Ozone Induced Alterations in the Metabolite Pools and Enzyme

Jul 23, 2009 - Environmental Protection Agency, 200 S.W. 35th Street, Corvallis, Ore. ... Ozone exposure also caused a decrease in the activity of the...
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4 Ozone Induced Alterations in the Metabolite Pools and Enzyme Activities of Plants D A V I D T. TINGEY

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Environmental Protection Agency, 200 S.W. 35th Street, Corvallis, Ore. 97330

Abstract A c u t e or chronic o z o n e e x p o s u r e may r e d u c e plant g r o w t h a n d c a u s e greater reductions in root g r o w t h t h a n in top g r o w t h . These g r o w t h reductions a r e associated with m e t a b o l i c alterations. When s o y b e a n l e a v e s a n d pine n e e d l e s were e x p o s e d to o z o n e , there was a n initial d e c r e a s e in the levels of soluble s u g a r s followed by a subsequent increase. Ozone e x p o s u r e also c a u s e d a d e c r e a s e in the activity of the glycolytic p a t h w a y a n d the d e c r e a s e in the activity was reflected in a l o w e r e d rate of nitrate reduction. Amino acids a n d protein also a c c u m u l a t e d in s o y b e a n leaves following e x p o s u r e . Ozone increased t h e activities of enzymes involved in phenol m e t a b o l i s m (phenylalanine ammonia lyase and polyphenoloxidase). T h e r e was also an increase in the levels of total p h e n o l s . L e a c h a t e s f r o m f e s c u e leaves e x p o s e d to o z o n e inhibited nodulation. O z o n e - i n d u c e d reductions in root g r o w t h resulted from altered foliage m e t a b o l i s m rather t h a n f r o m direct action upon t h e roots t h e m s e l v e s . More specifically t h e d e c r e a s e in root g r o w t h p r o b a b l y resulted f r o m a reduction in either translocation a n d / o r t h e quality of the p h o t o s y n t h a t e translocated to the roots. S o l u b l e c a r b o h y d r a t e s a n d starch were at l o w e r levels in roots of ponderosa p i n e exposed to low levels of o z o n e at the end of the g r o w i n g s e a s o n , while t h e levels of amino acids a n d Kjeldahl nitrogen were higher. N o d u l a t i o n of legumes e x p o s e d t o o z o n e was r e d u c e d s u g g e s t i n g a reduction in the amount of nitrogen fixed p e r plant. Root e x u d a t e s f r o m plants e x p o s e d to ozone also inhibited root growth and nodulation in other plants. Introduction P l a n t growth and development i s a c o o r d i n a t e d s e t o f i n t e r r e l a t e d e v e n t s . The g r o w t h o f v a r i o u s p l a n t p a r t s c a n i n f l u e n c e the growth o f o t h e r p a r t s through m e t a b o l i c a c t i v i t i e s ( 1 ) . 40

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

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4.

TiNGEY

41

Enzyme Activities of Plants

Leaves s u p p l y t h e b u l k o f t h e photosynthate t h a t s u p p o r t s p l a n t g r o w t h w h i l e r o o t s a b s o r b n e c e s s a r y w a t e r and m i n e r a l n u t r i e n t s . L e a v e s and r o o t s a l s o s y n t h e s i z e hormones t h a t i n f l u e n c e p l a n t g r o w t h and d e v e l o p m e n t . I t i s apparent t h a t reduced growth o f e i t h e r r o o t o r s h o o t w o u l d be r e f l e c t e d i n t h e g r o w t h and metabol i t e p o o l s o f o t h e r p l a n t p a r t s . An a i r p o l l u t a n t , s u c h a s o z o n e , w o u l d be e x p e c t e d t o r e d u c e p l a n t g r o w t h ( a ) i f i t i m p a i r e d a r a t e l i m i t i n g s t e p i n g r o w t h ; ( b ) i f i t made some p l a n t system r a t e - l i m i t i n g ; ( c ) i f i t reduced the a v a i l a b i l i t y o f a n e e d e d m e t a b o l i t e o r hormone a t t h e g r o w t h s i t e ; o r ( d ) i f i t c a u s e d t h e f o r m a t i o n o f a p h y t o t o x i c compound. The o b j e c t o f t h i s p a p e r i s t o show t h a t b o t h a c u t e a n d c h r o n i c o z o n e e x p o s u r e s c a n e x e r t s i m i l a r e f f e c t s on p l a n t s b y : ( a ) r e d u c i n g f o l i a g e and r o o t g r o w t h ; ( b ) d i r e c t l y a l t e r i n g m e t a b o l i t e p o o l s and enzyme a c t i v i t i e s i n f o l i a g e ; ( c ) i n d i r e c t l y a l t e r i n g metabolite pools i n roots. The p a p e r a l s o s u g g e s t s t h a t t h e s e a l t e r a t i o n s i n m e t a b o l i t e p o o l s i n l e a v e s and r o o t s c o u l d be a s s o c i a t e d w i t h o b s e r v e d r e d u c t i o n s i n p l a n t g r o w t h . E f f e c t s on P l a n t Growth P l a n t g r o w t h may be r e d u c e d b y s i n g l e , m u l t i p l e o r c h r o n i c ozone e x p o s u r e s . R a d i s h e s r e c e i v i n g a s i n g l e ozone exposure a t d i f f e r e n t s t a g e s o f p l a n t development e x h i b i t e d growth r e d u c t i o n s r a n g i n g f r o m 2 t o 1 5 % f o r f o l i a g e and 15 t o 37% f o r r o o t g r o w t h (2) (Table I ) . When r a d i s h e s r e c e i v e d m u l t i p l e o z o n e e x p o s u r e s , g r o w t h r e d u c t i o n s r a n g e d f r o m 10 t o 27% f o r f o l i a g e a n d f r o m 43 t o 75% f o r r o o t growth ( T a b l e I ) .

