f o r c o r r e c t i n t e r - p r e t a t i o n o f r e s u l t s w h i c h o c c u r when l e a v e s a r e f u m i g a t e d w i t h h i g h e r l e v e l s o f ozone. 4.3.2 E f f e c t s o f I n t e r m e d i a t e L e v e l s o f 0 3 F u m i g a t i o n o f l e a v e s exposed t o 710nm o r broad-band w h i t e l i g h t w i t h ozone l e v e l s r a n g i n g from 80 t o 250ppb causes changes i n t h e p h o t o s y n t h e t i c f r e e r a d i c a l s i f t h e f u m i g a t i o n i s extended beyond 2 h o u r s . F i g u r e s 35-39 p r e s e n t EPR s i g n a l s from Kentucky b l u e g r a s s l e a v e s f u m i g a t e d w i t h lOOppb 0 3 . F i g u r e 35 shows t h e s i g n a l s p r i o r t o f u m i g a t i o n , when t h e l e a f was h e l d i n t h e d a r k , under 710nm l i g h t and under broad-band w h i t e l i g h t , r e s p e c t i v e l y , a f t e r t h e i n i t i a l s t a n d a r d i z a t i o n p e r i o d . These s p e c t r a show a d e f i n i t e S i g n a l H u + S i n d a r k n e s s ( F i g u r e 35a), -112- 3236 3276 M a g n e t i c f i e l d (Gauss) 10 G J I I I L 3236 3276 M a g n e t i c f i e l d (Gauss) F i g u r e 35. EPR s i g n a l s from an i n t a c t , a t t a c h e d K e n t u c k y b l u e g r a s s l e a f p r i o r t o f u m i g a t i o n . a. i n d a r k ; b. i n 710nm l i g h t ; c. i n w h i t e l i g h t . Microwave f r e q u e n c y - 9.190. -113- a s t r o n g S i g n a l I superimposed upon S i g n a l I I U + S under 710nm l i g h t ( F i g u r e 35b) and a v e r y s m a l l S i g n a l I imposed upon S i g n a l I I U + S under broad-band w h i t e l i g h t ( F i g u r e 3 5 c ) . F i g u r e s 36a and 36c, r e c o r d e d a f t e r 2 hou r s o f f u m i g a t i o n w i t h lOOppb 0 3 , w i t h t h e l e a f h e l d i n 710nm l i g h t d u r i n g t h i s p e r i o d , show t h e appearance o f a new s i g n a l superimposed upon S i g n a l I I U + S , when t h e s p e c t r a were r e c o r d e d i n d a r k n e s s o r under broad-band w h i t e l i g h t , r e s p e c t i v e l y . F i g u r e 3 6b i n d i c a t e s l i t t l e change i n t h e h e i g h t o f t h e s i g n a l r e c o r d e d w i t h t h e l e a f h e l d under 710nm l i g h t a t t h i s t i m e . However, t h e peak-to-peak w i d t h o f t h i s l a t t e r s i g n a l has i n c r e a s e d from 8 gauss t o a p p r o x i m a t e l y 10.5 gauss and t h e g - v a l u e has s h i f t e d t o a p p r o x i m a t e l y 2.0035, s u g g e s t i n g t h a t t h i s , t o o , i s now a composi t e s i g n a l w h i c h i n c l u d e s S i g n a l I I U + S , S i g n a l I and a new f r e e - r a d i c a l s i g n a l . S u b t r a c t i o n o f t h e s p e c t r a i n F i g u r e 35 from t h e a p p r o p r i a t e s p e c t r a i n F i g u r e 36 r e v e a l e d t h e f o r m a t i o n o f a v e r y s m a l l new f r e e - r a d i c a l s i g n a l ( S i g n a l N Q 1 ) h a v i n g a g - v a l u e o f a p p r o x i m a t e l y 2.004 and a peak-to-peak l i n e w i d t h o f a p p r o x i m a t e l y 10 gauss ( d a t a n o t shown). F i g u r e 37 shows t h e s i g n a l s o b t a i n e d when f u m i g a t i o n was c o n t i n u e d f o r one more hour w i t h t h e b l u e g r a s s l e a f s t i l l m a i n t a i n e d under 710nm l i g h t . By t h i s t i m e t h e f r e e - r a d i c a l s i g n a l s o b t a i n e d from t h e l e a f under each l i g h t regime a r e i d e n t i c a l . The 710nm l i g h t - i n d u c e d S i g n a l I has d i s a p p e a r e d and t h e new f r e e - r a d i c a l s i g n a l ( S i g n a l N Q 1 ) w i t h a g - v a l u e o f -114- a M a g n e t i c f i e l d (Gauss) F i g u r e 36. EPR s i g n a l s from an i n t a c t , a t t a c h e d K e n t u c k y b l u e g r a s s l e a f a f t e r e x p o sure t o lOOppb ozone f o r 2 h o u r s . The l e a f was m a i n t a i n e d i n 710nm l i g h t d u r i n g t h e 2 h o u r s o f f u m i g a t i o n . a. i n d a r k ; b. i n 710nm l i g h t ; c. i n w h i t e l i g h t . Microwave f r e q u e n c y - 9.190. 1 3236 3276 M a g n e t i c f i e l d (Gauss) J I I I L 3236 3276 M a g n e t i c f i e l d (Gauss) F i g u r e 37. EPR s i g n a l s from an i n t a c t , a t t a c h e d K e n t u c k y b l u e g r a s s l e a f a f t e r e x p o sure t o lOOppb ozone f o r 3 h o u r s . The l e a f was m a i n t a i n e d i n 710nm l i g h t d u r i n g t h e 3 h o u r s o f f u m i g a t i o n e x c e p t f o r t h e 8 mi n u t e s when t h e s i g n a l s shown i n F i g u r e s 36a and 36c were r e c o r d e d . a. i n d a r k ; b. i n 710nm l i g h t ; c. i n w h i t e l i g h t . Microwave f r e q u e n c y - 9.190. -116- 2.0041 and a peak-to-peak l i n e w i d t h o f 10 gauss i s o b s e r v e d under a l l l i g h t r e g imes. The e x a c t c o n c e n t r a t i o n l e v e l and t i m e p e r i o d needed t o i n d u c e t h e s e changes v a r i e d from l e a f t o l e a f , and even between p a r t s o f any g i v e n l e a f . R y e g r a s s l e a v e s were g e n e r a l l y more s e n s i t i v e and u s u a l l y showed t h e change t o S i g n a l N Q 1 s h o r t l y a f t e r 2 h o u r s o f f u m i g a t i o n a t c o n c e n t r a t i o n s as low as 80ppb 0 3 . Kentucky b l u e g r a s s l e a v e s , on t h e o t h e r hand, g e n e r a l l y r e q u i r e d O 3 l e v e l s o f a t l e a s t lOOppb and a f u m i g a t i o n t i m e p e r i o d o f t e n a p p r o a c h i n g 3 h o u r s . These s i g n a l changes were r e v e r s i b l e once f u m i g a t i o n was t e r m i n a t e d . F i g u r e 38 i n d i c a t e s t h e s i g n a l s o b t a i n e d 20 m i n u tes a f t e r t h e end o f f u m i g a t i o n o f a b l u e g r a s s l e a f . D u r i n g t h i s 20 m inute p e r i o d f i l t e r e d a i r was p a s s e d t h r o u g h t h e c a v i t y and t h e l e a f was m a i n t a i n e d i n 710nm l i g h t . The s i g n a l o b t a i n e d w i t h 710nm l i g h t has r e v e r t e d back t o 8 gauss i n w i d t h and a g - v a l u e o f 2.0025 ( F i g u r e 38b). The s i g n a l s o b t a i n e d i n d a r k n e s s and under broad-band w h i t e l i g h t ( F i g u r e s 38a and 38c) show no s i g n o f t h e 0 3 - i n d u c e d f r e e - r a d i c a l s i g n a l ( S i g n a l N Q 1 ) , b u t a l s o show a somewhat d i s t o r t e d S i g n a l I I U + S when compared t o t h e comparable s i g n a l s ( F i g u r e s 35a and 35c) a t t h e s t a r t o f t h e e x p e r i m e n t . A f t e r t h e s i g n a l s shown i n F i g u r e 38 were r e c o r d e d , t h e l e a f -117- 3236 3276 3236 3276 M a g n e t i c f i e l d (Gauss) (Magnetic f i e l d (Gauss) F i g u r e 38. EPR s i g n a l s from an i n t a c t , a t t a c h e d Kentucky b l u e g r a s s l e a f 20 minutes a f t e r t e r m i n a t i o n o f a 3 hour exposure t o lOOppb ozone. The l e a f was m a i n t a i n e d i n 7l0nm l i g h t t h r o u g h o u t , e x c e p t f o r t h e 8 minute p e r i o d s d u r i n g w h i c h t h e s i g n a l s shown i n F i g u r e s 37a, 37c and 38a, 38c were r e c o r d e d , a. i n d a r k ; b. i n 710nm l i g h t ; c. i n w h i t e l i g h t . Microwave f r e q u e n c y - 9.190. -118- was removed from t h e c a v i t y b u t remained a t t a c h e d t o t h e h o l d e r t o p e r m i t p r e c i s e r e l o c a t i o n i n t h e c a v i t y . The p l a n t was h e l d i n d a r k n e s s f o r 8 hours f o l l o w e d by room l i g h t f o r 8 h o u r s . F o l l o w i n g r e p l a c e m e n t o f t h e l e a f i n t h e c a v i t y , t h e s i g n a l s were a g a i n r e c o r d e d . F i g u r e 39b i n d i c a t e s no f u r t h e r change i n t h e 710nm l i g h t - i n d u c e d s i g n a l w h i l e F i g u r e s 39a and 39c i n d i c a t e a near complete r e t u r n t o t h e o r i g i n a l S i g n a l H u + S , ( c f . F i g u r e s 35a,35c,) by t h i s t i m e . The amount o f t i m e n e c e s s a r y f o r complete r e c o v e r y o f t h e l e a f v a r i e d g r e a t l y among d i f f e r e n t l e a v e s b u t was g e n e r a l l y c o r r e l a t e d t o t h e l e n g t h o f t i m e t h e l e a f was exposed t o t h e p o l l u t a n t . I f t h e t i m e o f f u m i g a t i o n was extended t o p e r i o d s e x c e e d i n g 3 hour s (e.g. 5 h o u r s ) , t h e t i m e r e q u i r e d t o r e v e r s e t h e s i g n a l s i n c r e a s e d , i n some c a s e s , r e q u i r i n g 48 hours exposure t o c l e a n a i r . A c c u r a t e d o s e - r e s p o n s e and r e c o v e r y c u r v e s c o u l d n o t be c o n s t r u c t e d because o f t h i s extreme v a r i a b i l i t y i n s e n s i t i v i t y among l e a v e s . A t no t i m e d u r i n g t h e s e e x p e r i m e n t s were v i s i b l e s i g n s o f ozone i n j u r y a p p a r e n t . -119- M a g n e t i c f i e l d (Gauss) J I I 1 L 3236 3276 M a g n e t i c f i e l d (Gauss) F i g u r e 39. EPR s i g n a l s from an i n t a c t , a t t a c h e d Kentucky b l u e g r a s s l e a f 16 h o u r s and 20 mi n u t e s a f t e r a 3 hour exposure t o lOOppb ozone. A f t e r f u m i g a t i o n ended, t h e l e a f was m a i n t a i n e d i n 710nm l i g h t f o r 20 m i n u t e s , d a r k n e s s f o r 8 hours and room l i g h t f o r 8 h o u r s . a. i n d a r k ; b. i n 710nm l i g h t ; c. i n w h i t e l i g h t . Microwave f r e q u e n c y - 9.190. -120- 4.3.3 E f f e c t s o f H i g h L e v e l s o f 0 3 There was a marked c o n t r a s t i n f r e e - r a d i c a l s i g n a l r e s p o n s e s when l e a v e s were exposed t o h i g h l e v e l s o f 0 3 (2 50ppb t o lppm) f o r p e r i o d s e x c e e d i n g 1 hour. Data p r e s e n t e d h e r e i n d i c a t e changes w h i c h o c c u r r e d when Kentucky b l u e g r a s s l e a v e s were f u m i g a t e d w i t h 500ppb 0 3 f o r 1.5 h o u r s . F i g u r e 4 0a i n d i c a t e s t h e t r u e S i g n a l I a t t h e s t a r t o f t h e ex p e r i m e n t . T h i s s i g n a l , o b t a i n e d by s u b t r a c t i n g S i g n a l H u + S ( r e c o r d e d w i t h t h e l e a f i n dar k n e s s ) from t h e com p o s i t e o f S i g n a l I and H u + S ( r e c o r d e d w i t h t h e l e a f under 710nm l i g h t ) has t h e t y p i c a l g - v a l u e o f 2.0025 and a l i n e w i d t h o f 7.5 gauss. F i g u r e 40b i n d i c a t e s t h e new s i g n a l ( S i g n a l N 7 1 0 ) , d i s c u s s e d e a r l i e r i n S e c t i o n 3.3.4.1, wh i c h i s i n d u c e d by c o n s t a n t exposure t o 710 nm l i g h t . T h i s s i g n a l , o b t a i n e d by s u b t r a c t i n g t h e o r i g i n a l S i g n a l I shown i n F i g u r e 40a, from t h e 710nm l i g h t - i n d u c e d s i g n a l a f t e r 45 minutes exposure t o 710nm l i g h t has a g - v a l u e o f a p p r o x i m a t e l y 2.0054 and a l i n e w i d t h o f 8.5 gauss. S i g n a l I I U + S was s u b t r a c t e d i n each c a s e . I n l e a v e s , t h e t i m e o f appearance o f S i g n a l N 7 1 0 v a r i e s b u t i t i s alway s p r e s e n t w i t h i n one hour o f c o n s t a n t exposure t o 710nm l i g h t . F i g u r e 41 i n d i c a t e s t h e changes i n t h e t r u e 710nm l i g h t - i n d u c e d and d a r k s i g n a l s w h i c h o c c u r a f t e r 1.5 ho u r s o f -121- 10 G -1 I I I L 236 3276 M a g n e t i c f i e l d (Gauss) 3236 3276 M a g n e t i c f i e l d (Gauss) F i g u r e 40. 710nm l i g h t - i n d u c e d s i g n a l s from an i n t a c t , a t t a c h e d K e n t u c k y b l u e g r a s s l e a f p r i o r t o f u m i g a t i o n , a. True S i g n a l I ; b. True S i g n a l n 7 1 Q , i n d u c e d by exposure t o 1 hour o f 710nm l i g h t . Microwave f r e q u e n c y - 9.190. -122- b F i g u r e 41. D i f f e r e n c e s i n f r e e - r a d i c a l s i g n a l s i n i n t a c t , a t t a c h e d K e n t u c k y b l u e g r a s s l e a v e s a f t e r e x p o s u r e t o 0.5ppm ozone f o r 1.5 h o u r s , a. D i f f e r e n c e i n s i g n a l o b t a i n e d i n 710nm l i g h t ; b. D i f f e r e n c e i n s i g n a l i n d a r k n e s s ; c. True change i n 710nm l i g h t - i n d u c e d s i g n a l ( F i g u r e 41a - 41b). Microwave f r e q u e n c y - 9.190. -123- f u m i g a t i o n w i t h 500ppb 0 3 . F i g u r e 41a was o b t a i n e d by s u b t r a c t i n g t h e 710nm l i g h t - i n d u c e d s i g n a l o b t a i n e d a t t h e s t a r t o f t h e e x p e r i m e n t from t h e 710nm l i g h t - i n d u c e d s i g n a l a f t e r 1.5 h o u r s o f f u m i g a t i o n . However, t h i s d i f f e r e n c e i n s i g n a l i s composed o f t h e change i n f r e e r a d i c a l f o r m a t i o n w h i c h o c c u r s i n d a r k n e s s , and t h e change which o c c u r s under 710nm l i g h t . T h i s h i g h ozone t r e a t m e n t was found t o r e s u l t i n an a p p r e c i a b l e change i n t h e t r u e d a r k s i g n a l as shown i n F i g u r e 41b. T h i s f i g u r e was o b t a i n e d by s u b t r a c t i n g t h e s i g n a l o b t a i n e d i n d a r k n e s s a t t h e s t a r t o f t h e ex p e r i m e n t from t h e s i g n a l o b t a i n e d i n d a r k n e s s a f t e r 1.5 hour s o f f u m i g a t i o n . T h i s new s y m m e t r i c a l ' d a r k 7 f r e e - r a d i c a l s i g n a l ( S i g n a l N Q 2 ) has a g - v a l u e o f 2.0055 and a l i n e w i d t h o f 7-8 gauss. These p a r a m e t e r s a r e s i m i l a r t o t h e t h o s e o f S i g n a l N 7 1 Q d i s c u s s e d i n S e c t i o n 3.3.4.1, b u t t h e s i g n a l s a r e c l e a r l y d i f f e r e n t as f o r m a t i o n o f S i g n a l N 7 1 0 i s dependent upon i r r a d i a t i o n o f t h e l e a f w h i l e i n t h e s p e c t r o m e t e r c a v i t y , w h i l e t h i s 'dark' s i g n a l ( S i g n a l N 0 2 ) i s p r e s e n t i n t h e absence o f l e a f i r r a d i a t i o n a t t h e t i m e o f measurement. The r e a l d i f f e r e n c e i n t h e 710nm l i g h t - i n d u c e d s i g n a l i s shown i n F i g u r e 41c, o b t a i n e d by s u b t r a c t i n g F i g u r e 41b from F i g u r e 41a. However, t h e s i g n a l shown i n F i g u r e 41c s t i l l i n c l u d e s t h e 710nm l i g h t - i n d u c e d s i g n a l ( S i g n a l N 7 1 0 ) d i s c u s s e d i n S e c t i o n 3.3.4.1 (see F i g u r e 40b), which i s not dependent upon f u m i g a t i o n . When t h i s s i g n a l , shown i n F i g u r e 40b, i s a l s o -124- s u b t r a c t e d from t h e s i g n a l i n F i g u r e 41c, t h e s i g n a l shown i n F i g u r e 42a i s r e v e a l e d . T h i s l a t t e r s i g n a l ( S i g n a l N S o x ) i s a s y m m e t r i c a l , has a g - v a l u e o f 2.001, a l i n e w i d t h o f a p p r o x i m a t e l y 10 gauss and shows a s m a l l r i s e i n t h e g=2.08 r e g i o n . The l a t t e r f e a t u r e ( F i g u r e 42a) a l o n e , c o u l d be d i s m i s s e d as ' n o i s e ' , b u t combined t o g e t h e r w i t h t h e o t h e r i d e n t i f y i n g p a r a m e t e r s , i n d i c a t e s t h a t t h i s s i g n a l has t h e a t t r i b u t e s o f t h e s i g n a l a s s i g n e d t o t h e s u p e r o x i d e a n i o n r a d i c a l (Knowles e t a l . 1969; B a l l o u e t a l . 1969; Bray e t a l . 197 0 ) . F o r pur p o s e s o f compa r i s o n , an example o f t h e s u p e r o x i d e s i g n a l from t h e r e l e v a n t l i t e r a t u r e i s shown i n F i g u r e 42b ( F i g u r e 8c from B a l l o u e t a l . 1969). Once i t has been i n d u c e d , S i g n a l N S o x can a l s o be r e v e a l e d by s i m p l y s u b t r a c t i n g t h e s i g n a l o b t a i n e d i n d a r k n e s s a f t e r f u m i g a t i o n from t h e s i g n a l o b t a i n e d under broad-band w h i t e l i g h t a f t e r f u m i g a t i o n . T h i s i s shown i n F i g u r e 42c. There i s no e v i d e n c e f o r t h e d e t e c t i o n o f S i g n a l N S o x i n d a r k n e s s . I f f u m i g a t i o n i s c o n t i n u e d f o r a n o t h e r hour, w i t h t h e l e a f s t i l l m a i n t a i n e d i n 710nm l i g h t , S i g n a l I i s e l i m i n a t e d . Upon e l i m i n a t i o n o f S i g n a l I , t h e f r e e r a d i c a l changes a r e i r r e v e r s i b l e upon t e r m i n a t i o n o f f u m i g a t i o n , even though t h e r e a r e no v i s i b l e s i g n s o f 0 3 i n j u r y a t t h i s p o i n t i n t i m e . S i g n a l N S o x i s a l s o r e v e a l e d , u s i n g t h e same s u b t r a c t i o n t e c h n i q u e , when e x c i s e d p i e c e s o f r a d i s h l e a v e s a r e f u m i g a t e d -125- F i g u r e 42. L i g h t - d e p e n d e n t f r e e - r a d i c a l s i g n a l s i n i n t a c t , a t t a c h e d Kentucky b l u e g r a s s l e a v e s a f t e r 1.5 h o u r s o f f u m i g a t i o n w i t h 0.5ppm ozone. a. S i g n a l N S o x ( F i g u r e 41c - 40b) w i t h o r d i n a t e d o u b l e d t o f a c i l i t a t e c o m p arison w i t h F i g u r e 42b. b. F r e e r a d i c a l s i g n a l a t t r i b u t e d t o t h e s u p e r o x i d e a n i o n f r e e r a d i c a l by B a l l o u e t a l . 1969; c. D i f f e r e n c e o f w h i t e l i g h t - i n d u c e d s i g n a l minus d a r k s i g n a l a f t e r 1.5 h o u r s o f f u m i g a t i o n w i t h 0.5ppm ozone. Microwave f r e q u e n c y - 9.190. -126- w i t h lppm ozone f o r 30 minutes ( d a t a n o t p r e s e n t e d ) . S i m i l a r changes a l s o o c c u r i n p e r e n n i a l r y e g r a s s l e a v e s upon f u m i g a t i o n w i t h h i g h l e v e l s o f 0 3 . However, t h e s p e c t r a a r e more d i f f i c u l t t o i n t e r p r e t because o f t h e l a r g e r M n + + and F e + + s i g n a l s u n d e r l y i n g t h e p h o t o s y n t h e t i c s i g n a l s (see S e c t i o n 2.4). I n each spectrum shown i n F i g u r e 43, S i g n a l H u + S has been s u b t r a c t e d from t h e co m p o s i t e s i g n a l . These s p e c t r a show t h e o r i g i n a l S i g n a l I ( F i g u r e 4 3 a ) , t h e i n c r e a s e i n t h e combined d a r k and 710nm l i g h t - i n d u c e d s i g n a l a f t e r f u m i g a t i o n o f p e r e n n i a l r y e g r a s s l e a v e s w i t h lppm 0 3 f o r 3 0 m i n u t e s ( F i g u r e 43b), t h e d a r k s i g n a l i n c r e a s e a f t e r t h i s f u m i g a t i o n ( F i g u r e 43c) and t h e s m a l l i n c r e a s e i n t h e 710nm l i g h t - i n d u c e d s i g n a l a f t e r s u b t r a c t i o n o f t h e d a r k s i g n a l i n c r e a s e ( F i g u r e 43d). The new d a r k s i g n a l ( F i g u r e 43c) i s c l e a r l y t h e same as t h a t found i n Kentucky b l u e g r a s s l e a v e s ( S i g n a l N Q 2 ) ( F i g u r e 41b) b u t t h e s h i f t s i n t h e m e t a l s i g n a l s d i s t o r t t h e l i g h t - i n d u c e d s i g n a l ( F i g u r e 4 3d) t o t h e e x t e n t t h a t a t t r i b u t i o n t o t h e s u p e r o x i d e a n i o n r a d i c a l , o r any o t h e r s p e c i f i c f r e e - r a d i c a l s i g n a l , i s not p o s s i b l e from t h e s e r y e g r a s s l e a f s p e c t r a . -127- M a g n e t i c f i e l d (Gauss) F i g u r e 43. F r e e - r a d i c a l s i g n a l d i f f e r e n c e s i n i n t a c t , a t t a c h e d p e r e n n i a l r y e g r a s s l e a v e s f u m i g a t e d w i t h lppm ozone f o r 30 m i n u t e s a f t e r s u b t r a c t i o n o f i n i t i a l S i g n a l I I u + 5 . a. S i g n a l I p r i o r t o f u m i g a t i o n ; b. D i f f e r e n c e i n 710nm l i g h t - i n d u c e d s i g n a l a f t e r f u m i g a t i o n w i t h lppm ozone f o r 30 m i n u t e s ; c. D i f f e r e n c e i n d a r k s i g n a l a f t e r f u m i g a t i o n w i t h lppm ozone f o r 30 m i n u t e s ; d. Ap p a r e n t new s i g n a l i n d u c e d by ozone f u m i g a t i o n i n 710nm l i g h t ( F i g u r e 43b - 4 3 c ) . -128- 4.4 D i s c u s s i o n The s t u d i e s w i t h d i f f e r e n t l e v e l s o f 0 3 i n d i c a t e t h a t t o t a l e x p o s u r e t o t h e p o l l u t a n t s u b s t a n t i a l l y a f f e c t s t h e r e s p o n s e w i t h i n t h e p l a n t . F u m i g a t i o n w i t h low l e v e l s o f ozone (up t o 80ppb) f o r p e r i o d s up t o 8 hours f a i l e d t o r e v e a l any d i s t i n c t o r c o n s i s t e n t changes i n EPR s p e c t r a from t h o s e a s s o c i a t e d w i t h u n f u m i g a t e d l e a v e s . S t u d i e s w i t h i n t e r m e d i a t e l e v e l s o f 0 3 (80-250ppb) i n d i c a t e t h a t two independent e v e n t s o c c u r . F i r s t , t h e r e i s t h e i n i t i a t i o n o f t h e f o r m a t i o n o f a new f r e e r a d i c a l d e p i c t e d as S i g n a l N 0 1 , w i t h a g - v a l u e o f 2.0041 and a peak-to-peak w i d t h o f a p p r o x i m a t e l y 10 gauss. T h i s r a d i c a l i s n o t dependent upon l i g h t as i t i s formed i n d a r k n e s s , under broad-band w h i t e l i g h t and i n 710nm l i g h t . The i n i t i a l appearance o f t h i s s i g n a l i n d a r k n e s s and w h i t e l i g h t i s shown i n F i g u r e s 3 6a and 36c, r e s p e c t i v e l y . The p r e s e n c e o f t h i s s i g n a l i s a p p a r e n t i n F i g u r e 36b i n t h e l a r g e r peak-to-peak l i n e w i d t h and change i n g - v a l u e of t h e 710nm l i g h t - i n d u c e d s i g n a l . A t t h i s t i m e , S i g n a l I i s s t i l l p r e s e n t i n F i g u r e 36b. A f t e r a n o t h e r hour o f f u m i g a t i o n , S i g n a l I has d i s a p p e a r e d ( F i g u r e 37b) and S i g n a l N 0 1 has r e a c h e d a maximum ( F i g u r e s 37a,37b and 3 7 c ) , r e g a r d l e s s o f l i g h t r egime. A p p a r e n t l y , t h e l i g h t - i n d u c e d o x i d a t i o n o f t h e r e a c t i o n c e n t r e c h l o r o p h y l l a wh i c h r e s u l t s i n t h e f o r m a t i o n o f S i g n a l I has been i n t e r r u p t e d as a r e s u l t o f f u m i g a t i o n . The l o s s o f -129- S i g n a l I i s r e v e r s i b l e but t h e l e n g t h o f t i m e r e q u i r e d t o r e s t o r e i t a f t e r t e r m i n a t i o n o f f u m i g a t i o n i s dependent upon t o t a l e x p o s u r e . The i d e n t i t y o f t h e s p e c i e s g i v i n g r i s e t o S i g n a l N Q 1 , h a v i n g a g - v a l u e o f 2.0041 and a peak-to-peak w i d t h o f a p p r o x i m a t e l y 10 gauss, can not be a s c e r t a i n e d from t h e s e d a t a . However, i t i s p o s s i b l e t h a t t h e f r e e r a d i c a l c o n c e r n e d i s r e s p o n s i b l e f o r t h e e l i m i n a t i o n o f S i g n a l I . A t h i g h c o n c e n t r a t i o n s o f O 3 (500ppb) S i g n a l I i s a l s o l o s t a t a p p r o x i m a t e l y t h e same r a t e . However, p r i o r t o t h i s l o s s t h e r e i s t h e emergence o f a d i f f e r e n t f r e e - r a d i c a l s i g n a l ( S i g n a l N 0 2 ) , w h i c h i s a l s o n o t l i g h t - d e p e n d e n t . T h i s i s not t h e same s i g n a l ( S i g n a l N Q ^ ) w h i c h appears a t l o w e r c o n c e n t r a t i o n s , s i n c e S i g n a l N Q 2 has a g - v a l u e o f 2.0055 and a l i n e w i d t h o f 7-8 gauss. T h i s s i g n a l may be due t o t h e p r e c u r s o r o f t h e ' o r g a n i c ' f r e e - r a d i c a l s i g n a l found i n aged p l a n t m a t e r i a l ( M i s h r a e t a l . 