Photosensitizing Porphyrins as Herbicides - ACS Symposium Series

Chapter 26

Photosensitizing Porphyrins as Herbicides 1

Stephen O . Duke, Josfe M. Becerril , Timothy D . Sherman, and Hiroshi Matsumoto 2

Downloaded by PENNSYLVANIA STATE UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: January 9, 1991 | doi: 10.1021/bk-1991-0449.ch026

Southern Weed Science Laboratory, Agricultural Research Service, U . S . Department of Agriculture, P.O. Box 350, Stoneville, M S 38776

Several porphyrin intermediates of heme and/or chlorophyll biosynthesis are potent photosensitizers which generate high levels of singlet oxygen in the presence of molecular oxygen and light. Many compounds that affect the heme and/or chlorophyll pathways are strongly herbicidal due to accumulation of phytotoxic levels of these porphyrins in response to the chemical. For instance, several commercial and experimental herbicides inhibit protoporphyrinogen oxidase, the enzyme that converts protoporphyrinogen to protoporphyrin IX (PPIX). This leads to uncontrolled autooxidation of the substrate and results in massive accumulation of PPIX. In plants treated with these herbicides, damage is light dependent and closely correlated with the level of PPIX that accumulates. PPIX accumulation is apparently largely extraplastidic. Treatment with the porphyrin precursor δ-aminolevulinic acid (ALA), in combination with the heme and chlorophyll pathway inhibitor 2,2'-dypyridyl (DP), results in the accumulation of toxic levels of primarily Mg-PPIX monomethylester. DP deregulates porphyrin synthesis and ALA provides additional substrate. DP and other chlorophyll synthesis modulators in combination with ALA can increase the selectivity as well as enhance the efficacy of ALA as a herbicide. Exogenously applied porphyrins are far less effective as herbicides than treatment with compounds that cause plants to accumulate their own porphyrins. P h o t o d y n a m i c compounds, i n c l u d i n g many n a t u r a l p r o d u c t s , h a v e b e e n p r o p o s e d f o r u s e a s h e r b i c i d e s b y many r e s e a r c h e r s ( e . g . , 1 - 3 ) . Current address: Universidad del Pais Vasco-EHU, Facultad de Ciencias, Dept. Fisiologia Vegetal y Ecologia, Apartado 644, '18080 Bilbao, Spain 2

Current address: Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305, Japan This chapter not subject to U.S. copyright Published 1991 American Chemical Society In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

NATURALLY OCCURRING PEST BIOREGULATORS


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However, a problem w i t h t h e s e c h e m i c a l s i s t h e i r i n d i s c r i m i n a t e t o x i c i t y i n t h e p r e s e n c e o f l i g h t and m o l e c u l a r oxygen. This t o x i c i t y p r e c l u d e s t h e i r use a s p e s t i c i d e s . S a f e a l t e r n a t i v e s are to t r e a t t a r g e t organisms with chemicals that e i t h e r are s e l e c t i v e l y m e t a b o l i z e d t ophotodynamic compounds o r cause t h e t a r g e t o r g a n i s m t o produce t o x i c l e v e l s o f n a t u r a l photodynamic compounds w i t h i t s own b i o c h e m i c a l m a c h i n e r y . T h i s r e v i e w examines one a s p e c t o f t h e l a t t e r a l t e r n a t i v e - t r e a t m e n t o f p l a n t s w i t h compounds t h a t cause the accumulation o fh e r b i c i d a l l e v e l s o f photodynamic p o r p h y r i n s . C h l o r o p h y l l i s a p h o t o d y n a m i c compound when i t i s n o t a c o m p o n e n t o f t h e p h o t o s y n t h e t i c a p p a r a t u s . One w a y i n w h i c h t h e p l a n t p r o t e c t s i t s e l f from the photodynamic p r o p e r t i e s o f c h l o r o p h y l l i s through l i n k i n g c h l o r o p h y l l t oa biochemical complex that d i s s i p a t e s the energy o fl i g h t - e n e r g i z e d c h l o r o p h y l l through s p l i t t i n g o f w a t e r and e n e r g i z i n g o f e l e c t r o n s f r o m w a t e r f o r e n e r g y t r a n s d u c t i o n and p h o t o s y n t h e t i c r e d u c i n g power. I n t e r m e d i a t e s o f c h l o r o p h y l l b i o s y n t h e s i s are photodynamic a l s o . Since they cannot be u t i l i z e d i n p h o t o s y n t h e s i s and a r e p h o t o d y n a m i c , t h e r e i s s t r o n g s e l e c t i o n p r e s s u r e a g a i n s t accumulation o f these compounds. M u t a t i o n s t h a t cause the a c c u m u l a t i o n o f t h e s e compounds are d e l e t e r i o u s t othe p l a n t . For i n s t a n c e , y e l l o w mutants of maize have been d e s c r i b e d t h a t accumulate h i g h l e v e l s o fp r o t o p o r p h y r i n IX (PPIX) (4). A l t h o u g h t h e s e mutants have i m p a i r e d c h l o r o p h y l l s y n t h e s i s , p a r t o f t h e p h y t o t o x i c e f f e c t o f t h e m u t a t i o n i s due t o the photodynamic e f f e c t o f PPIX. Two a p p r o a c h e s t o s t i m u l a t i o n o f p o r p h y r i n a c c u m u l a t i o n i n p l a n t s have been t a k e n . The f i r s t i s t o s u p p l y t h e p l a n t w i t h t h e p o r p h y r i n p r e c u r s o r 6 - a m i n o l e v u l i n c a c i d (ALA) a l o n g w i t h compounds t h a t a f f e c t t h e p o r p h y r i n pathway. The second i s t o b l o c k porphyrin synthesis a tthe protoporphyrinogen oxidase step i n the pathway, t h e r e b y d e r e g u l a t i n g t h e pathway and c a u s i n g a c c u m u l a t i o n p r i m a r i l y o f PPIX. ALA a s a H e r b i c i d e R e b e i z e t a l . (5) i n t r o d u c e d t h e c o n c e p t o f ALA i n c o m b i n a t i o n w i t h various c h l o r o p h y l l synthesis modulators as a h e r b i c i d e . Previous l i t e r a t u r e had d e m o n s t r a t e d t h a t ALA t r e a t m e n t o f p l a n t t i s s u e s could cause the accumulation o fabnormally high l e v e l s o f c o p r o p o r p h y r i n , p r o t o c h l o r o p h y l l i d e ( P C h l i d e ) , PPIX, a n d M g - p r o t o p o r p h y r i n IX monomethyl e s t e r (MgPPIXME) (6, 7 ) . I n cucumber s e e d l i n g s s p r a y e d w i t h 1 0 t o 2 0 mM A L A , a n d t h e n p l a c e d i n t h e d a r k f o r 17 h t o a l l o w c h l o r o p h y l l p r e c u r s o r s t o a c c u m u l a t e , a two- t o f o u r - f o l d i n c r e a s e i n t o t a l p o r p h y r i n s ( p r i m a r i l y PChlide) was o b s e r v e d ( 5 ) . T h i s l e d t o 95 % photodynamic damage t o t h e s e e d l i n g s a f t e r t h e y were p l a c e d i n t h e l i g h t . The e f f e c t i v e l e v e l o f a p p l i e d ALA c o u l d b e r e d u c e d b y s p r a y i n g i t i n c o m b i n a t i o n w i t h 2 , 2 ' - d i p y r i d y l (DP), a r e l a t i v e l y i n e x p e n s i v e s y n t h e t i c compound t h a t s t i m u l a t e s p o r p h y r i n s y n t h e s i s b y p r e v e n t i n g heme s y n t h e s i s t h r o u g h c h e l a t i n g i r o n ( 8 ) . The p o r p h y r i n s y n t h e s i s pathway i s u n d e r s t r o n g f e e d b a c k c o n t r o l b y heme ( 9 , 1 0 ) . I n a d d i t i o n t o s t i m u l a t i n g p o r p h y r i n s y n t h e s i s , DP b l o c k s c o n v e r s i o n o f MgPPIXME t o P C h l i d e (8, 9 ) ( F i g . 1 ) . Thus, both t h e q u a n t i t y and t y p e o f porphyrins that accumulate are a f f e c t e d .

