Mechanisms of Ozone Injury to Plants

6 Mechanisms of Ozone Injury to Plants S A U L R I C H and

HARLEY TOMLINSON

Downloaded by CORNELL UNIV on October 23, 2016 | http://pubs.acs.org Publication Date: June 1, 1974 | doi: 10.1021/bk-1974-0003.ch006

Connecticut Agricultural Experiment Station, New Haven, Conn. 06504

Ozone and related oxidants a r e e s t i m a t e d to be responsible for a b o u t 95% o f t h e a n n u a l $130 million c r o p loss c a u s e d b y air pollutants in the United States. R e p o r t s have indicated that ozone can seriously damage i m p o r t a n t c r o p s s u c h as spinach, beans, petunias, citrus, t o b a c c o , s o y b e a n s , and alfalfa, and forest trees s u c h as Eastern w h i t e p i n e and P o n d e r o s a p i n e . Susceptible cultivars of most of t h e s e plants d e v e l o p s e v e r e leaf injury when e x p o s e d t o 2 to 5 pphm of o z o n e for 1 to 4 hr. This level of o z o n e is very common in u r b a n a r e a s and so a r e symptoms of ozone injury. However, s u c h symptoms have also b e e n reported f r o m plants g r o w i n g in s u c h rural states a s Maine and South Dakota. E v e n when no o b v i o u s injury c a n be s e e n , plants e x p o s e d t o l o w levels of ozone may n o t grow a s well or yield a s much a s plants growing in air free of o z o n e . On some plants, e . g . , spinach, t h e symptoms of o z o n e injury a r e quite distinctive; c o n s e q u e n t l y , plants like t h e s e a r e b e i n g u s e d in some places to detect and m o n i t o r air pollution. I n C o n n e c t i c u t , o z o n e i n j u r y was f i r s t s e e n on t o b a c c o 20 y e a r s ago. T h i s shade-grown c r o p u s e d f o r c i g a r w r a p p e r s has an a n n u a l c a s h v a l u e o f a b o u t $22 m i l l i o n . I n some y e a r s , o z o n e damage c a u s e d a l o s s o f up t o $5 m i l l i o n . P l a n t b r e e d e r s soon p r o d u c e d c u l t i v a r s b o t h h i g h l y r e s i s t a n t t o ozone and y e t w i t h the necessary commercial q u a l i t i e s . With the adoption o f these c u l t i v a r s , annual l o s s e s t o the C o n n e c t i c u t tobacco crop from o z o n e d r o p p e d t o o n l y a few t h o u s a n d d o l l a r s , e v e n t h o u g h t h e l e v e l o f p o l l u t i o n d i d not recede. This r e p o r t includes a possible anatomical basis f o r the f l e c k i n g symptom and a summary o f o u r s e a r c h f o r t h e mechanisms by which ozone i n j u r e s p l a n t s . Anatomical Basis of Flecking F l e c k i n g on t h e u p p e r s u r f a c e o f l e a v e s i s a common symptom o f o z o n e i n j u r y on d i c o t y l e d o n o u s p l a n t s . A s i n g l e f l e c k i s a s m a l l l i n e o f d e a d t i s s u e t h a t a p p e a r s w h i t e , y e l l o w , o r brown

