Phenols, Ozone, and Their Involvement in Pigmentation and

8 Phenols, Ozone, and Their Involvement in Pigmentation and Physiology of Plant Injury

Downloaded by CORNELL UNIV on October 23, 2016 | http://pubs.acs.org Publication Date: June 1, 1974 | doi: 10.1021/bk-1974-0003.ch008

R O B E R T K. H O W E L L

Air Pollution Laboratory, Agricultural Environmental Quality Institute, Northeastern Region, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Md. 20705 Abstract In plant cells, p h e n o l s a n d derivatives a r e located in chloroplasts a n d in vacuoles; enzymes that oxidize p h e n o l s a r e also located in chloroplasts and in cytoplasm b u t are maintained in s e p a r a t e c o m p a r t m e n t s b y membranes. Ozone impairs t h e integrity of cell membranes a n d thus p e r m i t s oxidative enzymes to oxidize of phenols to their respective quinones. o - Q u i n o n e s have a n E'o +1.9V a n d will p o l y m e r i z e with amino acids, a m i n e s a n d sulfhydryl g r o u p s of proteins to f o r m l o w molecular w e i g h t r e d d i s h - b r o w n p i g m e n t s in leaves of ozone-treated plants. T h i s i n v o l v e m e n t of p h e n o l s a p p e a r s to be the c a u s e of the visible necrotic lesions on injured leaves. C h e m i c a l l y , the p i g m e n t s or p o l y m e r s r e s e m b l e t h o s e f o r m e d in t o b a c c o leaves during t h e curing p r o c e s s e s . Hydrolzates of the p o l y m e r s contain several amino acids, m e t a l s a n d a p h e n o l . The p o l y m e r s a r e ligninor tannin-like and a s s u c h detract f r o m t h e esthetic and probably nutritional v a l u e of foliage f r o m i m p o r t a n t f o o d a n d feed crops. C o n c e n t r a t i o n s o f caffeoyl derivatives, caffeic and chlorogenic acids a r e increased in ozone-damaged tissues. B o t h o - d i p h e n o l s increase O consumption and reduce CO fixation. T h e r e f o r e , plant growth a n d quality could be r e d u c e d b y o z o n e ' s ( a ) impairing membrane integrity w h i c h w o u l d p r o m o t e cell d e g r a d a t i o n t h r o u g h reduction in synthesis o f products of primary m e t a b o l i s m a n d b y (b) increasing products of secondary metabolism. 2

2

Introduction One f a c t o r c o n t r i b u t i n g t o c r o p l o s s e s c a u s e d b y o z o n e i s t h e development o f abnormal pigments which a r e viewed a s d i s c o l o r e d areas on f o l i a g e o f o z o n e - s e n s i t i v e c u l t i v a r s . Such d i s c o l o r a t i o n i s e s t h e t i c a l l y u n a c c e p t a b l e on l e a f y f o o d c r o p s and p r o b a b l y c a u s e s a r e d u c t i o n i n t h e n u t r i t i o n a l v a l u e o f a l l f o l i a r components used f o r food and f e e d . B i o c h e m i c a l and p h y s i o l o g i c a l r e a c t i o n s r e s p o n s i b l e f o r o z o n e - i n d u c e d d i s c o l o r a t i o n s have n o t 94

Dugger; Air Pollution Effects on Plant Growth ACS Symposium Series; American Chemical Society: Washington, DC, 1974.


8.

