Biochemical Responses Induced by Herbicides 9780841206991, 9780841208742, 0-8412-0699-6

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Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.fw001

Biochemical Responses Induced by Herbicides

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.fw001

Biochemical Responses Induced by Herbicides Donald

E.

Moreland,

EDITOR

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.fw001

U.S. Department of Agriculture

Judith B. St. John,

EDITOR

U.S. Department of Agriculture

F. Dana Hess,

EDITOR

Purdue University

Based on a symposium sponsored by the Division of Pesticide Chemistry at the 181st ACS National Meeting, Atlanta, Georgia, March 29-April 3, 1981.

ACS SYMPOSIUM

AMERICAN WASHINGTON,

SERIES

CHEMICAL D. C .

181

SOCIETY 1982

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.fw001

Library of Congress

Data

Biochemical responses induced by herbicides. (ACS symposium series, ISSN 0097-6156; 181) Includes bibliographies and index. 1. Plants, Effect of herbicides on—Congresses. 2. Herbicides—Physiological effect—Congresses. I. Moreland, Donald E., 1919- . II. St. John, Judith, 1940- . III. Hess, F. Dana, 1946- . IV. American Chemical Society. Division of Pesticide Chemistry. V. Series. QK753.H45B56 581.2'4 81-20645 ISBN 0-8412-0699-6 AACR2 ACSMC8 181 1-274 1982

Copyright © 1982 American Chemical Society All Rights Reserved. The appearance of the code at the bottom of the first page of each article in this volume indicates the copyright owner's consent that reprographic copies of the article may be made for personal or internal use or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, for creating new collective work, for resale, or for information storage and retrieval systems. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission, to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. PRINTED IN THE UNITED STATES OF AMERICA

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.fw001

ACS Symposium Series M. Joan Comstock, Series Editor

Advisory Board David L. Allara

Marvin Margoshes

Robert Baker

Robert Ory

Donald D. Dollberg

Leon Petrakis

Robert E. Feeney

Theodore Provder

Brian M. Harney

Charles N. Satterfield

W. Jeffrey Howe

Dennis Schuetzle

James D. Idol, Jr.

Davis L. Temple, Jr.

Herbert D. Kaesz

Gunter Zweig

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.fw001

FOREWORD The ACS SYMPOSIUM SERIES was founded in 1974 to provide

a medium for publishing symposia quickly in book form. The format of the Series parallels that of the continuing ADVANCES IN CHEMISTRY SERIES except that in order to save time the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. Papers are reviewed under the supervision of the Editors with the assistance of the Series Advisory Board and are selected to maintain the integrity of the symposia; however, verbatim reproductions of previously published papers are not accepted. Both reviews and reports of research are acceptable since symposia may embrace both types of presentation.

PREFACE

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.pr001

T

he symposium at which the papers in this volume were presented provided an opportunity to present, discuss, and publish a comprehensive account of the current status of research concerned with basic biochemical and physiological responses associated with the phytotoxic action of herbicides. Several of the contributions consider interferences by herbicides with photoinduced electron transport in chloroplasts. These papers include such topics as sites of interference on the electron transport pathway, identification and biogenesis of the photosystem II (PS II) triazine-binding protein and lipid composition in triazine-susceptible and triazine-resistant interactions between herbicides and the PS II binding site, the relation between interference with photoinduced electron transport and the expression of phy to toxicity, differences between properties of the triazine-binding protein and lipid composition in triazine-susceptible and triazine-resistant bio types of weed species, and alterations to the permeability properties and lipid composition of organelle membranes induced by herbicides, some of which are PS II inhibitors. Other contributions report on the activation of diphenyl ether herbicides by light, effects of herbicides and growth regulators on pigment metabolism, the complex metabolic alterations induced by glyphosate, the use of unicellular green algae to differentiate between physiological modes of herbicidal action, and procedural details that can be used to differentiate between effects imposed on cellular division and cellular enlargement.

As evidenced by the contents of the papers, considerable progress has been made in recent years in the identification of mechanisms and modes of action of herbicides. However, the elucidation of the mechanisms of action of herbicides at the cellular and molecular levels, together with the translation of the primary perturbations to the mode of action that leads to the expression of phytotoxicity, will continue to challenge the ingenuity of investigators for many years. A clear insight into the physiological and biochemical mechanisms through which herbicides operate depends on the development of a better comprehension of growth, and the factors through which it is controlled, at the molecular level. Each year background information increases, as does knowledge of the regulation of subcellular metabolism, which helps clarify the action of herbicides. Consequently, future symposia can be expected to witness the report of substantial and exciting progress. ix

We would like to acknowledge the cooperation and enthusiasm provided by the speakers with their presentations, their participation in the symposium, and in the development of their manuscripts. We are especially grateful for the assistance in editing, typing, andfinalizationof the manuscripts that was provided by Linda M . Lynch and William P. Novitzky. DONALD E . MORELAND

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.pr001

U.S. Department of Agriculture Agricultural Research Service Departments of Crop Science and Botany North Carolina State University Raleigh, NC 27650 JUDITH B. ST. JOHN

U.S. Department of Agriculture Beltsville Agricultural Research Center Beltsville, MD 20705 F. DANA HESS

Purdue University Department of Botany and Plant Pathology West Lafayette, IN 47907 October 13, 1981

x

1 Photosystem II Inhibiting Chemicals Molecular Interaction Between Inhibitors and a Common Target

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch001

C. J. V A N ASSCHE and P. M . CARLES ROUSSEL U C L A F , C.R.B.A. PROCIDA, Crop Protection, Scientific Division, St. Marcel, F. 13011 Marseille, France

From several lines of evidence (fluorescence and chemical-triggered luminescence measurements, competition studies, and mild trypsin digestion) various chemical families of PS II inhibitors appear to act the same way and upon a common site associated with the reducing side of photosystem II and the Β protein, a specialized plastoquinone­ -protein complex. A model, using molecular orbital approaches, is proposed that is consistent with a hypothesis for the existence of hydrogen bonds between inhibitors and a simulated proteinaceous target. Short- and long-range intermolecular energies within the micro-environment of the "receptor" Β - protein complex show that in cal­ culating reciprocal interactions between inhibi­ tors and the simulated binding site, polarization and dispersion components are relatively more important than electrostatic energy. Many commercially a v a i l a b l e h e r b i c i d e s have been demon­ s t r a t e d to i n t e r f e r e with one or more steps of photosynthesis, by r e a c t i n g near the photosystem II (PS II) center [for a recent review see (_1 ) , among o t h e r s ] . DCMU (2) and other chemical f a m i l i e s of photosynthetic i n h i b i t o r s (3, 4·) were shown to s h i f t the p o t e n t i a l of the PS I I secondary e l e c t r o n acceptor B , a s p e c i a l i z e d plastoquinone molecule, bound to a p r o t e i n Ç5). PS II i n h i b i t o r s b i n d to a hydrophobic domain of a 32 Kdalton polypeptide considered e i t h e r to be the B - p r o t e i n or to be associated c l o s e l y with i t (6). The exact nature of the molecular i n t e r a c t i o n between i n h i b i t o r s and target has not been e l u c i d a t e d ; however, a model was proposed, using a molecular o r b i t a l approach (7), i n which support was given to the p o s t u l a t e that hydrogen bonds were i n v o l v e d i n the i n t e r a c t i o n between i n h i b i t o r s and a simulated t a r g e t , i . e . , a d i p e p t i d e u n i t . This paper w i l l describe r e s u l t s obtained with the approach presented

0097-6156/82/0181 -0001 $05.25/0 © 1982 American Chemical Society

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

2

p r e v i o u s l y (7) f o r DCMU-type i n h i b i t o r s and f o r compounds that e x h i b i t a d i f f e r e n t s i t e of a c t i o n . These s t u d i e s were conducted to determine i f the compounds i n t e r f e r e d w i t h the same molecular t a r g e t . A multimethodological approach w i l l be described h e r e i n f o r t h i s purpose. Evidence of a Common M o l e c u l a r Target f o r Various I n h i b i t o r Chemical F a m i l i e s I n d i r e c t Measurements o f E f f e c t s on PS I I Primary E l e c t r o n Acceptor. When photosynthetic o r g a n e l l e s (microalgae or c h l o r o p l a s t s ) are i l l u m i n a t e d , e l e c t r o n s e x t r a c t e d from water are t r a n s ­ ported to a f i n a l e l e c t r o n acceptor NADP which becomes reduced. Intermediary e l e c t r o n acceptors of the photosynthetic e l e c t r o n t r a n s p o r t chain undergo v a r i o u s redox t r a n s i e n t s . T h i s chain may be represented simply as f o l l o w s :

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch001

+

H C-^S — • S ^ IU ± 0

•S -^S -^S,-^PS I I — • Q -•B—*-PQ -•PS L J i\

1

0

0

I—•NADP

+

C h l o r o p h y l l α-j-j f l u o r e s c e n c e y i e l d w i l l depend not only on the redox s t a t e of the PS I I primary e l e c t r o n acceptor Q, but a l s o on the donor s i d e Ζ of PS I I , from which 4 o x i d i z i n g e q u i v a l e n t s are r e q u i r e d f o r the e v o l u t i o n o f O2 from water, and the PS I I secondary e l e c t r o n acceptor B. Therefore, fluorescence represents a meaningful probe f o r t e s t i n g i n t e r f e r e n c e w i t h the redox s t a t e of the PS I I complex. Chemicals l i s t e d i n Table I have been t e s t e d f o r t h e i r e f f e c t s on c h l o r o p h y l l f l u o r e s c e n c e . A l l chemicals show prompt fluorescence k i n e t i c s s i m i l a r to that of DCMU, except f o r n i t r o f e n and t r i f l u r a l i n which are excluded from the group of soc a l l e d "DCMU-type i n h i b i t o r s " . A q u a n t i t a t i v e r e l a t i o n s h i p between i n h i b i t o r c o n c e n t r a t i o n and s e l e c t e d fluorescence p a r a ­ meters has been researched. Steady-state f l u o r e s c e n c e or (see F i g u r e 1) i s reached when Q i s completely reduced, a t l e a s t i n Chlovella. Half-effect concentrations f o r each DCMU-type i n h i b i t o r , expressed as the concentration that gives r i s e to a 50% i n c r e a s e of Φ d termed ρ 3> ,are shown i n Table I I . The p I and ρ φ maxso values are of tne same order o f magnitude, although Φ ^ χ values are, on the average, higher than values f o r i n h i b i t i o n of the H i l l r e a c t i o n . These d i f f e r e n c e s might be caused by experimental c o n d i t i o n s that are not i d e n t i c a l i n both cases ( c h l o r o p h y l l c o n c e n t r a t i o n , e l e c t r o n acceptor, l i g h t i n t e n s i t y , e t c . ) . There­ f o r e , other fluorescence parameters have been s t u d i e d i n order to f i n d a b e t t e r c o r r e l a t i o n . Murata et_ a l . , (8) proposed that the area A (Figure 1) over the fluorescence r i s e normalized to φ , was p r o p o r t i o n a l a

1 Π 3 χ

max

0

5 Q

m a x

Μ

χ

n

1. VAN ASSCHE AND CARLES

3

PS II Inhibiting Chemicals TABLE I

CI

CL

CI/Q\NH6N£"

/Q\NH?ÔCHC=CHCI

3

2

DCMU (N-phenylurea)

Barban

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch001

CI

II J CH C ^ C H s H N ^ NH CH ^

°

( ( ))NHCCH CH \W/

3

2

1

(s-triazine)

Metribuzin

Propanil

(tviazinone)

Lenacil Ο

I o x y n i l (hydro xybenzonitrite)

F

3 \0/C

(N-Ohenylearbamate)

CI

Ν

Atrazine

2

N ( C H 2 C H

N0

2

C H

(N-aoylanilide)

(uracil)

(ipyridazinone)

Pyramin

3>

2

3

H C(CH ) 0^ /NCNHCH 3

2

2

s

3

2

T r i f l u r a l i n (dtnitroaniline)

RU 21731(carbamoyl-thiadiazoline)

CI

N i t r o f e n (dîphenyether)

r 4 > C

I-dinoseb

H

i s

„

(ainitroiphenol)

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch001

4

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

Figure 1. Definitions of the different fluorescence parameters on chloroplasts of Chlorella pyrenoidosa. Fluorescence intensity is expressed in arbitrary units.

P Chi(antenna) P680*

(reaction 2)

e l e c t r o n t r a n s f e r event takes p l a c e . The e l e c t r o n i c e x c i t a t i o n moves through the antenna pigments i n the form of s i n g l e t excitons (48,49). The e l e c t r o n t r a n s f e r chain on the reducing s i d e of PS I I c o n s i s t s of a t l e a s t P680, a pheophytin molecule (Pheo), and two plastoquinone molecules (Figure 1) Q and B. The ground s t a t e of the r e a c t i o n center i s s t a t e 1. When a s i n g l e t e x c i t o n reaches P680

Pheo

Q

Β

( s t a t e 1)

the r e a c t i o n center, P680 becomes e l e c t r o n i c a l l y e x c i t e d to P680* and s t a t e 2 i s populated. An e l e c t r o n i s then e j e c t e d from P680* P680*

Pheo

Q

Β

( s t a t e 2)

to a nearby Pheo molecule (50-55) with a p o s s i b l e intermediate t r a n s f e r across a C h i a molecule and s t a t e 3 i s populated. P680

+

Pheo"

Q

Β

( s t a t e 3)

Evidence f o r the p o s s i b l e p a r t i c i p a t i o n of an intermediate C h i a molecule comes from a recent ESR study (53) and an analogy with the p r o p e r t i e s of the corresponding photosystem (56) i n purple photosynthetic b a c t e r i a . When the e l e c t r o n t r a n s f e r chain on the

2.

SHiPMAN

Binding Sites

27

reducing s i d e of PS I I i s blocked, the r a d i c a l p a i r corresponding to (P680 Pheo") must c o l l a p s e back to a lower t r i p l e t s t a t e or to the ground s i n g l e t s t a t e . This c o l l a p s e d t r i p l e t s t a t e i s h i g h l y s p i n - p o l a r i z e d (53), i . e . , the middle t r i p l e t s u b l e v e l (m = 0) i s p r e f e r e n t i a l l y populated. The s p i n p o l a r i z a t i o n p a t t e r n i n d i c a t e s that e l e c t r o n i c exchange between the two r a d i c a l s i n the r a d i c a l p a i r i s s m a l l , which i n turn suggests that P680 and Pheo are not i n contact. Further study w i l l be necessary to prove or disprove the p a r t i c i p a t i o n of an i n t e r ­ mediary Chi a. A s i m i l a r s p i n - p o l a r i z e d t r i p l e t has been observed p r e v i o u s l y i n purple photosynthetic b a c t e r i a (57,58). An e l e c t r o n t r a n s f e r from Pheo" to Q populates s t a t e 4 from +

s

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch002

P680+

Pheo

Q"

Β

( s t a t e 4) +

s t a t e 3. At t h i s point P680 i s reduced v i a an e l e c t r o n t r a n s f e r from e l e c t r o n donor Z, an intermediary between P680 and the Mn-protein responsible f o r oxygen e v o l u t i o n . Upon reduction of P680 and t r a n s f e r of an e l e c t r o n from Q" to B, s t a t e 5 i s +

P680

Pheo

Q

B~

(state 5)

populated. I f another photon i s not absorbed by PS I I i n state 5, s t a t e 5 i s s t a b l e f o r many seconds (59,60). Inter­ e s t i n g l y , o p t i c a l s p e c t r a i n d i c a t e that reduced Β i s i n the form Β", not BH (60-63); the l o c a l p r o t e i n environment around Β must s t a b i l i z e the anion form over the n e u t r a l form and/or provide a b a r r i e r f o r the uptake of protons onto B". Absorption of a second photon by PS I I ( r e a c t i o n 1) and m i g r a t i o n of the r e s u l t a n t s i n g l e t e x c i t o n to P680 ( r e a c t i o n 2 ) leads to the population of s t a t e 6. State 7 i s populated from s t a t e 6 by the P680* P680

+

Pheo

Q

Pheo"

B" Q

B"

( s t a t e 6) ( s t a t e 7)

e j e c t i o n of an e l e c t r o n from P680* to a nearby Pheo (again with a p o s s i b l e intermediate t r a n s f e r across a Chi a molecule). State 8 i s populated from s t a t e 7 by the t r a n s f e r of an e l e c t r o n P680

+

from Pheo" to Q. P680

Pheo

Q"

P680 Pheo

+

B~

( s t a t e 8)

i s then reduced by Ζ to populate s t a t e 9.

Q"*

B"

( s t a t e 9)

E l e c t r o n i c absorption s p e c t r a of the plastoquinone show that the two e l e c t r o n s plus two protons r a p i d l y end up on the same plastoquinone molecule, PQH2. State 10 may be t r a n s i e n t l y

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

28

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch002

P680

Pheo

Q

2

B~

(state

10)

populated before the protons a r r i v e on plastoquinone B. The protons do not come d i r e c t l y from the e x t e r n a l aqueous media, they must pass through a b a r r i e r , probably a p r o t e i n . Proton uptake from the e x t e r n a l aqueous phase has been measured o p t i c a l l y by use of pH-sensitive dyes; t h i s proton uptake from aqueous media i s almost two orders of magnitude slower than e l e c t r o n t r a n s p o r t to PQ (64). The d e t a i l e d mechanism and k i n e t i c s f o r the two-electron, two-protein r e d u c t i o n of PQ to PQH2 have not, as y e t , been worked out and f u r t h e r s t u d i e s are i n d i c a t e d . From s t a t e 10, the reducing s i d e of PS I I r e c y c l e s to s t a t e 1. The o x i d i z i n g s i d e of PS I I , however, has a four photon c y c l e with an 02 molecule evolved on every c y c l e . The four photon c y c l e of the o x i d i z i n g s i d e i s to be contrasted with the two-photon c y c l e on the reducing s i d e . Requirements f o r H e r b i c i d a l A c t i v i t y As mentioned i n the i n t r o d u c t i o n , molecules from a number of d i f f e r e n t c l a s s e s of h e r b i c i d e s are PS I I i n h i b i t o r s . A t h e o r e t i c a l a n a l y s i s has been c a r r i e d out to i s o l a t e the mole­ c u l a r p r o p e r t i e s shared by a l l a c t i v e PS I I h e r b i c i d e s (36). When the r e s u l t s of t h i s t h e o r e t i c a l a n a l y s i s were combined with experimental data on t r a n s p o r t and metabolism, the f o l l o w i n g l i s t of r e q u i r e d p r o p e r t i e s was developed f o r a c t i v e PS I I h e r b i c i d e s . 1. The h e r b i c i d e molecule must have a f l a t p o l a r component approximately the s i z e of a phenyl r i n g . 2. The f l a t p o l a r component must have hydrophobic s u b s t i t u ­ ents such that i t i s p a r t i t i o n e d s t r o n g l y i n t o l i p i d . 3. The h e r b i c i d e molecule must be r e a d i l y absorbed and t r a n s ­ ported through many p a r t i t i o n s between the p o i n t of contact (roots or f o l i a g e ) and the s i t e of a c t i o n ( c h l o r o p l a s t ) . 4. The metabolism of the h e r b i c i d e must be very slow because the b i n d i n g to the a c t i v e s i t e i s noncovalent and r e v e r s i b l e , and the h e r b i c i d e must be bound long enough f o r the d e l e t e r i o u s a c t i o n of l i g h t and oxygen to take e f f e c t . Mechanisms of A c t i o n As p r e v i o u s l y d i s c u s s e d , the PS I I h e r b i c i d e s act by b i n d i n g noncovalently to a s p e c i a l proteinaceous s i t e (or overlapping s i t e s ) on the reducing s i d e of PS I I and i n h i b i t e l e c t r o n t r a n s f e r from Q~ to B. Because the a v a i l a b l e experimental evidence shows only that the e l e c t r o n stays on Q" and does not d i r e c t l y probe B, at l e a s t two mechanisms are c o n s i s t e n t with a v a i l a b l e data. Mechanism 1. I n h i b i t o r y h e r b i c i d e s d i s p l a c e plastoquinone Β from i t s proteinaceous b i n d i n g s i t e on the reducing s i t e of PS I I .

2.

SHiPMAN

29

Binding Sites

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch002

Evidence i n support of mechanism 1 i s ( i ) the displacement of ubiquinone by the i n h i b i t o r orthophenanthroline i n the analogous photosystem found i n purple photosynthetic b a c t e r i a (56) and ( i i ) the s i m i l a r i t y i n s i z e and shape between the f l a t p o l a r component of the PS I I h e r b i c i d e s and the quinone head of plastoquinone. Mechanism 1 can be studied d i r e c t l y through competitive d i s p l a c e ­ ment r e a c t i o n s between PS I I h e r b i c i d e s and PQ. Mechanism 2. Herbicide b i n d i n g s h i f t s the redox p o t e n t i a l f o r Β to more negative v a l u e s , i . e . , when the h e r b i c i d e i s bound, Β i s harder to reduce. T h i s s h i f t i n redox p o t e n t i a l could a r i s e from ( i ) a d i r e c t coulombic i n t e r a c t i o n between B~ and the p o l a r component of the bound h e r b i c i d e and/or ( i i ) i n h i b i t i o n of the r e l a x a t i o n of the B-binding p r o t e i n during the Β -> B~ transformation, and/or ( i i i ) i n h i b i t i o n of proton uptake by the p r o t e i n on the reducing s i d e of PS I I during the Β + B~ transformation. Further experimental work i s needed to choose between mechan­ isms 1 and 2. Blockage of e l e c t r o n transport on the reducing s i d e of PS I I i s j u s t the f i r s t i n a s e r i e s of steps that u l t i m a t e l y leads to plant death. Much of the energy from absorbed photons that i s normally d i r e c t e d i n t o e l e c t r o n transport i s r e d i r e c t e d i n t o f l u o ­ rescence and t r i p l e t formation when the h e r b i c i d e s are bound. The t r i p l e t s t a t e s are of s p e c i a l i n t e r e s t because of the d e s t r u c t i v e i n t e r a c t i o n between e x c i t e d c h l o r o p h y l l and molecular oxygen through the f o l l o w i n g four step mechanism, where 5

S

0

Chi + hV -> 5

T

1

Τ °Chl +

S

Chi + heat S ° 0 + °Chl + 2

S

0

S

(reaction

3)

(reaction

4)

(reaction

5)

derivatives (reaction

6)

s °0

2

+ heat

0

Chl + S

Chi

0

Chi -> Τ

l

C>2 Τ

oxidized chlorophyll s

Τ

°Chl, C h l , °Chl, ° 0 2 , and ° 0 2 are the ground s i n g l e s t a t e of C h l , the lowest e x c i t e d s i n g l e t s t a t e of C h l , the lowest t r i p l e t s t a t e of C h l , and ground t r i p l e t s t a t e of O2, and the lowest s i n g l e t s t a t e of O2, r e s p e c t i v e l y . F o r t u n a t e l y , the c h l o r o p h y l l s are somewhat protected in_ v i v o by the r a p i d t r a n s ­ f e r of t r i p l e t excitons from c h l o r o p h y l l s to carotenoids (65) v i a r e a c t i o n 7. This p r o t e c t i o n of Chl i s paid f o r by the o c c a s i o n a l Τ

S °Chl +

S °Car

°Chl +

Τ °Car + heat

(reaction

7)

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch002

30

photodestruction of carotenoids. Consistent with t h i s p i c t u r e i s the observation that carotenoid pigments tend to be bleached before c h l o r o p h y l l pigments i n the presence of PS I I h e r b i c i d e s , oxygen, and l i g h t (66). Although the mechanism i n v o l v i n g stoppage of e l e c t r o n t r a n s f e r i n PS I I followed by photodestruction i s c u r r e n t l y thought to be the major mode of a c t i o n f o r the PS I I h e r b i c i d e s , i t should be kept i n mind that many h e r b i c i d e s have m u l t i p l e modes of a c t i o n . Exogenous molecules such as h e r b i c i d e s can and do induce a v a r i e t y of p h y s i o l o g i c a l and biochemical changes i n the p l a n t . Of p a r t i c u l a r i n t e r e s t are the " i n h i b i t o r y uncouplers" (4) (e.g., n i t r o phenols) which not only bind s p e c i f i c a l l y to PS I I and i n h i b i t e l e c t r o n t r a n s f e r , but a l s o i n t e r a c t n o n s p e c i f i c a l l y with membranes and break down the pH gradient e s t a b l i s h e d across photosynt h e t i c and m i t o c h o n d r i a l membranes. Herbicide

S e l e c t i v i t y and

Resistance

The s e l e c t i v i t y of a p a r t i c u l a r h e r b i c i d e toward a s p e c i f i c p l a n t i s based upon d i f f e r e n c e s between p l a n t s f o r a number of f a c t o r s i n c l u d i n g , but not l i m i t e d t o , the f o l l o w i n g . 1. Rate of uptake. 2. Transport r a t e s and p a r t i t i o n i n g . 3. Metabolism r a t e . 4. Binding s i t e a f f i n i t y . For the p a r t i c u l a r case of t r i a z i n e - r e s i s t a n t weed biotypes found i n areas of the world where there has been frequent use of t r i a z i n e h e r b i c i d e s , the r e s i s t a n c e has been traced to a lowered b i n d i n g a f f i n i t y at the PS I I h e r b i c i d e b i n d i n g s i t e (17,19,25,26). Herbicide-Binding

Proteins

P r o t e i n s provide the framework upon which the l i g h t - a b s o r b i n g , e x c i t o n - t r a n s f e r r i n g , and e l e c t r o n - t r a n s f e r r i n g components are arranged. A random c o l l e c t i o n of manganese i o n s , c h l o r o p h y l l s , pheophytin, and plastoquinones would not have the p r o p e r t i e s that we a s s o c i a t e with PS I I . The p r o t e i n s h o l d the e l e c t r o n t r a n s f e r components i n proper alignment and separation with respect to each other so that a high quantum y i e l d f o r forward e l e c t r o n t r a n s f e r i s obtained while g i v i n g a low quantum y i e l d f o r the c a t i o n h o l e e l e c t r o n recombination. T r y p s i n i s a water-soluble protease that cleaves polypeptide chains p r e f e r e n t i a l l y at a r g i n i n e and l y s i n e r e s i d u e s . Thus, when t r y p s i n i s a p p l i e d to broken c h l o r o p l a s t s or PS I I p a r t i c l e s the surface exposed p r o t e i n s are digested f i r s t and the f u l l y b u r i e d p r o t e i n s are i n i t i a l l y l e f t alone. A number of s t u d i e s (10,22,29, 30,38,41,42,43,67) have been conducted to determine the e f f e c t s of d i g e s t i o n of the surface-exposed polypeptides on the f u n c t i o n of the reducing s i d e of PS I I . T r y p s i n treatment impairs the a b i l i t y of PS I I h e r b i c i d e s to i n h i b i t the r e d u c t i o n of water-soluble

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch002

2.

SHIPMAN

Binding Sites

31

e l e c t r o n acceptors such as f e r r i c y a n i d e by PS I I . Extensive d i g e s t i o n of the photosynthetic membrane e v e n t u a l l y destroys the herbicide binding s i t e ( s ) . T r y p s i n d i g e s t i o n of PS I I p a r t i c l e s leads to the l o s s of polypeptides of molecular weight 27 and 32 kDaltons (22); t h i s i s c o n s i s t e n t with the molecular weights of the h e r b i c i d e b i n d i n g p r o t e i n s as determined through p h o t o a f f i n i t y l a b e l i n g experiments. PS I I h e r b i c i d e s that are converted to p h o t o a f f i n i t y l a b e l s have proved q u i t e u s e f u l f o r the i d e n t i f i c a t i o n of the p r o t e i n s that p a r t i c i p a t e i n h e r b i c i d e b i n d i n g . In a t y p i c a l experiment, the p h o t o a f f i n i t y l a b e l i s bound noncovalently to the PS I I herb i c i d e b i n d i n g s i t e i n the dark. The system i s then i r r a d i a t e d with UV l i g h t and a f t e r i r r a d i a t i o n the p h o t o a f f i n i t y l a b e l i s bound c o v a l e n t l y at or near i t s o r i g i n a l binding s i t e . In p a r t i c ular, l^C-labelled azidoatrazine (4-azido-2-isopropylamino-6e t h y l a m i n o - s - t r i a z i n e ) has been shown to bind to a polypeptide of molecular weight 32 kDaltons (37,38,43). A H - l a b e l l e d azido d e r i v a t i v e of dinoseb (2-azido-4-nitro-6-isobutylphenol) was shown to bind to polypeptides i n the 30-40 kDalton range (40). I t may be concluded that the polypeptides i n PS I I that are respons i b l e f o r b i n d i n g the PS I I h e r b i c i d e s have molecular weights i n the 30-40 kDalton range; one of them having a molecular weight of 32 kDaltons. 3

T h e o r e t i c a l Studies of H e r b i c i d e - B i n d i n g

Site Interactions

In a recent t h e o r e t i c a l study, Shipman (36) proposed s e v e r a l s p e c i f i c models f o r the h e r b i c i d e b i n d i n g s i t e on PS I I . I t was proposed that the PS I I h e r b i c i d e s bind e l e c t r o s t a t i c a l l y at or near a p r o t e i n s a l t bridge or the terminus of an alpha h e l i x . The s a l t b r i d g e could be argininium a s p a r t a t e , argininium g l u t a mate, l y s i n i u m asparate, l y s i n i u m glutamate, a r g i n i n i u m l y s i n e carbamate, or l y s i n i u m lysine-carbamate. Both the s a l t bridges and the terminus of an alpha h e l i x generate very strong l o c a l e l e c t r i c f i e l d s with which the p o l a r component of the h e r b i c i d e molecules could bind e l e c t r o s t a t i c a l l y , perhaps with a d d i t i o n a l s t a b i l i z a t i o n from hydrogen bonds. The strong l o c a l e l e c t r i c f i e l d from the p r o t e i n , i f s u i t a b l y o r i e n t e d , could s t a b i l i z e B~ r e l a t i v e to B; t h i s would e x p l a i n , i n p a r t , the s t a b i l i t y of B" f o r many seconds. Adding to the b i n d i n g s i t e model the r e q u i r e ment that i t be b u r i e d i n a hydrophobic environment away from the aqueous phase, the dual requirements of l i p o p h i l i c i t y and p o l a r i t y f o r the PS I I h e r b i c i d e s are explained. P i - p i complex between a model f o r a p o l a r component of a h e r b i c i d e ( u r a c i l ) and a model f o r a s a l t b i n d i n g s i t e (guanidinium bicarbonate) i s shown i n Figure 2. The geometry was computed from ab i n i t i o c a l c u l a t i o n s (36). I t should be noted that i n the lowest energy geometry shown i n Figure 2, the d i p o l e moment of u r a c i l i s a n t i p a r a l l e l to the d i p o l e moment of the s a l t .

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch002

32

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

polar component of a PS II herbicide (uracil) and a salt model (guanidinium bicarbonate). The circles around the atoms were drawn at 20% of their van der Waals radii (36;.

2.

SHiPMAN

Binding Sites

33

C a r l e s and Van Assche (68,69) have proposed a d i p e p t i d e model f o r the h e r b i c i d e b i n d i n g s i t e and have used the CNDO/s semie m p i r i c a l quantum mechanical method to study h e r b i c i d e - d i p e p t i d e i n t e r a c t i o n s . Two hydrogen bonds are important f o r the energetics of h e r b i c i d e b i n d i n g to the d i p e p t i d e s . Coulombic i s o p o t e n t i a l maps were used as a guide to the way the h e r b i c i d e molecules might i n t e r a c t with p r o t e i n . Acknowledgment

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch002

Work supported by the D i v i s i o n of Chemical Sciences, O f f i c e of Basic Energy Sciences, U.S. Department of Energy, under Contract W-31-109-Eng-38.

Literature Cited 1. Ashton, F. M.; Crafts, A. S. "Mode of Action of Herbicides"; J. Wiley & Sons: New York, 1973, 504 pp. 2. Corbett, J. R. "The Biochemical Mode of Action of Pesticides"; Academic Press: New York, 1974, 330 pp. 3. Audus, L. J. "Herbicides"; Vol. 1; Academic Press: New York, 1976, 608 pp. 4. Moreland, D. E. Ann. Rev. Plant Physiol. 1980, 31, 597. 5. Hansch, N. C. in "Progress in Photosynthesis Research"; Metzner, H. Ed.; H. Loupp, Jr.: Tübingen. 1969; p. 1685. 6. Moreland, D. E. in "Progress in Photosynthesis Research"; Metzner, H. Ed.; H. Loupp, Jr.: Tübingen, 1969; p. 1693. 7. Trebst, Α.; Harth, Ε. Z. Naturforsch. 1974, 29C, 232. 8. Regitz, G.; Ohad, I. J. Biol. Chem. 1976, 251, 247. 9. Renger, G. FEBS Lett. 1976, 69, 225. 10. Renger, G. Biochim. Biophys. Acta 1976, 440, 287. 11. Renger, G.; Erixon, K.; Doring, G.; Wolff, C. Biochim. Bio­ phys. Acta 1976, 440, 278. 12. Trebst, Α.; Reimer, S.; Dallacker, F. Plant Sci. Lett. 1976, 6 21. 13. Izawa, S. in "Encyclopedia of Plant Physiology"; New Series, Vol. 5; Trebst, Α.; Avron, M. Ed.; Springer: Berlin, 1977; p. 266. 14. Renger, G. in "Bioenergetics of Membranes"; Packer, L. Ed.; Biomedical Press, Elsevier: Amsterdam, 1977; p. 339. 15. Tischer, W.; Strotmann, H. Biochim. Biophys. Acta 1977, 460, 113. 16. Draber, W.; Fedtke, C. in "Advances in Pesticide Science"; Geissbühler, H., Ed.; Pergamon: Oxford, 1979; p. 475. 17. Machado, V. S.; Arntzen, C. J.; Bandeen, J . D.; Stephensen, G. R. Weed Sci. 1978, 26, 318. 18. Van Rensen, J. J. S.; Wong, D.; Govindjee Z. Naturforsch. 1979, 34C, 951. ,

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BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

19. Arntzen, C. J.; Ditto, C. L . ; Brewer, P. E. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 278. 20. Brewer, P. E.; Arntzen, C. J.; Sliffe, F. W. Weed Sci. 1979, 27, 300. 21. Brugnoni. G. P.; Moser, P.; Trebst, A. Z. Naturforsch. 1979, 34C, 1028. 22. Croze, E.; Kelly, M.; Horton, P. FEBS Lett. 1979, 103, 22. 23. Govindjee; Jursinic, P. A. Photochem. Photobiol. Rev. 1979, 4, 125. 24. Pfister, K.; Radosevich, S. R.; Arntzen, C. J. Plant Physiol. 1979, 64, 995. 25. Pfister, K.; Arntzen, C. J. Z. Naturforsch. 1979, 34C, 996. 26. Radosevich. S. R.; Steinback, Κ. E.; Arntzen, C. J. Weed Sci. 1979, 27, 216. 27. Reimer, S.; Link, K.; Trebst, A. Z. Naturforsch. 1979, 34C, 419. 28. Renger, G.; Tiemann, R. Biochim Biophys. Acta 1979, 545, 316. 29. Renger, G. Z. Naturforsch. 1979, 34C, 1010. 30. Tischer, W.; Strotmann, H. Z. Naturforsch. 1979, 34C, 992. 31. Trebst, A. Z. Naturforsch. 1979, 34C, 986. 32. Urbach, W.; Lurz, G.; Hartmeyer, H.; Urbach, D. Z. Natur­ forsch. 1979, 34C, 951. 33. Van Rensen, J. J. S.; Hobe, J. H. Z. Naturforsch. 1979, 34C, 1021. 34. Wright, K.; Corbett, J. R. Z. Naturforsch. 1979, 34C, 966. 35. Moreland, D. E. Ann. Rev. Plant Physiol. 1980, 31, 597. 36. Shipman, L. L. J. Theoret. Biol. 1981, 90, 123. 37. Gardner, G. Science 1981, 211, 937. 38. Pfister, K.; Steinback, Κ. E.; Arntzen, C. J. in Proc. 5th Int. Congr. Photosynth., Halkidiki, Greece, 1981; in press. 39. Oettmeier, W.; Johanningmeier, U. in "First European Bioenergetics Conference"; Pàtron Editore: Bologna, 1980; p. 57. 40. Oettmeier, W.; Masson, K.; Johanningmeier, U. FEBS Lett. 1980, 118, 267. 41. Steinback, Κ. E.; Pfister, K.; Arntzen, C. J. Z. Naturforsch. 1981, 36C, 98. 42. Oettmeier, W.; Masson, K. Pesticide Biochem. Physiol. 1980, 14, 86. 43. Mullet, J. E.; Arntzen, C. J. Biochim. Biophys. Acta 1981, 635, 236. 44. Staehelin. L. Α.; Armond, P. Α.; Miller, K. R. in "Chloro­ phyll-Proteins, Reaction Centers, and Photosynthetic Mem­ branes"; Brookhaven Symposium in Biology No. 28; Brookhaven National Laboratory: Upton, Long Island, New York, 1976; p. 278. 45. Arntzen, C. J.; Armond, P. Α.; Briantais, J.-M.; Burke, J. J.; Novitzky, W. P. in "Chlorophyll-Proteins, Reaction Centers, and Photosynthetic Membranes"; Brookhaven Symposium in

2.