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

26.

DUKEETAL.

Photosensitizing Porphyrins as Herbicides

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A L A p l u s DP a c t e d s y n e r g i s t i c a l l y a s h e r b i c i d e s , d e s p i t e t h e f a c t t h a t t h e r e was g e n e r a l l y o n l y an a d d i t i v e e f f e c t o n t o t a l p o r p h y r i n a c c u m u l a t i o n ( T a b l e I ) . T h e s e r e s u l t s i n d i c a t e one o r more o f t h e f o l l o w i n g : (a) one o f t h e e a r l i e r c h l o r o p h y l l i n t e r m e d i a t e s (MgPPIXME o r PPIX) i s more h e r b i c i d a l t h a n P C h l i d e , (b) t h e p o r p h y r i n s a c t s y n e r g i s t i c a l l y , o r (3) t h a t t h e s y n e r g i s m o f A L A p l u s DP i s n o t b a s e d o n t h e i r e f f e c t s o n p o r p h y r i n s . T a b l e I . E f f e c t s o f A L A a n d DP, a l o n e o r i n c o m b i n a t i o n , o n p o r p h y r i n a c c u m u l a t i o n and h e r b i c i d a l damage. Cucumber s e e d l i n g s (6-day-old) were assayed f o r p o r p h y r i n s a f t e r being sprayed w i t h t h e h e r b i c i d e s a n d i n c u b a t e d i n d a r k n e s s f o r 17 h . H e r b i c i d a l d a m a g e was a s s e s s e d 10 d a y s a f t e r p o r p h y r i n s w e r e a s s a y e d a n d d u r i n g which they were exposed t o greenhouse l i g h t c o n d i t i o n s . Taken from r e f . ( 5 ) . Treatment Control 5 mM A L A 15 mM DP 5 mM A L A + 15 mM a * ,

P o r p h y r i n s ( n m o l e s / 1 0 0 mg PChlide MaPPIXME PPIX 0.0 17.3 0.6 0.0 100.7 1.6 24.0 2.6 12.3 DP 121.1 8.1 26.3

protein) D a m a a e (%) Total 0 17.9 30 101.3 10 38.9 80 155.5

The f i r s t and s e c o n d p o s s i b i l i t i e s a r e c o m p l i c a t e d by t h e p o s s i b i l i t y that these porphyrins are a l l d i f f e r e n t i a l l y p h o t o l a b i l e a n d p h o t o d e g r a d a t i o n p r o d u c t s may b e i n v o l v e d i n t h e i r p h o t o d y n a m i c action. For instance, PChlide disappears i n green t i s s u e r a p i d l y a f t e r e x p o s u r e t o l i g h t (5) ( F i g . 2 ) , a l t h o u g h t h e p r o p o r t i o n s c o n v e r t e d t o c h l o r o p h y l l o r p h o t o d e g r a d e d a r e n o t k n o w n . Some e v i d e n c e i n d i c a t e s t h a t most o f the A L A - s t i m u l a t e d P C h l i d e a c c u m u l a t i o n i s p h o t o d e g r a d e d ( 6 ) , a l t h o u g h t h i s may n o t a l w a y s b e t h e c a s e . MgPPIXME l e v e l s i n cucumber c o t y l e d o n s d e c l i n e l e s s r a p i d l y i n l i g h t t h a n do t h o s e o f P C h l i d e ( F i g . 2 ) . In normal e t i o l a t e d p l a n t s , complete PChlide phototransformation of chlorophyllide (Chlide) in bright light i s very rapid (usually less t h a n a m i n u t e ) and s u b s e q u e n t p h y t y l l a t i o n o f C h l i d e t o f o r m c h l o r o p h y l l ( C h i ) i s c o m p l e t e i n 30 m i n o r l e s s ( e . g . , H ) . The p r o l o n g e d d e c a y o f P C h l i d e i n t i s s u e t r e a t e d w i t h A L A p l u s DP c o u l d be due t o s y n t h e s i s o c c u r r i n g more r a p i d l y t h a n n o r m a l r a t e s o f c o n v e r s i o n t o C h i , t o slowed p h o t o t r a n s f o r m a t i o n and p h y t y l l a t i o n , o r t o a l a r g e component o f n o n - p h o t o t r a n s f o r m a b l e (NPTF) P C I i d e . Gassman (12) f o u n d t h a t extended t r e a t m e n t o f e t i o l a t e d bean l e a v e s w i t h 10 mM A L A r e s u l t e d i n a g r e a t e r p o r p o r t i o n o f N P T F P C I i d e a c c u m u l a t i o n t h a n i n u n t r e a t e d l e a v e s and t h a t NPTF P C I i d e i n h i b i t s a c c u m u l a t i o n o f p h o t o t r a n s f o r m a b l e (PTF) P C I i d e . T h u s , NPTF P C I i d e may p l a y a r o l e a s a p h o t o d y n a m i c p i g m e n t i n t h i s s y s t e m , a l t h o u g h R e b e i z et a / . ( 5 ) d i d n o t d i f f e r e n t i a t e b e t w e e n P T F a n d N P T F PCIide. I n t h e o r i g i n a l s y s t e m o f R e b e i z et al. ( 5 ) , a r a t h e r l o n g p o s t - s p r a y d a r k p e r i o d was r e q u i r e d f o r s u f f i c i e n t a c c u m u l a t i o n o f p o r p h y r i n s f o r h e r b i c i d a l a c t i v i t y o c c u r . The h e r b i c i d a l e f f e c t o f A L A p l u s DP was a g e a n d s p e c i e s d e p e n d e n t ; h o w e v e r , t h e r e was n o t a l w a y s a s t r o n g c o r r e l a t i o n b e t w e e n t h e e f f e c t on p o r p h y r i n


374

NATURALLY OCCURRING PEST

BIOREGULATORS

Gtutamate

Gabacullne

^ ALA

DHA

Levullnate

Membrane lipid peroxidation

Porphobilinogen Photobleaching

^


herbicides -

^ Protoporphyrin IX

Protoporphyrinogen-^

^

r

oxygen

'2 Protoporphyrin IX

8

Cytochromes

M g

+

^ ^

Light +

0

2

Mg Protoporphyrin IX M g PPIX M E ^^2,2-dlpyridyl Protochlorophyllide Chlorophyllide Chlorophyll a

F i g u r e 1. T h e p o r p h y r i n s y n t h e s i s p a t h w a y a n d s i t e s o f i n h i b i t i o n o f v a r i o u s i n h i b i t o r s and m o d u l a t o r s . Inhibitors are u n d e r l i n e d and s i t e s o f i n h i b i t i o n a r e i n d i c a t e d

F i g u r e 2. T i m e c o u r s e o f P C h l i d e a n d M g - P P I X M E d i s a p p e a r a n c e from ALA p l u s D P - t r e a t e d cucumber s e e d l i n g s i n d a y l i g h t a f t e r a 17-h a c c u m u l a t i o n p e r i o d . (Reprinted with permission from ref. 5. Copyright 1984 Butterworth Scientific, Ltd.)


