Use of Subtoxic Herbicide Pretreatments to Improve Crop Tolerance

5 Use of Subtoxic Herbicide Pretreatments to Improve Crop Tolerance to Herbicides G. R. STEPHENSON and G. EZRA

Downloaded by UNIV LAVAL on July 11, 2016 | http://pubs.acs.org Publication Date: April 26, 1985 | doi: 10.1021/bk-1985-0276.ch005

Department of Environmental Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1

Antagonistic combinations of herbicides can lead to the development of seed applied chemical safeners to protect crops from herbicide injury. Another approach has been the development of chloroacetamide compounds as s e l c t i v e safeners that can be added to the formulations of thiocarbamate herbicides to improve their s e l e c t i v i t y in corn. A promising new approach involves early pretreatments with subtoxic l e v e l s of a p a r t i c u l a r herbicide to increase crop tolerance to l a t e r , higher rates of that herbicide. In a series of growth room studies, 0.001 to 0.1X pretreatments with metribuzin in tomato and pyrazon in red beets increased the tolerance of these crops to later higher l e v e l s of these p a r t i c u l a r herbicides. Pretreatments with CDAA were a l s o highly e f f e c t i v e for increasing corn tolerance to CDAA but s i m i l a r studies with EPTC, metribuzin, atrazine, chlorosulfuron, and a l a c h l o r in corn, metribuzin in soybeans, atrazine in sorghum, and chlorsulfuron in oats were less promising. Elevated substrate, enzyme a c t i v i t i e s , and detoxication rates were involved in some of the cases of improved crop tolerance v i a the e a r l i e r subtoxic pretreatments with herbicides. Chemical herbicides have been a v a i l a b l e for more than a century but major impacts on crop production awaited the development of "truely" s e l e c t i v e herbicides or innovations that would permit use of non-selective herbicides in crop situations. We now have some form of s e l e c t i v e chemical weed control for most of our major crops. However, continuing problems with herbicide injury to crops as w e l l as poor control of weeds that are b o t a n i c a l l y s i m i l a r to crops remind us that further improvements in herbicide s e l e c t i v i t y are still needed. Introductions of new s e l e c t i v e herbicides will continue but the high costs of these new chemicals are stimulating efforts to make wider use of existing herbicide chemistry. One successful approach has been to g e n e t i c a l l y improve the tolerance of new crop c u l t i v a r s to major herbicides 0097-6156/85/0276-0069$06.00/0 © 1985 American Chemical Society