76

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


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Mechanisms of Ozone Injury

77

against the green l i v i n g t i s s u e t h a t surrounds i t . When t h e f l e c k i s e x a m i n e d c l o s e l y , t h e l e s i o n c a n be a s s o c i a t e d w i t h contiguous stomata i n the upper s u r f a c e . Often, t h e f i r s t v i s i b l e symptom o f o z o n e t o x i c i t y i s t h e d e a t h o f t h e p a l i s a d e parenchyma c e l l s t h a t l i n e t h e c a v i t i e s d i r e c t l y b e n e a t h the upper stomata. I n the case o f beans (Phaseolus v u l g a r i s L . ) o r t o b a c c o ( N i c o t i a n a t a b a c u m L.) a n d p e r h a p s o t h e r p l a n t s , the upper stomata l i e i n p a t t e r n s o f a r c s o r c i r c l e s , while the lower stomata are s c a t t e r e d randomly and r e g u l a r l y across the epidermis. A d j a c e n t upper stomata appear t o be connected b y a i r p a s s a g e s t h r o u g h t h e p a l i s a d e p a r e n c h y m a . These p a s s a g e s c a n be s e e n e a s i l y when a d e t a c h e d l e a f i s f i l l e d w i t h w a t e r u n d e r pressure by i n j e c t i n g i t through the p e t i o l e w i t h a hypodermic s y r i n g e . The w a t e r i s f o r c e d a l o n g t h e p a t h o f l e a s t r e s i s t a n c e and f i l l s t h e a i r s p a c e s c o n n e c t i n g t h e s u b s t o m a t a l chambers o f the upper stomata. Apparently the p a l i s a d e c e l l s l i n i n g the substomatal cavi t i e s a r e t h e c e l l s most s e n s i t i v e t o o z o n e i n t h e s e l e a v e s . As the c e l l s are k i l l e d b y ozone a l o n g t h e a i r passages c o n n e c t i n g t h e s u b s t o m a t a l c h a m b e r s , t h e e l o n g a t e d f l e c k i s f o r m e d . The random p a t t e r n o f s t o m a t a i n t h e l o w e r s u r f a c e , t h e d i f f e r e n t geometry o f t h e a i r passages t h r o u g h the spongy parenchyma, and perhaps t h e g r e a t e r r e s i s t a n c e o f spongy parenchyma c e l l s t o o z o n e make i t u n l i k e l y t h a t f l e c k i n g w o u l d a p p e a r o n t h e l o w e r surface o f these leaves. L e v e l s o f o z o n e t h a t do more t h a n f l e c k t h e u p p e r s u r f a c e s c a n c a u s e i r r e g u l a r s p o t s a n d b l o t c h e s o f dead t i s s u e o n t h e lower surface. S u f f i c i e n t ozone t o k i l l a r e a s i n t h e l o w e r s u r f a c e a r e u s u a l l y enough t o c a u s e t h e e n t i r e a r e a o f t h e l e a f t o c o l l a p s e , l e a d i n g t o the formation o f " b i f a c i a l " l e s i o n s . Biochemical

Studies

To d e t e r m i n e how p l a n t s a r e i n j u r e d , we s t u d i e d c h a n g e s i n c e r t a i n c e l l u l a r c o n s t i t u e n t s i n p l a n t s e x p o s e d t o o z o n e . The c e l l s o f these p l a n t s l e a k t h e i r contents and so i t i s probable t h a t t h e i n i t i a l damage i s t o c e l l u l a r membranes. The n o r m a l f u n c t i o n i n g o f t h e s e membranes depends o n l i p i d c o n s t i t u e n t s probably s t a b i l i z e d by s u l f h y d r y l groups i n a s s o c i a t e d p r o t e i n s . S u l f h y d r y l Groups. S u l f h y d r y l l i n k a g e s are c o n s i d e r e d t o i m p o r t a n t t o t h e s t r u c t u r a l i n t e g r i t y o f membrane p r o t e i n s . I n o u r f i r s t e x p e r i m e n t s ( 1 ) we s u b j e c t e d b e a n , s p i n a c h ( S p i n a c i a o l e r a c e a L . ) a n d t o b a c c o l e a v e s t o o z o n e a t 1 ppm f o r 30 t o 60 m i n . A t t h i s high concentration o f ozone, the s u l f h y d r y l c o n t e n t o f t h e l e a v e s was d i m i n i s h e d 15 t o 2 5 % ( T a b l e I ) . T h e r e was l i t t l e d i f f e r e n c e b e t w e e n t h e s u l f h y d r y l c o n t e n t o f o z o n e r e s i s t a n t and o z o n e - s u s c e p t i b l e tobacco e i t h e r b e f o r e o r a f t e r ozonation.

be


78

AIR

Table I .