1971; P r i e s t l e y e t a l . 1980) . The appearance of t h e s m a l l l i g h t - i n d u c e d s i g n a l ( S i g n a l N G O X ) w h i c h has t h e c h a r a c t e r i s t i c s o f t h e s i g n a l a s c r i b e d t o t h e s u p e r o x i d e a n i o n r a d i c a l i s l i k e l y an e f f e c t o f t h e damage caused by t h e o t h e r f r e e r a d i c a l s w h i c h have been c r e a t e d as a r e s u l t o f f u m i g a t i o n w i t h 0 3 . T h i s s i g n a l a ppears a f t e r t h e o t h e r f r e e - r a d i c a l s i g n a l s have been formed and i s p r o b a b l y t h e r e s u l t o f a c c e l e r a t e d senescence, caused by t h e o t h e r f r e e r a d i c a l s . The sequence o f e v e n t s makes i t u n l i k e l y t h a t t h e -130- f r e e r a d i c a l d e p i c t e d as S i g n a l N S o x i s t h e p r e c u r s o r o f damage. I n young, h e a l t h y l e a v e s , f r e e - r a d i c a l s c a v e n g e r s such as s u p e r o x i d e d i s m u t a s e (SOD), a - t o c o p h e r o l ( V i t a m i n E ) , a s c o r b i c a c i d ( V i t a m i n C) , c a t a l a s e and |3-carotene c o u n t e r t h e e f f e c t o f t o x i c s p e c i e s such as t h e s u p e r o x i d e and h y d r o x y l f r e e r a d i c a l s , and s i n g l e t oxygen. However, i n aged t i s s u e t h e c a p a c i t y o f p l a n t t i s s u e t o scavenge t h e s e d e l e t e r i o u s compounds d e c r e a s e s because a c t i v i t y o f t h e s c a v e n g e r s d e c l i n e s w i t h a g i n g (Thompson e t a l . 1987). Hence, i f t h e f o r m a t i o n o f f r e e r a d i c a l s d e p i c t e d as S i g n a l s N Q 1 and N Q 2 a c c e l e r a t e s t i s s u e s e nescence, t h e s c a v e n g i n g c a p a c i t y o f t h e p l a n t t i s s u e w i l l be reduced o v e r t i m e . A l t e r n a t i v e l y , t h e c u m u l a t i v e e f f e c t o f c o n t i n u e d f u m i g a t i o n w i t h h i g h l e v e l s o f 0 3 may overwhelm t h e c a p a c i t y o f t h e a n t i - o x i d a n t s c a v e n g e r s even i f t h e i r a c t i v i t y s t a y s c o n s t a n t o v e r t h e f u m i g a t i o n p e r i o d . I n e i t h e r c a s e , t h e l a g t i m e p r i o r t o t h e f o r m a t i o n o f S i g n a l N S o x seen i n t h i s s t u d y would be e x p e c t e d . The d i s c o v e r y o f t h e s m a l l 710nm l i g h t - i n d u c e d s i g n a l ( S i g n a l N 7 1 0 ) whi c h o c c u r s i n t h e f i r s t hour o f ex p o s u r e t o f a r - r e d l i g h t was c r u c i a l t o c o r r e c t s u b t r a c t i o n o f s i g n a l s i n t h e s e o z o n e - f u m i g a t i o n s t u d i e s . P r i o r t o d i s c o v e r y o f t h i s s i g n a l , d a t a from t h e s t a r t o f exposure t o 710nm l i g h t were used as t h e base w h i c h must be s u b t r a c t e d from d a t a a t any o t h e r t i m e t o e s t a b l i s h t h e change i n t h e 710nm l i g h t - i n d u c e d s i g n a l caused by e x p osure t o 0 3 . However, w i t h t h e d i s c o v e r y o f t h i s s i g n a l i t became a p p a r e n t t h a t i t was n e c e s s a r y t o i n c l u d e t h i s s i g n a l as a p a r t o f t h e b a s e - l i n e d a t a , s i n c e o t h e r w i s e i t s p r e s e n c e would confound r e s u l t s . The t r u e e f f e c t s o f f u m i g a t i o n w i t h 0 3 can be o b t a i n e d u s i n g t h e complex s e r i e s o f s u b t r a c t i o n s d e s c r i b e d i n S e c t i o n 4.3.3. A l t e r n a t i v e l y , t h e y can be r e v e a l e d by u s i n g t h e d a t a o b t a i n e d once t h e s m a l l s i g n a l has been i n d u c e d , as t h e base which needs t o be s u b t r a c t e d . T h i s l a t t e r a pproach i s o n l y v a l i d i f no changes have o c c u r r e d i n t h e d a r k s i g n a l i n t h e i n t e r v a l . -132- 5.0 SULPHUR DIOXIDE STUDIES 5.1 I n t r o d u c t i o n I n an e f f o r t t o i n c r e a s e knowledge o f t h e mechanisms t h r o u g h w h i c h S 0 2 a f f e c t s v e g e t a t i o n , numerous i n v e s t i g a t o r s have s t u d i e d t h e b i o c h e m i c a l (Ewald and S c h l e e , 1983; M a l h o t r a and H o c k i n g , 1976; M a l h o t r a and S a r k a r , 1979; R o b i n s o n and W e l l b u r n , 1983; W e l l b u r n e t a l . 1981) and p h y s i o l o g i c a l changes (Le Sueur-Brymer and Ormrod, 1984; M a j e r n i k , 1971; M a j e r n i k and M a n s f i e l d , 1971, 1972; T a y l o r and T i n g e y , 1981) w h i c h o c c u r i n p l a n t s exposed t o S 0 2 • One m e t a b o l i c p r o c e s s a f f e c t e d by e x p o sure t o S 0 2 i s p h o t o s y n t h e s i s ( B l a c k e t a l . 1982; C a r l s o n , 1979, 1983a, 1983b; Thomas and H i l l , 1937). The v a s t m a j o r i t y o f s t u d i e s i n d i c a t e t h a t exposure t o S 0 2 causes r e d u c t i o n s i n t h e p h o t o s y n t h e s i s o f p l a n t s (Lamoreaux and Chaney, 1978; Ormrod e t a l . 1981; Takemoto and Noble, 1982; Winner, and Mooney, 1980), a l t h o u g h l i t t l e i s known o f t h e mechanisms o f t o x i c i t y . A t t h e m o l e c u l a r l e v e l , t h e f o r m a t i o n o f t o x i c f r e e r a d i c a l s has been p o s t u l a t e d as one o f t h e p r i m a r y e v e n t s i n t h e p r o c e s s e s by w h i c h a i r p o l l u t a n t s such as S 0 2 may a d v e r s e l y a f f e c t p h o t o s y n t h e s i s (Tanaka and Sugahara, 1980). A g e n e r a l p o l l u t a n t - i n d u c e d p r o d u c t i o n o f t h e s u p e r o x i d e and o t h e r r a d i c a l s has been proposed as t h e p r i m a r y cause o f impairment o f membrane f u n c t i o n t h r o u g h o u t t h e c e l l a f t e r p o l l u t a n t uptake -133- (Mudd, 1973,1982). These s t u d i e s were c a r r i e d o u t w i t h l e a v e s o r c h l o r o p l a s t p r e p a r a t i o n s from l e a v e s a f t e r f u m i g a t i o n w i t h S 0 2 . I n o r d e r t o d e t e r m i n e t h e changes i n f r e e r a d i c a l s w h i c h o c c u r d u r i n g f u m i g a t i o n , t h e p r e s e n t s t u d y i n v e s t i g a t e d t h e i n v i v o EPR- d e t e c t a b l e changes i n S i g n a l s I and H u + S d u r i n g f u m i g a t i o n w i t h S 0 2 • 5.2 Methods P l a n t m a t e r i a l s , and t h e e x p e r i m e n t a l p r o t o c o l s used, were e s s e n t i a l l y t h e same as t h o s e d e s c r i b e d e a r l i e r ( S e c t i o n s 3.2 and 4.2). P r e l i m i n a r y i n v e s t i g a t i o n s showed t h a t , i n r a d i s h , t h e magnitude o f S i g n a l I , S i g n a l I I U + S and t h e M n + + s i g n a l v a r i e d from l e a f t o l e a f , even i f t h e c h r o n o l o g i c a l ages o f t h e l e a v e s were i d e n t i c a l . The l e a s t v a r i a t i o n i n s i g n a l i n t e n s i t y o c c u r r e d w i t h l e a v e s o f s i m i l a r age which were a l s o v i s u a l l y comparable i n terms o f gre e n n e s s . Subsequent i n v e s t i g a t i o n s showed t h a t g r e a t e r c o n s i s t e n c y c o u l d be o b t a i n e d by s e q u e n t i a l l y u s i n g d i f f e r e n t segments o f t h e same l e a f . Thus, t h e e x p e r i m e n t s u s i n g r a d i s h e s d e s c r i b e d below w h i c h i n c l u d e d s e v e r a l t r e a t m e n t s were done by u s i n g e x c i s e d segments from t h e same l e a f t o o b t a i n c o n t r o l and t r e a t m e n t d a t a . L e a f segments were c u t p a r a l l e l t o t h e m i d r i b . I n o r d e r t o m i n i m i z e a g i n g e f f e c t s and c o n c o m i t a n t s i g n a l p e r t u r b a t i o n ( S e c t i o n 3.3.1), t h e -134- l e a v e s from w h i c h t h e l a m i n a r segments were e x c i s e d were not d e t a c h e d from t h e r a d i s h p l a n t s . I n s t r u m e n t s e t t i n g s f o r a l l k i n e t i c t r a c e s were as f o l l o w s : t i m e c o n s t a n t 0.2 50s; m o d u l a t i o n 1.0 x 10 gau s s ; r e c e i v e r g a i n 5 . 0 x l 0 4 ; power lOmW. F o r o t h e r t r a c e s t h e i n s t r u m e n t s e t t i n g s were as d e s c r i b e d p r e v i o u s l y ( S e c t i o n 3.2). S 0 2 was c o n t r o l l e d by i n j e c t i n g a S 0 2 i n a i r m i x t u r e i n t o a m e d i c a l grade a i r stream f l o w i n g a t a r a t e o f 2 1/min t h r o u g h a m i x i n g chamber. S 0 2 c o n c e n t r a t i o n s i n t h e c a v i t y were m o n i t o r e d w i t h a T h e r m o e l e c t r o n Model 4 3 S 0 2 m o n i t o r . 5.3 R e s u l t s 5.3.1 Detached R a d i s h L e a f Segments 5.3.1.1 S i g n a l I S t u d i e s Numerous e x p e r i m e n t s were conducted u s i n g e x c i s e d p i e c e s o f r a d i s h l e a v e s . Because o f t h e l i m i t e d 60 minute p e r i o d a v a i l a b l e ( S e c t i o n 3.3.1) f o r s t u d y i n g s i g n a l dynamics i n d e t a c h e d l e a f p i e c e s , a wide range o f c o n c e n t r a t i o n s (up t o 400ppm) was used. Hence, t h e r e s u l t s p r e s e n t e d i n t h i s s e c t i o n s h o u l d be viewed w i t h c a u t i o n i n r e l a t i o n t o t h e i r r e l e v a n c e t o changes i n d u c e d by p o l l u t e d atmospheres. -135- F o r S i g n a l I ( a t t r i b u t e d t o Photosystem I) k i n e t i c s t u d i e s , changes i n t h e low f i e l d maximum were f o l l o w e d (see F i g u r e 8 ) . T h i s peak was chosen t o a v o i d p o s s i b l e c o n f u s i o n w i t h changes w h i c h may o c c u r i n S i g n a l I I U + S ( a t t r i b u t e d t o D, a component o f P h otosystem I I ) . S i g n a l H u + S i s p r e s e n t i n h e a l t h y l e a v e s , r e g a r d l e s s o f t h e immediate l i g h t regime, u n l e s s t h e l e a f has been h e l d i n a b s o l u t e d a r k n e s s f o r o v e r 24 h o u r s . However, i t does n o t i n t e r f e r e w i t h t r a c k i n g t h e low f i e l d S i g n a l I maximum a t t h i s p o i n t because t h i s i s t h e p o i n t a t whi c h t h e b a s e l i n e c r o s s o v e r o f S i g n a l I I U + S o c c u r s . P r e v i o u s l i g h t a d a p t a t i o n e f f e c t s (Tikhonov and Ruuge, 1975a) were s t a n d a r d i z e d by e x p o s i n g t h e l e a f segment t o 3 0 seconds o f 710nm l i g h t f o l l o w e d by 3 minutes o f d a r k n e s s i n t h e c a v i t y . The l e a v e s were t h e n i l l u m i n a t e d w i t h t h e a p p r o p r i a t e l i g h t . F i g u r e 44 shows k i n e t i c s o f s i g n a l s from u n f u m i g a t e d l e a v e s . S i g n a l I was not formed i n d a r k n e s s , a v e r y s m a l l i n c r e a s e o c c u r r e d under 650nm l i g h t , a l a r g e S i g n a l I was i n d u c e d by 710nm l i g h t and an i n t e r m e d i a t e s i z e s i g n a l was formed under i l l u m i n a t i o n w i t h broad-band w h i t e l i g h t . The k i n e t i c s o f t h e d a r k , 650nm and 710nm s i g n a l s were c o n s i s t e n t from l e a f t o l e a f , d i f f e r i n g o n l y i n s i g n a l magnitude. However, t h e m u l t i p h a s i c k i n e t i c s o f t h e w h i t e l i g h t s i g n a l v a r i e d c o n s i d e r a b l y . S i m i l a r l y , t h e i n i t i a l r i s e and i n c r e a s e i n s i g n a l i n t e n s i t y i n t h e second h a l f hour a f t e r detachment o f t h e l e a f from t h e p l a n t a l s o v a r i e d from l e a f t o l e a f . M a j o r -136- F i g u r e 44. K i n e t i c s o f S i g n a l I f o r m a t i o n i n e x c i s e d u n f u m i g a t e d r a d i s h l e a f p i e c e s i n d i f f e r e n t l i g h t r e g i m e s , a. d a r k ; b. 710nm l i g h t ; c. 650nm l i g h t ; d. w h i t e l i g h t . A l l t r a c e s b e g i n i n d a r k n e s s ; arrows ( A ) i n d i c a t e t i m e a t whi c h l i g h t was t u r n e d on. • -137- d i f f e r e n c e s o c c u r r e d even i n segments from t h e same l e a f . Thus, t h e w h i t e - l i g h t i n d u c e d t r a c e s shown i n F i g u r e 44d can o n l y be r e g a r d e d as a g e n e r a l o u t l i n e o f w h i t e l i g h t s i g n a l k i n e t i c s . F i g u r e 4 5 i n d i c a t e s changes i n S i g n a l I k i n e t i c s w h i c h o c c u r r e d upon f u m i g a t i o n w i t h a h i g h c o n c e n t r a t i o n o f S 0 2 , a p p r o x i m a t e l y 400ppm. F o l l o w i n g t h e s t a r t o f f u m i g a t i o n no changes o c c u r r e d i n t h e d a r k s i g n a l ( F i g u r e 4 5 a ) , b u t under 710nm l i g h t , ( F i g u r e 45b), needed t o i n v o k e S i g n a l I i n h e a l t h y l e a v e s , t h e r e was a s m a l l i n c r e a s e i n s i g n a l magnitude t h a t commenced a f t e r 3 0 t o 4 5 seconds o f f u m i g a t i o n . T h i s was superimposed upon t h e S i g n a l I n o r m a l l y i n d u c e d by 710nm l i g h t . However, t h i s d i f f e r e n c e i n s i g n a l was so s m a l l t h a t , even w i t h s i g n a l s u b t r a c t i o n , i t c o u l d n o t be p o s i t i v e l y a s c r i b e d t o e i t h e r an i n c r e a s e d S i g n a l I o r t o a new f r e e - r a d i c a l s i g n a l w h i c h o v e r l a p s S i g n a l I . Under 650nm l i g h t , w h i c h i n v o k e s v i r t u a l l y no S i g n a l I r e s p o n s e i n h e a l t h y l e a v e s ( F i g u r e 4 4 c ) , an i n c r e a s e i n s i g n a l i n t e n s i t y began a f t e r 30 t o 45 seconds o f f u m i g a t i o n , and t h e s i g n a l ( F i g u r e 4 5 c ) , a f t e r 50 m i n u t e s , was comparable i n magnitude t o t h e 710nm l i g h t - i n d u c e d s i g n a l . A s i m i l a r i n c r e a s e , i n d u c e d by S 0 2 , was o b s e r v e d i n t h e s i g n a l i n d u c e d by w h i t e l i g h t ( F i g u r e 45d). On t e r m i n a t i o n o f f u m i g a t i o n (50min S 0 2) t h e r e l a t i v e s i z e s o f t h e t h r e e l i g h t s i g n a l s had changed m a r k e d l y . The a b s o l u t e s i g n a l s v a r i e d from l e a f t o l e a f , b u t , i n a l l c a s e s , -138- b 0 I I | | | 60 t i m e (min) F i g u r e 45. K i n e t i c s o f S i g n a l I f o r m a t i o n i n S 0 2 - f u m i g a t e d (400ppm) r a d i s h l e a f p i e c e s i n d i f f e r e n t l i g h t r e g i m e s . a. d a r k ; b. 7l0nm l i g h t ; c. 650nm l i g h t ; d. w h i t e l i g h t . A l l t r a c e s b e g i n i n d a r k n e s s ; l a r g e arrows ( f ) i n d i c a t e t i m e a t w h i c h l i g h t was t u r n e d on; s m a l l arrows ( f ) i n d i c a t e t i m e a t w h i c h S 0 2 f u m i g a t i o n began. -139- were c o r r e l a t e d w i t h t h e s i z e o f t h e 710nm l i g h t - i n d u c e d s i g n a l from each l e a f p r i o r t o f u m i g a t i o n . A f t e r f u m i g a t i o n , t h e w h i t e l i g h t - i n d u c e d s i g n a l exceeded t h e 710nm l i g h t - i n d u c e d s i g n a l , w h i l e t h e s i g n a l i n d u c e d by 650nm l i g h t (which n o r m a l l y o n l y i n v o k e s S i g n a l I I U + S , a s s o c i a t e d w i t h Photosystem I I ) was now v i r t u a l l y i d e n t i c a l t o t h e 710nm l i g h t - i n d u c e d s i g n a l . F i g u r e 4 6 shows t h e s i g n a l s p e c t r a a f t e r 50 m i n u t e s o f f u m i g a t i o n i n 650nm l i g h t . I n a l l c a s e s t h e g - v a l u e s and peak-to-peak l i n e w i d t h s a r e i d e n t i c a l t o t h o s e a s s o c i a t e d w i t h t h e 710nm l i g h t - i n d u c e d S i g n a l I , i n d i c a t i n g t h a t t h e i n c r e a s e d 650nm and w h i t e l i g h t s i g n a l s a r e i n d i s t i n g u i s h a b l e from S i g n a l I , and a r e not unknown f r e e - r a d i c a l s i g n a l s w i t h s i m i l a r p a r a m e t e r s . These changes o c c u r r e d r e g a r d l e s s o f whether t h e f u m i g a t i o n t o o k p l a c e i n d a r k n e s s , i n 650nm l i g h t , i n 710nm l i g h t o r i n broad-band w h i t e l i g h t . The i n c r e a s e s i n t h e s i g n a l s appeared t o be i r r e v e r s i b l e ; c o n t i n u a t i o n o f t h e k i n e t i c t r a c e showed no d e c r e a s e i n t h e s i g n a l s d u r i n g t h e f o l l o w i n g hour. The i n c r e a s e i n s i g n a l i n t e n s i t y was d i r e c t l y r e l a t e d t o t h e up t a k e o f S 0 2 , as shown by e x p e r i m e n t s i n which t h e d u r a t i o n o f t h e ex p o s u r e t o S 0 2 was v a r i e d . A t t h i s h i g h l e v e l o f f u m i g a t i o n , s t e a d y - s t a t e l e v e l s o f t h e i n c r e a s e d s i g n a l s were a c h i e v e d a f t e r f u m i g a t i o n t i m e s r a n g i n g from 9 t o about 15 m i n u t e s , depending upon t h e t h e l e a f p i e c e used. F i g u r e 47 shows t h a t 2 seconds o f f u m i g a t i o n d i d not i n d u c e t h e r i s e i n -140- F i g u r e 46. EPR s i g n a l s i n d i f f e r e n t l i g h t r e gimes from a r a d i s h l e a f p i e c e a f t e r 50 m i n u t e s o f f u m i g a t i o n w i t h 400ppm S 0 2 • The c a v i t y was i l l u m i n a t e d w i t h 650nm l i g h t d u r i n g f u m i g a t i o n , a. 710nm l i g h t ; b. w h i t e l i g h t ; c. d a r k ; d. 650nm l i g h t . -141- F i g u r e 47. The e f f e c t o f 400ppm SO, f u m i g a t i o n d u r a t i o n on t h e i n t e n s i t y o f t h e w h i t e l i g h t - i n d u c e d S i g n a l I i n e x c i s e d r a d i s h l e a f p i e c e s . T r a c e s b e g i n i n d a r k n e s s ; l a r g e arrows (^ ) i n d i c a t e t i m e a t w h i c h l i g h t was t u r n e d on; s m a l l arrows i n d i c a t e t i m e a t which f u m i g a t i o n began ( f ) and ended ( I ) . F u m i g a t i o n d u r a t i o n ; a. 2 s e c ; b. 36 s e c ; c. 1.5 min; d. 3 min; e. 6 min; f . 