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

BIOREGULATORS FOR PEST CONTROL

70


(1). A n o t h e r approach has been t o i n c r e a s e the p h y s i o l o g i c a l t o l e r a n c e o f c r o p p l a n t s t o p a r t i c u l a r h e r b i c i d e s t h r o u g h the development of chemical a n t i d o t e s or s a f e n e r s . S i n c e the i n t r o d u c t i o n o f t h i s l a t t e r concept by O t t o Hoffman (2) i n the e a r l y 1960's, a t l e a s t f i v e a n t i d o t e s o r s a f e n e r s h a v e been developed commercially (Table I). R e s e a r c h on c h e m i c a l a n t i d o t e s o r s a f e n e r s has been summarized i n s e v e r a l r e v i e w s and p u b l i s h e d symposia (3.-9)· Most o f the major d e v e l o p m e n t s ( T a b l e I) h a v e r e s u l t e d from i m p i r i c a l s c r e e n i n g programs by I n d u s t r y t h a t may h a v e been s t i m u l a t e d by o b s e r v a t i o n s o f h e r b i c i d e antagonism i n p l a n t s (.3, 10). However, some o f t h e r e s e a r c h on mode o f a c t i o n o f a n t i d o t e s has been d i r e c t e d a t f i n d i n g new ways t o p r o t e c t crop p l a n t s from h e r b i c i d e s (3). The r e s e a r c h t o be d i s c u s s e d i n t h i s t e x t , namely the use o f s u b t o x i c h e r b i c i d e p r e t r e a t m e n t s to improve crop t o l e r a n c e to s e l e c t e d h e r b i c i d e s , a r i s e s i n p a r t from r e s e a r c h on the mode o f a c t i o n o f R-25788 as a s e l e c t i v e a n t i d o t e f o r EPTC o r b u t y l a t e i n corn. Mode o f A c t i o n o f R-25788 In some o f the e a r l i e s t r e s e a r c h , W i l k i n s o n (11) s u g g e s t e d t h a t o p p o s i n g a c t i o n s o f EPTC and R-25788 on l i p i d s y n t h e s i s i n p l a n t s c o u l d e x p l a i n t h e i r mechanisms as a h e r b i c i d e and a n t i d o t e , respectively. E z r a et a l . (12) h a v e p r o v i d e d s t r o n g support f o r t h i s i d e a w i t h t h e i r o b s e r v a t i o n s t h a t EPTC and R-25788 c o u l d h a v e m e a s u r a b l e and o p p o s i t e e f f e c t s on l i p i d s y n t h e s i s as e a r l y as 1 h r a f t e r treatment i n c o r n c e l l s u s p e n s i o n c u l t u r e s . Other r e s e a r c h by Lay and C a s i d a (13) has s u p p o r t e d the concept t h a t R25788 enhances the m e t a b o l i c d e t o x i c a t i o n o f EPTC and i t s s u l f o x i d e by e l e v a t i n g the g l u t a t h i o n e (GSH) l e v e l s and GSH-S.t r a n s f e r a s e a c t i v i t y i n v o l v e d i n the c o n j u g a t i o n o f EPTC-su I f o x i d e with glutathione. E a r l y r e s e a r c h i n our l a b o r a t o r y on a n t i d o t e s t r u c t u r e / a c t i v i t y r e l a t i o n s h i p s (14. 15) e s t a b l i s h e d t h a t acetamide and carbamate m o l e c u l e s more s i m i l a r i n s t r u c t u r e t o EPTC t h a n to R-25788 c o u l d h a v e e q u a l o r even g r e a t e r a n t i d o t e a c t i v i t y a g a i n s t EPTC i n c o r n w i t h s o i l f r e e b i o a s s a y systems. The s t r u c t u r e / a c t i v i t y s t u d i e s seemed to support the i d e a t h a t the a n t i d o t e R-25788 (or i t s a n a l o g u e s ) may a c t as a c o m p e t i t i v e i n h i b i t o r a t s i t e ( s ) o f EPTC a c t i o n i n c o r n . These l a t t e r two t h e o r i e s o f " a n t i d o t e enhanced h e r b i c i d e m e t a b o l i s m " v e r s u s " c o m p e t i t i v e i n h i b i t i o n " were h a r d to r e c o n c i l e w i t h each o t h e r . To r e s o l v e t h i s apparent d i s a g r e e m e n t , a s e r i e s o f acetamide a n a l o g u e s w i t h known a n t i d o t e a c t i v i t i e s were t e s t e d f o r t h e i r e f f e c t s on g l u t a t h i o n e l e v e l s i n c o r n ( F i g u r e 1). In t h i s c o m p a r a t i v e s t u d y , the d i c h l o r o d i a l l y l acetamide (R-25788) was s l i g h t l y more e f f e c t i v e as an a n t i d o t e f o r EPTC i n c o r n than was the m o n o c h l o r o d i a l l y l acetamide (CDAA) and the t r i c h l o r o and nonc h l o r i n a t e d a n a l o g u e s were l e a s t e f f e c t i v e ( F i g u r e l a ) . S u r p r i s i n g l y , t h i s r e l a t e d s e r i e s o f a c e t a m i d e s had v i r t u a l l y the same spectrum o f e f f e c t s on g l u t a t h i o n e l e v e l s i n c o r n s e e d l i n g s ( F i g u r e l b ) . The i n c l u s i o n o f CDAA, (Ν,Ν-diallyl-2c h l o r o a c e t a m i d e ) i n t h i s s t u d y was v e r y f o r t u n a t e . T h i s c h e m i c a l has been a w i d e l y u s e d h e r b i c i d e f o r weed c o n t r o l i n o n i o n s and at h i g h r a t e s i t can a l s o be t o x i c t o c o r n w i t h symptoms not u n l i k e


5.

Table I .

C o m m e r c i a l l y important h e r b i c i d e


C h e m i c a l name

Application

Crops

antidotes

protected

or

safeners.