46. 47. 48.

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49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69.

SHIPMAN

Binding Sites

35

Biology No. 28; Brookhaven National Laboratory: Upton, Long Island, New York, 1976; p. 316. Staehelin, L. Α.; Arntzen, C. J. in "Chlorophyll Organization and Energy Transfer in Photosynthesis"; Ciba Foundation Sym­ posium 61; Excerpta Medica: New York, 1979; p. 147. Amesz, J.; Duysens, L. Ν. M. in "Primary Processes of Photo­ synthesis"; Topics in Photosynthesis, Vol. 2; Barber, J . Ed.; Elsevier: New York, 1977; p. 149. Knox, R. S. in "Bioenergetics of Photosynthesis"; Govindjee Ed.; Academic Press: New York, 1975; p. 183. Shipman, L. L. Photochem. Photobiol. 1980, 31, 157. Klimov, V. V.; Dolan, E.; Ke, B. FEBS Lett. 1980, 112, 97. Klimov, V. V.; Dolan, E.; Ke, B. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 7227. Shuvalov, V. Α.; Klimov, V. V.; Dolan, E.; Parson, W. W.; Ke, B. FEBS Lett. 1980, 118, 279. Rutherford, A. W.; Paterson, D. R.; Mullet, J. E. Biochim. Biophys. Acta 1981, 635, 205. Fajer, J.; Davis, M. S.; Forman, Α.; Klimov, V. V.; Dolan, E.; Ke, B. J . Am. Chem. Soc. 1980, 102, 7143. Rutherford, A. W.; Mullet, J. E.; Crofts, A. R. FEBS Lett. 1981, 123, 235. "The Photosynthetic Bacteria"; Clayton, R. U.; Sistrom, W. R. Ed.; Plenum Press: New York, 1978. Dutton, P. L . ; Leigh, J. S.; Seibert, M. Biochem. Biophys. Res. Commun. 1971, 40, 406. Thurnauer, M. C.; Katz, J. J.; Norris, J . R. Proc. Natl. Acad. Sci. U.S.A. 1975, 72, 3270. Velthuys, B. R.; Amesz, J. Biochim. Biophys. Acta 1974, 333, 85. Pulles, M. P. J.; van Gorkom, H. J.; Willemsen, J . G. Biochim. Biophys. Acta 1976, 449, 539. Stiehl, H. H.; Witt, H. T. Z. Naturforsch. 1968, B23, 220. Witt, K. FEBS Lett. 1973, 38, 116. Van Gorkom, H. J. Biochim. Biophys. Acta 1974, 347, 439. Ausländer, W.; Junge, W. Biochim. Biophys. Acta 1974, 357, 285. Kramer, H.; Mathis, P. Biochim. Biophys. Acta 1980, 593, 319. Pallett, Κ. E.; Dodge, A. D. Pestic. Sci. 1979, 10, 216. Böger, P.; Kunert, K.-J. Z. Naturforsch. 1979, 34C, 1015. Carles, P. M.; Van Assche, C. J. in Proc. 5th Int. Congr. Photosynth., Halkidiki, Greece, 1981; in press. Van Assche, C. J.; Carles, P. Μ., Chapter 1 in this book.

RECEIVED

October 6, 1981.

3 Identification of the Receptor Site for Triazine Herbicides in Chloroplast Thylakoid Membranes 1

KATHERINE Ε. STEINBACK, KLAUS PFISTER and CHARLES J. ARNTZEN

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch003

Michigan State University, MSU-DOE Plant Research Laboratory, East Lansing, MI 48824

A broad range of inhibitors of photosynthesis act by blocking the same step in electron transport in chloroplast membranes. Inhibition occurs at the level of a protein-bound plastoquinone (B) that functions as the second stable electron acceptor for photosystem II (PS II). Studies based on the use of the proteolytic enzyme, tryp­ sin, indicate that the receptor site for PS II inhibitors is a protein of the structural PS II complex. Using a photoaffinity C-labeled derivative of atrazine, we have identified the specific receptor polypeptide for this inhibitor of PS II function. Analysis of membrane polypep­ tides by polyacrylamide gel electrophoresis and fluorographic techniques have shown that the photoaffinity triazine covalently binds to a polypeptide of 32-34 kilodaltons. We have fur­ ther shown that this polypeptide is surface expos­ ed; trypsin treatment of thylakoid membranes re­ sults in the stepwise alteration of the peptide to a 16 kilodalton species. The site for covalent attachment of the photoaffinity probe is located on the intrinsic 16 kilodalton fragment. Conse­ quently, the binding site appears to be deter­ mined, in part, by the intrinsic hydrophobic do­ main of the 32-34 kilodalton polypeptide. The biogenesis of the 32-34 kilodalton polypeptide is discussed with relation to genetic mechanisms that may be responsible for triazine resistance at the level of the chloroplast membrane. 14

1

Current address: University of Wurzburg, Botanical Institute, Wurzburg 87, West Ger­

many. 0097-6156/82/0181-0037$05.00/0 © 1982 American Chemical Society

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch003

38

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Approximately h a l f of a l l commercial h e r b i c i d e s act by i n h i b i t i n g photosynthesis by i n t e r a c t i n g with s p e c i f i c s i t e s along the photosynthetic e l e c t r o n t r a n s p o r t c h a i n . A number of d i v e r s e chemicals i n c l u d i n g the ureas, amides, t r i a z i n e s , t r i a z i n o n e s , u r a c i l s , p y r i d a z i n o n e s , q u i n a z o l i n e s , t h i a d i a z o l e s , and c e r t a i n phenols are thought to act s p e c i f i c a l l y at a common i n h i b i t o r y s i t e at the reducing s i d e of photosystem II (PS II) (1, 2 ) . Several l i n e s of evidence i n d i c a t e that t h i s i n h i b i t i o n occurs at the l e v e l of a p r o t e i n bound plastoquinone c a l l e d "B" (3). This e l e c t r o n c a r r i e r acts as the second s t a b l e e l e c t r o n acceptor of PS II (4^ 5 ) . It has been proposed that the common mode of a c t i o n of these chemical c l a s s e s i s v i a h i g h - a f f i n i t y binding to the PS II complex {6). Herbicide binding induces a change in the redox p o t e n t i a l of the quinone c o f a c t o r of B, thereby making the t r a n s f e r of e l e c t r o n s from Q (the f i r s t s t a b l e e l e c t r o n acceptor o f PS II) thermodynamically unfavorable (3, 5 ) . Establishment of 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 ï ï i p s within h e r b i c i d e c l a s s e s has been extremely useful in e l u c i d a t i n g important s t r u c t u r a l aspects of the i n h i b i t o r molecules themselves (_7). U n t i l r e c e n t l y , however, much l e s s emphasis has been d i r e c ted at understanding the biochemical c h a r a c t e r i s t i c s of the h e r b i c i d e receptor s i t e within the c h i o r o p l a s t membrane. Progress in t h i s d i r e c t i o n was i n i t i a t e d when Strotmann and T i s c h e r ( β , 8) introduced techniques f o r monitoring s p e c i f i c binding of h e r b i c i d e s to i s o l a t e d c h i o r o p l a s t t h y l a k o i d membranes. These and other workers (_3, j>, 9) have u t i l i z e d a v a r i e t y of r a d i o l a ­ beled i n h i b i t o r s of PS II f u n c t i o n f o r the c h a r a c t e r i z a t i o n of p r o p e r t i e s of the h e r b i c i d e binding s i t e ; the studies have r e s u l ­ ted in the demonstration of a competition f o r a s i n g l e binding domain per photosynthetic e l e c t r o n transport c h a i n . An understanding of the biochemical c h a r a c t e r i s t i c s of the PS II l o c a l i z e d h e r b i c i d e receptor domain is p a r t i c u l a r l y r e l e ­ vant because of the appearance of t r i a z i n e - r e s i s t a n t weed biotypes in the United S t a t e s , Canada, and Europe (3). I n i t i a l attempts at understanding the mechanism(s) of resisTance d i r e c t e d i n v e s t i ­ gators to evaluate a l t e r a t i o n s in uptake, t r a n s l o c a t i o n , or meta­ bolism of t r i a z i n e s . Only small d i f f e r e n c e s between s u s c e p t i b l e and r e s i s t a n t biotypes were e s t a b l i s h e d , these being i n s u f f i c i e n t to e x p l a i n the mechanism of extreme h e r b i c i d e r e s i s t a n c e . As the primary mechanism of action of the s - t r i a z i n e s i n v o l ­ ves i n h i b i t i o n of PS II e l e c t r o n t r a n s p o r t , a t t e n t i o n was also d i r e c t e d at a n a l y s i s of c h i o r o p l a s t r e a c t i o n s in r e s i s t a n t weed biotypes (10, 11, 12). These studies can be summarized as f o l ­ lows: (a) m ΤΓ1 cases studied to date, there is a m o d i f i c a t i o n in the c h i o r o p l a s t membranes of r e s i s t a n t biotypes that changes the c h a r a c t e r i s t i c s of s - t r i a z i n e b i n d i n g ; (b) t h i s m o d i f i c a t i o n r e s u l t s in a l t e r e d binding c h a r a c t e r i s t i c s of other c l a s s e s of h e r b i c i d e s , ( i . e . , only s l i g h t r e s i s t a n c e to ureas, but increased s e n s i t i v i t y to phenols) (see L3 f o r review), and (c) the a l t e r a ­ t i o n of the h e r b i c i d e receptor in r e s i s t a n t weeds is accompanied

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by a d e t e c t a b l e change in the k i n e t i c c h a r a c t e r i s t i c s of e l e c t r o n t r a n s f e r from Q" to Β in the native membranes even 1n the absence of a l l h e r b i c i d e s (3^ 14, Jj>), which i n d i c a t e s that the apoprotein o f Β may be a l t e r e d i n T h e r e s i s t a n t c h l o r o p l a s t s so that the bound quinone c o f a c t o r e x h i b i t s d i f f e r e n t redox p r o p e r t i e s . In t h i s paper, we have summarized our current understanding of the biochemical nature of the t r i a z i n e binding s i t e within the PS II complex. Studies using the p r o t e o l y t i c enzyme t r y p s i n as a s e l e c t i v e , s u r f a c e - s p e c i f i c m o d i f i e r of membrane polypep­ t i d e s and the use of a p h o t o a f f i n i t y t r i a z i n e have been u t i l i z e d s e p a r a t e l y to i d e n t i f y the t r i a z i n e receptor protein as a 32-34 k i l o d a l t o n (kDal) polypeptide of the PS II complex in peas (Pisum sativum L . ) . The nature of the covalent attachment of the p h o t o a f f i n i t y probe has also enabled us to i d e n t i f y the t r i a z i n e receptor p r o t e i n as a product of c h l o r o p l a s t - d i r e c t e d p r o t e i n s y n t h e s i s ; t h i s implies that the s t r u c t u r a l gene f o r the t r i a z i n e receptor polypeptide i s encoded on c h i o r o p l a s t DNA. This i s in agreement with r e p o r t s , based on c l a s s i c a l genetic a n a l y s i s , that t r i a z i n e r e s i s t a n c e in B r a s s i c a campestris L. (16) i s a maternally i n h e r i t e d t r a i t . Materials

and Methods

Chioroplast Isolation. C h l o r o p l a s t s of peas, spinach ( S p i n a c i a o l e r a c e a L . ) , and biotypes of Amaranthus hybridus L. suscept i b l e or r e s i s t a n t t o _ s - t r i a z i n e s were i s o l a t e d and stroma-free t h y l a k o i d s prepared as p r e v i o u s l y described (17). Intact c h l o r o ­ p l a s t s were obtained from pea leaves f o l l o w i n g the method of B l a i r and E l l i s (18). Trypsin Treatment. Trypsin incubations were c a r r i e d out at room temperature as p r e v i o u s l y described (JL9). Trypsin concen­ t r a t i o n s used are s p e c i f i e d in the t e x t . Photoaffinity Labeling. The p h o t o a f f i n i t y l a b e l i n g of pea thyla1 DCPIP). 14

2

Figure 2B. Comparison of data derived from studies of C-atrazine affinity for thylakoid membranes incubated with trypsin with that obtained from electron transport assays (H 0 -> DCPIP) in the presence of 0.25 μΜ atrazine. 14

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p r o t e o l y t i c enzyme). This was followed by a gradual change in a f f i n i t y over the next 15 min of treatment. This i s in c o n t r a s t to the gradual time-dependent decrease in binding s i t e s , implying at l e a s t a two-step a l t e r a t i o n in the p r o t e i n (or p r o t e i n s ) of the PS II complex that c o n s t i t u t e the a t r a z i n e binding s i t e . Mild t r y p s i n treatment has been shown also to a l t e r the a f f i n i t y f o r a number of other chemical f a m i l i e s of PS II d i r e c ted h e r b i c i d e s in a manner s i m i l a r to that of the s - t r i a z i n e s . Trypsin-mediated decreases in i n h i b i t o r y a c t i v i t y are found for u r a c i l (19), urea, pyridazinone ( 1 £ , 28) and t r i a z i n o n e (28) herbicides. In c o n t r a s t , p h e n o l - t y p e T e r b i c i d e s increasecTin i n h i b i t o r y a c t i v i t y f o l l o w i n g b r i e f t r y p s i n treatment {19_ 28), although the trend was reversed over longer treatment perioïïs. The d i s t i n c t l y d i f f e r e n t behavior of the phenol-type h e r b i c i d e s f o l l o w i n g t r y p s i n treatment suggests that d i f f e r e n t d e t e r minants within the PS II protein complex e s t a b l i s h the "domains" that r e g u l a t e the binding p r o p e r t i e s of these i n h i b i t o r s . In s p i t e of the f a c t that phenol-type h e r b i c i d e s w i l l d i s p l a c e bound r a d i o l a b e l e d h e r b i c i d e s such as d i u r o n , these i n h i b i t o r s show noncompetitive i n h i b i t i o n (29, 30). At present, there are three l i n e s of evidence which favor Tfie involvement of two domains within the PS II complex that p a r t i c i p a t e in c r e a t i n g the binding s i t e s f o r these h e r b i c i d e s : (a) i s o l a t e d PS II p a r t i c l e s can be s e l e c t i v e l y depleted of a polypeptide with p a r a l l e l loss of a t r a zine s e n s i t i v i t y , but not dinoseb i n h i b i t i o n a c t i v i t y (33); (b) in r e s i s t a n t weed b i o t y p e s , c h i o r o p l a s t membranes that e x h i b i t extreme t r i a z i n e r e s i s t a n c e have increased s e n s i t i v i t y to the phenol-type h e r b i c i d e s (13); and (c) experiments with azido (photoa f f i n i t y ) d e r i v a t i v e s of phenol and t r i a z i n e h e r b i c i d e s r e s u l t in the covalent l a b e l i n g of d i f f e r e n t PS II polypeptides (20, 31).

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I d e n t i f i c a t i o n of the T r i a z i n e Binding S i t e . An ultimate goal f o r the biochemical understanding of h e r b i c i d a l e f f e c t s on c h i o r o p l a s t membranes is to i d e n t i f y the s p e c i f i c membrane cons t i t u e n t that serves as h e r b i c i d e r e c e p t o r . For the s - t r i a z i n e s , i d e n t i f i c a t i o n of the s p e c i f i c receptor i s r e q u i r e d for understanding t r i a z i n e r e s i s t a n c e at the molecular l e v e l . Because t r y p s i n m o d i f i c a t i o n of membranes r e s u l t s in p r o t e i n - s p e c i f i c a l t e r a t i o n s of the membrane, the stepwise loss of h e r b i c i d e binding a f f i n i t y and binding s i t e s is a t t r i b u t e d to the stepwise a l t e r a t i o n of a protein that serves as the h e r b i c i d e r e c e p tor. One approach to the i d e n t i f i c a t i o n of h e r b i c i d e receptors has been the a n a l y s i s of peptide a l t e r a t i o n s f o l l o w i n g t r y p s i n treatment by using SDS-PAGE. A n a l y s i s of polypeptide a l t e r a t i o n in t h y l a k o i d membranes has been u n s a t i s f a c t o r y , however, because of the m u l t i p l i c i t y of p r o t e i n changes brought about by t r y p s i n (19). However, c h a r a c t e r i z a t i o n of membranes subfractionated f o l l o w i n g detergent treatment has been somewhat more i n f o r m a t i v e . When PS II enriched s u b f r a c t i o n s are i s o l a t e d from t r y p s i n treated membranes, the number of a l t e r e d polypeptides that could

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correspond to an a l t e r e d receptor p r o t e i n i s narrowed down to four candidates. Among these i s a polypeptide of 32 kDal (19). Highly p u r i f i e d PS II complexes i s o l a t e d by detergent f r a c t i o n a t i o n also contain a polypeptide of 32 kDal (32, 33). Trypsin, treatment of i s o l a t e d PS II complexes r e s u l t s in the degradation of the 32 kDal polypeptide with concomitant l o s s of h e r b i c i d e a c t i v i t y (32, 33). F i n a l l y , s e l e c t i v e removal of the 32 kDal polypeptide from PS II p a r t i c l e s by a d d i t i o n a l detergent t r e a t ments r e s u l t s in loss of both diuron and a t r a z i n e binding (33). These l i n e s of evidence a l l i m p l i c a t e a 32 kDal polypeptide as the receptor f o r t r i a z i n e and urea c l a s s e s of h e r b i c i d e s . The a s s o c i a t i o n of diuron and a t r a z i n e with c h i o r o p l a s t membranes i s v i a a h i g h - a f f i n i t y , but noncovalent b i n d i n g . Attempts at p h y s i c a l i s o l a t i o n of proteins labeled by r a d i o a c t i v e i n h i b i t o r s have f a i l e d because techniques such as detergent f r a c t i o n a t i o n or e l e c t r o p h o r e t i c separation r a p i d l y lead to a new e q u i l i b r i u m and the d i s s o c i a t i o n of the noncovalent r e c e p t o r - i n h i b i t o r complex. One approach that overcomes t h i s d i f f i c u l t y in i d e n t i f y i n g a h e r b i c i d e receptor i s to attach a r a d i o labeled p h o t o a f f i n i t y azido d e r i v a t i v e of the h e r b i c i d e to i t s h i g h - a f f i n i t y receptor p r o t e i n . A c t i v a t i o n of the azido f u n c t i o n of p h o t o a f f i n i t y compounds by UV i r r a d i a t i o n produces a n i t r e n e that i s h i g h l y r e a c t i v e (34). If the compound remains l o c a l i z e d at i t s h i g h - a f f i n i t y s i t e throughout the l i f e t i m e of the d e s t a b i l ized n i t r e n e group, covalent binding w i l l occur at the binding site. A z i d o a t r a z i n e has been shown to i n h i b i t photosynthetic e l e c t r o n t r a n s p o r t at a s i t e i d e n t i c a l to that of a t r a z i n e (20). We have used a z i d o a t r a z i n e as a p h o t o a f f i n i t y probe to ident~T7y the h e r b i c i d e - r e c e p t o r p r o t e i n in c h l o r o p l a s t s of _A. hybridus. In order to demonstrate the s p e c i f i c i t y of binding to a h i g h a f f i n i t y s i t e , membranes from both s u s c e p t i b l e and r e s i s t a n t biotypes were u t i l i z e d . A n a l y s i s of membrane polypeptides from s u s c e p t i b l e and r e s i s t a n t membranes by SDS-PAGE i s shown in Figure 3 (Coomassie blue stained polypeptides are in lanes A ) . No major d i f f e r e n c e s in polypeptide composition or s t a i n i n g i n t e n s i t y between the two samples are apparent. For both c a s e s , membranes were UV i r r a d i a t e d in the presence of a z i d o - ^ C a t r a z i n e p r i o r to e l e c t r o p h o r e s i s . A n a l y s i s of the gel by a f l u o r o g r a p h i c technique showed no detectable bound r a d i o l a b e l a s s o c i a t e d with the membrane sample from r e s i s t a n t c h l o r o p l a s t s . In the s u s c e p t i b l e membranes, however, the r a d i o l a b e l extended from a region corresponding to 34 kDal to that of a stained polypeptide at 32 kDal. We s h a l l present evidence in the f o l l o w i n g d i s c u s s i o n that t h i s pattern of l a b e l i n g i s due to covalent h e r b i c i d e a s s o c i a t i o n to a s i n g l e p r o t e i n which e x i s t s in e i t h e r of two molecular weight s p e c i e s : a developmental precursor form of 34 kDal which is s i z e - p r o c e s s e d to a 32 kDal form. For the remaining d i s c u s s i o n we s h a l l designate t h i s t r i a z i n e h e r b i c i d e receptor as the 32-34 kDal polypeptide.

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Proc. Natl. Acad. Sci.

Figure 3. Polyacrylamide slab gel elec­ trophoresis of thylakoid membrane poly­ peptides from susceptible and resistant biotypes of A. hybridus, stained for pro­ tein (lanes A) and by fluorography (lanes B). Susceptible and resistant membranes were incubated with 0.5 μΜ azido- Catrazine under UV light for 10 min prior to SDS solubilization. The predominant location of the radiolabel, as shown by fluorography, is over the 34- to 32-kDal size class. 14

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One a d d i t i o n a l l i n e of evidence that independently i m p l i c a t e s a 32 kDal polypeptide in t r i a z i n e binding comes from studies on c h l o r o p l a s t s i s o l a t e d from a maize mutant that s p e c i f i c a l l y lacks the s t a i n a b l e 32 kDal polypeptide and the r a p i d l y labeled 34 kDal polypeptide. C h l o r o p l a s t s of t h i s PS I I - l e s s l e t h a l mutant lack binding s i t e s f o r r a d i o a c t i v e a t r a z i n e (35). Step-wise M o d i f i c a t i o n of the Herbicide Binding Polypeptide In V i t r o . Evidence from a t r a z i n e binding studies described in a previous s e c t i o n suggested that trypsin-mediated a l t e r a t i o n s of surface exposed membrane polypeptides r e s u l t e d i n a sequential a l t e r a t i o n of a t r a z i n e binding s i t e s . F i r s t , a rapid alteration of h e r b i c i d e a f f i n i t y was detected followed by a more gradual loss of d e t e c t a b l e binding s i t e s . This stepwise a l t e r a t i o n was f u r t h e r i n v e s t i g a t e d at the l e v e l of polypeptide s t r u c t u r e by using the s e l e c t i v e membrane m o d i f i e r , t r y p s i n , against membranes that had been c o v a l e n t l y tagged with the r a d i o l a b e l e d photoa f f i n i t y t r i a z i n e probe. As shown in Figure 4A, the use of low c o n c e n t r a t i o n s of a z i d o - ^ C - a t r a z i n e r e s u l t e d in l a b e l i n g of polypeptide species at 34 kDal. We i n t e r p r e t these data as i n d i c a t i n g that the 34 kDal form of the 32-34 kDal polypeptide creates the h i g h - a f f i n i t y h e r b i c i d e binding s i t e . In membrane samples treated with 2 yg trypsin/ml f o r 15 min (see legend of Figure 4 f o r d e t a i l s ) , a t r y p s i n concentration shown p r e v i o u s l y to p r i n c i p a l l y bring about changes in a t r a z i n e a f f i n i t y (Figure 2B), the 34 kDal polypeptide l a b e l e d with a z i d o a t r a z i n e was a l t e r ed to a species that comigrated with a stained 32 kDal polypept i d e (Figure 4B). At higher t r y p s i n concentrations (Figures 4C and 4D; 10 and 40 ug t r y p s i n / m l , r e s p e c t i v e l y ) where loss of both e l e c t r o n t r a n s p o r t f u n c t i o n and i n h i b i t o r binding s i t e s were observed p r e v i o u s l y , the polypeptide tagged by a z i d o a t r a z i n e was f u r t h e r degraded to species at 18 and then 16 kDal in a s e q u e n t i a l , stepwise manner. [The experiment of Figure 4 u t i l i z e d membranes which were f i r s t l a b e l e d with a z i d o a t r a z i n e and then subjected to t r y p s i n treatment.] We conclude that the major covalent attachment s i t e f o r a z i d o a t r a z i n e is in a hydrophobic region of the membrane which is i n a c c e s s i b l e to t r y p s i n , thereby leaving an i n t r i n s i c 16 kDal fragment of the t r i a z i n e binding p r o t e i n associated with the membrane f o l l o w i n g t r y p s i n treatment. Further degradation of the 16 kDal polypeptide is not observed a f t e r prolonged t r y p s i n treatment. The data of Figure 4 i n d i c a t e d that a 34 kDal form of the 32-34 kDal polypeptide i s the h i g h - a f f i n i t y binding s i t e f o r triazines. To t e s t whether the t r y p s i n - d e r i v e d , membrane-bound fragments of t h i s p r o t e i n s t i l l bound h e r b i c i d e , a z i d o a t r a z i n e was used against t r y p s i n - t r e a t e d membranes. Figure 5 shows the fluorogram of e l e c t r o p h o r e t i c a l l y separated pea c h i o r o p l a s t polypeptides from c o n t r o l (A and B) and t r y p s i n - t r e a t e d (C and D) membranes that were tagged with a z i d o a t r a z i n e a f t e r the protease m o d i f i c a t i o n . When a z i d o a t r a z i n e was applied against

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Figure 4. Fluorogram of a polyacrylamide gel that shows trypsin sensitivity of the 34-kDal polypeptide of pea chioro­ plast membranes following covalent radiolabeling with azido- C-atrazine. Key: A, control; B, 2 μg trypsin/mL; C, 10 μ-g trypsin/mL; D, 40 pg trypsin/mL for 15 min at room temperature. Proteolysis was stopped by the addition of a 20-fold excess of trypsin inhibitor. Membranes were washed twice in PSNM buffer prior to electrophoresis. 14

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Figure 5. Fluorogram of electrophoretically separated pea chioroplast poly­ peptides from control and trypsin-treated membranes that were tagged with radio­ labeled azidoatrazine. Key: A, control membranes (50 fig Chl/mL) UV-irradi­ ated 10 min in the presence of 2.5 μΜ azido- C-atrazine; B, as in A, but with 25 μΜ azidoatrazine; C, treatment of chloroplasts with 2 μg trypsin/mL for 15 min prior to tagging with 25 μΜ azidoatrazine; D, as in C, but 40 μg tryp­ sin/mL for 15 min prior to tagging with azidoatrazine. 14

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c o n t r o l membranes at a concentration of 2.5 uM (equivalent to an a t r a z i n e concentration r e q u i r e d to i n h i b i t e l e c t r o n t r a n s p o r t by 90%) o n l y the 34 kDal polypeptide species was labeled (Figure 5A). When a 1 0 - f o l d higher concentration is u t i l i z e d (25 y M ) , equivalent to an a t r a z i n e concentration r e q u i r e d to i n h i b i t e l e c t r o n t r a n s p o r t by 90% in t r y p s i n - t r e a t e d membranes) both the 34 and 32 kDal forms of the 32-34 kDal polypeptide are labeled (Figure 5B). Mild t r y p s i n - t r e a t m e n t (2 yg t r y p s i n / m l , 15 min) followed by a z i d o a t r a z i n e tagging using 25 yM a z i d o a t r a z i n e (Figure 5C) r e s u l t e d in tagging of the 32 kDal species ( i n c o n t r a s t to l a b e l ­ ing both the 34 and 32 kDal forms of the polypeptide in c o n t r o l membranes). This supports the hypothesis that the presence of the t r y p s i n - d e r i v e d 32 kDal polypeptide provides both an a f f i n i t y s i t e and a binding s i t e f o r t r i a z i n e s . When thylakoids are tagged f o l l o w i n g high t r y p s i n treatment (40 yg t r y p s i n / m l , 15 min) no label is observed at 32 kDal, nor at the molecular weights of the expected breakdown products of 18 and 16 kDal. These data f u r t h e r suggest that a f f i n i t y and/or binding to the i n t r i n s i c fragments alone can not occur. It should be noted that in a l l cases where a high concentration of a z i d o a t r a z i n e i s u t i l i z e d (25 yM, Figures 5B, C, and D) a d e t e c t a b l e l e v e l of n o n s p e c i f i c binding of the compound to a number of t h y l a k o i d polypeptides i s observed. The most i n t e n s e l y l a b e l e d was a p o l y ­ peptide species of 25 kDal before t r y p s i n treatment or 23 kDal a f t e r protease d i g e s t i o n . This polypeptide was p r e v i o u s l y demon­ s t r a t e d to be the apoprotein of the l i g h t - h a r v e s t i n g c h l o r o p h y l l a/b pigment p r o t e i n which has a surface-exposed segment (23). The T r i a z i n e Receptor P r o t e i n i s a Product of C h l o r o p l a s t Directed P r o t e i η Synthesfs"] T r i a z i n e r e s i s t a n c e has been demon­ s t r a t e d from r e c i p r o c a l c r o s s i n g experiments to be i n h e r i t e d u n i p a r e n t a l l y through the female parent i n J3. campestri s (16). As the r e s i s t a n c e mechanism has been shown to r e s i d e at the l e v e l of the c h i o r o p l a s t membrane, a r o l e f o r the c h i o r o p l a s t genome i n c o n f e r r i n g r e s i s t a n c e is strongly implied (17). It was of i n t e r e s t to determine i f the c h i o r o p l a s t membrane p r o t e i n of 32-34 kDal that binds the p h o t o a f f i n i t y t r i a z i n e , and which appears to be required f o r t r i a z i n e binding in i s o l a t e d PS II p a r t i c l e s , i s a c h i o r o p l a s t gene product. In developing c h l o r o p l a s t s , in p a r a l l e l to the appearance of f u n c t i o n a l a c t i v i ­ t i e s , there i s r a p i d synthesis and accumulation of a major t h y l a ­ koid p r o t e i n of 34 kDal (36). This r a p i d l y synthesized c h i o r o ­ p l a s t p r o t e i n has been shown to be encoded by the c h i o r o p l a s t genome i n 1. mays (37). This s e c t i o n o u t l i n e s experiments that were c a r r i e d out to determine i f the c h i o r o p l a s t polypeptide, which serves as the t r i a z i n e binding s i t e , i s i d e n t i c a l to the chloroplast-encoded p r o t e i n of the same molecular weight. As was shown i n Figure 4, the 34 kDal polypeptide l a b e l e d by a z i d o a t r a z i n e shows a s p e c i f i c stepwise a l t e r a t i o n mediated

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

50

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch003

by the p r o t e o l y t i c enzyme t r y p s i n . We i n t e r p r e t that the s p e c i f i c stepwise a l t e r a t i o n o f t h i s polypeptide is regulated by (a) the presence of l y s i n e or a r g i n i n e residues in the primary sequence of t h i s peptide that are s t e r i c a l l y a c c e s s i b l e to the p r o t e o l y t i c probe and (b) the increased a c c e s s i b i l i t y of s p e c i f i c cleavage s i t e s caused by trypsin-mediated a l t e r a t i o n s of other surface-exposed peptides that share a common microenvironment at the membrane surface with the 32-34 kDal polypeptide. The s t e p wise t r y p s i n degradation of the 34 kDal form of the polypeptide to 32, 18,and 16 kDal d e f i n e s a "map" of i t s s t r u c t u r a l i n t e g r a t i o n within the membrane and can be used to i d e n t i f y the same p r o t e i n "tagged" by an independent method. The c h l o r o p l a s t - s y n t h e s i z e d p r o t e i n of the same molecular weight as the t r i a z i n e - b i n d i n g protein i s the most r a p i d l y synt h e s i z e d p r o t e i n in vivo of pea t h y l a k o i d membranes. Thus, i t s t r y p s i n s e n s i t i v i t y can be r e a d i l y evaluated by autoradiographic techniques. Pea seedlings were allowed to take up and i n c o r p o r ate 3 5 s _ e t h i o n i n e f r 4 h; t h i s r e s u l t e d in r a d i o l a b e l i n g of r a p i d l y synthesized p o l y p e p t i d e s . Control and t r y p s i n - t r e a t e d membrane samples were subjected to SDS-PAGE. Following s t a i n i n g for p r o t e i n , polypeptides were analyzed for r a d i o l a b e l i n c o r p o r a t i o n by X-ray fluorography. As shown in Figure 6A, i n c o r p o r a t i o n of 3 5 $ - r a d i o l a b e l was observed f o r two major molecular weight species — the apop r o t e i n of the l i g h t harvesting complex (LHC) at 25 kDal and a polypeptide of 34 kDal. Following t r y p s i n treatment (Figure 6B), the r a d i o l a b e l associated with the LHC polypeptides was a l t e r e d in e l e c t r o p h o r e t i c m o b i l i t y by 2 kDal f o l l o w i n g the expected a l t e r a t i o n f o r the Coomassie-stained peptide species ( 2 3 ) . The r a p i d l y synthesized 34 kDal polypeptide was also s u s c e p t i b l e to trypsin. Corresponding to i t s loss with t r y p s i n treatment, new r a d i o l a b e l e d bands appeared at 32 (Figure 6B), then 18, and 16 kDal (Figures 6C,D) i n an i d e n t i c a l , t r y p s i n - c o n c e n t r a t i o n dependent fashion to that of the p h o t o a f f i n i t y tagged 34 kDal p o l y peptide (see Figure 4 ) . From the i d e n t i c a l t r y p s i n s e n s i t i v i t y of the p h o t o a f f i n i t y tagged polypeptide and the r a p i d l y synthes i z e d c h i o r o p l a s t p r o t e i n of the same molecular weight, we conclude that the two polypeptides are one and the same. m

0

Evidence that the T r i a z i n e Binding Protein i s Present in r i a z i n e Resistant Weed B i o t y p e s . The u t i l i z a t i o n of a photoa f f i n i t y labeled t r i a z i n e h e r b i c i d e has been i n v a l u a b l e in the d e f i n i t i v e i d e n t i f i c a t i o n of one s p e c i f i c polypeptide of the PS II complex as the t r i a z i n e binding s i t e . The absence of covalent l a b e l i n g in r e s i s t a n t membranes suggests that e i t h e r the polypeptide i s missing from the membrane or that i t i s present, but g e n e t i c a l l y a l t e r e d , r e s u l t i n g in an a l t e r a t i o n in i t s primary s t r u c t u r e , and p o s s i b l y changing i t s conformation in the membranes. In s u s c e p t i b l e membranes, t r i a z i n e and urea h e r b i c i d e s com-

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch003

3. STEINBACK ET AL.

Receptor Site for Triazine Herbicides

51

Figure 6. Fluorogram of polyacrylamide gel showing trypsin sensitivity of the 34-kDal polypeptide of pea chioro­ plast membranes following in vivo incor­ poration of S-methionine in whole leaves. Key: A, control; B, 2 ^g trypsin/ mL; C, 10 ng trypsin/mL; D, 40 μg trypsin/mL. Treatments were as described in Figure 4. 35

52

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

pete f o r the same binding region as determined by d i r e c t compet i t i o n s t u d i e s (9). In r e s i s t a n t membranes, whereas t r i a z i n e s e n s i t i v i t y i s e x t e n s i v e l y d i m i n i s h e d , the a b i l i t y of diuron to i n h i b i t e l e c t r o n t r a n s p o r t i s not a l t e r e d s i g n i f i c a n t l y (3., 9., 13). This i n d i c a t e s an a l t e r a t i o n in the t r i a z i n e , but not the urea a f f i n i t y s i t e and f u r t h e r suggests that a common binding s i t e , i . e . , p r o t e i n , i s present in r e s i s t a n t membranes, but possesses an a l t e r e d a f f i n i t y f o r the t r i a z i n e h e r b i c i d e s . It was of i n t e r e s t to determine i f the polypeptide r e s p o n s i b l e for h e r b i c i d e s e n s i t i v i t y i n c h l o r o p l a s t s of normal, t r i a z i n e - s u s c e p t i b l e plants was also present, i . e . , s y n t h e s i z e d , in the r e s i s t a n t biotypes. Using the techniques f o r 3 5 $ - e t h i o n i n e i n c o r p o r a t i o n j j i v j v o described f o r peas, i n c o r p o r a t i o n of ^ S - r a d i o label i n t o t h y l a k o i d polypeptides of s u s c e p t i b l e and r e s i s t a n t biotypes of A. hybridus was i n v e s t i g a t e d . The data shown in Figure 7 demonstrate that the 34 kDal polypeptide i s synthesized in both h e r b i c i d e - s u s c e p t i b l e and r e s i s t a n t p l a n t s . Furthermore, the polypeptide in r e s i s t a n t membranes shows an i d e n t i c a l s e n s i t i v i t y to t r y p s i n - t r e a t m e n t as the 34 kDal polypeptide of the s u s c e p t i b l e membranes. Whereas i t is evident that the polypept i d e i s synthesized and present in both b i o t y p e s , there i s no apparent s i z e d i f f e r e n c e or change in membrane o r i e n t a t i o n , as r e f l e c t e d by t r y p s i n s e n s i t i v i t y , that can account for the extreme d i f f e r e n c e s in t r i a z i n e a f f i n i t i e s . The t r i a z i n e - r e s i s t a n c e mechanism, t h e r e f o r e , probably r e s i d e s in an a l t e r e d primary s t r u c t u r e of t h i s p r o t e i n . m

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch003

5

Acknowledgements: This research was supported, in p a r t , by a Grant from the United States - I s r a e l B i n a t i o n a l A g r i c u l t u r a l Research and Development Fund (BARD), DOE Contract DE-AC02-7ER01338 to Michigan State U n i v e r s i t y , and a Wellesley College F a c u l t y Aid Grant to K. Steinback. We also thank Dr. Gary Gardner of S h e l l A g r i c u l t u r a l Chemicals f o r his c o l l a b o r a t i o n and c o n s u l t a t i o n s in experiments using a z i d o - a t r a z i n e .