HOWELL

Pigmentation and Physiology

95

b e e n e x p l o r e d t o t h e same e x t e n t a s o z o n e - i n d u c e d p h y s i o l o g i c a l r e a c t i o n s associated with primary plant metabolism. Therefore, any d i s c u s s i o n o f t h e p h y s i o l o g y o f a b n o r m a l p i g m e n t a t i o n r e s u l t i n g from ozone a s s a u l t s has t o c o n s i d e r the l i m i t e d i n f o r m a t i o n a v a i l a b l e and r e l a t e the f i n d i n g s from other i n v e s t i g a t i o n s i n v o l v i n g p l a n t s t r e s s e s t o f i n d i n g s from ozone-induced changes i n plant metabolism. P h e n o l s a n d enzymes o f t h e p h e n o l a s e c o m p l e x , o - d i p h e n o l : O2 o x i d o r e d u c t a s e (E.C. 1.10.3.1.) a n d p e r o x i d a s e (E.C. 1.11.1.7.) c o n t r i b u t e t o secondary pigment f o r m a t i o n d u r i n g c u r i n g p r o c e s s e s (1) and d u r i n g s t r e s s e s t o p l a n t s caused b y ozone ( 2 ^ 3 ) , d i s e a s e s , c h e m i c a l s , p h y s i c a l wounding and adverse t e m p e r a t u r e s , m o i s t u r e and n u t r i e n t l e v e l s ( 4 , 5 ) . The r e s u l t i n g p i g m e n t s c o n t r i b u t e t o abnormal f o l i a r and f o o d c o l o r a t i o n (6) and f l a v o r s ( 6 , 7) o f p l a n t s and p l a n t products. Ozone s t i m u l a t e s t h e s y n t h e s i s o f a n t h o c y a n i n s ( 2 ) , c a f f e o y l d e r i v a t i v e s ( 8 ) , a n d t o t a l p h e n o l s (9_) i n p l a n t f o l i a g e . I t induces n e c r o t i c l e s i o n s and l o c a l i z e d d i s c o l o r e d areas o f y e l l o w , r e d o r brown o n f o l i a g e o f p l a n t s o f many s p e c i e s . The s i z e a n d number o f l e s i o n s a n d t h e i n t e n s i t y o f c o l o r a r e r e l a t e d t o p l a n t c u l t i v a r , maturity o f f o l i a g e , c u l t u r a l conditions, concentration o f ozone and d u r a t i o n o f t i m e a f t e r e x p o s u r e . Why p h e n o l i c c o n s t i t u e n t s i n c r e a s e i n o z o n e - i n j u r e d t i s s u e s o r what t h e y c o n t r i b u t e t o p l a n t i n j u r y i s n o t c l e a r . To s u p p o r t the concept t h a t t h e r e i s a r e l a t i o n s h i p between p l a n t p h e n o l i c c o n c e n t r a t i o n s and ozone i n j u r y , f o u r approaches are p r e s e n t e d : (a) a r r a y o f p l a n t s p e c i e s t h a t produce pigment i n response t o ozone t r e a t m e n t ; (b) t i m i n g o f pigment development a f t e r ozone t r e a t m e n t ; ( c ) p l a n t r e s i s t a n c e t o ozone and absence o f pigment a t i o n ; and (d) c o r r e l a t i o n between t o t a l p h e n o l s i n f o l i a g e and r e l a t i v e s e n s i t i v i t y o f peanut c u l t i v a r s t o ozone. S p e c i e s T h a t P r o d u c e P i g m e n t s i n R e s p o n s e t o Ozone. Ozone c a u s e s p i g m e n t c h a n g e s i n many p l a n t s p e c i e s . C h l o r o p h y l l degr a d a t i o n o c c u r s a n d c h l o r o s i s r e s u l t s . I n one p l a n t , Rumex c r i s p u s L., c h l o r o p h y l l c o n c e n t r a t i o n s d i d not decrease b u t a n t h o c y a n i n s y n t h e s i s was i n c r e a s e d a f t e r e x p o s u r e t o o z o n e (2)· In other p l a n t s , reddish-brown pigments are formed, b u t there i s a decrease i n c h l o r o p h y l l content simultaneous w i t h t h e i r format i o n (_). The r e d d i s h - b r o w n p i g m e n t s c a n be e a s i l y v i s u a l i z e d and l o c a t e d i n i n j u r e d l e a v e s a f t e r t h e r e m a i n i n g c h l o r o p h y l l a n d o t h e r e t h a n o l - s o l u b l e p i g m e n t s a r e washed f r o m l e a v e s . Certain c u l t i v a r s o f the f o l l o w i n g p l a n t s t y p i c a l l y e x h i b i t polymerized pigments a f t e r ozone t r e a t m e n t s : green beans, sunflower, potato, w h i t e and r e d c l o v e r , s p i n a c h , s a f f l o w e r , c u r l y d o c k , sweet c l o v e r , sweet a n d g r a i n sorghum, l i m a b e a n s , c h r y s a n t h e m u m , s y c a m o r e , dogwood, p e a n u t s , a l f a l f a , c o t t o n , r a d i s h , b u c k w h e a t a n d soybeans. C e r t a i n c u l t i v a r s o f green bean and a l f a l f a are r e s i s t a n t t o ozone and f a i l t o produce t h e p o l y m e r i z e d pigment f o l l o w i n g exposures t o ozone c o n c e n t r a t i o n s t h a t i n j u r e s e n s i t i v e


