Bioregulators for Pest Control - American Chemical Society

2 Interference by Herbicides with Photosynthetic Electron Transfer WALTER OETTMEIER

Downloaded by UNIV OF ARIZONA on May 2, 2017 | http://pubs.acs.org Publication Date: April 26, 1985 | doi: 10.1021/bk-1985-0276.ch002

Lehrstuhl Biochemie der Pflanzen, Ruhr-Universität, Postfach 10 21 48, D-4630 Bochum 1, Federal Republic of Germany

Herbicides that inhibit photosynthetic electron flow prevent reduction of plastoquinone by the photosystem II acceptor complex. The properties of the photosystem II herbicide receptor proteins have been investigated by binding and displacement studies with radiolabeled herbicides. The herbicide receptor proteins have been identified with herbicide-derived photoaffinity l a bels. Herbicides, similar in their mode of action to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) bind to a 34 kDa protein, whereas phenolic herbicides bind to the 43-51 kDa photosystem II reaction center proteins. At these receptor proteins, plastoquinone/herbicide interactions and plastoquinone binding sites have been studied, the latter by means of a plastoquinone-derived photoaffinity label. For the 34 kDa herbicide binding protein, whose amino acid sequence is known, herbicide and plastoquinone binding are discussed at the molecular level. Plastoquinone is one of the most important components of the photosynthetic electron transport chain. It shuttles both electrons and protons across the photosynthetic membrane system of the thylakoid. In photosynthetic electron flow, plastoquinone is reduced at the acceptor side of photosystem II and reoxidized by the cytochrome b /f-complex. Herbicides that interfere with photosynthesis have been shown to specifically and effectively block plastoquinone reduction. However, the mechanisms of action of these herbicides, i . e., how inhibition of plastoquinone reduction is brought about, has not been established. Recent developments have brought a substantial increase to our knowledge in this field and one objective of this article will be to summarize the recent progress. It was originally assumed that the herbicides bind to a protein component of photosystem II (named "B" or "R") (1,2). This protein component was assumed to contain a special bound plastoquinone whose midpoint potential is lowered due to herbicide binding. Consequently, electron flow is interrupted (1,2). The photosystem II 6

0097-6156/85/0276-0019$06.00/0 © 1985 American Chemical Society Hedin et al.; Bioregulators for Pest Control ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

20

BIOREGULATORS FOR PEST CONTROL

h e r b i c i d e b i n d i n g p r o t e i n component was l a t e r e s t a b l i s h e d t o f u n c t i o n as a " p r o t e i n a c e o u s s h i e l d " f o r p h o t o s y s t e m I I by Renger (3) . The " p r o t e i n a c e o u s s h i e l d " can be removed by t r e a t m e n t w i t h t h e p r o t e o l y t i c enzyme t r y p s i n and, s u b s e q u e n t l y , D C M U - s e n s i t i v i t y o f phot o s y n t h e t i c e l e c t r o n t r a n s p o r t i s l o s t (J3,4) . I n 1979, t h e c o n c e p t o f a p h o t o s y s t e m I I h e r b i c i d e b i n d i n g p r o t e i n with d i f f e r e n t but overlapping binding s i t e s f o r the various p h o t o s y s t e m I I h e r b i c i d e s was s i m u l t a n e o u s l y e s t a b l i s h e d by T r e b s t and Draber {5) and P f i s t e r and A r n t z e n ( 6 ) . T h i s i d e a o f a h e r b i c i d e r e c e p t o r p r o t e i n p r o v e d t o be extremely f r u i t f u l because t h e t e c h n i q u e s o f r e c e p t o r b i o c h e m i s t r y were now a p p l i c a b l e . T i s c h e r and Strotmann Ç7) were t h e f i r s t i n v e s t i g a t o r s t o study b i n d i n g o f radiolabeled herbicides i n isolated thylakoids.


Herbicide Binding

Experiments

T y p i c a l r e s u l t s o b t a i n e d i n a b i n d i n g experiment f o r two d i f f e r e n t photosysÇgm I I h e r b i c i d e s a r e p r e s e n t e d i n F i g u r e 1 f o r t h e t r i a z i n o n e [ c] m e t r i b u z i n ( r i g h t f o r m u l a ^ , a s o - c a l l e d "DCMU-type" h e r b i c i d e , and t h e p h e n o l i c h e r b i c i d e [ H ] 2 - i o d o - 4 - n i t r o - 6 - i s o b u t y l p h e n o l (8_) ( l e f t formula) . The term "DCMU-type" h e r b i c i d e does n o t denote a c h e m i c a l d e f i n i t i o n , b u t i s a f u n c t i o n a l d e f i n i t i o n because DCMU (diuron) i s t h e most w i d e l y u s e d p h o t o s y n t h e s i s i n h i b i t o r . I n b i n d i n g e x p e r i m e n t s m e t r i b u z i n seems t o s a t u r a t e a t r e l a t i v e l y low c o n c e n t r a t i o n s ( F i g u r e 1 ) . However, t h e b i n d i n g o f m e t r i b u z i n i s i n f a c t b i p h a s i c : i t has a s o - c a l l e d s p e c i f i c (high a f f i n i t y ) b i n d i n g and an u n s p e c i f i c (low a f f i n i t y ) b i n d i n g (Ί)· The l a t t e r shows a l i n e a r dependency on the c o n c e n t r a t i o n . T h i s ( e x t r a p o l a t e d ) unspe c i f i c b i n d i n g o f t h e p h e n o l i c h e r b i c i d e i s much h i g h e r , a s compared t o t h a t o f m e t r i b u z i n ( F i g u r e 1, upper dashed l i n e ) . T h i s i s j u s t one o f t h e many d i f f e r e n c e s t h a t can be f o u n d between "DCMU-type" and p h e n o l i c h e r b i c i d e s and which j u s t i f i e s t o view them as two d i f f e r e n t c l a s s e s o f h e r b i c i d e s ( f o r r e v i e w , see (JO ) . The b i n d i n g c u r v e s o f t h e h e r b i c i d e s i n F i g u r e 1, e s p e c i a l l y the one f o r m e t r i b u z i n , l o o k v e r y much l i k e M i c h a e l i s - M e n t e n enzyme k i n e t i c s . Indeed, h e r b i c i d e b i n d i n g can be t r e a t e d i n t h e same way Ç7). F i g u r e 2 p r e s e n t s a Lineweaver-Burk p l o t o f t h e b i n d i n g d a t a f o r 2 - i o d o - 4 - n i t r o - 6 - i s o b u t y l p h e n o l . C l e a r l y , t h e two t y p e s o f b i n d i n g , s p e c i f i c and u n s p e c i f i c b i n d i n g , can be r e c o g n i z e d . T h i s i s even more e v i d e n t i n t h e S c a t c h a r d p l o t o f t h e b i n d i n g d a t a ( i n s e t F i g u r e 2 ) . F u r t h e r m o r e , from these p l o t s , b i n d i n g p a r a m e t e r s , such as t h e b i n d i n g c o n s t a n t Κ, , and number o f b i n d i n g s i t e s , x , can be o b t a i n e d Ç 7 ) . These a r e a l s o l i s t e d i n F i g u r e 2. A c c o r d i n g t o T i s c h e r and Strotmann ( 7 ) , t h e b i n d i n g c o n s t a n t corresponds t o the i n h i b i t i o n c o n s t a n t , i . e. t h e I ^ v a l u e (the c o n c e n t r a t i o n n e c e s s a r y f o r 50% i n h i b i t i o n o f p h o t o s y n t h e t i c e l e c t r o n t r a n s p o r t ) , provided the I v a l u e i s e x t r a p o l a t e d t o z e r o c h l o r o p h y l l concen t r a t i o n . The v a l u e o f 527 m o l e c u l e s o f c h l o r o p h y l l p e r m o l e c u l e o f bound i n h i b i t o r i n d i c a t e s t h a t r o u g h l y one m o l e c u l e o f h e r b i c i d e b i n d s p e r e l e c t r o n t r a n s p o r t c h a i n , because about 400-600 m o l e c u l e s o f c h l o r o p h y l l a r e c o n s i d e r e d t o be a s s o c i a t e d w i t h each e l e c t r o n transport chain. t