Receptor Site for Triazine Herbicides

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch003

STEINBACK ET AL.

Figure 7. Fluorogram of a polyacrylamide gel showing trypsin sensitivity of the 34-kDal polypeptide of chioroplast thylakoid membranes isolated from susceptible (S) and resistant (R) biotypes of A. hybridus following in vivo incorporation of S-methionine in whole leaves. Isolated thylakoid membranes were treated with A, no trypsin; B, 2 fig trypsin/mL; C, 20 μg trypsin/mL for 15 min as described for Figure 4. 35

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch003

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BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

Literature Cited 1. Ashton, F. M.; Crafts, A. S. "Mode of Action of Herbicides"; Wiley: New York, 1973; pp. 69-99. 2. Wright, K.; Corbett, J. Κ. Z. Naturforsch. 1979, 34c, 966-72. 3. Pfister, K.; Arntzen, C. J. Z. Naturforsch. 1979, 34c, 996-1009. 4. Bouges-Bocquet, B. Biochim. Biophys. Acta 1973, 314, 250-6. 5. Velthuys, B. R.; Amesz, J. Biochim. Biophys. Acta 1974, 333, 85-94. 6. Tischer, W.; Strotmann, H. Biochim. Biophys. Acta 1977, 460, 113-25. 7. Trebst, Α.; Draber, W. "Advances in Pesticide Research"; Geissbuhler, H., Ed.; Pergamon Press: Oxford, 1979; pp. 223-34. 8. Strotmann, H.; Tischer, W.; Edelman, K. Ber. Deutsch Bot. Ges. 1974, 87, 457-63. 9. Pfister, K.; Radosevich, S. R.; Arntzen, C. J. Plant Physiol. 1979, 64, 995-9. 10. Radosevich, S. R.; DeVilliers, O. T. Weed Sci., 1976, 24, 229-32. 11. Souza-Machado, V.; Arntzen, C. J.; Bandeen, J. D.; Stephen­ son, G. R. Weed Sci., 1978; 26, 318-22. 12. Arntzen, C. J.; Ditto, C. L.; Brewer, P. Proc. Natl. Acad. Sci., U.S.A., 1979, 76, 278-82. 13. Pfister, K.; Watson, J.; Arntzen, C. J. Weed Sci. 1981 (in press). 14. Bowes, J.; Crofts, A. R.; Arntzen, C. J. Arch. Biochem. Biophys. 1980, 200, 303-8. 15. Arntzen, C. J.; Pfister, K.; Steinback, Κ. E. "Studies of Herbicide Resistance", LeBaron, H.; Gressel, J . , Eds.; CRC Press (in press). 16. Souza-Machado, V.; Bandeen, J. D.; Stephenson, G. R.; Lavigne, P. Can J. Plant Sci. 1978, 58, 977-81. 17. Darr, S.; Souza Machado, V.; Arntzen, C. J. Biochim. Biophys. Acta 1981, 634, 219-28. 18. "Blair, G. E.; Ellis, R. J. Biochim. Biophys. Acta 1973, 319, 223-34. 19. Steinback, K.E.; Pfister, K.; Arntzen, C. J. Z. Naturforsch. 1981, 36c, 98-108. 20. Pfister, K.; Steinback, Κ. E.; Gardner, G.; Arntzen, C. J. Proc. Natl. Acad. Sci., U.S.A. 1981, 78, 981-5. 21. Gardner, G. Science 1981, 211, 937-40. 22. Laemmli, U. K. Nature (London) 1970, 227, 680-5. 23. Steinback, Κ. E.; Burke, J. J.; Arntzen, C. J. Arch. Biochem. Biophys. 1979, 195, 546-57. 24. Lasky, R. Α.; Mills, A. D. Eur. J. Biochem. 1975, 56, 335-41. 25. Regitz, G.; Ohad, I. "Proc. 3rd. Int. Congr. on Photosynthe­ sis", Vol. III; Avron, M., Ed.; Elsevier: Amsterdam, 1975; p. 1615.

3.

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26. 27. 28. 29. 30. 31. 32. 33.

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34. 35. 36. 37.

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Renger, G. Z. Naturforsch. 1979, 34c, 1010-14. Trebst, A. Z. Naturforsch. 1979, 34c, 986-91. Böger, P.; Kunert, K. J. Z. Naturforsch. 1979, 34c, 1015-25. Reimer, S.; Link, K.; Trebst, A. Z. Naturforsch. 1979, 34c, 419-26. Oettmeier, W.; Masson, K. Pestic. Biochem. Physiol. 1980, 14, 86-97. Oettmeier, W.; Masson, K.; Johanningmeier, U. FEBS Lett. 1980, 118, 267-70. Croze, E.; Kelly, M.; Horton, P. FEBS Lett. 1979, 103, 22-6. Mullet, J. E.; Arntzen, C. J. Biochim. Biophys. Acta 1981, 635, 236-48. Bayley, H.; Knowles, J. R. "Methods in Enzymology", Vol. XLVI; Jackoby, W. B.; Wilchek, M., Eds.; Academic Press: New York, 1977; 69-114. Leto, K.; Keresztes, Α.; Arntzen, C. J. Plant Physiol. 1981, (in press). Grebanier, A. E.; Steinback, Κ. E.; Bogorad, L. Plant Physiol. 1979, 63, 436-9. Bedbrook, J. R.; Link, G.; Coen, D. M.; Bogorad, L.; Rich, A. Proc. Natl. Acad. Sci., U.S.A. 1978, 75, 3060-4.

RECEIVED

August 14, 1981.

4 The Role of Light and Oxygen in the Action of Photosynthetic Inhibitor Herbicides A L A N D. DODGE

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch004

University of Bath, School of Biological Sciences, Claverton Down, Bath, Avon BA2 7AY, United Kingdom

P h y t o t o x i c symptoms of i n j u r y caused by h e r b i c i d e s that inhibit c h l o r o p l a s t e l e c t r o n t r a n s p o r t , such as the ureas, t r i a z i n e s , and h y d r o x y b e n z o n i t r i l e s , are promoted by both light and oxygen. When carbon d i o x i d e fixation is prevented, excess excitation energy leads to the generation of longer lived triplet c h l o r o p h y l l . I f unquenched by c a r o t e n o i d s , this may directly induce proton a b s t r a c t i o n from u n s a t u r a t e d f a t t y a c i d s or s i n g l e t oxygen might be generated which induces lipid peroxide formation. Lipid p e r o x i d a t i o n leads to cellular d e s t r u c t i o n and death. H e r b i c i d e s that d i v e r t p h o t o s y n t h e t i c e l e c t r o n t r a n s p o r t , such as the bipyridyls paraquat and diquat, yield the superoxide a n i o n . Superoxide l e v e l s produced overtax the normal defense mechanisms. More t o x i c species such as hydroxyl free r a d i c a l s are a l s o probably produced which instigate lipid p e r o x i d a t i o n and lead to cellular d i s o r g a n i z a t i o n and death. James Franck wrote i n 1949 (1) " . . . i t i s one of the m i r a c l e s of photosynthesis that the p l a n t can use a dye able to f l u o r e s c e i n the presence of oxygen, predominantly f o r the purpose of r e d u c t i o n , and i s able to hold the process of photooxidation i n check so that damage i s prevented or minimized even under severe conditions". I t i s evident that the c h i o r o p l a s t i s endowed with p r o t e c t i v e devices that are able to l i m i t damage except under extreme c o n d i t i o n s . Such s i t u a t i o n s are promoted by the presence of photosynthetic i n h i b i t o r h e r b i c i d e s . In the normal c h i o r o p l a s t , l i g h t energy (hv) absorbed by pigments and i n p a r t i c u l a r by the c h l o r o p h y l l s (Chl) causes _g e x c i t a t i o n to the s i n g l e t s t a t e . This s h o r t - l i v e d s t a t e (^10 sec) i s quenched by r a p i d energy t r a n s f e r to c h l o r o p h y l l i n the reaction centers. I f unquenched, intersystem c r o s s i n g may lead to the generation of the longer l i v e d t r i p l e t s t a t e (^10~3 s e c ) .

0097-6156/82/0181 -0057$05.00/0 © 1982 American Chemical Society

58

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

ί

.S.C

l

Chl + hv

3

Chl

Chl

T r i p l e t c h l o r o p h y l l can be harmlessly quenched by c h i o r o p l a s t carotenoid pigments (2) d i s s i p a t i n g the e x c i t a t i o n energy (3). I f t r i p l e t c h l o r o p h y l l i s unquenched, energy t r a n s f e r from t h i s pigment t o ground s t a t e oxygen may generate s i n g l e t oxygen ( Û 2 ) . T h i s i s g e n e r a l l y termed a type 2 r e a c t i o n . 1

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch004

3

3

Chl + 0

^

2

Chl +

0

l

2

S i n g l e t oxygen may a l s o be quenched by cartenoids (4) and by f r e e r a d i c a l scavengers l o c a t e d w i t h i n the c h i o r o p l a s t t h y l a k o i d s , such as α-tocopherol (5). During a c t i v e photosynthesis, oxygen concentrations w i t h i n the c h i o r o p l a s t may be higher than the surrounding cytoplasm (6) and e l e c t r o n leakage from the thylakoids could y i e l d the superoxide anion r a d i c a l (θ£~) (7). I t i s p o s s i b l e that t h i s could be formed during normal p s e u d o c y c l i c e l e c t r o n t r a n s p o r t (8). Although the superoxide r a d i c a l i s apparently l e s s t o x i c than s i n g l e t oxygen, i t s t o x i c i t y may be r e l a t e d to the generation of more t o x i c species such as hydroxyl f r e e (OH*) r a d i c a l s . The c h i o r o p l a s t possesses an e f f i c i e n t superoxide scavenging system i n the form o f Cu-Zn superoxide dismutase (SOD) (9) to p r o t e c t against t h i s p o t e n t i a l t o x i c species. Of

+ 0 2 ~ + 2H

+

S

Q

D

»» H 0 2

2

+0

2

The dismutating a c t i v i t y of bound manganese ( 1 0 ) may a l s o be important. The generation o f hydrogen peroxide i n t h i s r e a c t i o n may be t o l e r a t e d by the presence of a d d i t i o n a l enzyme systems that c a t a l y s e the d e s t r u c t i o n of H2O2 (11): ( i ) ascorbate peroxidase, ( i i ) dehydroascorbate reductase, and ( i i i ) g l u t a t h i o n e reductase. Η

2θ2^ν

H 0^ 2

(i)

^.Ascorbate

^-^glutathione

*Dehydroascorbate>^glutathione (ii)

(ΟΧ)-ν.

•NADPH

( r e d ) i f *NADP

+

(iii)

The p o t e n t i a l hazards of oxygen and l i g h t might seem to be exacerbated by low carbon d i o x i d e concentrations w i t h i n the chioroplast. This might be minimized by p h o t o r e s p i r a t i o n (12) whereby under low carbon d i o x i d e concentrations, oxygen r e a c t s with the carbon d i o x i d e acceptor molecule r i b u l o s e bisphosphate to form 3-phosphoglycerate and 2-phosphoglycollate. Further metabolism of phosphoglycollate r e c y c l e s carbon d i o x i d e to the c h i o r o p l a s t (13).

4.

DODGE

Photosynthetic Inhibitor Herbicides

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch004

E l e c t r o n Transport

59

Inhibitors

A large number of h e r b i c i d e s i n c l u d i n g the phenylureas, ^ - t r i a z i n e s , u r a c i l s , triazinones, hydroxybenzonitriles, pyridazinones, and a c y l a n i l i d e s i n h i b i t photosynthetic e l e c t r o n t r a n s p o r t ( H i l l Reaction). The s i t e of a c t i o n i s g e n e r a l l y thought to be a p r o t e i n component l o c a t e d on the outside of the t h y l a k o i d membrane (14) and a f f e c t i n g a p o i n t between the h y p o t h e t i c a l Q and Β components of the e l e c t r o n transport chain. As a r e s u l t of t h i s i n t e r a c t i o n , e l e c t r o n transport ceases. This leads to a r a p i d i n h i b i t i o n of carbon d i o x i d e f i x a t i o n that proceeds at a s i m i l a r r a t e i r r e s p e c t i v e of whether the leaves are incubated i n darkness or l i g h t (15) (Table I ) . An i n t e r r u p t i o n of photosynthesis w i l l e v e n t u a l l y lead to a r e d u c t i o n of food reserves and subsequent s t a r v a t i o n . The appearance of phytot o x i c symptoms, such as c h l o r o p h y l l bleaching, i s c l e a r l y promoted by l i g h t (L5, _16, 17) (Figure 1). The i n h i b i t i o n of e l e c t r o n flow w i l l prevent photosynthetic oxygen e v o l u t i o n , but the c h i o r o p l a s t envelope probably provides l i t t l e r e s i s t a n c e to inward oxygen d i f f u s i o n . Experiments have demonstrated that with both monuron and i o x y n i l , i n c u b a t i o n of cotyledons under argon reduced, but d i d not prevent the r a p i d d e s t r u c t i o n of c h l o r o p h y l l (Figure 2) (18). Promotion of the t o x i c a c t i o n of photosynthetic i n h i b i t o r h e r b i c i d e s by l i g h t may be i n i t i a l l y a s c r i b e d to type 1 reactions. T r i p l e t c h l o r o p h y l l , without the involvement of oxygen, may d i r e c t l y i n i t i a t e e l e c t r o n or hydrogen a b s t r a c t i o n from p a r t i c u l a r l y s u s c e p t i b l e molecules (e.g., unsaturated f a t t y acids (LH)) to y i e l d l i p i d f r e e r a d i c a l s .

LH

L-

Once i n i t i a t e d , subsequent propagation r e a c t i o n s i n v o l v i n g oxygen may generate more f r e e r a d i c a l s that a t t a c k unsaturated l i p i d s i n a chain r e a c t i o n of gathering momentum.

L" + 0

L02*

2

L0 * + LH 2

»» L* + LOOH

The production of l i p i d hydroperoxides (LOOH) a l s o may be achieved by the d i r e c t i n t e r a c t i o n of s i n g l e t oxygen with s a t u r a t e d f a t t y acids (19).

LH +

0

L

2

LOOH

un­

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60

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

TABLE I Carbon d i o x i d e f i x a t i o n by f l a x cotyledons measured by an IRGA with i l l u m i n a t i o n of 115 Wm , p r e v i o u s l y incubated with 1 0 M monuron i n the dark or l i g h t of 5.25 or 30 Wm"", from P a l l e t t and Dodge (15) -2

_3

2

Incubation Time (Min)

ymol C02/g.F.Wt./h Dark

5.25

2

Wm'

2

30 WnT

0

68.0

68.0

68.0

15

18.6

11.1

15.4

30

3.2

3.5

2.2

60

2.3

1.9

0.9

120

0.0

0.0

0.0

Photosynthetic Inhibitor Herbicides

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch004

DODGE

Figure 1. The chlorophyll content of flax cotyledons incubated on water (A) or 10' M monuron (B), in the dark (Φ) or with light of 5.25 Wm' (A) or 30 Wrn «Π5λ 3

2

2

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BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

o-i

.

0

48

H 96

Incubation Time (h)

Figure 2. The effect of a 10 M solution of ioxynil (A) or monuron (B) on the chlorophyll content of flax cotyledons incubated in sealed conicalflaskscontaining air (%) or argon (O)with light of 30 Wm (IS). 3

2

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4.

DODGE

Photosynthetic Inhibitor Herbicides

63

C h i o r o p l a s t t h y l a k o i d membranes are composed of an almost equal percentage of p r o t e i n and l i p i d . The a c y l l i p i d s of the c h i o r o p l a s t membrane a r e h i g h l y unsaturated; f o r example, around 80% of the f a t t y a c i d component i s the 18:3 unsaturated l i n o l e n i c a c i d . The consequences of l i p i d p e r o x i d a t i o n r e a c t i o n s are (a) the promotion of membrane d e s t r u c t i o n which i n part may be v i s i b l y demonstrated as c h l o r o p h y l l l o s s , and observed by e l e c t r o n microscopy as a d i s o r g a n i z a t i o n of t h y l a k o i d s and other c e l l u l a r membranes (18); and (b) the l i p i d hydroperoxides undergo fragmentation producing short chain hydrocarbons such as ethane (20, 21). Experiments with both monuron and i o x y n i l showed that the l o s s of c h l o r o p h y l l i n f l a x cotyledons was preceded by the breakdown of carotenoid pigments, e s p e c i a l l y the carotenes, suggesting that t h i s p r o t e c t i v e system was over-taxed and destroyed. This was followed by a r a p i d formation of ethane (18) (Figures 3 and 4). Experiments i n which monuron t r e a t e d leaves were t r e a t e d with the s i n g l e t oxygen quencher DABCO ( d i a z o b i c y c l o octane) showed a l i m i t e d c o n t r o l of c h l o r o p h y l l breakdown (22) (Table I I ) . Many experiments i n t o the secondary e f f e c t s of photos y n t h e t i c i n h i b i t o r h e r b i c i d e s have been performed with i s o l a t e d chloroplasts. I s o l a t e d c h l o r o p l a s t s provide a convenient system f o r studying the generation and quenching o f s i n g l e t oxygen. Recent experiments with pea c h l o r o p l a s t s i l l u m i n a t e d i n the absence of an e l e c t r o n acceptor have shown that both c h l o r o p h y l l and l i n o l e n i c a c i d breakdown was retarded by the s i n g l e t quenchers DABCO and c r o c i n (23). Further work showed that l i n o l e n i c a c i d breakdown and ethane generation i n i s o l a t e d c h i o r o p l a s t t h y l a k o i d s was promoted by the a d d i t i o n of the s i n g l e t oxygen generator rose bengal immobilized on DEAE-sepharose (24). Divertors of Electron

Transport

The i n t e r a c t i o n of b i p y r i d y l h e r b i c i d e s (paraquat and diquat) with photosynthesis i s d i f f e r e n t from that of the e l e c t r o n transport i n h i b i t o r s . These compounds, with h i g h l y negative redox p o t e n t i a l s (paraquat E ^ " 446mV; diquat E Q 349mV), i n t e r a c t i n the v i c i n i t y of f e r r e d o x i n causing a d i v e r s i o n o f e l e c t r o n flow from the u l t i m a t e e l e c t r o n acceptor NADP . This was c l e a r l y seen i n paraquat-treated p l a n t m a t e r i a l as a progressive i n h i b i t i o n of carbon dioxide uptake (25) (Figure 5). Although carbon d i o x i d e uptake r a p i d l y ceased, e l e c t r o n flow from water continued f o r some time u n t i l t h i s system was t o t a l l y i n a c t i v a t e d by the general d e s t r u c t i o n of c e l l u l a r i n t e g r i t y (25). Paraquat (and/or diquat) i s reduced by a one e l e c t r o n t r a n s f e r t o give the paraquat f r e e - r a d i c a l . Under anaerobic c o n d i t i o n s t h i s r a d i c a l can accumulate (26, 27). However, i n the presence of oxygen, which should be p l e n t i f u l i n the v i c i n i t y o f the t h y l a k o i d i n the e a r l y stages of paraquat

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch004

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

Incubation time (h)

Figure 3. The effect of 10' M monuron on pigment content and ethane generation of flax cotyledons incubated at 30 Wm' . Pigment levels are expressed as a percent­ age of the levels at 0 h. Key: carotenes, ·; xanthophylls, A; chlorophyll, |; and ethane generation, Ο (IS). 3

2

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch004

DODGE

Photosynthetic Inhibitor Herbicides

Incubation time (h)

Figure 4. The effect of 10~ M ioxynil on pigment content and ethane generation of flax cotyledons incubated at 5.25 Wm . Pigment levels are expressed as a per­ centage of the levels at 0 h. Key: carotenes, ·; xanthophylls, A; chlorophyll, |/ and ethane generation, Ο (IS). 3

2

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch004

66

TABLE I I The e f f e c t o f DABCO ( 1 0 " % ) on the c h l o r o p h y l l content of f l a x cotyledons incubated on monuron ( 1 0 " % ) f o r 96 h a t 5.25 Wm", from Youngman e t a l . (22) 2

Treatment

mg c h l o r o p h y l l / g . f r e s h wt.

Water

1.735

Monuron

1.184

DABCO

1.610

Monuron + DABCO

1.598

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch004

4.

DODGE

Photosynthetic Inhibitor Herbicides

67

Hours Paraquat Treatment

Figure 5. Carbon dioxide uptake and evolution by paraquat-treatedflaxcotyledons, incubated on paraquat (10~ M) in either light of 5.25 Wm (O) or darkness (%). The uptake and evolution of the cotyledons were estimated after treatment 4

(25).

2

68

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

treatment, the reduced paraquat was superoxide : PQ

PQ'

+

2 +

+ 0

r a p i d l y r e o x i d i z e d to y i e l d

+ e"

2

PQ'

+

PQ

2 +

+

0 " 2

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch004

The a c t i v i t y of these h e r b i c i d e s i s known to be promoted by oxygen (28, 29, 30). I t has a l s o been demonstrated that c h l o r o s i s i s promoted by i n c r e a s i n g l i g h t (see Figure 11 f o r Conyza) but diminished i n the presence of an e l e c t r o n transport i n h i b i t o r (28) (Figure 6). A d d i t i o n a l l i n e s of evidence f u r t h e r i n d i c a t e the importance of superoxide i n the a c t i o n of these h e r b i c i d e s . I t has been shown with i s o l a t e d c h l o r o p l a s t s that the production of super­ oxide, as measured by the conversion of hydroxylamine to n i t r i t e , was promoted by paraquat and r e s t r a i n e d by superoxide dismutase (21) (Figure 7). The generation of superoxide was l i m i t e d i f an e l e c t r o n transport i n h i b i t o r such as monuron was present (Table I I I ) . Although the c h i o r o p l a s t contains superoxide dismutase enzymes, i t i s assumed that the r a p i d generation of 0 ~ overtaxes the c a p a b i l i t i e s of the enzymes. Further experiments with whole leaves and the superoxide scavenger copper p e n i c i l l a m i n e (31) showed that the presence of t h i s compound not only m i t i g a t e d the a c t i o n of the h e r b i c i d e i n promoting c h l o r o ­ p h y l l decay (32) (Figure 8), but a l s o l i m i t e d l i p i d p e r o x i d a t i o n as shown by the diminished r e l e a s e of ethane (33) (Figure 9). The f e a s i b i l i t y of the superoxide r a d i c a l being the i n i t i a l t o x i c species i n v i v o was p r e d i c t e d by F a r r i n g t o n et a l . , (34) using pulse r a d i o l y s i s s t u d i e s . I t was p o s t u l a t e d that the concentra­ t i o n of superoxide w i t h i n the p l a n t c e l l could remain constant at 1 μΜ up to 10 ym or more from the c h i o r o p l a s t t h y l a k o i d membrane. 2

Although i t was thought i n i t i a l l y that superoxide i t s e l f could i n t e r a c t with membrane l i p i d s to i n i t i a t e l i p i d per­ o x i d a t i o n , more recent evidence has expressed doubt on the p o t e n t i a l r e a c t i v i t y of t h i s molecule to induce t h i s process (35). Much d i s c u s s i o n has centered on the p o s s i b l e involvement of Fenton and Haber-Weiss type r e a c t i o n s o c c u r r i n g i n v i v o and generating more t o x i c species such as hydroxyl f r e e r a d i c a l s and s i n g l e t oxygen. At present i t i s not p o s s i b l e to a f f i r m that such systems operate (36, 37). However, i t might be p o s s i b l e f o r the reduced paraquat r a d i c a l to i n t e r a c t with superoxide to generate hydrogen peroxide, a w e l l known product of reduced paraquat r e o x i d a t i o n (38). 2H PQ*

+

+ 0

2

PQ

2 +

+ 0

2

+

H 0 2

2

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4.

DODGE

69

Photosynthetic Inhibitor Herbicides

Figure 6. The effect of monuron (ΙΟ M) on the chlorophyll level of paraquattreated (10~ M) cotyledons under light of 5.25 Wm' . Key: paraquat, O; and para­ quat plus monuron, |3

4

2

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch004

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

Illumination Time

(mins)

Figure 7. Nitrite formation from hydroxylamine. Reaction mixture contained in 3 mL: 50 mM phosphate buffer pH 7.8; 1 μτηοΐ of NH OH; chloroplasts with 75 ng of chlorophyll and where indicated 6.6 Μ paraquat; 50 units SOD. Illuminated at 275 Wm' at 22°C. Key: control, •; plus paraquat, A; plus SOD, •; and plus paraquat and SOD, Δ (22). 2

μ

2

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch004

4.

DODGE

71

Photosynthetic Inhibitor Herbicides

TABLE I I I The e f f e c t of monuron (lCT^M) n the a b i l i t y of paraquat to promote the formation of n i t r i t e from hydroxylamine i n i s o l a t e d chloroplasts. F o r f u r t h e r d e t a i l s see legend to Figure 7, from Youngman et_ a l . (32) Q

Treatment

Water (nmoles n i t r i t e / m g

Monuron chlorophyll/hr)

Control

100

17

Paraquat

195

28

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch004

72

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

24

48

Illumination Time (h)

Figure 8. The chlorophyll content of paraquat-treatedflaxcotyledons incubated in the presence or absence of copper-penicillamine (PA-Cu) under light of 5.25 Wm' . The final concentration of paraquat was 10 M and the PA-Cu represented 50 units of superoxide dismutase. Key: control, paraquat plus PA-Cu, A ; and paraquat, Φ (32). 2

s

Illumination Time (h)

Figure 9. Ethane generation byflaxcotyledons incubated inflasksunder light of 5.25 Wm' . Thefinalconcentration of paraquat was 10' M and the copper-peni­ cillamine (PA-Cu) represented 50 units of superoxide dismutase. Key: control, ·; paraquat, A / and paraquat plus PA-Cu, |f33J. 2

6

4.

DODGE

Photosynthetic Inhibitor Herbicides

73

Further i n t e r a c t i o n of reduced paraquat w i t h hydrogen peroxide could y i e l d the more r e a c t i v e hydroxyl f r e e r a d i c a l (39) which could i n i t i a t e l i p i d p e r o x i d a t i o n i n membrane f a t t y a c i d s .

+

PQ* + H 0

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch004

2

2

^PQ

2

+

+ 0H~ +

OH"

E l e c t r o n microscope s t u d i e s of paraquat-treated leaves showed that a f t e r a few hours, membranes had been d i s r u p t e d and c e l l u l a r compartmentalization was destroyed. Experiments demonstrating the r e l e a s e of potassium from t r e a t e d t i s s u e as an i n d i c a t i o n of plasmalemma and tonoplast d i s r u p t i o n (25) (Figure 10) corresponded i n time with the v i s i b l e demonstration of c e l l u l a r d i s o r g a n i z a t i o n (40). I f the i n i t i a l a c t i o n of the h e r b i c i d e s i s to i n d i r e c t l y promote l i p i d p e r o x i d a t i o n of membrane unsaturated f a t t y a c i d s and lead to c e l l u l a r d i s o r g a n i z a t i o n , then subsequent d e t e r i o r a t i v e changes w i l l occur because of the r e l e a s e of vacuolar contents. Not only w i l l there be a r a p i d change i n the osmotic p r o p e r t i e s of the c e l l , but the r e l e a s e of vacuolar h y d r o l y t i c enzymes w i l l cause f u r t h e r damage. In a d d i t i o n , type 1 and type 2 s e n s i t i z e d r e a c t i o n s may a l s o i n c r e a s e and c o l l e c t i v e l y these changes w i l l l e a d to r a p i d p l a n t death. Although paraquat i s used as a broad spectrum t o t a l - k i l l h e r b i c i d e , t o l e r a n c e has been found i n c e r t a i n l i n e s of Lolium perenne (41) and the weed Conyza (42) (Figure 11) . In both i n s t a n c e s , t o l e r a n t l i n e s showed a l a c k of e f f e c t on carbon d i o x i d e f i x a t i o n , i n d i c a t i n g reduced p e n e t r a t i o n of h e r b i c i d e (42, 43) (Figure 12). T o l e r a n t l i n e s of Lolium perenne were nevertheless shown to possess greater superoxide dismutase a c t i v i t y as w e l l as greater c a t a l a s e and peroxidase a c t i v i t i e s (44). T o l e r a n t Conyza biotypes showed approximately t h r e e - f o l d increases i n superoxide dismutase a c t i v i t y (42). Conclusion The p h y t o t o x i c a c t i o n of both e l e c t r o n transport i n h i b i t o r and e l e c t r o n d e v i a t o r h e r b i c i d e s i s promoted by l i g h t and i s l a r g e l y a response to the overtaxing of p r o t e c t i v e systems. In both i n s t a n c e s , type 1 r e a c t i o n s , i m p l i c a t i n g the d i r e c t a c t i o n of e x c i t e d t r i p l e t c h l o r o p h y l l , may be i n v o l v e d . However, of greater p o t e n t i a l importance i s the i n t e r a c t i o n of a c t i v e oxygen s p e c i e s . With e l e c t r o n t r a n s p o r t i n h i b i t o r s , s i n g l e t oxygen i s produced as an i n c i d e n t a l response to unquenched t r i p l e t c h l o r o p h y l l . With e l e c t r o n d e v i a t o r h e r b i c i d e s , the generation of superoxide i s a d i r e c t consequence of the d i v e r s i o n of e l e c t r o n flow (Figure 13). Once membrane d i s r u p t i o n i s

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch004

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

3

6

9

Hours Paraquat Treatment

Figure 10. Membrane permeability measured as potassium leakage from slices of paraquat-treated (10~ M)flaxcotyledons (25). 4

v

2.0

5 LL CD

—?4h ^ ^ B i o t y p e il

1.5

B

"48h

Ε

S

1.01

c Ο ϋ

] | 0.5

α ο ο .C ο

24 Biotype l

10 Light

Intensity

40

30

20 (W

m" ) 2

Figure 11. The chlorophyll content of leaf sections of two biotypes of Conyza treated for 24 or 48 h with paraquat at 10~ M , at various light intensities (41). 5

75

Photosynthetic Inhibitor Herbicides

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch004

4. DODGE

Figure 13. Summary scheme of the primary effects caused by A, photosynthetic inhibitor herbicides and B, photosynthetic deviator herbicides. 0 is triplet or ground state oxygen; PQ represents paraquat. 3

2

76

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

i n i t i a t e d by l i p i d p e r o x i d a t i o n , and death f o l l o w s .

rapid c e l l u l a r

disorganization

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch004

Literature Cited 1. Franck, J. "Photosynthesis in Plants"; The Iowa State College Press: Iowa, 1949; p 293. 2. Krinsky, N.I. Pure and Appl. Chem. 1979, 51, 649. 3. Wolff, Ch.; Witt, H.T. Z. Naturforsch. 1969, 246, 1031. 4. Foote, C.S.; Denny, R.W. J. Am. Chem. Soc. 1968, 90, 6233. 5·. Hughes, C.T.; Gaunt, J.K.; Laidman, D.L. Biochem. J . 1971, 124, 9p. 6. Steiger, H.M.; Beck, E.; Beck, R. Plant Physiol. 1977, 60, 903. 7. Marsho, T.V.; Behrens, P.W.; Radmer, R.J. Plant Physiol. 1979, 64, 656. 8. Elstner, E.F.; Stoffer, C.; Heupel, A. Z. Naturforsch. 1975, 30C, 53. 9. Asada, K.; Urano, M.; Takahashi, M. Eur. J. Biochem. 1973, 36, 257. 10. Foyer, C.H.; Hall, D.O. FEBS Lett. 1979, 101, 324. 11. Halliwell, B.; Foyer, C.H.; Charles, S.A. Proc. 5th Int. Congr. Photosynth.: Halkidiki, Greece, 1981; in press. 12. Heber, V.; Krause, G.H. TIBS 1980, 5, 32. 13. Chollet, R. TIBS, 1977, 2, 155. 14. Renger, G. Biochim. Biophys. Acta 1976, 440, 287. 15. Pallett, K.E.; Dodge, A.D. Z. Naturforsch. 1979, 34C, 1058. 16. Minshall, W.H. Weeds, 1957, 5, 29. 17. Oorschot, J.L.P. Van; Leeuwen, P.H. Van. Weed Res. 1974, 14, 81. 18. Pallett, K.E.; Dodge, A.D. J. Exp. Bot. 1980, 31, 1051. 19. Rawls, H.R.; Santen, P.J. Van. J . Am. Oil. Chem. Soc. 1970, 47, 121. 20. Riely, C.A.; Cohen, G.; Liebermann, M. Science 1974, 183, 208. 21. Elstner, E.F.; Konze, J.R. Nature 1976, 263, 351. 22. Youngman, R.J.; Pallett, K.E.; Dodge, A.D. "Chemical and biochemical aspects of superoxide and superoxide dismutase". Elsevier/North Holland: New York, 1980; p 402. 23. Percival, M.; Dodge, A.D. (in preparation). 24. Percival, M.; Dodge, A.D. Proc. 5th Int. Congr. Photosynth.: Halkidiki, Greece, 1981; in press. 25. Harris, N.; Dodge, A.D. Planta 1972, 104, 210. 26. Zweig, G.; Shavit, Ν.; Avron, M. Biochim. Biophys. Acta 1965, 109, 332. 27. Dodge, A.D. Endeavour 1971, 30, 130. 28. Mees, G.C. Ann. Appl. Biol. 1960, 48, 601. 29. Merkle, M.G.; Leinweber, C.L.; Bovey, R.W. Plant Physiol. 1965, 40, 832.

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4.