26.

DUKEETAL.

375



s y n t h e s i s a n d h e r b i c i d a l d a m a g e . No h e r b i c i d a l d a m a g e was o b s e r v e d w h e n t h e r e was l i t t l e o r no e f f e c t o n c h l o r o p h y l l p r e c u r s o r l e v e l s b u t , i n some t i s s u e s o f s o m e s p e c i e s , a c c u m u l a t i o n o f p o r p h y r i n s d i d not r e s u l t i n h e r b i c i d a l damage ( T a b l e I I ) . G e n e r a l l y , t h e y found g r a s s e s , i n c l u d i n g m a i z e , b a r l e y , o a t , and w h e a t t o be t o l e r a n t t o t h e s e h e r b i c i d e s , w h i l e a l l d i c o t weeds t e s t e d were h i g h l y s e n s i t i v e . Thus, t h i s h e r b i c i d e c o m b i n a t i o n showed promise f o r b r o a d l e a f weed c o n t r o l i n g r a i n c r o p s . T a b l e I I . E f f e c t s o f A L A (5mM) p l u s DP ( 1 5 mM) o n p o r p h y r i n accumulation in seedling tissues of several plant species. Plant were assayed f o r p o r p h y r i n s a f t e r being sprayed with the h e r b i c i d e s a n d i n c u b a t e d i n d a r k n e s s f o r 17 h . H e r b i c i d a l d a m a g e was a s s e s s e d 10 d a y s a f t e r p o r p h y r i n s w e r e a s s a y e d a n d s u b s e q u e n t e x p o s u r e t o greenhouse l i g h t c o n d i t i o n s . Taken from r e f . (5). Species ( S e e d l i n g age)

Porphyrins ( n m o l e s / 1 0 0 mg p r o t e i n ) Herbicidal PChlide MgPPIXME PPIX D a m a g e (%) Con T r e a t e d Con T r e a t e d Con T r e a t e d 90 ( 1 2 ) 30 24 29 36 201 12

Mustard leaves Cotton cotyledon (14) 18 C o t t o n stem (14) 4 Kidney bean l e a f (9) 117 Kidney bean stem (9) 37 G i a n t f o x t a i l (6) 8 M a i z e (9) 79 small n e c r o t i c areas

37 4

4 1

9 1

0 0

0 0

63 0

439

3

430

5

22

100

3 0 12

14 14 0

0 S.N. S.N.

82 79 85

4 0. ,4 5

76 12 15

a

R e b e i z e t a7. (13) c l a s s i f i e d s p e c i e s i n t o f o u r d i f f e r e n t g r e e n i n g g r o u p s , b a s e d on t h e i r C h i s y n t h e s i s h e t e r o g e n e i t y . The f o u r groups were c a t e g o r i z e d a c c o r d i n g t o the predominance o f d i v i n y l o r m o n o v i n y l p o r p h y r i n s y n t h e s i s and under what c o n d i t i o n s ( d a r k o r l i g h t ) s y n t h e s i s o f each p o r p h y r i n t y p e o c c u r r e d . The f o u r groups are described with r e p r e s e n t a t i v e species i n Table I I I . Table I I I . Greening groups

and r e p r e s e n t a t i v e p l a n t s p e c i e s .

Greening Group 1. D a r k d i v i n y l / 1 i g h t d i v i n y l

Representative

Species

(DDV/LDV)

Cucumber, mustard, common p u r s l a n e 2. D a r k m o n o v i n y l / 1 i g h t d i v i n y l ( D M V / L D V ) Maize, wheat, b a r l e y , common b e a n , s o y b e a n , pigweed 3. D a r k d i v i n y l / 1 i g h t m o n o v i n y l ( D D V / L M V ) Ginkgo 4. D a r k m o n o v i n v l / 1 i g h t m o n o v i n y l (DMV/LMV) A p p l e , . i o h n s o n q r a s s T h e y h y p o t h e s i z e d t h a t t h e s e n s i t i v i t y o f a s p e c i e s t o ALA p l u s c h l o r o p h y l l m o d u l a t o r s i s due t o both e x t e n t o f p o r p h y r i n s y n t h e s i s and t h e c h e m i c a l n a t u r e o f t h e a c c u m u l a t e d p o r p h y r i n s . T h u s , t h e greening group to which a s p e c i e s belongs c o u l d s t r o n g l y i n f l u e n c e



