Biochemical Responses Induced by Herbicides - ACS Publications

10 Biochemical Effects of Glyphosate [N-(Phosphonomethyl)glycine] ROBERT E. HOAGLAND and STEPHEN O. DUKE

Downloaded by MONASH UNIV on May 4, 2015 | http://pubs.acs.org Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch010

Southern Weed Science Laboratory, Stoneville, MS 38776

Glyphosate [N-(phosphonomethyl)glycine] i s i m p l i c a t e d i n the biochemical a l t e r a t i o n of various processes i n p l a n t s and microorganisms, however, its i n h i b i t i o n of aromatic amino a c i d b i o s y n t h e s i s i s the only w e l l e s t a b l i s h e d primary mode of a c t i o n of t h i s h e r b i c i d e . The enzyme 5-enolpyruvylshikimate-3-phosphate synthase i s i n h i b i t e d by p h y s i o l o g i c a l concent r a t i o n s of glyphosate and is the most s e n s i t i v e site of a c t i o n of glyphosate i n reducing aromatic amino a c i d l e v e l s . Aromatic amino a c i d d e p l e t i o n reduces or stops p r o t e i n s y n t h e s i s , causing c e s s a t i o n of growth and e v e n t u a l l y cellular d i s r u p t i o n and death. Supplemental aromatic amino a c i d s reverse glyphosate-caused growth inhibition i n m i c r o organisms and i n Lemna, however, they are not always a n t i d o t a l , e s p e c i a l l y with i n t a c t terrestrial plants. In higher p l a n t s , glyphosate increases e x t r a c t a b l e phenylalanine ammonial y a s e (PAL) activity which partially explains observed reduced phenylalanine and t y r o s i n e pools. Still, PAL inhibitor s t u d i e s and feeding experiments i n d i c a t e that the mode of a c t i o n of glyphosate i n i n t a c t higher p l a n t s cannot be s o l e l y explained by i n t e r f e r e n c e with phenolic metabolism. Glyphosate's d i v a l e n t metal c a t i o n c h e l a t i o n p r o p e r t i e s may a l s o be important i n many biochemical i n t e r a c t i o n s . Glyphosate a l s o d i s r u p t s c h l o r o p l a s t s , membranes, and cell w a l l s ; a l t e r s p r o t e i n and n u c l e i c a c i d s y n t h e s i s , photosynthesis, and r e s p i r a t i o n ; and reduces c h l o r o p h y l l , and other porphyrin compound synthesis. Whether these e f f e c t s are primary or secondary i s not yet e s t a b l i s h e d . The r a p i d

This chapter not subject to U.S. copyright. Published 1982 American Chemical Society. In Biochemical Responses Induced by Herbicides; Moreland, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

176

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

b i o c h e m i c a l e f f e c t s of glyphosate on many parameters, and its h i g h l y n o n - s p e c i f i c t o x i c i t y i n d i c a t e t h a t t h i s h e r b i c i d e may have m u l t i p l e primary a c t i o n s i t e s .

General aspects of glyphosate [N-(phosphonomethyl)glycine], i n c l u d i n g chemical and h e r b i c i d a l p r o p e r t i e s are important to c o n s i d e r , because they a s s i s t i n understanding g l y p h o s a t e s biochemical e f f e c t s . A b s o r p t i o n , t r a n s l o c a t i o n , and degradation of glyphosate and the e f f e c t s of glyphosate on growth and the a s s o c i a t e d p h y t o t o x i c symptoms w i l l be b r i e f l y presented, followed by an in-depth d i s c u s s i o n of the b i o c h e m i c a l a c t i o n of glyphosate. An attempt to p o i n t out what we c o n s i d e r to be primary a c t i o n s and secondary e f f e c t s w i l l be made i n the summary s e c t i o n .


1

Discovery and Development of

Glyphosate

Glyphosate has developed i n t o an extremely important h e r b i c i d e s i n c e i t s i n t r o d u c t i o n i n 1971 ( 1 ) . I t has a simple molecular s t r u c t u r e , a r e l a t i v e l y h i g h water s o l u b i l i t y , and a low molecular weight compared to most h e r b i c i d e s (Figure 1; Table I ) . Roundup (Monsanto s h e r b i c i d e f o r m u l a t i o n of the isopropylamine s a l t of glyphosate w i t h a s u r f a c t a n t ) i s now used e x t e n s i v e l y i n v a r i o u s crop and n o n - a g r i c u l t u r a l s i t u a t i o n s i n many c o u n t r i e s of the world. Glyphosate i s a n o n - s e l e c t i v e , broad spectrum, postemergence h e r b i c i d e and i s the only compound of t h i s chemical c l a s s which i s r e g i s t e r e d as a h e r b i c i d e ; however, an analog, glyphosine (Figure 2) i s a p l a n t growth r e g u l a t o r . About 1200 l i t e r a t u r e c i t a t i o n s ( i n c l u d i n g p u b l i s h e d a r t i c l e s and a b s t r a c t s ) e x i s t on v a r i o u s aspects of glyphosate, but there are few review a r t i c l e s on the compound. A d e s c r i p t i o n of the compound and i t s general h e r b i c i d a l p r o p e r t i e s was p u b l i s h e d soon a f t e r i t s i n t r o d u c t i o n ( 1 ) . Updates of the compound's s e l e c t i v i t y and c h a r a c t e r i s t i c s have s i n c e been p u b l i s h e d (2_, _3, *t). Franz (5) covered both general and s p e c i f i c aspects of glyphosate, i n c l u d i n g the r e l a t i o n s h i p between s t r u c t u r e and a c t i v i t y of glyphosate d e r i v a t i v e s and r e l a t e d compounds as w e l l as v a r i o u s proposed modes of a c t i o n . A b i b l i o g r a p h y of glyphosate l i t e r a t u r e to 1978 i s a v a i l a b l e (6). The H e r b i c i d e Handbook of the Weed Science S o c i e t y of America o u t l i n e s glyphosate's uses, p r o p e r t i e s , p r e c a u t i o n s , p h y s i o l o g i c a l and b i o c h e m i c a l behavior, s y n t h e s i s , a n a l y t i c a l methods and procedures, e t c . ( 7 ) . A very b r i e f g e n e r a l overview of glyphosate i n c l u d i n g b i o c h e m i c a l and p h y s i o l o g i c a l a c t i o n was p u b l i s h e d r e c e n t l y (8). A comprehensive review of glyphosate i s p r e s e n t l y i n p r e p a r a t i o n (9). A chapter that presents 1

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

10.

