Biochemical Responses Induced by Herbicides - American Chemical

8 Proposed Site(s) of Action of New Diphenyl Ether Herbicides GREGORY L . ORR

Downloaded by UNIV OF ARIZONA on May 3, 2017 | http://pubs.acs.org Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch008

Colorado State University, Department of Botany and Plant Pathology, Fort Collins, CO 80523 F. DANA HESS Purdue University, Department of Botany and Plant Pathology, West Lafayette, IN 47907

The new diphenylether (DPE) herbicides, e.g., acifluorfen-methyl {methyl 5-[2-chloro-4(trifluoromethyl)phenoxy]-2-nitrobenzoate} are proposed to be activated in light by carotenoids and then initiate radical chain reactions with membrane fatty acids. This hypothesis is based on the following: (a) DPE's are active in green and etiolated tissue; (b) damage does not occur after inhibition of carotenoid biosynthesis by fluridone {1-methyl-3-phenyl-5-[3-(trifluoromethyl)phenyl]4(1H)-pyridinone}; (c) incurred damage requires light and oxygen; (d) injury is expressed as a general increase in membrane permeability 10 to 15 min following light-activation; (e) membrane disruption can be verified by electron microscopy; (f) ultrastructural analysis revealed early injury to the chloroplast envelope; (g) ethane, ethylene, and thiobarbituric acid-reacting materials can be detected after treatment; (h) pretreatment with α-tocopherol can protect against DPE injury. To determine the p h y s i o l o g i c a l and biochemical mechanism of a c t i o n of a given group of h e r b i c i d e s , the primary s i t e of a c t i o n must be i d e n t i f i e d . This i s often d i f f i c u l t when the p h y s i o l o g i c a l e f f e c t requires long periods of time to detect or i s induced only by high concentrations. There are now s e v e r a l DPE s a v a i l a b l e that e x h i b i t a r a p i d and r e l a t i v e l y high degree of h e r b i c i d a l a c t i v i t y . Thus, following the development of bioassays capable of detecting subtle q u a n t i t a t i v e d i f f e r e n c e s between b i o l o g i c a l responses induced by these h e r b i c i d e s , i t became p o s s i b l e to study the primary h e r b i c i d a l mechanism. In the following d i s c u s s i o n , much information concerning the biochemical mechanism of a c t i o n of DPE's w i l l be from research on a c i f l u o r f e n - m e t h y l (AFM). However, we w i l l also review recent developments from studies of other compounds 1

0097-6156/82/0181-0131$05.25/0 © 1982 American Chemical Society Moreland et al.; Biochemical Responses Induced by Herbicides ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

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i n t h i s c l a s s , e.g., o x y f l u o r f e n [2-chloro-l-(3-ethoxy-4nitrophenoxy)-4-(trifluoromethyl)benzene], bifenox [methyl 5-(2,4dichlorophenoxy)-2-nitrobenzoate]. F i n a l l y , we s h a l l examine, from a t h e o r e t i c a l viewpoint, proposals f o r future research and speculate on p o s s i b l e outcomes and i n t e r p r e t a t i o n s . Structure-activity Relationships


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The r a t e of e f f l u x of ^^Rb from excised and preloaded cucumber (Cucumis s a t i v u s L.) cotyledons (Figure 1), was used to study 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 f o r s e v e r a l DPE h e r b i c i d e s (1). I t was apparent an R-group ortho to the n i t r o group on the £-nitrophenyl moiety was required f o r short-term (Rb e f f l u x from excised and preloaded cucumber cotyledons, AFM i n j u r y was detected at concentrations as low as 10 nM (_1 ). A l s o , because AFM has an absolute requirement of l i g h t f o r expression of h e r b i c i d a l a c t i v i t y , plant t i s s u e s can be pretreated i n darkness without injury. Then, f o l l o w i n g l i g h t - a c t i v a t i o n , damage can be detected i n r e l a t i v e l y short time periods (10 to 15 min) (1 ). We b e l i e v e these observations are i n d i c a t i v e of i t s primary biochemical mechanism of a c t i o n .


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L i g h t Requirement and Membrane D i s r u p t i o n . As mentioned above, DPE s have an absolute requirement of l i g h t f o r a c t i v i t y 3-12). The l i g h t - a c t i v a t e d form of the AFM molecule apparently has a r e l a t i v e l y short h a l f - l i f e , because f u r t h e r i n j u r y can be prevented by r e t u r n i n g the t i s s u e to darkness ( 1_). By decreasing l i g h t q u a n t i t y , the e f f e c t of AFM i s delayed, although magnitudes of the responses at d i f f e r e n t i n t e n s i t i e s are n e a r l y equal (_1 ). The same observations were made with o x y f l u o r f e n (10). DPE treatment appears to d i s r u p t c e l l membranes. Following l i g h t - a c t i v a t i o n o f AFM, i n j u r y can be detected as: (a) a general increase i n e l e c t r o l y t i c c o n d u c t i v i t y of the e x t e r n a l bathing s o l u t i o n o f treated t i s s u e s ; (b) e f f l u x of inorganic ions such as R b , C l " ( F i g u r e 2A), and C a ( F i g u r e 2B); (c) e f f l u x of a n e u t r a l organic molecule, 3-0-methy1-[^C]glucose (Figure 2C); and (d) e f f l u x of a charged organic molecule, [^C]methylamine (Figure 2D). 1

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P o t e n t i a l Resistance Mechanism(s). In studies designed to e l u c i d a t e the mechanism of a c t i o n of AFM, cucumber seedlings were grown i n the dark f o r 6 or 7 days. Cotyledons were excised, greened, and loaded with 8*>Rb £ i l i g h t (75 uE m~2 s""*) for 24 h. The cotyledons were subsequently exposed to h e r b i c i d e i n high l i g h t (600 μΕ m~2 s ~ l ) and found to be i n j u r e d r a p i d l y by micromolar concentrations of AFM ( 1_). However, excised cotyledons allowed to green i n low l i g h t f o r 48 h, before high l i g h t and h e r b i c i d e treatment, were not i n j u r e d . Resistance may be a t t r i b u t e d to decreased p e n e t r a t i o n r e s u l t i n g from an +

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Moreland et al.; Biochemical Responses Induced by Herbicides ACS Symposium Series; American Chemical Society: Washington, DC, 1982.