The

POLLUTION

EFFECTS

ON

PLANT

GROWTH

e f f e c t o f o z o n e on t h e c o n c e n t r a t i o n o f s u l f h y d r y l (SH) g r o u p s i n l e a v e s Tobacco

Time o f a n a l y s i s i n r e l a t i o n t o treatmentsBean

Ozoneresistant

Spinach


y m o l e s SH/g 1.20 1.10 0.90 0.90

Immediately b e f o r e Immediately a f t e r 30 M i n a f t e r 60 M i n a f t e r

1.65 1.50 1.35

fresh

Ozonesusceptible

tissue

0.67 0.60

0.74 0.67

0.56

0.63

" ^ P l a n t s were t r e a t e d w i t h 1 ppm o z o n e f o r v a r i o u s t i m e s : 30 m i n ; s p i n a c h , 45 m i n ; t o b a c c o , 60 m i n .

beans,

I n a n o t h e r e x p e r i m e n t ( 1 ) we t r e a t e d o z o n e - r e s i s t a n t and o z o n e - s u s c e p t i b l e v a r i e t i e s o f t o b a c c o w i t h t o x i c doses o f o c - i o d o a c e t i c a c i d , and c c - i o d o a c e t a m i d e , b o t h s u l f h y d r y l - b i n d i n g r e a g e n t s . The symptoms p r o d u c e d b y b o t h compounds were s i m i l a r t o t h o s e p r o d u c e d by o z o n e . The s e v e r i t y o f t h e i n j u r y a l s o p a r a l l e l e d o z o n e r e s i s t a n c e ( T a b l e I I ) . The d e g r e e o f i n j u r y c a u s e d by t h e s e two compounds a l s o p a r a l l e l e d t h e o z o n e s u s c e p t i b i l i t y o f l e a v e s o f d i f f e r e n t a g e s on t h e same p l a n t . The u p p e r m o s t , y o u n g e s t , l e a v e s a p p e a r t o be most r e s i s t a n t t o b o t h t h e s u l f h y d r y l - b i n d i n g r e a g e n t s and t o o z o n e .

Table I I .

Damage f r o m s u l f h y d r y l - b i n d i n g r e a g e n t s t o t h e d e t a c h e d l e a v e s o f two t o b a c c o v a r i e t i e s

Leaf p o s i t i o n l

% L e a f s u r f a c e showing Ozone-resistant variety

v i s i b l e damage Ozone-susceptible variety

a-iodoacetamide (10-2M) f o r 4 h r 1 2 3 4

10 20 40 75

40 oc-iodoacetic acid (10-3M) f o r 24 h r

4

50

85

P o s i t i o n 1 i s t h a t o f t h e youngest f u l l y expanded l e a f . P o s i t i o n 3 a n d 4 a r e c o n s e c u t i v e l y l o w e r down t h e s t e m .


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79

I n l a t e r e x p e r i m e n t s ( 2 ) , b e a n s were s u b j e c t e d t o a m i l d e r , l o n g e r e x p o s u r e t o ozone (25 pphm f o r 3 h r ) . T h i s t r e a t m e n t d i d not d i m i n i s h t h e s u l f h y d r y l c o n t e n t a p p r e c i a b l y , even though t h e o z o n a t e d l e a v e s showed i n j u r y 18 h r l a t e r . However, we were a b l e to d e t e c t newly produced d i s u l f i d e s (Table I I I ) . We c o n c l u d e d t h a t o z o n a t i o n changes p r o t e i n s s u f f i c i e n t l y t o expose and o x i d i z e a d d i t i o n a l s u l f h y d r y l groups.


