The Impact of Ozone on the Bioenergetics of Plant Systems

9 The Impact of Ozone on the Bioenergetics of Plant Systems EVA

J.

PELL

Downloaded by CORNELL UNIV on October 23, 2016 | http://pubs.acs.org Publication Date: June 1, 1974 | doi: 10.1021/bk-1974-0003.ch009

Department of Plant Pathology, Pennsylvania State University, University Park, Penn. 16802

Abstract Ozone h a s b e e n shown to initiate many physiological a n d biochemical changes in sensitive plant species. D e c r e a s e s in photosynthesis and increases a n d d e c r e a s e s in respiration h a v e occurred in r e s p o n s e to ozonation. The bioenergetic s t a t u s of mitochondria and chloroplasts is disturbed by ozone. Decreases in oxidativeand photophosphorylation h a v e b e e n reported a s have increases in a d e n o s i n e triphosphate a n d total a d e n y l a t e c o n t e n t of plant tissue. The variable physiological responses a p p e a r to be related to the stage of symptom d e v e l o p m e n t at the t i m e of analysis a n d to the mode of o z o n e e x p o s u r e , viz. in vivo and in vitro.

In r e c e n t y e a r s t h e r e h a s been i n c r e a s e d i n t e r e s t i n e l u c i d a t i n g t h e b i o c h e m i c a l and p h y s i o l o g i c a l responses o f p l a n t s to a i r p o l l u t a n t s . Ozone h a s r e c e i v e d p a r t i c u l a r s c r u t i n y b e c a u s e o f i t s i m p o r t a n c e i n p h o t o c h e m i c a l smog. The e f f e c t s o f ozone on t h e spectrum o f p h y s i o l o g i c a l and b i o c h e m i c a l systems has b e e n a n a l y z e d i n p l a n t s y s t e m s b o t h i n v i v o a n d i n v i t r o t o t r y t o d e t e r m i n e t h e mode o f a c t i o n o f t h e g a s a t b o t h t h e c e l l u l a r a n d s u b c e l l u l a r l e v e l ( 1 ) . The d a t a o b t a i n e d f r o m t h i s r e s e a r c h h a s a l l o w e d t h e c h a r a c t e r i z a t i o n o f o z o n e damage a t v a r i o u s s t a g e s o f symptom d e v e l o p m e n t a n d a t v a r i o u s c e l l u l a r levels. We now know t h a t o z o n e h a s t h e p o t e n t i a l t o damage memb r a n e s y s t e m s , a l t e r enzyme f u n c t i o n a n d m o d i f y c o r r e s p o n d i n g m e t a b o l i c p a t h w a y s ( 1 ) . However, b e c a u s e o f o z o n e ' s r a p i d r e a c t i o n r a t e , t h e p r i m a r y s i t e o f i t s a c t i o n i s s t i l l open t o s p e c u l a t i o n . W h i l e t h e p r i m a r y e f f e c t s o f ozone on t h e c e l l w i l l l e a d t o t h e a l t e r e d p l a n t growth u l t i m a t e l y observed, t h e c o n n e c t i o n may n o t b e d i r e c t . T h e r e f o r e , i t would be a p p r o p r i a t e t o c o n s i d e r t h e r e s p o n s e o f a r e g u l a t o r y system t o ozone e x p o s u r e . R e g a r d l e s s o f whether t h e r e s p o n s e t o ozone i s p r i m a r y o r s e c o n d a r y , t h e e f f e c t s o n t h e c e l l a n d e n t i r e p l a n t s y s t e m may b e 106

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

9.

PELL

Bioenergetics of fiant Systems

107

permanent. The s t u d y o f b i o e n e r g e t i c s d e a l s w i t h e n e r g y f o r m a t i o n a n d u t i l i z a t i o n i n l i v i n g s y s t e m s . I n most l i v i n g c e l l s e n e r g y i s found i n the form o f adenosine t r i p h o s p h a t e (ATP). Many b i o c h e m i s t s (2_, 3_) b e l i e v e t h a t t h e c e l l i s r e g u l a t e d , a t l e a s t i n p a r t , by the energy charge, i . e . , [ATP] energy charge = ]


t

A

T

P

t

+ %[ADP] [ a

d

p

]+ [

α

μ

ρ

]