Q

Hedin et al.; Bioregulators for Pest Control ACS Symposium Series; American Chemical Society: Washington, DC, 1985.


OETTMEIER

Photosynthetic Electron Transfer Inhibition

4-nitro-6-isobutylphenol

(•

•)

to i s o l a t e d

spinach thylakoids.


22



Herbicide-Resistant

Weeds

The i m p o r t a n c e o f b i n d i n g e x p e r i m e n t s w i t h r a d i o l a b e l e d h e r b i c i d e s became i m m e d i a t e l y e v i d e n t i n t h e case o f h e r b i c i d e - r e s i s t a n t weeds. The use o f c e r t a i n s - t r i a z i n e h e r b i c i d e s l i k e a t r a z i n e f o r more t h a n two decades had l e d t o b i o t y p e s t h a t a r e r e s i s t a n t t o n o r m a l l y a p p l i e d doses. B i n d i n g experiments with a t r a z i n e i n t h y l a k o i d s i s o l a t ed from r e s i s t a n t weed p l a n t s d e m o n s t r a t e d t h a t t h e s p e c i f i c b i n d i n g o f a t r a z i n e was c o m p l e t e l y a b s e n t and o n l y some u n s p e c i f i c b i n d i n g was l e f t (10,11). Thus, t h e r e s i s t a n c e i s due t o d e c r e a s e d b i n d i n g o f t h e h e r b i c i d e i n t h e t h y l a k o i d o f t h e r e s i s t a n t p l a n t , which does not i n h i b i t p h o t o s y n t h e t i c e l e c t r o n t r a n s p o r t a t concentrations that a r e l e t h a l t o the* s u s c e p t i b l e t y p e . S i m i l a r i l y , t h e s p e c i f i c b i n d i n g o f m e t r i b u z i n a l s o i s c o m p l e t e l y l o s t , whereas t h e b i n d i n g o f u r e a and b i s c a r b a m a t e h e r b i c i d e s i s o n l y s l i g h t l y a f f e c t e d (11) . I n c o n t r a s t , t h e b i n d i n g o f p h e n o l i c h e r b i c i d e s , i n g e n e r a l , i s more p r o nounced i n t h y l a k o i d s o f r e s i s t a n t p l a n t s than i n t h o s e o f t h e s u s c e p t i b l e types (11). H e r b i c i d e Displacement Experiments The p h o t o s y s t e m I I h e r b i c i d e s b i n d r e v e r s i b l y and n o n - c o v a l e n t l y t o t h e i r b i n d i n g s i t e . C o n s e q u e n t l y , a r a d i o l a b e l e d h e r b i c i d e can be d i s p l a c e d from t h e b i n d i n g s i t e by a n o t h e r h e r b i c i d e o r i n h i b i t o r , p r o v i d e d i t h a s an i d e n t i c a l b i n d i n g s i t e . Even a d i s p l a c e m e n t from a d i f f e r e n t b i n d i n g s i t e i s f e a s i b l e , i f both b i n d i n g s i t e s i n t e r a c t w i t h each o t h e r . A Çvpical d i s p l a c e m e n t e x p e r i m e n t i s shown i n F i g u r e 3. E v i d e n t l y , f c ] m e t r i b u z i n i s e a s i l y d i s p l a c e d from t h e t h y l a k o i d membrane by DCMU. S i n c e t h e P I C Q v a l u e s ( n e g a t i v e l o g a r i t h m o f c o n c e n t r a t i o n a c h i e v i n g 50% i n h i b i t i o n ) o f b o t h compounds a r e in t h e same o r d e r o f magnitude , about 50% o f t h e bound m e t r i b u z i n i s removed from t h e membrane a t t h e i s o m o l a r p o i n t , i . e. when t h e c o n c e n t r a t i o n s o f b o t h compounds a r e i d e n t i c a l . F o r t h e p h e n o l i c h e r b i c i d e d i n o s e b ( 2 , 4 - d i n i t r o - 6 - s e c . - b u t y l p h e n o l ) , a much h i g h e r c o n c e n t r a t i o n , 7 χ 10 M, i s n e c e s s a r y t o o b t a i n 50% r e m o v a l . T h i s i s a consequence o f t h e l o w e r p I ^ v a l u e o f d i n o s e b o f 5.5 ( 1 2 ) . Thus, t h e c o n c e n t r a t i o n n e c e s s a r y f o r 50% d i s p l a c e m e n t r o u g h l y c o r responds t o the P l ^ v a l u e . I t i s p o s s i b l e , t h e r e f o r e , t o assay the pl^ v a l u e o f a new compound j u s t by e x a m i n a t i o n o f i t s d i s p l a c e m e n t b e h a v i o u r . I t i s no l o n g e r n e c e s s a r y t o d e t e r m i n e t h e p I c v a l u e by t e s t i n g the i n h i b i t i o n o f a l i g h t - d r i v e n p h o t o r e d u c t i o n . Another very p o t e n t i n h i b i t o r o f p h o t o s y n t h e t i c e l e c t r o n t r a n s p o r t , DBMIB (2,5d i b r o m o - 3 - m e t h y l - 6 - i s o p r o p y l - l , 4 - b e n z o q u i n o n e ) (13) , a l m o s t c o m p l e t e l y f a i l s t o d i s p l a c e m e t r i b u z i n from t h e membrane ( F i g u r e 3 ) . T h i s i s due t o t h e f a c t t h a t DBMIB h a s a c o m p l e t e l y d i f f e r e n t s i t e o f a c t i o n a s compared t o t h e p h o t o s y s t e m I I h e r b i c i d e s , i . e. i t i n h i b i t s p l a s t o h y d r o q u i n o n e o x i d a t i o n by a c t i n g a t t h e cytochrome b / f - c o m p l e x Q