Glyphosate[N-(Phosphonomethyl)glycine]

HOAGLAND AND DUKE

O

H

177

O

HO-C-CH-N-CH-P-OH 2

2 I


OH N-(Phosphonomethyl)glycine Figure 1.

Glyphosate chemical structure.

HOOC-CH-N(CH-P0 H > 3

2

2

Glyphosine

HOOC-CH-NH-CH-P0 H 3

Glyphosate

2

—^Z^

NH-CH-P0 H 3

2

AminomethyIphosphonic

Acid

CH-NH-CH-COOH 3

2

Sarcosine

NH-CH-COOH 2

2

Glycine

Figure 2. Analogs, metabolites, and/or degradation products of glyphosate.


178


Table I .

General c h a r a c t e r i s t i c s and p r o p e r t i e s of glyphosate.


PHYSICAL AND CHEMICAL PROPERTIES Physical state Molecular weight Solubility Melting point Vapor pressure Density K 1, 2, 3 Chelation P

-

-

a

white odorless s o l i d 169.1 H 0 (1 to 8%, 25-100°C) 200°C w. decomposition negligible 0.5 g/cc 2.3, 5.9, 10.9 metal c a t i o n s 2

STABILITY ASPECTS Photodecomposition Shelf l i f e B i o d e g r a d a b i l i t y and s o i l persistence

negligible very s t a b l e r a p i d l y degraded by s o i l microoganisms T^ < 60 da.

UPTAKE AND METABOLIC ASPECTS Absorption Translocation Metabolism Specificity

r e a d i l y absorbed by r o o t s and f o l i a g e r a p i d l y t r a n s l o c a t e d from a p p l i c a t i o n point metabolism by p l a n t s i s extremely low, but r a p i d i n s o i l s n o n - s e l e c t i v e , broad spectrum

TOXICITY Rats Rabbits Fish



10.

HOAGLAND AND DUKE


179

v a r i o u s aspects of glyphosate has been r e c e n t l y included i n a t e x t on h e r b i c i d e mode of a c t i o n (10). The o v e r a l l success of glyphosate may be a t t r i b u t e d to s e v e r a l p r o p e r t i e s (Table I) i n a d d i t i o n to i t s p h y t o t o x i c i t y . I t s low molecular weight and high water s o l u b i l i t y are f a c t o r s that a i d i n i t s r a p i d absorption and t r a n s l o c a t i o n by p l a n t tissues. Glyphosate i s a white, odorless s o l i d , and as a phosphonic a c i d has the a b i l i t y t o c h e l a t e c e r t a i n d i v a l e n t and t r i v a l e n t c a t i o n s (11-14). Glyphosate i s r a p i d l y absorbed by f o l i a r t i s s u e s and r o o t s and can be r e l a t i v e l y r a p i d l y t r a n s l o c a t e d to v a r i o u s p l a n t organs, d i s t a n t from the a p p l i c a t i o n s i t e (15-18). Once i n s i d e the p l a n t , glyphosate does not break down nor i s i t metabolized to a s i g n i f i c a n t degree (16, 19-21). Thus, i t can maintain i t s phytotoxic a c t i o n w h i l e t r a n s l o c a t i n g to v a r i o u s p l a n t p a r t s . These f a c t o r s provide u t i l i t y i n c o n t r o l l i n g h a r d - t o - k i l l p e r e n n i a l weeds that are deep-rooted or ones that possess v e g e t a t i v e propagules. In s o i l s , however, the compound i s s t r o n g l y adsorbed, and i s r a p i d l y degraded by microorganisms t o non-phytotoxic products i n c l u d i n g carbon d i o x i d e (_7, 17, 22) . Glyphosate i s very s t a b l e and i s not subject t o photodecomposition or v o l a t i l i t y . Due t o i t s b i o d e g r a d a b i l i t y , i t has a h a l f - l i f e of l e s s than 60 days i n s o i l . Although the compound i s a h i g h l y e f f i c a c i o u s h e r b i c i d e , i t s t o x i c o l o g i c a l e f f e c t s on mammals, honeybees, and f i s h , are r e l a t i v e l y low (5_, 7) and i t has l i t t l e e f f e c t on diatom and Daphnia populations i n a q u a t i c environments (23, 24). Non-biochemical

Considerations

Phytotoxic Symptoms. In d i v e r s e species, the f i r s t v i s i b l e growth e f f e c t of glyphosate a f t e r a p p l i c a t i o n i s g e n e r a l l y the i n d u c t i o n of c h l o r o s i s , which i s u s u a l l y followed by n e c r o s i s (15, 16, 25-33). These symptoms commonly take from two t o t e n days t o occur and i n some perennials, i n j u r y symptoms may be evident i n the year f o l l o w i n g treatment. Morphological a b n o r m a l i t i e s of leaves and w i l t i n g have been observed under some c o n d i t i o n s (16, 27, 30, 34). Root and rhizome growth and s u r v i v a l are s t r o n g l y i n h i b i t e d by glyphosate (18, 25, 27, 3542). No p a r t i c u l a r p l a n t organ or t i s s u e has been c o n c l u s i v e l y shown t o be the primary s i t e of a c t i o n of glyphosate, however, the e f f e c t s on meristems are perhaps d i r e c t l y caused by the h e r b i c i d e because they have high metabolic a c t i v i t y and glyphosate accumulates there (40). C h l o r o s i s i s o f t e n induced more r a p i d l y under high than under low l i g h t i n t e n s i t y (43-46). Morphological d i f f e r e n c e s between sun and shade l e a v e s , however, can r e s u l t i n greater t o x i c i t y t o shaded than to unshaded p l a n t s sprayed with glyphosate (35). Increased l i g h t i n t e n s i t y has a l s o been