96

AIR

POLLUTION

EFFECTS

ON

PLANT

GROWTH


cultivars. Time o f P i g m e n t F o r m a t i o n A f t e r Ozone T r e a t m e n t s . I n g e n e r a l , pigmented l e s i o n s i n green l e a v e s o f t e s t p l a n t s are not v i s i b l e f o r a p p r o x i m a t e l y 20 h r o r more a f t e r o z o n e t r e a t m e n t s o f 10 pphm o r l e s s f o r 2-8 h r , d e p e n d i n g upon c u l t i v a r s u s e d . I f , h o w e v e r , a l c o h o l - s o l u b l e p i g m e n t s a r e removed f r o m s i m i l a r o z o n e t r e a t e d l e a v e s , the o x i d i z e d pigments are e a s i l y v i s i b l e w i t h i n 2 h r a f t e r f u m i g a t i o n s . Pigment f o r m a t i o n i s e v i d e n t i n Kent s o y b e a n l e a v e s ( F i g . 1 ) , w i t h i n 2 h r a f t e r o z o n e t r e a t m e n t , 10 pphm f o r 6 h r , and s p r e a d s t o n e a r l y t h e e n t i r e l e a f b y 4 h r a f t e r treatment. L i t t l e more p i g m e n t was d e t e c t a b l e d u r i n g t h e n e x t 20 h r . R e s i s t a n c e t o Ozone and F a i l u r e t o P r o d u c e A b n o r m a l P i g m e n t s . Tempo g r e e n b e a n s and a l f a l f a c l o n e s 11-1-1L a r e v e r y s e n s i t i v e t o o z o n e ; G r e e n p o d 4-07 and a l f a l f a c l o n e 2-1-2H a r e r e s i s t a n t t o o z o n e . I f t h e s e n s i t i v e and r e s i s t a n t c l o n e s o f t h e two s p e c i e s are c u l t u r e d i n ambient c o n c e n t r a t i o n s o f ozone, the l e a v e s o f r e s i s t a n t p l a n t s do n o t have any v i s i b l e l e s i o n s b u t t h o s e o f t h e s u s c e p t i b l e p l a n t s have e x t e n s i v e i n j u r y . A f t e r r e m o v i n g t h e a l c o h o l - s o l u b l e pigments from a l l l e a v e s , the l e a v e s from suscept i b l e p l a n t s a r e s i g n i f i c a n t l y more p i g m e n t e d t h a n t h o s e f r o m r e s i s t a n t p l a n t s , whose l e a v e s a r e e s s e n t i a l l y v o i d o f p o l y m e r i z e d pigments. C o r r e l a t i o n Between T o t a l P h e n o l s E x p r e s s e d a s P e r c e n t C a f f e i c A c i d E q u i v a l e n t s and Ozone I n j u r y . T w e l v e c u l t i v a r s o f p e a n u t s r a n g i n g f r o m l o w t o h i g h o z o n e s e n s i t i v i t y were e x a m i n e d . F o l i a g e o f e a c h o f t h e c u l t i v a r s was e v a l u a t e d f o r c o n t e n t o f t o t a l p h e n o l s b e f o r e and a f t e r o z o n e t r e a t m e n t . Because o f the 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 c a f f e i c a c i d i n peanut f o l i a g e , v a l u e s a r e e x p r e s s e d as p e r c e n t c a f f e i c e q u i v a l e n t s i n t h e t o t a l phenol assay. Experimental r e s u l t s used i n e s t a b l i s h i n g the c o r r e l a t i o n b e t w e e n c a f f e i c a c i d and o z o n e i n j u r y a r e shown i n Table I . Discussion I t i s a common phenomenon f o r p h e n o l i c m o i e t i e s t o a c c u m u l a t e i n p l a n t t i s s u e s a f t e r exposures t o s t r e s s e s . Concentrations o f phenols are increased i n ozone-treated p l a n t t i s s u e s ( 3 , Reddish-brown polymterized pigments a l s o develop i n a d i v e r s i t y o f p l a n t s i n j u r e d b y o z o n e . T h i s r e s p o n s e a p p e a r s t o be a g e n e r a l r e a c t i o n and n o t a s p e c i f i c r e s u l t o f ozone i n j u r y (_0. Similar p i g m e n t s have b e e n i d e n t i f i e d i n p l a n t t i s s u e s d u r i n g s e n e s c e n c e and by c u r i n g p r o c e s s e s ( 1 ) . The p r o d u c t s o f o x i d a t i o n a r e e a s i l y v i s u a l i z e d s h o r t l y a f t e r o z o n e t r e a t m e n t s b u t no c l a i m s h o u l d be made t h a t p h e n o l i c s o r t h e o x i d a t i v e enzymes, p o l y p h e n o l o x i d a s e , p h e n o l a s e o r p e r o x i d a s e


8.

HOWELL


97


are t h e p r i m a r y s i t e s o f ozone i n j u r y . The i n v o l v e m e n t o f p h e n o l s a n d enzymes o f t h e p h e n o l a s e comp l e x a p p e a r s t o b e s e c o n d a r y t o t h e i n d u c t i o n o f n e c r o s i s . The i n d u c t i o n must i n v o l v e a m o d i f i c a t i o n o f membrane s t r u c t u r e w h i c h l e a d s t o a l t e r e d membrane p e r m e a b i l i t y a n d l o s s o f c e l l c o m p a r t mentalization. I f t h i s occurs, r e g u l a t i o n o f c e l l u l a r metabolism i s l o s t , enzymes a r e a c t i v a t e d , a n d t h e s e a n d t h e i r s u b s t r a t e s t h a t a r e n o r m a l l y s e p a r a t e d b y membranes w o u l d r e a c t t o g e t h e r . The r e s u l t i n g o x i d a t i v e a c t i v i t i e s w o u l d t h e n l e a d t o t h e formation o f pigments o r l e s i o n s .