Table I .

R e d u c t i o n i n R a d i s h Growth from S i n g l e o r M u l t i p l e Ozone E x p o s u r e s 1

Time o f e x p o s u r e (days from seeding) 21 7 14 7 + 21 14 + 21 7 + 14 7 + 1 4 + 2 1

2/ Percent r e d u c t i o n f o l i a g e d r y wt 2 15 10 16 10 24 27

. 2/ Percent r e d u c t i o n r o o t d r y wt 15 23 37 43 54 63 75

P l a n t s were grown and e x p o s e d t o 785 \xg/m o z o n e f o r 1.5 h r i n a c o n t r o l l e d e n v i r o n m e n t f a c i l i t y a n d h a r v e s t e d a t 28 d a y s f r o m s e e d i n g . Means a r e b a s e d on 24 o b s e r v a t i o n s . Data from "Proceedings T h i r d I n t e r n a t i o n a l Clean A i r Congress" ( 2 ) a n d u n p u b l i s h e d d a t a f r o m T i n g e y and D u n n i n g .

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

AIR POLLUTION EFFECTS ON PLANT GROWTH

42

G r o w t h r e d u c t i o n s f r o m t h e m u l t i p l e e x p o s u r e s were a d d i t i v e t o the e f f e c t s o f t h e s i n g l e exposures. F o r a l l exposures t h e r e d u c t i o n i n r o o t g r o w t h was s u b s t a n t i a l l y l a r g e r t h a n t h a t i n f o l i a g e growth. C h r o n i c ozone exposures r e d u c e d t h e growth o f s e v e r a l p l a n t s ( T a b l e I I ) . Growth r e d u c t i o n s r a n g e d f r o m 5 t o 7 0 % f o r t o p g r o w t h a n d 16 t o 7 3 % f o r r o o t g r o w t h . Root g r o w t h was r e d u c e d more t h a n t o p g r o w t h i n a l l t r e a t m e n t s e x c e p t t h e t o b a c c o r e c e i v i n g t h e 200 yg/m o z o n e t r e a t m e n t where t h e r e d u c t i o n s were s i m i l a r i n m a g n i t u d e . 3

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Changes i n P l a n t

Metabolites

In g e n e r a l , t h e e f f e c t s o f an a c u t e ozone exposure on p l a n t m e t a b o l i s m w i l l be i l l u s t r a t e d u s i n g d a t a f r o m s o y b e a n , c v . D a r e . The s o y b e a n s were e x p o s e d t o o z o n e f o r two h r when t h e f i r s t t r i f o l i a t e l e a f was 50 t o 6 0 % expanded a n d a n a l y z e d f o r v a r i o u s m e t a b o l i t e s a n d enzyme a c t i v i t i e s a t 0, 2 4 , 48 a n d 72 h r f o l l o w i n g t e r m i n a t i o n o f e x p o s u r e . To i l l u s t r a t e t h e e f f e c t s o f c h r o n i c ozone e x p o s u r e s , d a t a from Ponderosa p i n e s e e d l i n g s w i l l be u s e d . P o n d e r o s a p i n e were grown f r o m s e e d u n d e r f i e l d c o n d i t i o n s a n d e x p o s e d t o 200 yg/m o f o z o n e 6 h r p e r d a y t h r o u g h o u t t h e g r o w i n g s e a s o n . H a r v e s t s were made a t m o n t h l y i n t e r v a l s a f t e r the i n i t i a t i o n o f exposure. 3