DODGE

Photosynthetic Inhibitor Herbicides

77

30. Rensen, J.J.S. van. Physiol. Plant 1975, 33, 42. 31. Lengfelder, E.; Elstner, E.F. Hoppe-Seylers Z. Physiol. Chem. 1978, 359, 751. 32. Youngman, R.J.; Dodge, A.D. Z. Naturforsch. 1979, 34C, 1032. 33. Youngman, R.J.; Dodge, A.D.; Lengfelder, E.; Elstner, E.F. Experientia. 1979, 35, 1295. 34. Farrington, J.A.; Ebert, M.; Land, E.J.; Fletcher, K. Biochim. Biophys. Acta 1973, 314, 372. 35. Bors, W.; Saran, M.; Lengfelder, E.; Spöttl, R.; Michel, C. Curr. Top. Radiat. Res. 1974, 9, 247. 36. Halliwell, B. FEBS Lett. 1976, 72, 8. 37. Koppenol, W.H.; Butler, J.; Leeuwen, J.W. van. Photochem. Photobiol. 1978, 28, 655. 38. Davenport, H.E. Proc. R. Soc. B. 1963, 157, 332. 39. Farrington, J.A. Proc. Brit. Crop. Prot. Conf. 1976, 225. 40. Harris, N.; Dodge, A.D. Planta 1972, 104, 201. 41. Harvey, B.M.R.; Muldoon, J.; Harper, D.B. PI. Cell Environ. 1978, 1, 203. 42. Youngman, R.J.; Dodge, A.D. Proc. 5th Int. Congr. Photosynth.: Halkidiki, Greece, 1981; in press. 43. Harvey, B.M.R.; Fraser, T.W. Pl. Cell. Environ. 1980, 3, · 44. Harper, D.B.; Harvey, B.M.R. P l . Cell. Environ. 1978, 1, 211. RECEIVED September 2, 1981. 1 0 7

5 Interaction of Herbicides with Cellular and Liposome Membranes DONALD E . MORELAND, STEVEN C. HUBER, and WILLIAM P. NOVITZKY

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch005

North Carolina State University, U.S. Department of Agriculture, Agricultural Research Service, Departments of Crop Science and Botany, Raleigh, NC 27650

Many h e r b i c i d e s inhibit c h l o r o p l a s t e l e c t r o n t r a n s p o r t by b i n d i n g to a p r o t e i n l o c a t e d on the reducing s i d e of PS II. Some of the h e r b i c i d e s (chlorpropham, dinoseb, p r o p a n i l , ioxynil), but not all (diuron, s-triazines, uracils), also interfere w i t h photophosphorylation and m i t o c h o n d r i a l e l e c t r o n transport and p h o s p h o r y l a t i o n . I n t h i s study, p r o p a n i l , dinoseb, ioxynil, and s e v e r a l c a r b a n i l a t e s , but not d i u r o n , a f f e c t e d the f o l l o w i n g responses, much like the uncoupler FCCP: (a) inhibited the light-dependent quenching of a t e b r i n fluorescence in t h y l a k o i d s ; (b) inhibited v a l i n o m y c i n ­ -induced s w e l l i n g of all o r g a n e l l e s ; (c) increased the p e r m e a b i l i t y of all o r g a n e l l e membranes to K+ in the absence of an ionophore; and (d) increased the p e r m e a b i l i t y of p h o s p h a t i d y l c h o l i n e liposomes to H. The structure/activity c o r r e l a t i o n s were similar f o r all o r g a n e l l e s , i.e., the responses were not membrane specific. R e s u l t s obtained suggested that when h e r b i c i d e s partition i n t o the lipid phases of o r g a n e l l e membranes, p e r t u r b a t i o n s are produced that l e a d to a l t e r a t i o n s in "fluidity" and p e r m e a b i l i t y to c a t i o n s . The a l t e r a t i o n s may be r e s p o n s i b l e f o r uncoupling of ATP generation i n mitochondria and c h l o r o p l a s t s , and inhibition of e l e c t r o n t r a n s p o r t in m i t o c h o n d r i a . +

A l a r g e number of commercial h e r b i c i d e s i n t e r f e r e with e l e c t r o n transport and ATP production i n i s o l a t e d c h l o r o p l a s t s and mitochondria (1). These h e r b i c i d e s can be d i v i d e d i n t o two groups: e l e c t r o n transport i n h i b i t o r s and i n h i b i t o r y uncouplers (1, 2 ) . The dimethylphenylureas, s u b s t i t u t e d u r a c i l s , s ~ t r i a z i n e s , and pyridazinones have been c l a s s i f i e d as e l e c t r o n

This chapter not subject to U.S. copyright. Published 1982 American Chemical Society.

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t r a n s p o r t i n h i b i t o r s , whereas the a l k y l a t e d d i n i t r o p h e n o l s , a c y l a n i l i d e s , halogenated b e n z o n i t r i l e s , and #-phenylcarbamates have been c l a s s i f i e d as i n h i b i t o r y uncouplers. A l l of the abovenamed h e r b i c i d e s i n h i b i t c h i o r o p l a s t e l e c t r o n t r a n s p o r t by b i n d i n g r e v e r s i b l y to a p r o t e i n ( s ) a s s o c i a t e d w i t h the Β complex (3, ^ , 5 ) . Β i s considered to f u n c t i o n as the secondary acceptor of e l e c t r o n s from PS I I . Binding a f f i n i t y of the h e r b i c i d a l i n h i b i ­ t o r s has been c o r r e l a t e d w i t h i n h i b i t i o n of PS I I . A d d i t i o n a l d e t a i l s of the b i n d i n g responses of the h e r b i c i d e s and chemical models proposed to e x p l a i n the i n t e r a c t i o n between i n h i b i t o r s and the receptor t a r g e t are provided i n other c o n t r i b u t i o n s published h e r e i n C5, 6^, 7) · The e l e c t r o n transport i n h i b i t o r s do not d i r e c t l y a f f e c t photophosphorylation or i n t e r f e r e w i t h m i t o c h o n d r i a l e l e c t r o n t r a n s p o r t and phosphorylation (1) . However, the i n h i b i t o r y un­ c o u p l e r s , i n a d d i t i o n to i n t e r f e r i n g w i t h e l e c t r o n t r a n s p o r t i n t h y l a k o i d s , uncouple photophosphorylation and o x i d a t i v e phosphory­ l a t i o n , and i n h i b i t m i t o c h o n d r i a l e l e c t r o n t r a n s p o r t . Whereas i n h i b i t i o n of c h i o r o p l a s t e l e c t r o n transport has been c o r r e l a t e d with b i n d i n g to a p r o t e i n ( s ) , the s i t e s and mechanisms through which h e r b i c i d e s i n t e r f e r e w i t h m i t o c h o n d r i a l and c h i o r o ­ p l a s t mediated phosphorylations remain to be i d e n t i f i e d . When l i p o p h i l i c h e r b i c i d e s p a r t i t i o n i n t o the l i p i d phases of mem­ branes, they could perturb l i p i d - l i p i d , l i p i d - p r o t e i n , and p r o t e i n - p r o t e i n i n t e r a c t i o n s that are r e q u i r e d f o r membrane func­ t i o n s such as e l e c t r o n t r a n s p o r t , ATP formation, and a c t i v e t r a n s ­ p o r t . Evidence f o r general membrane p e r t u r b a t i o n s caused by chlorpropham, 2 , 6 - d i n i t r o a n i l i n e s , p e r f l u i d o n e , and c e r t a i n phenylureas have been reported p r e v i o u s l y (8-11). The o b j e c t i v e s of the s t u d i e s reported here were to compare the a c t i o n of the compounds i d e n t i f i e d i n Table I (a) on c h i o r o ­ p l a s t and m i t o c h o n d r i a l e l e c t r o n t r a n s p o r t and phosphorylation, and (b) on the " f l u i d i t y " and p e r m e a b i l i t y to KT and H+ of c h i o r o p l a s t , m i t o c h o n d r i a l , and liposome membranes. For com­ p a r a t i v e purposes, dinoseb, i o x y n i l , p r o p a n i l , and 3-CIPC (chlorpropham) were i n c l u d e d to represent the i n h i b i t o r y un­ c o u p l e r s . Diuron represented the e l e c t r o n t r a n s p o r t i n h i b i t o r s , and FCCP was i n c l u d e d as a reference uncoupler. Three other c a r b a n i l a t e s (3-CHPC; 2,3-DCIPC; and 3,4-DCIPC) were s e l e c t e d to determine the e f f e c t of replacement of the i s o p r o p y l s i d e chain with a hexyl moiety and the e f f e c t of d i c h l o r i n a t i o n of the phenyl r i n g i n the 2,3- and 3 , 4 - p o s i t i o n s . In the phenylureas and a c y l a n i l i d e s , i n c r e a s i n g the length of the s i d e chain has been a s s o c i a t e d w i t h increased i n h i b i t o r y a c t i v i t y against the H i l l r e a c t i o n (12). A l s o , i n the phenylureas, phenylcarbamates, and a c y l a n i l i d e s , d i c h l o r i n a t i o n i n the 3,4-ring p o s i t i o n s i s more i n h i b i t o r y to c h i o r o p l a s t e l e c t r o n t r a n s p o r t than monochlorin a t i o n i n e i t h e r p o s i t i o n . A d d i t i o n a l l y , c h l o r i n a t i o n i n an ortho p o s i t i o n has been a s s o c i a t e d w i t h decreased i n h i b i t o r y a c t i v i t y (12).

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TABLE I Common names or d e s i g n a t i o n s and chemical names of compounds s t u d i e d .

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch005

Common name or d e s i g n a t i o n

Chemical name

FCCP Dinoseb Diuron Ioxynil Propanil 3-CIPC 3-CHPC 2.3- DCIPC 3.4- DCIPC

carbonylcyanide p-trifluromethoxyphenylhydrazone 2-S£c-butyl-4,6-dinitrophenol 3-(3,4-dichlorophenyl)-1,1-dimethylurea 4-hydroxy-3,5-diiodobenzonitrile 3 ,4'-dichloropropionanilide isopropyl m-chlorocarbanilate hexyl m-chlorocarbanilate isopropyl 2,3-dichlorocarbanilate isopropyl 3,4-dichlorocarbanilate 1

M a t e r i a l s and Methods Chloroplasts. I n t a c t c h l o r o p l a s t s were i s o l a t e d from f r e s h l y harvested growth chamber-grown spinach (Spinaoia oleraoea L.) as described by L i l l e y and Walker (13). Thylakoids were prepared by the method of Armond et al. (14). C h l o r o p h y l l concentrations were determined by the method of MacKinney (15). Reaction cuvettes were i l l u m i n a t e d at 25 C with a photon f l u e n c e r a t e of 7.5 X 10~^ mol/m^-s (PAR). Reduction of f e r r i c y a n i d e with water as the oxidant was measured s p e c t r o p h o t o m e t r i c a l l y at 420 nm i n a r e a c t i o n medium (5.0 ml volume) that contained 0.1 M s o r b i t o l , 50 mM tricine-NaOH (pH 8.0), 0.4 mM KH2PO4, 5 mM M g C l , 10 mM NaCl, 0.5 mM K Fe(CN)6, 1 mM ADP, and t h y l a k o i d s (100 yg c h l o r o p h y l l ) . E s t e r i f i c a t i o n of i n o r g a n i c phosphate was measured by the procedure of L a n z e t t a et al. (16). E f f e c t s on the l i g h t dependent quenching of a t e b r i n were measured i n a r e a c t i o n medium (2.0 ml volume) that contained 1.0 yM a t e b r i n , 0.1 M s o r b i t o l , 10 mM tricine-NaOH (pH 7.8), 10 mM NaCl, and t h y l a k o i d s (20 yg c h l o r o p h y l l ) . The t h y l a k o i d s were i l l u m i n a t e d w i t h red a c t i n i c l i g h t (Corning 2-64 f i l t e r ) . The a t e b r i n was e x c i t e d with 366 nm l i g h t and emission was observed at 505 nm. E f f e c t s imposed on the e f f l u x of K were measured by suspending i n t a c t c h l o r o p l a s t s (100 yg c h l o r o p h y l l ) i n a r e a c t i o n medium (1.0 ml volume) that contained 0.4 M s o r b i t o l , and 10 mM HepesNaOH (pH 7.1). A f t e r i n c u b a t i o n with t e s t chemicals f o r 2 min, the r e a c t i o n mixtures were c e n t r i f u g e d to p e l l e t the c h l o r o p l a s t s . id" content of the supernatants was measured by flame photometry. 2

3

+

Mitochondria. Mitochondria were prepared from 3-day-old dark-grown mung bean (Phaseolus aureus Roxb.) h y p o c o t y l s . The i s o l a t i o n procedure, measurements of oxygen u t i l i z a t i o n , and

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BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch005

e f f e c t s of the t e s t compounds on r e s p i r a t o r y s t a t e s were conducted as described p r e v i o u s l y ( 9 ) . Following the terminology of Chance and Williams (17), the ADP-stimulated r a t e of r e s p i r a t i o n w i l l be r e f e r r e d to as s t a t e 3 and ADP-limited r e s p i r a t i o n as s t a t e 4. The mung bean mitochondria had r e s p i r a t o r y c o n t r o l ( s t a t e 3/state 4) r a t i o s that averaged 3.9, 3.6, and 2.2; and c a l c u l a t e d ADP/O r a t i o s that averaged 2.3, 1.3, and 1.5, f o r the o x i d a t i o n of malate, NADH, and s u c c i n a t e , r e s p e c t i v e l y . Osmotic S w e l l i n g . Changes i n the osmotic s t a b i l i t y of the o r g a n e l l e s were monitored s p e c t r o p h o t o m e t r i c a l l y at 550 nm f o r c h l o r o p l a s t s and t h y l a k o i d s , and at 520 nm f o r mitochondria. The 2.0-ml r e a c t i o n mixture contained 0.15 M KSCN or KC1 and 10 mM Hepes-NaOH (pH 7.1). The i n i t i a l absorbance of the r e a c t i o n was adjusted to 0.8, 0.4, and 0.75 f o r c h l o r o p l a s t s (approximately 20 yg c h l o r o p h y l l ) , t h y l a k o i d s (approximately 20 yg c h l o r o p h y l l ) , and mitochondria (approximately 0.4 mg p r o t e i n ) , r e s p e c t i v e l y . Induction of p a s s i v e s w e l l i n g was measured with the t e s t compound being added 30 s a f t e r the i n t r o d u c t i o n of the o r g a n e l l e s . In s t u d i e s with valinomycin (0.1 yM), the t e s t compound was added 30 s p r i o r to the i n t r o d u c t i o n of the ionophore. Rates of s w e l l i n g were c a l c u l a t e d from the i n i t i a l phase of absorbance decrease. Liposomes. Liposomes were prepared by s o n i c a t i o n from egg y o l k phosphatidyl c h o l i n e (Sigma type X-E) according to the method of H i n k l e (18). The assay medium used to determine e f f e c t s of h e r b i c i d e s and FCCP contained 0.2 ml liposomes i n 1.8 ml of 0.3 M NaCl, 20 mM t r i s - H C l (pH 7.5), 5 mM Na-ascorbate, 80 yM ferrocene, and 80 yM tetraphenylboron. F e r r i c y a n i d e r e d u c t i o n was measured s p e c t r o p h o t o m e t r i c a l l y at 420 nm and 25 C. Test Chemicals. Stock s o l u t i o n s of the d e s i r e d concentrat i o n s of t e s t chemicals were prepared i n acetone. The f i n a l concentration of the s o l v e n t was h e l d constant at 1% (v/v) i n a l l assays i n c l u d i n g the c o n t r o l s . Data presented f o r the s e v e r a l s t u d i e s were averaged from determinations made w i t h a minimum of three separate r e p l i c a t i o n s and i s o l a t i o n s . Results

and

Discussion

Shown i n Table I I are I^Q values f o r i n h i b i t i o n of e l e c t r o n t r a n s p o r t i n spinach t h y l a k o i d s (water to f e r r i c y a n i d e ) and the a s s o c i a t e d phosphorylation r e a c t i o n by the t e s t compounds. Also i n c l u d e d are I 5 0 values f o r i n h i b i t i o n of the light-dependent quenching of a t e b r i n f l u o r e s c e n c e and the r a t i o obtained by d i v i d i n g the I C Q f o r the a t e b r i n response by the I 5 Q f o r i n h i b i t i o n of photophosphorylation. The r e l a t i v e order of i n h i b i t o r y potency f o r some of the compounds f o r i n h i b i t i o n of f e r r i c y a n i d e r e d u c t i o n has been reported p r e v i o u s l y (12). For the h e r b i c i d e s ,

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TABLE I I E f f e c t s of FCCP, s e l e c t e d h e r b i c i d e s , and c a r b a n i l a t e s on r e a c t i o n s mediated by spinach t h y l a k o i d s .

Compound

Ferricyanide PhosphoryReduction lation

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•I

FCCP Dinoseb Diuron Ioxynil Propanil 3-CIPC 3-CHPC 2,3-DCIPC 3,4-DCIPC

7

NE^ 7.5 0.07 0.3 0.7 150 62 130 12

5 0

Atebrin fluor.

Atebrin/ phosph. ratio

(PM)

0.35 4 0.08 0.2 0.6 140 15 125 8

0.4 23 NE 120 200 130 28 NE 70

— NE = no, or minimal, e f f e c t w i t h concentrations

1 6 —

600 333 1 2 —

9

up to 400 yM.

except diuron (a pure e l e c t r o n t r a n s p o r t i n h i b i t o r ) , the I 5 0 f o r i n h i b i t i o n of the coupled phosphorylation was lower than the I 5 0 f o r i n h i b i t i o n of f e r r i c y a n i d e r e d u c t i o n . A lower I^Q value f o r i n h i b i t i o n o f phosphorylation suggests that the compounds might be expressing an e f f e c t on the phosphorylation pathway that cannot be explained e n t i r e l y by i n t e r f e r e n c e w i t h e l e c t r o n t r a n s p o r t . The h e r b i c i d e s , except f o r d i u r o n , have been shown p r e v i o u s l y to i n ­ h i b i t c y c l i c photophosphorylation measured i n the absence of oxygen (1), and the l i g h t - i n d u c e d s y n t h e s i s of ATP mediated by d i t h i o t h r e i t o l and PMS (19). The l a t t e r r e a c t i o n s do not i n v o l v e the e n t i r e e l e c t r o n t r a n s p o r t chain and are i n s e n s i t i v e to diuron. An i n c r e a s e i n l i p o p h i l i c i t y of 3-CIPC w i t h the replacement of a h e x y l f o r the i s o p r o p y l s i d e chain (3-CHPC versus 3-CIPC) r e ­ s u l t e d i n increased i n h i b i t i o n of e l e c t r o n t r a n s p o r t and an even g r e a t e r i n h i b i t i o n of the coupled phosphorylation (Table I I ) . Increased r e d u c t i v e i n h i b i t o r y potency of 3,4-DCIPC over 3-CIPC a l s o i s shown. The i n h i b i t o r y uncouplers and the uncoupler FCCP, but not diuron and 2,3-DCIPC, a l s o i n h i b i t e d the l i g h t dependent quenching of a t e b r i n f l u o r e s c e n c e i n spinach t h y l a k o i d s (Table I I ) . I n h i ­ b i t i o n of the light-dependent quenching of a t e b r i n fluorescence has been a t t r i b u t e d to d i s s i p a t i o n o f the energized s t a t e (Δ pH) of the t h y l a k o i d membrane, which i s considered to provide the d r i v i n g f o r c e f o r phosphorylation (20, 21). The i n t e r f e r e n c e s observed support the suggestion that the i n h i b i t o r y uncoupler

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BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

h e r b i c i d e s do act as uncouplers. I n h i b i t i o n of l i g h t - i n d u c e d a t e b r i n fluorescence quenching by the c a r b a n i l a t e d e r i v a t i v e s a l s o was strengthened by an i n c r e a s e i n s i d e chain l i p o p h i l i c i t y (3-CIPC versus 3-CHPC) and d i c h l o r i n a t i o n (3-CIPC versus 3,4DCIPC), but negated by ortho s u b s t i t u t i o n (2,3-DCIPC). Of the p h e n o l i c h e r b i c i d e s , dinoseb was a good uncoupler, whereas i o x y n i l was a r e l a t i v e l y poor uncoupler. The i n h i b i t o r y un­ c o u p l e r s , but not the e l e c t r o n transport i n h i b i t o r s , a l s o stimulated e l e c t r o n t r a n s p o r t through PS I by i l l u m i n a t e d thy­ l a k o i d s (reduced DPIP as oxidant and methyl v i o l o g e n as reductant}, when e l e c t r o n flow through PS I I was blocked w i t h diuron (22). S t i m u l a t i o n o f oxygen uptake (22) and i n h i b i t i o n of the a t e b r i n induced fluorescence quenching (Table II) occurred over the same molar concentra t i o n ranges. Neither r e a c t i o n i n o v l v e d PS I I . The a t e b r i n / p h o s p h o r y l a t i o n r a t i o (Table I I , l a s t column) r e l a t e s i n h i b i t i o n of photophosphorylation to d i s s i p a t i o n of the p o s t u l a t e d energized s t a t e (Δ pH) o f the t h y l a k o i d membrane. A low r a t i o suggests that the two responses are c o r r e l a t e d . How­ ever, the high r a t i o s obtained f o r i o x y n i l and p r o p a n i l suggest that these two h e r b i c i d e s act as e l e c t r o n t r a n s p o r t i n h i b i t o r s r a t h e r than as uncouplers. M i t o c h o n d r i a l Responses. The h e r b i c i d e s r e f e r r e d to as i n ­ h i b i t o r y uncouplers were so named because a t low molar concentra­ t i o n s they s a t i s f y most, i f not a l l , o f the c r i t e r i a e s t a b l i s h e d f o r uncouplers o f o x i d a t i v e p h o s p h o r y l a t i o n . However, a t h i g h e r molar c o n c e n t r a t i o n s , they a l s o i n h i b i t m i t o c h o n d r i a l e l e c t r o n transport ( 1 ) . Uncoupling a c t i o n ( i n t e r f e r e n c e w i t h ATP generation) has been demonstrated by s t i m u l a t i o n o f s t a t e 4 r e s p i r a t i o n f o r the o x i d a t i o n of malate, s u c c i n a t e , and NADH; circumvention of o l i g o m y c i n - i n h i b i t e d s t a t e 3 r e s p i r a t i o n ; and i n d u c t i o n of ATPase a c t i v i t y (1). In a d d i t i o n , by using v a r i o u s s u b s t r a t e s , p a r t i a l r e a c t i o n s , and e l e c t r o n mediators, evidence has been presented that the h e r b i c i d e s i n h i b i t malate o x i d a t i o n and malate-PMS oxidoreductase by a c t i n g a t o r near complex I , succinate o x i d a t i o n and succinate-PMS oxidoreductase by a c t i n g at o r near complex I I , exogenous-NADH o x i d a t i o n by a c t i n g p r i o r t o the cytochrome chain, and c y a n i d e - r e s i s t a n t r e s p i r a t i o n ( a l t e r n a t e o x i d a s e ) . That i n h i b i t i o n of malate, s u c c i n a t e , and exogenous-NADH o x i d a t i o n i s not caused by i n t e r f e r e n c e a t a common s i t e , shared by the 3 sub­ s t r a t e s , i s i n d i c a t e d by the widely d i f f e r i n g I 5 0 values that a r e obtained ( 1 , J3, 9_> 1 0 ) . The pure e l e c t r o n transport i n h i b i t o r s of c h i o r o p l a s t e l e c t r o n t r a n s p o r t have only a m a r g i n a l , i f any, e f f e c t on m i t o c h o n d r i a l responses. For the most p a r t , w i t h the compounds included i n t h i s study, maximum uncoupling a c t i v i t y ( s t i m u l a t i o n o f s t a t e 4 r e s p i r a t i o n ) was obtained f o r the o x i d a t i o n o f succinate and i n h i b i t i o n of malate s t a t e 3 r e s p i r a t i o n was most s e n s i t i v e . Therefore, only data f o r s t a t e 4 s t i m u l a t i o n f o r the o x i d a t i o n of succinate and

5.

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85

TABLE I I I E f f e c t s of FCCP, s e l e c t e d h e r b i c i d e s , and c a r b a n i l a t e s on r e a c t i o n s mediated by mung bean mitochondria.

Compound

Succinate state 4 respiration (Concn.) (Stim.)

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch005

μΜ

FCCP Dinoseb Diuron Ioxynil Propanil 3-CIPC 3-CHPC 2,3-DCIPC 3,4-DCIPC

—

NE = no,

0.1 1 , NR§/ 10 200 200 20 NE 40

Malate state 3 respiration (Inhib.)

% 108 92 NE 90 80 71 71 NE 69

or marginal, e f f e c t with concentrations

I (uM) 50

50 55 410 150 170 100 28 90 47

up

to 400

μΜ.

i n h i b i t i o n of s t a t e 3 r e s p i r a t i o n f o r the o x i d a t i o n of malate are presented i n Table I I I . Only diuron and 2,3-DCIPC f a i l e d to stimulate s t a t e 4 r e s p i r a t i o n . None of the compounds was as e f f e c t i v e an uncoupler as FCCP. P r o p a n i l and 3-CIPC showed maxi­ mum s t i m u l a t o r y a c t i v i t y at a r e l a t i v e l y high molar concentration (200 μ Μ ) . Replacement of the i s o p r o p y l group of CIPC f o r the h e x y l group (3-CHPC) was a s s o c i a t e d with an increase i n uncou­ p l i n g a c t i v i t y as was the a d d i t i o n of a second c h l o r i n e i n the 4 - p o s i t i o n (3,4-DCIPC) of the r i n g . For some of the h e r b i c i d e s , s t i m u l a t i o n of s t a t e 4 r e s p i r a t i o n occurred at molar concentra­ t i o n s that a l s o i n h i b i t e d s t a t e 3 r e s p i r a t i o n , which can be a t t r i b u t e d to an e f f e c t on e l e c t r o n t r a n s p o r t . Hence, the e f f e c t on e l e c t r o n transport may have masked the f u l l expression of uncoupling a c t i v i t y . A l l of the compounds i n h i b i t e d s t a t e 3 r e s p i r a t i o n with malate as s u b s t r a t e , however, diuron was a r e l a t i v e l y weak i n ­ h i b i t o r . As i n the expression of uncoupling a c t i o n , the l e n g t h ­ ening of the s i d e chain (3-CHPC versus 3-CIPC) and the a d d i t i o n of a second c h l o r i n e to the phenyl r i n g i n the para p o s i t i o n (3,4-DCIPC versus 3-CIPC) was a s s o c i a t e d w i t h enhanced a c t i v i t y . The 2,3-DCIPC d e r i v a t i v e was s l i g h t l y l e s s a c t i v e than 3-CIPC, the reference c a r b a n i l a t e . Whereas i n h i b i t i o n of c h i o r o p l a s t e l e c t r o n transport has been c o r r e l a t e d with b i n d i n g to a p r o t e i n ( s ) , the mechanisms f o r

86

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch005

i n t e r f e r e n c e w i t h photophosphorylation and the m i t o c h o n d r i a l responses remain to be i d e n t i f i e d . The h e r b i c i d e s could act independently with s e v e r a l components of the m i t o c h o n d r i a l e l e c t r o n t r a n s p o r t and p h o s p h o r y l a t i o n pathway, or they could be p e r t u r b i n g the membranes i n such a way that m u l t i s i t e i n t e r f e r e n c e results. Swelling Responses. E f f e c t s of the h e r b i c i d e s on the " f l u i d i t y " and p e r m e a b i l i t y p r o p e r t i e s of membranes were measured. A l t e r a t i o n s to the f l u i d i t y and p e r m e a b i l i t y of o r g a n e l l e membranes can a l t e r the osmotic p r o p e r t i e s of the membranes. C h i o r o p l a s t , t h y l a k o i d , and m i t o c h o n d r i a l membranes are known to be r e l a t i v e l y impermeable to c a t i o n s such as K*~ and but f r e e l y permeable to l i p o p h i l i c anions such as SCN"" (23, 24). Hence, o r g a n e l l e s are o s m o t i c a l l y s t a b l e when suspended i n i s o t o n i c KSCN. K p e r m e a b i l i t y , however, can be induced a r t i f i ­ c i a l l y by ionophores such as valinomycin (Figure 1A). Valinomycin i s considered to form a l i p i d - s o l u b l e complex with ΚΓ*" and func­ t i o n s to t r a n s p o r t K across the membranes (25). An i n c r e a s e i n i n t e r n a l K+ w i l l be accompanied by the d i f f u s i o n of SCN", the counter i o n , across the membrane to maintain e l e c t r o n e u t r a l i t y . An i n c r e a s e i n i n t e r n a l s o l u t e c o n c e n t r a t i o n w i l l r e s u l t i n an i n f l u x of water and the o r g a n e l l e s w i l l s w e l l . Shown i n the lowest t r a c e s of F i g u r e IB, C, and D i s the r a t e and magnitude of s w e l l i n g obtained when valinomycin was added to i n t a c t c h l o r o p l a s t s , t h y l a k o i d s , and mitochondria, r e s p e c t i v e l y . A l l of the t e s t compounds except d i u r o n , i n h i b i t e d the r a t e and magnitude of valinomycin-induced s w e l l i n g i n the three o r g a n e l l e s suspended i n i s o t o n i c KSCN, as shown i n F i g u r e 1 f o r dinoseb. Dose/response curves were developed from the t r a c e s (Figure 1) and I^Q values obtained from the curves are presented i n Table IV. The e f f e c t was expressed at molar concentrations higher than those r e q u i r e d to i n h i b i t c h i o r o p l a s t e l e c t r o n t r a n s ­ port and p h o s p h o r y l a t i o n . However, the data demonstrate that i n h i b i t o r y uncouplers can a f f e c t the p r o p e r t i e s of o r g a n e l l e mem­ branes. The I 5 0 values f o r i n h i b i t i o n of valinomycin-induced s w e l l i n g i n mitochondria, w i t h the exception of FCCP, c o r r e l a t e d c l o s e l y with I 5 0 values f o r i n h i b i t i o n of malate s t a t e 3 r e s p i r a ­ t i o n ( c f . Table I I I ) . Two of the c a r b a n i l a t e s (3,4-DCIPC and 3-CHPC) were as i n h i b i t o r y , or were b e t t e r i n h i b i t o r s , as i n d i ­ cated by the lower I^Q v a l u e s , than the standard uncoupler FCCP, i n a l l three o r g a n e l l e s . +

+

The osmoticum used had a marked e f f e c t on the i n h i b i t o r y potency of FCCP and the non-carbanilates (Table I V ) . For the nonc a r b a n i l a t e h e r b i c i d e s and FCCP, valinomycin-induced s w e l l i n g of mitochondria suspended i n i s o t o n i c KC1 was i n h i b i t e d at much lower concentrations of the h e r b i c i d e s than when the mitochondria were suspended i n i s o t o n i c KSCN. For the c a r b a n i l a t e s , the molar c o n c e n t r a t i o n f o r 50% i n h i b i t i o n of valinomycin-induced s w e l l i n g of mitochondria suspended i n KSCN was about twice the

5.

Interaction of Herbicides

MORELAND ET A L .

@

©

MODEL

87

THYLAKOIDS

MEMBRANE • HjO

DINOSEB

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch005

•SCN

μ -

INTACT CHLOROPLASTS DINOSEB VAL

® MITOCHONDRIA DINOSEB

I—0.5 min-

Figure 1. Representative traces of absorbance changes that show inhibition by dinoseb of valinomycin-induced swelling of intact spinach chloroplasts (B), spinach thylakoids (C), and mung bean mitochondria (D) suspended in isotonic KSCN. The model system is presented diagrammatically in A. Swelling was initiated by the addition of 0.1 Μ valinomycin (VAL). μ

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

88

TABLE IV I n h i b i t i o n of valinomycin-induced s w e l l i n g of i n t a c t spinach c h l o r o p l a s t s , spinach t h y l a k o i d s , and mung bean mitochondria by FCCP, s e l e c t e d h e r b i c i d e s , and c a r b a n i l a t e s .

Osmoticum Compound Chloroplasts

KSCN Thylakoids

Mitochondria

KCl Mitochondria

I (uM)

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch005

50

FCCP Dinoseb Diuron Ioxynil Propanil 3-CIPC 3-CHPC 2,3-DCIPC 3,4-DCIPC a/ — NE = no,

19 220 NEâ' 310 185 180 3 120 28

or minimal,

65 170 NE 240 180 135 14 95 17

22 90 NE 330 200 185 33 170 33

e f f e c t w i t h concentrations

0.1 0.4 300 2 5 100 14 66 25 up to 400

μΜ.

concentration r e q u i r e d to i n h i b i t the response w i t h mitochondria suspended i n K C l . I n h i b i t i o n of valinomycin-induced s w e l l i n g would occur i f the t e s t compounds i n t e r f e r e d w i t h movement of e i t h e r K* or anion, or both. Because valinomycin i s a mobile c a r r i e r of i t s r a t e of movement i s dependent upon the f l u i d i t y of the membrane. One i n t e r p r e t a t i o n f o r the observed i n h i b i t i o n s i s that the compounds, by p a r t i t i o n i n g i n t o the o r g a n e l l e membranes, decrease the " f l u i d i t y " of the membrane, and, subsequently, the r a t e at which the v a l i n o m y c i n - K complex moves across i t . The g r e a t e r s e n s i t i v ­ i t y expressed w i t h KCl as the osmoticum suggested that FCCP and the i n h i b i t o r y uncouplers might a l s o i n t e r f e r e , i n some way, with CI" movement. The p o s s i b i l i t y e x i s t s t h a t , i n mung bean mito­ chondria, movement of C l ~ i s c a r r i e r - m e d i a t e d , whereas SCN", being a l i p o p h i l i c anion, d i f f u s e s f r e e l y across the membrane. The h e r b i c i d e s may, i n some manner, i n t e r f e r e with the endogenous t r a n s p o r t mechanism and, t h e r e f o r e , cause greater i n h i b i t i o n of swelling i n i s o t o n i c KCl. +

Not a l l endogenous membrane t r a n s p o r t systems are a f f e c t e d by the h e r b i c i d e s . For example, spontaneous m i t o c h o n d r i a l s w e l l i n g i n i s o t o n i c s o l u t i o n s of ammonium phosphate or n e u t r a l amino acids, was a f f e c t e d only m a r g i n a l l y by the compounds (data not shown). Swelling i n these systems i n v o l v e s the endogenous Pi"/0H~ a n t i p o r t e r (26) and amino a c i d p o r t e r (27) , r e s p e c t i v e l y . At t h i s

5.

MORELAND ET AL.

Interaction of Herbicides

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch005

time, there i s no ready e x p l a n a t i o n f o r the apparent of C I " t r a n s p o r t by the n o n - c a r b a n i l a t e s .