Herbicides

seed

corn sorghum wheat oats rice

carbamates chloroacetanilides thiocarbamates others

seed o r herbicide formulation

corn sorghum barley wheat rice

thiocarbamates acetanilides carbamates dithiocarbamates others

1,8-naphthalic anhydride (NA)

N,N-diallyl-2,2dichloroacetamide (R-25788)

71

Crop Tolerance to Herbicides

STEPHENSON AND EZRA

a-[(cyanomethoxy) seed imino b e n z a c e t o n i t r i l e ] (cyoxymetrinil)

sorghum wheat rice

chloroacetanilides

a-(l,3-dioxolan-2-ylmethoxy)iminobenzacetonitrile (CGA-92194)

seed

sorghum

chloroacetanilides

2-chloro-2-4seed (trifluoromethyl)-5thiazolecarboxylic acid, benzyl ester (f lurazole)

sorghum

chloroacetanilides

From Stephenson and E z r a

(3.) ·




0.9-

LSD 0.05 (a)

g 80o u. ο g60-

«»9 \ CO fo.6-

LSD 0.05 (b)

S2 40-J

8 20^

ζ

ο ο

x CO

8

ANTIDOTE TREATMENT (I0" M) CALL PLUS EPTC (I0" M)] 4

ANTIDOTE TREATMENT

(ΙΟ^Μ)

4

F i g u r e 1· C o m p a r a t i v e e f f e c t s o f f o u r d i a l l y l acetamides as a n t i d o t e s t o EPTC ( l e f t ) and on GSH l e v e l s ( r i g h t ) i n c o r n s e e d l i n g s . The v a r i o u s d i a l l y l acetamides [R-COlKCHjCH-CI^l were R=CH -, CH C1-, C H C 1 - and CCI3 f o r A, B, C, and D, r e s p e c t i v e l y . A n t i d o t e Β i s b e t t e r known as t h e h e r b i c i d e CDAA. A n t i d o t e C i s t h e a n t i d o t e R-25788 which has been d e v e l o p e d c o m m e r c i a l l y f o r EPTC o r b u t y l a t e . Reproduced w i t h p e r m i s s i o n from Ref. 17. C o p y r i g h t 1983 Pergamon P r e s s . 3

2

2



5.

STEPHENSON AND EZRA

73


t h o s e o f EPTC. However, a t lower r a t e s i t i s an e f f e c t i v e a n t i d o t e o r s a f e n e r f o r EPTC i n c o r n ( F i g u r e l a , Compound B). F u r t h e r m o r e , i n t h e s e s t u d i e s we l e a r n e d t h a t l i k e R-25788 i t would a l s o e l e v a t e g l u t a t h i o n e l e v e l s i n c o r n ( F i g u r e l b , Compound B). A l s o , l i k e EPTC, i t i s known t o be d e t o x i f i e d i n p l a n t s by c o n j u g a t i o n w i t h g l u t a t h i o n e (16). With t h i s c o m b i n a t i o n o f knowledge, i t seemed more l i k e l y t h a t CDAA was a c t i n g as a l e s s p h y t o t o x i c mimic o f EPTC i n s t e a d o f as a c o m p e t i t i v e i n h i b i t o r o f EPTC. I t seemed p o s s i b l e t h a t a t low r a t e s , b o t h CDAA and EPTC might be s t i m u l a t o r y on p r o c e s s e s such as l i p i d s y n t h e s i s , but a t h i g h r a t e s , they might b o t h be i n h i b i t o r y . I f the r e q u i r e d r a t e s f o r t h e two compounds were d i f f e r e n t , i t might be p o s s i b l e f o r a s t i m u l a t o r y c o n c e n t r a t i o n o f CDAA ( o r R-25788) t o overcome i n h i b i t o r y e f f e c t s o f EPTC. F u r t h e r m o r e , i f b o t h CDAA and EPTCs u I f o x i d e were c o n j u g a t e d w i t h g l u t a t h i o n e , i t seemed p l a u s i b l e t h a t t h e a n t i d o t e (CDAA) may a c t by enhancing t h e same pathway needed t o d e t o x i f y EPTC and i t s s u l f o x i d e - namely t h e GSH and G S H - S - t r a n s f e r a s e system. One o t h e r q u e s t i o n a l s o emerged. I f CDAA c o u l d e l e v a t e GSH l e v e l s c o u l d o t h e r h e r b i c i d e s , known t o be c o n j u g a t e d w i t h GSH, a l s o e l e v a t e GSH l e v e l s ? Some o f t h e s e q u e s t i o n s h a v e n o t y e t been examined b u t t h e l a t t e r s p e c u l a t i o n p r o v e d t o be c o r r e c t . I n a d d i t i o n t o CDAA, f i v e o t h e r h e r b i c i d e s were shown t o e l e v a t e g l u t a t h i o n e l e v e l s i n c o r n s e e d l i n g s ( T a b l e I I ) and t h e m e t a b o l i s m o f a l l t h e s e h e r b i c i d e s i s known t o i n v o l v e g l u t a t h i o n e c o n j u g a t i o n (18). Table