0

0

Q

fi

(13) . P h o t o a f f i n i t y Labeling of the Herbicide Binding

Proteins

As a l r e a d y s t r e s s e d , p h o t o s y s t e m I I h e r b i c i d e s b i n d r e v e r s i b l y t o t h e i r binding s i t e . Altough r a d i o l a b e l e d h e r b i c i d e s are a v a i l a b l e , i t i s impossible to i d e n t i f y the h e r b i c i d e receptor p r o t e i n without a chemical m o d i f i c a t i o n o f the h e r b i c i d e t h a t allows f o r covalent


2.


OETTMEIER

23

% bound


100< 80 60 40 20

10-8

10-

10"

7

6

10-

5

tog cone.

DCMU (7.4)

DINOSEB (5.5)

DBMIB (7.5)

r 1 τ "1 F i g u r e 3. D i s p l a c e m e n t o f L J m e t r i b u z i n from t h e t h y l a k o i d mem brane by DCMU ( · ·) , Dinoseb (• •) , and DBMIB (A A) . The numbers i n p a r a n t h e s i s below t h e s t r u c t u r a l f o r m u l a s o f t h e compounds c o r r e s p o n d t o t h e i r ρ Ι values. C

ς η



24


attachment o f t h e h e r b i c i d e . T h i s m o d i f i c a t i o n i s a c h i e v e d by i n t r o d u c t i o n o f an a z i d o f u n c t i o n i n t o t h e h e r b i c i d e m o l e c u l e . An o r g a n i c a z i d e upon i l l u m i n a t i o n w i t h v i s i b l e o r UV l i g h t r e a d i l y s p l i t s - o f f m o l e c u l a r n i t r o g e n and forms a n i t r e n e . N i t r e n e s are e x t r e m e l y e l e c t r o p h i l i c compounds and r e a c t i m m e d i a t e l y w i t h any n u c l e o p h i l i c groups i n t h e i r environment. I f the àzidoderivative o f the h e r b i c i d e i s as good an i n h i b i t o r as i t s p a r e n t compound, i t s s p e c i f i c b i n d i n g s h o u l d e x c l u s i v e l y o c c u r a t i t s r e c e p t o r p r o t e i n . C o n s e q u e n t l y , the n i t r e n e s h o u l d form a c o v a l e n t bond t o the r e c e p t o r p r o t e i n . S i n c e the àzidoderivative o f the h e r b i c i d e i s r a d i o l a b e l e d , the r e c e p t o r p r o t e i n can be e a s i l y i d e n t i f i e d because i t becomes r a d i o a c t i v e by the attachment o f the n i t r e n e . The common p r o c e d u r e f o r i d e n t i f i c a t i o n i n c l u d e s d i s r u p t i o n o f the t h y l a k o i d membrane system by d e t e r g e n t t r e a t m e n t , s e p a r a t i o n o f the t h y l a k o i d p r o t e i n s by p o l y a c r y l amide g e l e l e c t r o p h o r e s i s , and a s s a y i n g f o r r a d i o a c t i v i t y e i t h e r by c u t t i n g the g e l i n t o p,ieces, which a r e s o l u b i l i z e d and c o u n t e d i n a l i q u i d s c i n t i l l a t i o n c o u n t e r , o r by e x p o s u r e o f t h e g e l on X - r a y film. So f a r , t h r e e d i f f e r e n t p h o t o a f f i n i t y l a b e l s o f p h o t o s y s t e m I I h e r b i c i d e s are a v a i l a b l e (Figure 4): azidodinoseb (phenolic) (14), a z i d o a t r a z i n e (15), and a z i d o t r i a z i n o n e (16) (both "DCMU-type" h e r b i c i d e s ) . A z i d o a t r a z i n e i n i s o l a t e d t h y l a k o i d s from s p i n a c h , and the a l g a Ch1amydomonas r e i n h a r d t i i as w e l l l a b e l s a p r o t e i n w i t h an ap p a r e n t m o l e c u l a r weight o f 34 kDa (15,17). F u r t h e r m o r e , a z i d o a t r a z i n e i n the weed Amaranthus b i n d s t o the 34 kDa p r o t e i n o n l y i n t h y l a k o i d s from a t r a z i n e - s u s c e p t i b l e and not t o t h y l a k o i d s from a t r a z i n e - r e s i s t a n t p l a n t s (18). I t was c o n c l u d e d , t h e r e f o r e , t h a t t h e 34 kDa p r o t e i n i s the p h o t o s y s t e m I I h e r b i c i d e b i n d i n g p r o t e i n f o r "DCMU-type" h e r b i c i d e s . T h i s 34 kDa h e r b i c i d e b i n d i n g p r o t e i n i s i d e n t i c a l t o the "photogene" o r " r a p i d l y t u r n i n g o v e r " 34 kDa p r o t e i n t h a t s t a n d s o u t amongst a l l o f t h e t h y l a k o i d p r o t e i n s due t o i t s r a p i d d e s t r u c t i o n and de novo b i o s y n t h e s i s (19). The i d e a o f t h e 34 kDa h e r b i c i d e b i n d i n g p r o t e i n has met some c r i t i c i s m by G r e s s e l (20). T h i s c r i t i c i s m i s due t o the f a c t t h a t p h o t o a f f i n i t y l a b e l i n g e x p e r i m e n t s , i n g e n e r a l , a r e n o t unambiguous. I t i s f e a s i b l e , t h a t a p h o t o a f f i n i t y l a b e l does n o t b i n d t o the t a r get p r o t e i n , but to a neighbouring p r o t e i n i n s t e a d . S p e c i f i c a l l y , G r e s s e l * s c r i t i c i s m i s b a s e d on the f a c t t h a t i n t h e a z i d o a t r a z i n e m o l e c u l e , the a z i d o group and the s t r u c t u r a l element g e n e r a l l y r e c o g n i z e d f o r h e r b i c i d a l a c t i v i t y l i e on o p p o s i t e p a r t s o f the mole c u l e . T h e r e f o r e , t h e r e i s a p o s s i b i l i t y t h a t the 34 kDa p r o t e i n t h a t i s t a g g e d by a z i d o a t r a z i n e i s n o t the r e a l h e r b i c i d e b i n d i n g p r o t e i n . To c l a r i f y t h i s q u e s t i o n , we r e c e n t l y s y n t h e s i z e d a n o t h e r "DCMU-type" p h o t o a f f i n i t y l a b e l : a z i d o t r i a z i n o n e ( F j g u r e 4) (_16) . F i g u r e 5 shows r e s u l t s of a l a b e l i n g experiment with £ c ] a z i d o t r i a z i n o n e . Only one p r o t e i n i s h e a v i l y l a b e l e d . From the p o s i t i o n o f the marker p r o t e i n s , a m o l e c u l a r w e i g h t o f 34 kDa can be e s t i m a t e d . I f samples o f the t h y l a k o i d l a b e l e d by C] a z i d o a t r a z i n e o r £ c ] a z i d o t r i a z i n o n e a r e run i n a d j a c e n t l a n e s o f a g e l , r a d i o a c t i v i t y i n b o t h c a s e s i s f o u n d i n e x a c t l y the same p o s i t i o n . F u r t h e r m o r e , p r e l a b e l i n g w i t h i n a c t i v e ^ c ] a z i d o t r i a z i n o n e p r e v e n t s l a b e l i n g o f the 34 kDa p r o t e i n by £ c j a z i d o a t r a z i n e (16). A z i d o t r i a z i n o n e i s c o m p l e t e l y d i f f e r e n t i n i t s c h e m i c a l s t r u c t u r e from a z i d o a t r a z i n e . However, b o t h p h o t o a f f i n i t y l a b e l s b i n d t o an i d e n t i c a l 34 kDa p r o t e i n . I t has t o be c o n c l u d e d t h a t G r e s s e l s s u g g e s t i o n t h a t the 34 kDa p r o t e i n i s n o t the h e r b i c i d e b i n d i n g p r o t e i n i s not v a l i d . 1