I m b a l a n c e s i n e n e r g y c h a r g e w i t h i n a c e l l may b e due t o r e d u c e d a v a i l a b i l i t y a n d / o r i n c r e a s e d demand f o r ATP o r i t s p r e c u r s o r s . Reduced e n e r g y a v a i l a b i l i t y may r e s u l t f r o m i m p a i r m e n t o f t h e energy-generating u n i t and/or from d i s l o c a t i o n o f the h i g h energy m o l e c u l e f r o m t h e s i t e r e q u i r i n g ATP. I n t h e p l a n t c e l l t h e r e a r e t w o s i t e s o f ATP g e n e r a t i o n : t h e c h l o r o p l a s t s and t h e m i t o c h o n d r i a . Adenosine t r i p h o s p h a t e i s generated d u r i n g the p r o c e s s o f oxygen u t i l i z a t i o n , v i z . d u r i n g r e s p i r a t i o n and p h o t o s y n t h e s i s w h i c h r e s u l t s i n o x y g e n e v o l u t i o n . S i n c e o z o n e i s a n a l l o t r o p e o f o x y g e n , i t was l o g i c a l t o e x a m i n e t h e e f f e c t s o f t h e a i r p o l l u t a n t o n t h e u p t a k e and e v o l u t i o n o f o x y g e n b y t h e m i t o c h o n d r i a and c h l o r o p l a s t s , r e s p e c t i v e l y , a n d t o a n a l y z e these o r g a n e l l e s f o r c o r r e s p o n d i n g e f f e c t s on phospho rylation. I n t h e f o l l o w i n g pages I s h o u l d l i k e t o d i s c u s s r e s e a r c h w h i c h has b e e n c o n d u c t e d t o d e t e r m i n e t h e e f f e c t o f o z o n e o n b o t h ATP c o n c e n t r a t i o n s and t h e p h o s p h o r y l a t i n g a b i l i t i e s o f p l a n t species; then I w i l l c o r r e l a t e t h i s research w i t h s t u d i e s d e f i n i n g t h e p h o t o s y n t h e t i c and r e s p i r a t o r y b e h a v i o r o f p l a n t systems. R e s p i r a t i o n and p h o t o s y n t h e s i s were among t h e f i r s t r e s p o n s e s t o o z o n e e x a m i n e d (4-_9). R e s p i r a t i o n has d e c r e a s e d u n d e r some c i r c u m s t a n c e s (J5, 7T, b u t i n g e n e r a l i t has b e e n shown t o i n crease. These i n c r e a s e s have b e e n c o r r e l a t e d w i t h t h e a p p e a r a n c e o f v i s i b l e symptoms ( 8 ) . S i n c e t h e g e n e r a t i o n o f ATP i s s o c l o s e l y a s s o c i a t e d w i t h e l e c t r o n t r a n s p o r t and o x y g e n u p t a k e , t h e e f f e c t s o f o z o n e o n o x i d a t i v e p h o s p h o r y l a t i o n were e x a m i n e d . I n t a c t t o b a c c o p l a n t s were e x p o s e d t o 0.60-0.70 μΐ/ΐ o z o n e f o r 1 h r ; m i t o c h o n d r i a i s o l a t e d f r o m v i s i b l y i n j u r e d t i s s u e demon s t r a t e d an i n h i b i t i o n o f o x i d a t i v e phosphorylation i n c o n j u n c t i o n w i t h a n i n c r e a s e i n r e s p i r a t i o n ( 6 ) . However, when d e t a c h e d t o b a c c o l e a v e s were f u m i g a t e d w i t h 1.0 μΐ/ΐ o z o n e f o r 1-5 h r , t h e m i t o c h o n d r i a e x t r a c t e d f r o m t h e t i s s u e p r i o r t o symptom d e v e l o p ment e x h i b i t e d r e d u c e d o x y g e n u p t a k e and r e d u c e d o x i d a t i v e p h o s p h o r y l a t i o n ( 7 ) . I n a n e x p e r i m e n t o f s i m i l a r d e s i g n when o z o n e was b u b b l e d t h r o u g h a s o l u t i o n o f i s o l a t e d m i t o c h o n d r i a , b o t h r e s p i r a t i o n and o x i d a t i v e p h o s p h o r y l a t i o n were r e d u c e d ( 7 ) . As w i l l b e e l a b o r a t e d s u b s e q u e n t l y , i t a p p e a r s t h a t t h e e f f e c t s o f ozone on m i t o c h o n d r i a a r e s e c o n d a r y . Studies i n v e s t i g a t i n g the photosynthetic f u n c t i o n o f p l a n t s have demonstrated an immediate d e c r e a s e i n p h o t o s y n t h e t i c r a t e s f o l l o w e d b y a r e t u r n t o n o r m a l when symptoms a p p e a r ( 8 , 9 ) .