89 inhibition

E f f e c t s on membrane p e r m e a b i l i t y to i o n s . For the most p a r t , the compounds induced c h l o r o p l a s t s , t h y l a k o i d s , and mitochondria suspended i n i s o t o n i c KSCN t o s w e l l i n the absence of an ionophore. T y p i c a l r e s u l t s obtained w i t h dinoseb are presented i n F i g u r e 2. As shown i n the no-dinoseb c o n t r o l curves, some spontaneous s w e l l i n g occurred at a slow r a t e . However, the a d d i t i o n of dinoseb g r e a t l y i n c r e a s e d the r a t e and magnitude of the s w e l l i n g response i n a l l three o r g a n e l l e s . In these experiments, as i n the valinomycin s t u d i e s , s w e l l i n g would be expected to occur only i f the p e r m e a b i l i t y of the o r g a n e l l e membranes to was i n c r e a s e d by the t e s t compounds because the membranes are considered to be f r e e l y permeable to SCN" ( F i g u r e 2A). Dose/response curves were developed from t r a c e s such as those shown i n F i g u r e 2 f o r dinoseb. For comparative purposes, the conc e n t r a t i o n of compound r e q u i r e d to induce an i n c r e a s e i n the s w e l l i n g r a t e of 0.02 A i n 1 min r e l a t i v e to the n o - h e r b i c i d e cont r o l s i s shown i n Table V f o r c h l o r o p l a s t s , t h y l a k o i d s , and mitochondria. Thylakoids d i d not s w e l l as e x t e n s i v e l y as the o t h e r two o r g a n e l l e s , consequently, the values are f o r a change of 0.01 A. The r e l a t i v e order o f a c t i v i t y shown by the compounds i s s i m i l a r to that given i n Table IV f o r i n h i b i t i o n of v a l i n o m y c i n induced s w e l l i n g i n the three o r g a n e l l e s . Mitochondria suspended i n i s o t o n i c K C l were a l s o induced to s w e l l by a l l o f the compounds, except d i u r o n (Table V ) . However, as opposed to i n h i b i t i o n of valinomycin-induced s w e l l i n g (Table IV), the values obtained i n i s o t o n i c K C l were s l i g h t l y h i g h e r than those obtained i n i s o t o n i c KSCN, w i t h two exceptions (3-CHPC and 2,3-DCIPC). As i n the valinomycin-induced system, i n order f o r s w e l l i n g to occur, both the c a t i o n and anion must cross the membranes. Because SCN" i s a permeant anion and i t s movement i s c o n t r o l l e d e l e c t r o g e n i c a l l y , e f f e c t s imposed by the compounds on the movement of id" would determine the occurrence and extent of s w e l l i n g . Hence, with mitochondria suspended i n KSCN, data r e f l e c t e f f e c t s imposed on the movement of id". In mitochondria suspended i n K C l , the compounds could a f f e c t movement of e i t h e r or both id" and C l ~ because both are nonpermeant. I f the compounds were i n c r e a s i n g only the p e r m e a b i l i t y of the m i t o c h o n d r i a l membrane to K", then data obtained i n KSCN and K C l would be expected to be s i m i l a r . The higher values obtained i n K C l r e l a t i v e t o KSCN may r e f l e c t i n t e r f e r e n c e by the compounds with C l ~ movement. As f o r i n h i b i t i o n o f valinomycin-induced s w e l l i n g , some o f the c a r b a n i l a t e s (3-CHPC and 2,3-DCIPC) seem to be a c t i n g d i f f e r e n t l y i n t h a t values obtained i n i s o t o n i c K C l were lower than those obtained i n i s o t o n i c KSCN. The p e r m e a b i l i t y of c h i o r o p l a s t membranes to endogenous K+ was a l s o i n c r e a s e d by FCCP and the i n h i b i t o r y uncouplers, but not 4

90

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

(§)

MODEL

©

THYLAKOIDS

MEMBRANE

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch005

OUT/ SCN"

/

I/ /

IN

DINOSEB

• SCN"

HoO

INTACT CHLOROPLASTS

τ

0.05A

1 J-

M 0

®

MITOCHONDRIA

DINOSEB

DINOSEB

V

\ \ \

^°

V \ΝΛιοο \V ^ 2 0 0 \ 400 1—

1 min —

-0.5 min

Figure 2. Representative traces of absorbance changes that show induction of passive swelling by dinoseb of intact spinach chloroplasts (B), spinach thylakoids (C), and mung bean mitochondria (D) suspended in isotonic KSCN. The model system is presented diagrammatically in A.

5.

MORELAND ET AL.

Interaction of Herbicides

91

TABLE V Induction of p a s s i v e s w e l l i n g of i n t a c t spinach c h l o r o p l a s t s , spinach t h y l a k o i d s , and mung bean mitochondria by FCCP, s e l e c t e d h e r b i c i d e s , and c a r b a n i l a t e s . Osmoticum n

KSCN

A

Compound

KCl

C h l o r o p l a s t s ^ ^ Thylakoids-!?/ Mitochondria^/

_ Mitochondria*'

i (yM)

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch005

5 0

FCCP Dinoseb Diuron Ioxynil Propanil 3-CIPC 3-CHPC 2,3-DCIPC 3,4-DCIPC

110 200 NEC./ 330 160 110 14 71 15

31 230 NE 200 200 110 14 NE 57

9 31 230 45 59 105 34 180 18

30 110 NE 110 220 190 24 89 38

a/ — Concentration (μΜ) r e q u i r e d to induce an i n c r e a s e i n the s w e l l ­ ing r a t e of 0.02 A i n 1 min, r e l a t i v e to the no-herbicide controls. — / Concentration (μΜ) r e q u i r e d to induce an i n c r e a s e i n the s w e l l ­ ing r a t e o f 0.01 A i n 1 min, r e l a t i v e to the no-herbicide controls. SJ NE = no, or minimal, e f f e c t with concentrations up to 400 μΜ.

the e l e c t r o n t r a n s p o r t i n h i b i t o r s (Table V I ) . The c h l o r o p l a s t s used i n these s t u d i e s contained about 250 nmoles stromal K /mg c h l o r o p h y l l . A slow e f f l u x o f K+ was induced by acetone (1%) used as a s o l v e n t f o r the t e s t compounds. Data shown i n Table VI have been c o r r e c t e d f o r the n o - h e r b i c i d e c o n t r o l e f f l u x (40 nmoles K+/mg chlorophyll·min). Diuron d i d not i n c r e a s e the e f f l u x o f K " s i g n i f i c a n t l y above that induced by acetone. I o x y n i l , 3-CIPC, dinoseb, and 2,3-DCIPC induced s i g n i f i c a n t K+ e f f l u x over a s i m i l a r molar c o n c e n t r a t i o n range. P r o p a n i l had the weakest e f f e c t on K+ e f f l u x . The most a c t i v e compounds were 3-CHPC, which i n i t i a t e d e f f l u x a t concentrations below 2 μΜ, and 3,4-DCIPC, which was a s l i g h t l y b e t t e r inducer than FCCP. The r e s u l t s d i r e c t l y support the p o s t u l a t e that the h e r b i c i d e s i n c r e a s e d mem­ brane p e r m e a b i l i t y to K . +

4

+

The i n h i b i t o r y uncouplers, but not diuron and 2,3-DCIPC, a l s o a l t e r e d the p e r m e a b i l i t y t o protons o f a r t i f i c i a l , p u r e l y l i p o i d a l , liposome membranes. I n the system (Figure 3A), e l e c t r o n s flow

Figure 3. Effects of dinoseb on increasing the rate of reduction of ferricyanide, included within egg yolk phosphatidyl choline liposomes, by ferrocene (B). The model system is presented diagrammatically in A.

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TABLE VI E f f e c t s of FCCP, s e l e c t e d h e r b i c i d e s , and c a r b a n i l a t e s on the e f f l u x of potassium from i n t a c t spinach c h l o r o p l a s t s and the p e r m e a b i l i t y of l e c i t h i n liposomes

K

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch005

Compound

FCCP Dinoseb Diuron Ioxynil Propanil 3-CIPC 3-CHPC 2.3- DCIPC 3.4- DCIPC

+

e f f l u x from a/ chloroplasts^

22 110 NE£' 110 290 100 6 120 19

H

+

permeability

of liposomes-^

.05 1 NE 3 120 360 49 NE 140

a/ + — Concentration (yM) r e q u i r e d to induce e f f l u x of Κ at a r a t e of 100 nmoles/mg chlorophyll·2 min above the no-herbicide control rate. — ^ Concentration (yM) r e q u i r e d to i n c r e a s e the r a t e of f e r r i ­ cyanide r e d u c t i o n to 80 nmoles/min. The no-herbicide c o n t r o l r a t e was about 40 nmoles/min. c/ — NE = no, or minimal, e f f e c t with concentrations up to 400 yM.

from ascorbate (outside) v i a ferrocene ( i n the liposome membrane) to f e r r i c y a n i d e (included w i t h i n the liposome) i n the presence of tetraphenylboron (18). D i f f u s i o n of H+" across the liposome mem­ brane i s r e q u i r e d to maintain e l e c t r o n e u t r a l i t y . Dose/response curves were developed from t r a c e s such as shown i n F i g u r e 3B f o r dinoseb. For comparative purposes, the concen­ t r a t i o n of compound r e q u i r e d to i n c r e a s e the r a t e of f e r r i c y a n i d e r e d u c t i o n to twice that o f the no-herbicide c o n t r o l r a t e are shown i n the l a s t column of Table V I . Uncouplers such as FCCP a c c e l ­ erate the r a t e of f e r r i c y a n i d e r e d u c t i o n , presumably by s h u t t l i n g protons across the membrane i n response to the e l e c t r i c a l poten­ t i a l generated by the r e d u c t i o n of f e r r i c y a n i d e by ferrocene (28). In t h i s study, FCCP was the most e f f e c t i v e compound. The two phenolic h e r b i c i d e s (dinoseb and i o x y n i l ) were more a c t i v e than p r o p a n i l and chlorpropham. Among the c a r b a n i l a t e s , 3-CHPC and 3,4-DCIPC were more a c t i v e than the parent compound (3-CIPC), whereas 2,3-DCIPC was e s s e n t i a l l y i n a c t i v e . These r e s u l t s

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

94

demonstrate that the i n h i b i t o r y uncoupler h e r b i c i d e s , l i k e the uncoupler FCCP, can i n c r e a s e the p e r m e a b i l i t y of an a r t i f i c i a l , p u r e l y l i p o i d a l membrane to IT*". In the liposome experiments as reported here, the noh e r b i c i d e c o n t r o l r a t e was a f f e c t e d by the r e l a t i v e concentration of ferrocene and t e t r a p h e n y l boron i n the r e a c t i o n mixture. A d d i t i o n a l l y , responses induced by the compounds v a r i e d q u a n t i ­ t a t i v e l y with the source and p u r i t y of the phosphatidyl c h o l i n e from which the liposomes were prepared. However, the r e l a t i v e order of a c t i v i t y of the compounds remained constant. Data were reported f o r the egg y o l k p r e p a r a t i o n because i t provided a b e t t e r e v a l u a t i o n of the r e l a t i v e a c t i v i t i e s of the c a r b a n i l a t e s .

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch005

Conclusions From the s t u d i e s and r e s u l t s reported h e r e i n , the f o l l o w i n g general conclusions and e x t r a p o l a t i o n s can be made: a. The s t r u c t u r e / a c t i v i t y c o r r e l a t i o n s apply to c h l o r o p l a s t s , t h y l a k o i d s , mitochondria, and liposomes, i . e . , the responses observed were not membrane s p e c i f i c . b. The i n h i b i t i o n of valinomycin-induced s w e l l i n g ( a t t r i b u t e d to decreased membrane " f l u i d i t y " ) and i n d u c t i o n of passive s w e l l i n g ( a t t r i b u t e d to increased p e r m e a b i l i t y of the membranes to K ) are not o r g a n e l l e s p e c i f i c responses. c. The l i m i t e d s t r u c t u r e / a c t i v i t y s t u d i e s conducted with the c a r b a n i l a t e s suggest that a f f i n i t y f o r b i n d i n g to the Β p r o t e i n complex c o r r e l a t e s with i n c r e a s e d uncoupling a c t i v i t y i n PS I assays, increased uncoupling a c t i v i t y i n mitochondria, enhanced membrane p e r t u r b a t i o n s , enhanced p e r m e a b i l i t y of c h l o r o p l a s t s to K+, and enhanced p e r m e a b i l i t y of liposomes to H+. d. The a c t i o n on photophosphorylation may be separate and independent from b i n d i n g to the Β p r o t e i n complex. e. The i n h i b i t o r y uncouplers may conceivably perturb a l l c e l l u l a r membranes (plasmalemma, t o n o p l a s t , n u c l e a r , and endo­ plasmic r e t i c u l u m i n a d d i t i o n to the c h i o r o p l a s t and mitochondrial membranes). However, marker systems that can be monitored r e a d i l y which r e f l e c t the p e r t u r b a t i o n s remain to be i d e n t i f i e d . This physiochemical i n t e r a c t i o n of the i n h i b i t o r s with l i p i d s can be expected to a l t e r the many t r a n s p o r t , b i o s y n t h e t i c , and r e g u l a t o r y a c t i v i t i e s a s s o c i a t e d with c e l l u l a r membranes and could be i n ­ volved i n the expression of p h y t o t o x i c i t y . The r e l a t i o n between membrane p e r t u r b a t i o n s and the expres­ s i o n of p h y t o t o x i c i t y remains to be i d e n t i f i e d . In some of the experiments r e p o r t e d , concentrations i n the 1 0 0 to 2 0 0 yM range were r e q u i r e d to produce I 5 0 responses. However, i n many of these s t u d i e s , i n i t i a l e f f e c t s could be detected at concentrations of 1 to 1 0 yM f o r many of the compounds. At t h i s time, i t i s not p o s s i b l e to determine the impact of minor a l t e r a t i o n s to the p e r m e a b i l i t y and " f l u i d i t y " of o r g a n e l l e membranes on the p h y s i o ­ l o g i c a l s t a t u s of an organism. Conceivably, small changes, +

5.

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95

coupled w i t h a r e d u c t i o n i n the a v a i l a b i l i t y of c h i o r o p l a s t and m i t o c h o n d r i a l l y generated ATP, could have a s i g n i f i c a n t and d r a s t i c e f f e c t over a time span of s e v e r a l hours or days.

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch005

Acknowledgements This was a cooperative i n v e s t i g a t i o n of the North C a r o l i n a A g r i c u l t u r a l Research S e r v i c e and the United States Department of A g r i c u l t u r e , A g r i c u l t u r a l Research S e r v i c e , R a l e i g h , N . C . Paper No. 7079 of the J o u r n a l S e r i e s of the North C a r o l i n a A g r i c u l t u r a l Research S e r v i c e , R a l e i g h , N . C . The study was supported i n p a r t by P u b l i c H e a l t h S e r v i c e Grant ES 00044. A p p r e c i a t i o n i s ex­ tended to F . S. Farmer f o r t e c h n i c a l a s s i s t a n c e w i t h the mitochon­ d r i a l phases of the study.

Literature Cited 1. Moreland, D. E. Annu. Rev. Plant Physiol. 1980, 31, 597-638. 2. Moreland, D. E.; Hilton, J. L. in "Herbicides: Physiology, Biochemistry, Ecology", Vol. 1; Audus, L. J., Ed.; Academic Press: London, 1976; pp. 493-523. 3. Tischer, W.; Strotman, H. Biochim. Biophys. Acta 1977, 460, 113-25. 4. Pfister, K.; Arntzen, C. J . Z. Naturforsch. 1977, 34c, 9961009. 5. Steinback, Κ. E.; Pfister, K.; Arntzen, C. J. Chapter 3 in this book. 6. Shipman, L. L. Chapter 2 in this book. 7. Van Assche, C. J.; Carles, P. M. Chapter 1 in this book. 8. Moreland, D. E.; Huber, S. C. in "Plant Mitochondria"; Ducet, G.; Lance, C., Eds.; Elsevier/North Holland Biomedical Press: Amsterdam, 1978; pp. 191-8. 9. Moreland, D. E.; Huber, S. C. Pestic. Biochem. Physiol. 1979, 11, 247-57. 10. Moreland, D. E. Pestic. Biochem. Physiol. 1981, 15, 21-31. 11. Ducruet, J . M.; Gauvrit, C. Weed Res. 1978, 18, 327-34. 12. Moreland, D. E. in "Progress in Photosynthesis Research", Vol III; Metzner, H., Ed.; Int. Union Biol. Sci.: Tubingen, 1969; pp. 1693-1711. 13. Lilley, P. McC; Walker, D. A. Biochim. Biophys. Acta 1974, 368, 269-78. 14. Armond, P. Α.; Arntzen, C. J.; Briantais, J.-M.; Vernotte, C. Arch. Biochem. Biophys. 1976, 175, 54-63. 15. MacKinney, G. J . Biol. Chem. 1941, 140, 315-22. 16. Lanzetta, P. Α.; Alvarez, L. J.; Reinach, P. S.; Candia, O.A. Anal. Biochem. 1979, 100, 95-7. 17. Chance, B.; Williams, G. R. J . Biol. Chem. 1955, 217, 409-27. 18. Hinkle, P. Biochem. Biophys. Res. Commun. 1970, 41, 1375-81. 19. Alsop, W. R.; Moreland, D. E. Pestic. Biochem. Physiol. 1975, 5, 163-70.

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch005

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BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

20. Good, Ν. E. in "Encyclopedia of Plant Physiology, New Series", Vol. 5; Trebst, Α.; Avron, Μ., Eds.; Springer-Verlag: Berlin, 1977; pp. 429-36. 21. Fiolet, J. T. W.; Bakker, E. P.; Van Dam, K. Biochim. Biophys. Acta 1974, 368, 432-45. 22. Moreland, D. E.; Huber, S. C.; Novitzky, W. P. Proc. 5th Int. Congr. Photosynth.: Halkidiki, Greece, 1981; in press. 23. Chappel, J . B.; Crofts, A. R. in "Regulation of Metabolic Processes in Mitochondria"; Tager, J. M.; Pappa, S.; Quagliariello, E.; Slater, E. C., Eds.; Elsevier: Amsterdam, 1966; pp. 293-316. 24. Jagendorf, A. T. in "Bioenergetics of Photosynthesis"; Govindjee, Ed.; Academic Press: New York, 1975; pp. 413-92. 25. Pressman, B. C. Annu. Rev. Biochem. 1976, 45, 501-29. 26. Huber, S. C.; Moreland, D. E. Plant Physiol. 1979, 64, 115-9. 27. Cavalieri, A. J.; Huang, A. H. C. Plant Physiol. 1980, 66, 588-91. 28. Bakker, E. P.; Van Den Heuvel, E. J.; Wiechmann, A. H. C.; Van Dam, K. Biochim. Biophys. Acta 1973, 292, 78-87. RECEIVED

September 21, 1981.

6 Effects of Herbicides on the Lipid Composition of Plant Membranes J. B. ST. JOHN

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch006

U.S. Department of Agriculture, Beltsville Agricultural Research Center, Beltsville, M D 20705

Substituted pyridazinone herbicides directly inhibit photosystem II, chloroplast pigment biosynthesis, and membrane lipid biosynthesis. Depending on the substitution, pyridazinones can: specifically inhibit the synthesis of linolenic acid in galactolipids and phospholipids; preferentially alter the fatty acid composition of monogalactosyl diglycerides compared with digalactosyl diglycerides; and cause a build-up of saturated fatty acids in the chloroplast membranes. The differential responses of plant species and tissues to substituted pyridazinones suggest that control of linolenic acid biosynthesis may vary depending on plant species and even tissue. An interaction between triazine herbicides and the lipid composition of chloroplast membranes may influence sensitivity of weed biotypes to the triazine herbicides. Substituted pyridazinone h e r b i c i d e s may have a m u l t i f u n c t i o n a l mode of a c t i o n . I n h i b i t o r y a c t i o n by the s u b s t i t u t e d pyridazinones has been demonstrated at three separate s i t e s ÇL, 2, 3 ) . The f i r s t s i t e i n v o l v e s i n t e r f e r e n c e with photos y n t h e t i c functions (_1) . The second s i t e i n v o l v e s i n t e r f e r e n c e with the accumulation of c h i o r o p l a s t pigments and the t h i r d s i t e i n v o l v e s i n t e r f e r e n c e with the formation of c h i o r o p l a s t g l y c e r o l i p i d s (2, 3 ) . The molecular s t r u c t u r e s used most f r e q u e n t l y to evaluate a c t i o n at these s i t e s are depicted i n F i g u r e 1. Photosynthetic E l e c t r o n Transport I n h i b i t i o n Numerous h e r b i c i d e s are known to i n h i b i t photosystem I I (PS II) - dependent e l e c t r o n t r a n s p o r t . The values f o r i n h i b i t i o n by s e l e c t e d pyridazinones of e l e c t r o n transport i n i s o l a t e d b a r l e y c h l o r o p l a s t s are shown i n Table I . The value f o r diuron [ 3 - ( 3 , 4 d i c h l o r o p h e n y l ) - l , l - d i m e t h y l urea] may be used to r e l a t e these r e s u l t s to those i n other l i t e r a t u r e .

This chapter not subject to U.S. copyright. Published 1982 American Chemical Society.

98

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch006

PYRAZON

133-410 H

9785

9774

NORFLURAZON

6706 0

Figure 1.

CL

Structure of six substituted pyridazinones (2).

6.

Lipid Composition of Plant Membranes

ST. JOHN

99

Table I H e r b i c i d e i n h i b i t i o n of f e r r i c y a n i d e r e d u c t i o n by H i l l r e a c t i o n i n i s o l a t e d c h l o r o p l a s t s (1). Herbicide

Molar concn. f o r 50%

Pyrazon

the

inhibition

7.0

, -6 χ 10

1.4

χ 10

-5 San 9785 (BASF 13 338)

D

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch006

-5 San

9774

1.4

χ 10

San

6706

4.4

χ 10

1.4

χ 10

-6 -7

Diuron

I n h i b i t i o n of PS I I i s thought to occur at the l e v e l of a proteinaceous component l o c a t e d between p l a s t i q u i n o n e s Q and B, the primary and secondary acceptors of e l e c t r o n s from PS I I . The competitive binding experiments of T i s c h e r and Strotmann (4) suggest that the phenylureas, biscarbamates, t r i a z i n e s , t r i a zinones, and pyridazinones i n h i b i t e l e c t r o n transport by i n t e r ­ a c t i o n w i t h the same component of PS I I . A c t i o n at t h i s s i t e seemed to account f o r the p h y t o t o x i c i t y of pyrazon [5-amino-4chloro-2-phenyl-3(2H)-pyridazinone]. In a d d i t i o n to a c t i o n at t h i s s i t e , compounds with molecular s u b s t i t u t i o n s onto the s t r u c t u r e of pyrazon (Figure 1) a l s o i n t e r f e r e w i t h the formation of c h i o r o p l a s t membrane l i p i d s , namely the c h l o r o p h y l l s , c a r otenoids, and g l y c e r o l i p i d s . Chioroplast

Pigment I n h i b i t i o n

The c h l o r o p h y l l s and carotenoids are the pigmented l i p i d s a s s o c i a t e d with c h i o r o p l a s t membranes. S u b s t i t u t e d pyridazinones reduce the accumulation of these pigments (Table I I ) . Table I I E f f e c t of s u b s t i t u t e d pyridazinones on c h i o r o p l a s t pigment accumulation i n 4-day-old wheat shoots ( 2 ) . Treatment Chlorophyll Carotenoids % of c o n t r o l Pyrazon 88 93 60 San 133-410H 60 98 San 9785 (BASF 13 338) 102 57 San 9774 45 5-(4-methylphenylsulfonyl-oxy)1,3-dimethylpyrazole A d d i t i o n a l herbicides mentioned i n the text: Bentazon (Basagran), 3-isopropyl-2,1,3-benzothiadiazinone-(4)-2,2-dioxide (BASF AG) Diuron (DCMU), 3-(3,4-dichlorophenyl)-1,1-dimethylurea; Monuron i s the 4-chlorophenyl analog.

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch007

f

to rapid loss of water and/or ions. In contrast to algae# s u b s t a n t i a l bleaching cannot develop before death. Direct e f f e c t s of herbicides upon the electron transport and the membrane system are followed by secondary disturbances of plant metabolism, and many c o n f l i c t i n g data found i n the l i t e r a t u r e on primary or secondary mode of action can be resolved by c a r e f u l l y measuring the k i n e t i c s during herbicide treatment, using proper sublethal concentrations, d i f f e r e n t analogous d e r i v a t i v e s , and appropriate species. I n v e s t i gating modes of action of h e r b i c i d e s , s t e r i l e l i q u i d cultures of u n i c e l l u l a r microalgae have been quite advantageous. The e f f e c t s are seen very early (within 24 to 48 h) and the herbicides can be applied i n exact concentration to the target c e l l . P h y s i o l o g i c a l parameters (growth, pigments, oxygen evolution) can be determined more e a s i l y than with higher plants (see [13] for d e t a i l s ) . Interference with Carotene Biosynthesis As shown i n Table I, presence of both the phenylpyridazinone norflurazon and the diphenylether oxyf l u o r f e n decreased the pigment content. However, a d i f ference between these herbicides i s indicated by the

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

116

Table I Carotenoids and c h l o r o p h y l l bleaching i n autotrophic and heterotrophic cultures of the green microalga Scenedesmus acutus cultured f o r 24 h i n the presence of 1 μΜ norflurazon or oxyfluorfen Control

Culture Autotrophic

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch007

Oxyfluorfen

(1ight)

Chlorophyll Carotenoids Heterotrophic Chlorophyll Carotenoids

Norflurazon

19.54 0.053

16.92 0.023

10.22 0.015

3.58 0.011

3.76 0.005

2.33 0.008

(dark)

Data are mg/ml packed c e l l volume. For c u l t i v a t i o n and methods see (13). small decrease of c h l o r o p h y l l vs. carotenoids observed with norflurazon i n the l i g h t , with no change of chlo­ r o p h y l l i n the dark. In contrast, decrease of chloro­ p h y l l as well as carotenoids with oxyfluorfen present was independent of l i g h t . This dark experiment i s clear evidence against a simultaneous i n h i b i t i o n of both carotene and c h l o r o p h y l l formation by pyridazinones (14,15). Our f i n d i n g i s i n accordance with e.g. the r e ­ port of (16) that a photoflash s t i l l can induce c h l o ­ r o p h y l l synthesis i n e t i o l a t e d wheat leaves that were devoid o f carotenes because of pretreatment with nor­ flurazon. A l l data mentioned support the hypothesis that the c h l o r o p h y l l s are photooxidized i n p y r i d a z i n one-treated plants due to the absence of carotenoids which have a p r o t e c t i v e function as natural quenchers of s i n g l e t oxygen (V7) ; f o r a short overview see (18). L i t t l e ethane production i s observed during the e a r l y phase of the bleaching process caused by phenylpyridazinones (Table I I I ) . During the i n h i b i t i o n of carotene biosynthesis by substituted phenylpyridazinones i n algae (.12*20) wheat (21) and barley (22) , an accumulation of phytoene, an uncolored (triene) carotene precursor, could be observed. Other bleaching herbicides such as the experimen­ t a l phenylfuranone h e r b i c i d e , difunon, (20,23) and de­ r i v a t i v e s , or f l u r i d o n e (21) are thought to act on o

r

7.

SANDMANN AND BÔGER

Mode of Action of Herbicide Bleaching

Table I I Incorporation o f r a d i o a c t i v i t y (dpm χ 1000) from 1 μΟί of [2-14c]-DL-mevalonic acid by a Phycomyces extract i n the presence of 10 μΜ herbicides

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch007

Herbicide

Squalene

GGPP*)

Phytoene

β-Carotene

Control

32.1

22.8

10.3

2.1

+Norflurazon +BAS 13761 +0xyfluorfen

62.3 34.6 35.5

62.4 28.1 26.4

1.1 9.6 11.8

0.9 1.6 2.2

*) geranylgeranyl pyrophosphate, see (26) carotene biosynthesis very s i m i l a r l y to 2-phenyl p y r i ­ dazinones as suggested by the accumulation of phytoene. The action of aminotriazole seems to be somewhat d i f ­ ferent. With t h i s herbicide, 3 -carotene, γ-carotene, and lycopene are accumulated i n wheat seedlings i n ­ stead of phytoene (25). Consequently, an i n h i b i t i o n of the enzyxne(s) involved i n the dehydrogenation steps of carotene biosynthesis was concluded. In Pennisetum seedlings treated with difunon i n higher concentration (>10 μΜ), the bleaching of p i g ­ ments was reported to be p a r a l l e l e d by a decrease of the porphobilinogenase l e v e l whereas the contents of . These c e l l - f r e e assays should be investigated further with regard to possible d i f f e r e n t s e n s i t i v i t y against i n h i b i t o r s because of the species used. S t r u c t u r e / A c t i v i t y Relationship. The bleaching a c t i v i t y of 2-phenylpyridazinones i s dependent on the substituents and t h e i r p o s i t i o n on the molecule. In a s t r u c t u r e / a c t i v i t y i n v e s t i g a t i o n we have been able to c o r r e l a t e bleaching with e l e c t r o n i c f a c t o r s . Bleaching a c t i v i t y of 2-phenylpyridazinones i s improved by sub­ s t i t u e n t s with increasing 610 nm, l i n e 2) of 100 W/m according to (j>4) . Dark controls were below 5% of the data measured i n the l i g h t . 2

about the same degree (Table I ) , while ethane formation of the norflurazon-treated c e l l s was s t i l l low, a l ­ though i t had somewhat increased due to peroxidations and/or c h l o r o p h y l l - s e n s i t i z e d photooxidations develop­ ing i n the course of i n h i b i t e d carotene biosynthesis. As demonstrated further with i n t a c t c e l l s (Figure 3; Table I I I , l i n e 3) and i s o l a t e d chloroplasts as w e l l (Figure 4), ethane evolution induced by oxyfluorfen de­ creased when electron transport was blocked by simul­ taneous addition of diuron (1_2) . Ethane evolution was lowered likewise with oxyfluorfen given i n the dark. Noteworthy, red l i g h t beyond 610 nm gave r e s u l t s com­ parable to those obtained with white l i g h t (Table I I I , l i n e s 1,2) i n d i c a t i v e of carotenoids being not d i r e c t ­ l y involved i n diphenylether a c t i v a t i o n (38). The requirement of l i g h t for the a c t i v a t i o n of oxyfluorfen and n i t r o f e n has been known f o r years. How­ ever, the involvement of photosynthetic electron trans­ port was doubted (.39*40). At the moment, our hypothesis claims p-nitrophenylethers to be reduced to a (nitroanion?) r a d i c a l which subsequently i n i t i a t e s peroxida­ t i o n of (galactolipid) polyunsaturated f a t t y acids and other membrane components. Apparently, also r e s p i r a t o r y reactions are able to a c t i v a t e oxyfluorfen, since our h e t e r o t r o p h i c a l l y growing cultures e x h i b i t a small bleaching i n the dark (Table I ) . Possibly these

122

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

Light incubation

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch007

. Dark incubation

-/h-

'

'

'

10 50 100 500 (uM) A d d i t i o n of u n l a b e l e d n i t r o f e n 1

Figure 5. Addition of nitrofen to isolated spinach chloroplasts in the light and in the dark. Thylakoids equivalent to 100 μg of chlorophyll were incubated at pH 7.4 in the presence of 50 μΜ C-labeled nitrofen for 15 min. Aliquots of this preloaded material were then treated with increasing concentrations of cold nitrofen as indi­ cated (31). 14

r a d i c a l s also bind to the thylakoid membrane. Illumina­ t i o n of i s o l a t e d spinach chloroplasts i n the presence of 4 c - i a b e l e d n i t r o f e n l e d to a strong attachment of t h i s diphenylether (Figure 5); no replacement by un­ labeled n i t r o f e n was possible (32). Our experiments were performed with microalgae under optimum conditions of photosynthesis such as supply of l i g h t , C O 2 , minerals, e t c . I t i s conceivable that suboptimum conditions may allow f o r oxygen a c t i v a ­ t i o n at the pigments leading to subsequent a c t i v a t i o n of (bleaching) diphenylethers. Carotenes may then be functional f o r the action of diphenylethers as was r e ­ cently confirmed with higher plants treated with a c i fluorfen-methyl (41^) . Using Scenedesmus, photosynthetic electron transport was not necessary a f t e r a 2-hr oxy­ f l u o r f e n treatment 0 2 ) when photosynthesis started to decay anyway. Then surplus of l i g h t energy possibly may be diverted d i r e c t l y v i a piments as indicated above. 1

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7.

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Mode of Action of Herbicide Bleaching

123

B i p y r i d y l s . Biypridylium s a l t s l i k e paraquat also cause destruction of chioroplast organization (42). An early symptom i s the light-induced oxygen uptake and the strong i n h i b i t i o n of the photosystem-II region with photosystem I not being affected. Concurrently, malondialdehyde i s formed. A decrease of c h l o r o p h y l l i s not observed to occur concurrently (9). Ethane can be formed under the influence of paraquat (43,44) · In two recent publications i t was shown by f a t t y a c i d determinations that unsaturated f a t t y acids are preferent i a l l y degraded under the influence of paraquat (45, 46). The primary mode of action i s i t s one-electron r e duction and the subsequent reduction of the r a d i c a l by molecular oxygen Γ7)· The r e s u l t i n g superoxide anion i s assumed to give r i s e to other a c t i v e oxygen species (8). Apparently, light-induced paraquat reduction and subsequent peroxidation i s also achieved without e l e c ­ tron transport, e.g., i n subchloroplast p a r t i c l e s where endogenous substrates or c h l o r o p h y l l i t s e l f may be the electron donors (42). Similar peroxidative processes are induced by diquat (^8). Mechanism. Degradation of α-linolenic acid (ctl i n ) as proposed by (£9,^0,51_) i s demonstrated i n Figure 6. The i n i t i a l step i s a hydrogen abstraction from an a - l i n o l e n i c molecule by a r a d i c a l species that was formed as a r e s u l t of h e r b i c i d a l action. In the following r a d i c a l - c h a i n reaction the CJ-3 a l k y l per­ oxide i s formed v i a the peroxy r a d i c a l . Subsequently, t h i s peroxide i s decomposed i n a Fenton-type reaction to the co-3 alkoxy r a d i c a l i n the presence of t r a n s i ­ t i o n metals that can undergo one-electron t r a n s f e r r e ­ action, e.g., Cu(I/II), F e ( I I / I I I ) , T i ( I I l / I V ) , or Ce(III/IV). The CO-3 alkoxy r a d i c a l can s p l i t by βs c i s s i o n to an unsaturated aldehyde and the e t h y l r a ­ d i c a l . The l a t t e r i s e i t h e r oxidized to ethylene or reduced to ethane. Quantitative estimation indicates that only about 1% of peroxidized α-lin i s decomposed to ethane or ethylene (52!) . However, the high s e n s i t i v i t y o f gaschromatography for v o l a t i l e hydrocarbons and the ad­ vantage that t h i s determination can be done with i n t a c t organisms make short-chain hydrocarbons e x c e l l e n t markers to trace herbicide-induced peroxidation r e ­ actions. In addition to ethane/ethylene, other v o l a t i l e hydrocarbons can be formed i n s u b s t a n t i a l amounts. The xanthophycean alga B u m i l l e r i o p s i s f i l i f o r m i s produces mainly propane a f t e r treatment with paraquat (£4) or oxyfluorfen (V2), whereas l i p i d peroxidation i n animals r e s u l t s i n pentane formation (5^).

124

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

Peroxidation of a-linolenic I ) Chain initiation a - l i n + R*

acid

( hydrogen abstraction ) • α - l i n · • RH

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch007

II) Radical chain reaction r^a-lin« • 0

2

a-lin-0-0·

• a-lin-0-0· • a-lin

( peroxy radical)

• a - lin-0-OH •a-lin · /ω-3-alkyl \ I peroxide /

III) Degradation of peroxo compound a-lin-0-OH

a-lin-Ο·

(ω-3-alkoxy

radical)

CHO-CH = CH-R' CH = CH 2

2

CH -CH « 3

2

(ethyl

radical)

CH3-CH3

Figure 6. Peroxidation of α ω-3-polyunsaturated fatty acid. A radical R- gen­ erated, e.g., by the photosynthetic electron-transport system, can abstract a hydro­ gen from a-linolenic acid (α-lin). After conjugation of two double bonds, this leads to linolenic acid radical (α-lin·), which reacts with molecular oxygen giving peroxy radical. This again reacts with another linolenic acid molecule thereby starting the chain reaction (part II). The resulting ω-3-alkyl peroxide is reduced to ω-3-alkoxy radical through a Fenton-type reaction (see part III) mediated by metal ions like Fe(Il/III) or Cu(I/II). By β-scission this radical splits into the corresponding aldehyde and ethyl radical (R' denotes the fatty acid moiety carrying the car boxy I group). With copper ions present, the ethyl radical reacts with Cu(II) to give ethylene and with Cu(I) to yield ethane. With Cu(II) in excess, most of the hydro­ carbon produced is ethylene (51). We assume that most of the Cu(ll) is reduced by photosynthetic electron transport.