2.


OETTMEIER

[ H]Azido-dinoseb 3

w

[ c]Azido-atrazine

[ C]Azido-triazinone K


X

N^N '

F i g u r e 4. S t r u c t u r a l

H

11

NC H H 2

2

5

5

25

I

N(CH )

formulas o f h e r b i c i d a l p h o t o a f f i n i t y

3

2

labels.

F i g u r e 5. Photograph o f a L i - d o d e c y l s u l f a t e p o l y a c r y l a m i d e e l e c t r o p h o r e s i s g e l (10-15%) and r a d i o a c t i v i t y d i s t r i b u t i o n ^ h e r e i n o f s p i n a c h t h y l a k o i d s i s o l a t e d by 20 nmol/mg c h l o r o p h y l l £ c j a z i d o triazinone.



26


The p h e n o l i c p h o t o a f f i n i t y l a b e l a z i d o d i n o s e b ( F i g u r e 4) b i n d s l e s s s p e c i f i c a l l y t h a n e i t h e r a z i d o a t r a z i n e o r a z i d o t r i a z i n o n e (14). In a d d i t i o n t o o t h e r p r o t e i n s , i t l a b e l s p r e d o m i n a n t l y t h e p h o t o s y s tem I I r e a c t i o n c e n t e r p r o t e i n s ( s p i n a c h : 43 and 47 kDa; Chlamydomonas: 47 and 51 kDa) (17). Because o f the u n s p e c i f i c b i n d i n g o f a z i d o d i n o s e b , t h i s can b e s t be seen i n p h o t o s y s t e m I I p r e p a r a t i o n s (17). Thus, the p h e n o l i c h e r b i c i d e s b i n d p r e d o m i n a n t l y t o the p h o t o system I I r e a c t i o n c e n t e r , which might e x p l a i n many o f the d i f f e r e n ces o b s e r v e d between "DCMU-type" and p h e n o l i c h e r b i c i d e s (9) . The p h o t o s y s t e m I I r e a c t i o n c e n t e r p r o t e i n s and the 34 kDa h e r b i c i d e b i n d i n g p r o t e i n must be l o c a t e d c l o s e l y t o and i n t e r a c t w i t h each o t h e r i n o r d e r t o e x p l a i n t h e mutual displacement of both types of h e r b i c i d e s (8,12,21). F u r t h e r m o r e , i t s h o u l d be n o t e d t h a t f o r phe n o l i c h e r b i c i d e s , some e f f e c t s a t the donor s i d e o f p h o t o s y s t e m I I (22) and on c a r o t e n o i d o x i d a t i o n i n the p h o t o s y s t e m I I r e a c t i o n c e n t e r have been found (23). The h e r b i c i d a l p h o t o a f f i n i t y l a b e l s a r e a l s o u s e f u l t o o l s f o r e l u c i d a t i o n of h e r b i c i d e binding p r o p e r t i e s i n various photosynthet i c p r e p a r a t i o n s . In p h o t o s y s t e m I I p r e p a r a t i o n s w i t h an i n t a c t water s p l i t t i n g enzyme system b o t h , a z i d o a t r a z i n e and a z i d o d i n o s e b b i n d t o t h e i r r e s p e c t i v e p r o t e i n s (9). In c o n t r a s t , i n a p h o t o s y s t e m I I p a r t i c l e w i t h o u t the water s p l i t t i n g enzyme complex, a z i d o a t r a z i n e does n o t b i n d , whereas a z i d o d i n o s e b s t i l l does ( 1 7 ) . T h i s does not i n d i c a t e , however, t h a t the 34 kDa p r o t e i n i s not p r e s e n t i n t h i s p h o t o system I I p a r t i c l e . As a l r e a d y s t r e s s e d , the 34 kDa h e r b i c i d e b i n d i n g p r o t e i n has a h i g h t u r n o v e r r a | g (19). I f Chlamydomonas c e l l s are grown i n a medium c o n t a i n i n g [ c] a c e t a t e and p h o t o s y s t e m I I p a r t i c l e s a r e p r e p a r e d from t h e s e a l g a e , the maximum o f the r a d i o a c t i v i t y i n the g e l i s f o u n d e x a c t l y a t t h a t p o s i t i o n where the 34 kDa h e r b i c i d e b i n d i n g p r o t e i n m i g r a t e s (24). No b i n d i n g o f a z i d o a t r a z i n e i s o b s e r v e d i n n-hexane e x t r a c t e d t h y l a k o i d s , whereas a z i d o d i n o s e b b i n d i n g i s u n a f f e c t e d by t h i s p r o c e d u r e (24). These r e s u l t s i n d i c a t e t h a t the h e r b i c i d e b i n d i n g p r o p e r t i e s o f the 34 kDa p r o t e i n a r e v e r y s e n s i t i v e t o changes i n i t s p r o t e i n or l i p i d environment. Herbicide/Quinone