180



c o r r e l a t e d with i n c r e a s e d glyphosate accumulation i n untreated p l a n t t i s s u e p a r t s (47). Although s e e d l i n g growth i s i n h i b i t e d by glyphosate, t h i s h e r b i c i d e has no s i g n i f i c a n t e f f e c t on germination of a wide v a r i e t y of s p e c i e s (40, 44, 48-51). Because glyphosate i s t i g h t l y bound to s o i l (52) and i s a l s o r a p i d l y metabolized i n s o i l , i t s a v a i l a b i l i t y t o germinating seeds may be minimized. Absorption and T r a n s l o c a t i o n . Absorption and t r a n s l o c a t i o n of glyphosate i s summarized and d i s c u s s e d i n depth elsewhere (10). Glyphosate uptake and t r a n s l o c a t i o n i s r e l a t i v e l y r a p i d i n d i v e r s e s p e c i e s (15-19, 21, 53, 54). Environmental f a c t o r s such as temperature and r e l a t i v e humidity have been s t u d i e d w i t h r e s p e c t to glyphosate p h y t o t o x i c i t y , uptake, and t r a n s l o c a t i o n (20, 21, 35, 36, 43, 55-58). Growth stage and water s t r e s s e f f e c t s on g l y p h o s a t e s m o b i l i t y i n bermudagrass [Cynodon d a c t y l o n (L.) Pers.] has a l s o been i n v e s t i g a t e d (59). Although such f a c t o r s can a l t e r the r a t e s of a b s o r p t i o n and t r a n s l o c a t i o n of glyphosate, the h e r b i c i d e i s g e n e r a l l y r a p i d l y absorbed and t r a n s l o c a t e d to v a r i o u s p l a n t t i s s u e s . Meristems are known s i t e s of glyphosate accumulation (40). T r a n s l o c a t i o n to underground propagules of p e r e n n i a l species prevents regrowth from these s i t e s and r e s u l t s i n t h e i r subsequent d e s t r u c t i o n (19, 20). Information a v a i l a b l e from a b s o r p t i o n and t r a n s l o c a t i o n s t u d i e s suggests t h a t most glyphosate movement i s i n the symplast but there i s a l s o some evidence of a p o p l a s t i c t r a n s p o r t (15, 17, 18, 21, 38). 1

Degradation of Glyphosate. Glyphosate i s very s t a b l e i n higher p l a n t s (16, 19-21, 53), but i s degraded by microorganisms i n s o i l Ç7, 17, 22). Various metabolites or degradation products of glyphosate have been i d e n t i f i e d , t e n t a t i v e l y i d e n t i f i e d , or proposed (Figure 2). Aminomethylphosphonic a c i d i s the p r i n c i p l e product of glyphosate degradation i n s o i l s (60). T h i s compound has been found i n p l a n t s , but was absorbed from the s o i l and d i d not r e s u l t from metabolic a c t i o n on glyphosate i n the p l a n t . Sarcosine and g l y c i n e are other p o s s i b l e non-phytotoxic products of glyphosate degradation i n s o i l s (60). R a d i o l a b e l e d glyphosate has been shown to degrade completely i n the s o i l to carbon d i o x i d e (17, 22). Biochemical and P h y s i o l o g i c a l E f f e c t s E a r l y Work and Feeding S t u d i e s . Although many b i o c h e m i c a l and p h y s i o l o g i c a l i n v e s t i g a t i o n s have been conducted on glyphosate e f f e c t s and a c t i o n i n p l a n t s , the most promising of these have i m p l i c a t e d a d i s r u p t i o n of p h e n o l i c metabolism as the b a s i s f o r i t s molecular mode of a c t i o n . An o u t l i n e of these compounds and a s s o c i a t e d enzymes of p h e n o l i c metabolism i n h i g h e r p l a n t s i s presented i n F i g u r e 3. Enzymes 1, 2, 3,


10.


HOAGLAND AND DUKE

181

TANNINS

\

GALLIC ACID SINAPIC ACID FERULIC ACID

LIGNINS

\

SOFLAVONOIDS FLAVONOIDS CHALCONES

t CHLOROGENIC

CAFFEIC ACID

ACID

4 4


16)

p-COUMARATE

1-CINNAMATE (g) PROTEIN

VJ

— TRYPTOPHAN

5) ^

» AMMONIA «

"s.

PHENYLALANINE

PREPHENATE

»*— ®

Î© CHORISMATE

t© t® t© SHIKIMATE

© © 3-DEOXY-D-ARABINO-HEPTULOSONATE - 7 - P

© ERYTHROSE-4-

PHOSPHOENOL PYRUVATE

Figure 3. Schematic outline of various intermediates and products including enzymes of the phenolic pathway in plants. Enzymes: 1, 3-deoxy-2-oxo-O-arabinoheptulosate-7-phosphate synthase; 2, 5-dehydroquinate synthase; 3, shikimate dehydrogenase; 4, shikimate kinase; 5, 5-enolpyruvylshikimate-3-phosphate synthase; 6, chorismate synthase; 7, chorismate mutase; 8, prephenate dehydrogenase; 9, tyrosine aminotransferase; 10, prephenate dehydratase; 11, phenylalanine aminotransferase; 12, anthranilate synthase; 13, tryptophan synthase; 14, phenylalanine ammonia-lyase; 15, tyrosine ammonia-lyase; and 16, polyphenol oxidase.