Changes i n M e t a b o l i t e P o o l s i n F o l i a g e 3 I n s o y b e a n s a s i n g l e a c u t e o z o n e dose ( 0 , 490 o r 980 yg/m ozone f o r 2 h r ) s i g n i f i c a n t l y d e c r e a s e d t h e l e v e l o f r e d u c i n g sugars immediately f o l l o w i n g exposure, and t h e c o n c e n t r a t i o n s r e m a i n e d b e l o w t h e c o n t r o l l e v e l f o r 24 h r ( F i g . 1 ) . L e v e l s o f r e d u c i n g s u g a r s were n o t d i f f e r e n t among t r e a t m e n t s a t 48 h r ; h o w e v e r , a f t e r 72 h r t h e r e d u c i n g s u g a r l e v e l i n p l a n t s r e c e i v i n g 980 yg/m was h i g h e r t h a n t h e c o n t r o l l e v e l (j3). The o z o n e t r e a t m e n t h a d no e f f e c t o n s t a r c h c o n t e n t . S o l u b l e c a r b o h y d r a t e l e v e l s o f s e e d l i n g P o n d e r o s a p i n e were a l t e r e d by a c h r o n i c ozone exposure ( T i n g e y and W i l h o u r , unp u b l i s h e d ) . The l e v e l o f s o l u b l e c a r b o h y d r a t e s was 1 0 % b e l o w t h e c o n t r o l l e v e l one month a f t e r i n i t i a t i o n o f t h e e x p o s u r e ( F i g . 2 ) . A t t h e s e c o n d a n d s u b s e q u e n t h a r v e s t s , t h e r e were h i g h e r l e v e l s o f s o l u b l e carbohydrates i n t h e t o p s o f exposed plants than i n the c o n t r o l s . S t a r c h l e v e l s i n exposed f o l i a g e were e l e v a t e d above t h e c o n t r o l a f t e r two months o f e x p o s u r e a n d stayed t h e r e f o r t h e remainder o f t h e season. The o z o n e - i n d u c e d d e p r e s s i o n i n s o l u b l e s u g a r l e v e l s o b s e r v e d i n s o y b e a n a n d p i n e c o u l d have r e s u l t e d f r o m a d e p r e s s i o n in the photosynthetic rate. H i l l a n d L i t t l e f i e l d (M_) r e p o r t e d t h a t 785 t o 1175 yg/m o z o n e f o r 1/2 t o 1 1/2 h r r e d u c e d p h o t o s y n t h e s i s a b o u t 5 0 % i n s e v e r a l p l a n t s p e c i e s . Ozone h a s a l s o b e e n shown t o d e c r e a s e t h e p h o t o s y n t h e s i s o f P o n d e r o s a p i n e ( 5 ) . 3

3

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

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HOURS AFTER TERMINATION OF EXPOSURE Physiologie Planta rum

Figure 1.

Effects of a single ozone exposure on the levels reducing sugars in soybean leaves (3). Each mean is based on four observations.

90

40

I

1

1

1

1

2 3 4 MONTHS AFTER INITIATION OF EXPOSURE

»

5

Figure 2. Effect of chronic ozone exposure on the soluble carbohydrate leveh of seedling Ponderosa pine tops. Each mean is based on nine observations.

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

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AIR POLLUTION EFFECTS ON PLANT GROWTH

T a b l e II·

E f f e c t s o f C h r o n i c Ozone E x p o s u r e s

. Plant—

Ozone c o n e , (pg/m

1

Radish Cherry B e l l e

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Alfalfa Vernal

Soybean Dare

Soybean Hood

Tobacco B e l W3

)

Percent reduction t o p d r y wt

on P l a n t

Percent reduction r o o t d r y wt

0 100

0 10

0 50

0 100 200

0 13 36

0 22 57

0 200

0 28

0 200

0 16

0 100 200

0 10 70

Growth

0 41 73

D a t a f o r r a d i s h i s f r o m J . Amer. S o c . H o r t . S c i . ( 2 3 ) ; d a t a f o r t h e o t h e r p l a n t s i s u n p u b l i s h e d d a t a o f T i n g e y and R e i n e r t . R a d i s h e s were e x p o s e d f o r 5 w e e k s , a l f a l f a was e x p o s e d f o r 3 months and s o y b e a n and t o b a c c o were e x p o s e d 4 and 5 weeks. A l l p l a n t s were grown and e x p o s e d u n d e r g r e e n h o u s e c o n d i t i o n s .

The o z o n e - i n d u c e d i n c r e a s e i n t h e l e v e l s o f s o l u b l e c a r b o h y d r a t e s i n s o y b e a n and P o n d e r o s a p i n e f o l i a g e has a l s o b e e n r e p o r t e d b y o t h e r w o r k e r s (£, 6, 7 ) . The i n c r e a s e i n s o l u b l e c a r b o h y d r a t e s i n t h e f o l i a g e c o u l d have r e s u l t e d f r o m ( a ) r e duced sugar u t i l i z a t i o n ; (b) reduced sugar t r a n s l o c a t i o n , and/or ( c ) t h e f a c t t h a t t h e s u g a r was r e n d e r e d u n a v i l a b l e f o r metabol i s m i n p r o d u c t s such as g l y c o s i d e s . I n any c a s e , t h e r e t e n t i o n o f c a r b o h y d r a t e s i n f o l i a g e c o u l d cause a r e d u c t i o n i n photos y n t h e s i s b y f e e d b a c k i n h i b i t i o n and w o u l d r e d u c e t h e amount o f photosynthate a v a i l a b l e f o r t r a n s l o c a t i o n t o other p l a n t organs. The d e c r e a s e i n a v a i l a b l e p h o t o s y n t h a t e f o r t r a n s l o c a t i o n c o u l d r e s u l t i n reduced growth o f the o t h e r p l a n t organs. S u g a r s c a n be m e t a b o l i z e d b y e i t h e r g l y c o l y s i s o r b y t h e p e n t o s e p h o s p h a t e p a t h w a y . The a c t i v i t i e s i n t h e s e p a t h w a y s c a n be d e t e r m i n e d b y m e a s u r i n g t h e enzyme a c t i v i t i e s o f s e l e c t e d dehydrogenases i n each p a t h . I n soybean, ozone can a l t e r t h e

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

4.