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Mode of Action of Herbicide Bleaching

125

O r i g i n of Hydrocarbon Gases. In order to f i n d out which f a t t y acids are the sources of v o l a t i l e hydrocarbons we used thylakoid preparations from several blue-green algae that contain d i f f e r e n t f a t t y acids. The peroxidative reactions were mediated and sustained by Cu(II) ions i n the l i g h t . Table IV shows that f a t t y acids with at l e a s t 2 double bonds are necessary f o r hydrocarbon formation. As Anacystis lacks those f a t t y acids, no peroxidative v o l a t i l e hydrocarbons were produced. S p i r u l i n a e x c l u s i v e l y contains CO-6 f a t t y acids as endogenous polyunsaturated f a t t y acids and evolved C 5 hydrocarbons only. The t h i r d species, Anabaena, whose thylakoids contain Co-3 and Co-6 polyunsaturated f a t t y acids formed C 2 and C 5 hydrocarbons simultaneousl y . We conclude that Co-3 polyunsaturated f a t t y acids are the source of ethane and ethylene and that the CO-6 polyunsaturated f a t t y acids are the source of pentane and pentene i n herbicide-induced peroxidation r e actions. Furthermore, we obtained evidence that the propane measured with B u m i l l e r i o p s i s a f t e r an 18-h treatment with e i t h e r 10 μΜ oxyfluorfen or 50 μΜ Cu(II) o r i g i n a t e s from a CO-4 polyunsaturated f a t t y a c i d . We have recently i s o l a t e d and i d e n t i f i e d t h i s acid as 16:3co4 (Sandmann, Lambert, Boger; i n preparation). This b i o l o g i c a l plant system confirms and extends the report of Dumelin and Tappel (5j2) who were the f i r s t to prove the o r i g i n of ethane and pentane by a metal- or hematin-catalyzed chemical reaction. Further­ more, addition of appropriate f a t t y acids to i s o l a t e d thylakoids o f Anacystis according to Table IV, a f t e r short-term i l l u m i n a t i o n y i e l d e d hydrocarbon evolution that confirmed the r u l e explained above (54). Using the l a t t e r system we could also demonstrate that hydrocar­ bons do not o r i g i n a t e from carotenoids. Experiments with i s o l a t e d f a t t y acids, however, should be done i n short experimental times, otherwise unphysiological peroxidations of f a t t y acids may occur (comp. 55). A d d i t i o n a l Assays on H e r b i c i d a l Mode of Bleaching Direct evidence f o r degradation of l i p i d s can be obtained by p r e l a b e l i n g the thylakoid s u l f o l i p i d o f , e.g., microalgae with 35>S-sulfate. when oxyfluorfen or norflurazon were applied to cause a 90% decrease of a- and β-carotene over 2 culture days, more than 90% of the r a d i o a c t i v i t y of the s u l f o l i p i d disappeared i n the oxyfluorfen sample. Using norflurazon, only 20% of the s u l f o l i p i d was degraded during t h i s period (5j5 ).

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BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

Table IV Concentration of polyunsaturated f a t t y acids i n the thylakoids of blue-green algae and light-induced v o l a t i l e hydrocarbon production by spheroplasts i n the presence of 50 μΜ Cu(II) Anacystis nidulans

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch007

Fatty acids,

μ9/μ9

Anabaena variabilis

Spirulina platensis

chlorophyll

16:2co6

0

16:3ω3

0

1 8 : 2 to6

0

14.9

9.2

1 8 : 3 co3

0

19.9

0

18:3co6

0

0

2 0 : 4 0J6

0

0

0 0.32

0 0

12.8 0

Hydrocarbons produced, pmol/mg c h l o r o p h y l l χ h Ethane 0 0 109.8 Ethylene

0

113.2

Pentane

0

131 .3

92.5

Pentene

0

45.5

69.8

0

Spheroplasts prepared according to (J55) were shocked and illuminated f o r 2 hours, according to (51 ,54).

A rapid screening method to d i s t i n g u i s h between an i n h i b i t o r y mode of action on carotenoid biosynthesis and peroxidative bleaching i s the measurement of f l u o r ­ escence induction (52) . In the presence of both nor­ flurazon and oxyfluorfen, the v a r i a b l e fluorescence s i g n a l of Scenedesmus was abolished a f t e r an 8-h t r e a t ­ ment. Based on c h l o r o p h y l l , the maximum fluorescences i g n a l height of norflurazon-treated c e l l s rose about twice as high as the c o n t r o l , whereas that of the oxyfluorfen-treated c e l l s decreased to about 10%. Further­ more, only with the pyridazinone-treated c e l l s was the constant fluorescence increased, as was seen by a "spike"-type s i g n a l absent i n the oxyfluorfen sample. Undoubtedly, these fluorescence-signal changes are

7.

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Mode of Action of Herbicide Bleaching

111

caused by the h e r b i c i d e - i n d u c e d decay o f e l e c t r o n t r a n s p o r t a c t i v i t y . A c c o r d i n g t o the s i g n a l d i f f e r ­ ences, t h i s decay i s a p p a r e n t l y produced i n a d i f f e r e n t manner due t o d i s t u r b e d energy t r a n s f e r , pigment de­ s t r u c t i o n o r d e g r a d a t i o n o f redox c a r r i e r s .

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch007

Peroxidations

as Secondary H e r b i c i d a l E f f e c t s

As p o i n t e d out p r e v i o u s l y f o r c h l o r o p h y l l d e s t r u c ­ t i o n and ethane e v o l u t i o n w i t h a l g a e i n t h e p r e s e n c e o f n o r f l u r a z o n (Tables I , I I I ) , p e r o x i d a t i v e r e a c t i o n s may show up as a consequence o f a p r i m a r y i n h i b i t i o n o f c a r o t e n e s y n t h e s i s , which i t s e l f does not i n d u c e peroxidation-initiating radicals. In a d d i t i o n t o the b l e a c h i n g h e r b i c i d e s , e l e c t r o n t r a n s p o r t i n h i b i t o r s b i n d i n g a t the Β p r o t e i n ( F i g u r e 1) may i n i t i a t e p e r o x i d a t i o n a f t e r l o n g - t e r m t r e a t m e n t and under h i g h l i g h t i n t e n s i t y . As a consequence o f i n h i b i t e d e l e c t r o n t r a n s p o r t , the e x c i t e d c h l o r o p h y l l s t r a n s f e r t h e i r energy t o ground s t a t e oxygen y i e l d i n g h i g h l y r e a c t i v e s i n g l e t oxygen U 3 , 5 j * ) . S i n g l e t oxygen i s quenched by c a r o t e n e s . A p p a r e n t l y , c a r o t e n e s a l s o are broken down i n t h i s r e a c t i o n p r e c e d i n g the d e s t r u c ­ t i o n o f c h l o r o p h y l l ( H * 5 j > / 6 0 ) . When the c a r o t e n e l e v e l i s d e c r e a s e d , p h o t o d e s t r u c t i o n o f c h l o r o p h y l l as w e l l as l i p i d p e r o x i d a t i o n a r e s t a r t e d as o b s e r v e d w i t h p e r ­ o x i d a t i v e d i p h e n y l e t h e r s . Such pigment d e g r a d a t i o n s are known f o r monuron (59) , d i u r o n (^58,60) , and the ex­ p e r i m e n t a l p h e n y l - h y d r o x y - p y r r o l o n e h e r b i c i d e CGA 71884 (unpublished r e s u l t s ) . T h i s compound i s an e l e c t r o n t r a n s p o r t i n h i b i t o r w i t h I 5 0 = ·7 χ 10"7 M (6J_) . These secondary b l e a c h i n g phenomena g e n e r a l l y r e ­ q u i r e q u i t e u n p h y s i o l o g i c a l c o n d i t i o n s such as sub­ s t a n t i a l l y i n h i b i t e d e l e c t r o n t r a n s p o r t and/or a de­ c r e a s e d c a r o t e n e l e v e l i n the t h y l a k o i d s (induced, e.g., by p h e n y l p y r i d a z i n o n e s ) t o g e t h e r w i t h a h i g h quantum f l u x r e a c h i n g the pigments. By c a r e f u l l y c h o o s i n g the c u l t i v a t i o n c o n d i t i o n s , e.g., a l l o w i n g f o r some growth with s u b l e t h a l concentrations of s - t r i a z i n e s or t r i a z i n o n e h e r b i c i d e s p r e s e n t , even i n c r e a s e d l e v e l s o f c h l o r o p h y l l may be o b s e r v e d ("greening e f f e c t " , see [62] f o r h i g h e r p l a n t s , ^63] f o r a l g a e ) . Acknowledgment T h i s s t u d y was s u p p o r t e d by the Deutsche F o r s c h u n g s g e m e i n s c h a f t . The experiments were made p o s s i b l e t h r o u g h a generous s u p p l y o f pure h e r b i c i d e samples which were g i f t s from BASF AG, Limburgerhof and Lud-

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BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch007

wigshafen, Celamerck GmbH, Ingelheim, both Germany, Ciba-Geigy AG, Sandoz AG, both Basel, Switzerland, and Rohm and Haas, Spring House, Pennsylvania, USA (see legend of Figure 2). Literature Cited 1. Moreland, D.E. Annu. Rev. Plant Physiol. 1980, 31, 597-638. 2. Böger, P. Plant Res. Development (Tübingen) 1978, 8, 79-101. 3. P f i s t e r , K . ; Arntzen, C . J . Z. Naturforsch. 1979, 34c, 996-1009. 4. Trebst, Α.; Draber, W. i n "Advances i n Pesticide Science" Part 2; Ed. H. Geissbühler, Pergamon Press, Oxford-New York, 1979; pp. 223-234. 5. Böger, P. Z. Pflanzenkr. Pflanzenpathol. Pflanzenschutz (Plant Disease and Protection) 1981, spec. issue no. IX, 153-162. 6. Black, C.C Biochim. Biophys. Acta 1966, 120, 332340. 7. Farrington, J . A . ; Ebert, M . ; Land, E.J.; Fletcher, K. Biochim. Biophys. Acta 1973, 314, 373-381. 8. Dodge, A.D. i n "Herbicides and Fungicides"; Ed. N.R. McFarlane, Chemical Society, Special Publication 29, London, 1977; pp. 7-21. 9. Böger, P . ; Kunert, K . J . Z. Naturforsch. 1978, 33c, 688-694. 10. Lambert, R.; Kunert, K.J.; Böger, P. Pesticide Biochem. Physiol. 1979, 11, 267-274. 11. Kunert, K.J.; Böger, P. Z. Naturforsch. 1979, 34c, 1047-1051. 12. Kunert, K.J.; Böger, P. Weed S c i . 1981, 29, 169173. 13. Sandmann, G . ; Kunert, K.J.; Böger, P. Ζ. Natur­ forsch. 1979, 34c, 1044-1046. 14. Lichtenthaler, H . K . ; Kleudgen, H.K. Ζ. Naturforsch. 1979, 32c, 236-240. 15. Kleudgen, H.K. Pest. Biochem. Physiol. 1979, 12, 231-238. 16. Axelsson, L.; Klockare, B . ; Ryberg, H . ; Sandelius, A.S. Proc. 5th Int. Congr. Photosynth. H a l k i d i k i , Greece, 1981, i n press. 17. Foote, C.S. i n "Free Radicals i n Biology" V o l . II; Ed. W.A. P r i o r , Academic Press, New York, 1976; pp. 85-133. 18. Eder, F.A. Z. Naturforsch. 1979, 34c, 1052-1054. 19. Vaisberg, A.J.; Schiff, J . A . Plant Physiol. 1976, 57, 260-269.

7.

SANDMANN AND BÔGER

20. 21. 22. 23. 24.

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25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43.

Mode of Action of Herbicide Bleaching

Urbach, D . ; Suchanka, M . ; Urbach, W. Ζ. Natur­ forsch, 1976, 31c, 652-655. Bartels, P . G . ; Watson, C.W. Weed S c i . 1978, 26, 198-203. Ridley, S.M.; Ridley, J . Plant Physiol. 1979, 63, 392-398. Kunert, K.J.; Böger, P. Weed S c i . 1978, 26, 292296. Hampp, R.; Sankhla, N.; Huber, W. Physiol. Plant. 1975, 33, 53-57. Rudiger, W.; Benz, J.; Lempert, U . ; Schoch, S.; Steffens, D. Z. Pflanzenphysiol. 1976, 80, 131143. Sandmann, G.; Bramley, P . M . ; Böger, P. Pest. Biochem. Physiol. 1980, 14, 185-191. Beyer, P . ; Kreuz, K . ; K l e i n i g , H. Planta (Berl.) 1980, 150, 435-438. Sandmann, G.; Kunert, K.J.; Böger, P. Pest. Biochem. Physiol. 1981, 15, i n press. Feierabend, J.; Schubert, B. Plant Physiol. 1978, 61, 1017-1022. Burns, E . F . ; Buchanan, G.A.; Carter, M.G. Plant Physiol. 1971, 47, 144-148. Draber, W.; Fedtke, C. i n "Advances i n Pesticide Science"; Ed. H. Geissbühler, Pergamon Press, Oxford-New York, 1979, pp. 475-486. Devlin, R . M . ; Kisiel, M . J . ; Kostusiak, A . S . Weed Res. 1 979, 19, 59-61. Kawakubo, K . ; Shindo, J. Plant Physiol. 1979, 64, 774-779. St.John, J.B.; Christiansen, M.N. Plant Physiol. 1976, 57, 38-40. Willemot, C. Plant Physiol. 1977, 60, 1-4. Bugg, M.W.; Whitmarsh, J.; Rieck, C . E . ; Cohen,W.S. Plant Physiol. 1980, 65, 47-50. Lambert, R.; Böger, P. Z. Pflanzenkr. Pflanzenschutz (Plant Disease and Protection) 1981, spec, issue no. IX, 147-152. Lambert, R.; Sandmann, G . ; Böger, P. Weed Sci. 1982, submitted. Matsunaka, S. Residue Rev.1969, 25, 45-58. Fadayomi, O.; Warren, G.F. Weed S c i . 1976, 24, 598-600. Orr, G.K.; Hess, F.D. Abstracts 1981 Meeting WSSA, No. 248. Harris, N.; Dodge, A.D. Planta (Berl.) 1972, 104, 210-219. Elstner, E.F. Ber. Dtsch. Bot. Ges. 1978, 91, 569-577.

130

BIOCHEMICAL RESPONSES INDUCED B Y HERBICIDES

44.

Boehler-Kohler, B . A . ; Läpple, G . ; Hellmann, V.; Böger, P. Pestic. S c i . 1981, i n press. Youngman, R.J.; Dodge, A.D. Z. Naturforsch. 1979, 34c, 1032-1035. Boehler-Kohler, B . A . ; Schopf, M . ; Böger, P. Abstract, 5th Int. Congr. Photosynth. H a l k i d i k i , Greece 1980. Elstner, E . F . ; Lengfelder, E . ; Kwiatkowski, G. Z. Naturforsch. 1979, 35c, 303-307. van Rensen, J . J . S . Physiol. Plant. 1975, 33, 4246. H a l l i w e l l , B. i n "Strategies of Microbial Life i n Extreme Environments", Life Sciences Research Report 13; Ed. M. Shilo, Verlag Chemie, Weinheim, 1979, pp. 195-221. Elstner, E . F . ; Pils, I . Z. Naturforsch. 1979, 34c, 1040-1043. Sandmann, G . ; Böger, P. Plant Physiol. 1980, 66, 797-800. Dumelin, E . E . ; Tappel, A . L . Lipids 1977, 12, 894900. D i l l a r d , C.J.; Dumelin, E . E . ; Tappel, A . L . Lipids 1977, 12, 109-144. Sandmann, G . ; Böger, P. Lipids 1982, i n press. Schobert, B . ; Elstner, E.F. Plant Physiol. 1980, 66, 215-219. Sandmann, G . ; Böger, P. Plant S c i . Lett. 1981, i n press. Böhme, H . ; Kunert, K.J.; Böger, P. Weed S c i . 1981, i n press. Takahama, U . ; Nishimura, M. Plant C e l l Physiol. 1975, 16, 737-748. Pallett, K . E . ; Dodge, A.D. Z. Naturforsch. 1979, 34c, 1058-1061. Ridley, S.M. Plant Physiol. 1977, 59, 724-732. Brugnoni, G.P.; Moser, P . ; Trebst, Α. Ζ. Natur­ forsch. 1979, 34c, 1028-1031. Fedtke, C. Weed S c i . 1979, 27, 192-195. Böger, P . ; Schlue, U. Weed Res. 1976, 16, 149-154. Sandmann, G . ; Böger, P. Ζ. Pflanzenphysiol. 1980, 98, 53-59. S p i l l e r , H . ; Böger, P. Methods Enzymol. 1980, 69, 105-115.

45. 46. 47. 48.

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49.

50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65.

RECEIVED

September 15, 1981.

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

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

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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

8 6

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3 6

4 5

2 +

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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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New Diphenyl Ether Herbicides

ORR AND HESS

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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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v

e

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ORR AND HESS

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Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch008

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

8

+

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

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch008

1

1

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 .

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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.

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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

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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 .

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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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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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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). +

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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

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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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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

t

c

.

F

A

P

2

•0

2

2

·

d

p

P

Death

decompartmentalization

F

p

2

decomposition

Τ BARM ( e . g . , MDA)/SCHG ( e . g . ,

L i p i d peroxide

Term Rx

PUFA-02

k°

PUFA- + H

0

permeability)

A

» Prop Rx

(increased

(polymers)

- PUFA-O-PUFA

,

AFM

N

^- ,AAFM* F M * ^ f ^ PUFA-H PUFA-H

Plant Physiology

ethane)

2

2

PUFA-0 H

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).

Cellular

o

2

+ PUFA-O-PUFA

PUFA-0

J-°2

PUFA* + PUFA-0

Membrane d i s r u p t i o n

P U F A - 0 * 4- PUFA

n

U

« „ Prop Rx

2 PUFA-O

e

P

i

carotenoid

3

carotenoic* ^

" "\(

2l9 3) without i t s e l f being incorporated i n t o the carotene molecules (4). A s i m i l a r s t i m u l a t o r y e f f e c t of 3-ionone was observed on carotene production i n h e t e r o t h a l l i c c u l t u r e s of Phycomyces blakesleeanus (_5) . In Β lake s l e a t r i s p o r a . an e f f e c t somewhat s i m i l a r to that observed with 3-ionone was seen with t r i s p o r i c acid (6). T r i s p o r i c a c i d only stimulated caroteno­ genesis i n the (-) s t r a i n (7). Both 3-ionone and t r i s p o r i c a c i d do not appear to a f f e c t carotene b i o s y n t h e s i s i n higher p l a n t s (8). Ninet et a l . (9) i n v e s t i g a t e d the b i o s y n t h e s i s of c a r o ­ tenoids by 15. t r i s p o r a i n the presence of v a r i o u s nitrogenous compounds. P y r i d i n e , i m i d a z o l e , and some of t h e i r d e r i v a t i v e s were found to stimulate the s y n t h e s i s of trans-lycopene ( ψ , ψ c a r o t e n e ) , whereas i s o n i c o t i n o y l h y d r a z i n e , s u c c i n i m i d e , f

This chapter not subject to U.S. copyright. Published 1982 American Chemical Society.

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154

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

and 4-hydroxypyridine enhanced 3-carotene (3,3~carotene) syn­ thesis. In 1969, Knypl (10) found that an onium compound, chlormequat [(2-chloroethyl)-trimethylammonium c h l o r i d e ] , caused a l l trans-lycopene accumulation i n detached pumpkin (Curcubita pepo) cotyledons. However, no such response was observed when chlormequat was a p p l i e d to other p l a n t t i s s u e s (11). A major step forward was observed i n 1970 with the d i s c o v e r y that another onium compound, 2-(4-chlorophenylthio)-triethylammonium c h l o r i d e (CPTA), can cause the accumulation of lycopene i n a wide a r r a y of p l a n t t i s s u e s and microorganisms (11). Since the d i s c o v e r y o f CPTA, r e s e a r c h on onium compounds has generated a s u b s t a n t i a l amount of i n f o r m a t i o n r e l a t i v e to the 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 b i o r e g u l a t i o n of c a r o t e n o i d pigments i n higher p l a n t s and microorganisms. Both the t r a n s and c j i s - b i o s y n t h e t i c pathways can be r e g u l a t e d . The c a r o t e n o i d p a t t e r n observed i s determined e s s e n t i a l l y by the nature of the onium compounds employed. All-trans

Carotenes

General Formula. carotenogenic system, formula:

In the r e g u l a t i o n o f the t r a n s the onium compounds have the s p e c i a l

C

2H

5 x

N-CH -R 2

C H ^ 2

5

In g e n e r a l , the Ν,Ν-dimethyl analogs are l e s s e f f e c t i v e than the Ν,Ν-diethyl compounds i n promoting c a r o t e n o i d formation. The magnitude of the s t i m u l a t i o n , but not the general p a t t e r n of c a r o t e n o i d response, depends on R (12). The nature of R can cause m o d i f i c a t i o n s i n the amount o f the i n d i v i d u a l c a r o t e n o i d s . Thus, when R l a c k s an aromatic r i n g , t h e r e appears to be l e s s i n h i b i t i o n o f the c y c l a s e ( s ) ; consequently, more c y c l i c c a r o ­ tenoids accumulate. Examples are g i v e n i n Tables I - I I I . A l l of the compounds tested caused lycopene accumulation i n Marsh grape­ f r u i t (Citrus paradisi). The untreated f r u i t had the normal l i g h t y e l l o w c o l o r . A f t e r treatment, the c o l o r of the flavedo ranged from l i g h t orange to an i n t e n s e r e d . The flavedo of a l l t r e a t e d f r u i t showed lycopene accumulation (Tables I, I I , and III). Lycopene was not detected i n the untreated f r u i t s and i t i s not normally present i n mature g r a p e f r u i t (13). The b i o l o g i ­ c a l a c t i v i t y was a l s o c o r r e l a t e d with the l o g a r i t h m of the 1-octanol/water p a r t i t i o n c o e f f i c i e n t ( l o g P ) . D i e t h y l a l k y l a m i n e s . Treatments w i t h d i f f e r e n t d i e t h y l a l k y l amines (Table I) gave a f a i r l y c o n s i s t e n t response p a t t e r n as the length o f the a l k y l group was i n c r e a s e d . The amount of any given carotene remained about the same or increased s l i g h t l y w i t h

9.

Table I. Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch009

Bioregulation

YOKOYAMA ET AL.

Phytofluene ζ-Carotene Neurosporene Lycopene γ-Carotene α-Carotene 3-Carotene

37.3 2.25 1.72

Total Log Ρ

43.9

0.37 0.54 1.72

(C H5) N(CH2)4CH3 (C2H5)2N(CH2) CH (C H5)2N(CH ) CH3 2

2

5

2

155

Biosynthesis

E f f e c t of t r i a l k y l a m i n e s on carotene content of the flavedo of Marsh seedless g r a p e f r u i t (pg/g dry w t ) . Postharvest treatment of f r u i t s , and method of i s o l a ­ t i o n and i d e n t i f i c a t i o n o f pigments are described i n r e f e r e n c e 12. A p o r t i o n of ground flavedo was d r i e d i n vacuo a t 65 C to o b t a i n the dry wt. z~\ . t Control

*1. 2. 3.

of Pigment

2

6

3

4. 5.

Compound* ^

7

;

^

38.6 2.47 1.28 1.01 0.30 1.04 1.41

29.0 3.13 1.37 6.99 0.96 1.02 1.20

39.5 27.8 28.3 17.8 4.32 - 14.6 2.94 5.16 1.38 115 59.0 143 3.21 2.59 1.17 2.11 2.07 1.26 6.75 4.73 1.35

46.1 2.94

43.7 3.44

96.8 197.7 189.5 4.94 4.44 3.94

(C2H )2N(CH ) CH (C H ) N(CH )8CH 5

2

5

2

2

2

7

3

3

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156

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

Table I I . E f f e c t of diethylaminoalkylbenzenes on carotene content of f l a v e d o o f Marsh seedless g r a p e f r u i t (pg/g d r y wt). See c a p t i o n o f Table I f o r e x p e r i ­ mental d e t a i l s . Control

Phytofluene ζ-Carotene Neurosporene Lycopene γ-Carotene α-Carotene 3-Carotene

23.1 1.13 0.71

Total Log Ρ

26.5

*1. 2. 3.

0.57 0.95

(C2H5) NCH2C H (C H ) N(CH ) C H (C H ) N(CH ) C H 2

2

5

2

2

5

6

2

2

2

4. 5.

5

2

6

3

5

6

5

C o m

—j

2

d

P°™ *

^

25.3 1.38 0.83 8.62 0.55 0.77 0.56

29.3 37.9 38.7 86.2 1.38 6.77 27.5 53.8 0.83 1.11 7.16 12.0 8.62 188 104 153 0.55 1.07 0.59 1.67 0.77 0.61 2.19 0.76 0.56 0.95 t r a c e 0.68

37.8 3.07

37.8 236 180 308 3.07 4.07 4.57 5.07

(C H ) N(CH ) C H (C H ) N(CH ) C H 2

5

2

2

4

6

5

2

5

2

2

5

6

5

*1. 2. 3.

Total Log Ρ

5

6

2

2

2

49.9

0.27 0.35 1.35

44.9 2.74 0.33

C H OCH2CH2N(C H5)2 £-CH3~C H40CH CH N(C2H5)2 2-C2H5-C6H4OCH2CH2N(C2H5)2

6

Phytofluene ζ-Carotene Neurosporene Lycopene γ-Carotene α-Carotene 3-Carotene

Control

7

6

4

6

2

2

2

2

· £-iso-C H -C H OCH2CH N(C2H5)2 5. - t e r t - C 4 H 9 - C H 4 0 C H C H N ( C H 5 ) 3

2

245 4.05 323 3.55

79.5 3.05

255 4.25

£

95.6 4.65 303 4.35

44.9 13.7 0.93 182 2.13 0.38 0.82

50.9 19.0 0.84 249 2.48 0.41 0.40

48.1 5.45 0.91 22.2 1.01 0.57 1.26

42.6 10.1 1.21 199 1.90 0.23 0.39

4

33.7 4.57 0.63 54.4 1.04 0.63 0.58 51.1 20.6 1.62 226 2.74 0.40 0.70

Compound* Τ

ô

;

See c a p t i o n

on carotene

(ug/g dry w t ) .

CPTA

content o f flavedo o f Marsh seedless g r a p e f r u i t of Table I f o r experimental d e t a i l s .

Table I I I . E f f e c t of CPTA and analogs of diethylaminoethylphenylether

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158

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

d i e t h y l p e n t y l a m i n e and diethylhexylamine, whereas d i e t h y l h e p t y l amine caused a l a r g e r accumulation, D i e t h y l o c t y l a m i n e and d i e t h y l nonylamine caused very l a r g e i n c r e a s e s i n t o t a l carotenoid con­ t e n t . Lycopene accounted f o r most of the i n c r e a s e i n the t o t a l carotene content but the intermediates, ζ-carotene (7,8,7',8'tetrahydro-i//,ij;-carotene) and neurosporene (7,8-dihydro^,φcarotene) a l s o increased a p p r e c i a b l y . Of s i g n i f i c a n c e are the 3.5 and 4.6-fold increases i n the c y c l i c carotenes, γ-, α- and 3-carotene ( i . e . , 3,ψ-, 3,ε- and 3,3-carotene) caused by d i e t h y l o c t y 1 amine and diethylnonylamine, r e s p e c t i v e l y . The i n c r e a s e i n c y c l i c carotenes i s much l a r g e r than that caused by r e g u l a t o r s t h a t con­ tained an aromatic r i n g . B u t y l d i e t h y l a m i n e has a l s o been observed to cause the development of red c o l o r i n g r a p e f r u i t , but only with higher c o n c e n t r a t i o n s and longer treatment p e r i o d s . The higher members of t h i s s e r i e s , d i e t h y l d e c y l a m i n e , diethylundecylamine, and diethyldodecylamine caused i n c r e a s i n g p e e l i n j u r y as the length of the a l k y l chain i n c r e a s e d . The l a s t two damaged the peel whenever they were a p p l i e d . The c o l o r of the p e e l next to the damaged areas showed c o l o r enhancement, but to a l e s s e n i n g degree as the a l k y l c h a i n was lengthened. Diethylaminoalkylbenzene. The responses of f r u i t t r e a t e d w i t h compounds l i s t e d i n Table I I were s i m i l a r to that of f r u i t treated with d i e t h y l a l k y l a m i n e s (Table I ) . There was a much l a r g e r i n c r e a s e i n ζ-carotene and neurosporene f o r diethylaminobutylbenzene and diethylaminopentylbenzene, whereas the i n c r e a s e i n c y c l i c carotenes was modest. For the d i e t h y l a l k y l a m i n e s , l y c o ­ pene increased to a very h i g h l e v e l and then dropped f o r the l a s t number o f the s e r i e s (Table I ) . The drop i n lycopene accumulation was observed w i t h diethylaminobutylbenzene, but the amount of lyco­ pene increased with diethylaminopentylbenzene, although remaining l e s s than the maximum f o r t h i s s e r i e s ' o f b i o r e g u l a t o r s . The very l a r g e i n c r e a s e i n phytofluene (15 cis-7,8,11,12,7 ,8'-hexahydroψ,ψ-carotene), as w e l l as ζ-carotene, caused by diethylaminopentylbenzene r e s u l t e d i n a g r e a t e r i n c r e a s e i n the t o t a l carotene content. Whether t h i s was caused, i n p a r t , by some s i d e e f f e c t of the extensive p e e l damage or e n t i r e l y by the compound i s not c e r ­ t a i n . A s i m i l a r e f f e c t had been observed p r e v i o u s l y (14) a f t e r treatment w i t h [γ-(diethylamino)-propoxy]-benzene and [0.5 mM) propham a l s o i n h i b i t s RNA, DNA, and p r o t e i n s y n t h e s i s (54) as w e l l as prevents microtubule formation (53). 1

Compounds a s s o c i a t e d with a microtubule absence during m i t o s i s o f t e n reduce microtubule frequency when studied at short time i n t e r v a l s (0.5 h) or at low c o n c e n t r a t i o n s . For example, treatment of c e l l s with low concentrations of c o l c h i c i n e (55) or t r i f l u r a l i n (43, 45) r e s u l t s i n a p a r t i a l loss of microtubules. The remaining microtubules are sometimes d i s o r i e n t e d (45).

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

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222

J. Cell Biology

Figure 8. Multipole spindle apparatus (51). Microtubules are present but have functioned abnormally. Haemanthus katherinae liquid endosperm cells had been treated with 56 μΜ propham. Magnification = ΙΟ,ΟΟΟχ.

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch011

11.

HESS

Growth Inhibition Induced by Herbicides

223

Other h e r b i c i d e s are reported to d i s r u p t m i t o s i s ; however, in most instances the mechanism of d i s r u p t i o n i s unknown. DCPA (dimethyl t e t r a c h l o r o t e r e p h t h a l a t e ) at 30 μΜ was reported to i n t e r f e r e with nuclear d i v i s i o n i n corn roots (_56). D i a l l a t e treatment (60 μΜ) induced the formation of abnormal d i v i s i o n f i g u r e s and reduced metaphase, anaphase, and telophase i n w i l d oat (Avena f a t u a L.) stem t i s s u e (17)· Prasad and Blackman, who found (57^ 1 mM dalapon induced abnormal c e l l d i v i s i o n i n corn root t i p s , concluded m i t o s i s was a r r e s t e d at prophase. Carlson et a l . (58) reported pronamide [3,5-dichloro(N-l,1-dimethyl2-propynyl)benzamide] induced the occurrence of " a r r e s t e d metaphase" i n oat root seedlings w i t h i n 0.5 h a f t e r treatment with 20 μΜ. B a r t e l s and H i l t o n (44) found pronamide (100 μΜ) caused a " l o s s of both c o r t i c a l and s p i n d l e microtubules i n root cells". Two i n t e r e s t i n g observations are f r e q u e n t l y reported for h e r b i c i d e s that d i s r u p t m i t o s i s . F i r s t , t i s s u e d i f f e r e n t i a t i o n and maturation continues a f t e r m i t o t i c d i s r u p t i o n . As a r e s u l t , d i f f e r e n t i a t e d c e l l s occur abnormally c l o s e to the root t i p (e.g. 20, 50, 56, 59, 60). Second, compounds that d i s r u p t m i t o s i s o f t e n induce abnormal stem formation (e.g. 61-65). The xylem elements become shortened, twisted, and discontinuous. This abnormality r e s u l t s i n swollen hypocotyls that are o f t e n b r i t t l e . Compounds may not a f f e c t chromosome movement, but can d i s r u p t or prevent c e l l p l a t e formation at telophase. This e f f e c t i s c l a s s i f i e d as a c e l l d i v i s i o n d i s r u p t i o n rather than an i n h i b i t i o n because the e a r l y stages of m i t o s i s occur and appear normal. The c e l l d i v i s i o n sequence i s not d i s r u p t e d u n t i l c e l l p l a t e formation at telophase. Although no h e r b i c i d e s are known to d i s r u p t or i n h i b i t c e l l w a l l formation as t h e i r sole mode of a c t i o n , the compound c a f f e i n e (1,3,7-trimethylxanthine) is a s p e c i f i c i n h i b i t o r of c e l l p l a t e formation at telophase (35). Paul and Goff reported (66) that aminophylline, t h e o p h y l l i n e (1,3-dimethylxanthine), c a f f e i c a c i d , and c a f f e i n e blocked c e l l w a l l formation at telophase by i n h i b i t i n g v e s i c l e f u s i o n . The

Occurrence of M u l t i n u c l e a t e

Cells

M u l t i n u c l e i observed by l i g h t microscopy are almost u n i v e r s a l l y reported to occur i n c e l l s treated with m i t o t i c d i s r u p t i n g h e r b i c i d e s (e.g. 67^, 68, 6>9). Authors suggest that nuclear d i v i s i o n without c e l l p l a t e formation i s an i n j u r y response of m i t o t i c d i s r u p t i n g h e r b i c i d e s . They o f t e n report the absence of normal d i v i s i o n f i g u r e s and the presence of clumped chromosome arrangements. Observation of published micrographs reveals that most of the reported m u l t i n u c l e i are i n proximity. Observation of p a r a f f i n s e c t i o n s , with an average thickness of 6 to 10 μπι, may not r e s o l v e t h i n connections between the spheres; t h e r e f o r e , uninucleate c e l l s may be i n t e r p r e t e d as being

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BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

multinucleate. Studies of t h i n s e c t i o n s (1 um t h i c k ) o f t e n r e v e a l small connections between the proximally located s p h e r i c a l nuclear lobes (Figure 9). Walne (70) recognized that some of the m u l t i n u c l e a t e c e l l s induced by c o l c h i c i n e could be a m i s i n t e r p r e t a t i o n of polymorphic u n i n u c l e i . With s t a i n i n g techniques, Walne found small interconnections between many n u c l e i of Chlamydomonas eugametos c e l l s . In an e l e c t r o n microscope study, Pickett-Heaps (26) reported the occurrence of polymorphic n u c l e i a f t e r c o l c h i c i n e treatment. In root t i p t i s s u e treated with t r i f l u r a l i n , polymorphic n u c l e i have been observed by e l e c t r o n microscopy (Figure 10, 45). There are three explanations f o r m u l t i n u c l e a t e c e l l s . F i r s t , one or more blocked metaphase chromosomes may become separated from the main group (Figure 11). When the nuclear membrane forms, s e v e r a l n u c l e i could r e s u l t . This occurs i n c o l c h i c i n e treated t i s s u e (71). Second, the meristem contains c e l l s i n a l l stages of the c e l l c y c l e . I f treatment induces microtubule absence i n c e l l s at l a t e anaphase or e a r l y telophase, nuclear envelope reformation w i l l r e s u l t i n a binucleate c e l l . C e l l s c o n t a i n i n g two n u c l e i are at times termed "multinucleate". T h i r d , polymorphic n u c l e i may have very t h i n connections between lobes (Figures 9 and 10). These c e l l s may appear as m u l t i n u c l e a t e when i n f a c t they are uninucleate. To suggest a m i t o t i c d i s r u p t i n g h e r b i c i d e has induced a "multinucleate c o n d i t i o n " , a n a l y s i s must prove more than two d i s c r e t e n u c l e i are a c t u a l l y present i n the c e l l . Conclusion As shown i n Figure 1, i n h i b i t i o n of growth can be caused by numerous aberrant conditions w i t h i n the growth regions of p l a n t s . I f c h a r a c t e r i z a t i o n beyond "growth i n h i b i t i o n " i s attempted f o r a given h e r b i c i d e , the various p o s s i b i l i t i e s must be thoroughly investigated. To learn i f the e f f e c t i s on c e l l d i v i s i o n , c e l l enlargement, or both, experiments must be conducted at times and concentrations where growth i n h i b i t i o n f i r s t occurs. Then one must a s c e r t a i n whether the e f f e c t i s caused by an i n h i b i t i o n or a d i s r u p t i o n of the process. A f t e r t h i s i s determined, e v a l u a t i o n of the aberrancy may r e v e a l the p r e c i s e s i t e of a c t i o n . Growth i n h i b i t i o n should not be described beyond what i s a c t u a l l y known about the cause.