I I . E f f e c t s o f R-25788 and v a r i o u s h e r b i c i d e s on g l u t a t h i o n e (GSH) l e v e l s i n r o o t s o f 5 - d a y - o l d c o r n s e e d l i n g s .

3 Root (GSH)

Treatments

R-25788 EPTC alachlor propachlor atrazine barban

C o n t e n t i (u moles/g fw) 5

0

10" M

10" M

10-*M

0.19 0.19 0.30 0.20 0.18 0.19

0.31 0.29 0.41 0.29 0.22 0.41

0.48 0.45 0.49 0.35 0.31 0.48

0.71 0.41 0.71 0.42 0.34 0.86

6

From Stephenson e t . a l . ( 1 7 ) . * ^ 3

F o r a l l experiments LSD's were 0.05 u moles/g o r l e s s . H i g h r a t e o f barban caused a s i g n i f i c a n t r e d u c t i o n i n r o o t fw. The m e t a b o l i s m o f a l l o f t h e s e c h e m i c a l s except R-25788, i s known t o i n v o l v e c o n j u g a t i o n w i t h g l u t a t h i o n e i n p l a n t s (18).




I n c r e a s i n g Crop T o l e r a n c e t o H e r b i c i d e s w i t h H e r b i c i d e Pretreatments At t h i s p o i n t , e v i d e n c e t h a t s i m i l a r m o l e c u l e s a c t e d as e f f e c t i v e a n t i d o t e s by i n d u c i n g needed m e t a b o l i c pathways f o r h e r b i c i d e d e t o x i c a t i o n was a t most v e r y s p e c u l a t i v e . However, another h y p o t h e s i s emerged. C o u l d e a r l y h e r b i c i d e p r e t r e a t m e n t s i n c r e a s e crop t o l e r a n c e t o t h e s e h e r b i c i d e s by e l e v a t i n g t h e s u b s t r a t e s and enzymes needed f o r d e t o x i c a t i o n ? W h i l e n o t a new concept i n a n i m a l systems, such an i d e a has r e c e i v e d l i t t l e a t t e n t i o n i n p l a n t systems and i t c e r t a i n l y has n o t been e x p l o i t e d i n any p r a c t i c a l way. The w h o l e i d e a has seemed much more c r e d i b l e w i t h the study by J a c e t t a and R a d o s e v i c h (19) o f p h o t o s y n t h e t i c r e c o v e r y i n c o r n a f t e r treatment w i t h a t r a z i n e . More s p e c i f i c a l l y , t h e y showed t h a t i n h i b i t i o n o f p h o t o s y n t h e s i s was reduced and t h e r a t e o f r e c o v e r y enhanced i n c o r n p l a n t s t r e a t e d f o r t h e second o r t h i r d time w i t h a t r a z i n e compared t o " f i r s t exposed" p l a n t s ( F i g u r e 2). F u r t h e r m o r e , t h e f a s t e r r e c o v e r y was r e l a t e d t o enhanced r a t e s o f a t r a z i n e m e t a b o l i s m i n t h e p r e v i o u s l y treated plants (Table I I I ) . S t i m u l a t e d by the a n t i d o t e r e s e a r c h , as w e l l as by the work of J a c e t t a and R a d o s e v i c h (19). we d e c i d e d to examine t h e i n f l u e n c e of pretreatment with subtoxic r a t e s of s e v e r a l h e r b i c i d e s on l a t e r crop t o l e r a n c e t o t h e s e same h e r b i c i d e s . F o r t h e s e growth room s t u d i e s (25°C/18°C, 16h p h o t o p e r i o d , l i g h t i n t e n s i t y 400 uE/m / s e c , 75% r e l a t i v e h u m i d i t y ) , p r e t r e a t m e n t s a t c o n c e n t r a t i o n s o f 0.1% t o 10% o f t h e f i n a l h e r b i c i d a l r a t e were g i v e n as a r o o t drench o r seed treatment