108

AIR

POLLUTION

EFFECTS

ON

PLANT

GROWTH

T h i s d a t a has b e e n a c c r u e d f r o m o x y g e n e v o l u t i o n and c a r b o n d i o x i d e f i x a t i o n measurements c o n d u c t e d on i n t a c t l e a f t i s s u e h a r v e s t e d from p l a n t s exposed t o ozone i n v i v o . U n f o r t u n a t e l y , i s o l a t i o n o f c h l o r o p l a s t s f r o m t h e l e a f t i s s u e was n o t c o n d u c t e d t o d e t e r m i n e p h o t o s y n t h e t i c o r phot©phosphorylation r a t e s . T h e r e f o r e , we c a n n o t compare t h e p h o t o s y n t h e t i c d a t a s e c u r e d f r o m l e a f t i s s u e d e t e r m i n a t i o n s and t h e r e s p i r a t i o n d a t a o b t a i n e d f r o m s t u d i e s c o n d u c t e d on t h e m i t o c h o n d r i a . S u c h c o m p a r i s o n s i n s i t u a r e n o t p o s s i b l e b e c a u s e a change i n g a s e o u s e x c h a n g e i n l e a f t i s s u e c o u l d be a d i r e c t r e s u l t o f i m p a i r e d o r g a n e l l e f u n c t i o n o r an i n d i r e c t e f f e c t o f a l t e r e d g a s e o u s i n g r e s s due t o a n o t h e r c e l l u l a r m a l f u n c t i o n . S e v e r a l i n v i t r o s t u d i e s have b e e n c o n d u c t e d w i t h i s o l a t e d c h l o r o p l a s t s i n w h i c h t h e o r g a n e l l e s were e x p o s e d t o o z o n e ; p h o t o p h o s p h o r y l a t i o n r a t e s and p h o t o s y n t h e t i c p a r a m e t e r s were m e a s u r e d . I t was d e m o n s t r a t e d t h a t when i s o l a t e d s p i n a c h c h l o r o p l a s t s were e x p o s e d t o a t o t a l o f 900 n a n o m o l e s o r l e s s o f o z o n e , e l e c t r o n t r a n s p o r t and c y c l i c p h o t o p h o s p h o r y l a t i o n were r e d u c e d ( 1 0 ) . I n a n o t h e r e x p e r i m e n t when 40 u l / 1 o z o n e w e r e b u b b l e d t h r o u g h a p e a c h l o r o p l a s t s u s p e n s i o n , ATP f o r m a t i o n was r e d u c e d ( 1 1 ) . Adenosine t r i p h o s p h a t e i s u t i l i z e d i n p o r t i o n s o f the c e l l o t h e r t h a n t h e m i t o c h o n d r i a and c h l o r o p l a s t s ; t h e r e f o r e , t h e u t i l i z a t i o n as w e l l as t h e p r o d u c t i o n o f ATP i s o f i m p o r t a n c e t o t o t a l a d e n y l a t e s t a t u s . As a r e s u l t , i t became i m p o r t a n t t o c o n s i d e r t o t a l ATP c o n t e n t o f p l a n t s . When d e t a c h e d p i n t o b e a n l e a v e s were e x p o s e d t o 1.0 μΐ/ΐ o z o n e f o r 30 m i n t o t a l ATP c o n t e n t o f t h e l e a f d e c r e a s e d (12)· S i n c e ozone a l t e r e d l e a f ATP c o n t e n t i t c o u l d a l s o a l t e r t h e l e a f ' s a d e n y l a t e s t a t u s ; we wished t o determine i f a c o r r e l a t i o n e x i s t e d between a l t e r a t i o n i n a d e n y l a t e s and t h e change p r e v i o u s l y r e p o r t e d i n p h o t o s y n t h e s i s and r e s p i r a t i o n . S i n c e ATP i s r e a d i l y b r o k e n down b y a d e n o s i n e t r i p h o s p h a t a s e s , a r e l i a b l e method o f e x t r a c t i o n and q u a n t i t a t i v e method o f ATP a n a l y s i s was d e s i g n e d f o r t h e s t u d y (8).