T a b l e I . I n d e x o f o z o n e i n j u r y t o f o l i a g e o f 12 p e a n u t c u l t i v a r s and t o t a l p l a n t p h e n o l e x p r e s s e s a s % c a f f e i c a c i d equivalents i n assay T o t a l plant phenol expresses as % c a f f e i c a c i d equivalents i n assay Peanut cultivar

Injury score

Before ozone t r e a t m e n t

After ozone t r e a t m e n t

PI268661

96

0.042

0.108

Wilco

66

0.038

0.073

Florspan

69

0.034

0.065

NC4X

60

0.033

0.055

SER 56-15

70

0.039

0.080

Spancross

67

0.040

0.093

NC 4

65

0.035

0.066

T e n n e s s e e Red

69

0.031

0.070

Florunner

56

0.038

0.053

Tifspan

70

0.044

0.098

V i r g i n i a 61R

70

0.030

0.065

Florigiant

61

0.028

0.068

Mean r

0.036

= 98.6

A f t e r l o s s o f membrane i n t e g r i t y i n a f e w s u s c e p t i b l e c e l l s , c h a i n r e a c t i o n s i n v o l v i n g b o t h i n t r a - a n d e x t r a c e l l u l a r compon e n t s c o u l d commence. The p o s s i b i l i t y o f t h i s t y p e o f o v e r a l l r e s p o n s e i s made more p l a u s i b l e when we c o n s i d e r t h e work