376


i t s s u s c e p t i b i l i t y . I t f o l l o w e d t h a t s i n c e some s p e c i e s a c c u m u l a t e r e l a t i v e l y l a r g e amounts o f p o r p h y r i n s i n r e s p o n s e t o ALA, b u t d i s p l a y l i t t l e h e r b i c i d a l damage, some p o r p h y r i n s m i g h t b e r e l a t i v e l y poor p h o t o s e n s i t i z e r s . T h i s hypothesis was t e s t e d by u s i n g ALA i n c o m b i n a t i o n w i t h c h l o r o p h y l l s y n t h e s i s m o d u l a t o r s o t h e r t h a n DP ( 1 3 ) . Although the r e s u l t s of these extensive experiments are not e a s i l y i n t e r p r e t e d , t h e h y p o t h e s e s w e r e made (a) t h a t P C h l i d e i s t h e most i m p o r t a n t and u b i q u i t o u s p h o t o d y n a m i c s p e c i e s c a u s e d t o a c c u m u l a t e b y A L A - b a s e d t r e a t m e n t s , ( b ) t h a t MV P C h l i d e i s a m o r e e f f e c t i v e p h o t o d y n a m i c p i g m e n t t h a n DV P C h l i d e i n D D V / L D V a n d DMV/LDV s p e c i e s , a n d ( c ) t h a t b o t h D D V / L D V a n d DMV/LDV s p e c i e s a r e h i g h l y s u s c e p t i b l e t o a m i x t u r e o f Mg-PPIX and Mg-PPIXME ( 1 4 ) . T h e r e s u l t s are d i f f i c u l t t o i n t e r p r e t because equimolar l e v e l s o f d i f f e r e n t p o r p h y r i n s were n o t p r o d u c e d and t h e c o m b i n a t i o n s o f porphyrins produced by d i f f e r e n t modulators v a r i e d with s p e c i e s . P o t e n t i a l d i f f e r e n c e s i n t o l e r a n c e to t o x i c oxygen s p e c i e s between s p e c i e s were not c o n s i d e r e d . Others have attempted t o e x p l a i n d i f f e r e n t i a l s e n s i t i v i t y to p o r p h y r i n - g e n e r a t i n g h e r b i c i d e s between s p e c i e s (15) and between h e r b i c i d e - s e n s i t i v e b i o t y p e s w i t h i n s p e c i e s (16) b y d i f f e r e n c e s i n a b i l i t y t o d e t o x i f y t o x i c oxygen s p e c i e s . A s w i t h o t h e r h e r b i c i d e s , p e n e t r a t i o n o f t h e l e a f c u t i c l e b y ALA a n d / o r DP c a n a l s o p l a y a r o l e i n d i f f e r e n c e s i n e f f i c a c y o f t h i s h e r b i c i d e combination (17). The Chi s y n t h e s i s m o d u l a t o r s t h a t R e b e i z e t a/. (13, 1 4 ) u s e d i n c o n j u n c t i o n w i t h ALA c o u l d b e d i v i d e d i n t o t h r e e c a t e g o r i e s : A) e n h a n c e r s o f ALA c o n v e r s i o n t o p o r p h y r i n s ( 2 - p y r i d i n e a l d o x i m e , 2-pyridine aldehyde, p i c o l i n i c acid, 2,2'dipyridyl d i s u l f i d e , 2 , 2 ' - d i p y r i d y l amine, 4 , 4 ' d i p y r i d y l , and p h e n a n t h r i d i n e ) , B) i n d u c e r s o f ALA b i o s y n t h e s i s and p o r p h y r i n a c c u m u l a t i o n ( 2 , 2 ' - d i p y r i d y l a n d 1 , 1 0 - p h e n a n t h r o l i n e ) , a n d C ) i n h i b i t o r s o f MV PChlide synthesis (2,3-dipyridyl, 2,4-dipyridyl, 1,7-phenanthroline, and 4 , 7 - p h e n a n t h r o l i n e ) . Compounds i n group A d i d not cause s i g n i f i c a n t p o r p h y r i n accumulation alone; however, they enhanced d a r k c o n v e r s i o n o f e x o g e n o u s ALA t o p o r p h y r i n s . T h i s g r o u p w a s f u r t h e r s u b d i v i d e d i n t o compounds t h a t e n h a n c e d c o n v e r s i o n o f ALA t o MV P C h l i d e ( 2 - p y r i d i n e a l d o x i m e , 2 - p y r i d i n e a l d e h y d e , p i c o l i n i c a c i d , and 2 , 2 ' - d i p y r i d y l d i s u l f i d e ) and t h o s e t h a t s t i m u l a t e d c o n v e r s i o n t o DV P C h l i d e ( 4 , 4 ' d i p y r i d y l , 2 , 2 ' d i p y r i d y l a m i n e , a n d p h e n a n t h r i d i n e ) . T o q u a l i f y a s a n ALA b i o s y n t h e s i s and p o r p h y r i n a c c u m u l a t i o n i n d u c e r ( c a t e g o r y B ) , t h e compound had t o c a u s e t h e s e e f f e c t s i n t h e a b s e n c e o f ALA. Compounds i n c a t e g o r y C had t o i n h i b i t a c c u m u l a t i o n o f MV P C h l i d e w i t h o r w i t h o u t A L A . In most c a s e s , i n c o n j u n c t i o n w i t h A L A , t h e c o m p o u n d s s t i m u l a t e d DV P C h l i d e accumulation compared t othe ALA-treated c o n t r o l . With knowledge o f g r e e n i n g group c h a r a c t e r i s t i c s and m o d u l a t o r t y p e , one can t h e o r e t i c a l l y m a n i p u l a t e t h e s e l e c t i v i t y o f ALA-modulator combinations. T h i s approach was used to d e s i g n a c o m b i n a t i o n f o r c o n t r o l o f c r e e p i n g c h a r l i e (DDV/LDV) i n K e n t u c k y b l u e g r a s s ( D M V / L D V ) ( 1 3 ) . S i n c e t h e g r e e n i n g t y p e o f t h e two s p e c i e s d i v e r g e d a t n i g h t , a " d a r k s p r a y " a p p l i e d n e a r d u s k was t h e o r i z e d t o be m o s t s e l e c t i v e . H o w e v e r , a f i n a l c h o i c e o f t h e ALA p l u s DP c o m b i n a t i o n w a s made s i n c e t h i s c o m b i n a t i o n l e d t o t h e g e n e r a l l y l e t h a l a c c u m u l a t i o n o f DV M g P P I X M E a n d DV M g P P I X i n m o s t s p e c i e s .



26.

D U K E E T AL.


377

H o w e v e r , K e n t u c k y b l u e g r a s s w a s g e n e r a l l y much l e s s s e n s i t i v e , despite these accumulations. The mechanism o f t o l e r a n c e was not explained. R e c e n t l y , A v e r i n e t a/. (18) found t h a t more A L A s u p p l i e d t o barley accumulated as porphobilinogen, r a t h e r than being converted t o p o r p h y r i n s , than i n bean. A l s o , the p o r p h y r i n s i n b a r l e y were d e g r a d e d i n l i g h t much more r a p i d l y t h a n i n b e a n . T h u s , some o f t h e s e l e c t i v i t y o f A L A p l u s DP may b e i n f l u e n c e d b y many f a c t o r s o t h e r t h a n g r e e n i n g t y p e . T h e u l t r a s t r u c t u r a l d e v e l o p m e n t o f damage from t r e a t m e n t w i t h A L A p l u s DP i n b e a n c o t y l e d o n s o c c u r s r a p i d l y ( 1 9 ) . W i t h i n 1 h o f exposure t o l i g h t c h l o r o p l a s t s s w e l l e d a n d became s p h e r i c a l . Subsequently, grana andstromal t h y l a k o i d s swelled and d e s t r u c t i o n o f i n t r a c h l o r o p l a s t membranes was o b s e r v e d b y 2 h . W i t h i n 6 t o 24 h , c h l o r o p l a s t s and m i t o c h o n d r i a were b r o k e n . T o d a t e , o n l y s i x p u b l i c a t i o n s ( 5 , 13, 1 4 > 1Z-12) e x i s t o n A L A a s a h e r b i c i d e . A n o t h e r paper has been p u b l i s h e d o n A L A a s a n i n s e c t i c i d e (20). Although most o f t h e s e are h i g h l y s u b s t a n t i a l papers, several questions remain regarding r e s u l t s o f these s t u d i e s . The a c t u a l r e l a t i v e p h y t o t o x i c i t y o f v a r i o u s p o r p h y r i n s i s not clear. The i n t r a c e l l u l a r s i t e ( s ) o fporphyrin accumulation are also n o t known. F u r t h e r m o r e , t h e complex i n t e r a c t i o n s between g r e e n i n g t y p e , t o l e r a n c e t o t o x i c oxygen s p e c i e s , and c a p a c i t y t o s y n t h e s i z e porphyrins i s poorly understood. Although A L A i n combination with v a r i o u s Chi s y n t h e s i s modulators has been p a t e n t e d f o r h e r b i c i d e use, none o f t h e c o m b i n a t i o n s i s p r e s e n t l y c o m m e r c i a l l y a v a i l a b l e . However, a l a r g e number o f synthetic herbicides that act by causing the accumulation o f photodynamic porphyrins are sold throughout the world. Synthetic Herbicide-Induced Accumulation

o f Porphyrins

H i s t o r i c a l background. Diphenyl ethers o f the general s t r u c t u r e shown b e l o w were i n t r o d u c e d a s commercial h e r b i c i d e s i n t h e 1960's (21) a n d s i n c e t h a t t i m e many members o f t h i s h e r b i c i d e c l a s s h a v e been commercialized (22). A l l o f the commercialized v e r s i o n s have been p a r a - n i t r o s u b s t i t u t e d . _ ni