HESS

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11.

Figure 9. Section (1.5 μπι thick) from a cotton lateral root tip treated with tri­ fluralin for 24 h. Note the presence of a polymorphic nucleus. Magnification = 3,000χ.

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

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J. Cell Science

Figure 10. Electron micrograph of a polymorphic nucleus (45). There is a thin connection between each nuclear lobe (arrows). The micrograph is from cotton lateral root tissue treated with trifluralin for 24 h. Magnification = 5,500χ.

Growth Inhibition Induced by Herbicides

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HESS

Figure 11. Aberrant divisionfigurefrom a lateral root of cotton treated with an aqueous saturated solution of trifluralin for 24 h. Some chromosomes (arrows) are widely separated from the main group. Magnification = 3,000χ.

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Literature Cited 1. Parker, C. Weeds 1966, 14, 117-21. 2. Hoagland, D. R.; Arnon, D. I. Univ. California Agric. Exp. Stn. Circ. 347 1950, p 32. 3. Porter, F. E.; Nelson, I. S.; Wold, Ε. K. Crops & Soils 1966, 18, No 8, 10-2. 4. Bonner, J. J. Gen. Physiol. 1933, 17, 63-76. 5. Thimann, Κ. V.; Schneider, C. L. Amer. J. Bot. 1939, 26, 792-7. 6. Nitsch, J. P.; Nitsch, C. Plant Physiol. 1956, 31, 94-111. 7. Prendeville, G. N.; Warren, G. F. Weed Res. 1977, 17, 251-8. 8. Orr, G. L.; Hess, F. D. Pest. Biochem. Physiol. 1981, In Press. 9. Key, J. L. Plant Physiol. 1964, 39, 365-70. 10. Hurwitz, J . ; Furth, J. J . ; Malamy, M.; Alexander, M. Proc. Nat. Acad. Sci. USA 1962, 48, 1222-30. 11. Cleland, R. Plant Physiol. 1965, 40, 595-600. 12. Cleland, R. Symp. Soc. Exp. Biol. 1977, 31, 101-15. 13. Rayle, D. L.; Cleland, R. E. Plant Physiol. 1970, 46, 250-3. 14. Cleland, R. Proc. Nat. Acad. Sci. USA 1973, 70, 3092-3. 15. Shimabukuro, Μ. Α.; Shimabukuro, R. H. Weed Sci. Soc. Amer. Abstr. 1981, 111. 16. Tagawa, T.; Bonner, J. Plant Physiol. 1957, 32, 207-12. 17. Banting, J. D. Weed Sci. 1970, 18, 80-4. 18. Wilkinson, R. E. Weeds 1962, 10, 275-80. 19. Deal, L. M.; Hess, F. D. Weed Sci. 1980, 28, 168-75. 20. Cutter, E. G.; Ashton, F. M.; Huffstutter, D. Weed Res. 1968, 8, 346-52. 21. Cleland, R. Physiol. Plantarum. 1958, 11, 559-609. 22. Setterfield, G.; Bayley, S. T. J. Biophys. & Biochem. Cytol. 1958, 4, 377-82. 23. Northcote, D. H. Proc. R. Soc. Lond. ser. B, Biol Sci. 1969, 173, 21-30. 24. Roelofsen, P. Α.; Houwink, A. L. Acta Bot. Neerl. 1953, 2, 218-25. 25. Green, P. B.; Erickson, R. O.; Richmond, P. A. Ann. Ν. Y. Acad. Sci. 1970, 175, 712-31. 26. Pickett-Heaps, J. D. Dev. Biol. 1967, 15, 206-36. 27. Hepler, P. K.; Palevitz, B. A. Annu. Rev. Plant Physiol. 1974, 25, 309-62. 28. Hess, F. D. "The mechanism of action of trifluralin: influence on cell division, cell enlargement, and microtubule protein"; Ph.D. Dissertation: Univ. Calif., Davis, 1975; p 194. 29. Howard, Α.; Pelc, S. R. Heredity 1953, 6(Suppl.), 261-73.

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30. Hess, F. D. Weed Sci. 1980, 28, 515-20. 31. Jensen, William A. "Botanical Histochemistry"; W. H. Freeman & Company: San Francisco, California, 1962; p 408. 32. Sass, John E. "Botanical Microtechniques"; The Iowa State Univ. Press: Ames Iowa, 1958; p 228. 33. Van't Hof, J . "Methods in Cell Physiology", Vol III; Prescott, D., Ed.; Academic Press: New York, 1968; pp 95-117. 34. Rost, T. L.; Bayer, D. E. Weed Sci. 1976, 24, 81-7. 35. Rost, T.L. "Mechanisms & Control of Cell Division"; Rost, T. L . ; Gifford, Ε. Μ., Eds.; Dowden, Hutchinson & Ross, Inc: Stroudsburg, Pennsylvania, 1977; pp 111-43. 36. Barlow, P. W. Planta 1969, 88, 215-23. 37. Webster, P. L.; Van't Hof, J. Amer. J. Bot. 1970, 57, 130-9. 38. Canvin, D. T.; Friesen, G. Weeds 1959, 7, 153-6. 39. Dhillon, N. S.; Anderson, J. L. Weed Res. 1972, 12, 182-9. 40. Rost, T. L.; Morrison, S. L.; Sachs; E. S. Amer. J. Bot. 1977, 64, 780-5. 41. Ray, T. B. Proc. British Crop Prot. Conf. - Weeds 1980, 1, 7-14. 42. Wilson, L. Life Sci. 1975, 17, 303-10. 43. Jackson, W. T.; Stetler, D. A. Can. J. Bot. 1973, 51, 1513-8. 44. Bartels, P. G.; Hilton, J. L. Pest Biochem. Physiol. 1973, 3, 462-72. 45. Hess, F. D.; Bayer, D. E. J. Cell Sci. 1974, 15, 429-41. 46. Hess, F. D.; Bayer, D. E. J. Cell Sci. 1977, 24, 351-60. 47. Hess, F. D. Exp. Cell Res. 1979, 119, 99-109. 48. Hertel, C.; Quader, H.; Robinson, D. G.; Marme, D. Planta 1980, 149, 336-40. 49. Ennis, W. B. Amer. J. Bot. 1948, 35, 15-21. 50. Ennis, W. B. Amer. J. Bot. 1949, 36, 823. 51. Hepler, P. K.; Jackson, W. T. J. Cell Sci. 1969, 5, 727-43. 52. Jackson, W. T. J. Cell Sci. 1969, 5, 745-55. 53. Coss, R. Α.; Pickett-Heaps, J. D. J. Cell Biol. 1974, 63, 84-98. 54. Rost, T. L.; Bayer, D. E. Weed Sci. 1976, 24, 81-7. 55. Jokelainen, P. I. J. Cell Biol. 1968, 39, 68a-9a. 56. Bingham, S. W. Weed Sci. 1968, 16, 449-52. 57. Prasad, R.; Blackman, G. E. J. Exp. Bot. 1964, 15, 48-66. 58. Carlson, W. C.; Lignowski, E. M.; Hopen, H. J. Weed Sci. 1975, 23, 155-61. 59. Peterson, R. L.; Smith, L. W. Weed Res. 1971,11,84-7. 60. Scott, Μ. Α.; Struckmeyer, Β. E. Bot. Gaz. 1955, 117, 37-45. 61. Kirby, C. J.; Standifer, L. C.; Normand, W. C. Proc. Southern Weed Conf. 1968,21,343. 62. Nishimoto, R. K.; Warren, G. F. Weed Sci. 1971, 19, 343-6. 63. Andersen, J. L.; Shaybany, B. Weed Sci. 1972, 20, 434-9.

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64. Hess, B.; Bayer, D.; Ashton, F. Weed Sci. Soc. Amer. Abstr. 1974, 17. 65. Struckmeyer, Β. E.; Binning, L. K.; Harvey, R. G. Weed Sci. 1976, 24, 366-9. 66. Paul, D. C.; Goff, C. W. Exp. Cell Res. 1973, 78, 399-413. 67. Talbert, R. E. Proc. Southern Weed Conf. 1965, 18, 652. 68. Hacskaylo, J . ; Amato, V. A. Weed Sci. 1968, 16, 513-5. 69. Lignowski, Ε. M.; Scott, E. G. Weed Sci. 1972, 20, 267-70. 70. Walne, P. L. Amer. J. Bot. 1966, 53, 908-16. 71. Hindsmarsh, M. M. Proc. Linnean Soc. N. S. Wales 1953, 77, 300-6. Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch011

RECEIVED

September 28, 1981.

12 Modes of Herbicide Action as Determined with Chlamydomonas reinhardii and Coulter Counting C A R L FEDTKE

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch012

Pflanzenschutz, Biologische Forschung, Bayer A G 5090 Leverkusen, Federal Republic of Germany

Synchronously grown Chlamydomonas r e i n h a r d i i cells were used f o r the d i f f e r e n t i a t i o n between d i f f e r e n t p h y s i o l o g i c a l modes o f h e r b i c i d e a c t i o n . The cells were counted with a C o u l t e r Counter and the volume spectrum was obtained by an adapted Channelyzer. The a c t i o n o f the t e s t e d h e r b i c i d e s was c l a s s i f i e d as f o l l o w s : (a) growth i n h i b i t i o n i n the l i g h t without r a p i d p h y t o t o x i c i t y ( m e t r i b u z i n ) , (b) b l e a ching o f the cells without r a p i d p h y t o t o x i c i t y ( f l u r i d o n e ) , (c) r a p i d p h y t o t o x i c i t y i n the l i g h t ( n i t r o f e n , paraquat), (d) r a p i d p h y t o t o x i c i t y i n the dark (PCP), (e) d i r e c t and r a p i d i n h i b i t i o n o f cell d i v i s i o n but not o f cell growth (amiprophos methyl, chlorpropham, trifluralin) and (f) ind i r e c t (delayed) i n h i b i t i o n o f cell d i v i s i o n and growth ( a l a c h l o r ) . Depending on c o n c e n t r a t i o n , h e r b i c i d e s may i n h i b i t many d i f ferent p l a n t metabolic pathways i n v i t r o as w e l l as i n v i v o . However, often the relevance o f an i n h i b i t o r y a c t i v i t y i n v i t r o for the a c t i o n on the i n t a c t growing plant i s not c l e a r . T r i f l u r a l i n and chlorpropham e . g . a f f e c t c h l o r o p l a s t and mitochond r i a l a c t i v i t i e s i n v i t r o (J_). In the f i e l d , however, they i n h i b i t the growth o f s e e d l i n g s and provoke c h a r a c t e r i s t i c morp h o l o g i c a l responses which can not be traced to the e f f e c t s on the o r g a n e l l e s mentioned above. A simple t e s t system more c l o s e l y r e f l e c t i n g the primary s e n s i t i v e pathway under f i e l d c o n d i t i o n s would therefore c l e a r l y be h e l p f u l . U n i c e l l u l a r algae have repeatedly been used f o r the estimat i o n and c l a s s i f i c a t i o n o f h e r b i c i d e a c t i o n s (2, 3^, A ) . Chlamydomonas r e i n h a r d i i appeared to be most s u i t a b l e s i n c e "Ehis species proved to be s e n s i t i v e towards h e r b i c i d e s o f many d i f f e r e n t groups. Chlamydomonas r e i n h a r d i i s t r a i n 11 - 32 a/89 was obtained from the "Algensammlung G ô t t i n g e n " and was grown on T r i s - A c e t a t e Phosphate medium (j> ) . The c u l t u r e v e s s e l s (250 ml beakers

This chapter not subject to U.S. copyright. Published 1982 American Chemical Society.

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BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

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c o n t a i n i n g 150 ml growth medium) were kept on a r o t a r y shaker a t 100 rpm i n a 12/12 h l i g h t / d a r k regime. The i l l u m i n a t i o n was 5 000 l x from Osram HQI bulbs and the temperature was 24 C during the l i g h t and 18 C d u r i n g the dark phase. The maximum c e l l d e n s i t y at which the c u l t u r e s were d i l u t e d was 7 x 105 c e l l s / m l . Under these c o n d i t i o n s , the m u l t i p l i c a t i o n f a c t o r was ca. 4 to 5 i n 24 h. The h e r b i c i d e s were d i s s o l v e d i n methanol. Only c h e m i c a l l y pure compounds (>99 %) were used. The c e l l s were counted by a C o u l t e r Counter Model ZB equipped with a 50 yum aperture a f t e r a p p r o p r i a t e d i l u t i o n with growth medium. Simultaneously the s i z e d i s t r i b u t i o n was obtained by a C o u l t e r Channelyzer Model C-1000. The s i z i n g i s l i n e a r with volume and was s e t to span the range from 70 to 900 pm? (Figures 2, 3, e t c . ) . C e l l Cycle A n a l y s i s with the C o u l t e r Counter In the r e g u l a r c e l l c y c l e o f Chlamydomonas r e i n h a r d i i , c e l l s grow i n the l i g h t and d i v i d e i n the dark (Figure 1). At the beg i n n i n g o f the dark phase, the c e l l s shed t h e i r f l a g e l l a and s w e l l s l i g h t l y to g a i n a s p h e r i c a l form. During the dark phase, n u c l e a r , c h l o r o p l a s t , and c e l l d i v i s i o n s take p l a c e , and the daughter c e l l s are l i b e r a t e d . In c o n t r a s t to other s t r a i n s , i n these experiments the daughter c e l l s were l i b e r a t e d throughout the dark phase and not synchronously a t i t s end {6}. I n o c u l a t i o n o f new c u l t u r e s and C o u l t e r Counter measurements were u s u a l l y s t a r t e d a t the beginning o f the dark phase. At t h i s time, f u l l y grown c e l l s are present (Figure 2, t r a c e 0 ) . F i v e hours l a t e r (trace 5), most o f the c e l l s have swollen and entered d i v i s i o n . E i g h t hours i n t o the dark phase (trace 8 ) , many mother c e l l s have already l i b e r a t e d the daughter c e l l s , whereas a t the end o f the dark phase (trace 12) a homogenous p o p u l a t i o n o f s m a l l , young c e l l s i s obtained. These c e l l s grow d u r i n g the next 12 h l i g h t phase to a t t a i n the f u l l y grown s i z e again (Figure 3 ) . The h e r b i c i d e s s e l e c t e d f o r the present study are given i n Table I together with t h e i r r e s p e c t i v e chemical names. Table I I presents the a c t i v i t y data towards Chlamydomonas r e i n h a r d i i f o r these h e r b i c i d e s and a l s o i n d i c a t e s the metabolic pathway or process which i s presumably p r i m a r i l y a f f e c t e d by the a c t i o n o f the h e r b i c i d e and whose i n h i b i t i o n or a l t e r a t i o n most l i k e l y causes the observed e f f e c t s . The f l a g e l l a regeneration represents a process which depends on microtubule p o l y m e r i z a t i o n , and which i s , t h e r e f o r e , seen as a t e s t f o r some types o f a n t i m i t o t i c a c t i v i t y ( 7 ) . However, f l a g e l l a r regeneration i s an energy r e q u i r i n g process and i s , t h e r e f o r e , i n h i b i t e d a l s o by the r e s piratory uncoupler PCP. I n t e r e s t i n g l y , n i t r o f e n i s a l s o a c t i v e i n t h i s t e s t . Summarizing, two broad groups may be formed from the s e l e c t e d h e r b i c i d e s , one i n t e r f e r i n g with photosynthetic processes and a second i n t e r f e r i n g with c e l l d i v i s i o n , 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 (J_, 8, £ ) . These two groups w i l l be discussed s e p a r a t e l y .

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12. FEDTKE

Figure 1. Chlamydomonas growth and cell division during a 24-h cell cycle. In the cells, a cup-shaped chloroplast with a pyrenoid and the eye spot are seen. The twoflagellaprovide locomotion.

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

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234

Figure 2. Size distribution of control Chlamydomonas cell cultures during the dark phase. Numbers 0, 5, 8, and 12 mean after 0, 5, 8, or 12 hours in the dark.

FEDTKE

Herbicide Action

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12.

Figure 3. Size distribution of control Chlamydomonas cell cultures during the light phase. Numbers 12, 17, 20, and 24 mean after 0, 5, 8, or 12 hours in the light.

4 - A m i n o - 6 - t e r t - b u t y l - 3 - ( m e t h y l t h i o ) - a s - t r i a z i n - 5 ( 4 H )one 2,4-Dichlorophenyl p - n i t r o p h e n y l

Metribuzin

Nitrofen

1,1 -Dimethy1-4,4 -bipyridinium i o n Pentachlorophenol JL, A> ^L-Trifluoro-2,6-dinitro-N,N-dipropy1-p-toluidine

Paraquat

PCP

Trifluralin

ether

1- Methyl-3-phenyl-5-(3~( t r i f l u o r o m e t h y l ) phenyl])-4 ( 1 H )-pyridinone

Fluridone

1

Isopropyl m - c h l o r o c a r b a n i l a t e

Chlorpropham (=CIPC)

1

0- Methyl-0-(A-methyl-6-nitrophenyl-N-isopropyl-phosphorothioamidate)

acetanilide

Amiprophos-methyl (=APM)

f

2-Chloro-2 jo'-diethyl-N-fmethoxymethyl)

Chemical name

Alachlor

Common name

Table I . Common names, a b b r e v i a t i o n s ( i n parentheses), and chemical names o f h e r b i c i d e s used i n the present study

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Carotenoid b i o s y n t h e s i s

R e s p i r a t o r y energy conservation

M i c r o t u b u l e - c o n t r o l l e d processes ( c e l l growth, c e l l d i v i s i o n , etc.)

1

>

J

Fluridone

PCP

APM

Trifluralin

CIPC

0

Ip- = the c o n c e n t r a t i o n t h a t i n h i b i t s by 50 %.

division

Photosynthesis

Nitrofen

Cell

Photoreductions

Paraquat

Alachlor

Photosynthetic e l e c t r o n t r a n s p o r t

Metribuzin

Γ

Metabolic pathway o r process a f f e c t e d

Herbicide

> 500

52.4

1.0 2.6

0.4

0.3

4.8

121

3.2

34.1

> 500

Flagella

regeneration

reinhardii I^-values

2.9

0.3

5-6

6.1

0.2

0.1

0.5

Growth

Chlamydomonas

Table I I . A c t i o n o f the s e l e c t e d h e r b i c i d e s on Chlamydomonas growth and f l a g e l l a regeneration and the most important u n d e r l y i n g s e n s i t i v e metabolic pathways or processes.

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(yuMi

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BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

The s e n s i t i v i t y o f the a l g a l c e l l s v a r i e d c o n s i d e r a b l y among d i f f e r e n t t e s t s . Thorough s t a n d a r d i z a t i o n was t h e r e f o r e necessary. The c o n d i t i o n s a p p l i e d f o r the e s t i m a t i o n o f the I^o-values i n Table II (growth over 4 to 5 g e n e r a t i o n s , s t a r t i n g with low c e l l d e n s i t i e s ) d i f f e r e d from those i n the t e s t s recorded with the C o u l t e r Counter (growth over 2 g e n e r a t i o n s , s t a r t i n g with higher c e l l d e n s i t i e s ) . Moreover, d u r a t i o n o f p r e c u l t u r e o f the c e l l s i n aqueous medium or on agar and p r i o r d u r a t i o n o f synchroneous growth were other parameters which were observed to change the s e n s i t i v i t y . However, c o n t r o l l i n g these f a c t o r s d i d not e n t i r e l y e l i m i n a t e the v a r i a t i o n s i n s e n s i t i v i t y . The concentrations a p p l i e d i n the recorded experiments were s e l e c t e d f o r optimum e f f e c t i v i t y . In some cases, where the c h a r a c t e r i s t i c s o f the h e r b i c i d a l a c t i o n changed with the c o n c e n t r a t i o n , a lower and a higher one are r e c o r d e d . E f f e c t s o f H e r b i c i d e s I n t e r f e r i n g with

Photosynthesis

M e t r i b u z i n represents the h e r b i c i d e s that i n h i b i t photos y n t h e t i c e l e c t r o n t r a n s p o r t s h o r t l y a f t e r photosystem II (63). As expected, m e t r i b u z i n does not i n h i b i t c e l l d i v i s i o n but i n h i b i t s c e l l growth i n the l i g h t (Table III and F i g u r e 4 ) . P a r a quat, which i s t o x i c i n the l i g h t through the photosynthetic r e d u c t i o n o f oxygen to the superoxide anion (JJ , a l s o does not a f f e c t c e l l d i v i s i o n , but leads to r a p i d c e l l death i n the l i g h t . The a c t i v i t y o f n i t r o f e n a t low concentrations resembles that o f paraquat, i n d i c a t i n g a c t i v a t i o n or a c t i v i t y o f the h e r b i c i d e i n the l i g h t (JO). At higher c o n c e n t r a t i o n s , however, c e l l d i v i s i o n i s a l s o a f f e c t e d . F l u r i d o n e , an i n h i b i t o r o f carotenoid b i o s y n t h e s i s (11), n e i t h e r i n t e r f e r e s with c e l l d i v i s i o n nor i n h i b i t s c e l l growth. However, the c e l l s are bleached a f t e r 24 h and do not d i v i d e or grow f u r t h e r . M e t r i b u z i n and f l u r i d o n e at 10 times higher concentrations d i d not show increased a c t i v i t y when compared with the lower c o n c e n t r a t i o n s . E f f e c t s o f H e r b i c i d e s I n t e r f e r i n g with C e l l

Division

C e l l d i v i s i o n i s a complicated process, and many d i f f e r e n t primary i n t e r f e r e n c e s might e v e n t u a l l y l e a d to the i n h i b i t i o n o f c e l l d i v i s i o n . One such primary i n t e r f e r e n c e that i n d i r e c t l y blocks c e l l d i v i s i o n because the c e l l s are r a p i d l y k i l l e d i s the uncoupling o f o x i d a t i v e p h o s p h o r y l a t i o n , as demonstrated by PCP (Table I V ) . Many d i f f e r e n t i n h i b i t o r y agents have been t e s t e d p r e v i o u s l y f o r t h e i r e f f e c t on Chlamydomonas, and a c o r r e l a t i o n between i n h i b i t o r y mechanism and accumulating a l g a l stage has been obtained {12, 1 3 ) · study, four growth i n h i b i t i n g h e r b i c i d e s with a more d i r e c t i n h i b i t o r y a c t i o n on c e l l d i v i s i o n were s e l e c t e d . I

n

t

n

i

s

bleached

cells

70

heterogeneous s i z e d i s t r i b u t i o n

t

a

b

6.1

Fluridone

210

90

c e l l s dead

0.2 4.0

Nitrofen 250

160 200 t

b

t

a 170

-

90 t

220 70

95 90

90

140

70

75

250

75

+

0.1 2.0

Paraquat

300

a

0.5

-

b

Cone. Average p a r t i c l e volume i n jum^ (h a f t e r s t a r t ) μΜ 0 12 24 36

Metribuzin

Control

Herbicide

100

13

0 0

25 0

100 107 68 13

32

100

100 98

24 - 36 h

0 - 14 h

Increase o f p a r t i c l e number

Table I I I . Influence o f h e r b i c i d e s that i n t e r f e r e with photosynthetic processes on growth and m u l t i p l i c a t i o n o f Chlamydomonas r e i n h a r d i i . The algae were kept i n a synchronized 12/12 h l i g h t / d a r k c y c l e , with Oh a t the beginning o f a dark phase.

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(%)

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

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240

Figure 4. Size distribution of Chlamydomonas cell cultures treated with metribuzin at time 0 (compare with Figure 3).

19

2.6 37

CIPC

Alachlor

a

f

300

0

-

750 t a

300

330 a 400 a 600 a 160 t a 700 a 800 a

90 300 a

250 a 150 a 300 a 500 t a 450 t a

-

90 500 t a

75

75 270 t a

36

24

(h a f t e r s t a r t )

250

12

Average p a r t i c l e volume i n pm3

heterogeneous s i z e d i s t r i b u t i o n

ΐ c e l l s dead

2.9 29

6.7

37

-

r

Cone.

Trifluralin

ΑΡΜ

PCP

Control

Herbicide

42 10

30

27 0

0

27

100

0 - 14 h

0 0

0

100 0

0

0

100

24 - 36 h

Increase o f p a r t i c l e number (%)

Table IV. Influence o f h e r b i c i d e s that i n t e r f e r e with c e l l d i v i s i o n on growth and m u l t i p l i c a t i o n o f Chlamydomonas r e i n h a r d i i . The algae were kept i n a synchronized 12/12 h l i g h t / d a r k c y c l e , with 0 h a t the beginning o f a dark phase.

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242

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

The h e r b i c i d e APM blocks c e l l d i v i s i o n very r a p i d l y (Figure 5). Although the f l a g e l l a were l o s t , a normal s w e l l i n g r e a c t i o n d i d not even occur a t 8 hours i n t o the dark phase. At the concentration a p p l i e d , a few young (small) c e l l s hatched a t the end o f the dark phase. A f t e r an a d d i t i o n a l 12 h growth i n the l i g h t (Figure 6, t r a c e 24), both small and l a r g e c e l l s had grown c o n s i d e r a b l y . Although some c e l l s were dead a t t h i s time, others s u r v i v e d without d i v i s i o n and enlarged during the second l i g h t phase, g a i n i n g a tremendous s i z e . C e l l volumes up to 8000 μπ? have been measured i n the microscope a f t e r 48 hours i n the presence o f 1.3 /uM APM. T r i f l u r a l i n and CIPC are i n h i b i t o r s o f the microtubule system l i k e APM {9). I t may, however, be a n t i c i p a t e d that they i n t e r f e r e with d i f f e r e n t molecular s i t e s o f a c t i o n . T h e i r e f f e c t on Chlamydomonas resembles that o f APM i n that they block c e l l d i v i s i o n but not the growth o f the c e l l s (Table I V ) . The l i m i t e d a c t i o n o f low t r i f l u r a l i n concentrations may be explained by the f a c t that t r i f l u r a l i n tends to adsorb to g l a s s and other s u r ­ faces because o f i t s low water s o l u b i l i t y and, t h e r e f o r e , i s removed from s o l u t i o n . A l a c h l o r does not i n t e r f e r e with the microtubular system. However, t h i s h e r b i c i d e a l s o a c t s by i n h i b i t i o n o f c e l l d i ­ v i s i o n (J[4 ). In Chlamydomonas, a l a c h l o r provoked r a t h e r p e c u l i a r e f f e c t s : although normal c e l l d i v i s i o n was i n i t i a t e d i n the dark, few daughter c e l l s were l i b e r a t e d (Figure 7 ) . Instead, the c e l l u l a r aggregates enlarged considerably during the f o l l o w i n g l i g h t phase, forming high volume p a r t i c l e s (Figure 8 ) . In the microscope, c e l l u l a r aggregates of up to 2000 ρηβ have been measured. Further A n a l y s i s o f the I n h i b i t i o n by A l a c h l o r . L i t t l e i s known about the h e r b i c i d a l mode o f a c t i o n o f a l a c h l o r and Chlamydomonas might, t h e r e f o r e , represent an i n t e r e s t i n g system f o r f u r t h e r s t u d i e s . Figure 9 demonstrates that a l a c h l o r a c t s on c e l l d i v i s i o n , but not on growth as measured by the i n c r e a s e i n c h l o r o p h y l l . C h l o r o p h y l l content and p a r t i c l e number, r e ­ presenting c e l l growth and c e l l d i v i s i o n , r e s p e c t i v e l y , show an i n c r e a s e only i n the r e s p e c t i v e l i g h t or dark phase. When a l a c h l o r i s added a t the beginning o f the dark phase, c e l l growth as measured by the c h l o r o p h y l l content continues to increase nor­ mally f o r 24 h, whereas c e l l d i v i s i o n (more p r e c i s e l y c e l l se­ p a r a t i o n , Figures 7, 8) i s blocked. More information about the i n h i b i t i o n o f c e l l d i v i s i o n by a l a c h l o r was obtained from the experiment recorded i n Table V. S t a t i o n a r y algae (grown to a high c e l l d e n s i t y where l a c k o f l i g h t and n u t r i e n t s l i m i t s f u r t h e r growth and d i v i s i o n ) were used when s t a r t i n g t h i s experiment. S i m i l a r r e s u l t s were ob­ t a i n e d when algae were taken from the l o g a r i t h m i c growth phase. The lower c e l l volume and c e l l m u l t i p l i c a t i o n f a c t o r are caused by these s p e c i f i c c o n d i t i o n s . When a l a c h l o r i s given a t the

Herbicide Action

243

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12. FEDTKE

Figure 5. Size distribution of Chlamydomonas cell cultures treated with APM at time 0 (compare with Figure 2).

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

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244

Figure 6. Size distribution of Chlamydomonas cell cultures treated with APM at time 0 (compare with Figure 3).

FEDTKE

Herbicide Action

245

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12.

Figure 7. Size distribution of Chlamydomonas cell cultures treated with alachlor at time 0 (compare with Figure 2).

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch012

246

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

Figure 8. Size distribution of Chlamydomonas cell cultures treated with alachlor at time 0 (compare with Figure 3).

FEDTKE

Herbicide Action

247

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12.

Time (hours)

Figure 9. Idealized curves for increases of particle number (Φ) and chlorophyll content (O) per culture vessel, and for alachlor action (- · ·) on these increases. Alachlor application at arrow.

American Chemical Society Library 1155 16th St. N. w. Washington, 0. C. 20036

160

210

190

A

A

A

9

0 -

24 - 72

0 - 24

b

150

A

9 - 72

aggregates

440

80

230

220

440

84

24

500

a

580

a

of 2 - 5 c e l l s

270

150

230

D

156

32

a

flagella

200

A

0 - 72

s i n g l e c e l l s without

181

C

8

a

b

100

310

100

400

220

92

48

a

b

150 110

170 200

100

-

200

115

72

after h

183

56

Average p a r t i c l e volume i n μπβ

0

h

"ime-interval o f

690 20

30 310

70

0

60

0

30

184

30-54

10

268

0-30

250

40

220

0

0

210

54-'

Increase o f particle-number d u r i n g time-interval i n %

Table V. A c t i o n of 37 yuM a l a c h l o r on Chlamydomonas r e i n h a r d i i growth and m u l t i p l i c a t i o n when present f o r d i f f e r e n t t i m e - i n t e r v a l s during the growth c y c l e . I n o c u l a t i o n was with s t a t i o n a r y algae with a c e l l volume o f 90 jum^. L i g h t phases were from 0-10, 24 - 34 and 48 - 58 h . C = C o n t r o l , A = a l a c h l o r treated.

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch012

3

Ο

W

X

κ!

»

m α

n

C

Ό

2

Ο

m

ο >

g

m

χ

δ ο

to oo

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12.

FEDTKE

Herbicide Action

249

beginning o f the f i r s t l i g h t phase (Table V, 0 - 7 2 ) , no d i v i s i o n s occur during the f o l l o w i n g dark phase. However, when a l a c h l o r i s added 9 hours l a t e r a t the beginning o f the dark phase (Table V, 9-72) the a l r e a d y f a m i l i a r response i s obtained: d i v i s i o n occurs, but the daughter c e l l s do not separate (compare F i g u r e s 7, 8 ) . A l a c h l o r , t h e r e f o r e , c l e a r l y i s an i n h i b i t o r o f c e l l d i v i s i o n , a l b e i t i n d i r e c t l y . C e l l growth i s not a f f e c t e d , i . e . , the t r e a t e d s i n g l e c e l l s (Table V, 0-72) or c e l l aggregates (Table V, 9 - 72) continue to enlarge u n t i l they e v e n t u a l l y die. When a l a c h l o r i s removed from the a l g a l suspension a f t e r 9 h (Table V, 0 - 9 ) by simple sedimentation and resuspension i n f r e s h medium, no d i v i s i o n s occur i n the f i r s t c y c l e . A l a c h l o r , t h e r e f o r e , seems to block some metabolic process t h a t i s preparatory f o r c e l l d i v i s i o n . The c e l l s grow f u r t h e r and, a f t e r being r e l e a s e d from the i n h i b i t i o n o f c e l l d i v i s i o n , d i v i d e ext e n s i v e l y u n t i l the normal s i z e o f young c e l l s i s again a t t a i n e d . At t h a t time, c o n t r o l and t r e a t e d c u l t u r e s are again equal, and a l a c h l o r has done nothing e l s e but h a l t e d c e l l d i v i s i o n d u r i n g one c y c l e . When a l a c h l o r i s added a t the beginning o f the second l i g h t phase (Table V, 2 4 - 7 2 ) , the i n h i b i t o r y e f f e c t i s the same as i f given from the beginning. However, when a l a c h l o r i s removed 24 h a f t e r a d d i t i o n , i . e . , a t the beginning o f the second l i g h t phase (Table V, 0 - 2 4 ) , the i n h i b i t o r y e f f e c t i s much stronger than f o r Table V, 0 - 9 , and one e n t i r e growth c y c l e i s d e l e t e d . Even the second c y c l e i s p a r t i a l l y i n h i b i t e d under these cond i t i o n s . The experiments ( 0 - 9 ) and ( 0 - 2 4 ) i n Table V c l e a r l y demonstrate t h a t the i n h i b i t i o n by a l a c h l o r i s r e v e r s i b l e . Conclusions The r e s u l t s demonstrate the u s e f u l n e s s o f synchronized Chlamydomonas r e i n h a r d i i c e l l c u l t u r e s f o r h e r b i c i d e mode o f a c t i o n s t u d i e s . I n h i b i t o r y a c t i o n s i n the l i g h t (metribuzin) and t o x i c a c t i o n s i n the l i g h t (paraquat, n i t r o f e n ) are c l e a r l y d i s c e r n e d . S i m i l a r l y , b l e a c h i n g h e r b i c i d e s ( f l u r i d o n e ) and r e s p i r a t o r y uncouplers (PCP) may be c l a s s i f i e d from the a l g a l r e sponse. C e l l d i v i s i o n i n h i b i t i n g h e r b i c i d e s may be recognized as e i t h e r d i r e c t (APM, t r i f l u r a l i n , CIPC) or i n d i r e c t ( a l a c h l o r ) i n h i b i t o r s . The a l g a l responses do not allow f o r the formulat i o n o f a biochemical mode o f a c t i o n . However, the p h y s i o l o g i c a l type o f response and the knowledge o f the s e n s i t i v e growth phase give valuable information. In the case o f a l a c h l o r , i n t e r e s t i n g new i n f o r m a t i o n has been obtained. The i n h i b i t i o n by a l a c h l o r i s r e v e r s i b l e and concerns a metabolic r e a c t i o n or process that i s r e q u i r e d f o r normal c e l l d i v i s i o n . The h e r b i c i d e a l a c h l o r might s p e c i f i c a l l y i n t e r f e r e with the r e g u l a t i o n o f c e l l d i v i s i o n .

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BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

Acknowledgment. The experiments communicated here have been c a r r i e d out by Mr. J a n t z e n .