A d e n o s i n e t r i p h o s p h a t e was m e a s u r e d by t h e l u c i f e r i n l u c i f e r a s e a s s a y and t o t a l a d e n y l a t e s were m e a s u r e d by c o n v e r t i n g a d e n o s i n e d i p h o s p h a t e (ADP) and a d e n o s i n e monophosphate (AMP) t o ATP and m e a s u r i n g s i m i l a r l y . P h o t o s y n t h e s i s and r e s p i r a t i o n were m e a s u r e d m a n o m e t r i c a l l y ( 8 ) . I n t a c t p i n t o b e a n p l a n t s were e x p o s e d t o 0.30 μΐ/ΐ o z o n e f o r t h r e e h r and a n a l y z e d i m m e d i a t e l y a f t e r o z o n a t i o n b e f o r e v i s u a l symptoms d e v e l o p e d ; we o b s e r v e d a s i g n i f i c a n t i n c r e a s e i n ATP and t o t a l a d e n y l a t e c o n t e n t , an i n c r e a s e i n r e s p i r a t i o n r a t e s and a d e c r e a s e i n p h o t o s y n t h e s i s r a t e s ( T a b l e I ) . Due t o t h e s e e m i n g d i s p a r i t y b e t w e e n o u r r e s u l t s and t h o s e r e p o r t e d p r e v i o u s l y (16, 7_, 1 0 , 1 1 , 1 2 ) , we r e p e a t e d o u r e x p e r i m e n t s m e a s u r i n g t h e s e p a r a m e t e r s a t 0, 6, 2 1 , and 72 h r a f t e r o z o n a t i o n t o more t h o r o u g h l y c h a r a c t e r i z e t h e r e s p o n s e s w i t h r e g a r d t o symptom d e v e l o p m e n t . We a g a i n o b s e r v e d t h e s i g n i f i c a n t i n c r e a s e i n ATP i n t o t a l a d e n y l a t e c o n t e n t and i n r e s p i r a t i o n r a t e , and a d e c r e a s e i n p h o t o s y n t h e s i s i m m e d i a t e l y



8.28

3

Photosynthesis r e s u l t s b a s e d on t t e s t .

171.7

X

X X

17.58

5.83

4.25

0.67

Control

3.60 NS x3

5.25 NS 12.03

Control

3.43

0.72

4.00

4.33

3.38

NS 4.93 NS

4.48

NS

Ozone

Respiration

μΐ 02/hr-mg d r y wt

Ozone

Photosynthesis

were s i g n i f i c a n t a t t h e 5% l e v e l

replicates.

21.37 29.14

o f t h i s experiment

D a t a f r o m P e l l (_8). 2 E a c h number i s t h e mean o f f o u r

1

L S D 0.05 ^ L S D 0.01

X

149.4

X X

130.0

111.5

194.4

X

205.7

177.7 147.9

X X

X X

139.7 142.7

2

Ozone Control Ozone n m o l e / g f r e s h wt

T o t a l Adenylates

117.4

112.4

ATP

ATP a n d t o t a l a d e n y l a t e c o n t e n t a n d p h o t o s y n t h e s i s a n d r e s p i r a t i o n r a t e s o f p i n t o b e a n l e a v e s h a r v e s t e d i m m e d i a t e l y a f t e r a 3-hr e x p o s u r e t o 0.30 μΐ/ΐ o z o n e l

Control

Table I .