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Rb a f t e r 6 to 8 h of exposure. Because tocopherols are known to be synthesized by plants a f t e r exposure to l i g h t (15), t h i s short term p r o t e c t i o n against AFM may be caused by increased q u a n t i t i e s of r a d i c a l quenching compounds. This p r o t e c t i v e mechanism i s e s s e n t i a l f o r plant l i f e i n the presence of l i g h t and oxygen, which create the p o t e n t i a l f o r t i s s u e damage by f r e e r a d i c a l s . Therefore, treatment of the alga with p i c r y l h y d r a z y l may decrease the time required to observe h e r b i c i d a l i n j u r y symptoms. Once a-T quenches a r a d i c a l and becomes an a r o y l r a d i c a l , there i s evidence i t i s reconverted ( r e p a i r e d ) to a-T in v i v o by t h i o l s (18). Therefore, i t may be p o s s i b l e to decrease the i n d u c t i o n period f o r membrane damage introduced by i n v i v o a-T through d e s t r u c t i o n of g l u t a t h i o n e (a t h i o l thought to be involved i n the r e p a i r mechanism). This could be accomplished by p r e t r e a t i n g cucumber cotyledons greened for 48 h with a compound, such as d i e t h y l maleate, which has been shown to destroy g l u t a t h i o n e (_19). Therefore, by decreasing the l e v e l s of g l u t a t h i o n e , t i s s u e would lose the a b i l i t y to prevent the formation of l i p i d peroxides or to d e t o x i f y l i p i d peroxides once formed. Any compound i n c r e a s i n g the c e l l u l a r content of t h i o l s (e.g., g l u t a t h i o n e ) would be a candidate f o r an antidote of AFM. One p o s s i b l e p r o t e c t a n t against DPE i n j u r y to corn (Zea mays L.) i s R-25788 ( N , N - d i a l l y l - 2 , 2 - d i c h l o r o a c e t a m i d e ) . Tocopherols are unsuited f o r f i e l d use because they are degraded i n l i g h t and a i r (20). +

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P o s s i b l e DPE E f f e c t s on P h e n o l i c s . Phenolic l e v e l s were s i g n i f i c a n t l y increased i n AFM treated t i s s u e (unpublished r e s u l t s ) . This may be an i n d i c a t i o n of a wound response. However, i t i s a l s o p o s s i b l e these h e r b i c i d e s may r e g u l a t e the a c t i v i t y of one of the l i g h t - a c t i v a t e d enzymes involved i n phenolic a c i d s y n t h e s i s . The r e s u l t a n t increase i n high l e v e l s of f r e e r a d i c a l intermediates known to occur i n these pathways, could be the u l t i m a t e cause of c e l l u l a r destruction. Treatment of cucumber with f l u o r o d i f e n [j>-nitrophenyl (α,α,α-trif luoro-2-nitro-£-tolyl)urea] has been shown to increase phenylalanine ammonia lyase (PAL) a c t i v i t y i n v i v o (21). L i g h t - a c t i v a t i n g Mechanism(s) 1

The a c t i v a t i o n of DPE s by l i g h t appears to r e q u i r e n e i t h e r c h l o r o p h y l l nor photosynthetic e l e c t r o n transport ( 3 ) . AFM w i l l induce h e r b i c i d a l i n j u r y i n green and e t i o l a t e d cucumber cotyledons i n the presence of DCMU [ 3 - ( 3 , 4 - d i c h l o r o p h e n y l ) - l , 1-dimethylurea] and DBMIB (2,5-dibromo-3-methyl-6-isopropyl-£benzoquinone), i n v i v o i n h i b i t o r s of n o n - c y c l i c and c y c l i c photosynthetic e l e c t r o n t r a n s p o r t , r e s p e c t i v e l y . Recently, Bugg et al_. (22) obtained evidence that i n d i c a t e d the s i t e of photosynthetic e l e c t r o n transport i n h i b i t i o n by n i t r o f l u o r f e n [2-rchloro-l-(4-nitrophenoxy)-4-(trifluoromethyl)benzene], was a s s o c i a t e d with the plastoquinone-Cyt f region between PS I and PS I I . This i s i n agreement with previous research conducted on the mechanism of a c t i o n of DPE s (L3, 23-27). However, i n view of the above data from cucumber, these r e s u l t s are probably not i n d i c a t i v e of the primary h e r b i c i d a l s i t e of a c t i o n . A f t e r 2 to 3 h, e t i o l a t e d t i s s u e s exposed to AFM and high l i g h t (600 μΕ m~2 s~*) show t y p i c a l i n j u r y symptoms and s i g n i f i c a n t increases i n the r a t e of ^ R b e f f l u x (3), The c h o r o p h y l l content i n the e t i o l a t e d c o n t r o l t i s s u e s , even a f t e r 4 h i n l i g h t , was l e s s than 1% of the green t i s s u e s . Although t h i s a n a l y s i s i n d i c a t e d no q u a n t i t a t i v e l o s s of pigments, t i s s u e bleaching was apparent ( i . e . , the o x i d a t i o n and subsequent l o s s of pigments was v i s u a l l y evident on the periphery of the l e a f ) . E t i o l a t e d cucumber cotyledons examined with the e l e c t r o n microscope revealed only e t i o p l a s t s with large p r o l a m e l l a r bodies. 1