98

AIR

POLLUTION

EFFECTS

ON

PLANT

GROWTH

d e s c r i b i n g l e a f age and o z o n e s e n s i t i v i t y ( 1 0 , 1 1 ) . I n b o t h Poa a n n u a L. and N i c o t i a n a g l u t i n o s a L., l e a v e s show s e v e r a l gradations i n c e l l maturation. O n l y s p e c i f i c a r e a s on the- l e a v e s o f b o t h s p e c i e s were s e n s i t i v e t o o z o n e . The a u t h o r s c o n c l u d e d t h a t o z o n e s e n s i t i v i t y i s a f u n c t i o n o f c e l l u l a r d e v e l o p m e n t and m a t u r i t y . The same c o n c l u s i o n was r e p o r t e d by T i n g and Dugger ( 1 2 ) . T h i s i n f o r m a t i o n i n d i c a t e s t h a t an i n t a c t l e a f o r p l a n t i s a dynamic system complete w i t h c e l l s o r l e a v e s o f d i f f e r e n t m a t u r i t i e s and, t h e r e f o r e , o f d i f f e r e n t a b i l i t i e s t o r e s i s t ozone u p t a k e and s u b s e q u e n t b i o c h e m i c a l c h a n g e s . G e n e r a l l y , p h e n o l m e t a b o l i s m i s c o n s i d e r e d a s e c o n d a r y p h y s i o l o g i c a l p r o c e s s com p a r e d t o p h o t o s y n t h e s i s . T h e r e f o r e , t h e r e l a t i v e t i m i n g o f an o z o n e e f f e c t i v e c o n t a c t on t h e two p r o c e s s e s i n a d y n a m i c s y s t e m ( s u c h a s a l e a f ) may be o n l y a m a t t e r o f s e c o n d s and makes a d i s c u s s i o n o f what t h e p r i m a r y o r s e c o n d a r y s i t e o f o z o n e a t t a c k i n an i n t a c t s y s t e m p o i n t l e s s . A l e a f i s composed o f many c e l l s , e a c h a t t a c h e d t o an a d j a c e n t one. D u r i n g l e a f e x p a n s i o n , some c e l l s are mature, others are e n l a r g i n g while others are s t i l l d i v i d i n g ; t h e r e f o r e , i n a g i v e n l e a f , t h e r e s h o u l d be some c e l l s a t a p h y s i o l o g i c a l age t h a t w o u l d f a v o r a n a b o l i s m and p h o t o s y n t h e s i s , and some t h a t w o u l d f a v o r c a t a b o l i s m , o r p h e n o l p o l y m erization. I n any d i s c u s s i o n where an a t t e m p t i s made t o i d e n t i f y t h e p r i m a r y o r s e c o n d a r y s i t e s o f o z o n e r e a c t i o n , one has t o c o n s i d e r t h e l e a f as h a v i n g many c e l l s , e a c h o f w h i c h u n d e r n o r m a l c o n d i t i o n s w o u l d p r o v i d e p r o t e c t i o n f o r s u b c e l l u l a r o r g a n e l l e s . As c e l l s age, p r o d u c t s o f p r i m a r y metabolism, such as p r o t e i n s , c a r b o h y d r a t e s and l i p i d s a r e q u a n t i t a t i v e l y r e d u c e d ( 1 3 ) and t h e s t a b i l i t y o f membranes d i m i n i s h e d , t h u s a l l o w i n g t h e b i o c h e m i c a l r e a c t i o n s and p r o d u c t s o f s e c o n d a r y m e t a b o l i s m t o d o m i n a t e . In a t i s s u e w i t h c e l l s o f d i f f e r e n t a g e s , o z o n e c o u l d be a f f e c t i n g many m e t a b o l i c r e a c t i o n s i n v o l v i n g t h e s e c o m p o n e n t s : ( a ) g l u c o s e ( 1 4 ) , ( b ) p r o t e i n (15), ( c ) l i p i d ( 1 6 ) , and ( d ) p h e n o l s ( 8 ) a t t h e same t i m e . So t h e f i r s t s i t e o f o z o n e d e g r a d a t i o n i s most p r o b a b l y i n membranes. Ozone c h a n g e s p l a n t c e l l membrane ( 1 7 , 18) and c h l o r o p l a s t membrane p e r m e a b i l i t y ( 1 9 ) . L o s s o f membrane i n t e g r i t y i s a l s o c a u s e d by p l a n t p a t h o g e n s (2Ό), d r o u g h t ( 2 1 ) , h e r b i c i d e s ( 2 2 ) and frost (21). P h e n o l s a r e p r e s e n t i n c h l o r o p l a s t s (23_, 24) and i n v a c u o l e s (25) o f p l a n t c e l l s . The enzyme p o l y p h e n o l o x i d a s e and o t h e r enzymes o f t h e p h e n o l a s e c o m p l e x a r e bound t o t h e c h l o r o p l a s t l a m e l l a e o r s t r o m a (2£, 27) and i n t h e c y t o p l a s m (26)· Although t h e enzyme and s u b s t r a t e a r e i n c l o s e p r o x i m i t y , t h e y f a i l t o react u n t i l a p h y s i c a l d i s r u p t i o n or n a t u r a l aging process a l t e r s membrane p e r m e a b i l i t y and p e r m i t s them t o r e a c t . o - P h e n o l s a r e r a p i d l y o x i d i z e d t o o - q u i n o n e s i n t h e p r e s e n c e o f o x y g e n . Ozone c o u l d c o n t r i b u t e t o q u i n o n e f o r m a t i o n i n two ways: by ( a ) d e s t r o y i n g membrane i n t e g r i t y t o p e r m i t s u b s t r a t e and enzyme t o r e a c t , and ( b ) p r o v i d i n g m o l e c u l a r o x y g e n f o r p h e n o l o x i d a t i o n .



8.

