2 Effect of Ozone on Plant Cell Membrane Permeability IRWIN P. TING, JOHN PERCHOROWICZ,
and LANCE EVANS
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Department of Biology, University of California, Riverside, Calif. 92502
General E f f e c t s Ozone, a strong oxidant component o f photochemical smog i s r e a d i l y formed under c o n d i t i o n s o f high r a d i a t i o n and hydrocarbon waste. Oxides o f n i t r o g e n , mainly n i t r o g e n d i o x i d e a r i s i n g from high temperature combustion processes, r e a c t with molecular oxygen i n the atmosphere i n the presence o f u l t r a v i o l e t s u n l i g h t t o produce n i t r o g e n oxide and ozone. When excess hydrocarbons are present the n i t r o g e n oxide product (NO) will be converted to n i t r o g e n d i o x i d e , r e s u l t i n g i n high ozone l e v e l s ( 1 ) .
The t o x i c e f f e c t s o f ozone i n p l a n t systems have been studied f o r some time, y e t the a c t u a l mechanisms o f i n j u r y are not fully understood. In a d d i t i o n t o visible n e c r o s i s which appears l a r g e l y on upper l e a f s u r f a c e s , many other p h y s i o l o g i c a l and biochemical e f f e c t s have been recorded ( 2 ) . One o f the first e a s i l y measurable e f f e c t s is a s t i m u l a t i o n o f r e s p i r a t i o n . Frequently, however, r e s p i r a t i o n may not increase without concomitant v i s i b l e i n j u r y . Furthermore, photosynthesis in green leaves as measured by CO2 a s s i m i l a t i o n , may decrease. I t is w e l l known that ozone exposure i s accompanied by a dramatic i n c r e a s e i n f r e e p o o l amino a c i d s ( 3 ) . Ordin and h i s co-workers (4) have c l e a r l y shown the e f f e c t o f ozone on cell w a l l b i o s y n t h e s i s . In a d d i t i o n , ozone is known to o x i d i z e c e r t a i n lipid components o f the cell ( 5 ) , to a f f e c t ribosomal RNA (6) and to a l t e r the f i n e s t r u c t u r e o f chloroplasts (7). In a d d i t i o n t o these e f f e c t s , there i s much evidence to suggest that ozone s i g n i f i c a n t l y a l t e r s the p e r m e a b i l i t y properties of cell membranes. In f a c t , Rich (8) proposed that a primary
8
Dugger; Air Pollution Effects on Plant Growth ACS Symposium Series; American Chemical Society: Washington, DC, 1974.
2.
TING ET AL.
Cell Membrane
Permeability
θ
e f f e c t o f o z o n e was t h e d e s t r u c t i o n o f membrane s e m i - p e r m e a b i l i t y . As e a r l y a s 1 9 5 5 , Wedding a n d E r i c k s o n ( 9 ) o b s e r v e d a n a l t e r e d phosphate and water p e r m e a b i l i t y i n p l a n t c e l l s exposed t o ozona t e d h e x e n e . Dugger a n d P a l m e r ( 1 0 ) , s t u d y i n g r o u g h l e m o n l e a v e s exposed t o ozone, found a d e f i n i t e a l t e r a t i o n i n p e r m e a b i l i t y t o g l u c o s e ; a f t e r a c u t e e x p o s u r e t o o z o n e , t h e r e was a s t e a d y i n c r e a s e d u p t a k e o f g l u c o s e b y t r e a t e d l e a v e s compared t o t h a t i n controls. The e x p o s e d l e a v e s a b s o r b e d a p p r o x i m a t e l y t w i c e a s much g l u c o s e a s u n t r e a t e d l e a v e s a f t e r s i x d a y s . The i n c r e a s e d u p t a k e a t t r i b u t a b l e t o o z o n e e x p o s u r e was due s o l e l y t o a n i n c r e a s e i n g l u c o s e - U - * C p e r m e a b i l i t y a n d was n o t t h e r e s u l t o f i n t e r n a l i n c r e a s e d g l u c o s e u t i l i z a t i o n s i n c e t h e r e was n o d i f f e r e n c e i n ^*C02 r e l e a s e f r o m c o n t r o l s a n d e x p o s e d t i s s u e s . In a d d i t i o n t o s t u d i e s w i t h whole c e l l s and t i s s u e s , i t has a l s o b e e n shown t h a t t h e p e r m e a b i l i t y o f i s o l a t e d o r g a n e l l e s c a n b e a l t e r e d b y o z o n e e x p o s u r e . F o r e x a m p l e , L e e ( 1 1 ) showed t h a t ozone a l t e r e d t h e p e r m e a b i l i t y o f tobacco m i t o c h o n d r i a , and C o u l s o n a n d H e a t h ( 1 2 ) r e p o r t e d membrane p e r m e a b i l i t y c h a n g e s o f i s o l a t e d c h l o r o p l a s t s a f t e r ozone e x p o s u r e .