™ CF3,

CI

R

2

= CI. NC*

R

3

-

R4 -

OCH3. COOCH3. OCjHs. H NO2. CI. I. NO

T h e s e h e r b i c i d e s c a u s e r a p i d b l e a c h i n g and d e s s i c a t i o n o f g r e e n t i s s u e s , s i m i l a r t o the e f f e c t s o fparaquat. Like paraquat, l i g h t i s r e q u i r e d f o r a c t i v i t y (22), however, u n l i k e paraquat, p h o t o s y n t h e s i s i s n o t a r e q u i r e m e n t f o r a c t i v i t y ( 2 3 - 2 7 ) , e x c e p t when p h o t o s y n t h e s i s i s i n d i r e c t l y r e q u i r e d f o r s u b s t r a t e (28) o r g e n e r a t i o n o f oxygen f o r l i p i d p e r o x i d a t i o n ( 2 7 ) . The d e v e l o p m e n t o f i n j u r y t o p l a n t t i s s u e s a f f e c t e d b y t h e s e compounds i s much l i k e t h a t c a u s e d b y p h o t o d y n a m i c pigments. Thef i r s t measureable e f f e c t i s c e l l u l a r leakage, followed s e q u e n t i a l l y by i n h i b i t e d photosynthesis, ethylene e v o l u t i o n , ethane and m a l o n d i a l d e h y d e e v o l u t i o n , a n d f i n a l l y b l e a c h i n g o f c h l o r o p l a s t



378


p i g m e n t s - a l l c h a r a c t e r i s t i c o f p h o t o d y n a m i c membrane l i p i d peroxidation (29). Furthermore, a f t e r a s u f f i c i e n t l ylong incubation in darkness, the h e r b i c i d a l a c t i v i t y i s almost e n t i r e l y independent o f temperature, l i k e a photodynamic dye (30). I t was obvious from several s t u d i e s t h a t the h e r b i c i d e i t s e l f i s not the photodynamic dye. The most c o m p e l l i n g e v i d e n c e f o r t h i s i s t h a t t h e a c t i o n s p e c t r u m i n d i c a t e d t h a t t h e p h o t o r e c e p t o r f o r p h o t o d y n a m i c damage i s a v i s i b l e pigment (31-33). Diphenyl ether h e r b i c i d e s absorb i n the u l t r a v i o l e t r a t h e r than the v i s i b l e spectrum. Furthermore, t h e r e was no strong evidence that the diphenyl ether herbicide acted a s a l i p i d p e r o x i d i z i n g r a d i c a l a s the r e s u l t o f energy t r a n s f e r from a p h o t o r e c e p t o r , e v e n t h o u g h some n i t r o d i p h e n y l e t h e r h e r b i c i d e s c a n b e photoreduced t on i t r o r a d i c a l anions by ^-carotene (34). This, coupled with apparent evidence that carotenoids are involved in the m o d e o f a c t i o n o f t h e s e h e r b i c i d e s ( e . g . , 2 3 , 24, 2 6 , 3 5 - 3 7 ) , l e d some t o h y p o t h e s i z e that a carotenoid-diphenyl ether exciplex might be i n v o l v e d i n t h e m e c h a n i s m o f a c t i o n o f t h e s e h e r b i c i d e s ( 3 8 ) . I n f a c t , o x y f l u o r f e n - t r e a t e d t h y l a k o i d membranes w i l l g e n e r a t e s i n g l e t o x y g e n when e x p o s e d t o l i g h t ( 3 9 ) . A l t h o u g h some o f t h e s e c o m p o u n d s can form r a d i c a l s , t h e r e are d i p h e n y l e t h e r h e r b i c i d e s t h a t d o not form r a d i c a l s t h a t are q u i t e e f f e c t i v e a s 1 i p i d - p e r o x i d i z i n g h e r b i c i d e s (40-42). D e s p i t e i n v e s t i g a t i o n s b y many l a b o r a t o r i e s , t h e n a t u r e o f t h e p h o t o r e c e p t o r f o r t h e p h o t o d y n a m i c damage r e m a i n e d a n enigma f o r more t h a n two d e c a d e s . S t u d i e s d e m o n s t r a t i n g t h a t t h e r e w a s a m e t a b o l i c requirement before the herbicide could cause e f f e c t s l i k e a p h o t o d y n a m i c d y e ( 2 4 , 28, 3 0 , 4 3 ) s h o u l d h a v e p r o v i d e d a c l u e t o t h e actual mechanism - the induction of the accumulation o fa natural photodynamic compound. S i t e o f a c t i o n . M a t r i n g e a n d S e a l l a (44> 4 5 ) , f o l l o w e d c l o s e l y b y o t h e r s (46, 47) r e p o r t e d t h a t d i p h e n y l e t h e r h e r b i c i d e - t r e a t e d t i s s u e s accumulated abnormally high l e v e l s o f PPIX. Furthermore, s p e c i f i c i n h i b i t o r s o fporphyrin synthesis could completely o r almost c o m p l e t e l y p r e v e n t h e r b i c i d a l damage from d i p h e n y l e t h e r h e r b i c i d e s ( 4 4 - 4 8 ) ( F i g s . 1 and 3 ) and t h e a b s o r p t i o n s p e c t r u m o f PPIX r o u g h l y f i t t h e a c t i o n s p e c t r a f o r t h e l i g h t - i n d u c e d damage b y t h e s e h e r b i c i d e s (31-33, 4 4 ) . N o n - d i p h e n y l e t h e r h e r b i c i d e s t h a t had been observed t oact in a s i m i l a r fashion to diphenyl ethers (oxadiazon, the p y r i d i n e d e r i v a t i v e LS 82-556, the novel phenylpyrazole T N P P - e t h y l , and t h e c y c l i c i m i d e c h l o r o p h t h a l i m - see b e l o w ) a l s o c a u s e d t r e a t e d p l a n t s t o a c c u m u l a t e h i g h l e v e l s o f PPIX (44-46> 4 8 - 5 2 ) . The c y c l i c i m i d e s , d i p h e n y l e t h e r s , and o x a d i a z o l e s had p r e v i o u s l y been demonstrated t o i n h i b i t c h l o r o p h y l l s y n t h e s i s (53-56), however, the connection t ot h e i r l i p i d - p e r o x i d i z i n g a c t i o n was n o t c l e a r . T o d a t e , common 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 s between these d i v e r s e h e r b i c i d e groups have not been determined. P r o t o p o r p h y r i n IX-magnesium c h e l a t a s e s y n t h e s i z e s Mg-PPIX from PPIX. Thus, i t seemed l i k e l y t h a t i n h i b i t i o n o f t h i s enzyme would l e a d t o a c c u m u l a t i o n o f P P I X ( 4 7 , 4 9 ) . H o w e v e r , M a t r i n g e et a / . ( 5 7 , 5 8 ) f o u n d t h a t t h e a c c u m u l a t i o n o f P P I X was d u e , i n f a c t , t o s t r o n g i n h i b i t i o n o f p r o t o p o r p h y r i n o g e n o x i d a s e ( P r o t o x ) , the enzyme t h a t c o n v e r t s p r o t o p o r p h y r i n o g e n t o PPIX ( F i g . 1). T h e s e r e s u l t s


26.

D U K E ET AL.