Interactions

R e c e n t developments have l e d t o a r e v i s i o n o f the o r i g i n a l i d e a t h a t t h e "B" o r "R" p r o t e i n (see above) which i s i d e n t i c a l t o the 34 kDa h e r b i c i d e b i n d i n g p r o t e i n c o n t a i n s a bound p l a s t o q u i n o n e . Velthuys (25) from f l a s h - i n d u c e d absorbance changes o f p l a s t o s e m i q u i n o n e i n the p r e s e n c e o f v a r i o u s i n h i b i t o r s , and Lavergne (26) from f l u o r e s cence e x p e r i m e n t s , i n f e r r e d t h a t t h e r e i s an e l e c t r o n - d e p e n d e n t d i r e c t c o m p e t i t i o n between p l a s t o q u i n o n e and h e r b i c i d e . A p l a s t o q u i n o n e m o l e c u l e from the p l a s t o q u i n o n e p o o l g e t s bound t o the a c c e p t o r com p l e x o f p h o t o s y s t e m I I t o become Q . Q^, i n t u r n , g e t s r e d u c e d by v i a the semiquinone a n i o n r a d i c a l t h e p r i m a r y a c c e p t o r o f pho tosystem I I , again another s p e c i a l plastoquinone molecule. Q - s t a b i l i z e s upon b i n d i n g t o Q . In a subsequent second s t e p , Q - g e t s r e d u c e d t o p l a s t o h y d r o q u i n o n e which i s exchanged w i t h a n o t h e r p l a s t o quinone from the p o o l . P h o t o s y s t e m I I h e r b i c i d e s compete w i t h p l a s t o q u i n o n e f o r b i n d i n g t o the h e r b i c i d e / q u i n o n e environment. Urbach et_ a l . (27) r e c e n t l y d e m o n s t r a t e d a f l a s h - i n d u c e d b i n a r y o s c i l l a t i o n o f h e r b i c i d e b i n d i n g . H e r b i c i d e b i n d i n g i s h i g h e r i n t h e dark or a t an


2.

OETTMEIER


27

even number o f f l a s h e s , i . e when Q i s o x i d i z e d , t h a n a t an odd number, when i s s i n g l y reduced. T h e r e f o r e , h e r b i c i d e b i n d i n g can o n l y take p l a c e when t h e b i n d i n g s i t e i s v a c a n t , i . e. n o t o c c u p i e d by Q " I t i s worthy o f s p e c i a l i n t e r e s t t o study d i r e c t l y t h e d i s p l a c e ment o f a h e r b i c i d e by p l a s t o q u i n o n e o r i t s a n a l o g u e s . I n normal t h y l a k o i d s , almost no d i s p l a c e m e n t o f DCMU even by a m i l l i o n - f o l d e x c e s s o f t h e s h o r t - c h a i n p l a s t o q u i n o n e a n a l o g u e p l a s t o q u i n o n e - 1 c a n be o b s e r v e d (28). T h i s may be due t o t h e h i g h endogenous p l a s t o q u i n o n e c o n t e n t o f t h e t h y l a k o i d membrane. I f t h e t h y l a k o i d s a r e d e p l e t e d o f p l a s t o q u i n o n e by means o f n-hexane e x t r a c t i o n , a c o m p e t i t i v e d i s p l a c e m e n t o f DCMU by p l a s t o q u i n o n e - 1 i s o b s e r v e d ( 2 8 ) . T h i s r e s u l t e s t a b l i s h e s a d i r e c t i n t e r a c t i o n between h e r b i c i d e and p l a s t o q u i n o n e , though n o t n e c e s s a r i l y ' a t an i d e n t i c a l b i n d i n g s i t e . From t h e d i s p l a c e m e n t e x p e r i m e n t s , a b i n d i n g c o n s t a n t f o r p l a s t o q u i n o n e - 1 o f 51± 19 μΜ i n p l a s t o q u i n o n e - d e p l e t e d t h y l a k o i d s c a n be c a l c u l a t e d ( 2 8 ) . As compared t o DCMU ( b i n d i n g c o n s t a n t 34 nM (24)) t h e a f f i n i t y o f p l a s t o q u i n o n e - 1 i s more than t h r e e o r d e r s o f magnitude l e s s . I n a s i m i l a r d i s p l a c e m e n t experiment o f b r o m o x y n i l by p l a s t o q u i n o n e - 1 i n t r i a z i n e - r e s i s t a n t t h y l a k o i d s , Vermaas e t a_l. (29) found a p l a s t o quinone-1 b i n d i n g c o n s t a n t o f 20 μΜ which i s i n t h e same o r d e r o f magnitude as o u r v a l u e ( 2 8 ) . f