9 min. -142- t h e w h i t e l i g h t - i n d u c e d s i g n a l , w hich became d i s t i n c t w i t h i n 3 6 seconds o f f u m i g a t i o n . The i n c r e a s e i n s i g n a l s i z e became l a r g e r as f u m i g a t i o n t i m e i n c r e a s e d from 3 6 seconds t o 9 m i n u t e s . F i g u r e 47 a l s o p r o v i d e s some i n d i c a t i o n o f t h e v a r i a b i l i t y among d i f f e r e n t l e a f p i e c e s i n t h e i r r e s p o n s e t o w h i t e l i g h t , p r i o r t o f u m i g a t i o n , as r e v e a l e d by t h e d i f f e r e n t magnitudes o f t h e i n c r e a s e s o b s e r v e d w i t h t h e o n s e t o f i l l u m i n a t i o n . I f f u m i g a t i o n was t e r m i n a t e d a f t e r l e s s t h a n 9 m i n u t e s , t h e r i s e o f S i g n a l I under w h i t e ( o r 650nm l i g h t ) was h a l t e d and a new s t e a d y s t a t e was a t t a i n e d . S u b s e q u e n t l y , i f , a f t e r achievement o f t h i s new s t e a d y - s t a t e i n t h e s i g n a l , f u m i g a t i o n recommenced, t h e n s i g n a l i n t e n s i t y i n c r e a s e d t o a new l e v e l , up t o a maximum, f o r any g i v e n l e a f ( F i g u r e 4 8 ) . S i m i l a r i n c r e a s e s i n t h e w h i t e and 650nm l i g h t - i n d u c e d s i g n a l s were found t o o c c u r a t l o w e r S 0 2 c o n c e n t r a t i o n s , down t o lOppm. However, a t t h e l o w e r c o n c e n t r a t i o n s , t h e r e was a d i s t i n c t l a g p e r i o d d u r i n g which t h e s i g n a l remained unchanged. The d a t a shown i n F i g u r e 49 a r e r e p r e s e n t a t i v e o f t r i a l s w i t h numerous l e a f p i e c e s . Exposure t o lOOppm S 0 2 r e s u l t e d i n a l a g p e r i o d o f a p p r o x i m a t e l y 17.5 minutes b e f o r e t h e i n c r e a s e d s i g n a l c o u l d be d e t e c t e d , w h i l e a t a f u m i g a t i o n l e v e l o f lOppm, t h e i n c r e a s e i n s i g n a l d i d not o c c u r u n t i l a f t e r 90 minutes o f f u m i g a t i o n had t a k e n p l a c e . F o r t h e l a t t e r o b s e r v a t i o n t h e -143- t i m e (min) F i g u r e 48. The e f f e c t o f i n t e r r u p t i o n o f 400ppm S 0 2 f u m i g a t i o n on t h e k i n e t i c s o f t h e w h i t e l i g h t - i n d u c e d f o r m a t i o n o f S i g n a l I i n e x c i s e d r a d i s h l e a f p i e c e s . T r a c e b e g i n s i n d a r k n e s s ; l a r g e arrow ( A ) i n d i c a t e s t i m e a t w h i c h l i g h t was t u r n e d on; s m a l l a rrows i n d i c a t e t i m e s a t w h i c h f u m i g a t i o n began ( \ ) and ended ( j ) . -144- 90 80 70 60 t i m e (min) 50 40 30 20 10 10 50 100 200 S 0 2 c o n c e n t r a t i o n (ppm) 400 500 F i g u r e 49. The l a g t i m e p r i o r t o t h e i n d u c t i o n o f t h e w h i t e l i g h t - i n d u c e d S i g n a l I i n r e l a t i o n t o S 0 2 c o n c e n t r a t i o n . -145- k i n e t i c s c a n was extended p a s t 1 hour, b u t , d e s p i t e t h e g r a d u a l i n c r e a s e i n t h e w h i t e l i g h t s i g n a l w h i c h o c c u r r e d because o f a g i n g (see S e c t i o n 3.4.1), t h e e f f e c t o f t h e S 0 2 was d i s c e r n i b l e as a d i s t i n c t jump i n s i g n a l i n t e n s i t y a t t h e 90 m i n u t e mark. L o w e r i n g t h e S 0 2 c o n c e n t r a t i o n below lOppm d i d n o t cause a d e t e c t a b l e s i g n a l i n c r e a s e w i t h i n 2 h o u r s and t h e c o n f o u n d i n g a g i n g e f f e c t s d i s c u s s e d i n S e c t i o n 3.3.1 p r e c l u d e d t h e use o f a l o n g e r s c a n p e r i o d . The e f f e c t o f a i r f l o w on t h e k i n e t i c s o f t h e r i s e i n t h e w h i t e l i g h t - i n d u c e d s i g n a l was a l s o s t u d i e d . The same i n c r e a s e o c c u r r e d a t i d e n t i c a l S 0 2 c o n c e n t r a t i o n s , r e g a r d l e s s o f a i r f l o w r a t e o v e r t h e range o f 1 1/min t o 3 1/min ( v e l o c i t y lm/s t o 3m/s). These changes i n S i g n a l I were c o n f i r m e d by l o c k i n g t h e s p e c t r o m e t e r on t h e h i g h f i e l d minimum (see F i g u r e 8 ) . Data from t h i s peak a r e p o t e n t i a l l y confounded by t h e a d d i t i o n a l p r e s e n c e o f S i g n a l I I U + S , b u t k i n e t i c s from t h i s peak were found t o be n e a r l y i d e n t i c a l t o t h o s e o b t a i n e d from t h e low f i e l d peak ( d a t a not shown). The g r a d u a l d e c r e a s e i n S i g n a l I I U + S d i s c u s s e d below ( S e c t i o n 5.3.1.3) was a l m o s t c o m p l e t e l y o b s c u r e d by t h e much l a r g e r i n c r e a s e i n S i g n a l I . -146- 5.3.1.2 Mn"1"1" S i g n a l I n h e a l t h y r a d i s h l e a v e s t h e Mn s i g n a l i s v e r y s m a l l r e g a r d l e s s o f l i g h t regime. However, upon f u m i g a t i o n w i t h 400ppm S 0 2 t h e s i g n a l was found t o i n c r e a s e g r a d u a l l y o v e r 1 hour. The M n + + s i g n a l s from h e a l t h y and f u m i g a t e d r a d i s h l e a v e s a r e shown i n F i g u r e 50. The M n + + s i g n a l k i n e t i c s were m o n i t o r e d by l o c k i n g t h e s p e c t r o m e t e r onto t h e f a r low f i e l d peak (see F i g u r e 50) o r onto t h e minimum a s s o c i a t e d w i t h t h e second peak from t h e h i g h f i e l d end o f t h e spectrum. The k i n e t i c s o f t h e i n c r e a s e o f t h e o v e r a l l M n + + s i g n a l i n t h e l e a v e s f u m i g a t e d w i t h S 0 2 a r e shown i n F i g u r e 51a, o b t a i n e d by t r a c k i n g t h e r i s e i n t h e low f i e l d peak. 5.3.1.3 S i g n a l H u + S F o r s i g n a l I I U + S s t u d i e s t h e s p e c t r o m e t e r was l o c k e d onto t h e h i g h f i e l d minimum o f S i g n a l I I U + S (see F i g u r e 8 ) . I n d a r k n e s s , S i g n a l H u + S i s not confounded w i t h S i g n a l I a t t h i s p o s i t i o n because l i g h t i s r e q u i r e d t o i n d u c e S i g n a l I . S i g n a l I I U + S was a l s o f o l l o w e d a t t h e low f i e l d maximum (see F i g u r e 8) under a l l l i g h t r e g imes. K i n e t i c s were i d e n t i c a l , r e g a r d l e s s o f l i g h t ; t h u s o n l y t h e h i g h f i e l d minimum k i n e t i c s r e c o r d e d i n d a r k n e s s a r e shown i n F i g u r e 51b. Unfumigated l e a v e s h e l d i n d a r k n e s s showed a v e r y s m a l l change i n S i g n a l I I U + S d u r i n g one hour ( F i g u r e 4 4 a ) . I n c o n t r a s t , l e a v e s f u m i g a t e d w i t h 400ppm -147- 100 G F i g u r e 50. The Mn s i g n a l i n h e a l t h y and f u m i g a t e d r a d i s h l e a f p i e c e s , a. H e a l t h y l e a f ; b. Same l e a f a f t e r f u m i g a t i o n w i t h 400ppm SO, f o r 50 m i n u t e s . The c a v i t y was i l l u m i n a t e d w i t h 710nm l i g h t d u r i n g f u m i g a t i o n and r e c o r d i n g o f t h e t r a c e s . -148- M n + + S i g n a l b a s e l i n e b a s e l i n e S i g n a l I l u + S 30 t i m e (min) 60 F i g u r e 51. Comparative k i n e t i c s o f t h e i n c r e a s e i n t h e Mn 1 s i g n a l and t h e d e c r e a s e i n S i g n a l I I U + S i n e x c i s e d r a d i s h l e a f p i e c e s d u r i n g f u m i g a t i o n w i t h 400ppm SO,. The c a v i t y was n o t i l l u m i n a t e d d u r i n g f u m i g a t i o n and r e c o r d i n g o f t r a c e s . Arrows ( j ) i n d i c a t e t i m e a t which f u m i g a t i o n began. -149- S 0 2 , showed a g r a d u a l d e c r e a s e i n S i g n a l I I U + S d u r i n g t h e c o u r s e o f f u m i g a t i o n . A f t e r 1 hour o f f u m i g a t i o n S i g n a l I I U + S e s s e n t i a l l y d i s a p p e a r e d , l e a v i n g o n l y t h e s m a l l q u i n o i d a l s i g n a l shown i n F i g u r e s 8c,9c. A p o i n t o f i n t e r e s t was t h e f i n d i n g t h a t c u r v e s o f S i g n a l I I U + S d e c r e a s e and M n + + s i g n a l i n c r e a s e had s i m i l a r s l o p e s ( F i g u r e s 51a and b ) . 5.3.2 A t t a c h e d , I n t a c t G r a s s Leaves 5.3.2.1 H i g h S 0 2 L e v e l s A t h i g h (100-500ppm) S 0 2 l e v e l s , changes i d e n t i c a l t o t h o s e w h i c h o c c u r e d i n S i g n a l I under w h i t e and 650nm l i g h t i n e x c i s e d p i e c e s o f r a d i s h l e a v e s , a l s o o c c u r r e d i n i n t a c t , a t t a c h e d Kentucky b l u e g r a s s and p e r e n n i a l r y e g r a s s l e a v e s . The t o p t r a c e s i n F i g u r e 52 shows EPR s i g n a l s from a h e a l t h y p e r e n n i a l r y e g r a s s l e a f h e l d i n t h e d i f f e r e n t l i g h t r egimes p r i o r t o t h e s t a r t o f f u m i g a t i o n . The bottom t r a c e s i n F i g u r e 52b show comparable s i g n a l s from t h e same p o r t i o n o f t h e l e a f a f t e r 4 0 m i n u t e s o f f u m i g a t i o n w i t h lOOppm S 0 2 . The l e a f was i l l u m i n a t e d w i t h 7l0nm l i g h t d u r i n g t h e f u m i g a t i o n p e r i o d . The s i g n a l r e c o r d e d i n d a r k n e s s ( S i g n a l I I U + S ) has o n l y changed m a r g i n a l l y , w h i l e l a r g e d i f f e r e n c e s have o c c u r r e d i n t h e s i g n a l s r e c o r d e d i n 650nm, 710nm and broad-band w h i t e l i g h t . The -150- 10 G F i g u r e 52. EPR s i g n a l s from h e a l t h y and S 0 2 - f u m i g a t e d a t t a c h e d , i n t a c t p e r e n n i a l r y e g r a s s l e a v e s . Top = h e a l t h y l e a f ; Bottom = same l e a f a f t e r 4 0min f u m i g a t i o n w i t h lOOppm S 0 2 • S p e c t r a r e c o r d e d i n a. d a r k n e s s ; b. i n 650nm l i g h t ; c. i n 710nm l i g h t ; d. i n w h i t e l i g h t . The c a v i t y was i l l u m i n a t e d w i t h 710nm l i g h t d u r i n g f u m i g a t i o n . -151- k i n e t i c s o f t h e changes c o u l d n o t be a c c u r a t e l y f o l l o w e d because o f s h i f t s i n t h e M n + + and F e + + s i g n a l s w h i c h u n d e r l y t h e p h o t o s y n t h e t i c s i g n a l s . However, i t was a p p a r e n t t h a t v e r y h i g h c o n c e n t r a t i o n s o f S 0 2 caused a more r a p i d i n c r e a s e i n t h e s i g n a l s t h e n c o m p a r a t i v e l y low c o n c e n t r a t i o n s . As w i t h r a d i s h , s i m i l a r changes i n t h e 650nm and broad-band w h i t e l i g h t - i n d u c e d s i g n a l s o c c u r r e d w i t h S 0 2 c o n c e n t r a t i o n s o f 100, 200, 300, 400 and 500ppm i f t h e p l a n t s were f u m i g a t e d i n d a r k n e s s , 650nm l i g h t , 710nm l i g h t o r broad-band w h i t e l i g h t ( d a t a n o t shown). 5.3.2.2 Low S 0 2 L e v e l s The use o f i n t a c t l e a v e s a l l o w s f o r l o n g e r t i m e p e r i o d s o f f u m i g a t i o n a t l o w e r c o n c e n t r a t i o n s w i t h o u t t h e c o n f o u n d i n g o f r e s u l t s caused by t h e a r t i f i c i a l a g i n g (see S e c t i o n 3.3.1) wh i c h o c c u r s w i t h e x c i s e d l e a f p i e c e s . R e s u l t s w i t h Kentucky b l u e g r a s s and p e r e n n i a l r y e g r a s s were i d e n t i c a l . The s p e c t r a p r e s e n t e d h e r e were o b t a i n e d from Kentucky b l u e g r a s s l e a v e s , w h i c h r e v e a l t h e s m a l l e r M n + + s i g n a l s , t h u s a l l o w i n g more r e l i a b l e c a l c u l a t i o n o f g - v a l u e s and l i n e w i d t h s . F i g u r e 53 shows t h e 710nm l i g h t - i n d u c e d EPR s i g n a l s o f a Kentucky b l u e g r a s s l e a f r e c o r d e d p r i o r t o f u m i g a t i o n ( F i g u r e 53a) and a f t e r 120 mi n u t e s f u m i g a t i o n w i t h 600ppb S 0 2 ( F i g u r e 53b). I t a l s o shows t h e s i g n a l i n d u c e d by w h i t e l i g h t a f t e r 120 min u t e s o f f u m i g a t i o n w i t h 600ppb S 0 2 ( F i g u r e 5 3 c ) . The -152- 3236 3276 M a g n e t i c f i e l d (Gauss) F i g u r e 53. L i g h t - i n d u c e d EPR s i g n a l changes i n a t t a c h e d , i n t a c t K e n t u c k y b l u e g r a s s l e a v e s f u m i g a t e d w i t h 600ppb S 0 2 - The c a v i t y was i l l u m i n a t e d w i t h 710nm l i g h t d u r i n g f u m i g a t i o n , e x c e p t when t r a c e s were r e c o r d e d i n d a r k n e s s and i n w h i t e l i g h t . A l l t r a c e s i n d i c a t e t h e s i g n a l o b t a i n e d by s u b t r a c t i n g S i g n a l I I U + , o b t a i n e d i n d a r k n e s s , from t h e s i g n a l o b t a i n e d i n l i g h t , a. I n 710nm l i g h t , p r i o r t o f u m i g a t i o n ; b. I n 710nm l i g h t , a f t e r 12 0min f u m i g a t i o n ; c. I n w h i t e l i g h t ; a f t e r 120min f u m i g a t i o n . Microwave f r e q u e n c y - 9.190. -153- t r a c e s i n t h e s e f i g u r e s were o b t a i n e d by s u b t r a c t i n g S i g n a l I I U + S , o b t a i n e d i n d a r k n e s s , from t h e combined s i g n a l s o b t a i n e d i n 710nm l i g h t and i n w h i t e l i g h t . The l e a f was m a i n t a i n e d i n 710nm l i g h t d u r i n g t h e f u m i g a t i o n p e r i o d e x c e p t d u r i n g t h e r e c o r d i n g o f t h e s i g n a l s o b t a i n e d i n d a r k n e s s and i n w h i t e l i g h t . F i g u r e 53b i n d i c a t e s a d i m i n u a t i o n o f S i g n a l I a f t e r 120 minutes o f f u m i g a t i o n as t h e g - v a l u e and peak-to-peak w i d t h o f t h i s s i g n a l a r e i d e n t i c a l t o t h o s e o f S i g n a l I shown i n F i g u r e 53a. I n c o n t r a s t , F i g u r e 53c i n d i c a t e s t h e f o r m a t i o n o f a new f r e e r a d i c a l when t h e f u m i g a t e d l e a f i s exposed t o broad-band w h i t e l i g h t . T h i s new w h i t e l i g h t - i n d u c e d s i g n a l ( S i g n a l Ng^) appears t o be s i m i l a r t o S i g n a l I b u t has a h i g h e r g - v a l u e and s l i g h t l y l a r g e r l i n e w i d t h . The peak-to-peak l i n e w i d t h s o f S i g n a l I and S i g n a l Ng^ a r e 7.5 and 8.0 gauss, r e s p e c t i v e l y . Such a s m a l l d i f f e r e n c e c o u l d be a t t r i b u t e d t o r e c o r d e r e r r o r , e s p e c i a l l y as S i g n a l I l i n e w i d t h s r a n g i n g from 7.5 t o 9.0 gauss a r e r e p o r t e d i n t h e l i t e r a t u r e , b u t t h e g - v a l u e s a r e c l e a r l y d i f f e r e n t : S i g n a l I has a g - v a l u e o f 2.0025 w h i l e S i g n a l N g l has a g - v a l u e o f 2.0032. Thus, i t i s c l e a r t h a t t h i s i s a new s i g n a l , n o t a w h i t e l i g h t - i n d u c e d S i g n a l I . I f f u m i g a t i o n i s c o n t i n u e d beyond 120 m i n u t e s , f u r t h e r changes o c c u r . F i g u r e 54 shows t h e EPR d i f f e r e n c e s i g n a l s when f u m i g a t i o n i s c o n t i n u e d f o r an a d d i t i o n a l 60 m i n u t e s . A f t e r 180 -154- m i n u t e s o f f u m i g a t i o n w i t h 600ppb S 0 2 , S i g n a l I i s no l o n g e r p r e s e n t when t h e l e a f i s i l l u m i n a t e d w i t h 710nm l i g h t and a d i f f e r e n t f r e e - r a d i c a l s i g n a l ( S i g n a l N S 2 ) i s r e v e a l e d ( F i g u r e 5 4 a ) . F i g u r e 54b shows t h e w h i t e l i g h t - i n d u c e d s i g n a l a f t e r 180 m i n u t e s o f f u m i g a t i o n . Once a g a i n , t o o b t a i n t h e s i g n a l s shown i n F i g u r e s 54a and 54b, S i g n a l I I U + S , r e c o r d e d w i t h t h e l e a f i n d a r k n e s s , has been s u b t r a c t e d out from t h e combined s p e c t r a o b t a i n e d w i t h t h e l e a f h e l d i n 710nm and broad-band w h i t e l i g h t . F i g u r e 54c, o b t a i n e d by s u b t r a c t i n g t h e s pectrum i n F i g u r e 53c from t h a t i n F i g u r e 54b, shows t h e change i n t h e w h i t e l i g h t - i n d u c e d s i g n a l w h i c h o c c u r s i n t h e i n t e r v a l between 120 and 180 m i n u t e s o f f u m i g a t i o n w i t h 600ppb S 0 2 . T h i s s i g n a l i s i n d i s t i n g u i s h a b l e from S i g n a l N S 2 shown i n F i g u r e 54a. S i g n a l N S 2 , shown i n F i g u r e s 54a and 54c, i s n e a r l y i d e n t i c a l i n l i n e shape and peak-to-peak w i d t h t o t h e r a d i c a l p a i r s i g n a l f i r s t shown by M c i n t o s h and B o l t o n (1979). The p u b l i s h e d g - v a l u e s f o r t h i s s i g n a l v a r y from 2.0026 t o 2.0051, depending upon t h e o r i e n t a t i o n o f t h e sample ( H o f f , 1984). The g - v a l u e o f S i g n a l N S 2 i s 2.0042. These changes i n t h e 710nm and w h i t e l i g h t - i n d u c e d s i g n a l s a r e l o n g - l i v e d , b u t not permanent. I f t h e l e a f i s exposed t o c l e a n a i r f o r 1 hour a f t e r t h e end o f f u m i g a t i o n t h e r e i s no change i n t h e new s i g n a l s ( s p e c t r a not shown), b u t i f t h e l e a f -155- 10 G a b c 3226 3276 M a g n e t i c f i e l d (Gauss) F i g u r e 54. L i g h t - i n d u c e d EPR s i g n a l changes i n a t t a c h e d , i n t a c t K e ntucky b l u e g r a s s l e a v e s f u m i g a t e d w i t h 600ppb S 0 2 . The c a v i t y was i l l u m i n a t e d w i t h 710nm l i g h t d u r i n g f u m i g a t i o n , e x c e p t when t r a c e s were r e c o r d e d i n d a r k n e s s and w h i t e l i g h t . A l l t r a c e s i n d i c a t e t h e s i g n a l o b t a i n e d by s u b t r a c t i n g S i g n a l I I U + , o b t a i n e d i n d a r k n e s s , from t h e s i g n a l o b t a i n e d i n l i g h t , a. I n 710nm l i g h t , a f t e r 180min f u m i g a t i o n ; b. I n w h i t e l i g h t , a f t e r 180min f u m i g a t i o n ; c. Change i n w h i t e l i g h t s i g n a l between 120 and 180 min f u m i g a t i o n ( F i g u r e 54b - 5 3 c ) . Microwave f r e q u e n c y - 9.190. -156- J I J I L 236 3276 M a g n e t i c f i e l d (Gauss) F i g u r e 55. EPR 710nm l i g h t - i n d u c e d and w h i t e l i g h t - i n d u c e d s i g n a l s i n i n t a c t , a t t a c h e d Kentucky b l u e g r a s s l e a v e s 16 h o u r s a f t e r t e r m i n a t i o n o f f u m i g a t i o n w i t h 600ppb SCu. T r a c e s i n d i c a t e t h e d i f f e r e n c e s a f t e r S i g n a l H u + S , o b t a i n e d i n d a r k n e s s , was s u b t r a c t e d from t h e s i g n a l s o b t a i n e d i n 710nm and broad-band w h i t e l i g h t . The c a v i t y was i l l u m i n a t e d w i t h 710nm l i g h t d u r i n g t h e 16 hour exposure t o c l e a n a i r . a. I n 710nm l i g h t ; b. I n w h i t e l i g h t . Microwave f r e q u e n c y - 9.190. -157- i s m a i n t a i n e d i n c l e a n a i r f o r 16 h o u r s , under 710nm l i g h t , most S 0 2 - c a u s e d changes have d i s a p p e a r e d ( F i g u r e 5 5 ) . A p p r o x i m a t e l y 95 p e r c e n t o f t h e 710nm l i g h t - i n d u c e d S i g n a l I has r e c o v e r e d ( c f . F i g u r e s 55a and 53a) w h i l e a l l o f t h e w h i t e l i g h t - i n d u c e d s i g n a l caused by S 0 2 ( S i g n a l N g l ) has d i s a p p e a r e d , b e i n g r e p l a c e d by a s m a l l w h i t e l i g h t - i n d u c e d S i g n a l I ( c f . F i g u r e s 54b and 55b). S i g n a l N S 2 has a l s o d i s a p p e a r e d . However, i f f u m i g a t i o n i s c o n t i n u e d beyond 180 m i n u t e s , t h e s i g n a l w h i c h has p a r a m e t e r s s i m i l a r t o t h e s u p e r o x i d e a n i o n r a d i c a l s i g n a l d i s c u s s e d i n S e c t i o n 4.3 i s formed. F i g u r e 56 shows t h i s s i g n a l ( S i g n a l N S o x ) a f t e r 240 m i n u tes o f f u m i g a t i o n w i t h S 0 2 . T h i s s i g n a l was o b t a i n e d t h r o u g h s u b t r a c t i o n o f t h e w h i t e l i g h t - i n d u c e d s i g n a l a f t e r 180 m i n u t e s o f f u m i g a t i o n from t h e w h i t e l i g h t - i n d u c e d s i g n a l a f t e r 240 m i n u t e s o f f u m i g a t i o n . Once t h i s s t a g e i s r e a c h e d S i g n a l s N S 1 and N S 2 appear t o be i r r e v e r s i b l e as subsequent exposure t o 24 h o u r s o f c l e a n a i r does n o t r e s u l t i n t h e i r e l i m i n a t i o n . However, d e s p i t e t h e a p p a r e n t d i s r u p t i o n o f e l e c t r o n t r a n s p o r t , v i s i b l e s i g n s o f c h l o r o s i s and subsequent n e c r o s i s a r e not a p p a r e n t a t t h i s t i m e . The r e s u l t s d i s c u s s e d above were o b t a i n e d a t f u m i g a t i o n l e v e l s r a n g i n g from 600 t o 2000ppb S 0 2 . I n a l l c a s e s , t h e changes i n t h e s e s i g n a l s were f i r s t n o t i c e a b l e between 100 and 150 m i n u t e s o f f u m i g a t i o n , depending upon t h e i n d i v i d u a l l e a f . -158- 10 G 3236 3276 M a g n e t i c f i e l d (Gauss) F i g u r e 56. S i g n a l N S o x , found i n a t t a c h e d , i n t a c t Kentucky b l u e g r a s s l e a v e s a f t e r f u m i g a t i o n w i t h 600ppb S 0 2 f o r 4 h o u r s . O b t a i n e d by s u b t r a c t i o n o f t h e l i g h t - i n d u c e d s i g n a l a f t e r 180 m i n u t e s o f f u m i g a t i o n from t h e l i g h t - i n d u c e d s i g n a l a f t e r 240 m i n u t e s o f f u m i g a t i o n . ( I r r e s p e c t i v e o f l i g h t r e g i m e ) . Microwave f r e q u e n c y - 9.190. -159- 5.4 D i s c u s s i o n EPR S i g n a l I , whi c h i s found i n p l a n t l e a v e s i l l u m i n a t e d w i t h f a r - r e d (710nm) l i g h t , i s i n d u c e d w i t h l i g h t o f low i r r a d i a n c e and r e a c h e s s a t u r a t i o n a t an i r r a d i a n c e o f a p p r o x i m a t e l y 1W m - 2 ( B l u m e n f e l d e t a l . 