110

AIR

POLLUTION

EFFECTS

ON

PLANT

GROWTH

a f t e r ozonation (Table I I ) . These c h a n g e s p e r s i s t e d f o r 6 h r . Twenty-one h r a f t e r o z o n e e x p o s u r e , t h e ATP l e v e l and p h o t o s y n t h e t i c r a t e had r e t u r n e d t o normal. S e v e n t y two h r a f t e r o z o n a t i o n , r e s p i r a t i o n r a t e s were s t i l l i n c r e a s e d b u t p h o t o s y n t h e s i s r a t e s and t o t a l a d e n y l a t e l e v e l s had r e t u r n e d t o n o r m a l . The ATP c o n t e n t o f t h e o z o n a t e d b e a n p l a n t s showed a s e c o n d a r y i n c r e a s e o v e r c o n t r o l p l a n t s a f t e r 72 h r . T h e r e a r e s e v e r a l o b s e r v a t i o n s w h i c h can be drawn f r o m t h e s e studies. Ozone a l t e r s r e s p i r a t i o n , p h o t o s y n t h e s i s and p h o s p h o r y l a t i o n r a t e s as w e l l as t h e g e n e r a l a d e n y l a t e s t a t u s o f p l a n t s . I n e a c h r e s e a r c h p a p e r d i s c u s s e d a b o v e , t h e r e were r e p o r t s o f c h a n g e s i n v a r i o u s b i o e n e r g e t i c s y s t e m s . The d i r e c t i o n and m a g n i t u d e o f t h e s e c h a n g e s were n o t a l w a y s t h e same. T h e r e a r e many p o s s i b l e e x p l a n a t i o n s f o r t h e s p e c t r u m o f r e s u l t s r e p o r t e d , i n c l u d i n g e n v i r o n m e n t a l c o n d i t i o n s b e f o r e , d u r i n g and a f t e r o z o n a t i o n and t h e u s e o f d i f f e r e n t e x p e r i m e n t a l methods t o t e s t any o f t h e c e l l f u n c t i o n s . However, t h e r e a r e two b a s i c d i f f e r e n c e s i n e x p e r i m e n t a l d e s i g n w h i c h may be r e s p o n s i b l e f o r t h e variable results: ( a ) u s e o f d i f f e r e n t o z o n e d o s a g e r e g i m e s and (b) use o f i n v i v o o r i n v i t r o p l a n t systems. The d o s e o f o z o n e , i . e . t h e gas c o n c e n t r a t i o n e x p o s u r e t i m e , w i l l d e t e r m i n e t h e s e v e r i t y and t h e r a t e o f symptom d e v e l o p m e n t . The r a t e o f d e v e l o p m e n t i s v e r y i m p o r t a n t when p h y s i o l o g i c a l a n d / o r b i o c h e m i c a l a n a l y s e s a r e c o n d u c t e d on p l a n t t i s s u e . If d i f f e r e n t d o s e s a r e u s e d ( a s was t h e c a s e a b o v e ) t h e r a t e s o f symptom d e v e l o p m e n t w i l l be d i f f e r e n t . The t i m e s o f t i s s u e h a r v e s t f o r f u n c t i o n a l a n a l y s e s w i l l p r o b a b l y n o t be c o m p a r a b l e a n d , t h e r e f o r e , t h e r e s u l t s may v a r y f o r t i s s u e a n a l y z e d a t d i f f e r e n t t i m e s d u r i n g o z o n e symptom d e v e l o p m e n t . S i n c e a l l t h e o z o n e c o n c e n t r a t i o n s u s e d i n t h e e x p e r i m e n t s d i s c u s s e d above l e a d t o c e l l d e a t h , i t i s l i k e l y t h a t t h e f u n c t i o n a l changes r e p o r t e d do o c c u r . I t i s t h e sequence i n which t h e s e changes o c c u r t h a t i s s i g n i f i c a n t , and t h i s i s d e p e n d e n t upon when measurements were made w i t h r e g a r d t o symptom d e v e l o p m e n t . When p l a n t t i s s u e was exposed t o h i g h ozone c o n c e n t r a t i o n s , r a p i d i n d u c t i o n o f c e l l u l a r and o r g a n e l l a r d e s t r u c t i o n c o u l d have o c c u r r e d . Any a n a l y s i s c o n d u c t e d s u b s e q u e n t t o t h e o z o n a t i o n w o u l d p r o b a b l y r e f l e c t an a d v a n c e d s t a g e o f c e l l damage. As a r e s u l t , any e a r l y e v e n t i n damage d e v e l o p m e n t w o u l d be o v e r l o o k e d . F o r e x a m p l e , an i n c r e a s e i n a d e n y l a t e c o n c e n t r a t i o n c o u l d o c c u r e a r l y i n the development o f symptoms f o l l o w e d by a r u p t u r i n g o f t h e i n t e g r i t y o f t h e c o u p l e d membrane s y s t e m . T h i s u n c o u p l i n g c o u l d l e a d t o i n c r e a s e d r e s p i r a t i o n and d e c r e a s e d o x i d a t i v e p h o s p h o r y l a t i o n . During r a p i d symptom d e v e l o p m e n t an e a r l y p h y s i o l o g i c a l change c o u l d p a s s b e f o r e t h e r e was an o p p o r t u n i t y f o r a n a l y s i s . B o t h i n v i v o and i n v i t r o s y s t e m s h a v e b e e n u t i l i z e d t o t r y t o e l u c i d a t e t h e e f f e c t s o f o z o n e on v e g e t a t i o n , b u t one must be c a u t i o u s about assuming t h a t t h e y are e q u a l . Ill v i t r o studies r e v e a l t h e p o t e n t i a l o f o z o n e t o i n j u r e o r g a n e l l e s , enzyme s y s t e m s



65.56

72

81.75

X

0.52 0.69

replicates.