B

In an attempt t o l e a r n more about t h e n a t u r e o f t h e p l a s t o q u i none b i n d i n g s i t e , we have a n a l y z e d t h e d i s p l a c e m e n t b e h a v i o u r o f 25 d i f f e r e n t 1,4-benzoquinones t o DCMU. A q u a n t i t a t i v e s t r u c t u r e a c t i v i t y r e l a t i o n s h i p r e v e a l e d t h a t t h e d i s p l a c i n g a c t i v i t y o f a quinone toward DCMU i s g o v e r n e d by t h e redox p o t e n t i a l and t h e g e o m e t r i c a l c o n f o r m a t i o n o f t h e quinone ( 3 0 ) . An A z i d o p l a s t o q u i n o n e P h o t o a f f i n i t y

Label

To i d e n t i f y p l a s t o q u i n o n e b i n d i n g p r o t e i n s , we have r e c e n t l y s y n t h e s i z e d an a z i d o p l a s t o q u i n o n e p h o t o a f f i n i t y l a b e l ( 3 1 ) . F i g u r e 6 shows a t y p i c a l l a b e l i n g p a t t e r n o f a spinach photosystem I I p r e p a r a t i o n . Only one major p r o t e i n i n t h e 32-34 kDa m o l e c u l a r weight range i s h e a v i l y t a g g e d . A s i m i l a r p i c t u r e i s o b t a i n e d , i f normal t h y l a k o i d s a r e u s e d ( 3 2 ) . L a b e l i n g o f t h e 32-34 kDa p r o t e i n i s p r e v e n t e d , i f t h e samples a r e p r e i n c u b a t e d e i t h e r w i t h DCMU, t h e p h e n o l i c h e r b i c i d e 2 - i o d o - 4 - n i t r o - 6 - i s o b u t y l p h e n o l , o r the photosystem I I i n h i b i t o r tetraiodo-1,4-benzoquinone (33). The q u e s t i o n a r i s e s a s t o whether t h e 3 2 - 3 4 kDa p r o t e i n , a s l a b e l e d by a z i d o p l a s t o q u i n o n e , and t h e 34 kDa p r o t e i n , as l a b e l e d by a z i d o a t r a z i n e o r a z i d o t r i a z i n o n e , a r e i d e n t i c a l . I f samples l a b e l e d by e i t h e r a z i d o p l a s t o q u i n o n e o r a z i d o atrazine are run i n adjacent lanes of a g e l , the R -values o f the s p o t s w i t h t h e maximum amount o f r a d i o a c t i v i t y d i f f e r by 0.05 (2£) . T h i s experiment h a s been r e p e a t e d s e v e r a l t i m e s . I f 4 M u r e a i s i n c l u d e d i n t h e g e l , t h e maxima o f r a d i o a c t i v i t y c o i n c i d e ( 2 4 ) . T h e r e a r e two p o s s i b l e e x p l a n a t i o n s . One i s t h a t t h e p r o t e i n s l a b e l e d by t h e two d i f f e r e n t p h o t o a f f i n i t y l a b e l s a r e i d e n t i c a l . The d i f f e r e n c e s i n t h e R - v a l u e s i n one g e l system may be due t o t h e attachment o f the two d i f f e r e n t m o i e t i e s o r i g i n a t i n g from t h e l a b e l t o t h e p r o t e i n . The second p o s s i b i l i t y would be t h a t t h e two p r o t e i n s a r e r e a l l y d i f f e r e n t , i . e. h e r b i c i d e and p l a s t o q u i n o n e b i n d i n g s i t e s a r e on d i f f e r e n t p r o t e i n s , b u t t h e two p r o t e i n s have v e r y s i m i l a r m o l e c u l a r w e i g h t s . Most r e c e n t l y , e v i d e n c e i s a c c u m u l a t i n g t h a t i n a d d i t i o n t o f


28


t h e 34 kDa h e r b i c i d e b i n d i n g p r o t e i n and a 33 kDa l y s i n e - r i c h p r o t e i n , which i s p a r t o f p h o t o s y s t e m I I , b u t i s a s s o c i a t e d w i t h the water s p l i t t i n g enzyme complex, y e t a n o t h e r u n i d e n t i f i e d 32-34 kDa p r o t e i n may p l a y a r o l e i n h e r b i c i d e and p l a s t o q u i n o n e b i n d i n g (34, 35).