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ll
Cell Permeability Age E f f e c t s . We have f o u n d i t u s e f u l t o d i s t i n g u i s h b e t w e e n t h e t e r m s " s u s c e p t i b l e " a n d " s e n s i t i v e " a n d t h u s , have d e f i n e d t h e a g e o f maximum i n j u r y p o t e n t i a l a s t h e s u s c e p t i b l e age. V a r i a t i o n s i n the degree o f i n j u r y d u r i n g the s u s c e p t i b l e stage are r e f e r r e d t o i n terms o f s e n s i t i v i t y . Susceptible leaves w i l l vary i n s e n s i t i v i t y p r i m a r i l y as a f u n c t i o n o f environmental differences. The a g e - s u s c e p t i b i l i t y phenomenon t o o x i d a n t i n j u r y i s shown i n F i g u r e 1. Young e x p a n d i n g l e a v e s p a s s t h r o u g h a s t a g e at which they are maximally s u s c e p t i b l e t o o x i d a n t i n j u r y . N e i t h e r young nor o l d l e a v e s a r e p a r t i c u l a r l y a f f e c t e d b y ozone, though o n l y young l e a v e s a r e s u s c e p t i b l e t o p e r o x y a c e t y l n i t r a t e (PAN) i n j u r y ( 2 ) . S e n s i t i v i t y o f a s u s c e p t i b l e l e a f c a n be markedly a l t e r e d by s h i f t s i n environmental c o n d i t i o n s such a s l i g h t , water and n u t r i t i o n . D e s p i t e the l a c k o f v i s i b l e n e c r o s i s o f young a n d o l d l e a v e s , i t i s p o s s i b l e t o demonstrate a l t e r a t i o n s i n c e l l p e r m e a b i l i t y by f o l l o w i n g m e t a b o l i t e uptake a f t e r ozone e x p o s u r e . I f A c a l a SJ-1 c o t t o n l e a v e s a r e e x p o s e d t o o z o n e a n d a l l o w e d t o t a k e up s u c r o s e 24 h r l a t e r , t h e r e i s n e a r l y a d o u b l i n g o f t h e r a t e o f s u c r o s e uptake b y l e a v e s which are maximally s u s c e p t i b l e t o ozone. However, b o t h y o u n g a n d o l d l e a v e s w h i c h show no v i s i b l e i n j u r y do show a b o u t a 5 0 % i n c r e a s e i n s u c r o s e u p t a k e ( T a b l e I ) . T h e s e d a t a s u g g e s t t h a t b o t h y o u n g a n d o l d t i s s u e a r e i n j u r e d t o some e x t e n t by ozone.
Dugger; Air Pollution Effects on Plant Growth ACS Symposium Series; American Chemical Society: Washington, DC, 1974.
10
AIR
Table I .