TINGEY

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Enzyme Activities of Pfonts

a c t i v i t i e s o f s e l e c t e d enzymes i n t h e g l y c o l y t i c a n d p e n t o s e p h o s p h a t e p a t h w a y s ( 8 ) . A s i n g l e o z o n e e x p o s u r e ( 9 8 0 yg/m o z o n e f o r 2 h r ) d e p r e s s e d t h e a c t i v i t y o f g l y c e r a l d e h y d e 3-phosphate d e h y d r o g e n a s e (GPD) 2 0 % a n d t h e a c t i v i t y o f t h e enzyme r e m a i n e d s i g n i f i c a n t l y b e l o w t h e c o n t r o l l e v e l f o r a t l e a s t 72 h r ( F i g . 3 A ) . C o n v e r s e l y , t h e a c t i v i t y o f g l u c o s e 6-phosphate d e h y d r o g e n a s e (G6PD) was u n a f f e c t e d i m m e d i a t e l y f o l l o w i n g t h e o z o n e e x p o s u r e ; b u t w i t h i n 24 h r , t h e a c t i v i t y o f G6PD was s i g n i f i c a n t l y above t h e c o n t r o l l e v e l a n d i t remained t h e r e f o r a t l e a s t 72 h r ( F i g . 3 B ) . A n o t h e r enzyme i n t h e p e n t o s e p h o s p h a t e p a t h way, 6 - p h o s p h o g l u c o n a t e d e h y d r o g e n a s e , showed s i m i l a r t r e n d s t o G6PD ( T i n g e y , u n p u b l i s h e d ) . The o z o n e t r e a t m e n t a p p a r e n t l y i n i t i a t e d c h a n g e s i n t h e pathways o f c a r b o h y d r a t e m e t a b o l i s m , w i t h g l y c o l y s i s b e i n g r e d u c e d w h i l e t h e a c t i v i t y o f t h e p e n t o s e p h o s p h a t e p a t h w a y was i n c r e a s e d . Rat l u n g t i s s u e exposed t o ozone a l s o e x h i b i t e d t h e o z o n e - i n d u c e d d e p r e s s i o n o f GPD a n d enhancement o f G6PD a c t i v i t y (9j 1 0 ) . The a c t i v a t i o n o f t h e p e n t o s e p h o s p h a t e p a t h w a y i s a characteristic feature o fdiseased plants (11, 12). I n p l a n t l e a v e s , n i t r a t e r e d u c t i o n r e q u i r e s NADH p r o d u c e d b y g l y c o l y t i c a c t i v i t y ( 1 3 ) . The o z o n e - i n d u c e d d e p r e s s i o n o f g l y c o l y t i c m e t a b o l i s m i n s o y b e a n l e a v e s was a l s o r e f l e c t e d i n a d e p r e s s e d r a t e o f n i t r a t e r e d u c t i o n (3_). A s i n g l e o z o n e e x p o s u r e d e p r e s s e d t h e i n v i v o n i t r a t e r e d u c t a s e (NR) a c t i v i t y about 60% ( T a b l e I I I ) . To d e t e r m i n e i f o z o n e a f f e c t e d t h e NR p r o t e i n d i r e c t l y , t h e i n v i t r o NR a c t i v i t y was d e t e r m i n e d i n l e a f e x t r a c t s f r o m p l a n t s e x p o s e d t o 0 a n d 980 yg/m o z o n e . The o z o n e t r e a t m e n t h a d n o s i g n i f i c a n t e f f e c t o n t h e i n v i t r o NR a c t i v i t y , i n d i c a t i n g t h a t i t d i d n o t i n a c t i v a t e t h e NR p r o t e i n (Table I I I ) . L e a f e x t r a c t s t h a t would couple t h e o x i d a t i o n o f f r u c t o s e - 1 , 6 - d i p h o s p h a t e t o n i t r a t e r e d u c t i o n were p r e p a r e d f r o m l e a v e s e x p o s e d t o e i t h e r 0 o r 980 yg/m o z o n e (*3). Ozone d e p r e s s e d t h e i n v i t r o c o u p l e d NR a c t i v i t y 5 8 % ( T a b l e I I I ) , i n d i c a t i n g t h a t t h e o b s e r v e d ozone d e p r e s s i o n o f n i t r a t e r e d u c t i o n i n the i n v i v o l e a f d i s k assay r e s u l t e d from a d e p r e s s i o n i n t h e r a t e o f NADH f o r m a t i o n b y GPD. I n soybean l e a v e s exposed t o a s i n g l e a c u t e ozone dose ( 0 , 4 9 0 o r 980 yg/m o z o n e f o r 2 h r ) t h e amino a c i d l e v e l was d e p r e s s e d i m m e d i a t e l y f o l l o w i n g e x p o s u r e ( F i g . 4 A ) (3_). T h i s d e p r e s s i o n o f amino a c i d s was s i m i l a r t o t h a t o b s e r v e d i n l e v e l s o f r e d u c i n g sugars immediately f o l l o w i n g exposure and p r o b a b l y r e s u l t e d f r o m a d e p r e s s i o n i n p h o t o s y n t h e s i s . W i t h i n 24 h r a f t e r e x p o s u r e , t h e amino a c i d l e v e l i n p l a n t s e x p o s e d t o 4 9 0 yg/m had r e t u r n e d t o t h e c o n t r o l l e v e l . However, i n p l a n t s e x p o s e d t o 980 y g / m , t h e amino a c i d l e v e l i n c r e a s e d above t h a t o f t h e control. C o n c u r r e n t w i t h t h e r i s e i n f r e e amino a c i d s was a n i n c r e a s e i n p r o t e i n l e v e l i n e x p o s e d f o l i a g e ( F i g . 4 B ) . The i n c r e a s e i n amino a c i d s f o l l o w i n g a n o z o n e e x p o s u r e h a s b e e n r e p o r t e d b y o t h e r workers ( 6 , 1 4 , 1 5 ) ; however, i n t h e s e c a s e s , t h e r i s e i n f r e e amino a c i d l e v e l was a s s o c i a t e d w i t h a d e c l i n e i n