182


5, 7, 8, 10, 14, 15, and 16 have r e c e i v e d the most a t t e n t i o n with r e s p e c t to glyphosate i n t e r a c t i o n (Table I I ) . In e a r l y i n v e s t i g a t i o n s of glyphosate on the a q u a t i c p l a n t , duckweed (Lemna gibba L.)» and the microorganism, Rhizobium japonicum, Jaworski i n d i c a t e d that the h e r b i c i d e caused decreased l e v e l s of aromatic amino acids (61). Feeding supplemental aromatic amino a c i d s r e s u l t e d i n r e v e r s a l of glyphosate-caused i n h i b i t i o n of growth. These s t u d i e s i n d i c a t e d that glyphosate caused d e f i c i t s of p h e n y l a l a n i n e , t y r o s i n e , and to a l e s s e r degree, tryptophan. The theory was thus proposed that glyphosate i n h i b i t e d the s y n t h e s i s of these amino a c i d s by i n h i b i t i n g or r e p r e s s i n g the enzymes chorismate mutase (Figure 3, No. 7) and prephenate dehydratase (Figure 3, No. 10) i n Lemna gibba (Figure 4). In a d d i t i o n to these two enzymes, prephenate dehydrogenase (Figure 3, No. 8) was a l s o proposed to be i n h i b i t e d i n Rhizobium japonicum (Figure 4 ) . D e p l e t i o n of aromatic amino acids pools could l e a d to reduced p r o t e i n s y n t h e s i s , r e s u l t i n g i n c e s s a t i o n of growth, c e l l u l a r d i s r u p t i o n , and e v e n t u a l l y death. T h i s o r i g i n a l theory has been s t r o n g l y supported i n most cases by subsequent experiments w i t h other microorganisms, p l a n t c e l l t i s s u e c u l t u r e s , and i s o l a t e d p l a n t t i s s u e systems. P a r t i a l or complete r e v e r s a l of glyphosate-caused growth i n h i b i t i o n by aromatic amino a c i d s has been shown i n the u n i c e l l u l a r organisms IS. c o l i (62, 63), Rhizobium japonicum (61), and Chlamydomonas (63); and i n t i s s u e c u l t u r e s of c a r r o t (Daucus c a r o t a L.) (63, 64), soybeans [ G l y c i n e max (L.) Merr.] (63), and tobacco ( N i c o t i a n a tabacum L.) (65). In i s o l a t e d soybean l e a f c e l l s , supplemental a d d i t i o n of aromatic amino a c i d s p a r t i a l l y prevented glyphosatereduced p r o t e i n s y n t h e s i s (66). Feeding aromatic amino a c i d s has a l s o reversed glyphosate-induced basal-stem s w e l l i n g and bud r e l e a s e i n g r a i n sorghum (Sorghum b i c o l o r L.) (67), g l y p h o s a t e - i n h i b i t e d t r a n s p i r a t i o n i n bean (Phaseolus v u l g a r i s L.) shoots (68), and g l y p h o s a t e - i n h i b i t e d anthocyanin s y n t h e s i s (69) i n buckwheat (Fagopyrum esculenturn Moench). There a r e , however, only a few r e p o r t s [duckweed (61), mouseearcress (Arabidopsis t h a l i a n a L.) (70), and g r a i n sorghum (67)] i n which s i g n i f i c a n t p r e v e n t i o n of glyphosate e f f e c t s on growth has been obtained by f e e d i n g i n t a c t higher p l a n t s supplemental amino a c i d s . Glyphosate i n h i b i t i o n of growth i s only m a r g i n a l l y prevented, or not prevented at a l l , by aromatic amino a c i d f e e d i n g i n s t u d i e s with maize (Zea mays L.) (39) soybean (71), wheat ( T r i t i c u m aestivum L.) (72) and bean s e e d l i n g s (73), as w e l l as quackgrass (Agropyron repens L. Beauv.) nodes (72). Furthermore, there i s l i t t l e or no e f f e c t of glyphosate on aromatic amino a c i d pools i n some cases with h i g h e r p l a n t t i s s u e c u l t u r e s (64) and g l y p h o s a t e - i n h i b i t e d growth cannot always be reversed with supplemental amino a c i d s (74).



CH

a

b CM

Prephenate

0

HO H

HOOCCH,-CO-COOH

Phenylpyruvate

CH-CO-COOH

p-Hydroxyphenylpyruvate

OH

NH,

Phenylalanine

CH-CH-COOH 2 Ν ι H

Tyrosine

0' OH

2 ι

CH-CH-COOH

Figure 4. Enzymes of Rhizobium (a) and Lemna (b) proposed as sites of glyphosate inhibition of aromatic amino acid synthesis. Abbreviations: CM, chorismate mutase; PDH, prephenate de hydrogenase; and PD, prephenate dehydratase.

2

-COOH

Chorismate

OH

COOH

0

CH-CO-COOH



Effects

Aerobacter

Chorismate synthase (EC 4.6.1.4)

Chorismate mutase (EC 5.4.99.5)

Aerobacter aerogenes JE. c o l i Vigna r a d i a t a

E. c o l i T r i t i c u m aestivum

aerogenes

elevated elevated

-

no

effect

strong i n h i b i t i o n inhibited inhibited

effect

no

5-Enolpyruylshikimate-3phosphate synthase (EC 2.5.1.19)

aerogenes

Aerobacter

Shikimate kinase (EC 2.7.1.71)

effect

no

T r i t i c u m aestivum G l y c i n e max

Shikimate dehydrogenase (EC 1.1.1.25)

elevated no e f f e c t

weak i n h i b i t i o n

no e f f e c t

coli

E.