t o seeds p l a n t e d i n m o i s t v e r m i c u l i t e i n s t y r o f o a m cups. H e r b i c i d a l t r e a t m e n t s were l a t e r a p p l i e d t o t h e r o o t s . P l a n t s were h a r v e s t e d 8-14 days a f t e r h e r b i c i d e treatment ( T a b l e I V ) . P o s s i b l y t h e most s u r p r i s i n g r e s u l t s from t h e s e s t u d i e s were those with a t r a z i n e . E v e n though J a c e t t a and R a d o s e v i c h (19) had o b s e r v e d reduced p h o t o s y n t h e t i c i n j u r y and enhanced a t r a z i n e m e t a b o l i s m w i t h a t r a z i n e p r e t r e a t m e n t s , i n our s t u d i e s , p r e t r e a t m e n t s w i t h a t r a z i n e d i d not p r e v e n t l a t e r d r y weight r e d u c t i o n s from h i g h r a t e s o f a t r a z i n e . We were a l s o s u r p r i s e d t h a t , i n s p i t e o f e f f e c t s on GSH l e v e l s , EPTC p r e t r e a t m e n t s f a i l e d to reduce l a t e r EPTC i n j u r y t o c o r n . Thus f a r , a n t i d o t e r e s e a r c h has had l i t t l e impact on i m p r o v i n g t h e t o l e r a n c e o f b r o a d l e a v e d crops to h e r b i c i d e s . On t h a t b a s i s we were q u i t e encouraged by the e f f e c t i v e n e s s o f m e t r i b u z i n p r e t r e a t m e n t s f o r r e d u c i n g l a t e r m e t r i b u z i n i n j u r y t o tomatoes ( T a b l e I V , F i g u r e 3) and soybeans ( T a b l e I V ) . P y r a z o n p r e t r e a t m e n t s were s l i g h t l y more e f f e c t i v e f o r p r e v e n t i n g pyrazon i n j u r y t o r e d b e e t s ( T a b l e I V ) , but t h e most p r o m i s i n g r e s u l t s were o b t a i n e d w i t h CDAA ( T a b l e IV, F i g u r e s 4, 5). Corn shoot d r y weights were r e d u c e d 40% by CDAA a p p l i e d a t c o n c e n t r a t i o n s o f 200 uM, 5 days a f t e r p l a n t i n g i n v e r m i c u l i t e nutrient culture. Optimum p r e v e n t i o n o f CDAA i n j u r y was o b s e r v e d w i t h a CDAA p r e t r e a t m e n t o f 5 uM, a p p l i e d 2.5 days e a r l i e r . However, s i g n i f i c a n t p r e v e n t i o n o f i n j u r y was a l s o o b s e r v e d w i t h the 1 and 10 uM p r e t r e a t m e n t s ( F i g u r e 4).



STEPHENSON AND EZRA

0

J

1

1


4

1

1

1

1

\

1

1

1

8 12 16 20 TIME (xlOOmin)

1

1

24

1

1

1

28

F i g u r e 2. I n h i b i t i o n and r e c o v e r y o f p h o t o s y n t h e t i c r a t e i n c o r n w i t h 3 s u c c e s s i v e exposures t o a t r a z i n e [ I l l u s t r a t e d by one r e p r e s e n t a t i v e i n f r a r e d gas a n a l y z e r t r a c e , + and - r e f e r t o p r e s e n c e o r absence o f a t r a z i n e ] . "Reproduced w i t h p e r m i s s i o n from Ref. 19. C o p y r i g h t 1981, 'Weed S c i e n c e S o c i e t y of America ". 1

F i g u r e 3. P r e v e n t i o n o f m e t r i b u z i n (0.5 mg/L) i n j u r y t o tomatoes w i t h m e t r i b u z i n p r e t r e a t m e n t s (1.0 ug/L). Left to r i g h t , c o n t r o l , 1.0 ug/L p r e t r e a t m e n t f o l l o w e d by 0.5 ng/L h e r b i c i d a l treatment 14 days l a t e r .