1974). T h i s s i g n a l can a l s o be i n d u c e d by broad-band w h i t e l i g h t o f " s u f f i c i e n t " i n t e n s i t y (Tikhonov and Ruuge, 1975a). Upon l e a f i l l u m i n a t i o n w i t h r e d l i g h t (634nm), S i g n a l I i s not i n d u c e d a t i r r a d i a t i o n i n t e n s i t i e s w hich s a t u r a t e t h e s i g n a l when 710nm l i g h t i s used as t h e i r r a d i a t i o n s o u r c e . When h i g h e r i n t e n s i t i e s o f 634nm l i g h t a r e used f o r i r r a d i a t i o n , t h e n S i g n a l I i s formed, b u t s a t u r a t i o n i s n o t a c h i e v e d a t i n t e n s i t i e s as h i g h as 100W m ( B l u m e n f e l d e t a l . 1974). The e l e c t r o n i n t e r s y s t e m t r a n s p o r t c h a i n i n c h l o r o p l a s t s u s p e n s i o n s can be b l o c k e d by i n h i b i t o r s such as DCMU, whic h b l o c k r e o x i d a t i o n o f QA by QB ( V e l t h u y s and Amesz, 1974). Thus, when c h l o r o p l a s t s u s p e n s i o n s a r e t r e a t e d w i t h DCMU, S i g n a l I can be i n d u c e d by r e d (Babcock and Sauer, 1975a) and w h i t e (Babcock and Sauer, 1975b) l i g h t . S i m i l a r l y , when l e a f p i e c e s a r e i n f i l t r a t e d w i t h DCMU, comparable i n t e n s i t i e s o f S i g n a l I a r e a c h i e v e d a t i r r a d i a t i o n i n t e n s i t i e s o f 1.2W m - 2 w i t h i r r a d i a t i o n by e i t h e r 710 o r 634nm l i g h t ( B l u m e n f e l d e t a l . 1974). Data p r e s e n t e d h e r e , based on f u m i g a t i o n o f p i e c e s o f r a d i s h l e a v e s , s u g g e s t t h a t a s i m i l a r b l o c k a g e o f t h e e l e c t r o n -160- t r a n s p o r t c h a i n i s caused by f u m i g a t i o n o f l e a v e s w i t h l e v e l s o f S 0 2 e x c e e d i n g lOppm. I n t h e absence o f f u m i g a t i o n , broad-band w h i t e and 650nm r e d l i g h t o n l y i n d u c e d a m i n i m a l S i g n a l I f o r m a t i o n i n h e a l t h y l e a v e s ( F i g u r e 4 4 ) . A f t e r f u m i g a t i o n , t h e w h i t e - l i g h t i n d u c e d s i g n a l exceeded t h a t o f t h e 710nm l i g h t - i n d u c e d s i g n a l w h i l e t h e 650nm l i g h t - i n d u c e d s i g n a l was comparable t o t h e 710nm l i g h t - i n d u c e d s i g n a l ( F i g u r e 4 4 ) . T h i s i s r e a d i l y e x p l a i n e d by t h e f a c t t h a t t h e w h i t e l i g h t used i n t h i s s t u d y was o f c o n s i d e r a b l y h i g h e r i n t e n s i t y t h a n t h e 710nm l i g h t . I n c o n t r a s t , t h e 650 and 7l0nm l i g h t beams were o f more comparable i n t e n s i t y ( T a b l e 1 ) . S i g n a l f o r m a t i o n was l i g h t - dependent, b u t t h e b l o c k a g e o f t h e i n t e r s y s t e m t r a n s p o r t c h a i n was not l i g h t dependent, as t h e r e l a t i v e p o s t - f u m i g a t i o n s i g n a l i n t e n s i t i e s were s i m i l a r upon i r r a d i a t i o n w i t h 710nm, 650nm and broad-band w h i t e l i g h t , r e g a r d l e s s o f whether t h e f u m i g a t i o n o c c u r r e d i n any l i g h t regime o r i n d a r k n e s s . F i g u r e s 47-49 i n d i c a t e t h a t t h e b l o c k a g e i s dependent upon t o t a l e x p o s u r e t o S 0 2 . A f t e r an i n i t i a l l a g p e r i o d o f j u s t o v e r 3 0 seconds, S i g n a l I i n c r e a s e d s t e a d i l y as f u m i g a t i o n c o n t i n u e d , u n t i l a maximum s t e a d y - s t a t e s i g n a l was a t t a i n e d a f t e r 9 t o 15 mi n u t e s o f f u m i g a t i o n , depending upon t h e l e a f . I f f u m i g a t i o n o c c u r r e d f o r l e s s t h a n 9 m i n u t e s , a s t e a d y s t a t e s i g n a l o f l e s s t h a n maximum i n t e n s i t y was a t t a i n e d . Maximum and l e s s t h a n maximum s t e a d y - s t a t e s i g n a l s were i r r e v e r s i b l e . Once a s p e c i f i c l e v e l o f b l o c k a g e had o c c u r r e d , t h e subsequent -161- t e r m i n a t i o n o f f u m i g a t i o n d i d not r e v e r s e t h e p r o c e s s . S i g n a l I o b t a i n e d w i t h w h i t e o r 650nm l i g h t remained a t t h e h e i g h t a t t a i n e d d u r i n g f u m i g a t i o n . S i m i l a r e f f e c t s o f S 0 2 on S i g n a l I were o b s e r v e d a t c o n c e n t r a t i o n s down t o lOppm. However, t h e l a g p e r i o d p r i o r t o i n d u c t i o n o f t h e w h i t e and 650nm l i g h t - i n d u c e d S i g n a l I p r o g r e s s i v e l y i n c r e a s e d as S 0 2 c o n c e n t r a t i o n was d e c r e a s e d . The t o t a l e xposure r e q u i r e d t o i n i t i a t e t h e i n c r e a s e i n S i g n a l I was s i m i l a r , c o n s i d e r i n g t h e v a r i a b i l i t y w i t h i n i n d i v i d u a l l e a f segments, a t c o n c e n t r a t i o n s o f 50, 100 and 2 00ppm S 0 2 . However, when t h e c o n c e n t r a t i o n was r e d u c e d t o lOppm t h e exposure r e q u i r e d t o i n d u c e S i g n a l I w i t h w h i t e o r 650nm l i g h t was r e d u c e d by a p p r o x i m a t e l y 50 p e r c e n t . T h i s i s a t t r i b u t a b l e t o t h e l o w e r i n g o f t h e r e l a t i v e h u m i d i t y caused by t h e passage o f t h e d r y a i r s t r e a m t h r o u g h t h e c a v i t y f o r t h e 90 minute p e r i o d . R a d i s h stomata have been shown t o open as r e l a t i v e h u m i d i t y d e c r e a s e s ( B l a c k and Unsworth, 1980) and t h u s t h e a c t u a l f l u x o f t h e p o l l u t a n t may w e l l have i n c r e a s e d o v e r t i m e . T h i s i s s i g n i f i c a n t s i n c e i t i s t h e a c t u a l f l u x o f t h e p o l l u t a n t i n t o t h e p l a n t w h i c h d e t e r m i n e s t h e e x t e n t o f damage, not t h e ambient a i r p o l l u t a n t c o n c e n t r a t i o n ( R u n e c k l e s , 1974). I n c r e a s e d o r d e c r e a s e d a i r f l o w , when t h e c o n c e n t r a t i o n o f S 0 2 was m a i n t a i n e d a t i d e n t i c a l l e v e l s , caused no change i n t h e l a g p e r i o d needed t o i n d u c e t h e s i g n a l i n c r e a s e . Ashenden and M a n s f i e l d (1977) showed t h a t p l a n t s were more s u s c e p t i b l e t o -162- a i r p o l l u t a n t s as t h e wind speed i n c r e a s e d . T h i s was a t t r i b u t e d t o a breakdown o f t h e boundary l a y e r component o f p l a n t r e s i s t a n c e t o p o l l u t a n t s . The changed a i r f l o w i n t h i s s t u d y d i d n o t have a s i m i l a r e f f e c t , t h u s s u g g e s t i n g t h a t even t h e l o w e s t a i r f l o w o f 1 1/min ( v e l o c i t y 1 m/s) was adequate t o r e d uce boundary l a y e r r e s i s t a n c e and t h a t t h e a c t u a l f l u x o f t h e p o l l u t a n t was comparable a t a l l t h r e e a i r f l o w r a t e s . The p r e c i s e mechanism t h r o u g h w h i c h S 0 2 b l o c k s e l e c t r o n t r a n s p o r t from P S I I t o PSI cannot be e l u c i d a t e d from r e s u l t s o f t h i s s t u d y , b u t most o f t h e d a t a a r e c o n s i s t e n t w i t h t h e f r e q u e n t l y s t a t e d h y p o t h e s i s t h a t a i r p o l l u t a n t s a f f e c t v e g e t a t i o n t h r o u g h t h e i n d u c t i o n o f v a r i o u s t o x i c a c t i v e oxygen s p e c i e s such as t h e s u p e r o x i d e and h y d r o x y l f r e e r a d i c a l s , and s i n g l e t oxygen (Tanaka and Sugahara, 1980; S h i m a z a k i e t a l . 1980; S a k a k i e t a l . 1983). The h y d r o x y l f r e e r a d i c a l has been i m p l i c a t e d i n t h e d e s t r u c t i v e p h o t o o x i d a t i o n o f c h l o r o p h y l l (Harbour and B o l t o n , 1978) w h i l e s u p e r o x i d e f o r m a t i o n was shown i n i l l u m i n a t e d c h l o r o p l a s t s u s p e n s i o n s under e l e v a t e d l e v e l s o f oxygen (Harbour and B o l t o n , 1975). McRae and Thompson (1983) found e l e v a t e d l e v e l s o f s u p e r o x i d e as p l a n t l e a v e s aged w h i l e Dupont and S i e g e n t h a l e r (1986) s u g g e s t e d t h a t t h e p a r a l l e l breakdown o f cytochromes and b l e a c h i n g o f pigments i n a g i n g t h y l a k o i d membranes may o c c u r as t h e r e s u l t o f f r e e r a d i c a l a c t i v i t y . -163- The r e s u l t s p r e s e n t e d i n F i g u r e s 47-49 a r e c o n s i s t e n t w i t h t h e h y p o t h e s i s t h a t p l a n t l e a v e s a r e a b l e t o t o l e r a t e f r e e r a d i c a l p r o d u c t i o n up t o a t h r e s h o l d t h r o u g h t h e p r o t e c t i v e mechanisms o f endogenous f r e e r a d i c a l s c a v e n g e r s such as s u p e r o x i d e d i s m u t a s e , c a t a l a s e , and - t o c o p h e r o l . However, once t h i s t h r e s h o l d i s r e a c h e d , t h e s c a v e n g e r s can no l o n g e r p r o t e c t t h e l e a v e s , and damage o c c u r s . Support f o r t h i s h y p o t h e s i s i s shown i n F i g u r e 49 as an i n c r e a s e i n l a g t i m e w i t h d e c r e a s i n g c o n c e n t r a t i o n s o f S 0 2 • However, w h i l e a c t i v e oxygen s p e c i e s , o r o t h e r f r e e r a d i c a l s , may have some i n v o l v e m e n t i n t h i s b l o c k i n g o f e l e c t r o n t r a n s p o r t , t h e s t u d i e s w i t h e x c i s e d p i e c e s o f r a d i s h l e a v e s s u b j e c t e d t o h i g h l e v e l s o f s u l p h u r d i o x i d e do no t p r o v i d e any e v i d e n c e f o r t h e i r p r e s e n c e . There i s no s p e c t r o m e t r i c e v i d e n c e i n t h e s t u d i e s u s i n g e x c i s e d r a d i s h l e a f p i e c e s and h i g h l e v e l s o f S 0 2 f o r t h e p r e s e n c e o f t h e s u p e r o x i d e a n i o n r a d i c a l . The f o r m a t i o n o f S i g n a l I under 650nm and broad-band w h i t e l i g h t o c c u r s even i f t h e f u m i g a t i o n t o o k p l a c e i n d a r k n e s s . However, s t u d i e s (Harbour and B o l t o n , 1975,1978; van G i n k e l and R a i s o n , 1980) i n d i c a t e t h a t l i g h t i s n e c e s s a r y f o r t h e i n d u c t i o n o f t h e s u p e r o x i d e a n i o n r a d i c a l i n c h l o r o p h y l l - c o n t a i n i n g systems. Thus, i t must be c o n c l u d e d t h a t , i f a c t i v e oxygen s p e c i e s p l a y a r o l e i n t h i s impairment o f e l e c t r o n t r a n s f e r caused by h i g h l e v e l s o f S 0 2 , i t i s o n l y a minor r o l e , i n r e l a t i o n t o e f f e c t s i n d u c e d by S 0 2 i t s e l f o r t h e b i s u l p h i t e i o n . S a k a k i and Kondo (1983) showed t h a t i n t r a c e l l u l a r s u l p h i t e a c c u m u l a t i o n i n p r o t o p l a s t s i n an -164- u n m e t a b o l i z e d s t a t e was r e s p o n s i b l e f o r t h e i n h i b i t i o n o f p r o t o p l a s t p h o t o s y n t h e s i s . The b l o c k a g e o f e l e c t r o n t r a n s f e r may o c c u r as a r e s u l t o f p e r t u r b a t i o n o f QB, r e s u l t i n g i n a b l o c k a g e o f t h e p l a s t o q u i n o n e b i n d i n g s i t e a f f e c t e d by DCMU and r e l a t e d h e r b i c i d e s . D a n i e l l and S a r o j i n i (1981) s u g g e s t e d t h a t t h e mechanism o f s u l p h i t e a c t i o n i n c h l o r o p l a s t s u s p e n s i o n s i s p e r t u r b a t i o n o f t h e Q p o o l (QA+QB). A l t e r n a t i v e l y , t h e p l a s t o q u i n o n e , cytochrome complex o r p l a s t o c y a n i n components o f t h e t r a n s f e r c h a i n may be a f f e c t e d o r some component o f Photosystem I I , o t h e r t h a n QB, may be a l t e r e d . However, t h e s i m i l a r i t y o f re s p o n s e when l e a v e s a r e t r e a t e d w i t h e i t h e r DCMU ( B l u m e n f e l d e t a l . 1974) o r S 0 2 ( t h i s s t u dy) s u g g e s t s t h a t t h e s i t e o f p e r t u r b a t i o n i s QB, b u t t h e e v i d e n c e i s n o t c o n c l u s i v e . The l o n g e r - t e r m low l e v e l S 0 2 s t u d i e s w i t h a t t a c h e d , i n t a c t K entucky b l u e g r a s s l e a v e s d i s c u s s e d i n S e c t i o n 5.3.2.2 i n d i c a t e d t h a t a f r e e - r a d i c a l s i g n a l w i t h p a r a m e t e r s s i m i l a r t o t h e s u p e r o x i d e a n i o n r a d i c a l i s d e t e c t a b l e i f f u m i g a t i o n i s extende d . Thus, i t must be c o n c l u d e d t h a t i f s u p e r o x i d e i s formed d u r i n g b r i e f p e r i o d s o f f u m i g a t i o n w i t h h i g h l e v e l s o f S 0 2 , t h e n i t s s i g n a l i s so t r a n s i e n t t h a t i t i s u n d e t e c t a b l e . The l a c k o f d e t e c t a b l e s u p e r o x i d e f r e e - r a d i c a l s i g n a l i n t h e p r e s e n t s t u d i e s p r o v i d e s s u p p o r t f o r t h e i n v e s t i g a t i o n by C o v e l l o and Thompson (1985). These i n v e s t i g a t o r s found n e i t h e r -165- t h e s p i n - t r a p p e d s i g n a l from t h e s u p e r o x i d e f r e e r a d i c a l n o r t h a t from t h e h y d r o x y l f r e e r a d i c a l w i t h t h e use o f t h e s p i n t r a p s , T i r o n and DMPO, r e s p e c t i v e l y , when c h l o r o p l a s t s u s p e n s i o n s which c o n t a i n e d b i s u l p h i t e i o n s a t c o n c e n t r a t i o n s r a n g i n g from 1 t o 5mM were i l l u m i n a t e d . However, t h e a d d i t i o n o f SOD t o t h e s e amended c h l o r o p l a s t s u s p e n s i o n s e l i m i n a t e d t h e f o r m a t i o n o f t h e s p i n - t r a p p e d s u l p h u r t r i o x i d e f r e e - r a d i c a l s i g n a l . These r e s u l t s s u g g e s t t h a t t h e s u p e r o x i d e f r e e r a d i c a l i s n e c e s s a r y f o r t h e i n i t i a t i o n o f s u l p h i t e o x i d a t i o n b u t does no t p a r t i c i p a t e i n t h e c h a i n r e a c t i o n o f s u l p h i t e o x i d a t i o n by c h l o r o p l a s t s ( C o v e l l o and Thompson, 1985). Thus, i t i s more l i k e l y t h a t t h e demonstrated e f f e c t on f r e e r a d i c a l f o r m a t i o n i n t h e h i g h - l e v e l S 0 2 e x p e r i m e n t s o f t h e p r e s e n t s t u d y i s t h e r e s u l t o f a g r a d u a l e l i m i n a t i o n o f b i n d i n g s i t e s on QB, not an immediate i n c r e a s e i n s u p e r o x i d e f o r m a t i o n . The d a t a i n F i g u r e s 47-49 can be v a l i d l y i n t e r p r e t e d t o s u p p o r t t h i s h y p o t h e s i s . The r e s u l t s have c o n f i r m e d t h e e a r l i e r i n v e s t i g a t i o n s o f Rowlands e t a l . (1970) w i t h r e g a r d t o t h e i n c r e a s e i n t h e M n + + s i g n a l and t h e d e c r e a s e i n S i g n a l H u + S a f t e r f u m i g a t i o n w i t h h i g h c o n c e n t r a t i o n s o f S 0 2 f o r 1 o r more h o u r s . F i g u r e 51 shows t h e p a r a l l e l i n c r e a s e i n t h e M n + + s i g n a l and d e c r e a s e i n S i g n a l H u + S w h i c h o c c u r when l e a v e s a r e f u m i g a t e d w i t h S 0 2 . The d i s a p p e a r a n c e o f S i g n a l H u + S i n d i c a t e s t h a t t h e o x i d a t i o n o f t h e unknown s p e c i e s needed t o produce t h i s s i g n a l has been e l i m i n a t e d . S i n c e S i g n a l H u + S i s a t t r i b u t e d t o D -166- (Boussac and E t i e n n e , 1982a, 1982b), a sec o n d a r y donor t o P 6 8 0 + , w h i c h i s l o c a t e d near t h e r e a c t i o n c e n t r e o f P S I I ( G o v i n d j e e e t a l . 1985), t h e d i s a p p e a r a n c e o f S i g n a l H u + S may be i n d i c a t i v e o f damage t o t h e r e a c t i o n c e n t r e o f P S I I as h y p o t h e s i z e d by S h i m a z a k i e t a l . (1984b). The e x a c t manganese component o f t h e l e a f w h i c h g i v e s r i s e t o t h e i n c r e a s e d M n + + s i g n a l can not be d e f i n i t i v e l y i d e n t i f i e d from t h e d a t a a v a i l a b l e . However, t h e p a r a l l e l i n v e r s e k i n e t i c s o f t h e M n + + s i g n a l and S i g n a l H u + S shown i n F i g u r e 51 s t r o n g l y s u g g e s t t h a t t h e g r a d u a l d e c r e a s e i n S i g n a l I I U + S i s r e l a t e d t o t h e change i n s t a t e o f t h e manganese p o o l r e p r e s e n t e d by t h e M n + + s i g n a l . I t has been shown t h a t t h e environment o f M n + + p r o t e i n s can g r e a t l y a f f e c t b o t h t h e i n t e n s i t y and shape o f t h e M n + + EPR spectrum (Reed and Cohn, 1970,; Reed and Ray, 1971; Reed e t a l . 1971). Thus, i t i s p o s s i b l e t h a t changes i n t h e environment o f e i t h e r t h e weakly o r v e r y s t r o n g l y bound p o o l s o f M n + + a r e r e s p o n s i b l e f o r t h e M n + + s i g n a l i n c r e a s e . A l t e r n a t i v e l y , t h e i n c r e a s e i n t h e M n + + s i g n a l may be a t t r i b u t e d t o a l o o s e n i n g o f t h e s t r o n g l y bound p o o l o f M n + + a c t i v e l y a s s o c i a t e d w i t h oxygen e v o l u t i o n . S 0 2 may t h u s a c t i n a manner s i m i l a r t o T r i s ( B l a n k e n s h i p and Sauer, 1974) o r hea t t r e a t m e n t (Wydrzynski and Sauer, 1980), w h i c h r e l e a s e t h e s t r o n g l y bound M n + + t o t h e i n t e r i o r o f t h e t h y l a k o i d membrane and r e s u l t i n t h e c o n c o m i t a n t appearance o f t h e Mn EPR s i g n a l . The Mn EPR s i g n a l may a l s o r e s u l t from a s h i f t i n manganese v a l e n c e from t h e h i g h e r o x i d a t i o n s t a t e s , w h i c h a r e EPR s i l e n t , t o M n + + . T h i s c o u l d o c c u r from a S 0 2 - i n d u c e d b l o c k i n t h e S - s t a t e model o f Kok e t a l . (1970). I n e i t h e r c a s e , S 0 2 - c a u s e d p e r t u r b a t i o n o f t h e o x y g e n - e v o l v i n g complex i s i n d i c a t e d . The p a r a l l e l k i n e t i c s s u g g e s t t h a t n o t o n l y does t h e M n + + donate an e l e c t r o n t o Z +, t h e p r i m a r y donor t o P 6 8 0 + , b u t a l s o t o D, t h e a u x i l i a r y donor r e p r e s e n t e d by S i g n a l I I U + S . P r e v i o u s l y , S h i m a z a k i and Sugahara (1980) p r e s e n t e d e v i d e n c e from f l u o r e s c e n c e s t u d i e s which i n d i c a t e d t h a t S 0 2 f u m i g a t i o n caused a d e c r e a s e i n t h e s i z e o f t h e Q p o o l . T h i s was a t t r i b u t e d t o a S 0 2 - c a u s e d d e c r e a s e i n p h o t o r e d u c t i o n o f Q r e s u l t i n g from S 0 2 i n a c t i v a t i o n o f t h e p r i m a r y e l e c t r o n donor o r t h e r e a c t i o n c e n t r e o f P S I I . S u b s e q u e n t l y , S h i m a z a k i e t a l . (1984b), a g a i n u s i n g f l u o r e s c e n c e s t u d i e s , showed r e a c t i o n c e n t r e damage t o t h e w a t e r - s p l i t t i n g enzyme system. The e v i d e n c e p r e s e n t e d h e r e , showing t h e d e c r e a s e i n EPR S i g n a l I I U + S and c o n c o m i t a n t i n c r e a s e i n t h e M n + + s i g n a l i s c o n s i s t e n t w i t h t h e s e c o n c l u s i o n s o f a t l e a s t an i n d i r e c t p e r t u r b a t i o n o f t h e o x y g e n - e v o l v i n g system and c o n c o m i t a n t impairment o f t h e r e a c t i o n c e n t r e o f P S I I . However, s i n c e t h e EPR s p e c t r a do n o t i n d i c a t e w h i c h M n + + p o o l i s r e p r e s e n t e d by t h e enhanced s i g n a l , and, as t h e r o l e o f D, whi c h i s r e p r e s e n t e d -168- by S i g n a l I I U + S , i s o n l y m a r g i n a l l y u n d e r s t o o d a t p r e s e n t , t h e EPR d a t a p r e s e n t e d h e r e i n a r e n o t c o n c l u s i v e . N o n e t h e l e s s , t h e d a t a o f F i g u r e 51 s u g g e s t a d e f i n i t e i n t e r a c t i o n between t h e s p e c i e s r e s p o n s i b l e f o r S i g n a l I I U + S and t h e M n + + s i g n a l . The Japanese i n v e s t i g a t o r s d i d n o t f i n d any e v i d e n c e f o r impairment o f t h e i n t e r s y s t e m t r a n s p o r t c h a i n ( S h i m a z a k i e t a l . 