5-Dehydroquinate synthase (EC 4.6.1.3)

(62, 88) (72)

(87)

(86, 87) (87) (87)

(87)

(Zi)

(72)

(62, 88)

(62, 88)

weak i n h i b i t i o n

Reference

In v i t r o activity

elevated

Extractable activity

(see F i g u r e 1 f o r pathway).

E. c o l i

Source

of glyphosate on enzymes of p h e n o l i c metabolism

3-Deoxy-2-oxo-D-arabinoheptulosate-7-phosphate synthase (EC 4.6.1.3)

Enzyme

Table I I .


In Biochemical Responses Induced by Herbicides; Moreland, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982. none none

elevated elevated elevated

elevated elevated no e f f e c t elevated

elevated elevated

Zea mays G l y c i n e max Gossypium hirsutum

Agropyron repens T r i t i c u m aestivum Fagopyrum esculentum Vigna r a d i a t a

T r i t i c u m aestivum G l y c i n e max

Phenylalanine ammonial y a s e (EC 4.3.1.5)

Polyphenol oxidase (EC 1.10.3.2)

no e f f e c t weak i n h i b i t i o n

elevated

aerogenes

Aerobacter E. c o l i

A n t h r a n i l a t e synthase (EC 4.1.3.27)

none

elevated

coli

E.

Prephenate dehydratase (EC 4.2.1.51)

none

elevated

coli

In v i t r o activity

Extractable activity

E.

Source

(Continued)

Prephenate dehydrogenase (EC 1.3.1.12)

Enzyme

Table I I .


(72) (Hoagland & Duke, unpub.)

(39, 78) (41, 42, 79, 80) (Duke & Hoagland, unpub.) (72) (72) (69) (Hoagland, unpub·)

(87) (88)

(62, 88)

(62, 88)

Reference

186


The e f f e c t s of glyphosate on s e v e r a l IS. c o l i enzymes of the aromatic amino a c i d b i o s y n t h e t i c pathway have been s t u d i e d (62) (Table I I ) . In v i t r o t e s t s i n d i c a t e d that 3-deoxy-2-oxoD-arabinoheptonic acid-7-phosphate synthetase (Figure 3, No. 1) and 5-dehydroquinic a c i d synthetase (Figure 3, No. 2) were i n h i b i t e d by glyphosate, but only at 10 mM. Both of these i n h i b i t o r y e f f e c t s were removed by the a d d i t i o n of C o 2 Chorismate mutase, prephenate dehydrogenase, and prephenate dehydratase (Figure 3, Nos. 7, 8, and 10) were not a f f e c t e d . H e r b i c i d e c o n c e n t r a t i o n s r e q u i r e d f o r i n v i t r o enzyme e f f e c t s were higher than apparent p h y s i o l o g i c a l l e v e l s ( i . e . , growthi n h i b i t i n g c o n c e n t r a t i o n s ) of glyphosate. These r e p o r t s i n d i c a t e that the mode of a c t i o n hypothesis of Jaworski does not adequately e x p l a i n the p h y t o t o x i c a c t i o n of glyphosate i n a l l p l a n t systems. +


e

E f f e c t s of Glyphosate on PAL. Because of inadequate s u b s t a n t i a t i o n of the above theory, we i n i t i a l l y p o s t u l a t e d that lowered phenylalanine and t y r o s i n e pools caused by glyphosate might a d d i t i o n a l l y be a t t r i b u t e d to i n d u c t i o n of phenylalanine ammonia-lyase [(PAL) F i g u r e 3, Nos. 14 and 15] activity. There was some evidence from other r e p o r t s that h i g h PAL a c t i v i t y could r e t a r d growth through aromatic-amino a c i d d e p l e t i o n (75). PAL deaminates t y r o s i n e to some extent i n most p l a n t systems (76), thus, i n c r e a s e d PAL a c t i v i t y c o u l d r e s u l t i n less-than-adequate l e v e l s of phenylalanine and t y r o s i n e r e q u i r e d f o r normal p r o t e i n s y n t h e s i s . PAL, by a c t i n g on p h e n y l a l a n i n e , p l a y s a key r o l e i n phenylpropanoid b i o s y n t h e s i s and r e g u l a t e s the formation of a v a r i e t y of p h e n o l i c compounds ( F i g u r e 3). T h i s enzyme has been shown to be r e g u l a t e d by a number of environmental f a c t o r s (water s t r e s s , wounding, i n f e c t i o n , l i g h t , chemical a c t i o n , e t c . ) (76). Accumulation of p h e n o l i c s c o u l d cause an a u t o a l l e l o p a t h i c or p h y t o t o x i c e f f e c t on the p l a n t s . Furthermore, the non-oxidative deamination of phenylalanine c o u l d y i e l d t o x i c l e v e l s of ammonia i f deaminat i o n enzymes d i d not provide p r o t e c t i o n (Figure 5 ) . In t e s t s of t h i s hypothesis with maize and soybean seedlings, we found that glyphosate caused pronounced i n c r e a s e s i n e x t r a c t a b l e PAL a c t i v i t y (39, 41, 42) (Figures 6 and 11). In v i t r o t e s t s showed that glyphosate had no d i r e c t e f f e c t on PAL. Later, Cole et_ a l . (72) found that glyphosate i n c r e a s e d PAL a c t i v i t y i n s i n g l e node buds of quackgrass rhizomes and i n root t i p s of wheat (Table I I I ) . H o l l a n d e r and Amrhein (69), however, found no e f f e c t of glyphosate on e x t r a c t e d PAL of buckwheat hypoc o t y l s . We found a good c o r r e l a t i o n between glyphosate-caused PAL a c t i v i t y i n c r e a s e s , and s u b s t r a t e (phenylalanine) and product (hydroxyphenolic) decreases caused by glyphosate i n maize (Figure 7) and soybean s e e d l i n g s (Figure 8 ) . N i l s s o n ' s l a b o r a t o r y a l s o showed that glyphosate decreased aromatic amino a c i d l e v e l s i n wheat r o o t s (77). P h e n o l i c l e v e l s were


10.