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P o t e n t i a l Involvement of Carotenoids. The pigment involved i n the l i g h t - a c t i v a t i n g mechanism of the DPE molecule may be a carotenoid (_5, 6 7)· The absorption spectrum of a crude pigment e x t r a c t taken from e t i o l a t e d cucumber cotyledons very c l o s e l y matches that of the xanthophyll l u t e i n (28), the primary pigment present i n e t i o l a t e d cucumber cotyledons (29), Other carotenoids (e.g., carotene, probably β-carotene, and one or more xanthophylls) are a l s o present (unpublished r e s u l t s ) . 3



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Obtaining a w e l l executed a c t i o n spectrum should make i t p o s s i b l e to match wavelengths most e f f i c i e n t i n i n i t i a t i n g the h e r b i c i d a l response with those of a s p e c i f i c plant pigment. Results from c h l o r o p h y l l o u s mutants of r i c e (Oryza s a t i v a L.) ( 5 ) , corn, and soybeans ( G l y c i n e max L. Merr.) ( 4 ) , support the contention that a carotenoid i s involved i n a c t i v a t i n g the DPE molecule. Yellow and green mutants were e q u a l l y s u s c e p t i b l e to h e r b i c i d a l i n j u r y ; however, the a l b i n o mutants were r e s i s t a n t . Cucumber seedlings pretreated with f l u r i d o n e , a known carotenoid b i o s y n t h e s i s i n h i b i t o r (30), were a l s o r e s i s t a n t (3). By q u a n t i t a t i v e l y examining h e r b i c i d a l a c t i v i t y d i f f e r e n c e s i n t i s s u e s treated with compounds (e.g., CPTA [ 2 - ( 4 - c h l o r o p h e n y l t h i o ) - t r i e t h y l a m i n e h y d r o c h l o r i d e ] , onium compounds, etc.) capable of r e g u l a t i n g the r e l a t i v e amounts of carotenoids present (31^, 32, 33), the pigment(s) involved i n the l i g h t - a c t i v a t i n g mechanism may be i d e n t i f i e d . Algal carotenoid mutants or various Neurospora species known to have s p e c i f i c carotenoids may a l s o a i d i n the study of DPE-pigment interactions. DPE-Carotenoid I n t e r a c t i o n s i n V i t r o . Observations of s p e c t r a l changes o c c u r r i n g upon i l l u m i n a t i o n of DPE-treated crude pigment e x t r a c t s have been attempted (34, 35). These experiments were s u c c e s s f u l only with n i t r o f e n (35). Nitrofen attacks the 4th f r e e r i n g of the c h l o r o p h y l l molecule i n methanol and carbon t e t r a c h l o r i d e , thus decreasing absorbance i n the v i s i b l e region (3>5). There was an apparent a s s o c i a t i o n between the s o l u t e and solvent molecules i n p y r i d i n e that e f f e c t i v e l y slowed the r e d u c t i o n i n absorbance. Because l i g h t - a c t i v a t i o n of the DPE molecule i n v i v o does not r e q u i r e c h l o r o p h y l l , s i m i l a r studies were conducted with carotenoids (unpublished r e s u l t s , 34). To date, none of these experiments have been s u c c e s s f u l . P r o t e i n - c a r o t e n o i d complexes destroyed by the organic e x t r a c t i o n procedures used might be required f o r h e r b i c i d a l a c t i v a t i o n . For carotenoids to f u n c t i o n p r o p e r l y they may e x i s t as a protein-pigment complex imbedded w i t h i n the hydrophobic matrix of c e l l membranes. The involvement of t h i s complex i s p a r t l y based on the d i f f i c u l t y i n demonstrating carotenoid fluorescence (37). One of the functions of carotenoids i n photosynthesis i s the t r a n s f e r of energy to c h l o r o p h y l l s . Perhaps, DPE s d i r e c t l y or i n d i r e c t l y i n t e r c e p t the e l e c t r o n or energy t r a n s f e r s normally o c c u r r i n g between carotenoids and c h l o r o p h y l l s (_38, 39^, 40). 1

The outer c h i o r o p l a s t and e t i o p l a s t envelope contains carotenoids (36). By i l l u m i n a t i n g t h i s s u b c e l l u l a r f r a c t i o n at various wavelengths i n a spectrophotometer and o b t a i n i n g a d i f f e r e n c e spectra i n the presence of DPE h e r b i c i d e s , the 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 pigment can be evaluated. The wavelength at which the i n t e r a c t i o n i s observed w i l l i m p l i c a t e the pigment i n v o l v e d .