T a b l e I I I . The s u l f h y d r y l c o n t e n t ( y m o l e s / g f r e s h w t ) o f o p p o s i t e b e a n l e a v e s , A a n d B, b e f o r e a n d a f t e r e x p o s u r e t o o z o n e (25 pphm f o r 3 h r ) Control

1

Ozonated

1

Ozonated, then Leaf A

a

Leaf B

Leaf A

Leaf Β

Leaf A

Leaf Β

1.45

1.45

1.35

1.50

1.20

1.35

1.45

1.50

1.35

1.60

1.35

1.50

1.50

1.55

1.50

1.60

1.40

1.50

1.60

1.55

1.70

1.90

1.45

1.60

1.70

1.70

1.75

1.95

1.50

1.70 1.80

1.75

1.75

1.75

1.95

1.50

1.75

1.80

1.80

1.95

1.60

1.90

1.85

2.00

1.65

1.85

Md= 0.014*

Md= 0 . 1 7 5

X

dark

Md=1.875

1

a

Y

L e a f A g r o u n d i n p o l y v i n y l p y r r o l i d o n e medium w i t h o u t s o d i u m sulfite. L e a f Β g r o u n d i n same medium w i t h s o d i u m s u l f i t e . C o n t r o l l e a v e s n o t exposed t o ozone o r s u b j e c t e d t o d a r k . Dark p e r i o d i s 18 h r f o l l o w i n g e x p o s u r e t o o z o n e , χ Not s i g n i f i c a n t . y

S i g n i f i c a n t b e y o n d 1% l e v e l .

L i p i d Metabolism. N e x t we e x p l o r e d c h a n g e s i n l i p i d m e t a b o l i s m i n l e a v e s exposed t o ozone. S t e r o l s and s t e r o l d e r i v a t i v e s were p a r t i c u l a r l y i n t e r e s t i n g t o u s b e c a u s e t h e y have b e e n a s s o c i a t e d w i t h m e m b r a n e - c o n t a i n i n g f r a c t i o n s o f l e a v e s (3_). Changes p r o d u c e d i n t h e s e compounds may be e a r l y e v e n t s i n t h e t o x i c i t y o f ozone t o p l a n t c e l l s . We f i r s t s t u d i e d t h e c h a n g e s i n f r e e s t e r o l s o f l e a v e s a n d c h l o r o p l a s t s o f b e a n s a n d s p i n a c h e x p o s e d t o o z o n e (4_). We f o u n d ( T a b l e IV) t h a t ozonated bean l e a v e s and c h l o r o p l a s t s had 25% and 12% l e s s f r e e s t e r o l s r e s p e c t i v e l y t h a n bean l e a v e s and c h l o r o -


80

AIR

POLLUTION

EFFECTS

ON

PLANT

GROWTH

p l a s t s t h a t had n o t b e e n o z o n a t e d . O z o n a t e d s p i n a c h l e a v e s and c h l o r o p l a s t s had 44% and 37% l e s s f r e e s t e r o l s r e s p e c t i v e l y t h a n t h e i r non-ozonated c o u n t e r p a r t s .

T a b l e IV.

Changes i n f r e e s t e r o l c o n t e n t o f w h o l e t i s s u e and c h l o r o p l a s t s o f b e a n and s p i n a c h l e a v e s e x p o s e d t o ozone (50 pphm f o r 1 h r ) Free s t e r o l content


Plant

( y m o l e s / 3 mg

part

Control

chlorophyll) Ozonated

Bean Whole l e a v e s Chloroplasts

0.83 0.33

0.62 0.26

1.16 0.43

0.65 0.27

Spinach Whole l e a v e s Chloroplasts

E a c h v a l u e r e p r e s e n t s t h e mean o f t h r e e e x p e r i m e n t s . Each e x p e r i m e n t c o n s i s t e d o f d u p l i c a t e r e a d i n g s on l e a f e x t r a c t s o f two p l a n t s . X

L o s s o f f r e e s t e r o l s i g n i f i c a n t b e y o n d 1%

level.