HOWELL

99


Ozone d o e s a f f e c t p h o t o s y n t h e s i s a s m e a s u r e d b y CO2 f i x a t i o n (28, 29). S i n c e l o s s o f membrane i n t e g r i t y and o x i d i z e d p i g m e n t f o r m a t i o n o c c u r w i t h i n 2 h r a f t e r ozone t r e a t m e n t , t h e r e i s r e a s o n t o s u s p e c t t h a t e i t h e r endogenous o - p h e n o l s o r t h e i r o x i d i z e d p r o d u c t s , f o r m e d a f t e r membrane d i s r u p t i o n , may e x p l a i n t h e r e l a t i o n s h i p o f o z o n e , p h e n o l s and r e d u c e d CO2 f i x a t i o n , a n d O2 evolution. C h l o r o p l a s t s o f p l a n t s p e c i e s d i f f e r i n t h e i r endog enous q u a n t i t i e s o f o - p h e n o l s ( 2 3 ) and p o l y p h e n o l o x i d a s e enzymes (26). B a l d r y ert a l . ( 2 3 ) h a v e compared t h e photosynthetic a b i l i t i e s o f c h l o r o p l a s t s i s o l a t e d f r o m s u g a r cane a n d s p i n a c h b y m e a s u r i n g CO2 f i x a t i o n a n d O2 e v o l u t i o n . CO2 f i x a t i o n a n d O2 e v o l u t i o n were b o t h s i g n i f i c a n t l y more s u p p r e s s e d b y c h l o r o p l a s t s f r o m s u g a r cane t h a n b y t h o s e f r o m s p i n a c h . I f spinach chloro p l a s t s were s u s p e n d e d i n e x t r a c t s f r o m s u g a r cane c h l o r o p l a s t s w h i c h c o n t a i n e d m a i n l y o - d i p h e n o l s , C 0 2 - d e p e n d e n t O2 e v o l u t i o n was r e d u c e d b y 1 0 0 % o f t h e c o n t r o l and p h o t o f i x a t i o n o f CO2 was r e d u c e d t o o n l y 3% o f t h a t o b s e r v e d f o r n o n t r e a t e d s p i n a c h c h l o r o plasts. The p r e d o m i n a n t o - d i p h e n o l s were i d e n t i f i e d a s c h l o r o genic andc a f f e i c a c i d s . Both o-diphenols a t concentrations o f 2 χ ΙΟ"* M s u p p r e s s e d CO2 f i x a t i o n a n d O2 e v o l u t i o n ; h o w e v e r , o f t h e t w o , c a f f e i c a c i d was t h e more e f f e c t i v e . An e x o g e n o u s e l e c t r o n a c c e p t o r added t o the c h l o r o p l a s t p r e p a r a t i o n negated t h e e f f e c t o f t h e o - d i p h e n o l a n d O2 e v o l u t i o n o c c u r r e d . Poly p h e n o l o x i d a s e i s bound t o c h l o r o p l a s t l a m e l l a e , and T o l b e r t ( 2 6 ) showed t h a t t h e a b i l i t y o f c h l o r o p l a s t p r e p a r a t i o n s t o o x i d i z e dihydrophenylalanine d i f f e r s d e p e n d i n g upon p l a n t s p e c i e s , i n t e n s i t y and q u a l i t y o f l i g h t , a n d a g i n g o f c h l o r o p l a s t s . Chloro p l a s t s f r o m s u g a r b e e t , S w i s s c h a r d , a n d s p i n a c h had l o w Km v a l u e s i n i t i a l l y ; b u t t h e v a l u e s i n c r e a s e d t o a maximum o f 2-6 mmole · mg*" c h l o r o p h y l l · h r " " a f t e r a g i n g f o r 1-5 d a y s . C h l o r o p l a s t s i s o l a t e d from a l f a l f a , wheat, o a t s , peas and sugar cane l e a v e s had maximum Km v a l u e s o f 1 1 - 120 y m p l e s · mg"" c h l o r o p h y l l · h r " * and d i d n o t r e q u i r e a g i n g f o r maximum o x i d a t i o n rates. T h u s , p l a n t enzyme p r o p e r t i e s d i f f e r and s u c h d i f f e r e n c e s have t o b e known b e f o r e o z o n e a n d i t s e f f e c t o n b i o c h e m i c a l s y s t e m s can b e a d e q u a t e l y i n t e r p r e t e d . Suppression o f photo s y n t h e s i s c o u l d b e due t o o - d i p h e n o l s and o - q u i n o n e s : (a) a c t i n g as e l e c t r o n d o n o r s o r a c c e p t o r s w h i c h u n c o u p l e t h e n o r m a l p a t h way o f p h o t o s y n t h e t i c e l e c t r o n f l o w o r ( b ) c h a n g i n g t h e r e d o x (EQ) p o t e n t i a l s o f i n t e r m e d i a t e s o f the e l e c t r o n t r a n s p o r t c h a i n . P a r t i a l photoreduction o f the o-diquinones a l s o produces h i g h l y r e a c t i v e and i n h i b i t o r y semi-quinones ( 2 3 ) . 3

1

1

1

1

Mason ( 3 0 ) a n d P i e r p o i n t ( 3 1 ) have d e s c r i b e d t h e i n v o l v e m e n t o f o - d i p h e n o l s i n p l a n t s and how t h e y c o n t r i b u t e t o a b n o r m a l p l a n t pigmentation. o-Diphenols are o x i d i z e d t o o-quinones by enzymes o f t h e p h e n o l a s e c o m p l e x ( o - d i p h e n o l : O2 o x i d o r e d u c t a s e , E.C.

1.10.3.1) and by p e r o x i d a s e

(E.C.

1.11.1.7).

o-Quinones

r e a c t w i t h amino a c i d s , p r o t e i n s , a m i n e s and t h i o l g r o u p s o f p r o t e i n s t o polymerize and from reddish-brown pigments. Concen t r a t i o n s o f c a f f e i c a c i d are doubled i n b o t h bean ( 8 ) and peanut


AIR

POLLUTION

EFFECTS

ON PLANT

GROWTH


Figure 1-1. Kent soybean leaves: a. No ozone but cleared in 95Ψο ethanol. b-g. Treated with ozone 10 pphm 6 hr, removed from phnts 0,1, 2, 4, 6, or 24 hrs after treatment and cleared, h. Ozone injury 24 hrs after treatment but not cleared.

Figure 1-2. Tifspan pea nut leaves: a. No ozone and cleared, b. Treated with ozone and cleared.