1

0 12 12 12 12 12

Recovery period

4 4 4

1

1

Atrazine exposure

Treatment

0 12 12 12

Recovery period

4

1

Atrazine exposure

0 12

Recovery period

1

a

S u p p l i e d as ^ C - a t r a z m e .

1

V a l u e s a d j a c e n t t o t h e same l e t t e r l e v e l of significance.

extracted

3

i n a column a r e n o t d i f f e r e n t

43.4a 54.6a 49.3a 67.9b 59.3a 72.7b

56.6a 45.4a 50.7a 32.1b 40.7a 27.3b

a t t h e 5%

%

Aqueous Chloroform fraction fraction

DPM

Occurrence o f i n c h l o r o f o r m and aqueous f r a c t i o n s o f c o r n s h o o t s a f t e r 1, 2, and 3 s u c c e s s i v e a t r a z i n e t r e a t m e n t s .

"Reproduced w i t h p e r m i s s i o n from Ref. 19. C o p y r i g h t 1981, 'Weed Science Society of America ."

4 4 4 4 4

Atrazine exposure

Table I I I .


Ο

73

m C/3 Η ο Ο Η

73

Ο

τι

73

I

73 m Ο a r

δ

00

ON

5.

STEPHENSON AND EZRA

Table

IV.

E f f e c t s o f h e r b i c i d e p r e t r e a t m e n t s on subsequent h e r b i c i d e i n j u r y to crops.


Herbicide

Treatment Concentrations PreHerbicidal-

20uM

76uM

CDAA

5uM

200uM

Atrazine

5uM

50mM

5uM

50mM

50pM

ΙΟηΜ

Alachlor

Chlorsulfuron

8.5nM 15uM

EPTC Metribuzin

Pyrazon

11


50uM 200uM

4.6nM

2uM

4.6nM

luM

0.5uM

lOOuM

ΙΟ.ΟηΜ

20uM

Crop

Prevention of i n j u r y by s u b t o x i c pretreatment Dry Wt. Height

corn ( c v PAG SX-111) corn ( c v PAG SX-111) sorghum ( c v De K a l b E59+) corn (cv PAG SX-111) corn ( c v PAG SX-111) oats (cv c e n t i n n e l ) corn ( c v PAG SX-111) tomatoes ( c v H2653) soybeans (cv EVANS) corn ( c v PAG SX-111) beets (cv D e t r o i t Dark Red)

++

+ +++

+

-

0

0

0

+

0

+

0

0

+

-

+ 0

+

++

-

R e s u l t s a r e from a t l e a s t two experiments. 0 = l e s s than 5%, + - 5-10% r e d u c t i o n i n i n j u r y , ++ - 11-20% r e d u c t i o n i n i n j u r y , +++ « g r e a t e r than 20% r e d u c t i o n i n i n j u r y . Not r e c o r d e d i n t h e s e e x p e r i m e n t s .



PLANT DRY WEIGHT

PLANT HEIGHT

100-

1*1

ii

80

til

ii


60-

PRETREATMENT 0 0 0.5 1.0 50 10.0 HERBICIDE 0 200200200200200

0 0

0 0.5 1.0 5.0 10.0 200200200200 200

CDAA CONCENTRATION (μΜ) F i g u r e 4. P r e v e n t i o n o f CDAA i n j u r y t o c o r n w i t h CDAA p r e t r e a t m e n t s a p p l i e d 2.5 days e a r l i e r . No i n j u r y was o b s e r v e d w i t h t h e pretreatmentβ a p p l i e d a l o n e except a t t h e h i g h e s t c o n c e n t r a t i o n (50.0 uM).

F i g u r e 5. P r e v e n t i o n o f i n j u r y t o c o r n from CDAA a t 200 uM w i t h 5 uM CDAA p r e t r e a t m e n t s . L e f t t o r i g h t , c o n t r o l , 200 uM CDAA a l o n e , 200 uM CDAA f o l l o w i n g 5 uM CDAA, and 5 uM CDAA alone.


5.