379


were c o n f i r m e d by Witkowski and H a i l i n g ( 5 9 ) . A p p a r e n t l y , b l o c k a g e at t h i s s i t e l e a d s t o a u t o o x i d a t i o n o f t h e s u b s t r a t e t o form PPIX, a s has b e e n o b s e r v e d when t h i s enzyme i s i n a c t i v e d u e t o g e n e t i c l e s i o n s i n y e a s t and humans ( 6 0 - 6 2 ) .

OXADIAZON

M ft B 39279

US 82-556

CHLOROPHTHAUM

TNPP—ETHYL

A c i f l u o r f e n - m e t h y l and LS 8 2 0 3 4 0 , r j - n i t r o a n d p _ - c h l o r o d i p h e n y l e t h e r s , r e s p e c t i v e l y , h a d U n ' s o f l e s s t h a n 1 /iM f o r t h e s y n t h e s i s o f Mg-PPIX f r o m ALA i n a m a i z e e t i o p l a s t p r e p a r a t i o n ( 5 7 ) . A h e r b i c i d a l l y i n a c t i v e a n a l o g o f a c i f l u o r f e n - m e t h y l (RH 5 3 4 8 ) h a d a n I q almost three orders o f magnitude higher than t h a t o f a c i f l u o r f e n - m e t h y l . None o f t h e compounds i n h i b i t e d Mg-PPIX f o r m a t i o n from PPIX. The I c q ' S f o r maize e t i o p l a s t P r o t o x were a b o u t 1 0 nM f o r t h e h e r b i c i d a l d i p h e n y l e t h e r . S i m i l a r r e s u l t s w e r e o b t a i n e d w i t h p o t a t o , y e a s t , and mouse l i v e r m i t o c h o n d r i a . No s i g n i f i c a n t i n h i b i t i o n o f t h e PPIX f e r r o c h e l a t a s e was m e a s u r e d . S i m i l a r r e s u l t s w e r e o b s e r v e d w i t h o x a d i a z o n , L S 8 2 - 5 5 6 , a n d M&B 3 9 2 7 9 ( 5 8 ) . M&B 3 9 2 7 9 ( s e e a b o v e ) i s a p h e n y l p y r a z o l e t h a t a p p e a r s to have a mechanism o f a c t i o n s i m i l a r t o t h a t o f d i p h e n y l e t h e r s ( 6 3 ) . The r e s u l t s o f Witkowski and H a i l i n g (59) w i t h a c i f l u o r f e n - m e t h y l o ncucumber Protox were s i m i l a r , a l t h o u g h they f o u n d a n I q o f a b o u t 3 0 nM. T o d a t e , n o s t u d i e s h a v e b e e n p u b l i s h e d on t h e t y p e ( s ) o f i n h i b i t i o n o f P r o t o x c a u s e d b y t h e s e h e r b i c i d e s o r on w h e t h e r t h e b i n d i n g s i t e s f o r t h e d i f f e r e n t c h e m i c a l types overlap. 5

5

Mode o f a c t i o n . PPIX i s a p h o t o l a b i l e compound, s o t h e q u e s t i o n o f w h e t h e r s u f f i c i e n t l e v e l s o f i t c a n e x i s t in vivo f o r i t t o e x e r t i t s e f f e c t i s i m p o r t a n t . We f o u n d t h e h a l f - l i f e o f P P I X i n a c i f l u o r f e n - t r e a t e d cucumber t i s s u e d u r i n g exposure t o b r i g h t l i g h t t o b e a b o u t 2.5 h ( 4 1 ) - s u f f i c i e n t t i m e f o r i t t o b e a n e f f e c t i v e h e r b i c i d e . F u r t h e r m o r e , PPIX a c c u m u l a t e d r a p i d l y i n b r i g h t l i g h t and d i d n o t b e g i n t o d e c r e a s e u n t i l c e l l u l a r damage was n e a r l y c o m p l e t e . Others have found l i t t l e c o r r e l a t i o n between t h e h e r b i c i d a l e f f e c t s and t h e amount o f PPIX a c c u m u l a t e d b y P r o t o x - i n h i b i t i n g h e r b i c i d e s (50, 6 4 ) - In cucumber ( F i g . 4 ) , pigweed, and v e l v e t l e a f , we f o u n d a s t r o n g c o r r e l a t i o n b e t w e e n t h e a m o u n t o f P P I X a c c u m u l a t e d i n r e s p o n s e t o a c i f l u o r f e n and t h e amount o f e n s u i n g h e r b i c i d a l d a m a g e ( 4 1 ) . A l s o , t h e r e was a n e x c e l l e n t c o r r e l a t i o n b e t w e e n t h e PPIX and t h e r e s u l t i n g h e r b i c i d a l damage c a u s e d b y a v a r i e t y o f d i p h e n y l e t h e r and o x a d i a z o l e h e r b i c i d e s ( 4 1 ) .


380


10 /xM AF & 1 mM DA O - control A - 1 mM DA • - 10 /iM AF -

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A -

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-

100

c o o

5

6

0

1

2 3

4

5

time (h)

F i g u r e 3. E f f e c t s o f g a b a c u l i n e and d i o x o h e p t a n o i c a c i d (DA) o n e f f i c a c y o f a c i f l u o r f e n (AF) on c e l l u l a r leakage a smeasured by e l e c t r o l y t e i n c r e a s e i n t h e bathing media o f cucumber c o t y l e d o n d i s c s i n c u b a t e d i n t h e v a r i o u s treatment s o l u t i o n s f o r 20 h i n darkness before exposure t o l i g h t (time 0 ) . (Reprinted with permission from ref. 46. Copyright 1988 Academic Press.)

E o o E

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D TJ C o o

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

protoporphyrin IX ( n m o l e s / 5 0 discs)

F i g u r e 4. R e l a t i o n s h i p between c e l l u l a r damage i n g r e e n cucumber c o t y l e d o n d i s c s and PPIX a c c u m u l a t i o n caused b y e x p o s u r e t o v a r i o u s c o n c e n t r a t i o n s o f a c i f l u o r f e n . C e l l u l a r damage was a s s a y e d a s i n F i g . 3, 1 h a f t e r exposure t o l i g h t . PPIX was assayed j u s t before exposure t o l i g h t and a f t e r a 20 h i n c u b a t i o n in darkness. (Reprinted with permission from ref. 41. Copyright 1989 Plant Physiology.)



26.