25.90

0.49

X X

4.43 3.58 3.30 NS

3.21

112.33 NS

120.33

4.50» 3.00

3.31 NS

3.23

155.88»

119.06

3.75»

2.91

3.34

3.88

181.54»

124.55

126.00»

X

4.23»

5.05

3.63

3.95XX

159.98»

99.56 NS

Respiration Control Ozone

μΐ 02/hr.mg d r y wt

Photosynthesis Control Ozone

113.58

vv

D a t a from P e l l ( 8 ) . 2 E a c h number i s t h e mean o f e i g h t

1

Total Adenylates Control Ozone

n m o l e / g f r e s h wt

Ozone

123.06

ATP

14.68 19.64

91.28

21

LSD 0.05 L S D 0.01

83.23

6

X X

82.81

2

Control

Mean v a l u e s f o r ATP a n d t o t a l a d e n y l a t e c o n t e n t a n d p h o t o s y n t h e s i s and r e s p i r a t i o n r a t e s o f p i n t o b e a n l e a v e s h a r v e s t e d o v e r a 7 2 - h r p e r i o d f o l l o w i n g a 3-hr o z o n a t i o n l

0

Time hr

Table I I .



112

AIR

POLLUTION

EFFECTS

ON

PLANT

GROWTH

e t c . , b u t t h e d a t a c a n n o t be e x t r a p o l a t e d t o t h e e f f e c t s o f o z o n e on t h e s e s y s t e m s i n v i v o b e c a u s e t h e e n v i r o n m e n t o f a s u b c e l l u l a r s t r u c t u r e and/or system i n v i v o i s q u i t e d i f f e r e n t from t h a t i n an a r t i f i c a l , i n v i t r o , medium. T h i s e n v i r o n m e n t may be c r u c i a l t o the system's response t o the gas. For example, o r g a n e l l e s a r e n o r m a l l y f o u n d b a t h e d i n c y t o p l a s m and a r e i n f l u e n c e d by the metabolic pools e x i s t i n g t h e r e i n . I f an o r g a n e l l e were damaged by o z o n e and r e q u i r e d p r e c u r s o r s f r o m t h e m e t a b o l i c p o o l f o r r e p a i r , t h i s r e s p o n s e w o u l d n o t be m e a s u r e d i n an i n v i t r o s y s t e m ; i n f a c t , a more s e v e r e o r d i f f e r e n t r e s p o n s e t h a n o c c u r s i n v i v o may be m e a s u r e d . One s u c h example d e a l s w i t h t h e dec r e a s e i n o x i d a t i v e - and p h o t o - p h o s p h o r y l a t i o n r a t e s o b s e r v e d when m i t o c h o n d r i a and c h l o r o p l a s t s were t r e a t e d and a n a l y z e d i n vitro. These d e c r e a s e s c o u l d have b e e n due t o i m p a i r m e n t o f s t r u c t u r e s necessary f o r phosphorylation or t o leakage o f necessary p r e c u r s o r s from the o r g a n e l l e i n t o the b u f f e r e d s o l u tion. The l a t t e r i s a d i s t i n c t p o s s i b i l i t y s i n c e t h e r e i s e v i d e n c e o f damaged membrane f u n c t i o n ( 1 3 ) . I n f a c t , i t has b e e n r e p o r t e d t h a t when i s o l a t e d m i t o c h o n d r i a a r e e x p o s e d t o o z o n e there i s increased leakage o f n u c l e o t i d e s d i r e c t l y p r o p o r t i o n a l t o l e n g t h o f o z o n e e x p o s u r e ( 1 4 ) . I f m e t a b o l i t e s and p r e c u r s o r s a r e l e a k i n g i n t o t h e c y t o p l a s m , c o n s i d e r a t i o n s h o u l d be g i v e n t o t h e i m p a c t w h i c h t h e s e m e t a b o l i t e s m i g h t have on c e l l u l a r functions. For example, i f adenylates l e a k out o f m i t o c h o n d r i a l and c h l o r o p l a s t membranes i n t o t h e c y t o p l a s m o f i n t a c t c e l l s , a demand f o r i n c r e a s e d s y n t h e s i s o f a d e n y l a t e s c o u l d o c c u r w i t h i n t h e s e o r g a n e l l e s . T h i s s y n t h e s i s w o u l d t h e n r e s u l t i n an i n c r e a s e i n n e t a d e n y l a t e c o n t e n t o f t h e e n t i r e t i s s u e . The e n zymes n e c e s s a r y f o r t h i s s y n t h e s i s a r e b e l i e v e d t o be c y t o p l a s m i c i n o r i g i n ; hence, i f the cytoplasm i s not p r e s e n t — a s i s the case i n an i n v i t r o e x p e r i m e n t — t h e i n c r e a s e c o u l d n o t o c c u r . From t h e d i s c u s s i o n above we c o n c l u d e t h a t o u r o b s e r v a t i o n s are not n e c e s s a r i l y i n c o n s i s t e n t w i t h those o f other r e p o r t s . B o t h t h e t i m e o f a n a l y s i s and e x p e r i m e n t a l d e s i g n may a f f e c t t h e results. An e x p l a n a t i o n f o r t h e i n c r e a s e i n a d e n y l a t e s u n d e r t h e c o n d i t i o n s o f o u r e x p e r i m e n t i s s t i l l n e e d e d . S i n c e b o t h ATP a l o n e and t o t a l a d e n y l a t e c o n c e n t r a t i o n s have i n c r e a s e d , i t does not appear t h a t a s h i f t i n p h o s p h o r y l a t i o n can account f o r t h e i n c r e a s e s . The d e c r e a s e i n p h o t o s y n t h e s i s and i n c r e a s e i n a d e n y l a t e s o c c u r d u r i n g t h e same t i m e p e r i o d and b o t h f a c t o r s r e t u r n t o n o r m a l a f t e r 21 h r . From p r e v i o u s r e s e a r c h we know t h a t the p h o t o s y n t h e t i c l e v e l s o f ozonated p i n t o bean f o l i a g e d e c r e a s e i m m e d i a t e l y a f t e r o z o n e e x p o s u r e e v e n when symptoms do n o t d e v e l o p (8_). T h i s d o e s n o t h o l d t r u e f o r t h e a d e n y l a t e o r r e s p i r a t i o n responses. T h e r e f o r e , i t appears t h a t the ozonei n i t i a t e d i n c r e a s e i n adenylates i s not c o r r e l a t e d d i r e c t l y t o the photosynthetic response. The i n c r e a s e i n r e s p i r a t i o n p e r s i s t s when a d e n y l a t e c o n t e n t and p h o t o s y n t h e t i c r a t e s have r e t u r n e d t o normal. I m p a i r e d m i t o c h o n d r i a l f u n c t i o n a p p e a r s t o be a s e c o n d a r y r e s p o n s e more c l o s e l y r e l a t e d t o symptom d e v e l o p m e n t .