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Carotenoids vs. F l a v i n s . To date, c o n s i d e r a b l e evidence supports the contention that carotenoids are involved i n the l i g h t - a c t i v a t i n g mechanism of DPE s (3, 4, 5_, 7-10). From a purely p h y s i c a l view, a f l a v i n can be perceived as a more l i k e l y candidate for a c t i v a t i o n of DPE s than can a c a r o t e n o i d . The f o l l o w i n g molecular p r o p e r t i e s that favor r i b o f l a v i n over β-carotene are from a review by Song, Moore, and Sun (37): (a) R i b o f l a v i n i s capable of i n t e n s e l y f l u o r e s c i n g , whereas the fluorescence of β-carotene i s weak and anomolous. (b) R i b o f l a v i n w i l l phosphoresce i n the v i s i b l e r e g i o n , whereas β-carotene w i l l not. (c) (η, π * ) s t a t e s are a v a i l a b l e f o r r i b o f l a v i n but, not f o r β-carotene. (d) R i b o f l a v i n w i l l decompose to y i e l d photoproducts and can a l s o i n i t i a t e various i n t e r m o l e c u l a r photooxidations. β-carotene can undergo c i s Φ trans photoisomerizations but i s incapable of i n t e r m o l e c u l a r photooxidations. (e) R i b o f l a v i n w i l l generate s i n g l e t oxygen by t r i p l e t energy t r a n s f e r and y i e l d hydrogen peroxide with subsequent r e s t o r a t i o n of f l a v i n . β-carotene quenches s i n g l e t oxygen and carotene i s consumed, ( f ) R i b o f l a v i n has the photochemical a b i l i t y to form r a d i c a l s , whereas β-carotene probably does not. Examination of e f f e c t s of DPE s on t i s s u e s [e.g., cucumber hypocotyl (41_) ] with r e l a t i v e l y high r i b o f l a v i n to carotenoid r a t i o s should be i n f o r m a t i v e . 1


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Spin-trapping Experiments. The paraquat ( 1 , 1 ' - d i m e t h y l ^ , 4 * - b i p y r i d i n i u m ion) r a d i c a l generated f o l l o w i n g r e d u c t i o n by PS I y i e l d s superoxide anions i n the presence of oxygen (20. The superoxide r a d i c a l was detected with s p i n - t r a p p i n g techniques and ESR spectroscopy (42). In these experiments, i s o l a t e d c h l o r o p l a s t s were i l l u m i n a t e d i n the presence of a spin adduct and paraquat. AFM-induced i n j u r y a l s o appears to r e q u i r e oxygen. However, pretreatment of c h l o r o p l a s t s with DABCO [ 1 , 4 - d i a z o b i c y c l o (2,2,2)-octane], a widely used quenching agent of s i n g l e t oxygen d i d not prevent damage by o x y f l u o r f e n (43). Although superoxide may not be involved i n DPE i n j u r y , these same s p i n - t r a p p i n g techniques may prove u s e f u l i n i d e n t i f y i n g the types of r e a c t i o n s o c c u r r i n g i n i l l u m i n a t e d c h l o r o p l a s t s or e t i o p l a s t s treated with DPE's. Reaction Mechanism(s) of DPE's. DPE's could p o t e n t i a l l y be degraded during l i g h t - a c t i v a t i o n r e a c t i o n s or r a d i c a l r e a c t i o n sequences. I d e n t i f i c a t i o n of DPE breakdown products from s u s c e p t i b l e plants kept i n the dark or i n the l i g h t would test this p o s s i b i l i t y . I f the products obtained under these two c o n d i t i o n s are d i f f e r e n t , i d e n t i f i c a t i o n of these products may a s s i s t i n i d e n t i f y i n g the l i g h t - a c t i v a t i o n mechanism. A free r a d i c a l mechanism was proposed f o r the expression of p h y t o x i c i t y by i o x y n i l ( 4 - h y d r o x y - 3 , 5 - d i i o d o b e n z o n i t r i l e ) (44). I o x y n i l undergoes r a d i c a l r e a c t i o n s with benzene upon i l l u m i n a t i o n with UV l i g h t . I d e n t i f i c a t i o n of the r e a c t i o n


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products by t h i n - l a y e r chromatography determined the types of r a d i c a l r e a c t i o n s o c c u r r i n g under these c o n d i t i o n s . Similar experiments may y i e l d information concerning l i g h t r e a c t i o n s of DPE's in v i v o .


I n j u r y to C e l l Membranes According to the fluid-mosaic model of membrane s t r u c t u r e (4^5), c e l l membranes c o n s i s t of a f l u i d phospholipid b i l a y e r . Embedded w i t h i n t h i s b i l a y e r are g l o b u l a r p r o t e i n s e s s e n t i a l to membrane f u n c t i o n . A large c l a s s of phospholipids present i n membranes are phosphoglycerides ( L5). The f a t t y a c i d i n the number 2 p o s i t i o n i s often unsaturated (46). In p l a n t s , the unsaturated f a t t y a c i d i s f r e q u e n t l y l i n o l e n i c a c i d (15); with 3 double bonds (18:3 9, > ! 5 ) . Δ

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L i p o p h i l i c Free R a d i c a l Reactions. The d i v i n y l methane s t r u c t u r e present i n PUFA i s s u s c e p t i b l e to hydrogen a b s t r a c t i o n with subsequent formation of a f a i r l y s t a b l e f r e e r a d i c a l (47). In the presence of the proper i n i t i a t i o n f a c t o r s , these r e a c t i o n s can be induced w i t h i n the hydrophobic matrix of the membrane (48). Once these r e a c t i o n s have started, there can be considerable c e l l damage (49). The o r d e r l y array of f a t t y acids present i n membranes permits maximum i n t e r a c t i o n of the i n d i v i d u a l molecules and thus, r e a d i l y propagates f r e e r a d i c a l reactions. Propagation r e a c t i o n s occur to the greatest extent when oxygen i s abundant (48); however, the rates of such r e a c t i o n s are p r o p o r t i o n a l to oxygen content only at low p a r t i a l pressures. To t e s t whether DPE's might be involved i n i n i t i a t i n g propagation r e a c t i o n s , a c t i v i t y of AFM was examined i n t i s s u e s kept i n an atmosphere of e i t h e r oxygen or n i t r o g e n (3). The a c t i v i t y o f AFM was s i g n i f i c a n t l y reduced when t i s s u e s were maintained i n a n i t r o g e n atmosphere. A r e l a t e d compound, f l u o r o d i f e n , was a l s o found to r e q u i r e oxygen and l i g h t f o r maximum a c t i v i t y (21). L i p i d Analyses. V e r i f i c a t i o n o f PUFA r a d i c a l chain r e a c t i o n s induced by l i g h t - a c t i v a t e d DPE molecules can be made by examining changes i n a polar l i p i d f r a c t i o n c o l l e c t e d from i n j u r e d t i s s u e s . R a d i c a l chain r e a c t i o n s can be terminated through c r o s s - r e a c t i o n s of the endoperoxides formed (48). Therefore, e i t h e r an appearance of l i p i d polymers or disappearance of c e r t a i n phospholipids should occur. I f only the PUFA moieties are involved i n these r e a c t i o n s , a l o s s of PUFA i n the DPE-treated t i s s u e or an increase i n the r a t i o o f saturated to unsaturated f a t t y acids should occur. E l e c t r o n micrographs from cucumber cotyledons r e v e a l the presence of large numbers of l i p i d bodies (oleosomes). L i p i d s i n oleosomes are comprised mostly, i f not e n t i r e l y , of t r i a c y l g l y c e r o l s (46). When studying DPE i n j u r y symptoms i t i s