Figure 1-3. King's Ran som chrysanthemum leaves: a. Three on the left— treated with ozone and cleared, b. Two on the right—no ozone and cleared.

Figure 1-4. Alfalfa leaves exposed to ambient concen trations of ozone: a. From a resistant clone and shows no injury or pigment accu mulation, b. From a sus ceptible plant and shows both leaf injury and pig ment accumulation.



8.

HOWELL


Figures 1-5 and 1-6. Green bean leaves exposed to ambient concentrations of ozone: a. Resistant and shows no pigment, b. Sensitive and shows that pigment is accumulated.

Figure 1-7. Greenpod 407 leaf with bean rust pustules.

Figure 1-8. Spinach leaves: a. Treated with ozone and cleared to expose pigment. b. No ozone but cleared.


102

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l e a v e s a f t e r o z o n e e x p o s u r e s . Ozone e x p o s u r e s a l s o i n c r e a s e d p e r o x i d a s e a c t i v i t y i n bean l e a v e s (32, 33). Increased concent r a t i o n s o f t o t a l p h e n o l s o c c u r i n t o b a c c o l e a v e s as a r e s u l t o f o x i d a n t e x p o s u r e s ( 9 ) . The r e d d i s h - b r o w n p o l y m e r s w h i c h f o r m i n o z o n e - i n j u r e d l e a v e s are presumably products t h a t form from r e a c t i o n s o f o z o n e ( E +2.07V) w i t h p h e n o l i c m o i e t i e s w h i c h a r e o x i d i z e d t o o - q u i n o n e s ( E +1.90V) and w h i c h t h e n p o l y m e r i z e w i t h amino a c i d s , p r o t e i n s , a m i n e s o r s u l f h y d r y l compounds. P l a n t s u s c e p t i b i l i t y t o o z o n e as d e t e r m i n e d by v i s i b l e i n j u r y may be v e r y c l o s e l y r e l a t e d t o q u a n t i t i e s o f o - d i p h e n o l s a s s o c i a t e d w i t h t h e c h l o r o p l a s t s and s p e c i f i c r e q u i r e m e n t s f o r a c t i v a t i o n o f p o l y p h e n o l o x i d a s e enzymes. T h e r e i s a s i g n i f i c a n t c o r r e l a t i o n b e t w e e n o z o n e i n j u r y and c o n c e n t r a t i o n s o f t o t a l p h e n o l s e x p r e s s e d as p e r c e n t c a f f e i c a c i d e q u i v a l e n t s i n p e a n u t cultivars. This concept i s not intended t o underestimate the i m p o r t a n c e o f membranes t h a t s e p a r a t e p h e n o l s and enzymes. Perh a p s f u t u r e r e s e a r c h w i l l d e m o n s t r a t e t h a t membranes o f r e s i s t a n t a l f a l f a , g r e e n b e a n and o t h e r s p e c i e s d i f f e r b o t h q u a l i t a t i v e l y and q u a n t i t a t i v e l y f r o m t h o s e o f s u s c e p t i b l e p l a n t s o f t h e s e species. Phenols are r e s p o n s i b l e f o r p l a n t r e s i s t a n c e t o c e r t a i n p a t h o g e n s (5_, 20, 3 4 ) . Because o f i n c r e a s e d i n t e r e s t i n b i o l o g i c a l pest c o n t r o l , s e l e c t i o n of plants with high concentrat i o n s o f p h e n o l s may p r o v i d e p r o t e c t i o n f r o m p l a n t p e s t s , b u t on the b a s i s o f our knowledge o f the d i s t r i b u t i o n o f o x i d a n t p o l l u t a n t s and t h e i r e f f e c t s on p l a n t m e t a b o l i s m s u c h a h y p o t h e s i s w o u l d be u n d e s i r a b l e . Ozone i s w i d e s p r e a d and d o e s c o n t r i b u t e t o t h e o x i d a t i o n o f p h e n o l s i n f o l i a g e o f many p l a n t s p e c i e s . R e s u l t i n g p o l y m e r s a r e l i g n i n - o r t a n n i n - l i k e , and d e t r a c t f r o m b o t h t h e e s t h e t i c and p r o b a b l y t h e n u t r i t i o n a l v a l u e s o f f o l i a g e f r o m s u c h c r o p s a s a l f a l f a , c l o v e r and s p i n a c h . Products of o x i d a t i o n i n Tempo b e a n l e a v e s p r o v i d e d p r o t e c t i o n a g a i n a g a i n s t a n a t u r a l b e a n r u s t (Uromyces p h a s e o l i ( P e r s . ) W i n t . ) i n f e c t i o n . No f u n g a l c o l o n i e s were d e t e c t e d on i n j u r e d Tempo b e a n l e a v e s . The c u l t i v a r G r e e n Pod 407 t h a t d i d n o t p r o d u c e t h e p o l y m e r ( F i g . 1) was w e l l i n o c u l a t e d . The b e a n r u s t p a t h o g e n i s an o b l i g a t e p a r a s i t e ; t h e r e f o r e , i t i s d i f f i c u l t t o d e t e r m i n e i f an e s s e n t i a l g r o w t h f a c t o r was i m m o b i l i z e d and n o t a v a i l a b l e t o t h e r u s t o r g a n i s m o r w h e t h e r t h e p a t h o g e n was i n h i b i t e d by p h e n o l s or o-quinones. P h e n o l i c compounds n a t u r a l l y o c c u r r i n g i n p l a n t s h a v e i n d u c e d many p h y s i o l o g i c a l r e s p o n s e s t h a t d u p l i c a t e t h o s e r e p o r t e d f o r ozone and/or p e r o x y a c e t y l n i t r a t e (PAN). C h l o r o g e n i c a c i d i s a c o m p e t i t i v e i n h i b i t o r o f I A A - o x i d a s e ( 3 5 ) and p l a n t g r o w t h i s a d v e r s e l y a f f e c t e d by i n c r e a s e d c o n c e n t r a t i o n s o f a u x i n s ( 3 6 ) . Concentrations of chlorogenic a c i d are increased i n tobacco t i s s u e e x p o s e d t o o z o n e ( 9 ) . P h e n o l s i n h i b i t ATP s y n t h e s i s ( 3 7 ) , o x i d a t i v e p h o s p h o r y l a t i o n ( 3 7 ) and SH~enzyme a c t i v i t y ( 2 7 ) ; t h e y i n c r e a s e r e s p i r a t i o n ( 3 8 ) , r e d u c e CO2 f i x a t i o n ( 2 2 ) , m o d i f y b o t h membrane p e r m e a b i l i t y T i b ) and o x i d a t i o n r a t e o f r e d u c e d NADH Q