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

AIR POLLUTION EFFECTS ON PLANT GROWTH

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24 48 HOURS AFTER TERMINATION OF EXPOSURE

72

Ozone alteration of dehydrogenases active in sugar oxidation in soybean leaves. Each mean is based on eight observations.

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

TINGEY

Enzyme Activities of Pfonts

3.0 A.

AMINO ACIDS

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Physiologie Plantarum

Figure 4. Effect of ozone on foliar levels of amino acids and protein in soybean leaves (3). Each mean is based on four observations.

American Chemical Society Library 1155 16th St., N.W. Washington, DC 20036 Dugger; Air Pollution Effects on Plant Growth ACS Symposium Series; American Chemical Society: Washington, DC, 1974.

AIR

48

POLLUTION EFFECTS ON PLANT GROWTH

foliar protein.

T a b l e I I I . E f f e c t s o f Ozone on N i t r a t e i n Soybean Leaves-*NR a c t i v i t y ymol/g f r e s h w t / h r )

(N0~

Reduction

Ozone c o n e . (yg/m ) 3

0

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I n v i v o NR Leaf Disc In V i t r o NADH d e p e n d e n t NR In V i t r o coupled assay NADH d e p e n d e n t NR

980

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7.3

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P l a n t s were e x p o s e d t o o z o n e f o r 2 h r a n d t h e 1 s t t r i f o l i a t e l e a v e s were a s s a y e d f o r enzyme a c t i v i t y i m m e d i a t e l y f o l l o w i n g e x p o s u r e . E a c h mean i s b a s e d o n 9 o b s e r v a t i o n s . Source : P h y s i o l . Plantarurn ( 3 ) .

P r e l i m i n a r y s t u d i e s w i t h f i e l d - g r o w n a l f a l f a exposed t o c h r o n i c o z o n e l e v e l s ( 2 0 0 yg/m t h r o u g h o u t t h e g r o w i n g s e a s o n ) i n d i c a t e d t h a t t h e l e v e l s o f amino a c i d s a n d p r o t e i n were i n c r e a s e d 19 a n d 28% r e s p e c t i v e l y ( N e e l y a n d T i n g e y , u n p u b l i s h e d ) . The a c t i v i t i e s o f enzymes a s s o c i a t e d w i t h p h e n o l m e t a b o l i s m i n s o y b e a n l e a v e s c a n be a l t e r e d b y a s i n g l e o z o n e e x p o s u r e ( 0 o r 980 yg/m o f o z o n e f o r 2 h r ) ( T i n g e y , u n p u b l i s h e d ) . Imme­ d i a t e l y f o l l o w i n g e x p o s u r e , p h e n y l a l a n i n e ammonia l y a s e ( P A L ) a c t i v i t y was s u p p r e s s e d b y o z o n e ( F i g . 5 ) . W i t h i n 24 h r , t h e PAL l e v e l i n e x p o s e d l e a v e s was s i g n i f i c a n t l y above t h a t o f t h e control. The a c t i v i t y o f p o l y p h e n o l o x i d a s e (ΡΡ0) i s s u p p r e s s e d 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 ( F i g . 5 ) . A f t e r 24 h r , PPO a c t i v i t y i n e x p o s e d l e a v e s was g r e a t e r t h a n t h a t i n t h e c o n t r o l and r e m a i n e d a t a h i g h e r l e v e l f o r t h e d u r a t i o n o f t h e e x ­ p e r i m e n t . PAL i s a k e y exzyme i n p h e n o l b i o s y n t h e s i s a n d i t s a c t i v i t y i s u s u a l l y a s s o c i a t e d w i t h an i n c r e a s e i n p h e n o l l e v e l s . The i n c r e a s e d enzyme a c t i v i t i e s s u g g e s t e d t h a t o z o n e - e x p o s e d l e a v e s s h o u l d c o n t a i n e l e v a t e d l e v e l s o f p h e n o l i c compounds a n d phenol o x i d a t i o n products. P o n d e r o s a p i n e s e e d l i n g s e x p o s e d t o a c h r o n i c ozone d o s e (200 yg/m , 6 h r / d a y t h r o u g h o u t t h e g r o w i n g s e a s o n ) a l s o e x ­ h i b i t e d changes i n p h e n o l metabolism (Tingey and W i l h o u r , u n ­ p u b l i s h e d ) . Between t h e f i r s t a n d s e c o n d months o f e x p o s u r e , t o t a l p h e n o l c o n c e n t r a t i o n began t o i n c r e a s e i n f o l i a g e o f e x ­ posed Ponderosa p i n e s e e d l i n g s ( F i g . 6 ) . Throughout t h e remain3

3

3

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

TINGEY

Enzyme Activities of Pfonts

oc χ

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Figure 5.