1984b). The r e a s o n f o r t h i s d i s c r e p a n c y between t h e i r r e s u l t s and r e s u l t s o f t h i s s t u d y i s r e l a t e d t o t h e t o t a l e x p o s ure r e q u i r e d t o cause i n t e r s y s t e m t r a n s p o r t breakdown. They s t u d i e d c h l o r o p l a s t s i s o l a t e d from l e a v e s f u m i g a t e d w i t h 2ppm S 0 2 f o r 45 m i n u t e s . I n c o n t r a s t , t h e s t u d i e s w i t h l e a f p i e c e s r e p o r t e d i n t h e p r e s e n t i n v e s t i g a t i o n i n d i c a t e d t h a t 90 m i n u t e s o f f u m i g a t i o n a t lOppm S 0 2 was r e q u i r e d t o i n d u c e t h e b l o c k a g e o f e l e c t r o n t r a n s p o r t from P S I I t o P S I . The f a c t t h a t s i m i l a r r e s u l t s were o b t a i n e d w i t h e x c i s e d r a d i s h l e a f p i e c e s and i n t a c t , a t t a c h e d Kentucky b l u e g r a s s and p e r e n n i a l r y e g r a s s l e a v e s i n d i c a t e s t h a t t h e changes i n s i g n a l s a r e a f u n c t i o n o f t h e h i g h c o n c e n t r a t i o n s o f S 0 2 , n o t o f t h e e x c i s i o n . I t appears t h a t t h e p l a n t s s u f f e r a m a s s i v e 'shock' when exposed t o t h e s e h i g h l e v e l s o f S 0 2 . However, when t h e r e s u l t s from t h e s e h i g h l e v e l S 0 2 s t u d i e s a r e c o n t r a s t e d w i t h r e s u l t s o b t a i n e d from s t u d i e s u s i n g l o w e r , more r e a l i s t i c c o n c e n t r a t i o n s o f fumigant and i n t a c t l e a v e s , i t i s r e a d i l y a p p a r e n t t h a t t h e r e s u l t s w i t h t h e h i g h gas c o n c e n t r a t i o n s can -169- not be e x t r a p o l a t e d t o most r e a l w o r l d s i t u a t i o n s because such h i g h c o n c e n t r a t i o n s o f S 0 2 a r e l i k e l y t o o c c u r o n l y i n b r i e f e p i s o d e s caused by i n d u s t r i a l a c c i d e n t s . The d a t a from t h e s t u d i e s i n v o l v i n g l o w e r c o n c e n t r a t i o n s o f S 0 2 a r e much more r e l e v a n t t o r e a l i s t i c S 0 2 p o l l u t i o n e p i s o d e s . Here, t h e e l i m i n a t i o n o f S i g n a l I s u g g e s t s t h a t f u m i g a t i o n w i t h S 0 2 e l i m i n a t e s t h e l i g h t - c a t a l y z e d o x i d a t i o n o f t h e c h l o r o p h y l l a o f t h e r e a c t i o n c e n t r e o f P S I . T h i s may o c c u r because t h e S 0 2 d e s t r o y s t h e c h l o r o p h y l l o f e i t h e r t h e r e a c t i o n c e n t r e o r t h e l i g h t - h a r v e s t i n g complex. The d e s t r u c t i o n o f c h l o r o p h y l l by b i s u l p h i t e has been de m o n s t r a t e d i n i n v i t r o systems ( P e i s e r and Yang, 1977). The new w h i t e - l i g h t i n d u c e d s i g n a l ( S i g n a l N S 1 ) , w h i l e n o t c o n c l u s i v e l y i d e n t i f i a b l e , may w e l l be t h e EPR m a n i f e s t a t i o n o f t h e appearance o f t h e S 0 3 ~ f r e e r a d i c a l . The g - v a l u e o f S i g n a l N S 1 i n t h i s s t u d y i s 2.0032 w h i l e r e p o r t e d g - v a l u e s f o r t h e S 0 3 ~ f r e e - r a d i c a l s i g n a l range from 2.0030 t o 2.0033 (Behar and Fessenden, 1972: F l o c k h a r t e t a l . 1971; M o t t l e y e t a l . 1982b). The DMPO-spin-trapped s i g n a l from t h e S 0 3 f r e e r a d i c a l has been shown t o o c c u r i n i l l u m i n a t e d c h l o r o p l a s t s u s p e n s i o n s amended w i t h b i s u l p h i t e ( C o v e l l o and Thompson, 1985) and i n t h e p r o s t a g l a n d i n h y d r o p e r o x i d a s e - c a t a l y z e d o x i d a t i o n o f b i s u l p h i t e ( M o t t l e y e t a l . 1982a). S i g n a l N S 1 i s n o t t h e EPR m a n i f e s t a t i o n o f e i t h e r t h e S 0 2 ~ o r S 0 4 ~ f r e e r a d i c a l as t h e s e have g - v a l u e s o f 2.0058 (Norman and S t o r e y , 1971) and 2.0125 (Er s h o v e t a l . 1971), r e s p e c t i v e l y . The appearance o f t h e r a d i c a l p a i r s i g n a l ( F i g u r e s 54a, 54c) a f t e r e xtended f u m i g a t i o n i s e x p l a i n a b l e i f i t assumed t h a t SC>2, w h i c h i s a r e d u c t a n t , i s c a p a b l e o f r e d u c i n g e l e c t r o n a c c e p t o r s X, B and A. The p r e s e n c e o f t h e i n i t i a l a c c e p t o r s , a Q and a^, has o n l y been shown i n s t r o n g l y r e d u c i n g c o n d i t i o n s (Gast e t a l . 1983; B o n n a r j e a and Evans, 1982) o r w i t h t h e l a t e r a c c e p t o r s p r e v i o u s l y i n a c t i v a t e d ( B a l t i m o r e and M a l k i n , 1980). Under t h e s e c o n d i t i o n s a back r e a c t i o n o c c u r s between P7 00 and t h e e a r l y a c c e p t o r s , and t h r o u g h t h e r a d i c a l p a i r mechanism ( H o f f , 1984) a s i g n a l s i m i l a r i n l i n e - s h a p e and g - v a l u e t o t h a t shown i n F i g u r e s 54a and 54c a p p e a r s . The p r e s e n c e o f d e t e c t a b l e amounts o f t h e s u p e r o x i d e a n i o n f r e e r a d i c a l a f t e r 4 h o u r s o f f u m i g a t i o n i s t h e s t a r t o f t h e p r o c e s s o f n e c r o s i s i n p l a n t s d i s c u s s e d e a r l i e r i n S e c t i o n 4.0. T h i s appears t o be not u n i q u e l y a t t r i b u t a b l e t o S 0 2 p o l l u t i o n b u t o c c u r s whenever t h e d e f e n s e s o f p l a n t s a r e overcome t h r o u g h e x p o s u r e t o d i f f e r e n t s t r e s s e s , i n c l u d i n g S 0 2 • -171- 6.0 GENERAL DISCUSSION The g e n e r a l purpose o f t h e s e s t u d i e s was t h e i n v e s t i g a t i o n , u s i n g X-band EPR s p e c t r o s c o p y a t room t e m p e r a t u r e , o f f r e e r a d i c a l changes i n p l a n t l e a v e s , w h i c h a r e caused by e x p o sure t o t h e gaseous a i r p o l l u t a n t s , ozone and s u l p h u r d i o x i d e . T h i s e n t a i l e d i n v e s t i g a t i o n o f changes i n t h e e s t a b l i s h e d p h o t o - s y n t h e s i s - a s s o c i a t e d f r e e r a d i c a l s r e p r e s e n t e d by S i g n a l I and S i g n a l I I U + S , and i n v e s t i g a t i o n o f t h e f o r m a t i o n and decay of o t h e r f r e e r a d i c a l s w hich o c c u r as a r e s u l t o f e xposure o f l e a v e s t o t h e s e a i r p o l l u t a n t s . P r e v i o u s i n v e s t i g a t i o n s o f such changes were l i m i t e d (Rowlands e t a l . 1970; S h i m a z a k i e t a l . 1984b), and were c o n f i n e d t o t h e d e s c r i p t i o n o f changes i n f r e e - r a d i c a l s i g n a l s a f t e r c o m p l e t i o n o f f u m i g a t i o n . However, t h e s e s t u d i e s c o u l d not r e v e a l t h e c h r o n o l o g y o f any changes i n f r e e r a d i c a l f o r m a t i o n w h i c h might o c c u r d u r i n g f u m i g a t i o n o f p l a n t l e a v e s . To a t t e mpt t o i n v e s t i g a t e such changes, i t was n e c e s s a r y t o d e v e l o p a methodology whereby i n s i t u f r e e - r a d i c a l s i g n a l s c o u l d be d e t e c t e d and f o l l o w e d d u r i n g t h e c o u r s e o f f u m i g a t i o n . C o n s e q u e n t l y , t e c h n i q u e s were d e v e l o p e d t o p e r m i t t h e i n v e s t i g a t i o n o f f r e e - r a d i c a l s i g n a l changes d u r i n g f u m i g a t i o n o f , f i r s t l y , s m a l l e x c i s e d l e a f segments and, s u b s e q u e n t l y , o f i n t a c t , a t t a c h e d l e a v e s o f l o n g , n a r r o w - l e a v e d p l a n t s such as g r a s s e s . P r i o r t o t h e i n v e s t i g a t i o n o f f r e e - r a d i c a l s i g n a l changes -172- caused by f u m i g a t i o n w i t h ozone and s u l p h u r d i o x i d e , s e v e r a l i s s u e s r e l a t i n g t o t h e p o t e n t i a l f o r f r e e r a d i c a l f o r m a t i o n i n s i t u i n p l a n t l e a v e s needed i n v e s t i g a t i o n . These were: a) t h e e l u c i d a t i o n o f c o n s t r a i n t s imposed by d i f f e r e n t p l a n t m a t e r i a l s ; b) t h e e f f e c t o f l e a f a d a p t a t i o n t o l i g h t on f r e e - r a d i c a l s i g n a l f o r m a t i o n ; c) t h e e f f e c t o f t h e c o m p o s i t i o n o f t h e a i r i n t h e c a v i t y on t h e f o r m a t i o n o f S i g n a l s I and I I U + S ; d) t h e e f f e c t o f s p e c t r o m e t e r o p e r a t i o n on f r e e r a d i c a l s i n l e a f t i s s u e ; and e) t h e p o t e n t i a l f o r i d e n t i f y i n g i n d i v i d u a l f r e e - r a d i c a l s i g n a l s i n mixed s p e c t r a by t h e use o f s u b t r a c t i o n t e c h n i q u e s . I n i t i a l i n v e s t i g a t i o n s r e v e a l e d two problems r e l a t e d t o t h e p l a n t m a t e r i a l a v a i l a b l e f o r use i n EPR s t u d i e s o f f r e e r a d i c a l f o r m a t i o n and decay i n p l a n t l e a v e s . The f i r s t o f t h e s e p e r t a i n s t o t h e use o f e x c i s e d l e a f p i e c e s . The p r e s e n t s t u d i e s w i t h e x c i s e d l e a f p i e c e s p r o v i d e c o n f i r m a t o r y e v i d e n c e t h a t an unknown f r e e r a d i c a l ( s ) i s formed upon a g i n g o f p l a n t l e a v e s . P r e v i o u s s t u d i e s had i n d i c a t e d t h a t a f r e e r a d i c a l y i e l d i n g a s i g n a l h a v i n g a g - v a l u e o f a p p r o x i m a t e l y 2.000 i s formed i n p l a n t l e a v e s s u b j e c t e d t o s a l t o r w a t e r s t r e s s ( K h a r i t o n e n k o v and K a l i c h a v a , 1966; C h e t v e r i k o v e t a l . 1967) and upon n a t u r a l a g i n g ( M i s h r a e t a l . 1971; P r i e s t l e y e t a l . 1980). Rowlands e t a l . (1970) a l s o showed t h e f o r m a t i o n o f a s i m i l a r f r e e - r a d i c a l s i g n a l upon extended f u m i g a t i o n o f soybean and p i n t o bean l e a v e s w i t h h i g h l e v e l s o f ozone. U n f o r t u n a t e l y , i n none o f t h e above -173- s t u d i e s was t h e f r e e - r a d i c a l s i g n a l c h a r a c t e r i z e d more p r e c i s e l y ( g = a p p r o x i m a t e l y 2.000). I n t h e p r e s e n t s t u d y , t h e f r e e - r a d i c a l s i g n a l a s s o c i a t e d w i t h s e n e s c e n c e was shown, by s u b t r a c t i o n o f t h e spectrum o b t a i n e d p r i o r t o a g i n g from t h e spectrum o b t a i n e d a f t e r a g i n g , t o have a g - v a l u e o f 2.0046 and a l i n e w i d t h o f a p p r o x i m a t e l y 10 gauss ( F i g u r e l O d ) . T h i s s i g n a l may a l s o be p r e s e n t i n t h e spectrum o b t a i n e d from a c h e r r y l e a f ( F i g u r e 1 7). The s i g n a l from t h e c h e r r y l e a f was not i n v e s t i g a t e d f u r t h e r , b u t younger c h e r r y l e a v e s d i d not show a s i m i l a r s i g n a l . R e g a r d l e s s o f i t s i d e n t i t y , t h e appearance o f t h i s s e n e s c e n c e - r e l a t e d f r e e - r a d i c a l s i g n a l n egates t h e use o f e x c i s e d l e a f p i e c e s f o r f u m i g a t i o n s t u d i e s w h i c h exceed one hour. A f t e r t h a t t i m e i t becomes i m p o s s i b l e t o d e t e r m i n e i f changes i n t h e v i c i n i t y o f t h e 2.000 g - v a l u e a r e a r e s u l t o f t r e a t m e n t o r a r i s e from t h e f o r m a t i o n o f t h i s f r e e r a d i c a l . F u r t h e r m o r e , i t i s n o t p o s s i b l e t o c o r r e c t f o r t h i s s i g n a l by s u b t r a c t i o n because t h e degree o f f r e e r a d i c a l f o r m a t i o n i s unknown, and appears t o v a r y from l e a f t o l e a f . Once formed, t h e s i g n a l i s permanent, and hence i t i s i m p o s s i b l e t o f i r s t r e c o r d i t and t h e n r e t u r n t h e l e a f t o a h e a l t h y s t a t e t o a s c e r t a i n t h e e f f e c t s o f t r e a t m e n t . T h e r e f o r e , i n o r d e r t o u n d e r t a k e s t u d i e s w h i c h i n v o l v e r e a l i s t i c , low l e v e l s o f a i r p o l l u t a n t s and t h u s r e q u i r e t i m e p e r i o d s e x c e e d i n g 1 hour, i t -174- became n e c e s s a r y t o use i n t a c t l e a v e s a t t a c h e d t o p l a n t s . The d i m e n s i o n s o f t h e c a v i t y dewar i n s e r t l i m i t s u ch s t u d i e s t o p l a n t s such as g r a s s e s w h i c h have l o n g , narrow l e a v e s . However, n e i t h e r o f t h e g r a s s e s i n v e s t i g a t e d , K e n t u c k y b l u e g r a s s (Poa p r a t e n s i s L . ) , and p e r e n n i a l r y e g r a s s ( L o l i u m perenne L . ) , p e r m i t t e d i n v e s t i g a t i o n o f t h e k i n e t i c s o f t h e EPR s i g n a l s r e l a t e d t o p h o t o s y n t h e s i s as a f f e c t e d by t h e a i r p o l l u t a n t s . I n each o f t h e s e s p e c i e s , k i n e t i c t r a c k i n g o f changes i n t h e p h o t o s y n t h e t i c s i g n a l s may be masked by changes i n t h e s t r o n g u n d e r l y i n g M n + + and F e + + s i g n a l s , s i n c e t h e s e l a t t e r s i g n a l s a r e a l s o a f f e c t e d by t r e a t m e n t w i t h l i g h t and f u m i g a n t s ( F i g u r e 1 8 ) . I n a d d i t i o n t o t h e s e s p e c i e s used e x t e n s i v e l y f o r t h e p r e s e n t s t u d i e s o f a i r p o l l u t a n t e f f e c t s , o t h e r g r a s s s p e c i e s such as b a r n y a r d g r a s s f E c h i n o c h l o a c r u s g a l l i (L.) Beauv.), r e d f e s c u e ( F e s t u c a r u b r a L . ) , b e n t g r a s s ( A g r o s t i s t e n u i s S i b t h . ) and a n n u a l b l u e g r a s s (Poa annua L.) a l s o r e v e a l e d pronounced i r o n and manganese s i g n a l s . However, k i n e t i c t r a c k i n g i s p o s s i b l e i n l e a v e s w h i c h r e v e a l o n l y m i n i m a l M n + + and F e + + u n d e r l y i n g s i g n a l s . Hence, i n s p i t e o f t h e l i m i t a t i o n s imposed when u s i n g e x c i s e d l e a f p i e c e s , t h e low l e v e l s o f t h e M n + + and F e + + s i g n a l s p r e s e n t i n r a d i s h l e a f t i s s u e p e r m i t t e d t h e f o l l o w i n g o f t h e changes i n p h o t o s y n t h e t i c s i g n a l s i n d u c e d by s u l p h u r d i o x i d e , a l b e i t a t r e l a t i v e l y h i g h c o n c e n t r a t i o n s o f t h e p o l l u t a n t . -175- A second problem r e l a t e d t o t h e s e l e c t i o n o f p l a n t m a t e r i a l c o n c e r n s t h e l i g h t i n t e n s i t y o f t h e ambient environment o f t h e l o c a t i o n where t h e p l a n t s were grown. Leaves from p l a n t s grown i n t h e greenhouse from September t o May were found t o r e v e a l t h e e s t a b l i s h e d p h o t o s y n t h e s i s - r e l a t e d S i g n a l s I and I I U + S ( F i g u r e 2 ) , w h i c h a r e a l s o found i n c h l o r o p l a s t and s u b c e l l u l a r p r e p a r a t i o n s . However, l e a v e s o f p l a n t s grown on greenhouse benches o r o u t s i d e i n f u l l s u n l i g h t from June t o August, r e v e a l e d a new f r e e - r a d i c a l s i g n a l ( S i g n a l N p i ) superimposed upon S i g n a l I I U + S . Depending upon t h e l e a f , S i g n a l I may, o r may n o t , be r e v e a l e d upon i r r a d i a t i o n o f such l e a v e s w i t h 710nm l i g h t . I n most l e a v e s (see s e c t i o n 3.3.5) S i g n a l N p i c o u l d be e l i m i n a t e d upon exposure t o microwave r a d i a t i o n i n t h e s p e c t r o m e t e r c a v i t y f o r 15 m i n u t e s , b u t , upon e l i m i n a t i o n o f t h i s s i g n a l , many l e a v e s r e v e a l e d d i f f e r e n t r e s p o n s e s i n f r e e - r a d i c a l f o r m a t i o n t o i r r a d i a t i o n w i t h t h e d i f f e r e n t l i g h t r egimes t h a n t h e r e s p o n s e s a s s o c i a t e d w i t h l e a v e s from p l a n t s grown under l o w e r l i g h t i n t e n s i t i e s . These changes were a t t r i b u t e d t o p h o t o i n h i b i t i o n damage caused by l e a f exposure t o h i g h photon f l u x d e n s i t y . S i n c e l e a v e s o f p l a n t s grown i n p a r t i a l shade t e n d e d t o respond more u n i f o r m l y and p r e d i c t a b l y , subsequent s t u d i e s i n summertime were c a r r i e d o ut w i t h l e a v e s o f p l a n t s grown i n p a r t i a l shade under greenhouse benches. These d i d n o t r e v e a l S i g n a l N p i , b u t d i d show t h e t y p i c a l p h o t o - s y n t h e t i c f r e e - r a d i c a l s i g n a l s , and were t h u s presumed t o be undamaged by t h e h i g h l i g h t i n t e n s i t y o f u n s h e l t e r e d a r e a s . -176- However, t h e d i s c o v e r y o f t h i s p r e v i o u s l y u n r e p o r t e d S i g n a l Npj r a i s e s an i n t e r e s t i n g p o i n t . S i n c e t h e appearance o f S i g n a l Np-j- i n p l a n t s grown i n f u l l s u n l i g h t p r e v e n t s t h e d e t e c t i o n o f S i g n a l s I and H u + S , as d i s c u s s e d i n S e c t i o n 3.3.5, t h e q u e s t i o n i s r a i s e d as t o whether t h e f r e e r a d i c a l s g i v i n g r i s e t o t h e s e s i g n a l s can s t i l l be i n d u c e d i n s uch t i s s u e s by d i f f e r e n t l i g h t t r e a t m e n t s , o r whether t h e y o n l y o c c u r i n t i s s u e s grown a t reduced l i g h t i n t e n s i t i e s . The o n l y p o s s i b l e e v i d e n c e f o r t h e i r o c c u r r e n c e i n t i s s u e grown i n h i g h l i g h t i n t e n s i t i e s comes i n d i r e c t l y from t h e v a r i o u s s t u d i e s on i s o l a t e d c h l o r o p l a s t s and c h l o r o p l a s t f r a c t i o n s p r e p a r e d from p l a n t s grown under a wide, b u t u n s p e c i f i e d . range o f l i g h t i n t e n s i t i e s (Commoner e t a l . 1956,1957). These s t u d i e s c o n s i s t e n t l y r e v e a l S i g n a l s I and I I U + S , i d e n t i c a l w i t h t h o s e found i n t h e p r e s e n t s t u d i e s w i t h i n t a c t t i s s u e s . I f , as s u g g e s t e d by t h e s t u d i e s d i s c u s s e d i n S e c t i o n 3.3.5, t h e a c c e p t e d e l e c t r o n t r a n s p o r t system o f t h e p h o t o s y n t h e t i c p r o c e s s i s changed under c o n d i t i o n s o f h i g h l i g h t i n t e n s i t y , why do p l a n t s c o n t i n u e t o grow d u r i n g t h e summer? I t appears t h a t an unknown compensatory mechanism may o c c u r i n t h e summer. A n o t h e r f a c t o r r e l e v a n t t o t h e use o f EPR s p e c t r o s c o p y f o r t h e s t u d y o f f r e e r a d i c a l f o r m a t i o n i n l e a f t i s s u e c o n c e r n s t h e e f f e c t o f e x p o sure t o l i g h t o f t h e d i f f e r e n t w a v e l e n g t h s needed t o d i f f e r e n t i a t e between S i g n a l s I and I I U + S , on t h e f o r m a t i o n o f t h e r a d i c a l s r e s p o n s i b l e f o r t h e s e s i g n a l s . F o r example, p r o l o n g e d e xposure t o d a r k n e s s o r broad-band w h i t e l i g h t i n f l u e n c e s t h e f o r m a t i o n o f S i g n a l I when t h e l e a f i s s u b s e q u e n t l y exposed t o f a r - r e d l i g h t . I f l e a v e s a r e k e p t i n d a r k n e s s p r i o r t o i r r a d i a t i o n w i t h f a r - r e d l i g h t t h e r e i s a l a g p e r i o d p r i o r t o t h e f o r m a t i o n o f S i g n a l I . The t i m e p e r i o d i n d a r k n e s s r e q u i r e d f o r i n d u c t i o n o f t h i s l a g p e r i o d v a r i e s g r e a t l y among s p e c i e s , b u t can be as s h o r t as 5 m i n u t e s f o r C h i n e s e r o s e ( H i b i s c u s sp.) l e a v e s (Ruuge and T i k h o n o v , 1975). The l a g p e r i o d i n c r e a s e s as t h e t i m e t h e l e a f i s h e l d i n d a r k n e s s i s i n c r e a s e d . The cause o f t h i s d e l a y i n S i g n a l I f o r m a t i o n has been a t t r i b u t e d t o a b u i l d - u p o f r e d u c t a n t s i n t h e d a r k n e s s (Khangulov and G o l ' d f e l ' d , 1977). However, a more r e a s o n a b l e e x p l a n a t i o n was proposed by Tikhonov and Ruuge (1975a). A f t e r i n f i l t r a t i o n o f l e a v e s w i t h an a r t i f i c a l e l e c t r o n a c c e p t o r , m e t h y l v i o l o g e n , t h e y found t h a t t h e l a g p e r i o d d i d not o c c u r i n d a r k - a d a p t e d l e a v e s . I n c o n t r a s t , when l e a v e s were i n f i l t r a t e d w i t h monuron, w h i c h b l o c k s e l e c t r o n t r a n s p o r t from P S I I t o P S I , t h e l a g p e r i o d s t i l l o c c u r r e d i n d a r k - a d a p t e d l e a v e s . C o n s e q u e n t l y , t h e y s u g g e s t e d t h a t t h e l a g p e r i o d i n d a r k - a d a p t e d l e a v e s i s a s s o c i a t e d w i t h a l t e r e d redox s t a t e s o f t h e e l e c t r o n a c c e p t o r s o f P S I . A s i m i l a r l a g p e r i o d a l s o o c c u r s when l e a v e s a r e exposed t o broad-band w h i t e l i g h t o r r e d l i g h t (Tikhonov and Ruuge, 1975b). However, i n t h i s c a s e t h e l a g p e r i o d o c c u r s as a r e s u l t o f t h e a c t i o n o f t h e e l e c t r o n t r a n s p o r t c h a i n c a r r i e r s w h i c h -178- were re d u c e d d u r i n g exposure t o t h e w h i t e o r r e d l i g h t , and t h u s p r o v i d e e l e c t r o n s f o r immediate r e d u c t i o n o f t h e s p e c i e s r e p r e s e n t e d by S i g n a l I . C o n s e q u e n t l y , i f one w i s h e s t o i n v e s t i g a t e t h e e f f e c t o f e x t e n d e d f u m i g a t i o n on t h e f o r m a t i o n o f f a r - r e d l i g h t - i n d u c e d S i g n a l I f o r any g i v e n d u r a t i o n o f f u m i g a t i o n , i t i s n e c e s s a r y t o m a i n t a i n t h e l e a f i n f a r - r e d l i g h t d u r i n g t h e f u m i g a t i o n p e r i o d . I f t h i s i s not done, t h e f o r m a t i o n o f S i g n a l I w i l l be d e l a y e d , and i f changes i n S i g n a l I w h i c h a r e i n d u c e d by f u m i g a t i o n a r e r e v e r s i b l e , t h e t i m e r e q u i r e d t o r e s t o r e S i g n a l I a f t e r t e r m i n a t i o n o f f u m i g a t i o n may r e s u l t i n t h e r e v e r s a l o f t h e s e changes. I t i s a l s o n e c e s s a r y t o be aware o f t h e p r e v i o u s l y u n r e p o r t e d s i g n a l ( S i g n a l N 7 1 0 ) formed i n undamaged l e a v e s upon p r o l o n g e d exposure t o 710nm l i g h t . The i d e n t i t y o f t h e s p e c i e s r e p r e s e n t e d by S i g n a l N 7 1 Q i s u n c e r t a i n b u t t h e s i g n a l has s i m i l a r i t i e s i n g - v a l u e t o t h a t o f t h e e a r l y e l e c t r o n a c c e p t o r a-^ o f P S I , b u t i s n a r r o w e r i n peak-to-peak l i n e w i d t h . However, t h e p r e c i s e a t t r i b u t e s o f a-^ and a Q have not been c o n c l u s i v e l y e s t a b l i s h e d ; v a r i o u s i n v e s t i g a t o r s have r e p o r t e d s l i g h t l y d i f f e r e n t g - v a l u e s and peak-to-peak l i n e w i d t h s ( S h u v a l o v e t a l . 