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Some l a b o r a t o r i e s ( 6 4 - 6 6 ) h a v e f o u n d P C h l i d e t o b e t h e p r i m a r y p o r p h y r i n t o a c c u m u l a t e i n d i p h e n y l e t h e r - t r e a t e d t i s s u e s . In o u r g r e e n c u c u m b e r c o t y l e d o n d i s c s y s t e m , we h a v e f o u n d o n l y P P I X l e v e l s t o be i n c r e a s e d by t h e s e h e r b i c i d e s ( 6 7 ) , h o w e v e r , i n i n t a c t c u c u m b e r s e e d l i n g s ( F i g . 5) a n d t e n t o x i n - a f f e c t e d c u c u m b e r c o t y l e d o n d i s c s ( 6 8 ) we f o u n d d i p h e n y l e t h e r - e n h a n c e d P C h l i d e l e v e l s . P P I X l e v e l s a c c u m u l a t e t o many ( a s much as s e v e r a l h u n d r e d ) t i m e s t h e c o n t r o l l e v e l s i n h e r b i c i d e - t r e a t e d t i s s u e s , w h e r e a s t h e maximum P C h l i d e accumulation i s only f o u r - f o l d that of the c o n t r o l . Since the m e t a b o l i c b l o c k i s a t P r o t o x , t h e r e i s no i n h i b i t i o n o f c o n v e r s i o n o f P C h l i d e t o Chi a f t e r e x p o s u r e t o l i g h t . T h e r e f o r e , i t seems u n l i k e l y that PChlide plays a s i g n i f i c a n t p a r t i n the mechanism of a c t i o n of t h e s e h e r b i c i d e s . Indeed, o f the Chi p r e c u r s o r s assayed, o n l y PPIX s i g n i f i c a n t l y c o r r e l a t e d w i t h h e r b i c i d a l damage c a u s e d by a s e v e r a l d i f f e r e n t acifluorfen/DP/ALA treatment combinations (67). No s i g n i f i c a n t c o r r e l a t i o n s were found with accumulated P C h l i d e , Mg-PPIX, o r Mg-PPIXME. F u r t h e r m o r e , n e i t h e r o f t h e p h o t o d y n a m i c p r e c u r s o r s o f PPIX, c o p r o p o r p h y r i n o g e n nor uroporpophyrinogen ( e x t r a c t e d as c o p r o p o r p h y r i n I I I and u r o p o r p h y r i n I I I ) a c c u m u l a t e i n diphenyl ether herbicide-treated plant tissues (Table IV). A l l of our d a t a a r e c o n s i s t e n t w i t h t h e view t h a t PPIX i s t h e p r i m a r y photodynamic pigment i n v o l v e d i n the mechanism o f a c t i o n o f these herbicides. T a b l e I V . E f f e c t o f 10 /xM a c i f l u o r f e n o n a c c u m u l a t i o n o f P P I X i t s i m m e d i a t e p r e c u r s o r s i n g r e e n c u c u m b e r c o t y l e d o n d i s c s d u r i n g 20 h o f d a r k i n c u b a t i o n . P r e v i o u s l y u n p u b l i s h e d d a t a o f Matsumoto and Duke. Porphyrin Uroporphyrin III Coproporphyrin III P r o t o D o r D h v r i n IX

Control -(nmoles/g 0.016 ± 0 . 0 0 5 0 . 0 0 4 ± 0.001 0 . 0 0 8 ± 0.001

Treated fresh weight)0 . 0 2 5 ± 0..001 0.014 ± 0,,004 0 . 2 0 7 ± 0,,015

Our l a b o r a t o r y (46) and t h a t o f Yanase and Andoh (52) f o u n d t h e h e r b i c i d a l e f f e c t o f ALA t o be s h o r t - l i v e d i n t h e l i g h t c o m p a r e d t o t h a t o f P r o t o x - i n h i b i t i n g h e r b i c i d e s . T h i s i s p r o b a b l y due t o t h e f a c t t h a t P C h l i d e l e v e l s a r e r a p i d l y r e d u c e d by c o n v e r s i o n t o C h i o r by p h o t o d e s t r u c t i o n i n t h e l i g h t i n A L A - t r e a t e d p l a n t t i s s u e s . The h a l f - l i f e o f P C h l i d e i n c u c u m b e r i n l i g h t was a h a l f h o u r o r l e s s ( F i g . 2 ) , whereas, the h a l f - l i f e o f PPIX i n cucumber c o t y l e d o n s d i s c s was m o r e t h a n 2 h ( 4 1 ) . The t o t a l amount o f p o r p h y r i n s t h a t accumulate i n d i p h e n y l e t h e r - t r e a t e d t i s s u e s i s c o n s i d e r a b l y higher than t h a t which accumulates in untreated t i s s u e s (67). Therefore, these h e r b i c i d e s a p p e a r t o i n c r e a s e c a r b o n f l o w i n t o t h i s p a t h w a y . Heme i s k n o w n t o f e e d b a c k i n h i b i t t h i s p a t h w a y a n d P P I X i s r e q u i r e d f o r heme s y n t h e s i s ( 9 ) . F e e d i n g heme t o d i p h e n y l e t h e r - t r e a t e d p l a n t c e l l s r e d u c e s t h e amount o f p o r p h y r i n - c a u s e d l i p i d p e r o x i d a t i o n o f c e l l h o m o g e n a t e s as m e a s u r e d by o x y g e n c o n s u m p t i o n (10). Why P P I X f o r m e d b y n o n e n z y m a t i c o x i d a t i o n o f p r o t o p o r p h y r i n o g e n d o e s n o t i m m e d i a t e l y r e e n t e r t h e c h l o r o p h y l l pathway i s p r o b a b l y due to a requirement f o r accumulation of a threshold level or saturation o f a n o n - a v a i l a b l e pool before r e e n t r y v i a a non-pathway r o u t e can