Ozone alteration of PAL and PPO activity in soybean leaves (8). Each mean is based on eight observations.

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

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50

ABR POLLUTION EFFECTS ON PLANT GROWTH

der o f the growing season, phenol l e v e l s i n exposed p l a n t s exceeded l e v e l s i n the c o n t r o l s . T h i s i n c r e a s e i n p h e n o l concent r a t i o n s was a s s o c i a t e d w i t h an i n c r e a s e i n t h e l e v e l o f s o l u b l e c a r b o h y d r a t e s ( F i g . 2) i n e x p o s e d p l a n t s . The c o n c u r r e n t i n c r e a s e i n s o l u b l e c a r b o h y d r a t e and p h e n o l s s u g g e s t e d t h a t o z o n e may have s t i m u l a t e d g l y c o s i d e f o r m a t i o n i n P o n d e r o s a p i n e foliage. Ozone t r e a t m e n t i s known t o i n c r e a s e g l y c o s i d e and t o t a l p h e n o l l e v e l s i n p l a n t s ( 1 6 , 1 7 , 1 8 , 19) The i n c r e a s e i n enzymes o f p h e n o l m e t a b o l i s m and i n g l y c o s i d e s s u g g e s t s t h a t some o f the pigments observed i n ozone-injured t i s s u e are p h e n o l i c products. H o w e l l and Kremer ( 1 8 ) have i s o l a t e d a r e d d i s h brown pigment complex from ozone t r e a t e d bean l e a v e s t h a t c o n t a i n e d c a f f e i c a c i d , amino a c i d s , s u g a r s and some m e t a l s . The o z o n e induced s y n t h e s i s o f g l y c o s i d i c pigments would a l s o reduce the amount o f p h o t o s y n t h a t e a v a i l a b l e f o r t r a n s l o c a t i o n and g r o w t h . The e f f e c t s o f o z o n e a r e n o t l i m i t e d t o a l t e r i n g m e t a b o l i c p o o l s i n p l a n t f o l i a g e ; t h e y can a l s o induce t h e f o r m a t i o n o f t o x i c compounds. L e a c h a t e was c o l l e c t e d f r o m f e s c u e l e a v e s 2 weeks f o l l o w i n g a 2 h r e x p o s u r e t o 590 yg/m o z o n e and u s e d t o i r r i g a t e c l o v e r p l a n t s ( 2 0 ) . Top and r o o t d r y w e i g h t s o f l a d i n o c l o v e r were u n a f f e c t e d b y l e a c h a t e f r o m o z o n e - t r e a t e d f e s c u e l e a v e s . However, t h e l e a c h a t e f r o m t h e o z o n e - e x p o s e d f e s c u e l e a v e s r e d u c e d t h e n o d u l e number o f l a d i n o c l o v e r ( T a b l e I V ) . 3

Table

IV.

E f f e c t o f F e s c u e L e a f L e a c h a t e s on t h e o f Ladino Clover

Treatment

Top wt

dry (g)

Root d r y wt (g)

Growth

Nodule number

Fescue l e a c h a t e

1.08

0.27

169

Ozone t r e a t e d fescue leachate

1.02

0.24

78

The c l o v e r p l a n t s were 1 month o l d when t h e a p p l i c a t i o n s o f f e s c u e l e a f l e a c h a t e s were i n i t i a t e d . The l e a c h a t e s w e r e a p p l i e d t o t h e s o i l d a i l y f o r 1 month when t h e c o v e r was h a r v e s t e d . E a c h mean i s b a s e d on 10 o b s e r v a t i o n s . D a t a f r o m K o c h h a r ( 2 0 ) .

The l e a c h a t e f r o m o z o n e - e x p o s e d l e a v e s c o u l d a f f e c t p l a n t - p l a n t i n t e r a c t i o n s and i n f l u e n c e t h e p o p u l a t i o n o f s o i l microorganisms. These m e t a b o l i c a l t e r a t i o n i n p l a n t f o l i a g e c o u l d a l s o m o d i f y l i t t e r turnover r a t e s .

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

4.