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important to examine changes i n a polar l i p i d f r a c t i o n and not a t o t a l l i p i d e x t r a c t . The reasons are t h r e e f o l d . F i r s t , i f the primary s i t e of a c t i o n of DPE s r e s i d e s i n c e l l membranes, i t i s l o g i c a l to look f o r changes only i n the s t r u c t u r a l components of those membranes; the polar phospholipids. Second, i n t e r a c t i o n s of the l i g h t - a c t i v a t e d DPE molecule with PUFA's i n l i p i d bodies would probably not r e s u l t i n any r e a l c e l l u l a r damage. P r e l i m i n a r y experiments with AFM i n d i c a t e there i s l i t t l e i n t e r a c t i o n between the h e r b i c i d e and fats of oleosomes (unpublished r e s u l t s ) . T h i r d and most important, the high q u a n t i t i e s of n e u t r a l l i p i d s i n oleosomes may mask the d e t e c t i o n of any h e r b i c i d e e f f e c t on membrane l i p i d s . Even i f s i g n i f i c a n t changes i n phospholipids cannot be detected, the p o s s i b i l i t y of r a d i c a l reactions being i n i t i a t e d as a r e s u l t of DPE-treatment cannot be discounted. Small perturbations i n the f a t t y a c i d moieties would l i k e l y r e s u l t i n large changes in membrane permeability c h a r a c t e r i s t i c s observed. This would r e s u l t i n c e l l u l a r decompartmentalization and death.


1

Products of L i p i d Peroxide Decomposition. In view of the p o t e n t i a l problems with d i r e c t l i p i d a n a l y s i s , a simpler and more s e n s i t i v e assay, the d e t e c t i o n of t h i o b a r b i t u r i c a c i d - r e a c t i n g m a t e r i a l s (TBARM), was used to detect DPE i n j u r y to membranes Ο). Various n o n - v o l a t i l e precursors of malonyl dialdehyde (MDA) are products of l i p i d peroxide decomposition in v i t r o and in v i v o (48). These products r e s u l t from f r e e r a d i c a l chain r e a c t i o n s i n v o l v i n g plant PUFA with three double bonds; e.g. l i n o l e n i c acid (50). The MDA-precursor materials can be detected using a c o l o r i m e t r i c r e a c t i o n with t h i o b a r b i t u r i c a c i d (TBA) (50-53). Detection of TBARM f o l l o w i n g the l i g h t - a c t i v a t i o n of AFM i n treated cucumber cotyledons i n d i c a t e s there i s d i r e c t p h y s i c a l damage to the membranes (3). Plasma membrane, tonoplast, and c h l o r o p l a s t envelope d i s r u p t i o n has been v e r i f i e d by e l e c t r o n microscopy (Figure 3). More important, the presence of TBARM i n damaged t i s s u e provides the f i r s t r e a l evidence that i n j u r y to the membranes r e s u l t s from the formation of h i g h l y r e a c t i v e and d e s t r u c t i v e l i p o p h i l i c free r a d i c a l s _52, 54-58). Because fluorescence measures various products of l i p i d peroxidation (_59), t h i s technique could be used to f u r t h e r examine DPE-induced i n j u r y to c e l l membranes. The e v o l u t i o n of short chain hydrocarbon gases (SCHG) can a l s o be used as an i n d i c a t i o n of l i p i d peroxidation (Jx4, 60, 61, 62). Kunert and Btfger (43) detected ethane e v o l u t i o n from oxy f l u o r fen-treated Scenedesmus c e l l s w i t h i n 1 to 3 h. I s o l a t e d c h l o r o p l a s t s evolve ethane w i t h i n 15 to 30 min f o l l o w i n g treatment and l i g h t - a c t i v a t i o n of o x y f l u o r f e n . Cucumber cotyledons treated with 1 μΜ AFM f o r 6 h i n darkness showed a s i g n i f i c a n t increase i n the amount of ethylene produced 30 min a f t e r exposure to l i g h t (unpublished r e s u l t s ) . Longer chain hydrocarbons (e.g., pentane) have not been detected (unpublished




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Figure 3. Electron micrographs of cells from greened cucumber cotyledons pretreated with 1 Μ AFM for 6 h prior to light (600 μΕ/m s, PAR) exposure. μ

2

A, Ultrastructure prior to light exposure (3). Glutaraldehyde plus osmium tetroxide fixation. Magnification = 20,000χ. Β, Cellular disruption after a 45-min light exposure. Potassium permanganate fixa tion. Magnification — 8,000χ.