Age
1 4
U p t a k e o f s u c r o s e - U - C by c o t t o n leaf discs
of cotyledon (days) 7 14 21
POLLUTION EFFECTS ON PLANT GROWTH
(Acala
Ratio 0 /control* ο
SJ-1)
Visible injury**
1.49 2.13 1.48
No Yes No
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* R a t i o o f s u c r o s e u p t a k e by ozone exposed d i s c s t o u p t a k e control discs.
by
* * V i s i b l e i n j u r y a s s e s s e d by o b s e r v a t i o n o f n e c r o s i s on l e a f surface. P l a n t s were e x p o s e d t o 0.5 ppm 0 f o r 1 h r , 24 h r s p r i o r t o the experiment. 3
Amino A c i d U p t a k e . I f s u s c e p t i b l e l e a v e s a r e e x p o s e d t o o z o n e and t h e n i n c u b a t e d i n l e u c i n e - U - ^ C , t r e a t e d l e a v e s a b s o r b t h i s amino a c i d a t a f a s t e r r a t e t h a n c o n t r o l s ( F i g . 2 ) . I f , a f t e r 4 hr o f continuous uptake the ^ C - l e u c i n e i s f o l l o w e d w i t h nonlabelled leucine, a s i g n i f i c a n t d i f f e r e n t i a l u t i l i z a t i o n of the i n c o r p o r a t e d l e u c i n e i s not observed ( F i g . 2 ) . T h i s suggests t h a t t h e g r e a t e r u p t a k e i s n o t a c o n s e q u e n c e o f more r a p i d u t i l i z a t i o n and t h a t i t i s p r o b a b l y a t t r i b u t a b l e t o i n c r e a s e d permeability. D e s p i t e a s i g n i f i c a n t l y g r e a t e r u p t a k e o f - ^ C - l e u c i n e by ozonated t i s s u e , greater i n c o r p o r a t i o n of l e u c i n e i n t o p r o t e i n does not appear t o o c c u r . When i n c o r p o r a t i o n i n t o p r o t e i n i s e x p r e s s e d a s a p e r c e n t a g e o f l ^ C a c t u a l l y t a k e n u p , t h e r e i s no d i f f e r e n c e b e t w e e n t h e u n t r e a t e d and o z o n e - t r e a t e d t i s s u e ( T a b l e I I ) . These d a t a f u r t h e r s u p p o r t t h e n o t i o n t h a t enhanced u p t a k e i s s o l e l y a f u n c t i o n o f i n c r e a s e d t r a n s p o r t and n o t o f increased i n t e r n a l u t i l i z a t i o n . S i n c e t h e k i n e t i c s o f l e u c i n e u p t a k e a p p e a r t o be e x p o n e n t i a l , a s t r a i g h t l i n e i s o b t a i n e d when t h e l o g o f u p t a k e i s p l o t t e d a g a i n s t t h e l o g o f t i m e ( F i g . 3 ) . The c o n s t a n t s l o p e and the lower y - i n t e r c e p t i n d i c a t e a higher uptake i n ozone-treated t i s s u e compared w i t h u n t r e a t e d t i s s u e . C a r b o h y d r a t e Uptake. Immediately a f t e r ozone e x p o s u r e , t r e a t e d b e a n l e a v e s a r e o b s e r v e d t o t a k e up l e s s g l u c o s e - U - ^ C than untreated leaves. A f t e r a few h o u r s , h o w e v e r , t h e o z o n e t r e a t e d l e a v e s b e g i n t o a s s i m i l a t e s i g n i f i c a n t l y more g l u c o s e . S i m i l a r l y , i f the nonmetabolizable carbohydrate 2-deoxyglucose i s s u p p l i e d t o o z o n e - t r e a t e d l e a v e s , enhanced uptake o c c u r s ( F i g . 4 ) . I m m e d i a t e l y f o l l o w i n g o z o n e e x p o s u r e 25% l e s s 2 - d e o x y g l u c o s e i s t a k e n up by o z o n e - t r e a t e d t i s s u e , b u t a f t e r 4 h r n e a r l y 50% more
Dugger; Air Pollution Effects on Plant Growth ACS Symposium Series; American Chemical Society: Washington, DC, 1974.
2.
TING ET AL.
Cell Membrane Permeability
11
20
100
80
Ο
H60 S
ω ζ
OC
< 10
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3