Enzyme Activities of Plants

TINGEY

51

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Changes i n M e t a b o l i t e P o o l s i n R o o t s The o b s e r v e d o z o n e - i n d u c e d g r o w t h r e d u c t i o n s o f r o o t s c o u l d r e s u l t f r o m ( a ) a d i r e c t t o x i c e f f e c t o f o z o n e on t h e r o o t , ( b ) an o z o n e m o d i f i c a t i o n o f t h e f o l i a g e m e t a b o l i s m w h i c h a l t e r s t h e q u a n t i t y and/or q u a l i t y o f m e t a b o l i t e s t r a n s l o c a t e d t o t h e r o o t s , or ( c ) an a l t e r a t i o n i n s o i l c h e m i s t r y . Several f a c t o r s suggested t h a t t h e observed r e d u c t i o n s i n r o o t g r o w t h were t h e r e s u l t o f o z o n e a l t e r i n g f o l i a g e m e t a b o l i s m r a t h e r t h a n o f i t s d i r e c t e f f e c t o n t h e r o o t . Ozone i s v e r y r e ­ a c t i v e and would be u n l i k e l y t o p e n e t r a t e i n t o s o i l t h a t i s e i t h e r m o i s t o r c o n t a i n s o r g a n i c m a t t e r . An e x p e r i m e n t was c o n ­ d u c t e d t o d e t e r m i n e i f a i r c o n t a i n i n g o z o n e c o u l d be drawn t h r o u g h s o i l columns (Blum a n d T i n g e y , u n p u b l i s h e d ) . An a i r s t r e a m c o n t a i n i n g 980 ug/m o z o n e was drawn t h r o u g h s o i l columns and t h e n a n a l y z e d t o d e t e r m i n e t h e o z o n e c o n c e n t r a t i o n i n a i r l e a v i n g t h e columns. D u r i n g t h e 2 h r e x p o s u r e p e r i o d , 120 μ g o f o z o n e was a p p l i e d t o s o i l columns w i t h t h e s e c h a r a c t e r i s t i c s : a s u r f a c e a r e a o f 78.5 cm2 a n d 2 o r 4 cm d e e p ; c o n t a i n i n g g r a v e l , sand, j i f f y mix o r j i f f y mix: g r a v e l ( 1 : 2 , V / V ) . However, n o ozone c o u l d be d e t e c t e d i n t h e a i r s t r e a m e x i t i n g t h e c o l u m n s . To d e t e r m i n e t h e d e p t h t h a t o z o n e c o u l d d i f f u s e i n t o t h e s o i l s , r e d u c e d 2, 6 - d i c h l o r o p h e n o l - i n d o p h e n o l was i m p r e g n a t e d o n t o s a n d . The s a n d was p l a c e d i n a n a t m o s p h e r e c o n t a i n i n g 980 yg/m o z o n e f o r 2 h r a n d t h e d e p t h o f o z o n e p e n e t r a t i o n was m e a s u r e d a s t h e d e p t h t h a t t h e r e d u c e d d y e o n t h e s a n d was o x i d i z e d . B y t h i s m e a s u r e , ozone p e n e t r a t e d l e s s t h a n 20 mm i n t o s a n d ( B l u m a n d Tingey, unpublished). The a b i l i t y o f o z o n e t o i n d i r e c t l y a l t e r r o o t g r o w t h c a n b e shown b y t h e u s e o f r o o t e x u d a t e s . Root e x u d a t e s f r o m f e s c u e p l a n t s e x p o s e d t o a s i n g l e o z o n e dose ( 5 9 0 ug/m f o r 2 h r ) i n ­ h i b i t e d t h e t o p and r o o t growth o f l a d i n o c l o v e r and a l s o reduced n o d u l e numbers ( T a b l e V ) ( 2 0 ) . 3

3

3

T a b l e V.

E f f e c t s o f Root E x u d a t e s f r o m F e s c u e P l a n t s E x p o s e d t o Ozone o n t h e Growth o f L a d i n o C l o v e r

Treatment Fescue r o o t exudate Ozone t r e a t e d fescue r o o t exudate

Top d r y wt ( g )

Root d r y wt (g)

Nodule number

1.40

0.40

149

1.19

0.31

101

The f e s c u e p l a n t s were 1 month o l d when e x p o s e d t o 0 o r 590 pg/m ozone f o r 2 h r . The 1-month-old c l o v e r p l a n t s were e x ­ p o s e d t o t h e f e s c u e r o o t e x u d a t e s f o r 1 month a n d t h e n h a r v e s t e d . Means a r e b a s e d on 10 o b s e r v a t i o n s . D a t a f r o m K o c h h a r ( 2 0 ) . 3

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

AIR

POLLUTION

EFFECTS

O N PLANT

100

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Ο

20" I

1 " 1 2 3 4 MONTHS AFTER INITIATION OF EXPOSURE

J 5

Figure 6. The influence of chronic ozone exposure on the levels of total phenoh in the foliage of seedling Ponderosa pine. Each mean is based on nine observations. 801

1



1

Figure 7. The effect of chronic ozone exposure on the hvels of carbohydrates in the roots of Ponderosa pine seedlings. Each mean is based on nine observations.

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

GROWTH

4.