9.

PELL

Bioenergetics of Plant Systems

113

I f p h o t o s y n t h e t i c a n d r e s p i r a t o r y changes c a n n o t a c c o u n t f o r t h e i n c r e a s e s i n a d e n y l a t e c o n c e n t r a t i o n , which system i s responsible? I t h a s b e e n r e p o r t e d t h a t ADP and ATP c o n c e n t r a t i o n s o f E h r l i c h a s c i t e s tumor c e l l s i n c r e a s e i n t h e presence o f a d e n i n e ( 1 5 ) . Whether t h i s w o u l d h o l d t r u e f o r p l a n t c e l l s i s n o t known, b u t i t seems p l a u s i b l e t h a t e q u i l i b r i u m s h i f t s w o u l d i n i t i a t e s i m i l a r r e s p o n s e s . An i n c r e a s e i n a d e n i n e c o n c e n t r a t i o n s c o u l d o c c u r i f t h e r e was any b r e a k d o w n o f n u c l e i c a c i d s . T h e r e i s one r e p o r t t h a t t h e number o f r i b o s o m e s i n t h e c h l o r o p l a s t d o e s d e c r e a s e i n r e s p o n s e t o o z o n e ( 1 6 ) . An i n c r e a s e i n s y n t h e s i s o f p u r i n e s i s a l s o p o s s i b l e b u t t h e r e i s no evidence t o e i t h e r support o r r e f u t e t h i s hypothesis. I n c r e a s e s i n a d e n y l a t e c o n t e n t a s have b e e n d i s c u s s e d h e r e are not unique responses o f p l a n t s t o ozone. Other s t r e s s e s , i n c l u d i n g t h e a i r p o l l u t a n t hydrogen f l u o r i d e and t o b a c c o m o s a i c v i r u s , h a v e b e e n r e p o r t e d t o i n d u c e s i m i l a r i n c r e a s e s i n ATP a n d / o r a d e n y l a t e s (17_, 1 8 ) . A l t h o u g h ATP i s c o n s i d e r e d a n e n e r g y source and, t h e r e f o r e , a v a l u a b l e b i o c h e m i c a l c e l l c o n s t i t u e n t , i t s p r o d u c t i o n a t t h e e x p e n s e o f o t h e r c e l l u l a r components w o u l d have d e l e t e r i o u s e f f e c t s . Furthermore, i t i s not the t o t a l conc e n t r a t i o n s o f adenylates per se, but the d i s t r i b u t i o n which i s i m p o r t a n t . Some r e s e a r c h e r s have s u g g e s t e d t h a t t h e movement o f adenylates from the p r o d u c t i o n s i t e t o s y n t h e s i s s i t e s c o n t r o l s c e l l u l a r metabolism (19, 20). I f the d i s t r i b u t i o n s h i f t s , metabolism w i l l be a l t e r e d . A t k i n s o n (2) has s t a t e d t h a t the e n e r g y c h a r g e may b e r e s p o n s i b l e f o r c o n t r o l l i n g o f gene f u n c t i o n . S i n c e t h e r e a r e a t l e a s t t h r e e s i t e s o f f u n c t i o n a l gene a c t i v i t y ( n u c l e i c a c i d s ) v i z . c h l o r o p l a s t , m i t o c h o n d r i a and n u c l e u s , t h e energy charge would be c r u c i a l i n a l l t h e s e a r e a s . I t would be important t o determine whether t h e i n c r e a s e i n t o t a l a d e n y l a t e s r e f l e c t s a n y change i n e n e r g y c h a r g e i n any o r a l l s i t e s o f gene f u n c t i o n . T h i s w o u l d b e a c c o m p l i s h e d b y m e a s u r i n g a b s o l u t e concentrations as w e l l as r a t i o s . The i m p a c t o f a l t e r e d e n e r g y c h a r g e i s open t o s p e c u l a t i o n s i n c e we d o n o t f u l l y u n d e r s t a n d t h e r o l e o f e n e r g y c h a r g e i n the healthy c e l l . Many r e s e a r c h e r s h a v e s u g g e s t e d t h a t o z o n e i n d u c e s premature senescence ( 2 1 , 2 2 ) . Normal senescence i s a f u n c t i o n o f p l a n t g r o w t h r e l a t e d d i r e c t l y t o gene f u n c t i o n . Perhaps ozone-induced senescence c o u l d be a t t r i b u t e d t o t h e e f f e c t s o f a l t e r e d a d e n y l a t e s t a t u s o n gene f u n c t i o n .