The

34 kDa

H e r b i c i d e B i n d i n g P r o t e i n : The

Molecular

Level

The 34 kDa h e r b i c i d e b i n d i n g p r o t e i n i s a p l a s t i d encoded p r o t e i n . I n the p r o c e s s o f s e q u e n c i n g the p l a s t i d genome, the DNA sequence o f the gene c o d i n g f o r t h e 34 kDa h e r b i c i d e b i n d i n g p r o t e i n became known and, hence, a l s o i t s amino a c i d sequence ( 3 6 ) . One g r e a t s u r prise a r i s i n g from the amino a c i d sequence i s the complete l a c k o f l y s i n e r e s i d u e s . E x c e p t f o r c o m p a r a t i v e s t u d i e s , the knowledge o f the amino a c i d sequence o f the 34 kDa h e r b i c i d e b i n d i n g p r o t e i n does n o t seem t o y i e l d v e r y much a d d i t i o n a l i n f o r m a t i o n . However, r e c e n t l y K y t e and D o o l i t t l e (31) and A r g o s e t a l . (38) have d e v i s e d a p r o gram t h a t p r o g r e s s i v e l y e v a l u a t e s the h y d r o p h i l i c i t y and h y d r o p h o b i c i t y o f a p r o t e i n a l o n g i t s amino a c i d sequence. T h i s approach i s v e r y i m p o r t a n t f o r membrane bound p r o t e i n s l i k e the 34 kDa h e r b i c i d e b i n d i n g p r o t e i n because i t a l l o w s one t o p r e d i c t r e g i o n s o f the p r o t e i n t h a t may be embedded w i t h i n the membrane system. I n the K y t e and D o o l i t t l e p r o c e s s (37), each amino a c i d i s a s s i g n e d a h y d r o p a t h y ("strong f e e l i n g about water") v a l u e which ranges from 4.5 f o r i s o l e u c i n e (as tl*«j most h y d r o p h o b i c amino a c i d ) t o -4.5 f o r a r g i n i n e (as t h e most h y d r o p h i l i c amino a c i d ) . Now a c e r t a i n "window", i . e. a c e r t a i n l e n g t h o f t h e amino a c i d sequence i s s e l e c t e d . Assuming a window o f 11 amino a c i d s , the sum o f the h y d r o pathy v a l u e s o f the amino a c i d sequence f o r amino a c i d s 1 t o 11 i s c a l c u l a t e d . Next t h e window i s moved one amino a c i d ahead w i t h i n the sequence, and the sum o f t h e h y d r o p a t h y v a l u e s f o r amino a c i d s 2 t o 12 i s c a l c u l a t e d . T h i s p r o c e s s i s r e p e a t e d f o r sequence 3-13, 4-14 e t c . u n t i l t h e end o f the sequence i s r e a c h e d . The sum o f the h y d r o p a t h y v a l u e s o v e r the number o f the amino a c i d r e s i d u e i s p l o t t e d . Such a h y d r o p a t h y p l o t f o r the 34 kDa h e r b i c i d e b i n d i n g p r o t e i n i s p r e s e n t e d i n F i g u r e 7. ( I t s h o u l d be n o t e d t h a t f o r the h y d r o p a t h y p l o t , the o r i g i n a l amino a c i d sequence as r e p o r t e d by Z u r a w s k i e t a l . (36) was n o t u s e d . I n s t e a d , a sequence s h o r t e r by 36 amino a c i d s which s t a r t s a t the second Met was used ( 3 9 ) ) . The p o s i t i v e (shaded) a r e a s i n F i g u r e 7 c o r r e s p o n d t o r e g i o n s o f h i g h h y d r o p a t h y , the ne g a t i v e a r e a s t o r e g i o n s o f low h y d r o p a t h y . Only r e g i o n s o f h i g h hy d r o p a t h y o f the p r o t e i n a r e t h o u g h t t o be b u r i e d w i t h i n the membrane system, p r o v i d e d they a r e a p p r o x i m a t e l y 20 amino a c i d s l o n g t o a c count f o r a h e l i c a l span t h r o u g h the l i p i d b i l a y e r o f the membrane. Based on t h i s assumption, seven h e l i c a l spans a r e p r e d i c t e d f o r the 34 kDa h e r b i c i d e b i n d i n g p r o t e i n ( F i g u r e 7 ) . A v e r y s i m i l a r h y d r o pathy p l o t f o r the 34 kDa h e r b i c i d e b i n d i n g p r o t e i n was p r e s e n t e d by A r g o s s group (40) c a l c u l a t e d a c c o r d i n g t o t h e i r method (38). I t d i f f e r s from t h a t i n F i g u r e 7 by the assignments o f spans V and VI (41) . 1

A s c h e m a t i c p i c t u r e o f the 34 kDa h e r b i c i d e b i n d i n g p r o t e i n as i t i s t h o u g h t t o be l o c a t e d i n the membrane i s g i v e n i n F i g u r e 8. The seven h e l i c a l spans t h r o u g h the membrane a r e i n d i c a t e d . F u r t h e r more, F i g u r e 8 p r o v i d e s t h r e e a d d i t i o n a l r e l e v a n t f a c t s or sugges t i o n s . The f i r s t one d e a l s w i t h the p o s s i b l e b i n d i n g s i t e o f a z i d o -



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29

F i g u r e 6. Photograph o f a L i - d o d e c y l s u l f a t e p o l y a c r y l a m i d e elec t r o p h o r e s i s g e l (10-15%) and r a d i o a c t i v i t y d i s t r i b u t i o n t h e r e i n o f a s p i n a c h p h o t o s y s t e m I I p r e p a r a t i o n l a b e l e d by 2 nmol/mg c h l o r o p h y l l [ ËT| a z i d o p l a s t o q u i n o n e .

H N-

-COOH

2

1

M I I

II

I I I

I

I I I I

I

I I I I

1

I M I

100 200 Nr. Amino Acid Residue (window: 11)

Figure

Iιιι ιh 1 300

7. Hydropathy p l o t o f t h e 34 kDa h e r b i c i d e b i n d i n g


protein.

30


Possible Plastoquinone Binding Sites:


Met-His 158.159

Phe-His-Met 161 , 163

Azido-atrazine Binding Site

Met-His 178,179

Ser 2

2

8

Resistance Gly, Ala

F i g u r e 8. Schematic drawing o f t h e p o s s i b l e l o c a t i o n o f t h e 34 kDa h e r b i c i d e b i n d i n g p r o t e i n w i t h i n t h e t h y l a k o i d membrane.



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atrazine on the 34 kDa herbicide binding protein. From their recent work on proteolytic digestion after azidoatrazine labeling, Wolber and Steinback (42) have concluded that the azidoatrazine binding site presumably is located in the region Pro 105 to Arg 189 of the amino acid sequence (spans IV to VI, Figure 8). The second suggestion relates to herbicide resistance. By sequencing the DNA of the 34 kDa herbicide binding protein from atrazine-resistant Amaranthus, Hirschberg and Mcintosh (39) have found 4 nucleotide differences as compared to the susceptible type. Only one of the differences leads to a change in the amino acid sequence: Ser in position 228 is replaced by Gly. Similarily, in an atrazine-resistant mutant from Chlamydomonas the same Ser in position 228 is exchanged, but this time against Ala (43). The third suggestion concerns possible plastoquinone binding sites in the 34 kDa herbicide binding protein, if there are any. According to a proposal by Hearst and Sauer (^4), the sequence MetHis is a possible quinone binding site, probably with an additional Phe. There are three consecutive Met-His sequences in the 34 kDa herbicide binding protein (Figure 8). It should be pointed out, however, that the arginine residues are also possible candidates for quinone binding (45). Indeed, the arginine-modifying reagent phenylglyoxal was found to decrease atrazine binding (46). In conclusion, observations made in the last few years, especially the binding studies with radiolabeled herbicides, the photoaffinity labeling technique, and the advances of molecular biology have substantially added to our knowledge of the mechanism of action of photosynthetic herbicides. However, many questions also remain to be answered. Acknowledgements This work was supported by Deutsche Forschungsgemeinschaft. I am indebted to Eva Neumann, Dr. Udo Johanningmeier, Klaus Masson, and Hans-Joachim Soli for their help in the experiments. Literature Cited 1. Bouges-Bocquet, B. Biochim. Biophys. Acta 1973, 314, 250-6. 2. Velthuys, B.R.; Amesz, J . Biochim. Biophys. Acta 1974, 333, 85-94. 3. Renger, G. Biochim. Biophys. Acta 1976, 440, 287-300. 4. Regitz, G; Ohad, I. J . Biol. Chem. 1976, 251, 247-52. 5. Trebst, A; Draber, W. In "Advances in Pesticide Science"; Geissbühler, H., Ed.; Pergamon Press: Oxford, New York, 1979; Part 2, p. 223. 6. Pfister, K; Arntzen, C.J. Z. Naturforsch. 1979, 34c, 996-1009. 7. Tischer, W; Strotmann, H. Biochim. Biophys. Acta 1977, 460, 113-25. 8. Oettmeier, W.; Masson, K.; Johanningmeier, U. Biochim. Biophys. Acta 1982, 679, 376-83. 9. Oettmeier, W.; Trebst, A. In "The Oxygen Evolving System of Photosynthesis"; Inoue, Y., et a l . , Eds.; Academic Press: Tokyo, 1983, p. 411. 10. Pfister, K.; Radosevich, S.R.; Arntzen, C.J. Plant Physiol. 1979, 64, 995-9.