In another experiment (Table V ) , the f r e e s t e r o l content of o z o n a t e d c h l o r o p l a s t s f r o m b e a n s was f o u n d t o be 32% l e s s and t h e c o n t e n t o f s t e r o l d e r i v a t i v e s 37% more t h a n t h a t o f n o n - o z o n a t e d chloroplasts. What happens t o t h e f r e e s t e r o l s ( F S ) , s t e r o l g l y c o s i d e s (SG) and a c e t y l a t e d s t e r o l g l y c o s i d e s (ASG) can be seen i n Table V I . I n t h e s e e x p e r i m e n t s (5_) w i t h w h o l e l e a v e s o f b e a n s , FS i n t h e o z o n a t e d l e a v e s was 2 1 % l e s s , SG 32% m o r e , and ASG 4 1 % more t h a n i n n o n - o z o n a t e d l e a v e s . One o f t h e i n t e r e s t i n g e f f e c t s o f ozone i s t h e 56% increase i n t h e l i n o l e n i c a c i d c o n t e n t o f ASG f r o m o z o n a t e d b e a n l e a v e s (5_). T h i s l e d us t o e x p l o r e t h e s o u r c e o f t h e a d d i t i o n a l l i n o lenic acid. Ongun and Mudd ( 6 ) had r e p o r t e d t h a t SG and ASG n o r m a l l y formed a t the expense o f f r e e s t e r o l s i n non-ozonated plants. What happens i n o z o n a t e d p l a n t s ? U s i n g bean l e a f d i s c s f e d l - i ^ C - a c e t a t e , we showed t h a t t h e r a d i o a c t i v e l y l a b e l e d d i g l y c e r i d e content of ozonated d i s c s became c o n s i s t e n t l y l e s s , and t h a t t h i s r e d u c t i o n was a c c o m p a n i e d by an i n c r e a s e i n t h e r a d i o a c t i v i t y o f ASG ( u n p u b l i s h e d ) . We p r o p o s e d t h a t t h i s was c a u s e d by an i n c r e a s e i n t h e r a t e o f l i p i d h y d r o l y s e s i n o z o n a t e d d i s c s . To s u p p o r t t h i s p r o p o s a l , we showed t h a t f l u o r e s c e i n d i l a u r a t e was h y d r o l y z e d 3 t o 4 t i m e s


6.

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AND TOMLINSON

81


f a s t e r i n ozonated d i s c s than i n non-ozonated d i s c s

T a b l e V.

Changes i n f r e e s t e r o l a n d s t e r o l d e r i v a t i v e o f c h l o r o p l a s t s i n bean l e a v e s exposed t o ozone (50 pphm f o r 1 h r ) μ

Sterol Component

m

o

l

e

s

/

3

m

S chlorophyll

Control

Free s t e r o l Downloaded by CORNELL UNIV on October 23, 2016 | http://pubs.acs.org Publication Date: June 1, 1974 | doi: 10.1021/bk-1974-0003.ch006

(unpublished).

1

Ozonated

0.38

0.39

0.35

0.24

0.25

0.26

X

0.10

0.11

0.09

0.14

0.15

0.17

y

Sterol derivatives 1

of

Each value leaves.

i s t h e mean o f d u p l i c a t e r e a d i n g s

made f r o m 3 g

S i g n i f i c a n t l y d i f f e r e n t beyond 1% l e v e l . y

S i g n i f i c a n t l y d i f f e r e n t b e y o n d 5% l e v e l .

T a b l e V I . E f f e c t o f o z o n a t i o n ( 2 5 pphm f o r 2.5 - 3.0 h r ) o n f r e e s t e r o l s ( F S ) , s t e r o l g l y c o s i d e s (SG), and a c e t y l a t e d s t e r o l g l y c o s i d e s (ASG) i n b e a n l e a f t i s s u e Sterol Before

0

Cone. ( p m o l e s / 1 0 D i s c s ) After 0

ο

Diff.