STEPHENSON AND EZRA

79


E f f e c t o f CDAA p r e t r e a t m e n t s

on subsequent CDAA m e t a b o l i s m

Corn seeds were germinated between m o i s t paper t o w e l s and were then t r e a t e d w i t h 5 uM CDAA e i t h e r 1 o r 2.5 days p r i o r t o a s s a y f o r GSH a f 6 days a f t e r p l a n t i n g ( T a b l e V). U s i n g t h e r e a g e n t DTNB [2,2 - d i t h i o b i s ( 2 - n i t r o b e n z o i c a c i d ) ] , r o o t GSH c o n t e n t s were assayed s p e c t r o p h o t o m e t r i c a 1 l y as p r e v i o u s l y d e s c r i b e d (20). Treatment w i t h CDAA ( a t 5 uM) f o r e i t h e r 1 o r 2.5 days e l e v a t e d GSH l e v e l s more than 60% ( T a b l e V).

T a b l e V.

E f f e c t o f CDAA on GSH l e v e l s

i n 6 day o l d c o r n s e e d l i n g s .


GSH c o n t e n t i n c o r n r o o t s (ug/g

Control

5 uM CDAA f o r 2.5 days

5 uM CDAA f o r 1 day

65+10

106+6

Reproduced w i t h Press.

fw)

109+11

p e r m i s s i o n from R e f . 20.

Copyright

1985 A c a d e m i c

CDAA a l s o e l e v a t e d G S H - S - t r a n s f e r a s e a c t i v i t y i n c o r n r o o t s o r shoots ( T a b l e V I ) when assayed s p e c t r o p h o t o m e t r i c a 1 l y w i t h CDNB ( l - c h l o r o - 2 , 4 - d i n i t r o b e n z e n e ) (20). However, i n t h i s c a s e , treatment w i t h CDAA f o r 2.5 days produced t h e g r e a t e s t i n c r e a s e i n enzyme a c t i v i t y i n t h e r o o t s o f 6 - d a y - o l d c o r n s e e d l i n g s .

T a b l e V I . E f f e c t o f CDAA p r e t r e a t m e n t s on GSH-Sj-transferase a c t i v i t y i n r o o t s or shoots o f 6-day-old c o r n s e e d l i n g s (c.v. PAG SX-111). 5 uM CDAA time

Pretreatment (days)

0 1 2.5 6

GSH-S-transferase s p e c i f i c a c t i v i t y (nmol/min/mg P r o t e i n ) Shoots Roots 1094 1486 1638 1371

276 315 258 316

A l t h o u g h CDAA p r e t r e a t m e n t s e l e v a t e d b o t h GSH and GSH-S.t r a n s f e r a s e a c t i v i t y (as assayed by CDNB), i t was e s s e n t i a l t o determine whether t h e s e e f f e c t s would a c t u a l l y r e s u l t i n g r e a t e r CDAA m e t a b o l i s m . F o r t h e s e s t u d i e s , we examined [ C]CDAA m e t a b o l i s m i n v i t r o and i n e x c i s e d c o r n r o o t t i p s [ d e t a i l e d methods d e s c r i b e d by E z r a e t . a l . (20)1. The i n v i t r o a s s a y r e v e a l e d a s i g n i f i c a n t l e v e l o f non-enzymatic [ C] CDAA



80

d e g r a d a t i o n [as determined by p a r t i t i o n i n g o f water s o l u b l e m e t a b o l i t e s from m e t h y l e n e c h l o r i d e i n t o water and v e r i f i c a t i o n by t h i n l a y e r chromatography]. However, t h e enzymatic r a t e was d o u b l e t h a t o f t h e non-enzymatic r a t e . Furthermore, a 2.5 day p r e t r e a t m e n t w i t h 5uM CDAA i n c r e a s e d t h e GSH-Sr-transferase a c t i v i t y f o r [ C ] C D A A m e t a b o l i s m from 62.9+4.6 t o 78.9+.5.3 nmol/min/mg p r o t e i n (20). M e t a b o l i s m o f [ C] CDAA was v e r y r a p i d i n e x c i s e d r o o t t i p s from c o n t r o l o r p r e t r e a t e d r o o t t i p s . However, i n a l l experiments, p r e t r e a t e d r o o t s took up 2 - f o l d more [ C]CDAA and m e t a b o l i z e d i t t w i c e as f a s t as n o n - p r e t r e a t e d r o o t tissue. 14