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11. Oettmeier, W.; Masson, K.; Fedtke, C . ; Konze, J.; Schmidt, R.R. Pestic. Biochem. Physiol. 1982, 18, 357-67. 12. Oettmeier, W.; Masson, K. Pestic. Biochem. Physiol. 1980, 14, 86-97. 13. Trebst, A; Harth, E . ; Draber, W. Z. Naturforsch. 1970, 25b, 1157-9. 14. Oettmeier, W.; Masson, K.; Johanningmeier, U. FEBS Lett.1980, 118, 267-70. 15. Gardner, G. Science 1981, 211, 937-40. 16. Oettmeier, W.; Masson, K.; Soll, H . J . ; Draber, W. Biochim. Biophys. Acta 1984, in press. 17. Johanningmeier, U.; Neumann, E.; Oettmeier, W. J . Bioenerg. Biomembr. 1983, 15, 43-66. 18. Pfister, K.; Steinback, K . E . ; Gardner, G.; Arntzen, C . J . Proc. Natl. Acad. Sci.USA 1981, 78, 981-5. 19. Mattoo, A . K . ; Pick, U.; Hoffmann-Falk, H . ; Edelman, M. Proc. Natl. Acad. Sci. USA 1981, 78, 1572-6. 20. Gressel, J . Plant Sci. Lett. 1982, 25, 99-106. 21. Laasch, H.; Pfister, K.; Urbach, W. Z. Naturforsch. 1982, 37c, 620-31. 22. Pfister, K.; Schreiber, U. Z. Naturforsch. 1984, 39c, 389-92. 23. Mathis, P.; Rutherford, A.W. Biochim. Biophys. Acta 1984, in press. 24. Oettmeier, W.; Soll, H . J . ; Neumann, Ε. Z. Naturforsch. 1984, 39c, 393-6. 25. Velthuys, B.R. FEBS Lett. 1981, 126, 277-81. 26. Lavergne, J . Biochim. Biophys. Acta, 1982, 682, 345-53. 27. Urbach, W.; Laasch, H . ; Schreiber, U. Z. Naturforsch. 1984, 39c, 397-401. 28. Oettmeier, W.; Soll, H.J. Biochim. Biophys. Acta 1983, 724, 287-90. 29. Vermaas, W.F.J.; Renger, G.; Arntzen, C . J . Z. Naturforsch. 1984, 39c, 368-73. 30. Soll, H . J . ; Oettmeier, W. In "Advances in Photosynthesis Re search"; Sybesma, C . , Ed.; Martinus Nijhoff/Dr. W. Junk Pub lishers: The Hague, 1984, Vol. 4, p. 5. 31. Oettmeier, W.; Masson, K.; Soll, H . J . ; Hurt, E . ; Hauska, G. FEBS Lett. 1982, 144, 313-7. 32. Oettmeier, W.; Masson, K.; Soll, H . J . ; Olschewski, E. In "Advan ces in Photosynthesis Research"; Sybesma, C., Ed.; Martinus Nijhoff/ Dr. W. Junk Publishers: The Hague, 1984, Vol. 1, p.469. 33. Oettmeier, W.; Soll, H.J. in preparation. 34. Satoh, K.; Nakatani, H.Y.; Steinback, K . E . ; Watson, J.; Arntzen, C.J. Biochim. Biophys. Acta 1983, 724, 142-50. 35. Renger, G.; Hagemann, R.; Vermaas, W.F.J. Z. Naturforsch. 1984, 39c, 362-7. 36. Zurawski, G.; Bohnert, H . J . ; Whitfield, P.R.; Bottomley, W. Proc. Natl. Acad. Sci. USA 1982, 79, 7699-703. 37. Kyte, J ; Doolitlle, R.F. J . Mol. Biol. 1982, 157, 105-32. 38. Argos, P.; Rao, J.K.M.; Hargrave, P.A. Eur. J . Biochem. 1982, 128, 565-75. 39. Hirschberg, J ; McIntosh, L. Science 1983, 222, 1346-9. 40. Rao, J.K.M.; Hargrave, P.Α.; Argos, P. FEBS Lett. 1983, 156, 165-9.


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41. Trebst, A. In "Proceedings of the Southern Section, American Society of Plant Physiology, 1984, in press. 42. Wolber, P.K.; Steinback, K.E. Z. Naturforsch. 1984, 39c, 425-9. 43. Erickson, J.M; Rahire, M.; Bennoun, P.; Delepelaire, P.; Diner, B.; Rochaix, J.D. Proc. Natl. Acad. Sci. USA 1984, 81, 3617-21. 44. Hearst, J . E . ; Sauer, Κ. Z. Naturforsch. 1984, 39c, 421-4. 45. Shipman, L . L . J . Theor. Biol. 1981, 90, 123-48. 46. Gardner, G.; Allan, C.D.; Paterson, D.R. FEBS Lett. 1983, 164, 191-4.


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