HOAGLAND AND DUKE


187

NH CH=CH-COOH

CH-CH-COOH PAL


Phenylalanine

NH

t.-Cinnamic

acid

Figure 5. Nonoxidative deamination of phenylalanine by PAL.

Plant Science Letters

GLYPHOSATE EXPOSURE

Figure 6. Increased extractable PAL, in maize roots caused by root-feeding of glyphosate to intact plants (39). Darkgrown, 3-day-old maize seedlings were transferred to 1 mM glyphosate (Φ), or water (O) and enzyme activity was moni tored over a 3-day time course during dark growth.




188

Figure 8. Correlation of extractable PAL activity increases with decreases in PAL substrate (phenylalanine) and products (hydroxyphenolics) during glyphosate treatment in axes of soybean seedlings (41, 42, SO).


10.


HOAGLAND AND DUKE


g e n e r a l l y higher on a f r e s h weight b a s i s but were lower on a p l a n t organ b a s i s i n g l y p h o s a t e - t r e a t e d t i s s u e s (39, 41, 42, 78). Although s e v e r a l equivocations can be made concerning the hydroxyphenolic data due t o techniques used (78), the lowering of anthocyanin l e v e l s (79) i n d i c a t e s that these r e s u l t s a r e q u a l i t a t i v e l y accurate. The r e s u l t s of our i n i t i a l s t u d i e s (39, 41, 42, 78) i n d i c a t e d that although glyphosate has profound e f f e c t s on e x t r a c t a b l e PAL, PAL s u b s t r a t e ( s ) , and PAL end products, i n c r e a s e d PAL a c t i v i t y was probably a secondary e f f e c t o f decreased feedback c o n t r o l due t o decreased s u b s t r a t e and, thus, decreased product.

Table I I I .

E f f e c t o f 0.5 mM glyphosate on e x t r a c t a b l e a c t i v i t y of four enzymes of p h e n o l i c metabolism from wheat root t i p s . Adapted from Cole et a l . (72).

Enzyme a c t i v i t y ( u n i t s mg p r o t e i n ) a f t e r 24 h Enzyme

Chorismate mutase Shikimate dehydrogenase PAL Polyphenol oxidase

1

Control

Glyphosate

2.3 4.5 3.3 6.1

5.9 11.5 19.1 14.1

We conducted f u r t h e r experiments t o determine i f g l y p h o s a t e s e f f e c t s c o u l d be reversed i n higher p l a n t s by i n c r e a s i n g aromatic amino a c i d l e v e l s u s i n g PAL i n h i b i t o r s (80) ( F i g u r e 9 ) . We reasoned that i f i n c r e a s e d PAL a c t i v i t y was i n v o l v e d i n g l y p h o s a t e s mode of a c t i o n , then b l o c k i n g PAL i n v i v o , with a PAL i n h i b i t o r might reduce o r reverse glyphosate s toxic e f f e c t s . The PAL i n h i b i t o r , a-aminooxy-βp h e n y l p r o p i o n i c a c i d (AOPP) had p r e v i o u s l y been shown t o e f f e c t i v e l y i n h i b i t PAL and t o i n h i b i t anthocyanin accumu l a t i o n while having l i t t l e or no e f f e c t on growth (81, 82). At 0.1 mM, AOPP had no s i g n i f i c a n t e f f e c t on soybean growth u n t i l 96 hours, but d i d p r o v i d e marginal growth r e v e r s a l (10%) of 0.5 mM g l y p h o s a t e s i n h i b i t i o n (80) (Figure 10). T h i s l e v e l of AOPP i n c r e a s e d phenylalanine and t y r o s i n e l e v e l s i n glyphosate-treated t i s s u e s t o c o n t r o l l e v e l s . PAL a c t i v i t y from axes was completely i n h i b i t e d i n v i t r o by 10 yM AOPP. E x t r a c t a b l e PAL a c t i v i t y was i n c r e a s e d by AOPP (Figure 11) by a l l measurement c r i t e r i a . T h i s i n c r e a s e was probably because of decreased feedback i n h i b i t i o n o f PAL by i t s products and 1

1

1

1



190


α-Aminooxyacetate

.

AO A

a-Aminooxy->9-phenylpropionate .

NH-O-CH-COOH

NH-O-CH-COOH 2 ι

AOPP

Figure 9. PAL inhibitors, a-aminooxy-fi-phenylpropionic acid (AOPP) and aminooxyacetic acid (A OA ).


10.

HOAGLAND AND DUKE



Plant Physiology

Figure 10. The effect of AOPP and glyphosate on soybean axis growth when supplied to roots in liquid culture (SO). Seedlings were exposed to continuous white light and root-fed various chemicals after 3 days of dark growth. Key: · , control; |, 0.5 mM glyphosate; A, 0.1 mM AOPP; and X ,glyphosate (0.5 mM) plus AOPP (0.1 mM).

TIME(h)

TIME

(h) Plant Physiology

Figure 11. The effect of AOPP and glyphosate on extractable PAL activity in light-grown soybean axes (SO). Seedlings were exposed to continuous white light and root-fed various chemicals after 3 days of dark growth. Key: O, control; 0.5 mM glyphosate; A , 0.1 mM AOPP; and Î3, glyphosate (0.5 mM) plus AOPP (0.1 mM).