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Figure 3. (continued) Electron micrographs of cells from greened cucumber co tyledons pretreated with 1 μΜ AFM for 6 h prior to light (600 μΕ/m s, PAR) exposure. 2

C, Vesiculation of plasmalemma after a 60-min light exposure. Glutaraldehyde plus osmium tetroxide fixation. Magnification = 21,000χ. D, Cytoplasmic damage following tonoplast disruption. Potassium permanganate fixation. Magnification — 5,000χ.



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r e s u l t s , 43). TBARM detected i n AFM-treated cucumber cotyledons supports the contention that SCHG evolved from these t i s s u e s was the r e s u l t of d i r e c t i n t e r a c t i o n of h e r b i c i d e and PUFA, and not merely an i n d i c a t i o n of c e l l u l a r death. Spin-trapping Experiments. As mentioned e a r l i e r , some of the r a d i c a l chain r e a c t i o n s i n i t i a t e d by l i g h t - a c t i v a t e d DPE s can terminate with the subsequent formation of PUFA polymers (48). Spin-trapping techniques have been used to study r e a c t i o n s of lipoxygenase with l i n o l e i c acid (63). I t was proposed that the formation of dimeric l i n o l e i c a c i d r e q u i r e d the involvement of h y d r o p e r o x y l i n o l e i c a c i d (64). The ESR spectrum from the i n t e r a c t i o n s of the PUFA r a d i c a l and the s p i n - t r a p i n d i c a t e d t h i s involvement occurred. Similar spin-trapping techniques could be used to i n v e s t i g a t e the p o s s i b i l i t y that DPE s may u l t i m a t e l y induce formation of various f a t t y acid dimers (e.g., dimers of l i n o l e n i c a c i d ) . Formation of such polymers should d r a m a t i c a l l y a f f e c t membrane f u n c t i o n .


1

1

Involvement o f Polyunsaturated F a t t y A c i d s . To f u r t h e r t e s t the importance of PUFA, the r e l a t i v e a c t i v i t y of DPE s f o l l o w i n g t i s s u e pretreatment with drugs (e.g., s u b s t i t u t e d pyridazinones) known to change the degree of u n s a t u r a t i o n w i t h i n the membrane (65) should be s t u d i e d . Compounds i n c r e a s i n g the degree of s a t u r a t i o n might a f f o r d the t i s s u e some p r o t e c t i o n against DPE i n j u r y , whereas, compounds i n c r e a s i n g the degree of u n s a t u r a t i o n should make the t i s s u e more s u s c e p t i b l e . The r e l a t i v e amounts of saturated and unsaturated f a t t y acids i n the membrane can be a l t e r e d by maintaining t i s s u e i n d i f f e r e n t temperature regimes (46). Organisms growing i n c o o l climates have increased l e v e l s of unsaturated f a t t y acids i n t h e i r membranes. As a r e s u l t , t h e i r membranes are more f l u i d (the melting point of phospholipids decreases with i n c r e a s i n g numbers of unsaturated f a t t y acid components). These t i s s u e s should be more s u s c e p t i b l e to DPE-injury. E t i o l a t e d cucumber cotyledons showed dramatic increases i n f a t t y acid desaturase a c t i v i t y f o l l o w i n g exposure to l i g h t (66), Consequently, green cotyledons have s i g n i f i c a n t l y more unsaturated f a t t y acids than e t i o l a t e d cotyledons. However, AFM appears to be as a c t i v e i n e t i o l a t e d t i s s u e as i n green t i s s u e s Ο). This can be explained by increased l e v e l s of r a d i c a l scavengers i n greened t i s s u e . 1

E f f e c t s of A n t i o x i d a n t s . The most important evidence i m p l i c a t i n g f r e e r a d i c a l s i n DPE membrane i n j u r y i s the a b i l i t y to p r o t e c t against damage with a known r a d i c a l scavenger. Compounds with the p o t e n t i a l a b i l i t y to provide l i m i t e d p r o t e c t i o n against DPE i n j u r y by f r e e r a d i c a l scavenging and other mechanisms include BHA ( b u t y l a t e d hydroxyanisole), BHT (butylated hydroxytoluene), EDU { N - [ 2 - ( 2 - o x o - l - i m i d a z o l i d i n y l ) -