ο

FS

0.93

0.73

-0.20

SG

0.25

0.33

+0.08

ASG

0.16

0.27

+0.11

At t h i s p o i n t , i t i s worth c o n s i d e r i n g t h e importance o f l i p i d p e r o x i d a t i o n a s a t o x i c mechanism i n c e l l s e x p o s e d t o ozone. S c o t t and L e s h e r ( 7 ) proposed t h a t ozone i n j u r e s c e l l membranes b y o x i d i z i n g u n s a t u r a t e d l i p i d s . G o l d s t e i n and Balchum (8) r e p o r t e d t h a t ozone r e a c t s w i t h u n s a t u r a t e d l i p i d s t o produce organic peroxides which, they suggested, poison c e l l s . They u s e d a t h i o b a r b i t u r i c a c i d - m a l o n y l d i a l d e h y d e (MDA) method t o measure l i p i d p e r o x i d a t i o n . U s i n g t h i s m e t h o d , we c o u l d f i n d n o i n c r e a s e i n MDA u n t i l a f t e r v i s i b l e i n j u r y a p p e a r e d o n b e a n l e a v e s ( £ ) . We c o n c l u d e d t h a t l i p i d p e r o x i d a t i o n may r e s u l t f r o m ozone i n j u r y t o bean l e a v e s r a t h e r t h a n b e i n g t h e cause o f i n jury. The o b j e c t i o n h a s b e e n r a i s e d t h a t a l t h o u g h t h e MDA t e s t d o e s


82

AIR

POLLUTION

EFFECTS

ON

PLANT

GROWTH


d e t e c t l i p i d p e r o x i d a t i o n , i t c a n a l s o be p r o d u c e d b y t h e p e r o x i d a t i o n o f f a t t y a c i d s . However, t h e i n c r e a s e i n MDA o n l y a f t e r v i s i b l e i n j u r y on o z o n a t e d l e a v e s a l l o w s u s t o c o n c l u d e ( a ) that l i p i d peroxidation occurs a f t e r the i n i t i a l t o x i c event, or ( b ) t h a t l i p i d p e r o x i d a t i o n does n o t o c c u r a t a l l . I n e i t h e r c a s e , l i p i d p e r o x i d a t i o n c a n be m i n i m i z e d a s a c a u s e o f o z o n e i n j u r y t o t h e p l a n t s t h a t we s t u d i e d . F i n a l l y , we c o n c l u d e t h a t t o x i c r e a c t i o n s i n p l a n t c e l l s i n j u r e d by ozone p r o b a b l y t a k e p l a c e i n t h e f o l l o w i n g sequence: sulfhydryl oxidation, l i p i d hydrolysis, cellular leaking, l i p i d p e r o x i d a t i o n , and t h e n c e l l u l a r c o l l a p s e .

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9.

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 ) 58:808-810. T o m l i n s o n , H. a n d Rich, S. P h y t o p a t h o l o g y ( 1 9 7 0 ) 60:1842-1843. G r u n w a l d , C. P l a n t Physiol. ( 1 9 7 0 ) 45:663-666. T o m l i n s o n , H. a n d Rich, S. P h y t o p a t h o l o g y ( 1 9 7 3 ) 63:903-906. T o m l i n s o n , H. a n d Rich, S. P h y t o p a t h o l o g y ( 1 9 7 1 ) 61:1404-1405. Ongun, A. a n d Mudd, J. B. P l a n t Physiol. ( 1 9 7 0 ) 45:255-262. Scott, D. Β. M. and L e s h e r , E. C. J . Bacteriology (1963) 85:567-576. Goldstein, B. D. and B a l c h u m , O. J. S o c . E x p . Biol. Med. P r o c . ( 1 9 6 7 ) 126:356-358. 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 7 0 ) 60:1531-1532.


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