114

AIR

POLLUTION

EFFECTS

ON

PLANT

GROWTH

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D u g g e r , W. M. a n d Ting, I. P. Ann. Rev. Plant Physiol. ( 1 9 7 0 ) 21:215. Atkinson, D. E. I n " H o r i z o n s of Bioenergetics." A. San Pietro, G e s t , H. E d s . , p p . 83-97. Atkinson, D. E. Ann. Rev. B i o c h e m . ( 1 9 6 6 ) 35:85. T o d d , G. W. Plant Physiol. ( 1 9 5 8 ) 33:416. T o d d , G. W. Physiol. Plantarum (1963) 16:57. M a c d o w a l l , F. D. H. Can. J. B o t . (1965) 43:419. L e e , T. T. Plant Physiol. (1967) 42:691. Pell, E. J. and B r e n n a n , E. P l a n t Physiol. ( 1 9 7 3 ) 51:378. Hill, A. C. a n d Littlefield, N. Environ. Sci. Technol. ( 1 9 6 9 ) 3:52. C o u l s o n , C. a n d H e a t h , R. L. P l a n t Physiol. ( 1 9 7 4 ) 53:32. N o b e l , P. S. and Wang, C. A r c h . B i o c h e m . B i o p h y s . (1973) 157:388. T o m l i n s o n , H. and Rich, S. P h y t o p a t h o l o g y ( 1 9 6 8 ) 58:808. S p o t t s , R., Lukezic, F. and H a m i l t o n , R. Phytopathology ( 1 9 7 4 ) in Press. L e e , T. T. Plant Physiol. (1968) 43:133. S n y d e r , F. F. and Henderson, J. F. Can. J. B i o c h e m . ( 1 9 7 3 ) 51:943. C h a n g , C. W. P h y t o c h e m i s t r y (1971) 10:2863. L o r d s , J. L. and M c N u l t y , I . B. U t a h A c a d . Sci. Arts Lett. ( 1 9 6 5 ) 42:163. S u n d e r l a n d , D. W. a n d Merrett, M. J. Physiol. P l a n t a r u m ( 1 9 6 7 ) 20:368. H e l d t , H. W. FEBS Letters ( 1 9 6 9 ) 5:11. Santarius, K. A. a n d H e b e r , U. B i o c h i m . B i o p h y s . A c t a (1965) 102:39. H e g g e s t a d , H. E. Amer. P o t a t o J. ( 1 9 7 3 ) 50:315. R i c h , S. Ann. Rev. P h y t o p a t h . ( 1 9 6 4 ) 2:253.


The Impact of Ozone on the Bioenergetics of Plant Systems

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