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New

Diphenyl

Ether

Herbicides

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1

ethyl]-N -phenylurea}, DPPD (N,N -dime thy l-£-phenylenediamine), DABCO, diphenylamine, sodium benzoate, SOD (superoxide dismutase) and α - τ . However, except f o r a-T ( F i g u r e 4 ) , the compounds tested have a f f o r d e d l i t t l e or no p r o t e c t i o n (unpublished data, 43). The i n a b i l i t y of DABCO and SOD to reduce o x y f l u o r f e n i n j u r y i n d i c a t e s s i n g l e t oxygen i s not the d e s t r u c t i v e agent (43). However, the a b i l i t y of a-T, a known i n v i v o scavenger of l i p o p h i l i c free r a d i c a l s , to p r o t e c t against AFM i n j u r y suggests the h e r b i c i d e i n i t i a t e s a f r e e r a d i c a l chain r e a c t i o n with PUFA moieties (e.g., l i n o l e n i c a c i d ) of the phospholipid molecules making up c e l l membranes Ο ) . In our experiments, l i m i t e d p r o t e c t i o n of AFM-induced i n j u r y to cucumber cotyledons was obtained with BHA and BHT. These two compounds showed some s y n e r g i s t i c c h a r a c t e r i s t i c s . However, the concentrations necessary f o r p r o t e c t i o n were high (400 μΜ) and caused some i n j u r y to the c o n t r o l s . The concentrations of BHA and BHT needed to p r o t e c t the t i s s u e should be higher than f o r a-T. BHA and BHT each have an a n t i o x i d a n t s t o i c h i o m e t r i c f a c t o r (n) equal to 1, whereas f o r α-Τ, η can be equal to 2 (67). An η of 2 f o r a-T means t h i s molecule has the a b i l i t y to quench up to 2 r a d i c a l s before being destroyed. DPPD has been reported to have a n t i o x i d a n t a c t i v i t y (68). However, i n our t e s t s DPPD was t o x i c at high concentrations (400 μΜ) and i n e f f e c t i v e at lower concentrations (e.g., 50 μ Μ ) . In V i t r o Assays. The proposed DPE mechanism of i n i t i a t i n g and propagating r a d i c a l chain r e a c t i o n s should be i n v e s t i g a t e d . For example, the e f f e c t s of " a c i d s y n e r g i s t s " , such as EDTA ( e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d ) , c i t r a t e , and ascorbate, should be examined with respect to t h e i r a b i l i t y to decrease the r a t e of l i p i d o x i d a t i o n by preventing r a d i c a l i n i t i a t i o n or propagation (48). A f t e r inducing i n j u r y 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 , one could study the e f f e c t s of t r a n s i t i o n metals (e.g., Cu or Fe) on the metal-catalyzed decomposition of the hydroperoxides formed. Kunert and Bbger (43) detected ethane e v o l u t i o n from o x y f l u o r f e n treated c h l o r o p l a s t s i n the presence of Fe-EDTA. However, the a d d i t i o n of f e r r e d o x i n to t h i s f r a c t i o n instead of Fe-EDTA y i e l d e d only 20% of the ethane evolved with i r o n present. Further s t u d i e s on the more i n t r i c a t e d e t a i l s of DPE-induced r a d i c a l i n i t i a t i o n and propagation must await the development of an i n v i t r o assay s e n s i t i v e to these h e r b i c i d e s . For example, u s i n g various s u b c e l l u l a r f r a c t i o n a t i o n techniques, a l l of the separated c e l l u l a r components r e q u i r e d f o r expression of h e r b i c i d a l a c t i v i t y can be recombined. I n j u r y may occur by combining an outer e t i o p l a s t envelope f r a c t i o n (the source of carotenoids) with plasma membrane (or microsomal f r a c t i o n ) obtained from oat (Avena s a t i v a L.) r o o t s , and exposing them to l i g h t and h e r b i c i d e . DPE-induced i n j u r y could be monitored by many of the standard methods f o r determining a u t o x i d a t i o n of l i p i d s (e.g., d e t e c t i o n of TBARM, e v o l u t i o n of SCHG, consumption




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4

TIME (h)

Plant Physiology

Figure 4. Efflux of Rb from cucumber cotyledons treated with 1 μΜ AFM in the presence of various concentrations (0, 50,100, and 200 μΜ) of a-tocopherol (3). A t time zero, cotyledons were exposed to herbicide and α-tocopherol in the dark in a nitrogen atmosphere. After 1 h, the atmosphere was changed to air and at 2 h the cotyledons were exposed to light (600 μΕ/m s, PAR). Closed circles are effluxes from control tissue treated with 1.0% ethanol or 1.0% ethanol plus 200 μΜ a- tocopherol. 86

+

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of O2, or measurement of various f l u o r e s c e n t products of l i p i d peroxidation). In t h i s p a r t i c u l a r system, i t may be p o s s i b l e to detect i n j u r y by measuring a c t i v i t y of the enzyme marker for oat root plasma membrane (^9); K - s t i m u l a t e d MgATPase. Another in v i t r o system f o r studying d e t a i l s of the mechanism of a c t i o n of DPE s involves the b i n d i n g of commercially a v a i l a b l e PUFA (e.g., l i n o l e n i c acid) to s i l i c a p l a t e s (48) and adding the various s u b c e l l u l a r components thought to be required for h e r b i c i d a l a c t i v i t y . The D P E - i n i t i a t e d r a d i c a l r e a c t i o n s i n the PUFA monolayers could be followed by d e t e c t i o n of TBARM or by measurement of oxygen consumption (59). +


1

C e l l u l a r Compartmentation of H e r b i c i d e s . For d e t a i l e d mechanism of a c t i o n research, the i n s i t u t i s s u e d i s t r i b u t i o n of DPE s should be known. One method to determine l o c a t i o n i s to t r e a t with r a d i o l a b e l e d h e r b i c i d e and, at some l a t e r time, f r a c t i o n a t e the t i s s u e i n t o i t s various s u b c e l l u l a r components (7Ό). The r e l a t i v e amount of r a d i o a c t i v i t y present i n the various organelles i n d i c a t e s the c e l l u l a r l o c a t i o n of the herbicide. There are, however, contamination problems a s s o c i a t e d with t h i s technique. An a l t e r n a t i v e i s to u t i l i z e the technique developed by MacRobbie and Dainty (71), and Pitman (72), f o r i n t r a c e l l u l a r l o c a t i o n of ions: ( i . e . , compartmental a n a l y s i s ) . The c e l l u l a r compartment containing h e r b i c i d e can be determined by loading the t i s s u e with r a d i o l a b e l e d h e r b i c i d e f o r r e l a t i v e l y long periods of time and then studying the k i n e t i c s of e f f l u x once the e x t e r n a l l a b e l i s removed. This technique has been used by P r i c e and Balke (73) to determine the c e l l u l a r compartmentation of ^ C - a t r a z i n e [2-chloro-4-(ethylamino)-6-(isopropylamino)s:-triazine] . Compartmental a n a l y s i s assumes t i s s u e i s i n a steady-state e q u i l i b r i u m with the r a d i o a c t i v e l a b e l . The a n a l y s i s would be i n v a l i d i f the h e r b i c i d e was e x e r t i n g i t s e f f e c t during the experiment. Because DPE s r e q u i r e l i g h t f o r a c t i v i t y , t h i s problem can be circumvented by doing the a n a l y s i s i n darkness. 1