TINGEY

53

Enzyme Activities of Pfonts

Ozone e i t h e r i n d u c e d t h e f e s c u e p l a n t s t o f o r m a p h y t o t o x i n o r c a u s e d some m e t a b o l i t e n e c e s s a r y f o r g r o w t h o r n o d u l a t i o n t o be removed from t h e r o o t exudate s t r e a m . I n e i t h e r c a s e , t h e ozone exposure o f t h e f o l i a g e i n f l u e n c e d t h e m e t a b o l i t e p o o l s i n t h e root. Root exudates from ozone-exposed p l a n t s c o u l d a l s o i n f l u e n c e t h e t y p e o r number o f s o i l m i c r o o r g a n i s m s i n t h e r h i z o sphere and a l t e r p l a n t - p l a n t i n t e r a c t i o n s . The l e v e l s o f s t o r e d c a r b o h y d r a t e s i n P o n d e r o s a p i n e r o o t s were r e d u c e d b y c h r o n i c o z o n e e x p o s u r e ( 2 0 0 pg/m , 6 h r / d a y throughout t h e growing season) (Tingey and W i l h o u r , u n p u b l i s h e d ) . The l e v e l o f s o l u b l e c a r b o h y d r a t e s i n r o o t s o f P o n d e r o s a p i n e s e e d l i n g s exposed t o ozone t e n d e d t o be l e s s t h a n t h o s e i n t h e c o n t r o l l e v e l t h r o u g h o u t most o f t h e s e a s o n ( F i g . 7 A ) . S t a r c h l e v e l s i n t h e r o o t s o f e x p o s e d a n d c o n t r o l p l a n t s were s i m i l a r u n t i l f a l l when s t a r c h s t o r a g e b e g a n . A t t h a t t i m e , t h e exposed p l a n t s accumulated s t a r c h a t a s i g n i f i c a n t l y slower r a t e than the c o n t r o l s ( F i g . 7B). This reduction i n food reserves could h i n d e r t h e i n i t i a t i o n o f growth t h e f o l l o w i n g s p r i n g . Wardlaw ( 2 1 ) h a s i n d i c a t e d t h a t t h e g r o w t h o f e s t a b l i s h e d s h o o t s appears t o have p r i o r i t y o v e r t h e growth o f buds and r o o t s when t h e a s s i m i l a t e i s d e f i c i e n t . Roots a n d buds appear t o be t h e p o o r r e l a t i o n s among t h e p l a n t o r g a n s , r e c e i v i n g o n l y t h e p h o t o s y n t h a t e i n e x c e s s o f t h e r e q u i r e m e n t s o f o t h e r p a r t s . The ozone-induced depression o f photosynthesis coupled w i t h a r e t e n t i o n o f carbohydrates i n t h e f o l i a g e and a r e d u c t i o n i n t r a n s l o c a t i o n o f photosynthate t o the roots could explain the reduct i o n i n both storage m e t a b o l i t e s and r o o t growth. L e v e l s o f m e t a b o l i t e s o t h e r t h a n c a r b o h y d r a t e s were a l s o a l t e r e d i n r o o t s o f Ponderosa p i n e s e e d l i n g s a s a r e s u l t o f c h r o n i c ozone exposure ( T i n g e y and W i l h o u r , u n p u b l i s h e d ) . A f t e r t h r e e months o f e x p o s u r e , w h i c h c o i n c i d e d w i t h t h e a p p e a r a n c e o f v i s u a l i n j u r y , amino a c i d l e v e l s i n r o o t s o f e x p o s e d s e e d l i n g s were e l e v a t e d above t h o s e o f t h e c o n t r o l s ( F i g . 8 A ) . The amino a c i d l e v e l s r e m a i n e d above t h a t o f t h e c o n t r o l f o r t h e r e m a i n d e r o f t h e s e a s o n . A l s o t h e l e v e l s o f K j e l d a h l n i t r o g e n were h i g h e r i n r o o t s o f exposed than o f c o n t r o l p l a n t s throughout t h e season (Fig. 8B). A l t e r a t i o n s i n r o o t m e t a b o l i s m a s a r e s u l t o f ozone t r e a t ment a r e a l s o r e f l e c t e d i n t h e n o d u l a t i o n o f s o y b e a n s ( 2 1 ) . When s o y b e a n s were e x p o s e d t o a s i n g l e a c u t e o z o n e d o s e ( 1 4 7 0 pg/m ozone f o r 1 h r ) and h a r v e s t e d a t w e e k l y i n t e r v a l s f o l l o w i n g e x p o s u r e , n o d u l e number i n c r e a s e d more s l o w l y i n e x p o s e d p l a n t s (2 n o d u l e s / w k ) t h a n i n c o n t r o l p l a n t s ( 8 n o d u l e s / w k ) ( F i g . 9 ) . A s i m i l a r t r e n d was s e e n i n t h e n o d u l e w e i g h t p e r p l a n t a n d l e g h e m o g l o b i n c o n t e n t p e r n o d u l e was n o t a f f e c t e d . This indicat e d t h a t t h e e f f e c t o f t h e o z o n e t r e a t m e n t was t o r e d u c e n o d u l e number; t h e r e d u c t i o n o f n o d u l e number c a u s e d a c o n c u r r e n t r e d u c t i o n i n t o t a l nodule weight and leghemoglobin p e r p l a n t . The d e c r e a s e i n l e g h e m o g l o b i n , a n i n d i c a t o r o f n i t r o g e n f i x a t i o n c a p a c i t y (22) suggested t h e n i t r o g e n f i x a t i o n would a l s o be

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3

3

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

POLLUTION

EFFECTS

ON PLANT

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AIR

Figure 8. Ozone alteration of amino acid and Rjeldahl nitrogen levels in the roots of Ponderosa pine seedlings exposed to chronic ozone doses. Each mean is based on nine observations.

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

GROWTH

TINGEY

Enzyme Activities of Plants

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4.