382


o c c u r . I f s u b s t r a t e c h a n n e l i n g , a s i n heme s y n t h e s i s i n m o u s e mitochondria (69), occurs with conversion of protoporphyrinogen to P P I X , P P I X f o r m e d b y a u t o o x i d a t i o n may b e o u t s i d e t h e n o r m a l m e t a b o l i c c h a n n e l . Thus, o n l y a f t e r a s u f f i c i e n t c o n c e n t r a t i o n o f PPIX b u i l d s up o u t s i d e t h e normal m e t a b o l i c channel w i l l i t r e e n t e r t h e pathway. The s i m u l t a n e o u s k i n e t i c s o f PPIX and P C I i d e accumulation in acifluorfen-treated, yellow (tentoxin-treated) cucumber t i s s u e s s u p p o r t t h i s h y p o t h e s i s ( 6 8 ) . High l e v e l s o f PPIX accumulate before the h e r b i c i d e enhances PCIide accumulation. P r o t o x i s thought t o b e a membrane-bound enzyme; p r o b a b l y i n o r on t h e p l a s t i d e n v e l o p e ( F i g . 6 ) . F l u o r e s c e n c e m i c r o s c o p y o f achlorophyllous t i s s u e s treated with acifluorfen in darkness, results i n s t r o n g p o r p h y r i n f l u o r e s c e n c e i n both p l a s t i d s and c y t o p l a s m , w h e r e a s , f l u o r e s c e n c e was l o c a l i z e d a l m o s t e x c l u s i v e l y i n p l a s t i d s o f u n t r e a t e d c e l l s ( 6 8 ) . PPIX c o n c e n t r a t i o n s were a l m o s t 2 0 0 - f o l d g r e a t e r i n t r e a t e d than untreated t i s s u e s . These data suggest t h a t PPIX l e a k s from p l a s t i d s o r p l a s t i d e n v e l o p e s o f t r e a t e d t i s s u e s and t h a t t h i s l e a k a g e i s i n d e p e n d e n t o f membrane damage due t o l i p i d p e r o x i d a t i o n . T h u s , a s i n o u r p r e v i o u s model (38)> t h e c e l l u l a r s i t e o f a c t i o n o f t h e s e h e r b i c i d e s may b e t h e p l a s t i d e n v e l o p e . The a c t i o n s p e c t r a f o r h e r b i c i d a l damage c a u s e d b y P r o t o x i n h i b i t o r s has a s t r o n g component i n t h e r e d (31, 32, 7 0 ) , w h e r e a s , PPIX a b s o r b s r e l a t i v e l y weakly i n t h i s r e g i o n o f t h e spectrum. Sato (70) has s p e c u l a t e d t h a t , i n g r e e n cucumber t i s s u e , i n a c t i v a t i o n o f p h o t o s y n t h e s i s a s a s e c o n d a r y e f f e c t o f p e r o x i d a t i v e damage r e s u l t s in c h l o r o p h y l l s being i n v o l v e d i n the photodynamic process. Sato (70) found a p o r p h y r i n - p r o t e i n complex i n c h l o r o p l a s t s o f cucumber t i s s u e s t r e a t e d w i t h S-23142, a c h l o r o p h t h a l i m a n a l o g . The complex was n o t f o u n d i n u n t r e a t e d p l a n t s . T h e p r o t e i n was a 6 3 - 6 6 k D m e m b r a n e p r o t e i n a n d P P I X was i d e n t i f i e d a s o n e o f t w o p o r p h y r i n s t h a t complex i t . A p p a r e n t l y t h i s p r o t e i n i s not P r o t o x , s i n c e i t has been d e t e r m i n e d t o h a v e a m o l e c u l a r mass o f 3 6 kD ( H ) . The r o l e o f t h i s p o r p h y r i n - p r o t e i n c o m p l e x i n t h e mode o f a c t i o n o f P r o t o x - i n h i b i t i n g h e r b i c i d e s has y e t t o b e d e t e r m i n e d . A p p l i e d a l o n e , PPIX i s r e l a t i v e l y i n e f f e c t i v e a s a h e r b i c i d e ( 4 6 ) . Even i f i t were h e r b i c i d a l l y e f f e c t i v e , i t s c o s t and t o x i c o l o g i c a l p r o p e r t i e s would p r o b a b l y p r o h i b i t s i t s use. The low a c t i v i t y o f PPIX s u p p l i e d e x o g e n o u s l y c o u l d b e due t o p o o r c e l l u l a r a b s o r p t i o n o f t h i s r e l a t i v e l y complex molecule. The s e l e c t i v i t y o f P r o t o x - i n h i b i t i n g h e r b i c i d e s i s p r o b a b l y due t o many f a c t o r s , i n c l u d i n g : d i f f e r e n t i a l d e g r a d a t i o n o f t h e h e r b i c i d e s (72), d i f f e r e n t i a l s e n s i t i v i t y t o t o x i c oxygen s p e c i e s (15, 16, 73), and, perhaps, d i f f e r e n t i a l s u s c e p t i b i l i t y a t t h e s i t e o f a c t i o n . No d a t a a r e a v a i l a b l e t o s u p p o r t t h e l a t t e r p o s s i b i l i t y . However, mutants o f the u n i c e l l u l a r green a l g a Chlamvdomonas r e i n h a r d t i i . produced bys e l e c t i o n with a Protox i n h i b i t o r a f t e r mutagenesis, were c r o s s r e s i s t a n t to a v a r i e t y o f P r o t o x - i n h i b i t i n g h e r b i c i d e s , but not t o PSII i n h i b i t o r s or t o paraquat, i n d i c a t i n g resistance a the molecular s i t e of action (70). Summary and

Conclusions

P o r p h y r i n s c a n n o t b e used d i r e c t l y as p h o t o s e n s i t i z i n g h e r b i c i d e s b e c a u s e o f t h e c o s t and p o s s i b l e t o x i c o l o g i c a l d a n g e r s . However, a



D U K E ET AL.

383


2I

. 10

• 100 Acifluorfen (/zM)

1

— 1000

F i g u r e 5. E f f e c t o f v a r i o u s c o n c e n t r a t i o n s o f a c i f l u o r f e n o n PPIX and P C h l i d e a c c u m u l a t i o n i n c o t y l e d o n s o f i n t a c t , g r e e n cucumber s e e d l i n g s . The p l a n t s were sprayed t o r u n o f f w i t h t h e h e r b i c i d e i n 0.5 % T w e e n 8 0 ( v / v ) a n d i n c u b a t e d i n d a r k n e s s f o r 12 h b e f o r e a s s a y s w e r e c o n d u c t e d . Previously unpublished data o f B e c e r r i l and Duke. INSIDE /

-4—» |

ALA

PROTOXJ

^

HERBICIDE INHIBITION

O

PLASTID ENVELOPE

MG-CHELATASE ~

S

W

E

PROTOPORPHYRINOGEN IX AUTOOXIDATION J ^ — 0 PPIX

2

SINGLET OXYGEN LIPID PEROXIDATION

I MEMBRANE DESTRUCTION

F i g u r e 6. H y p o t h e t i c a l m o d e l o f t h e p h y t o t o x i c m e c h a n i s m o f action f o r herbicides that inhibit Protox. Protoporphyrinogen which accumulated as a r e s u l t o f Protox i n h i b i t i o n l e a v e s t h e membrane-bound, c h a n n e l e d p o r p h y r i n pathway and i s a u t o o x i d i z e d to PPIX. Redrawn from ( 5 9 ) .



384


v a r i e t y o f b o t h n a t u r a l l y - o c c u r r i n g and s y n t h e t i c compounds can e f f e c t i v e l y s t i m u l a t e p l a n t s t o s y n t h e s i z e l e t h a l amounts o f photosensitizing porphyrins. ALA, a p o r p h y r i n p r e c u r s o r , i s r e a d i l y a b s o r b e d and c o n v e r t e d t o p o r p h y r i n s i n p l a n t t i s s u e s . Compounds t h a t modulate the c h l o r o p h y l l s y n t h e s i s pathway can b e used t o s y n e r g i z e ALA's e f f e c t s onporphyrin accumulation. Several classes o f c o m m e r c i a l h e r b i c i d e s ( o x a d i a z o l e s , /V-phenyl i m i d e s , a n d diphenyl ethers) i n h i b i t Protox, causing deregulation o f the p o r p h y r i n p a t h w a y and a u t o o x i d a t i o n o f p r o t o p o r p h y r i n o g e n t o f o r m p h o t o s e n s i t i z i n g , d e s t r u c t i v e l e v e l s o f PPIX. These compounds are much more e f f i c i e n t a s h e r b i c i d e s t h a n ALA p l u s m o d u l a t o r s . Future r e s e a r c h o n 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 s between these h e r b i c i d e s and i n h i b i t i o n o f P r o t o x c o u l d i m p r o v e t h e a c t i v i t y a n d / o r s e l e c t i v i t y of these herbicides. Acknowledgments We t h a n k Rohm a n d H a a s C o . f o r p r o v i d i n g t e c h n i c a l g r a d e c h e m i c a l s used i n our s t u d i e s d e s c r i b e d h e r e . The t e c h n i c a l a s s i s t a n c e o f Al Lane was i n v a l u a b l e i n the our e x p e r i m e n t s r e p o r t e d h e r e . J . L . W i c k l i f f , S.H. D u k e , a n d G.W. E l z e n p r o v i d e d h e l p f u l c r i t i c i s m s . T h i s w o r k was s u p p o r t e d i n p a r t b y t h e F u l b r i g h t / M i n i s t r y o f E d u c a t i o n and S c i e n c e , M a d r i d , S p a i n and t h e J a p a n e s e M i n i s t r y o f E d u c a t i o n , S c i e n c e , a n d C u l t u r e . Ryo S a t o g e n e r o u s l y p r o v i d e d a c o p y o f h i s Ph.D. Thesis. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

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