192


the f a c t that our p u r i f i c a t i o n and e x t r a c t i o n procedures f o r PAL removed AOPP. AOPP, glyphosate, and AOPP plus glyphosate reduced t o t a l hydroxyphenolic accumulation. Similar experiments were performed with aminooxyacetic a c i d (Figure 9 ) . A summary of the e f f e c t s o f these two PAL i n h i b i t o r s and glyphosate on growth and metabolism of soybean s e e d l i n g s i s presented i n Table IV. Cole et^ a l . (72) found no r e v e r s a l of glyphosate e f f e c t s with the PAL i n h i b i t o r s , D-phenylalanine, cinnamic a c i d , and AOPP. From these data, i t can be concluded that g l y p h o s a t e s enhancement of PAL a c t i v i t y p l a y s a r o l e i n d e p l e t i o n of phenylalanine and t y r o s i n e , but that PAL s r o l e i n g l y p h o s a t e s mode of a c t i o n , taken alone, may not be of major importance i n i t s p h y t o t o x i c e f f e c t s . Moreover, these r e s u l t s s t r o n g l y suggest that i n t e r f e r e n c e with aromatic amino a c i d metabolism does not f u l l y e x p l a i n g l y p h o s a t e s mode of a c t i o n i n higher p l a n t s . 1

1


1

1

Table IV.

Treatment

Glyphosate AOPP AOA

Summary of e f f e c t s of glyphosate (0.5 mM) and two PAL i n h i b i t o r s , AOPP (0.01 mM) and AOA (0.01 mM), on growth and metabolism of soybean s e e d l i n g s .

Growth

0

Soluble Protein

0

Soluble Phenolics

Extracted PAL

Phenylalanine

+ + +

+ +

These c o n c l u s i o n s are supported by s t u d i e s i n our l a b o r a t o r y i n which we found only a minimal r e v e r s a l of glyphosate-caused growth i n h i b i t i o n by r o o t - f e d aromatic amino a c i d s (71). Feeding aromatic amino a c i d s before glyphosate exposure d i d not enhance r e v e r s a l . On a fresh-weight b a s i s , glyphosate had no i n h i b i t o r y e f f e c t on uptake or i n c o r p o r a t i o n of these amino a c i d s i n t o p r o t e i n or secondary p h e n o l i c s . These data suggest that e i t h e r r o o t - f e d aromatic amino a c i d s are compartmentalized d i f f e r e n t l y than the endogenous pools that a r e a f f e c t e d by glyphosate, or that r o o t - f e d glyphosate e x e r t s most of i t s e f f e c t on soybean growth through means other than i n h i b i t i o n of aromatic amino a c i d b i o s y n t h e s i s . E f f e c t s of Glyphosate Analogs on Secondary P h e n o l i c Compound S y n t h e s i s . To t e s t the s p e c i f i c i t y of g l y p h o s a t e s a c t i o n and/or the e f f e c t o f analogs and p o s s i b l e degradation products (Figure 2), a study was conducted that used these compounds on 1


10.

HOAGLAND AND DUKE


the soybean system (79). Glyphosine was not as e f f e c t i v e i n i n c r e a s i n g a c t i v i t y of PAL as was glyphosate. The other compounds had no e f f e c t , except f o r aminomethylphosphonic a c i d , which s l i g h t l y reduced e x t r a c t a b l e PAL a c t i v i t y . Hydroxyphenolic l e v e l s were analyzed, but only glyphosine caused a decrease i n p h e n o l i c s and the other compounds had no e f f e c t . Anthocyanin l e v e l s were reduced by 50% i n glyphosatet r e a t e d s e e d l i n g s , but were decreased to a much l e s s e r degree by glyphosine. Hollander and Amrhein (69) obtained almost i d e n t i c a l r e s u l t s with buckwheat h y p o c o t y l s . Glyphosine was g e n e r a l l y not as e f f e c t i v e i n i n h i b i t i n g c h l o r o p h y l l accumul a t i o n as was glyphosate. Thus, g l y p h o s a t e s e f f e c t on PAL and the other parameters are r a t h e r s p e c i f i c when compared to those of i t s s t r u c t u r a l analogs.


1

1

S p e c i f i c i t y of G l y p h o s a t e s E f f e c t on PAL. Glyphosate's e f f e c t s on PAL are r a t h e r s p e c i f i c when compared to other h e r b i c i d e s . The e f f e c t s of 16 h e r b i c i d e s r e p r e s e n t i n g 14 h e r b i c i d e c l a s s e s on e x t r a c t a b l e PAL a c t i v i t y from both l i g h t and dark-grown soybean s e e d l i n g t i s s u e s were determined (83). Only 2 compounds i n c r e a s e d e x t r a c t a b l e PAL a c t i v i t y : a m i t r o l e ( 3 - a m i n o - s - t r i a z o l e ) , to about 30% of that of glyphosate on a per a x i s b a s i s ; and paraquat ( l , l - d i m e t h y l - 4 , 4 - b i p y r i d i n i u m i o n ) , which showed a somewhat greater enhancement at e a r l y exposure times w h i l e l a t e r lowering e x t r a c t a b l e PAL l e v e l s . These r e s u l t s support the view that glyphosate-caused PAL i n c r e a s e s are s p e c i f i c and are not a secondary e f f e c t of stress. ,