Q


GROWTH


8.

HOWELL


103

( 3 9 ) , a n d f o r m a d d u c t s t h a t i n c r e a s e o x i d a t i o n r a t e s o f amino a c i d s ( 4 1 ) . Dugger a n d T i n g r e l a t e i n t h e i r r e c e n t r e v i e w ( 4 2 ) t h a t o z o n e a n d / o r PAN e l i c i t t h e same p h y s i o l o g i c a l r e s p o n s e s i n s u p p r e s s e d growth r e s u l t i n g from ozone t r e a t m e n t s c o u l d r e s u l t f r o m i n t e r a c t i o n s o f o z o n e , membrane components a n d p h e n o l s , a n d s p e c i f i c enzymes o f t h e p h e n o l a s e c o m p l e x .


Conclusions Phenols, n a t u r a l l y found i n p l a n t s modify p i g m e n t a t i o n , q u a l i t y and growth o f those p l a n t s . N a t u r a l l y o c c u r r i n g p l a n t p h e n o l s , such as d e r i v a t i v e s o f c a f f e i c o r c h l o r o g e n i c a c i d , e l i c i t many p h y s i o l o g i c a l r e s p o n s e s i n p l a n t a s s a y s y s t e m s . When s i m i l a r assay systems are t r e a t e d w i t h ozone, responses s i m i l a r t o t h o s e s t i m u l a t e d by p h e n o l s a r e o b s e r v e d . T h e r e f o r e , ozone i s s u s p e c t e d o f i n t e r a c t i n g w i t h c e l l u l a r membranes w h i c h p e r m i t p h e n o l o x i d a s e a n d p h e n o l s t o r e a c t . As a r e s u l t o f s u c h r e a c t i o n s , ozone maximizes t h e e f f e c t s on p i g m e n t a t i o n , and perhaps on q u a l i t y a n d g r o w t h o f p l a n t p a r t s . C r o p s d e v e l o p e d f o r c e r t a i n areas o f the country, e s p e c i a l l y those subject t o high o x i d a n t c o n c e n t r a t i o n s , s h o u l d be s e l e c t e d on t h e b a s i s o f e i t h e r low phenol content o r phenolase a c t i v i t y . Acknowledgment. G r a t e f u l a p p r e c i a t i o n i s e x p r e s s e d t o D i a n e F. Kremer f o r h e r t e c h n i c a l a s s i s t a n c e . T h i s work was s u p p o r t e d i n p a r t u n d e r C o n t r a c t EPA-IAG-D4-0479, E n v i r o n m e n t a l P r o t e c t i o n Agency, N a t i o n a l E c o l o g i c a l Research L a b o r a t o r y , N a t i o n a l E n v i r o n m e n t a l R e s e a r c h C e n t e r , C o r v a l l i s , Oregon 97330.


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