1

U l t r a s t r u c t u r a l Analyses U l t r a s t r u c t u r a l analyses have been conducted to i d e n t i f y s t r u c t u r a l changes i n the c e l l u l a r membrane system of green and e t i o l a t e d cucumber cotyledons treated with AFM (_3, H*) · P r i o r to p r e p a r a t i o n f o r microscopy, cotyledons treated with 1 μΜ AFM f o r 6 h i n the dark were exposed to high l i g h t (600 μΕ m"^ s""*) for periods up to 1 h. The u l t r a s t r u c t u r e of the t i s s u e treated with AFM f o r 6 h i n darkness (Figure 3A) was the same as untreated t i s s u e . However, massive c e l l u l a r and membrane damage was apparent i n the AFM-treated t i s s u e w i t h i n 30 to 45 min f o l l o w i n g l i g h t - a c t i v a t i o n of the h e r b i c i d e (Figure 3B). Some e t i o p l a s t s and c h l o r o p l a s t s

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Society 1155 16th St. N. W. Moreland et al.; Biochemical Responses Induced by Herbicides Washington, D. C. 20039 ACS Symposium Series; American Chemical Society: Washington, DC, 1982.



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were swollen, which i s c h a r a c t e r i s t i c of an uncoupled o r g a n e l l e ; i . e . , the o r g a n e l l e i s i n d i s c r i m i n a t e l y permeable to s o l u t e s ( i n p a r t i c u l a r , protons) (75). Very often, large holes occurred i n the plasma membrane, t o n o p l a s t , and some c h l o r o p l a s t s and e t i o p l a s t s . A l s o apparent were i n v a g i n a t i o n s of membranes and, r e v e s i c u l a t i o n o f the i n v a g i n a t i o n s once they had broken away from the continuous membrane system (Figure 3C). Most of the other c e l l u l a r o r g a n e l l e s were s e v e r e l y damaged or destroyed. E a r l y signs of i n j u r y appeared i n the outer c h l o r o p l a s t or e t i o p l a s t envelope and i n the t o n o p l a s t . This information supports the proposed mechanism of a c t i o n of DPE h e r b i c i d e s . Carotenoids are known to be present i n the outer p l a s t i d envelope (36), and there i s a l s o a s u b s t a n t i a l amount of l i n o l e n i c a c i d e s t e r i f e d to the g a l a c t o l i p i d s and phospholipids of t h i s membrane ( 1_5 ). I n t e r e s t i n g l y , there was only l i m i t e d observable damage to the t h y l a k o i d s of t i s s u e exposed to l i g h t f o r short periods of time. This i s reasonable because tocopherols and tocopherylquinones are located i n the t h y l a k o i d s ( 1 5 ) . A secondary e f f e c t i n the sequence of events leading to c e l l u l a r death r e s u l t s from the d i s r u p t i o n of the tonoplast. The r e l e a s e of various h y d r o l y t i c enzymes from the vacuole i s extremely d e t r i m e n t a l to the surrounding cytoplasm (Figure 3D). Summary A model of the proposed mechanism of a c t i o n of a DPE, such as AFM, i s o u t l i n e d diagrammatically i n F i g u r e 5. L i g h t absorbed by yellow plant pigments (carotenoids) a c t i v a t e s the AFM molecule. The carotenoid molecule involved appears to be destroyed f o l l o w i n g the a c t i v a t i o n o f the h e r b i c i d e . The l i g h t - a c t i v a t e d form of the AFM molecule i s then i n v o l v e d , e i t h e r d i r e c t l y or i n d i r e c t l y , i n the i n i t i a t i o n of a r a d i c a l chain r e a c t i o n through the a b s t r a c t i o n of a hydrogen atom from the d i v i n y l methane s t r u c t u r e i n PUFA. This r e l a t i v e l y s t a b l e f r e e r a d i c a l subsequently r e a c t s with molecular oxygen to form a l i p i d peroxide that r e a d i l y propagates throughout the hydrophobic matrix of the membrane. The propagation r e a c t i o n s can be terminated i n a number of ways. One termination sequence involves cross r e a c t i o n s o f f a t t y a c i d moieties r e s u l t i n g i n the formation of polymers (48). The formation of these polymers would profoundly a f f e c t the f l u i d i t y and, t h e r e f o r e , the p e r m e a b i l i t y c h a r a c t e r i s t i c s of the membrane. The propagation r e a c t i o n s could a l s o be terminated through decomposition of the l i p i d peroxides formed, r e s u l t i n g i n a p h y s i c a l d i s i n t e g r a t i o n of some portions o f the membrane. Some of the products of l i p i d peroxide decompositon (e.g., MDA) are known to undergo S c h i f f s - b a s e - t y p e r e a c t i o n s with the amino acids of p r o t e i n s (^6). However, the d e t r i m e n t a l e f f e c t of c o v a l e n t l y c r o s s - l i n k i n g p r o t e i n s i s probably secondary i n nature. The r a d i c a l chain can a l s o be terminated i n a 1



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permeability)

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Plant Physiology

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2

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Figure 5. Model outlining a proposed mechanism of action for AFM (3). Abbreviations: caro tenoid*, activated carotenoid; carotenoid , photooxidized carotenoid; AFM*, activated AFM; PUFA-0 -, PUFA peroxide radical; Prop Rx, propagation reaction; Term Rx, termination reac tion; and α-Τ-, α-tocopherol radical (3).

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carotenoid

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Biochemical Responses Induced by Herbicides - American Chemical

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