T

E f f e c t s of Glyphosate on Enzymes of Aromatic Amino A c i d Synthesis. Although e a r l y work on glyphosate e f f e c t s on enzymes of aromatic amino a c i d s y n t h e s i s were i n c o n c l u s i v e (62), recent work i n Amrhein s l a b o r a t o r y has r e v e a l e d the apparent s i t e of a c t i o n of t h i s compound. They found that anthocyanin s y n t h e s i s i n i l l u m i n a t e d , e x c i s e d hypocotyls of buckwheat was s e v e r e l y depressed by glyphosate (69). T h i s occurred when glyphosate was a p p l i e d v i a root uptake, to f l o a t e d e x c i s e d h y p o c o t y l s , or by s p r a y i n g s e e d l i n g s . Lphenylalanine was the only aromatic amino a c i d that e f f e c t i v e l y reversed i n h i b i t i o n of anthocyanin s y n t h e s i s . When anthocyanin s y n t h e s i s was allowed to proceed f o r 10 h and then continued i n the presence of 3 mM glyphosate, i t s r a t e was reduced w i t h i n l e s s than 1 h. They suggested that t h i s r a p i d i n h i b i t i o n was due to a d i r e c t i n h i b i t i o n by glyphosate of a metabolic step i n the pathway l e a d i n g to anthocyanin r a t h e r than to the induced appearance (or disappearance) of an enzyme. I n v e s t i g a t i o n of the r a t e s of i n h i b i t i o n of c h l o r o p h y l l and anthocyanin formation by glyphosate i n d i c a t e d that i n h i b i t i o n of anthocyanin formation was more s e n s i t i v e than that of c h l o r o p h y l l by an order of magnitude (Figure 12). 1


193


194


Fresh

Weight

orophyl I

Anthocy

0 HI-

6

5

4

G l y p h o s a t e ( - log

3

2

Molarity) Plant Physiology

Figure 12. Effect of glyphosate on fresh weight increase, chlorophyll content, and anthocyanin content in excised buckwheat cotyledons (69). Cotyledons of 6-day-old etiolated seedlings were incubated at the indicated glyphosate concentrations for 24 h in the light.



10.

HOAGLAND AND DUKE

Glyphosaîe[N-(Phosphonomeîhyl)glycine]

195

Although phenylalanine alone was a b l e to a l l e v i a t e glyphosate i n h i b i t i o n of anthocyanin formation, f e e d i n g t y r o s i n e i n a d d i t i o n to phenylalanine was r e q u i r e d to achieve p a r t i a l a l l e v i a t i o n of the i n h i b i t i o n of c h l o r o p h y l l formation. A d d i t i o n a l tryptophan d i d not f u r t h e r i n c r e a s e the c h l o r o p h y l l content of cotyledons. F u r t h e r work with buckwheat i n d i c a t e d that a 24-h l i g h t or dark i n c u b a t i o n of untreated e x c i s e d hypocotyls had l i t t l e e f f e c t on endogenous shikimate content (84). Glyphosate, however, (1 mM) caused about a 2 0 - f o l d i n c r e a s e i n the shikimate c o n c e n t r a t i o n i n darkness and a g r e a t e r than 5 0 - f o l d i n c r e a s e i n the l i g h t . The glyphosate and l i g h t treatment r a i s e d the shikimate c o n c e n t r a t i o n i n the t i s s u e to n e a r l y 2 mM. Greater glyphosate-caused shikimate accumulation i n the l i g h t than i n the dark i n d i c a t e d that l i g h t i n c r e a s e d aromatic amino a c i d s y n t h e s i s (85). Glyphosate, even at 1 mM, d i d not i n h i b i t the growth of G. moHugo c e l l s i n a modified B5 medium i f the medium was f o r t i f i e d w i t h c a s e i n h y d r o l y s a t e (84). In the absence of exogenous amino a c i d s , 0.3 mM glyphosate i n h i b i t e d c e l l growth by 55%. Concentrations of glyphosate higher than 0.1 mM i n h i b i t e d anthraquinone production and produced an enormous accumulation of shikimate i n the c e l l s (Figure 13). G l y p h o s a t e s i n h i b i t i o n of anthraquinone production was p a r t i a l l y a l l e v i a t e d by 1 mM chorismate and 1 mM o-succinyl-benzoate, but not by 1 mM phenylalanine or t y r o s i n e , e i t h e r alone or i n combination. Chorismate alone, and the combination of phenylalanine and t y r o s i n e , i n h i b i t e d anthraquinone formation s l i g h t l y , whereas σ-succinyl-benzoate i n c r e a s e d pigment formation. Glyphosate was found to be a powerful i n h i b i t o r of the formation of a n t h r a n i l a t e from shikimate; 50% i n h i b i t i o n was achieved w i t h 5 to 7 mM exogenous glyphosate. Glyphosine, aminomethylphosphonate and i m i n o d i a c e t a t e showed no i n h i b i t o r y a c t i v i t y . A d d i t i o n a l work i n Amrhein s l a b o r a t o r y (86, 87) i n v e s t i gated the e f f e c t of glyphosate on the conversion of shikimate to a n t h r a n i l a t e . The a c t i v i t i e s of shikimate kinase (Figure 3, No. 4 ) , 5-enolpyruvylshikimate-3-phosphate synthase (Figure 3, No. 5), chorismate synthase (Figure 3, No. 6 ) , and a n t h r a n i l a t e synthase (Figure 3, No. 12) i n c e l l - f r e e e x t r a c t s of Aerobacter were s t u d i e d . Of the four enzymes i n v o l v e d i n t h i s t r a n s f o r m a t i o n , only 5-enolpyruvylshikimate-3-phosphate synthase was i n h i b i t e d by glyphosate (Table I I , F i g u r e 14). A h i g h l y s i g n i f i c a n t c o r r e l a t i o n between the accumulation of shikimate and r e d u c t i o n of anthocyanin formation i n buckwheat hypocotyls i n the presence of v a r i o u s c o n c e n t r a t i o n s of glyphosate was found. Shikimate-3-phosphate was i d e n t i f i e d as the product that accumulated i n a c e l l - f r e e g l y p h o s a t e - t r e a t e d system that e n z y m a t i c a l l y converted shikimate to a n t h r a n i l a t e . Roisch and Lingens (88) r e c e n t l y found 3-dehydroquinate synthase (Figure 3, No. 2 - a l t e r n a t e nomenclature) and phospho2-oxo-3-deoxyhepton-acetaldolase (Figure 3, No. 1 - a l t e r n a t e 1

1



196


800 4

1

1

l H 600

\

Biochemical Responses Induced by Herbicides - ACS Publications

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