1979; H e a t h c o t e and Evans, 1980; B a l t i m o r e and M a l k i n 1980; B o n n e r j e a and Evans, 1982; Gast and H o f f , 1979; Gast e t a l . 1983). N o n e t h e l e s s , r e g a r d l e s s o f t h e -179- i d e n t i t y o f t h e s p e c i e s w h i c h g i v e s r i s e t o t h i s s i g n a l , t h e f o r m a t i o n o f S i g n a l N 7 1 0 w i t h i n 1 hour o f exposure t o 710nm l i g h t r e q u i r e s t h e i n c l u s i o n o f t h i s s i g n a l i n t h e b a s e l i n e d a t a w h i c h must be s u b t r a c t e d i f new f r e e - r a d i c a l s i g n a l s r e v e a l e d d u r i n g l e a f exposure t o gaseous p o l l u t a n t s a r e t o be c h a r a c t e r i z e d a c c u r a t e l y . The t r a n s i t i o n from one l i g h t regime t o a n o t h e r causes b r i e f , b u t complex, s h i f t s i n t h e k i n e t i c c u r v e o f S i g n a l I i n d u c t i o n . The e x a c t shape o f t h e k i n e t i c c u r v e i s dependent upon t h e i n t e n s i t y and s p e c t r a l c o m p o s i t i o n o f t h e l i g h t , t h e p l a n t s p e c i e s and i t s p h y s i o l o g i c a l s t a t e (Tikhonov and Ruuge, 1975a). When a l e a f a l r e a d y i l l u m i n a t e d w i t h f a r - r e d l i g h t i s exposed t o a d d i t i o n a l r e d (Tikhonov and Ruuge, 1975a) o r broad-band w h i t e (Andreyeva e t a l . 1980) l i g h t , t h e i n t e n s i t y o f S i g n a l I r a p i d l y d r o p s , q u i c k l y r i s e s t o an o v e r b u r s t , and r a p i d l y d e c r e a s e s a g a i n , b e f o r e a t t a i n i n g an i n t e r m e d i a t e s t e a d y - s t a t e l e v e l . The e n t i r e sequence o c c u r s i n l e s s t h a n one minute. The p r e s e n t s t u d i e s u s i n g t h e V a r i a n E-109 s p e c t r o m e t e r and a t t a c h e d H e w l e t t - P a c k a r d 9835B computer r e q u i r e d a p p r o x i m a t e l y 4 m i n u t e s f o r t h e s t o r a g e o f t h e s i g n a l on t a p e f o r subsequent r e t r i e v a l . Hence t h e s t e a d y - s t a t e w h i t e l i g h t s i g n a l c o u l d be o b t a i n e d i f t h e l e a f was i l l u m i n a t e d by w h i t e l i g h t d u r i n g t h e 4 minute d a t a t r a n s f e r p e r i o d and s u b s e q u e n t l y d u r i n g t h e s c a n p e r i o d . -180- The p r e c i s e cause o f t h e s e r a p i d s h i f t s i n S i g n a l I i n t e n s i t y i s unknown a t p r e s e n t . T i k honov and Ruuge (1975a) i n f i l t r a t e d l e a v e s w i t h N H 4 C l , an u n c o u p l e r o f p h o t o - p h o s p h o r y l a t i o n , and found a monotonic d e c r e a s e t o t h e s t e a d y - s t a t e l e v e l o f S i g n a l I upon a d d i t i o n o f r e d l i g h t i l l u m i n a t i o n t o a l e a f a l r e a d y exposed t o f a r - r e d l i g h t . They h y p o t h e s i z e t h a t t h e r a p i d s h i f t s i n t h e k i n e t i c c u r v e o c c u r as a r e s u l t o f r e d i s t r i b u t i o n o f energy between t h e photosystems due t o p h o t o - i n d u c e d rearrangement o f t h e membranes o f t h e c h l o r o p l a s t s , c o u p l e d w i t h t h e p r o c e s s o f p h o t o p h o s p h o r y l a t i o n . The a d d i t i o n o f u n c o u p l e r s may p r e v e n t t h i s r e arrangement. The p r e s e n t s t u d y shows t h a t s i m i l a r s h i f t s i n t h e k i n e t i c t r a c e o f S i g n a l I , o b t a i n e d from u n f u m i g a t e d r a d i s h l e a f p i e c e s , o c c u r r e d when t h e l i g h t regime was changed from 710 o r 650nm l i g h t t o broad-band w h i t e l i g h t ( F i g u r e 1 1 ) . I n c o n t r a s t , when l e a f p i e c e s were exposed t o 400ppm S 0 2 f o r 10 m i n u t e s , t h e s e r a p i d k i n e t i c s h i f t s d i d n o t o c c u r , and a s t e a d y - s t a t e s i g n a l was a c h i e v e d w i t h o u t t h e appearance o f a m u l t i - p h a s i c k i n e t i c t r a c e ( F i g u r e 12). Thus, h i g h l e v e l s o f S 0 2 may a c t i n a manner a n a l o g o u s t o t h e u n c o u p l e r s o f p h o s p h o r y l a t i o n d i s c u s s e d above. The s u b t r a c t i o n o f s i g n a l s o b t a i n e d a t d i f f e r e n t p o i n t s i n t i m e was used t o c h a r a c t e r i z e new f r e e - r a d i c a l s i g n a l s r e s u l t i n g from f u m i g a t i o n o f l e a v e s w i t h ozone and s u l p h u r d i o x i d e , and -181- o t h e r new s i g n a l s , such as S i g n a l N 7 1 0 and S i g n a l N p i , caused by extended exposure t o 710nin l i g h t and e x p o sure t o h i g h photon f l u x d e n s i t y , r e s p e c t i v e l y . The use o f t h i s t e c h n i q u e i s n e c e s s a r y because t h e s i g n a l s d i s c u s s e d i n t h e p r e s e n t s t u d y o c c u r i n a v e r y narrow band and a r e superimposed upon each o t h e r . S p e c i f i c a l l y , i t can be used t o c l a r i f y t h e t r u e changes i n l i g h t - i n d u c e d f r e e - r a d i c a l s i g n a l s i f u n d e r l y i n g s i g n a l s p r e s e n t i n d a r k n e s s a r e a l s o changed by t r e a t m e n t . T h i s e n t a i l s f i r s t l y t h e s u b t r a c t i o n o f t h e s i g n a l o b t a i n e d i n t h e a p p r o p r i a t e l i g h t regime p r i o r t o t r e a t m e n t , from t h e s i g n a l o b t a i n e d d u r i n g o r a f t e r t r e a t m e n t i n t h e i d e n t i c a l l i g h t regime. Then t h e l i g h t i s t u r n e d o f f , and t h e s i g n a l o b t a i n e d i n d a r k n e s s p r i o r t o t r e a t m e n t i s s u b t r a c t e d from t h e s i g n a l o b t a i n e d i n d a r k n e s s d u r i n g o r a f t e r t r e a t m e n t . T h i s i s f o l l o w e d by t h e s u b t r a c t i o n o f t h e d i f f e r e n c e s i g n a l o b t a i n e d i n d a r k n e s s from t h e d i f f e r e n c e s i g n a l o b t a i n e d i n l i g h t , t o r e v e a l t h e t r u e change i n t h e l i g h t - i n d u c e d s i g n a l . I m p l i c i t i n t h e use o f t h i s t e c h n i q u e i s t h e a s s u m p t i o n t h a t t h e s i g n a l o b t a i n e d i n l i g h t , p r i o r t o s u b t r a c t i o n , i s a c o m p o s i t e s i g n a l w h i c h i n c l u d e s t h e s i g n a l o b t a i n e d i n d a r k n e s s . T h i s a s s u m p t i o n has been shown t o be v a l i d f o r t h e p h o t o s y n t h e t i c - r e l a t e d s i g n a l s ( S i g n a l s I and I I U + S ) shown i n F i g u r e 2. I f PSI p r e p a r a t i o n s a r e i l l u m i n a t e d , o n l y S i g n a l I i s r e v e a l e d , ( M a l k i n and Bearden, 1974; Warden and B o l t o n , 1974b) w h i l e P S I I p r e p a r a t i o n s r e v e a l o n l y S i g n a l H u + S (Warden and B o l t o n , -182- 1974a; Boussac and E t i e n n n e , 1982a). I d e n t i c a l s i g n a l s a r e r e v e a l e d when c h l o r o p l a s t s u s p e n s i o n s c o n t a i n i n g b o t h p hotosystems a r e examined, and t h e s i g n a l o b t a i n e d i n d a r k n e s s ( S i g n a l I I U + S ) i s s u b t r a c t e d from t h e combined s i g n a l r e v e a l e d upon i r r a d i a t i o n o f t h e s u s p e n s i o n w i t h f a r - r e d l i g h t (Weaver, 1968; H aehnel, 1984). The l a c k o f c a p a b i l i t y t o a c c u r a t e l y t r a c k t h e k i n e t i c s o f f o r m a t i o n and decay o f f r e e - r a d i c a l s i g n a l s w h i c h a r e s u p e r - imposed upon t h e s t r o n g M n + + and F e + + s i g n a l s o f g r a s s s p e c i e s , w h i c h a l s o change upon t r e a t m e n t , can be m i n i m i z e d by t h e use o f s i g n a l s u b t r a c t i o n t e c h n i q u e s . The r a t e o f i n c r e a s e o r d e c r e a s e o f t h e f r e e - r a d i c a l s i g n a l under s t u d y can n o t be c o n t i n u o u s l y f o l l o w e d w i t h c o n f i d e n c e because t h e f r e e - r a d i c a l s i g n a l s a r e m i n i m a l compared t o t h e p a r a m a g n e t i c m e t a l s i g n a l s , and a s m a l l change i n t h e l a t t e r s i g n a l s would o b s c u r e major changes i n t h e f r e e - r a d i c a l s i g n a l s . However, by s u b t r a c t i o n o f t h e i n i t i a l combined s i g n a l from t h e combined s i g n a l a t v a r i o u s p o i n t s i n t i m e , i t i s p o s s i b l e t o measure t h e h e i g h t ( i n t e n s i t y ) o f t h e f r e e - r a d i c a l s i g n a l under s t u d y s i n c e changes i n t h e m e t a l s i g n a l s w h i c h would a f f e c t t h e h e i g h t o f a k i n e t i c t r a c e o f t h e f r e e - r a d i c a l s i g n a l a r e a c c o u n t e d f o r i n t h e combined s i g n a l . C o n f u s i o n between t h e m e t a l s i g n a l s and t h e f r e e - r a d i c a l s i g n a l s i s e x t r e m e l y u n l i k e l y as t h e m e t a l s i g n a l s exceed 4 00 gauss i n w i d t h w h i l e f r e e - r a d i c a l s i g n a l s i n t h e p r e s e n t s t u d y a r e under 25 gauss i n w i d t h . I f c o m p o s i t e s i g n a l s -183- were r e c o r d e d a t s h o r t i n t e r v a l s , a r e a s o n a b l e k i n e t i c r e c o r d o f t h e f o r m a t i o n and decay o f t h e f r e e - r a d i c a l s i g n a l i n q u e s t i o n c o u l d be o b t a i n e d t h r o u g h measurement o f t h e h e i g h t o f t h e s i g n a l a f t e r s u b t r a c t i o n o f t h e c o n t r o l s p e c t r a a t each p o i n t i n t i m e . A f i n a l p o i n t r e g a r d i n g methodology o f EPR i n v e s t i g a t i o n o f p l a n t l e a v e s p e r t a i n s t o t h e r e q u i r e m e n t f o r oxygen. The r a t e s o f f o r m a t i o n o f EPR S i g n a l s I and H u + S have been shown t o change as s e e d l i n g s d e v e l o p e d i n d i f f e r e n t l i g h t r egimes ( C h e t v e r i k o v e t a l . 1982, 1984) and two t y p e s o f r e d u c t i o n p r o c e s s e s have been demonstrated i n t h e r e a c t i o n c e n t r e s o f P +700 i n t h e l e a v e s o f C h i n e s e r o s e p l a n t s (Khangulov and G o l ' d f e l ' d , 1977). A l s o , t h e i n d u c t i o n c u r v e o f f a r - r e d l i g h t - i n d u c e d S i g n a l I i s b i p h a s i c (Tikhonov and Ruuge, 1975a, 1975b; Ruuge and T i k honov, 1975) and t h e r e i s a 40% b i p h a s i c decay i n P700 and i r o n - s u l p h u r c e n t r e A a t 150K (Bearden and M a l k i n , 1972b). C o n s e q u e n t l y , i t has been s u g g e s t e d t h a t t h e s p e c i e s g i v i n g r i s e t o S i g n a l I i s heterogeneous a t c r y o g e n i c t e m p e r a t u r e s (Bearden and M a l k i n , 1972a, 1972b; L o z i e r and B u t l e r , 1974). The p r e s e n t s t u d i e s which i n v o l v e d r e p l a c e m e n t o f t h e a i r f l o w i n g t h r o u g h t h e dewar i n s e r t w i t h n i t r o g e n complement t h e s e e a r l i e r s t u d i e s w h i c h s u g g e s t e d a l a c k o f homogeneity i n t h e s p e c i e s r e p r e s e n t e d by S i g n a l I . The d a t a i n S e c t i o n 3.4.1.2 -184- i n d i c a t e t h a t S i g n a l I , once formed i n 710nm l i g h t , i s n o t a f f e c t e d by t h e removal o f oxygen. However, t o i n d u c e t h e f o r m a t i o n o f S i g n a l I , oxygen i s r e q u i r e d as o n l y 40% o f S i g n a l I can be formed i f t h e l e a f i s b r i e f l y h e l d i n d a r k n e s s under n i t r o g e n and i s t h e n i r r a d i a t e d w i t h 710nm l i g h t w h i l e s t i l l u nder n i t r o g e n . These d a t a s u p p o r t t h e v i e w t h a t t h e s p e c i e s g i v i n g r i s e t o S i g n a l I i s heterogeneous and s u g g e s t t h a t 60% o f t h i s s i g n a l i s oxygen-dependent. The p r e s e n t d a t a c o u l d a l s o be i n t e r p r e t e d as e v i d e n c e f o r t h e r e q u i r e m e n t o f C 0 2 f o r S i g n a l I f o r m a t i o n s i n c e C 0 2 i s t h e t e r m i n a l p h y s i o l o g i c a l a c c e p t o r o f t h e p h o t o s y n t h e t i c e l e c t r o n t r a n s p o r t c h a i n . The appearance o f d i f f e r e n t new f r e e - r a d i c a l s i g n a l s when d i f f e r e n t a i r p o l l u t a n t s a r e used as t h e fu m i g a n t s u g g e s t s t h a t S 0 2 and 0 3 cause damage t o p l a n t l e a v e s v i a d i f f e r e n t mechanisms. T h i s i s n o t unexpected as S 0 2 i s a r e d u c t a n t w h i l e O3 i s an o x i d a n t . The i d e n t i t y o f t h e f r e e r a d i c a l s r e s p o n s i b l e f o r S i g n a l s N Q 1 and N Q 2 , wh i c h a r e r e v e a l e d upon ex p o s u r e t o ozone, i s not known s i n c e t h e i r g - v a l u e s and peak-to-peak l i n e w i d t h s do n o t appear t o match t h o s e o f any s i g n a l s r e p o r t e d i n t h e r e l e v a n t l i t e r a t u r e . On t h e o t h e r hand, S i g n a l N S 1 , w h i c h i s r e v e a l e d upon p r o l o n g e d e x p o s u r e t o s u l p h u r d i o x i d e , may be r e p r e s e n t a t i v e o f t h e S 0 3 ~ f r e e r a d i c a l s i n c e i t s g - v a l u e and l i n e w i d t h a r e s i m i l a r t o t h o s e r e p o r t e d by M o t t l e y e t a l . (1982b) f o r t h e S 0 3 ~ f r e e - r a d i c a l s i g n a l (Appendix B ) . -185- The subsequent appearance o f S i g n a l N g 2 a f t e r c o n t i n u e d f u m i g a t i o n w i t h s u l p h u r d i o x i d e s u g g e s t s t h a t t h e r e d u c t i o n o f e l e c t r o n a c c e p t o r s X, B and A o c c u r s as a r e s u l t o f e x t e n d e d f u m i g a t i o n w i t h S 0 2 • I f t h e s e l a t e r e l e c t r o n a c c e p t o r s a r e reduced (Gast e t a l . 1983) o r o t h e r w i s e i n a c t i v a t e d ( B a l t i m o r e and M a l k i n , 1980), t h e p r e s e n c e o f t h e e a r l y e l e c t r o n a c c e p t o r s a Q and a-̂  i s r e v e a l e d a t room t e m p e r a t u r e ( B o n n a r j e a and Evans, 1982). A s i g n a l s i m i l a r i n l i n e - s h a p e t o S i g n a l N g 2 was d e t e c t e d by M c i n t o s h and B o l t o n (1979) and was a t t r i b u t e d t o PSI r a d i c a l s . Subsequent work (Gast e t a l . 1983; H o f f , 1984) i n d i c a t e d t h a t t h i s a r i s e s from t h e combined P 7 0 0 + and r a d i c a l . D e f i n i t e i d e n t i f i c a t i o n o f t h e r a d i c a l r e s p o n s i b l e f o r S i g n a l N g 2 i s n o t p o s s i b l e , b u t i t s 2.0042 g - v a l u e i s w i t h i n t h e range o f g - v a l u e s (2.0026-2.0051) r e p o r t e d , depending upon t h e o r i e n t a t i o n o f t h e combined P 7 0 0 + a 1 ~ s i g n a l ( H o f f , 1984). The s t u d i e s w i t h h i g h l e v e l s o f S 0 2 w h i c h r e v e a l e d t h e f o r m a t i o n o f S i g n a l I i n broad-band w h i t e l i g h t ( F i g u r e 46) s u g g e s t t h a t e i t h e r t h e e l e c t r o n t r a n s p o r t c h a i n i s b l o c k e d o r P S I I has been damaged by t h e f u m i g a t i o n w i t h S 0 2 . A s i m i l a r s i t u a t i o n o c c u r s as one o f t h e a l t e r n a t i v e r e s p o n s e s i n l e a v e s grown under h i g h photon f l u x d e n s i t y a f t e r t h e l e a v e s were exposed t o microwave r a d i a t i o n i n t h e s p e c t r o m e t e r c a v i t y ( F i g u r e 34a). I n b o t h c a s e s i t i s c l e a r t h a t t h e e l e c t r o n t r a n s p o r t from P S I I t o PSI needed t o reduce S i g n a l I i n w h i t e -186- l i g h t i n undamaged l e a v e s i s n o t o c c u r r i n g . However, i t i s by no means c e r t a i n t h a t t h e s i t e o f damage t o t h e l e a v e s i s i d e n t i c a l i n t h e s e two c a s e s . I t i s p o s s i b l e t h a t S 0 2 c a u s e s a b l o c k a g e o f t h e e l e c t r o n t r a n s p o r t c h a i n a t QB s i m i l a r t o t h a t caused by DCMU o r T r i s ( V e l t h u y s and Amesz, 1974) w h i l e exposure t o h i g h PFD i m p a i r s t h e r e a c t i o n c e n t r e o f P S I I (Powles and Borkman, 1983). K y l e e t a l . (1983) s u g g e s t e d t h a t t h e l o s s o f t h e 32kDa h e r b i c i d e - b i n d i n g p o l y p e p t i d e o f QB i s t h e p r i m a r y cause o f i n t e r r u p t i o n o f e l e c t r o n t r a n s p o r t and p h o t o i n h i b i t i o n o f P S I I . However, C l e l a n d and C r i t c h l e y (1985) were u n a b l e t o c o n f i r m t h i s , and su g g e s t e d t h a t t h e l o s s o f t h e 32kDa p o l y p e p t i d e i s a consequence, n o t a cause, o f p h o t o i n h i b i t i o n o f P S I I . From t h e EPR s t a n d p o i n t t h e r e s u l t would be i d e n t i c a l , as S i g n a l H u + S would n o t be a f f e c t e d s i n c e i t r e p r e s e n t s D, which p r e c e d e s t h e r e a c t i o n c e n t r e o f P S I I i n t h e e l e c t r o n t r a n s p o r t c h a i n ( F i g u r e 1 ) , and S i g n a l I would be formed i n w h i t e l i g h t i f e l e c t r o n t r a n s f e r from P S I I i s n o t o c c u r r i n g . The h y p o t h e s i s t h a t t h e f o r m a t i o n o f o x y - f r e e r a d i c a l s i s t h e i n i t i a l s t e p i n a i r p o l l u t a n t - c a u s e d damage t o v e g e t a t i o n ( S a k a k i e t a l . 1983) i s s e d u c t i v e , b u t can n e i t h e r be v e r i f i e d n or r e f u t e d from t h e p r e s e n t s t u d i e s . The p r e s e n t s t u d i e s u s i n g ozone ( S e c t i o n 4.3.3) i n d i c a t e d t h a t l e a v e s f u m i g a t e d w i t h ozone show t h e p r e s e n c e o f a new, s m a l l s i g n a l ( S i g n a l N S o x ) i n i n t a c t p l a n t l e a v e s a f t e r s e v e r a l h o u r s o f f u m i g a t i o n . The s i g n a l , w h i c h i s permanent once i n d u c e d , b u t o n l y r e s o l v a b l e -187- a f t e r a s e r i e s o f s i g n a l s u b t r a c t i o n s , appears t o be i d e n t i c a l t o t h a t a t t r i b u t e d t o t h e s u p e r o x i d e a n i o n r a d i c a l i n x a n t h i n e - x a n t h i n e o x i d a s e r a p i d f l o w systems (Palmer e t a l . 1964; B r a y e t a l . 1964; Knowles e t a l . 1969; B a l l o u e t a l . 1969). A l s o , t h e l o n g e r - t e r m s t u d i e s w i t h low l e v e l s o f S 0 2 ( S e c t i o n 5.3.2.2) d i s c l o s e d t h e f o r m a t i o n o f t h e same s i g n a l a f t e r extended f u m i g a t i o n . T h e r e f o r e , r e s u l t s from b o t h t h e ozone and s u l p h u r d i o x i d e s t u d i e s s u g g e s t t h a t i t i s p o s s i b l e t o d e t e c t t h e s u p e r o x i d e a n i o n f r e e r a d i c a l i n p l a n t l e a v e s . However, t h e c o n d i t i o n s under w h i c h t h i s s i g n a l , a t t r i b u t e d t o t h e s u p e r o x i d e a n i o n f r e e r a d i c a l , can be d e t e c t e d , s u g g e s t t h a t i t s f o r m a t i o n , i n amounts d e t e c t a b l e by EPR s p e c t r o s c o p y , o c c u r s as a r e s u l t o f damage caused by t h e o t h e r f r e e r a d i c a l s w h i c h have been c r e a t e d as a r e s u l t o f f u m i g a t i o n w i t h e i t h e r 0 3 o r S 0 2 • S i g n a l N S o x appears a f t e r S i g n a l s N Q 1 , N Q 2 , N S 1 and N g 2 have been formed, and i s p r o b a b l y t h e r e s u l t o f a c c e l e r a t e d senescence, caused by t h e f r e e r a d i c a l s r e p r e s e n t e d by t h e s e l a t t e r f o u r s i g n a l s . The sequence o f e v e n t s s u g g e s t s t h a t , i f t h e f o r m a t i o n o f t h e s u p e r o x i d e a n i o n f r e e r a d i c a l i s t h e p r i m a r y e v e n t i n a i r p o l l u t a n t damage t o p l a n t l e a v e s , t h e n t h e f o r m a t i o n and decay o f t h i s f r e e r a d i c a l i n t h e i n i t i a l s t a g e s o f a i r p o l l u t a n t - i n d u c e d damage t o p l a n t l e a v e s i s so r a p i d t h a t i t s E P R - d e t e c t a b l e f r e e - r a d i c a l s i g n a l can n o t be r e c o r d e d a t t h e s e i n i t i a l s t a g e s , u s i n g t h e methodology o f t h i s s t u d y . -188- On t h e o t h e r hand, i t s a c c u m u l a t i o n t o t h e l e v e l o f d e t e c t a b i l i t y may r e f l e c t damage t o t h e endogenous d e f e n s e mechanisms found w i t h i n p l a n t l e a v e s . The d e f e n s e mechanisms a g a i n s t f r e e r a d i c a l damage can be grouped i n t o two c a t e g o r i e s ; p r e v e n t i o n o f f r e e r a d i c a l f o r m a t i o n , and s c a v e n g i n g o r q u e n c h i n g o f p r e s e n t f r e e r a d i c a l s (Leshem, 1981). W i t h i n p l a n t l e a v e s t h e most p r e v a l e n t a n t i o x i d a n t s w h i c h p r e v e n t t h e f o r m a t i o n o f f r e e r a d i c a l s a r e a - t o c o p h e r o l ( V i t a m i n E ) , a s c o r b i c a c i d ( V i t a m i n C ) , ^ - c a r o t e n e , c a t a l a s e and g l u t a t h i o n e p e r o x i d a s e . The most prominent f r e e r a d i c a l s c a v e n g e r i s s u p e r o x i d e d i s m u t a s e , w h i c h combines w i t h s u p e r o x i d e t o form m o l e c u l a r oxygen (see S e c t i o n 2.1). I n r e a l i t y , SOD i s a f a m i l y o f enzymes d i f f e r e n t i a t e d by t h e i r m e t a l c o - f a c t o r ( F r i d o v i c h , 1976a, 1976b). E u k a r y o t e s have enzymes wh i c h i n c l u d e b o t h copper and z i n c (CuZnSOD) w h i l e p r o k a r y o t e s have enzymes wh i c h c o n t a i n e i t h e r i r o n (FeSOD) o r manganese (MnSOD) (Baum and S c a n d a l i o s , 1981; B r i d g e s and S a l i n , 1981; E l s t n e r , 1979; G i a n n o p o l i t i s and R i e s , 1977; Puget and M i c h e l s o n , 1974). C y t o k i n i n s , w h i c h a r e known t o r e t a r d senescence ( R u n e c k l e s and Resh, 1975), may a l s o a c t as f r e e r a d i c a l s c a v e n g e r s (Leshem, 1981; Leshem e t a l . 