E f f e c t s o f R-25788 and EPTC on t h e GSH/GSH-S-transferase System In a s e r i e s o f experiments, s i m i l a r t o t h o s e w i t h CDAA, R-25788 and EPTC were examined as p r e t r e a t m e n t s t o e i t h e r enhance t h e i r own m e t a b o l i s m by GSH-S_-transferase o r p r e v e n t l a t e r i n j u r y (from t o x i c doses) i n c o r n s e e d l i n g s . The e f f e c t s o f R-25788 on e l e v a t i o n o f GSH and GSH-St r a n s f e r a s e h a v e been w e l l documented (Γ3). However, u n l i k e CDAA, R-25788 p r e t r e a t m e n t s d i d not p r e v e n t o r reduce i n j u r y t o c o r n s e e d l i n g s from l a t e r h i g h e r doses o f R-25788 ( F i g u r e 6). T h i s may be e x p l a i n e d by t h e f a c t t h a t even though R-25788 e l e v a t e s GSH and GSH-S_-t r ans f e r a s e , i t i s n o t a c t u a l l y m e t a b o l i z e d by t h i s system. I n f a c t , R-25788 has been shown t o be d e t o x i f i e d i n c o r n , p r e d o m i n a n t l y t o Ν,Ν-diallyloxamic a c i d . However, t h e r e i s some N - d e a l k y l a t a t i o n and h y d r o l y s i s to d i c h l o r o a c e t i c a c i d and some g l y c o s i d e f o r m a t i o n (21). Even though EPTC p r e t r e a t m e n t s were i n e f f e c t i v e f o r p r e v e n t i n g EPTC i n j u r y ( T a b l e I V ) , EPTC d i d i n f l u e n c e t h i s same system. A t 10 ppm (5 χ 10"^ M), EPTC e l e v a t e d b o t h t h e GSH c o n t e n t ( F i g u r e 7) and G S H - S - t r a n s f e r a s e a c t i v i t y ( F i g u r e 8). However t h e s e e l e v a t i o n s were n o t as g r e a t and were not as p e r s i s t e n t as those o b t a i n e d w i t h R-25788. More s p e c i f i c a l l y , GSH and GSH-S_-transferase a c t i v i t y were e l e v a t e d by more than 50% f o r a t l e a s t 4-days w i t h R-25788 ( F i g u r e 8), whereas t h i s magnitude o f e l e v a t i o n o c c u r r e d o n l y b r i e f l y , 2-days a f t e r EPTC p r e t r e a t m e n t and then d e c l i n e d . I n o t h e r s t u d i e s ( u n p u b l i s h e d ) we h a v e o b s e r v e d no e f f e c t o f EPTC p r e t r e a t m e n t s on [ C ] E P T C metabolism i n excised corn root t i p s . E f f e c t s o f EPTC p r e t r e a t m e n t s on [ C]EPTC s u l f o x i d e m e t a b o l i s m h a v e not y e t been examined · One p o s s i b l e e x p l a n a t i o n f o r t h e i n e f f e c t i v e n e s s o f EPTC p r e t r e a t m e n t s f o r enhancing EPTC m e t a b o l i s m and f o r r e d u c i n g EPTC i n j u r y i s t h a t i t s c o n j u g a t i o n w i t h g l u t a t h i o n e may be p r i m a r i l y non-enzymatic as suggested by o t h e r r e s e a r c h e r s (22). However i n o t h e r r e c e n t r e s e a r c h ( u n p u b l i s h e d ) we h a v e shown t h a t a known GSH-jr-transferase i n h i b i t o r i s s y n e r g i s t i c a 1 l y p h y t o t o x i c w i t h EPTC i n c o r n , i n d i c a t i n g t h a t non-enzymatic c o n j u g a t i o n may n o t be t h a t important i n c o r n . A more l i k e l y e x p l a n a t i o n f o r the l a c k o f p r o t e c t i o n w i t h EPTC p r e t r e a t m e n t s , i s t h a t e f f e c t s on GSH and GSH-S_-traneferase a r e t o o t r a n s i e n t i n n a t u r e , and/or t h a t s u l f o x i d a t i o n i s an important and p o s s i b l y r a t e - l i m i t i n g s t e p . 1 4


STEPHENSON AND EZRA


PLANT DRY WEIGHT

PLANT HEIGHT ιοο-

ο ο £r or

Use of Subtoxic Herbicide Pretreatments to Improve Crop Tolerance

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