1981), w h i l e c h e m i c a l s such as p r o p y l g a l l a t e , sodium benzoate (Leshem, 1981) and e t h y l e n e d i u r e a (EDU) (Carnahan e t a l . 1978; L e g a s s i c k e and Ormrod, 1981; B i s e s s a r and Palmer, 1984)) can p r o v i d e p r o t e c t i o n a g a i n s t f r e e r a d i c a l f o r m a t i o n and ozone t o x i c i t y , r e s p e c t i v e l y . -189- The e x a c t r o l e o f any o f t h e s e p r o t e c t i v e a g e n t s cannot be e s t a b l i s h e d from EPR s t u d i e s a l o n e . However, i t i s p r o b a b l e t h a t t h e y p l a y a major r o l e i n p l a n t d e f e n s e mechanisms a g a i n s t a i r p o l l u t a n t s such as ozone and s u l p h u r d i o x i d e . The t i m e l a g i n t h e f o r m a t i o n o f S i g n a l N S o x d u r i n g f u m i g a t i o n w i t h e i t h e r p o l l u t a n t s u g g e s t s t h a t endogenous d e f e n s e mechanisms e x i s t t o c o u n t e r t h e d e l e t e r i o u s e f f e c t s o f t h e p o l l u t a n t s . Extended f u m i g a t i o n appears t o overcome t h e p r o t e c t a n t s and v a r i o u s f r e e r a d i c a l s r e p r e s e n t e d by S i g n a l s N Q 1 , N 0 2 , N S 1 , N S 2 and N S o x a r e f ° r r n e d a f t e r d i f f e r e n t f u m i g a t i o n t i m e p e r i o d s . The p o s s i b l e i n v o l v e m e n t o f f r e e - r a d i c a l s c a v e n g e r s was a l s o i n d i c a t e d i n t h e h i g h - l e v e l S 0 2 s t u d i e s w h i c h r e v e a l e d t h e f o r m a t i o n o f S i g n a l I i n broad-band w h i t e l i g h t a f t e r a l a g p e r i o d w h i c h d e c r e a s e d as t h e S 0 2 c o n c e n t r a t i o n was i n c r e a s e d ( F i g u r e 4 9 ) . The t o t a l e x t e r n a l exposure ( c o n c e n t r a t i o n x t i m e ) , and p r o b a b l e uptake o f t h e p o l l u t a n t , n e c e s s a r y t o i n d u c e t h e f o r m a t i o n o f t h e w h i t e l i g h t - d e p e n d e n t S i g n a l I was s i m i l a r a t c o n c e n t r a t i o n s o f 50, 100 and 200ppm S 0 2 . Thus, i t appears t h a t t h e endogenous s c a v e n g e r s can c o u n t e r t h e e f f e c t o f t h e S 0 2 u n t i l a t h r e s h h o l d has been r e a c h e d , b u t a f t e r t h i s l i m i t has been r e a c h e d t h e y a r e overcome, and m e t a b o l i c pathways a r e a f f e c t e d by a d d i t i o n a l a b s o r p t i o n o f t h e p o l l u t a n t . The d e l a y e d appearance o f S i g n a l N S o x i s one p o s s i b l e e x p l a n a t i o n f o r t h e l a c k o f s u c c e s s w i t h t h e s p i n t r a p s as -190- e x c i s e d l e a f p i e c e s cannot be m a i n t a i n e d i n t h e s p e c t r o m e t e r c a v i t y f o r t h e t i m e needed t o g e n e r a t e t h e f r e e r a d i c a l r e p r e s e n t e d by t h e S i g n a l N S o x . Fr e e r a d i c a l s c a v e n g e r s , such as SOD, w h i c h p r o t e c t t h e l e a v e s from t o x i c f r e e r a d i c a l s , may be s u c c e s s f u l i n c o u n t e r i n g t h e e f f e c t s o f t h e f r e e r a d i c a l s d u r i n g t h e t i m e p e r i o d (1 hour) t h a t e x c i s e d l e a f p i e c e s can be m a i n t a i n e d i n t h e s p e c t r o m e t e r c a v i t y w i t h o u t t h e appearance o f c o n f o u n d i n g e f f e c t s caused by a r t i f i c i a l a g i n g . A l t e r n a t i v e l y , t h e l a c k o f s p i n adduct f o r m a t i o n may be a f u n c t i o n o f i n a b i l i t y t o s u c c e s s f u l l y i n c o r p o r a t e t h e s p i n t r a p s i n t o t h e l e a f c e l l s . A l s o , i t i s p o s s i b l e t h a t f r e e r a d i c a l s such as t h e s u p e r o x i d e a n i o n f r e e r a d i c a l a r e formed i n t h e i n i t i a l s t a g e s o f e x p o sure t o gaseous a i r p o l l u t a n t s , b u t a r e so t r a n s i e n t t h a t t h e y can n o t be d e t e c t e d u s i n g t h e methodology o f t h i s s t u d y . F o r i n s t a n c e , C o v e l l o and Thompson (1985) showed t h a t t h e s p i n - t r a p p e d s u l p h u r t r i o x i d e f r e e - r a d i c a l s i g n a l was formed when c h l o r o p l a s t s u s p e n s i o n s amended w i t h s u l p h i t e were i l l u m i n a t e d . They found no e v i d e n c e f o r t h e f o r m a t i o n o f t h e s p i n - t r a p p e d s u p e r o x i d e f r e e - r a d i c a l s i g n a l , b u t when SOD was added t o t h e s u s p e n s i o n , t h e s u l p h u r t r i o x i d e f r e e - r a d i c a l s i g n a l was n o t formed. From t h e s e d a t a t h e y c o n c l u d e d t h a t s u p e r o x i d e f o r m a t i o n was n e c e s s a r y t o i n i t i a t e t h e o x i d a t i o n o f s u l p h i t e b u t s u p e r o x i d e was n o t i n v o l v e d i n t h e p r o p a g a t i o n o f t h e c h a i n r e a c t i o n o f s u l p h i t e o x i d a t i o n . I t may be p o s s i b l e t o d e t e c t t h i s t r a n s i e n t f o r m a t i o n o f t h e s u p e r o x i d e f r e e - r a d i c a l -191- s i g n a l t h r o u g h t h e use o f r a p i d scan s p e c t r o m e t r y w h i c h a l l o w s f o r t h e s c a n n i n g o f t h e magnetic f i e l d i n m i l l i s e c o n d s . However, t h i s would r e q u i r e f u r t h e r m o d i f i c a t i o n o f t h e p r e s e n t system as t h e E-271A r a p i d s c a n c a v i t y i s n o t c o m p a t i b l e w i t h t h e dewar i n s e r t used t o a i d i n s t a b i l i z a t i o n o f t h e l e a f and t o g u i d e t h e f l o w o f t r e a t m e n t p o l l u t a n t gases. A l l o f t h e p r e s e n t s t u d i e s have c o n f i r m e d t h a t when p l a n t s a r e s u b j e c t e d t o s t r e s s such as exposure t o s u l p h u r d i o x i d e o r ozone, f r e e r a d i c a l s , l i k e l y d e l e t e r i o u s , a r e formed. The i d e n t i f i c a t i o n , t h r o u g h t h e use o f EPR a l o n e , o f t h e s e s t r e s s - i n d u c e d f r e e r a d i c a l s i s tenuous a t b e s t . Changes a l s o o c c u r i n S i g n a l s I and H u + S when p l a n t s a r e s u b j e c t e d t o s t r e s s a s s o c i a t e d w i t h exposure t o gaseous p o l l u t a n t s , t h e r e b y s u g g e s t i n g t h a t b o t h Photosystems I and I I a r e i m p a i r e d , and normal e l e c t r o n t r a n s p o r t from H2O t o NADP i s no l o n g e r p o s s i b l e . C o n s e q u e n t l y , t h e growth r e d u c t i o n s r e p o r t e d i n t h e l i t e r a t u r e ( B e l l and Clough, 1972; R o b e r t s , 1976; M i l c h u n a s e t a l . 1981; A y a z l o o e t a l . 1982;) a r e t o be e x p e c t e d even i f symptoms o f s t r e s s a r e n o t a p p a r e n t . -192- 7.0 SUMMARY AND CONCLUSIONS 1. Methodology was d e v e l o p e d w h i c h a l l o w s f o r t h e m o n i t o r i n g o f EPR f r e e - r a d i c a l s i g n a l changes o c c u r r i n g i n v i v o i n a t t a c h e d , i n t a c t l e a v e s o f n a r r o w - l e a f e d p l a n t s , such as g r a s s e s , w h i c h a r e s u b j e c t e d t o gaseous a i r p o l l u t a n t s o r o t h e r t r e a t m e n t s . 2. The same t e c h n i q u e p e r m i t s t h e use o f e x c i s e d l e a f p i e c e s i n s t e a d o f i n t a c t , a t t a c h e d l e a v e s i f t h e e x p e r i m e n t t i m e l e n g t h does n ot g r e a t l y exceed one hour. I n t h e second hour a f t e r e x c i s i o n , e x c i s e d l e a f segments r e v e a l a l a r g e f r e e - r a d i c a l s i g n a l w i t h a g - v a l u e o f 2.0046. The f o r m a t i o n o f t h i s 'senescence' ( M i s h r a e t a l . 1971) o r ' o r g a n i c ' ( P r i e s t l e y e t a l . 1980) f r e e - r a d i c a l s i g n a l b e g i n s t o confound any f r e e - r a d i c a l s i g n a l changes i n d u c e d by t r e a t m e n t i n t h e second hour a f t e r e x c i s i o n . I n t h e p r e s e n t s t u d y t h e f o r m a t i o n o f t h i s s i g n a l i s a t t r i b u t a b l e t o t h e a r t i f i c a l a g i n g caused by wounding; n ot t o d e s i c c a t i o n . 3. C o n t i n u a l e x p osure o f t h e l e a f t o f a r - r e d (7l0nm) l i g h t r e s u l t s i n t h e f o r m a t i o n , w i t h i n 1 hour, o f a new EPR f r e e - r a d i c a l s i g n a l ( S i g n a l N 7 1 0 ) , w h i c h has a g - v a l u e o f 2.0054 and an app r o x i m a t e peak-to-peak l i n e w i d t h o f 8.5 gauss. The s p e c i e s g i v i n g r i s e t o t h i s s i g n a l may be t h e e a r l y e l e c t r o n a c c e p t o r a-,, b u t t h e e v i d e n c e i s not c o n c l u s i v e . -193- 4. K i n e t i c s t u d i e s o f f r e e - r a d i c a l s i g n a l f o r m a t i o n and decay d u r i n g t r e a t m e n t a r e p o s s i b l e i n s p e c i e s w h i c h have m i n i m a l M n + + and F e + + s i g n a l s u n d e r l y i n g t h e p h o t o s y n t h e t i c s i g n a l s . U n f o r t u n a t e l y , a l l g r a s s s p e c i e s i n v e s t i g a t e d r e v e a l e d l a r g e M n + + and F e + + s i g n a l s w h i c h a l s o change w i t h t r e a t m e n t , t h u s c o n f o u n d i n g k i n e t i c t r a c k i n g o f t h e p h o t o s y n t h e t i c o r o t h e r f r e e - r a d i c a l s i g n a l changes w h i c h o c c u r w i t h t r e a t m e n t . 5. When a i r i s r e p l a c e d by N 2 i n t h e s p e c t r o m e t e r c a v i t y t h e r e i s a m i n i m a l d e c r e a s e i n S i g n a l I and S i g n a l H u + S , i f t h e l e a f i s c o n t i n u a l l y i r r a d i a t e d w i t h 710nm l i g h t . However, i f t h e l e a f i s h e l d i n d a r k n e s s f o r a b r i e f p e r i o d under N 2, t h e n , when t h e l e a f i s r e t u r n e d t o 710nm l i g h t t h e r e i s o n l y a 40 p e r c e n t r e s t o r a t i o n o f S i g n a l I a f t e r 1 hour o f ex p o s u r e t o 710nm l i g h t , t h e r e b y s u g g e s t i n g t h a t S i g n a l I i s h e t e r o g e n e o u s . 6. No s p i n a d d u c t s were r e v e a l e d when a t t e m p t s were made t o i n c o r p o r a t e s p i n t r a p s i n t o e x c i s e d l e a f p i e c e s . T h i s i s a t t r i b u t a b l e t o a l a c k o f s u c c e s s i n t h e i n c o r p o r a t i o n o f t h e s p i n t r a p s i n t o t h e l e a f p i e c e s o r t o a l a c k o f new f r e e r a d i c a l f o r m a t i o n i n t h e 1 hour p e r i o d d u r i n g w h i c h e x c i s e d l e a f p i e c e s can be v a l i d l y s t u d i e d . 7. Leaves o f p l a n t s grown i n i n t e n s e s u n l i g h t r e v e a l an u n r e p o r t e d f r e e - r a d i c a l s i g n a l ( S i g n a l N p i ) , w h i c h has a g - v a l u e o f 2.0056 and a peak-to-peak l i n e w i d t h o f 9 gauss. -194- T h i s s i g n a l i s not l i g h t - d e p e n d e n t as i d e n t i c a l s i g n a l s a r e r e v e a l e d when t h e l e a v e s a r e h e l d i n d a r k n e s s , under 710nm l i g h t o r broad-band w h i t e l i g h t . I n most l e a v e s S i g n a l N p i i s e l i m i n a t e d upon exposure o f t h e l e a f t o microwave r a d i a t i o n i n t h e c a v i t y f o r 15 m i n u t e s . The i d e n t i t y o f t h e s p e c i e s r e p r e s e n t e d by S i g n a l N p i i s unknown. 8. Once S i g n a l N p I i s e l i m i n a t e d by exposure o f t h e l e a f t o microwave r a d i a t i o n , t h e l e a f may r e v e a l ; a. t h e t y p i c a l p h o t o s y n t h e t i c s i g n a l s from undamaged p l a n t s ; b. a w h i t e l i g h t - i n d u c e d S i g n a l I i n d i c a t i v e o f damage t o P h o tosystem I I o r ; c. no i n d u c t i o n o f S i g n a l I upon i r r a d i a t i o n w i t h f a r - r e d l i g h t , i n d i c a t i v e o f damage t o Photosystem I . 9. Leaves f u m i g a t e d w i t h low l e v e l s o f ozone (up t o 80ppb) do not r e v e a l f r e e - r a d i c a l s i g n a l changes d u r i n g f u m i g a t i o n t i m e p e r i o d s o f up t o 8 h o u r s . 10. A f t e r 3 h o u r s o f f u m i g a t i o n , l e a v e s exposed t o i n t e r m e d i a t e l e v e l s o f ozone (80-250ppb) r e v e a l t h e f o r m a t i o n o f a new f r e e - r a d i c a l s i g n a l ( S i g n a l N Q 1 ) h a v i n g a g - v a l u e o f 2.0041 and a peak-to-peak l i n e w i d t h o f 10 gauss. Concomitant t o t h e f o r m a t i o n o f S i g n a l N Q 1 i s t h e d i s a p p e a r a n c e o f a l l o f S i g n a l I and a d e c r e a s e i n S i g n a l I I U + S i n t e n s i t y . These changes a r e r e v e r s i b l e as S i g n a l N Q 1 decays and S i g n a l I i s r e f o r m e d w i t h i n 2 0 m i n u t e s o f t h e t e r m i n a t i o n o f f u m i g a t i o n . S i g n a l -195- I I U + S i s p a r t i a l l y r e s t o r e d w i t h i n 16 h o u r s o f t h e end o f f u m i g a t i o n . 11. Leaves f u m i g a t e d w i t h h i g h l e v e l s o f ozone ( e x c e e d i n g 250ppb) r e v e a l t h e f o r m a t i o n o f a d i f f e r e n t new f r e e - r a d i c a l s i g n a l ( S i g n a l N 0 2 ) i n d a r k n e s s w i t h i n 1.5 h o u r s o f f u m i g a t i o n . T h i s s i g n a l has a g - v a l u e o f 2.0055 and a peak-to-peak l i n e w i d t h o f 7-8 gauss. 12. Leaves f u m i g a t e d w i t h h i g h l e v e l s o f s u l p h u r d i o x i d e (10-500ppm) r e v e a l a l a r g e S i g n a l I upon exposure t o 650nm o r broad-band w h i t e l i g h t . T h i s i n d i c a t e s a b l o c k a g e o f e l e c t r o n t r a n s p o r t from P S I I t o PS I , p o s s i b l y by p e r t u r b a t i o n o f QB, s i m i l a r t o t h a t caused by t h e a d d i t i o n o f D C M U t o c h l o r o p l a s t p r e p a r a t i o n s . T h i s b l o c k a g e i s dependent upon t o t a l e x t e r n a l dose as b o t h S 0 2 c o n c e n t r a t i o n l e v e l and l e n g t h o f exposure a f f e c t t h e i n t e n s i t y o f t h e s i g n a l and t h e t i m e l a g p r i o r t o i t s f o r m a t i o n . 13. H i g h l e v e l s o f s u l p h u r d i o x i d e a l s o p e r t u r b t h e o x i d i z i n g s i d e o f Photosystem I I , as shown by t h e g r a d u a l d i s a p p e a r a n c e o f S i g n a l I I U + S and c o n c o m i t a n t i n c r e a s e i n t h e M n + + s i g n a l d u r i n g f u m i g a t i o n . The e x a c t s i t e o f S 0 2 damage i s unknown, as t h e p r e c i s e r o l e s o f t h e M n + + d e t e c t a b l e by EPR s p e c t r o s - copy and D , r e p r e s e n t e d by S i g n a l H u + S , have y e t t o be c l a r i f i e d , b u t i t l i k e l y i n v o l v e s a t l e a s t p e r i p h e r a l damage t o -196- t h e o x y g e n - e v o l v i n g complex o f P S I I . 14. Leaves f u m i g a t e d w i t h low l e v e l s o f s u l p h u r d i o x i d e (60-200pphm) r e v e a l t h e decay o f S i g n a l I i n 710nm l i g h t a f t e r 3 h o u r s o f f u m i g a t i o n . T h i s i s i n d i c a t i v e o f PSI damage as t h e l i g h t - o x i d a t i o n o f t h e c h l o r o p h y l l a o f t h e r e a c t i o n c e n t r e o f PSI needed f o r t h e f o r m a t i o n o f S i g n a l I i s no l o n g e r o c c u r r i n g . 15. These l e a v e s a l s o r e v e a l t h e f o r m a t i o n o f a new f r e e - r a d i c a l s i g n a l ( S i g n a l N S 1 ) under broad-band w h i t e l i g h t . S i g n a l N S 1 has a g - v a l u e o f 2.0032 and a peak-to-peak l i n e w i d t h o f 8.0 gauss, w h i c h a r e s i m i l a r t o t h e par a m e t e r s o f t h e s i g n a l a t t r i b u t e d t o t h e S 0 3 ~ f r e e - r a d i c a l . I f f u m i g a t i o n w i t h S 0 2 i s c o n t i n u e d , an u n i d e n t i f i e d new f r e e - r a d i c a l s i g n a l ( S i g n a l N S 2 ) i s formed under 710nm and w h i t e l i g h t . 16. Upon extended f u m i g a t i o n w i t h e i t h e r ozone o r s u l p h u r d i o x i d e , a f r e e - r a d i c a l s i g n a l ( S i g n a l N S o x ) w i t h p a r a m e t e r s i d e n t i c a l t o t h e s u p e r o x i d e a n i o n f r e e r a d i c a l s i g n a l , i s formed i n p l a n t l e a v e s i r r a d i a t e d w i t h broad-band w h i t e o r 710nm l i g h t . The t i m i n g o f t h e f o r m a t i o n o f t h i s s i g n a l , a f t e r t h e f o r m a t i o n o f S i g n a l s N Q 1 and N Q 2 i n t h e ozone s t u d i e s and S i g n a l s N S 1 and N S 2 i n t h e s u l p h u r d i o x i d e s t u d i e s , s u g g e s t s t h a t i t i s formed as a r e s u l t o f s t r e s s and premature a g i n g o f t h e l e a f caused by t h e p o l l u t a n t s - not as a d i r e c t r e s u l t o f exposure t o t h e p o l l u t a n t s . -197- 8.0 LITERATURE CITED A l l e n , H. and 0. H i l l . 1978. 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E l e c t r o n s p i n r e s o n a n c e s i g n a l s and c e l l s t r u c t u r e o f C h l o r e l l a p y r e n o i d o s a grown under d i f f e r e n t l i g h t i n t e n s i t i e s . J . M o l . B i o l . 10:57-62. Van G i n k e l , G. and J.K. R a i s o n . 1980. L i g h t - i n d u c e d f o r m a t i o n o f 0 2 ~ oxygen r a d i c a l s i n systems c o n t a i n i n g c h l o r o p h y l l . Photochem. P h o t o b i o l . 32:793-798. Van Gorkum, H.J., R.F. M e i b u r g and R.J. Van Dorssen. 1983. The e f f e c t s o f an e l e c t r i c a l f i e l d on t h e p r i m a r y r e a c t i o n s o f p h o t o s y s t e m 2. p. 204 A b s t r . 6 t h I n t . Congr. P h o t o s y n t h . , B r u s s e l s . V e l t h u y s , B.R. and J . Amesz. 1974. Charge a c c u m u l a t i o n a t t h e r e d u c i n g s i d e o f system 2 o f p h o t o s y n t h e s i s . B i o c h i m . B i o p h y s . A c t a 333:85-94. -221- V i s s e r , J.W.M., J . Amesz and F. Van G e l d e r . 1974. EPR s i g n a l s o f o x i d i z e d p l a s t o c y a n i n i n i n t a c t a l g a e . B i o c h i m . B i o p h y s . A c t a 333:279-287. Warden, J.T. and J.R. B o l t o n . 1974a. The r e l a t i o n o f t h e ESR S i g n a l I I t o e l e c t r o n t r a n s p o r t i n P hotosystem I I o f s p i n a c h c h l o r o p l a s t s . Photochem. P h o t o b i o l . 20:245-250. Warden, J.T. and J.R. B o l t o n . 1974b. F l a s h p h o t o l y s i s - e l e c t r o n s p i n r esonance s t u d i e s o f t h e dynamics o f p h o t o s y s t e m I i n g r e e n - p l a n t p h o t o s y n t h e s i s - I I . I n t a c t and b r o k e n s p i n a c h c h l o r o p l a s t s . Photochem. P h o t o b i o l . 20:263-269. Warden, J.T., R.E. B l a n k e n s h i p and K. Sauer. 1976. A f l a s h p h o t o l y s i s ESR s t u d y o f p h otosystem I I S i g n a l H v r , t h e p h y s i o l o g i c a l donor t o P-680 +. B i o c h i m . B i o p h y s . A c t a 423:462-478. Warden, J.T. and J.H. G olbeck. 1987. E l e c t r o n - s p i n r e s o n a n c e s t u d i e s o f t h e bound i r o n - s u l f u r c e n t e r s i n P h o tosystem I . I I . C o r r e l a t i o n o f P-700 t r i p l e t p r o d u c t i o n w i t h u r e a / f e r r i c y a n i d e i n a c t i v a t i o n o f t h e i r o n - s u l f u r c l u s t e r s . B i o c h i m . B i o p h y s . A c t a 891:286-292. Warner, C.W. and C a l d w e l l , M.M. 1983. I n f l u e n c e o f photon f l u x d e n s i t y i n t h e 400-700 nm waveband on i n h i b i t i o n o f p h o t o s y n t h e s i s by UV-B (280-320 nm) i r r a d i a t i o n i n soybean l e a v e s : S e p a r a t i o n o f i n d i r e c t and immediate e f f e c t s . Photochem. P h o t o b i o l . 37: W a s i e l e w s k i , M.R., J.R. N o r r i s , L.L. Shipman, C. L i n , and W. Svec. 1981. Monomeric c h l o r o p h y l l a e n o l : E v i d e n c e f o r i t s p o s s i b l e r o l e as t h e p r i m a r y e l e c t r o n donor i n p h o t o s y s t e m I o f p l a n t p h o t o s y n t h e s i s . P r o c . N a t l . Acad. S c i . USA 78:2957-2961. Weaver, E.C. 1962. P o s s i b l e i n t e r p r e t a t i o n o f t h e s l o w - d e c a y i n g ESR s i g n a l i n a l g a l s o l u t i o n s . A r c h . B i o c h i m . B i o p h y s . 99:193-196. Weaver, E.C. 1968. EPR s t u d i e s o f f r e e r a d i c a l s i n p h o t o s y n t h e t i c systems. Ann. Rev. P l a n t P h y s i o l . 19:283-294. Weiss, J . 1935. I n v e s t i g a t i o n on t h e r a d i c a l H0 2 i n s o l u t i o n . T r a n s . Faraday Soc. 31:668-681. W e l l b u r n , A.R., C. H i g g i n s o n , D. R o binson and C. Walmsley. 1981. B i o c h e m i c a l e x p l a n a t i o n s o f more t h a n a d d i t i v e i n h i b i t o r y e f f e c t s o f low a t m o s p h e r i c l e v e l s o f s u l p h u r d i o x i d e p l u s n i t r o g e n d i o x i d e upon p l a n t s . New P h y t o l . 88:223-237. -222- Whitelam, G.C. and G.A. Codd. 1983. P h o t o i n h i b i t i o n o f p h o t o s y n t h e s i s i n t h e c y a n o b a c t e r i u m M i c r o c y s t i s a e r u g i n o s a . P l a n t a 157:561-566. W i l t o n , A.C., J . J . Murray, H.E. Heggestad and F.V. J u s k a . 1972. T o l e r a n c e and s u s c e p t i b i l i t y o f Kentucky b l u e g r a s s (Poa p r a t e n s i s L.) c u l t i v a r s t o a i r p o l l u t i o n , i n t h e f i e l d and i n an ozone chamber. J . E n v i r o n . Q u a l . 1:112-114. Winner, W.E. and H.A. Mooney. 1980. E c o l o g y o f S 0 2 r e s i s t a n c e : I I . P h o t o s y n t h e t i c changes o f s h r u b s i n r e l a t i o n t o S 0 2 a b s o r p t i o n and s t o m a t a l b e h a v i o u r . O e c o l o g i a . 44:296-302. W y d r z y n s k i , T. and K. Sauer. 1980. P e r i o d i c changes i n t h e o x i d a t i o n s t a t e o f manganese i n p h o t o s y n t h e t i c oxygen e v o l u t i o n upon i l l u m i n a t i o n w i t h f l a s h e s . B i o c h i m . B i o p h y s . A c t a 589:56-70. Yamamoto, Y., M. D o i , N. Tamura and M. N i s h i m u r a . 1981. R e l e a s e o f p o l y p e p t i d e s from h i g h l y a c t i v e 0 2 e v o l v i n g p h o t o s y s t e m 2 p r e p a r a t i o n by T r i s t r e a t m e n t . FEBS L e t t . 133:265-268. Yamamoto, Y., S. Shimada and M. N i s h i m u r a . 1983. P u r i f i c a t i o n and m o l e c u l a r p r o p e r t i e s o f 3 p o l y p e p t i d e s r e l e a s e d from a h i g h l y a c t i v e 0 2 e v o l v i n g p h o t o s y s t e m - I I p r e p a r a t i o n by T r i s - t r e a t m e n t . FEBS L e t t . 151:49-53. Yocum, C.F. and G.T. Babcock. 1981. Amine-induced i n h i b i t i o n o f p h o t o s y n t h e t i c oxygen e v o l u t i o n . FEBS L e t t . 130:99-102. Yocum, C.F., C.T. Y e r k e s , R.E. B l a n k e n s h i p , R.R. Sharp and G.T. Babcock. 1981. S t o i c h i o m e t r y , i n h i b i t o r s e n s i t i v i t y , and o r g a n i z a t i o n o f manganese a s s o c i a t e d w i t h p h o t o s y n t h e t i c oxygen e v o l u t i o n . P r o c . N a t l . Acad. S c i . USA 78:7507-7511. Zimmermann, J . L . and A.W. R u t h e r f o r d . 1984. EPR s t u d i e s on t h e 0 2 - e v o l v i n g enzyme o f Photosystem I I . B i o c h i m . B i o p h y s . A c t a 767:160-167. Zimmermann, J . L . and A.W. R u t h e r f o r d . 198 6. P h o t o r e d u c t a n t - i n d u c e d o x i d a t i o n o f Fe i n t h e e l e c t r o n - a c c e p t o r complex o f Photosystem I I . B i o c h i m . B i o p h y s . A c t a 851:416-423. -223- APPENDIX A The use o f e l e c t r o n p a r a m a g n e t i c resonance (EPR) s p e c t r o s c o p y e n a b l e s t h e i n v e s t i g a t o r t o s t u d y t h e f o r m a t i o n o f f r e e r a d i c a l s i n t h e e l e c t r o n t r a n s p o r t c h a i n o f p h o t o - s y n t h e s i s . EPR i s a form o f absorbance s p e c t r o s c o p y d i s c o v e r e d by a R u s s i a n p h y s i c i s t ( Z a v o i s k y , 1945). EPR i s s i m i l a r t o o t h e r forms o f absorbance s p e c t r o s c o p y i n t h a t i t r e l i e s upon a t r a n s i t i o n i n t h e energy l e v e l o f m o l e c u l e s upon a b s o r p t i o n o f r a d i a t i o n . I n t h i s c a s e t h e t r a n s i t i o n e n e r g i e s i n m a g n e t i c resonance caused by microwave r a d i a t i o n c o r r e s p o n d t o t h e i n t e r a c t i o n o f u n p a i r e d e l e c t r o n s w i t h a m a g n e t i c f i e l d . As t h e t e c h n i q u e i s dependent upon t h e d e t e c t i o n o f u n p a i r e d e l e c t r o n s i t i s q u i t e s p e c i f i c . I t i s o n l y a p p l i c a b l e t o t h e s t u d y o f f r e e r a d i c a l s and m e t a l l o c o m p l e x e s w h i c h have p a r a m a g n e t i c m e t a l components. I n b i o l o g i c a l systems, copper, i r o n and manganese a r e t h e most p r e v a l e n t p a r a m a g n e t i c m e t a l s b u t molybdenum, c o b a l t , vanadium and t u n g s t e n a l s o show EPR s i g n a l s i n some o f t h e i r v a l e n c e s t a t e s . I n EPR s p e c t r o m e t r y t h e m a g n e t i c resonance a b s o r p t i o n i s d i s p l a y e d as i t s f i r s t d e r i v a t i v e ; i . e . maximum a b s o r p t i o n o c c u r s a t t h e f i e l d s t r e n g t h a t w h i c h t h e s p e c t r a l l i n e c r o s s e s t h e b a s e l i n e . EPR s i g n a l s a r e p r i m a r i l y i d e n t i f i e d by t h e i r g - v a l u e s , l i n e w i d t h s and h y p e r f i n e s p l i t t i n g s . The -224- g - v a l u e i s a d i m e n s i o n l e s s c o n s t a n t d e t e r m i n e d e x p e r i m e n t a l l y from t h e f i r s t d e r i v a t i v e s i g n a l as t h e v a l u e a t t h e p o i n t o f t h e maximum downward s l o p e o f t h e c u r v e , i . e . t h e f i e l d s t r e n g t h f o r maximum a b s o r p t i o n . I t i s d e f i n e d as: hv = gBH where h i s P l a n c k ' s c o n s t a n t , v i s t h e r a d i a t i o n f r e q u e n c y , B i s t h e Bohr magneton ( n a t u r a l u n i t o f e l e c t r o n m a g n e t i c moment) and H i s t h e m a g n e t i c f i e l d as measured i n gauss (1 gauss = 9 . 3 5 x l 0 " 5 c m ) . I n a d d i t i o n t o t h e use o f t h e above e q u a t i o n , g - v a l u e s can be o b t a i n e d by com p a r i s o n t o a s t a n d a r d such as DPPH ( d i p h e n y l - p i c r y l h y d r a z y l ) f o r w h i c h t h e g - v a l u e (2.0037) i s known. The unknown g - v a l u e , g x , i s c a l c u l a t e d from t h e e q u a t i o n : where g s i s t h e g - v a l u e o f t h e s t a n d a r d and H s and H x a r e t h e m a g n e t i c f i e l d s f o r t h e s t a n d a r d and unknown, r e s p e c t i v e l y . O r g a n i c f r e e r a d i c a l s have a g - v a l u e o f a p p r o x i m a t e l y 2.0023, w i t h d e v i a t i o n s o n l y o c c u r r i n g a t t h e t h i r d d e c i m a l p l a c e , t h e r e b y making i d e n t i f i c a t i o n based o n l y upon t h e g - v a l u e d i f f i c u l t , and a l s o c a u s i n g an o v e r l a p o f s i g n a l s i n many c a s e s . The h y p e r f i n e s p l i t t i n g s a s s o c i a t e d w i t h EPR s i g n a l s a r e caused by t h e i n t e r a c t i o n o f t h e u n p a i r e d e l e c t r o n s w i t h n e i g h b o u r i n g n u c l e i - u s u a l l y p r o t o n s o r n i t r o g e n atoms. These s p l i t t i n g s a r e f r e q u e n t l y r e s o l v a b l e i n s i m p l e b i o l o g i c a l -225- systems bu t a r e e x c e e d i n g l y complex i n whole t i s s u e . O t h e r a i d s i n i d e n t i f i c a t i o n o f EPR s i g n a l s i n c l u d e l i n e shape, peak-to-peak l i n e w i d t h and l i n e a m p l i t u d e (Knowles e t a l . 1976) . I n many c a s e s t h e p o s i t i o n s and s p l i t t i n g s o f t h e l i n e s , as s p e c i f i e d by t h e g - v a l u e s and h y p e r f i n e c o n s t a n t s , a r e dependent upon t h e d i r e c t i o n o f t h e magnetic f i e l d r e l a t i v e t o t h e m o l e c u l a r axes. T h i s s p e c t r a l a n i s o t r o p h y i s n o t e n c o u n t e r e d i n systems where t h e f r e e e l e c t r o n i s e x t e n s i v e l y d e l o c a l i z e d . I n such systems t h e a n i s o t r o p h y i s s m a l l and t h e r a p i d random r o t a t i o n o f t h e r a d i c a l s i n f r e e s o l u t i o n a v e r a g e s out t h e r e m a i n i n g s m a l l a n i s o t r o p h i c s p l i t t i n g s and s h i f t s . However, i n systems where t h e f r e e e l e c t r o n i s not d e l o c a l i z e d , such as c r y s t a l l i n e systems, t h e a n i s o t r o p h y i s n o r m a l l y c o m p l e t e l y s p e c i f i e d by t h e t h r e e g - v a l u e s and h y p e r f i n e c o n s t a n t s o b t a i n e d p a r a l l e l t o t h e p r i n c i p l e axes. The g - v a l u e s a r e u s u a l l y c i t e d as g 2 , g x and g v . I n c a s e s where t h e m o l e c u l a r system i s a x i a l l y s y m m e t r i c a l , t h e p r i n c i p a l g - v a l u e s a r e d e s i g n a t e d as ^ p a r a l l e l a n d ^ p e r p e n d i c u l a r (Knowles e t a l . 1976). O p t i m a l s i g n a l s t r e n g t h w i t h minimum n o i s e i s o b t a i n e d t h r o u g h e x p e r i m e n t a t i o n w i t h v a r y i n g t h e scan t i m e and t h e t i m e c o n s t a n t . A l o n g t i m e c o n s t a n t w i l l improve t h e s i g n a l - t o - n o i s e r a t i o b u t i f t h e s c a n t i m e i s l i m i t e d , t h e l i m i t i n g r e s p o n s e o f -226- t h e t i m e c o n s t a n t w i l l cause d i s t o r t i o n s i n t h e l i n e s h a p e . Thus, t h e s e t t i n g s chosen a r e a compromise between t h e t i m e t a k e n t o s c a n t h e spectrum and t h e t h e e x t e n t t o w h i c h n o i s e i s f i l t e r e d o u t . The c h o i c e o f m o d u l a t i o n a m p l i t u d e i s a l s o a compromise, between t h e m o d u l a t i o n r e q u i r e d t o d e t e c t a s t r o n g s i g n a l and l i n e s h a p e d i s t o r t i o n , known as m o d u l a t i o n b r o a d e n i n g , w h i c h o c c u r s as a r e s u l t o f e x c e s s i v e l y h i g h m o d u l a t i o n a m p l i t u d e s . S e t t i n g s a r e o p t i m i z e d by v a r y i n g t h e t i m e c o n s t a n t , s c a n t i m e and m o d u l a t i o n a m p l i t u d e u n t i l no f u r t h e r change i s produced i n t h e l i n e s h a p e . S p i n t r a p p i n g i s a t e c h n i q u e whereby s h o r t - l i v e d r e a c t i v e f r e e r a d i c a l s may be t r a n s f o r m e d i n t o p e r s i s t e n t p a r a m a g n e t i c s p e c i e s . C o n s e q u e n t l y , EPR t e c h n i q u e s can be used t o d e t e c t r a d i c a l s a t c o n c e n t r a t i o n s below t h e normal d e t e c t i o n l i m i t s ( F i n k e l s t e i n e t a l . 1980). The t e c h n i q u e i s dependent upon t h e i n c o r p o r a t i o n o f a d i a m a g n e t i c s u b s t a n c e , c a l l e d t h e s p i n t r a p , w h i c h has a h i g h a f f i n i t y f o r a r e a c t i v e r a d i c a l , t o t h e r e a c t i o n system c o n t a i n i n g t h e r a d i c a l . The p r o d u c t o f t h i s c o m b i n a t i o n , c a l l e d t h e s p i n a d d u c t , must be a p e r s i s t e n t f r e e r a d i c a l whose c o n c e n t r a t i o n w i l l b u i l d t o r e a d i l y d e t e c t a b l e l e v e l s ( P e r k i n s , 1980). U s e f u l s p i n t r a p s i n c l u d e DMPO ( 5 , 5 - d i m e t h y l - p y r r o l i n e - N - o x i d e ) , w h i c h forms s p i n a d d u c t s w i t h c h a r a c t e r i s t i c EPR s i g n a l s from d i f f e r e n t f r e e r a d i c a l s such as t h e s u p e r o x i d e , h y d r o x y l and s u l p h u r t r i o x i d e f r e e r a d i c a l s , and T i r o n ( ( 1 , 2 - d i h y d r o x y b e n z e n e - 3 , 5 - d i s u l p h o n a t e ) , w h i c h i s v e r y -227- s p e c i f i c , as i t forms a s p i n adduct w i t h s u p e r o x i d e and does not combine w i t h h y d r o x y l o r s i n g l e t oxygen ( M i l l e r and MacDowell, 197 5 ) . However, t h e i n t e n s i t y o f s p i n adduct s i g n a l s o b t a i n e d w i t h DMPO as t h e s p i n t r a p i s i n f l u e n c e d by t h e p r e s e n c e o f i r o n ( B u e t t n e r e t a l . 1978), w h i l e t h e i r h a l f - l i f e v a r i e s , depending upon pH ( B u e t t n e r and O b e r l e y , 1978). A l s o , t h e s p e c i f i c i t y o f T i r o n t o t h e s u p e r o x i d e f r e e r a d i c a l i s not u n i v e r s a l l y a c c e p t e d as Van G i n k e l and R a i s o n (1980) s t a t e t h a t T i r o n scavenges t h e h y d r o x y l f r e e r a d i c a l , not t h e s u p e r o x i d e f r e e r a d i c a l . -228- LITERATURE CITED B u e t t n e r , G.R. and L.W. O b e r l e y . 1978. C o n s i d e r a t i o n s i n t h e s p i n t r a p p i n g o f s u p e r o x i d e and h y d r o x y l r a d i c a l i n aqueous systems u s i n g 5 , 5 - d i m e t h y l - l - p y r r o l i n e - l - o x i d e . Biochem. B i o p h y s . Res. Commun. 83:69-74. B u e t t n e r , G.R., L.W. O b e r l e y and S.W.H. Chan L e u t h a u s e r . 1978. The e f f e c t o f i r o n on t h e d i s t r i b u t i o n o f s u p e r o x i d e and h y d r o x y l r a d i c a l s as seen by s p i n t r a p p i n g and on t h e s u p e r o x i d e d i s m u t a s e a s s a y . Photochem. P h o t o b i o l . 28:693-695. F i n k e l s t e i n , E., G.M. Rosen and E . J . Rauckman. 1980. S p i n t r a p p i n g o f s u p e r o x i d e and h y d r o x y l r a d i c a l : p r a c t i c a l a s p e c t s . A r c h . Biochem. B i o p h y s . 200:1-16. Knowles, P.F., D. Marsh and H.W.E. R a t t l e . 1976. M a g n e t i c Resonance o f B i o m o l e c u l e s . John W i l e y and Sons. London, New York, Sydney, T o r o n t o . 34 3pp. M i l l e r , R.W. and F.D.H. MacDowell. 1975. The T i r o n f r e e r a d i c a l as a s e n s i t i v e i n d i c a t o r o f c h l o r o p l a s t i c p h o t o o x i d a t i o n . B i o c h i m B i o p h y s . A c t a 387:176-187. P e r k i n s , M.J. 1980. S p i n t r a p p i n g . pp. 1-64 I n Advances i n P h y s i c a l O r g a n i c C h e m i s t r y . V o l . 17. V. G o l d and D. B e t h e l l ( E d s . ) . Academic P r e s s (London L t d . Van G i n k e l , G. and J.K. R a i s o n . 1980. L i g h t - i n d u c e d f o r m a t i o n o f 0 2 ~ oxygen r a d i c a l s i n systems c o n t a i n i n g c h l o r o p h y l l . Photochem. P h o t o b i o l . 32:793-798. Z a v o i s k y , E. 1945. Para m a g n e t i c r e l a x a t i o n o f l i q u i d s o l u t i o n s f o r p e r p e n d i c u l a r f i e l d s . J . Phys. USSR 9:211-216. -229- APPENDIX B E s t a b l i s h e d EPR S i g n a l s R e l e v a n t t o T h i s Study P h o t o s y n t h e t i c S i g n a l s Name g - v a l u e Width F u n c t i o n o f F r e e R a d i c a l S i g n a l I 2.0025 S i g n a l I I 2.0046 (u+s) 7.5-9.0g 18-20g 2.0054 10.8g 2.0017 11.5g P r i m a r y donor t o Photosystem I U n i d e n t i f i e d s e c o n d a r y donor t o Photosystem I I U n i d e n t i f i e d e a r l y a c c e p t o r o f Photosystem I U n i d e n t i f i e d e a r l y a c c e p t o r o f Photosystem I Othe r S i g n a l s Name g - v a l u e Width Other R e l e v a n t I n f o r m a t i o n DPPH SO^ 2.0037 2 . 001 2 . 0030- 2.0033 ' O r g a n i c ' 2.00 Fr e e R a d i c a l 4g Used as a marker t o e s t a b l i s h g- v a l u e s o f o t h e r f r e e r a d i c a l s lOg P r e s e n t i n s u p e r o x i d e - f o r m i n g enzyme systems; a s y m m e t r i c a l P r e s e n t i n S 0 3 ~ - f o r m i n g enzyme systems 9 - l l g P r e s e n t i n aged p l a n t m a t e r i a l -230- APPENDIX B (Continued) P r e v i o u s l y U n r e p o r t e d S i g n a l s R e v e a l e d i n T h i s Study Name q - v a l u e Width O t h e r C h a r a c t e r i s t i c s N 7 1 Q 2.0054 8.5g Occurs i n p l a n t l e a v e s w i t h i n 1 hour o f exposure t o 710nm l i g h t Npj 2.0056 9.0g P r e s e n t i n l e a v e s exposed t o h i g h photon f l u x d e n s i t y N Q J 2.0041 10.Og Occurs i n l e a v e s exposed t o more t h a n 2 hou r s o f i n t e r m e d i a t e l e v e l s o f ozone (80-250ppb) N Q 2 2.0055 7-8.Og Occurs i n d a r k n e s s i n l e a v e s exposed t o h i g h l e v e l s o f ozone N S o x 2.001 10.Og A s y m m e t r i c a l ; s i m i l a r t o s u p e r - + 2.08 o x i d e a n i o n r a d i c a l s i g n a l ; o c c u r s i n l e a v e s s u b j e c t e d t o p r o l o n g e d f u m i g a t i o n w i t h S 0 2 o r 0 3 N S 1 2.0032 8.0g Occurs under broad-band w h i t e l i g h t i n l e a v e s f u m i g a t e d w i t h low l e v e l s o f S 0 2 N S 2 2.0042 11.0g Occurs under w h i t e o r 7l0nm l i g h t i n l e a v e s f u m i g a t e d w i t h S 0 2 f o r more t h a n 3 h o u r s -231- APPENDIX C In a d d i t i o n t o t h e p h o t o s y n t h e t i c s i g n a l s c e n t e r e d around t h e 2.000 g - v a l u e , p l a n t l e a v e s a l s o r e v e a l a s i x peak M n + + s i g n a l , and a b r o a d F e + + s i g n a l w h i c h s l o p e s down from low t o h i g h f i e l d . The magnitude o f t h e s e s i g n a l s v a r i e s from s p e c i e s t o s p e c i e s and w i t h t h e age o f t h e l e a f , b u t i n most c a s e s , t h e s e m e t a l s i g n a l s a r e much l a r g e r t h a n t h e p h o t o s y n t h e t i c s i g n a l s . P h o t o s y n t h e t i c S i g n a l s I and I I , and o t h e r f r e e - r a d i c a l s i g n a l s w h i c h have a g - v a l u e c l o s e t o 2.000, a r e shown as a s p l i t i n t h e 4 t h manganese s i g n a l peak from t h e low f i e l d end o f t h e br o a d spectrum ( F i g u r e l a ) . I n s h o r t - t e r m s t u d i e s t h e m e t a l s i g n a l s can be e l i m i n a t e d by s i g n a l s u b t r a c t i o n s i n c e t h e y change more s l o w l y t h a n t h e p h o t o s y n t h e t i c s i g n a l s upon l e a f e xposure t o gaseous a i r p o l l u t a n t s . However, i f s i g n a l s i n t h e r e g i o n o f i n t e r e s t ( g = a p p r o x i m a t e l y 2.000) a r e p r e s e n t e d as r e v e a l e d p r i o r t o s u b t r a c t i o n , t h e p r e s e n c e o f t h e m e t a l s i g n a l s s u g g e s t s a s l o p i n g b a s e l i n e . The d i r e c t i o n and degree o f t h i s s l o p e i s a f u n c t i o n o f t h e s i z e o f t h e M n + + and F e + + s i g n a l s . I f t h e M n + + s i g n a l i s l a r g e t h e b a s e l i n e o f t h e p h o t o s y n t h e t i c s i g n a l s w i l l appear t o s l o p e upward from low f i e l d t o h i g h f i e l d ( F i g u r e l b ) because t h e p h o t o s y n t h e t i c s i g n a l s b e g i n and end on t h e f o u r t h M n + + peak ( F i g u r e l a ) . I n c o n t r a s t , i f t h e M n + + -232- s i g n a l i s s m a l l , b u t t h e Fe s i g n a l i s l a r g e , t h e b a s e l i n e i n t h e r e g i o n o f i n t e r e s t w i l l appear t o s l o p e down from low t o h i g h f i e l d . The s l o p i n g b a s e l i n e a t t r i b u t a b l e t o t h e F e + + s i g n a l can u s u a l l y be c o r r e c t e d f o r t h r o u g h t h e use o f t h e b a s e l i n e a d j u s t m e n t i n t h e V a r i a n s o f t w a r e package b u t t h e l a r g e r upward s l o p e caused by t h e M n + + peak i s d i f f i c u l t t o c o r r e c t . I n most l e a v e s t h e r e c e i v e r g a i n r e q u i r e d t o s u c c e s s f u l l y c h a r a c t e r i z e s m a l l s i g n a l s around t h e 2.000 g - v a l u e i s such t h a t t h e l a r g e r M n + + s i g n a l can not be r e c o r d e d i n i t s e n t i r e t y , t h u s p r o h i b i t i n g e s t i m a t i o n o f t h e degree o f c o r r e c t i o n n e c e s s a r y t o c r e a t e an a c c u r a t e n o n - s l o p i n g b a s e l i n e . C o n s e q u e n t l y , t h e b a s e l i n e was not a d j u s t e d on t h e s e s i g n a l s . -233- 2450 3250 4050 M a g n e t i c f i e l d (Gauss) 3230 3250 3270 M a g n e t i c f i e l d (Gauss) F i g u r e l . W h i t e - l i g h t i n d u c e d s i g n a l from an u n f u m i g a t e d p e r e n n i a l r y e g r a s s l e a f . a. Broad s i g n a l o v e r 1600 g a u s s ; b. S i g n a l i n r e g i o n o f i n t e r e s t . Microwave f r e q u e n c y - 9.188. -234-

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