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch012

Literature Cited. 1. Moreland, D.E. Ann. Rev. Pl. Physiol. 1980,31,597. 2. Kratky, B.A.; Warren, G.F. Weed Sci. 1971,19,658. 3. Sandmann, G.; Kunert, K.-J.; Böger, P. Z. Naturforsch. 1979, 34 c, 1044. 4. Hess, F.D. Weed Sci. 1980, 28, 515. 5. Amrhein, N.; Filner, P. Proc. Nat. Acad. Sci U.S.A. 1973, 70, 1099. 6. Mihara, S.; Hase, Ε. Plant Cell Physiol. 1971, 12, 225. 7. Quader, H.; Filner, P. Eur. J . Cell Biol. 1980, 21, 301. 8. Trebst, A. Proc. V. Int. Photosynthesis Congr. 1980. 9. Draber, W.; Fedtke, C. Proc. 4th Int. Congr. of Pesticide Chemistry (IUPAC) 1979; p. 475. 10. Fadayomi, O.; Warren, G.F. Weed Sci. 1976, 24, 598. 11. Bartels, P.G.; Watson, C.W. Weed Sci. 1978, 26, 198. 12. Howell, S.H.; Blaschke, W.J.; Drew, C.M. J . Cell. Biol. 1975, 67, 126. 13. Mihara, S.; Hase, Ε. Plant & Cell Physiol. 1978,19,83. 14. Deal, L.M.; Hess, F.D. Weed Sci. 1980, 28, 168. RECEIVED September 29, 1981.

13 Use of Chlorella to Identify Biochemical Modes of Action of Herbicides S. SUMIDA and R. YOSHIDA

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch013

Sumitomo Chemical Co., Ltd., Institute for Biological Science, 4-2-1 Takatsukasa, Takarazuka, Hyogo 665, Japan

Synchronized cultures of C h l o r e l l a e l l i p s o i d e a were used as a model system to i d e n t i f y biochemical modes of action of two h e r b i c i d e s , butamiphos ( O - e t h y l -0-(3-methyl-6-nitrophenyl)-N-sec-butyl-phosphorothioamidate, formerly coded S2846), and chlorpropham ( i s o p r o p y l m- c h l o r o c a r b a n i l a t e ) . C e l l cycle studies showed that butamiphos i n h i b i t e d cell division, whereas its effects on r e s p i r a t i o n and general biosynthesis were slight. Confirmatory experiments with onion root apices showed that m i t o s i s was blocked at the metaphase and that the spindle apparatus was d i s r u p t e d . Butamiphos seems to be a h i g h l y s p e c i f i c inhibitor of mitosis like c o l c h i c i n e . Chlorpropham i n h i b i t e d cell d i v i s i o n of a synchronized C h l o r e l l a c u l t u r e . Respir a t i o n was not significantly affected. P r o t e i n synthesis was more s e n s i t i v e to chlorpropham than the other b i o s y n t h e t i c processes tested, but a possibility remains that chlorpropham may directly inhibit mitosis without i n t e r f e r i n g with protein synthesis. The h i g h e r p l a n t i s a m u l t i c e l l u l a r s y s t e m t h a t c o n s i s t s o f v a r i o u s o r g a n s s u c h as l e a v e s , s t e m s , r o o t s , and f l o w e r s . C o m p a r a t i v e l y , the u n i c e l l u l a r a l g a i s a simpler system. T a x o n o m i c a l l y , the green a l g a i s r e l a t i v e l y c l o s e l y r e l a t e d to the h i g h e r p l a n t . Because o f t h i s r e l a t i o n s h i p , u n i c e l l u l a r green algae s u c h as C h l o r e l l a h a v e l o n g b e e n u s e d by b i o c h e m i s t s and p l a n t p h y s i o l o g i s t s as s i m p l e m o d e l s y s t e m s f o r 0097-6156/82/0181 -0251 $05.00/0 © 1982 American Chemical Society

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BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch013

h i g h e r p l a n t s , p a r t i c u l a r l y to study the biochemistry of p h o t o s y n t h e s i s . U n i c e l l u l a r green algae are suit­ a b l e t o g e n e r a t e q u a n t i t a t i v e and c l e a r - c u t r e s u l t s under r i g o r o u s l y d e f i n e d e x p e r i m e n t a l c o n d i t i o n s with­ in a short p e r i o d of time. Furthermore, the growth of u n i c e l l u l a r g r e e n a l g a e c a n be s y n c h r o n i z e d w i t h r e l a ­ t i v e ease, which p r o v i d e s a p a r t i c u l a r l y u s e f u l system for a biochemical study of c e l l d i v i s i o n i n c o n t r a s t t o most h i g h e r p l a n t t i s s u e i n w h i c h c e l l d i v i s i o n i s asynchronous. T h i s paper d e s c r i b e s the s u c c e s s f u l use of C h l o r e l l a e l l i p s o i d e a ( a b b r e v i a t e d as C h l o r e l l a ) as a s i m p l e model system t o study b i o c h e m i c a l modes o f a c t i o n o f t h e h e r b i c i d e s , b u t a m i p h o s and chlorpropham, which are i n h i b i t o r s of c e l l division. Butamiphos Butamiphos i s a phosphorothioamidate h e r b i c i d e [O-ethyl-0-(3-methy1-6-nitropheny1)-N-sec-butylphosp h o r o t h i o a m i d a t e ] formerly r e f e r r e d t o as S-2846 (see Figure 1). B u t a m i p h o s c o n t r o l s a n n u a l weeds by pree m e r g e n c e a p p l i c a t i o n (1). B u t a m i p h o s a c t s on the r o o t a p e x and c a u s e s i t t o s w e l l n o t i c e a b l y . When C h l o r e l l a i s t r e a t e d w i t h butamiphos, the c e l l p o p u l a ­ tion increase i s i n h i b i t e d . The a v e r a g e s i z e o f t h e t r e a t e d c e l l s becomes l a r g e r r e l a t i v e t o the untreated c o n t r o l s (2). By a s s u m i n g t h a t s w e l l i n g o f t h e root a p i c e s o f h i g h e r p l a n t s and t h e e n l a r g e d average s i z e o f C h l o r e l l a c e l l s a r e c a u s e d by a common m e c h a n i s m o f a c t i o n , we d e c i d e d t o u s e C h l o r e l l a a s a m o d e l system to l o c a l i z e a b i o c h e m i c a l s i t e of a c t i o n of butamiphos. P r e v i o u s r e s e a r c h h a s shown t h a t t h e c e l l p o p u l a ­ t i o n i n c r e a s e o f C h l o r e l l a was i n h i b i t e d 50% a t a b o u t 2 μΜ o f b u t a m i p h o s and n e a r l y 100% a t a b o u t 6 μΜ (2) . E f f e c t s o f b u t a m i p h o s on r e s p i r a t i o n and c e l l membrane p e r m e a b i l i t y were e x a m i n e d . B u t a m i p h o s a t 150 μΜ showed no s i g n i f i c a n t e f f e c t s on r e s p i r a t i o n as mea­ s u r e d by o x y g e n c o n s u m p t i o n {2). T h i s same c o n c e n t r a ­ t i o n d i d n o t a f f e c t s i g n i f i c a n t l y c e l l membrane p e r m e a b i l i t y as m e a s u r e d by l e a k a g e o f r a d i o l a b e l e d intracellular materials (2^) . T h i s means t h a t t h e s e biochemical p r o c e s s e s w e r e n o t a f f e c t e d by b u t a m i p h o s at a c o n c e n t r a t i o n 25 t i m e s g r e a t e r t h a n t h a t w h i c h inhibited c e l l population increase nearly completely. E f f e c t s o f b u t a m i p h o s on b i o s y n t h e t i c p r o c e s s e s w e r e examined. B i o s y n t h e s e s w e r e m e a s u r e d by incorporation o f an a p p r o p r i a t e radiolabeled precursor i n t o the r e ­ spective fraction. A t 30 and 150 μΜ, b u t a m i p h o s showed s l i g h t e f f e c t s on b i o s y n t h e s i s o f c e l l w a l l m a t e r i a l and RNA a f t e r a 6 0 - m i n t r e a t m e n t (2). However,

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b u t a m i p h o s h a d a l m o s t no i n h i b i t o r y e f f e c t a t 6 μΜ, t h e c o n c e n t r a t i o n at which the c e l l p o p u l a t i o n i n c r e a s e was a l m o s t c o m p l e t e l y i n h i b i t e d . E f f e c t s of 24-h t r e a t m e n t w i t h 6 μΜ o f b u t a m i p h o s on b i o s y n t h e t i c p r o ­ c e s s e s were s t u d i e d . B u t a m i p h o s h a d no i n h i b i t o r y e f f e c t s on b i o s y n t h e s e s o f p r o t e i n , l i p i d , c e l l w a l l p o l y s a c c h a r i d e , RNA,and DNA on a p e r c e l l b a s i s . I n s t e a d , p e r c e l l b i o s y n t h e s e s t e s t e d were s t i m u l a t e d 160 t o 300% (2). D u r i n g the course of the i n v e s t i g a ­ t i o n , a C h l o r e l l a c u l t u r e was a c c i d e n t a l l y contami­ n a t e d by b a c t e r i a . B a c t e r i a were a c t i v e l y p r o p a g a t i n g at the b u t a m i p h o s c o n c e n t r a t i o n where C h l o r e l l a cell p o p u l a t i o n i n c r e a s e was c o m p l e t e l y i n h i b i t e d . In order to c o n f i r m t h i s o b s e r v a t i o n , e f f e c t s of butami­ p h o s on a p u r e c u l t u r e o f E s c h e r i c h i a c o l i was examined as a m o d e l b a c t e r i u m . E . c o l i was more t h a n 150 times more r e s i s t a n t t o b u t a m i p h o s t h a n C h l o r e l l a (2^) . U n d e r t h e s e c i r c u m s t a n c e s , i t seemed p o s s i b l e t h a t b u t a m i p h o s a c t e d on a p r o c e s s p e c u l i a r t o e u k a r y o t i c cell division. In o r d e r t o f u r t h e r t e s t the e f f e c t o f butamiphos on C h l o r e l l a , a s y n c h r o n i z e d c u l t u r e was u s e d . In the c e l l c y c l e o f C h l o r e l i a , a young c e l l grows i n b i o m a s s . A f t e r n u c l e a r d i v i s i o n s , t h e c e l l d i v i d e s t o g i v e an e v e n number o f d a u g h t e r c e l l s . The d a u g h t e r c e l l s r e p e a t t h e same c y c l e . I t t a k e s a b o u t 24 h f o r one complete c e l l c y c l e . In a s y n c h r o n i z e d c u l t u r e , i n d i ­ v i d u a l c e l l s i n the whole p o p u l a t i o n are s y n c h r o n i z e d w i t h r e s p e c t t o t h e i r c e l l s t a g e s so t h a t t h e y grow i n b i o m a s s and u n d e r g o c e l l d i v i s i o n a p p r o x i m a t e l y simul­ taneously. S y n c h r o n i z a t i o n c a n be a c c o m p l i s h e d quite e a s i l y by c u l t u r i n g C h l o r e l l a a t 24 C i n an 18-h light and a 6-h d a r k p e r i o d (3) . Using a s y n c h r o n i z e d c u l t u r e , the e f f e c t of b u t a m i p h o s on c e l l d i v i s i o n was s t u d i e d ( F i g u r e 2 ) . I n t h e u n t r e a t e d c o n t r o l , t h e c e l l number was constant u n t i l c e l l d i v i s i o n s t a r t e d a t 18 h and t h e n t h e c e l l number i n c r e a s e d a t an a b r u p t l y r a p i d r a t e . The t o t a l volume o f the c e l l p o p u l a t i o n i n c r e a s e d c o n t i n ­ uously throughout t h e c e l l c y c l e as t h e i n d i v i d u a l c e l l s grew i n b i o m a s s . The a v e r a g e v o l u m e p e r cell g r a d u a l l y i n c r e a s e d u n t i l c e l l d i v i s i o n s t a r t e d , and t h e n i t became a b r u p t l y s m a l l e r . This pattern i s c h a r a c t e r i s t i c of a synchronized c u l t u r e . I f butami­ phos s p e c i f i c a l l y i n h i b i t s the c e l l d i v i s i o n p r o c e s s w i t h o u t a f f e c t i n g o t h e r s y n t h e t i c p r o c e s s e s s u c h as r e s p i r a t i o n and p h o t o s y n t h e s i s , t h e n t h e c e l l number s h o u l d be c o n s t a n t t h r o u g h o u t the c e l l c y c l e , the t o t a l c e l l volume o f the p o p u l a t i o n s h o u l d i n c r e a s e c o n t i n u ­ o u s l y (as i n t h e c a s e o f t h e u n t r e a t e d c o n t r o l ) , and

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3 Figure 1.

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch013

BUTAMIPHOS ( S-28A6 )

Chemical structure of butamiphos.

BUTAMIPHOS AND CELL CYCLE

t i m e i n hr

Figure 2. Effect of butamiphos on cell division of Chlorella grown in synchro­ nized culture. Butamiphos in methanol was added to the culture after 6 h. Aliquots were taken for the measurement of cell number and packed cell volume at various time intervals. The dark period was between the 18th and the 24th h. Key: O , untreated control; and Φ, butamiphos-treated (30 μΜ).

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the a v e r a g e volume p e r c e l l s h o u l d i n c r e a s e i n a cont i n o u s manner. As shown h e r e , t h e b u t a m i p h o s - t r e a t e d c e l l s b e h a v e d e x a c t l y as e x p e c t e d ( F i g u r e 2 ) . E f f e c t o f b u t a m i p h o s on a v e r a g e DNA content per c e l l was e x a m i n e d by u s i n g s y n c h r o n i z e d c u l t u r e s . The average DNA c o n t e n t p e r c e l l t r e a t e d a t 30 μ Μ f o r 24 h as m e a s u r e d by a m o d i f i c a t i o n o f S c h m i d t - T h a n n h a u s e r s p r o c e d u r e (3) became 3.9 t i m e s g r e a t e r t h a n t h a t o f the u n t r e a t e d c o n t r o l . T h i s seemed r e a s o n a b l e b e c a u s e one u n t r e a t e d c e l l y i e l d e d 4 d a u g h t e r c e l l s a f t e r division. This i n d i c a t e s that butamiphos d i d not act on t h e DNA d u p l i c a t i o n step. I t seemed l i k e l y t h a t b u t a m i p h o s a c t e d on a c e r t a i n p r o c e s s o c c u r r i n g a f t e r DNA synthesis. I f b u t a m i p h o s a c t s on a s p e c i f i c step i n the c e l l d i v i s i o n p r o c e s s , then there would e x i s t a butamiphos-sensitive p e r i o d t h a t s h o u l d be t r a n s i e n t in nature. Treatment of c e l l s with butamiphos before t h i s p e r i o d would b l o c k the c e l l c y c l e , but the t r e a t ­ ment a f t e r t h i s p e r i o d w o u l d a l l o w the c y c l e t o p r o ­ c e e d u n t i l t h e c e l l s come t o t h e butamiphos-sensitive p e r i o d i n the n e x t c y c l e . I n F i g u r e 3, t h e e f f e c t o f butamiphos treatment at d i f f e r e n t times i n the cell cycle i s given. When c e l l s w e r e t r e a t e d w i t h b u t a m i ­ p h o s a t t h e 6 t h , 1 0 t h , o r 1 5 t h h, c e l l d i v i s i o n was com­ pletely inhibited. When c e l l s w e r e t r e a t e d a t t h e 1 7 t h h , a b o u t 10% o f t h e c e l l s d i v i d e d . When t r e a t e d a t t h e 1 8 t h h , more t h a n 50% o f t h e c e l l s d i v i d e d . This i n d i c a t e d a butamiphos-sensitive p e r i o d ended a r o u n d t h e 1 7 t h t o 1 8 t h h. According to e l e c t r o n microscopic s t u d i e s by T a k a b a y a s h i et_ a l . (4) , m i t o s i s of C h l o r e l l a e l l i p s o i d e a s t a r t s around the 16th h. This approximately c o i n c i d e s w i t h the butamiphossensitive period. The a b o v e o b s e r v a t i o n l e d t o an e x p e r i m e n t t o a s c e r t a i n i f b u t a m i p h o s a c t e d d i r e c t l y on m i t o s i s . Unfortunately, C h l o r e 11a was n o t an a p p r o p r i a t e system t o s t u d y m i t o s i s by o p t i c a l m i c r o s c o p y . Therefore, i t s e e m e d d e s i r a b l e t o use a h i g h e r p l a n t s y s t e m . O n i o n r o o t t i p t i s s u e was s e l e c t e d for t h i s purpose. A t y p i c a l d i v i s i o n f i g u r e found i n butamiphos-treated t i s s u e s was t h a t , u n l i k e the u n t r e a t e d c e l l s , chromo­ somes were n o t a l i g n e d a l o n g t h e e q u a t o r i a l p l a t e , b u t d i s p e r s e d i n the c y t o p l a s m , g i v i n g r i s e to a s o - c a l l e d a r r e s t e d metaphase (5). I t i s w e l l known t h a t c o l c h i ­ c i n e i n h i b i t s m i t o s i s a t the metaphase. Butamiphost r e a t e d o n i o n c e l l s h a d t h e same p a t t e r n o f m i t o t i c i n h i b i t i o n as c o l c h i c i n e - t r e a t e d c e l l s u s e d as a reference (_5) . B u t a m i p h o s - t r e a t e d t i s s u e s w e r e com­ p a r e d w i t h the u n t r e a t e d c o n t r o l w i t h r e s p e c t t o p e r ­ centage d i s t r i b u t i o n of m i t o t i c c e l l s i n each m i t o t i c 1

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z -ι

uj

I

ι

5

10

ι

ι

15 20 TIME IN HR

ι

I

24

5

Figure 3. Effect of butamiphos (30 μΜ) on cell division of Chlorella grown in synchronized culture and treated at different time intervals. Butamiphos was added at one of the times indicated by arrows. Aliquots were taken for the measurement of cell number at various time intervals. The dark period was between the 18th and 24th h. Key: O , untreated control; · , treated at the 18th h; A > treated at the 17th h; and U> treated at the 6th, 10th, or 15th h.

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phase. In t h e u n t r e a t e d c o n t r o l , c e l l s i n the p r o ­ phase p l u s metaphase a c c o u n t e d f o r a b o u t 60%, and those i n the anaphase p l u s t e l o p h a s e f o r about 40%. On t h e o t h e r h a n d , i n t h e b u t a m i p h o s - t r e a t e d t i s s u e s , c e l l s i n the p r o p h a s e p l u s metaphase a c c o u n t e d f o r more t h a n 99%, and a n a p h a s e p l u s t e l o p h a s e c e l l s were nil (_5) . The r e d u c t i o n i n p e r c e n t a g e o f a n a p h a s e p l u s telophase c e l l s was a t t r i b u t e d to the i n c r e a s e i n p e r c e n t a g e o f a r r e s t e d metaphase c e l l s . I t i s known t h a t i f c o l c h i c i n e - t r e a t e d metaphase c e l l s are subjec­ t e d t o c e n t r i f u g a t i o n a t 1500 χ g f o r 5 min, chromo­ somes move i n t h e d i r e c t i o n o f c e n t r i f u g a l f o r c e , because s p i n d l e f i b e r s are broken (6). Butamiphost r e a t e d c e l l s were s u b j e c t e d t o t h i s c e n t r i f u g a t i o n experiment. As e x p e c t e d , c h r o m o s o m e s moved i n t h e d i r e c t i o n of c e n t r i f u g a l force suggesting that butami­ p h o s i n h i b i t e d m i t o s i s a t t h e m e t a p h a s e by d i s r u p t i n g s p i n d l e f i b e r s (_5) . Wanka r e p o r t e d t h a t c o l c h i c i n e p r e v e n t e d cell d i v i s i o n in synchronized c u l t u r e o f a C h l o r e 11a s p e c i e s (7)· c e l l s became p o l y p l o i d w i t h regard t o t h e i r DNA content. RNA and p r o t e i n s y n t h e s e s were not i n h i b i t e d . The t a r g e t o f c o l c h i c i n e a c t i o n was considered t o be s h o r t d e v e l o p m e n t a l s t a g e s d u r i n g successive nuclear duplication steps. Compared w i t h o u r r e s u l t s , t h e r e seems t o be a s t r i k i n g s i m i l a r i t y i n t i m e and mode o f a c t i o n b e t w e e n b u t a m i p h o s and c o l c h i c i n e , although t h e i r chemical s t r u c t u r e s are entirely different. S i m i l a r i t y i n mode o f a c t i o n was a l s o o b s e r v e d i n the o n i o n r o o t apex where b u t a m i p h o s b l o c k e d m i t o s i s a t t h e m e t a p h a s e by d i s r u p t i n g t h e spindle apparatus. The o v e r a l l r e s u l t s i n d i c a t e t h a t butamiphos i s a h i g h l y s p e c i f i c i n h i b i t o r of m i t o s i s . I t d o e s n o t a c t on r e s p i r a t i o n , membrane p e r m e a b i l i t y , or major b i o s y n t h e t i c processes. T

n

e

Chlorpropham C h l o r p r o p h a m i s i s o p r o p y l i n - c h l o r o c a r b a n i l a t e as shown i n F i g u r e 4. A number o f r e p o r t s h a v e a p p e a r e d concerning biochemical responses of higher p l a n t s to chlorpropham. C h l o r p r o p h a m was reported to i n h i b i t m i t o s i s i n o n i o n r o o t (60 . Mann e_t a l . r e p o r t e d that chlorpropham i n h i b i t e d p r o t e i n synthesis in b a r l e y c o l e o p t i l e s and s e s b a n i a h y p o c o t y l s (9_) . M o r e l a n d ejt a l . o b s e r v e d t h a t c h l o r p r o p h a m i n h i b i t e d t h e b i o s y n t h e s i s o f RNA and p r o t e i n i n s o y b e a n h y p o ­ c o t y l s and RNA s y n t h e s i s by c o r n m e s o c o t y l s (10). R o s t and B a y e r r e p o r t e d t h a t s y n t h e s i s o f p r o t e i n was r e d u c e d b e f o r e DNA a n d RNA s y n t h e s i s were i n h i b i t e d i n p e a r o o t (11) . L o t l i k a r e_t a_l. f o u n d t h a t

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chlorpropham i n h i b i t e d o x i d a t i v e p h o s p h o r y l a t i o n i n cabbage m i t o c h o n d r i a ( 1 2 ) . S t . John r e p o r t e d t h a t c h l o r p r o p h a m r e d u c e d t h e in_ v i v o l e v e l o f ATP i n C h l o r e l l a s o r o k i n i a n a ( 1 3 ) . The i n v e s t i g a t o r s c o n ­ ducted these studies using d i f f e r e n t plant tissues and d i f f e r e n t c o n c e n t r a t i o n s o f c h l o r p r o p h a m . Because c h l o r p r o p h a m i s known t o i n h i b i t c e l l d i v i s i o n , i t i s necessary to i d e n t i f y the r e l a t i v e c o n t r i b u t i o n s of these b i o c h e m i c a l e f f e c t s to i n h i b i t i o n o f c e l l divi­ sion. Does c h l o r p r o p h a m d i r e c t l y a f f e c t o x i d a t i v e p h o s p h o r y l a t i o n , which l e a d s t o i n h i b i t i o n o f c e l l division? Does i t d i r e c t l y a f f e c t m a j o r b i o s y n t h e t i c p r o c e s s e s , which lead to i n h i b i t i o n o f c e l l division? Or, d o e s i t d i r e c t l y a c t on a c e l l d i v i s i o n p r o c e s s w i t h o u t a f f e c t i n g e n e r g y m e t a b o l i s m and b i o s y n t h e s e s ? In o r d e r t o make d i r e c t c o m p a r i s o n s among t h e s e b i o ­ c h e m i c a l e f f e c t s o f c h l o r p r o p h a m , C h l o r e 11a was u s e d a g a i n as a m o d e l s y s t e m . E f f e c t s o f c h l o r p r o p h a m on t h e c e l l c y c l e was s t u d i e d (_3) ( S e e F i g u r e 5) . Cell population increase was c o m p l e t e l y s u p p r e s s e d by c h l o r p r o p h a m a t 14.4 μΜ w h i c h was 3.6 t i m e s t h e L C ^ ^ c o n c e n t r a t i o n . Average c e l l v o l u m e became c o m p a r a t i v e l y l a r g e r a f t e r one complete c e l l c y c l e . A v e r a g e DNA c o n t e n t p e r c e l l was i n c r e a s e d 3 . 0 - f o l d (_3) · These o b s e r v a t i o n s i n d i c a t e that chlorpropham d i r e c t l y i n h i b i t e d c e l l d i v i s i o n i n Chlorella. E f f e c t s o f c h l o r p r o p h a m on r e s p i r a t i o n w e r e s t u d i e d (_3) . O x y g e n u p t a k e was n o t s i g n i f i c a n t l y a f f e c t e d by c h l o r p r o p h a m a t a c o n c e n t r a t i o n up t o 47 μΜ w h i c h was a b o u t 40 t i m e s t h e QEffects of c h l o r p r o p h a m on b i o s y n t h e s e s o f p r o t e i n , c e l l w a l l p o l y s a c c h a r i d e , RNA, a n d l i p i d w e r e e x a m i n e d ( 3 ) . A t 47 μΜ, b i o s y n t h e s e s o f p r o t e i n a n d c e l l w a l l p o l y ­ s a c c h a r i d e s were s u b s t a n t i a l l y i n h i b i t e d . RNA s y n ­ t h e s i s was n o t a f f e c t e d . A t 4.7 μΜ, a s l i g h t inhibi­ t i o n o f p r o t e i n s y n t h e s i s was o b s e r v e d , b u t an i n h i b i ­ t o r y e f f e c t on c e l l w a l l s y n t h e s i s was n o t o b s e r v e d . S t i m u l a t o r y e f f e c t s on l i p i d s y n t h e s i s w e r e e r r a t i c , a n d , t h e r e f o r e , were n o t p u r s u e d f u r t h e r . Effect of c h l o r p r o p h a m on p r o t e i n s y n t h e s i s was e x a m i n e d a t l o w e r c o n c e n t r a t i o n s ( 2 0 . A t 2.4 μΜ o r l o w e r , b i o ­ s y n t h e s i s o f p r o t e i n was n o t i n h i b i t e d e v e n a f t e r a 24-h t r e a t m e n t . C e l l p o p u l a t i o n i n c r e a s e was inhib­ i t e d 50% a t 1.3 μ Μ . Among t h e b i o s y n t h e t i c p r o c e s s e s s t u d i e d , p r o t e i n s y n t h e s i s was t h e m o s t s e n s i t i v e t o chlorpropham. H o w e v e r , c o n s i d e r i n g t h e LC mentioned a b o v e , i t w o u l d be p r e m a t u r e t o c o n c l u d e t h a t p r o t e i n s y n t h e s i s i s the primary s i t e o f a c t i o n of c h l o r ­ propham. l c

5

13.

Biochemical

SUMIDA AND YosHiDA

Modes of Action

of Herbicides

259

-NH-C-OCH(CH )

3 2

0

Figure 4. Chemical structure of chlor­ propham.

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ch013

CHLORPROPHAM

CHLORPROPHAM AND CELL CYCLE

9

16 time in hr

2A

Figure 5. Effects of chlorpropham on cell division of Chlorella grown in synchro­ nized culture. Chlorpropham in methanol was added after 5 h. Aliquots were taken for the measurement of cell number and packed cell volume at various time inter­ vals. The dark period was between the 12th and 18th h. Key: O, untreated con­ trol; and · , chlorpropham-treated (14.1 μΜ).

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

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260

Conclusions Synchronized cultures of C h l o r e l l a ellipsoidea w e r e u s e d as a m o d e l s y s t e m t o s t u d y b i o c h e m i c a l modes o f a c t i o n o f b u t a m i p h o s and c h l o r p r o p h a m , b o t h o f which are i n h i b i t o r s o f c e l l d i v i s i o n . Cell cycle s t u d i e s showed t h a t b u t a m i p h o s was a h i g h l y specific i n h i b i t o r of mitosis l i k e c o l c h i c i n e . As f o r c h l o r ­ p r o p h a m , p r o t e i n s y n t h e s i s was i n h i b i t e d m o s t sensi­ t i v e l y , b u t a p o s s i b i l i t y was s u g g e s t e d t h a t c h l o r ­ propham c o u l d d i r e c t l y i n h i b i t m i t o s i s w i t h o u t inter­ f e r i n g with p r o t e i n synthesis. I t w i l l be interesting t o a s c e r t a i n how p l a n t m i t o s i s i s a f f e c t e d by h e r b i ­ c i d a l o r g a n o p h o s p h o r a m i d a t e s and c a r b a m a t e s at the m o l e c u l a r l e v e l i n more d e t a i l as w i l l be s t u d i e s i n v o l v i n g i n s e c t i c i d a l organophosphates and c a r b a m a t e s . Acknowledgements The a u t h o r s w i s h t o e x p r e s s t h e i r t h a n k s Sumitomo C h e m i c a l C o . , L t d . f o r p e r m i s s i o n t o t h i s work.

to publish

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Ueda, M. Japan P e s t i c Info. 1975, 23, 23-5. Kohn, G. K. Ed.; "Mechanism of P e s t i c i d e A c t i o n " ; American Chemical S o c i e t y : Washington, DC., 1974; p. 156-68. Sumida, S; Yoshida, R . ; Ueda, M. Plant C e l l P h y s i o l . 1977, 18, 9-16. Takabayashi, A; Nishimura, T . ; Iwamura, T. J. Gen Appl. M i c r o b i o l . 1976, 22, 183-96. Sumida, S . ; Ueda, M. Plant C e l l P h y s i o l . 1976, 17, 1351-4. Lingnowski, Ε. M . ; S c o t t , E. G. Weed Sci. 1970, 20, 267-70. Wanka, F. Arch. M i k r o b i o l . 1965, 52, 305-18. Ashton, F . M . ; C r a f t s , A. S. "Mode of Action of H e r b i c i d e s " ; John Wiley & Sons: New York, 1973; p. 200-20. Mann, J. D . ; Jordan, L . S . ; Day, Β. E. Weeds 1965, 13, 63-6. Moreland, D. E.; Malhotra, S. S . ; Gruenhagen, R. D . ; S h o k r a i i , Ε. H. Weed Sci. 1969, 17, 556-63. Rost, T. L.; Bayer, D . ; Weed Sci. 1976, 24, 81-7. L o t l i k a r , P. D . ; Remmert, L . F.; Freed, V. H. Weed Sci. 1968, 16, 161-5. S t . John, J. B. Weed Sci. 1971, 19, 274-6.

R E C E I V E D September 1 4 , 1 9 8 1 .

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ix001

INDEX

Absorbance changes that show induc­ tion of passive swelling by dinoseb 90/ Absorption, glyphosate 180 Acifluorfen-methyl 131 effect on efflux of radiolabeled substances from cucumber cotyledons 135/ electron micrograph of cells from greened cucumber cotyledons pretreated with 142/, 143/ model for mechanism of action 149/ N-Acylanilide, interaction maps be­ tween a proton and 16/ site of action 20 structure 3* A F M (see Acifluorfen-methyl) Alachlor action 247/ on Chlamydomonas reinhardii.... 248* inhibition 242-249 -treated Chlamydomonas, size distribution 245/, 246/ Algae use 231-260 ΛΓ-AlkyljiV-methylbenzylamines effect on carotene content 170* Alpha helix terminus 31 Amino acid, aromatic, effects of glyphosate 182, 183/, 193-198 a-Aminooxy-/?-phenylpropionic acid 189,190/ effect on P A L activity 191/ effect on soybean axis growth 191/ Aminooxyacetic acid 190/ Amiprophos-methyl 236*, 242 -treated Chlamydomonas, size distribution 243/, 244/ Anisotropic cells 215/ Anthocyanin content, effect of glyphosate 193,194/ Anthraquinone production, glyphosate effect 195,196/ Antioxidants effects 144-145 AOPP (see a-Aminooxy-£-phenylpropionic acid) A P M (see Amiprophos-methyl) Atebrin-phosphorylation ratio 84 ATP generation, interference 84 ATP synthetase 112/

Atrazine binding studies 41, 42/ -resistant biotypes, lipid composi­ tion of chloroplasts isolated from 106* structure 3* -susceptible biotypes, lipid composi­ tion of chloroplasts isolated from 106* Autoradiographic techniques 50, 218 Azidoatrazine 44 C-labeled 31 covalent attachment site 46 14

Β

B-protein complex 11 Barban structure 3* BASF 13 338 application rate effect on ratio of linoleic to linolenic acid 105/ Benzylamines 165, 170* Binding of C-atrazine in presence of photosynthetic inhibitors 12/, 13/, 19/ Binding site(s) associated with inhibition of PS II 23-25 chloroplastic 18 herbicide(s) interactions 31-33 modified by enzyme trypsin 40-43 triazine 39, 43-46 Biochemical effects of glyphosate .175-206 Bioregulation of pigment biosynthesis by onium compounds 153-173 Bioregulators, lipophilic-hydrophilic properties 163 Bioregulators, mode of action of *ran.y-carotenoid 165 Biosynthesis of carotenoids, regulation 153 chlorpropham effect 258 interference with carotene 115-119 pigment, bioregulation by onium compounds 153-173 Biosynthetic processes, effect of butamiphos 253 Bipyridyl herbicides, interaction 63 Bipyridylium salts 113,123

263

14

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264

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

Bleaching Carotene(s) (continued) additional assay on herbicidal content, effect of (continued) mode 125-127 steric position of methyl group in carotenoids and chlorophyll in pres­ diethylaminoethylmethylence of norflurazon or oxyphenylether 164* substituted dibenzylamines 167* fluorfen 116* trialkylamines 155* herbicidal mode of action .111-130, 112/ cyclic, accumulation 159 of 2-phenylpyridazinones, struc­ production, stimulatory effect of ture-activity relationship 118 /?-ionone 153 Blockage of electron transport 29 159-162 Bron-Oppenheimer approximation .... 14 β-Carotenes, inducers 165-171 Butamiphos 252-257 c/s-Carotenes 154-165 action on mitosis 255, 257 *ra«.y-Carotenes structural features for induction 163-165 effect on biosynthetic processes .253, 255 26 effect on cell division 252, 253-255 Carotenoids bleaching in presence of nor­ flurazon or oxyfluorfen 116* C - D P E interactions in vitro 138 Calcium uptake inhibition 221 vs. flavins 139 in light-activating mechanism of Carbamoyl-thiadiazoline structure .... 3* DPE molecule, involvement 137-138 Carbamoyl-l,3,4-thiadiazoline, site of regulation of biosynthesis 153 action 20 Carbanilates 80 c/s-Carotenoid bioregulators, mode of effects on efflux of potassium and action 171 permeability 93* *ra>ty-Carotenoid bioregulators, mode induction of passive swelling 91* of action 165 inhibition of valinomycin-induced Cell(s) swelling 88* cycle, chlorpropham effects 258 effect on reactions mediated by cycle, analysis with Coulter counter 232-238 mung bean mitochondria 85* division 214-223 effect on reactions mediated by effect of butamiphos 253-255 spinach thylakoids 83* disruption 219-223 Carbon dioxide herbicides interfering with 238-242 concentrations 58 effect on Chlamydomonas fixation by flax cotyledons reinhardii 241* previously incubated with inhibition 218-219 monuron 59, 60* by alachlor 242 uptake and evolution by paraquatmethods used to study 214-218 treated flax cotyledons 63, 67/ effects, types 218 Carbon-14 atrazine 11 elongation binding to trypsin-treated herbicide inhibition 212, 213 membranes 41 osmotic concentration 212 binding in the presence of photo­ synthetic inhibitors 12/, 13/, 19/ turgor pressure 212 wall-loosening factor 212 Carbon-14-labeled azidoatrazine 31 water conductivity potential 212 Carotene (s) enlargement 209 biosynthesis disruption 213-214 cell-free systems 117-118 inhibition 209-213 interference with 113, 115-119 internode in Chara 211/ content, effect of -free systems, carotene bio­ aryl and arylaliphatic esters of synthesis 117-118 2-diethylaminoethanol 162* membranes, injury 140-147 dibenzylamine and substituted 211/ dibenzylamines 166* Chara internode cell enlargement 200 diethylaminoalkylbenzenes 156* Chelation studies, glyphosate Chemical-triggered luminescence 7-9 2-diethylaminoethylesters of short chain aliphatic acids 161* Chlamydomonas growth and cell division 233/ furfurylamine analogs 168* oxyfluorfen 120/ Chlamydomonas, herbicides effect .... 237*

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ix001

INDEX

Chlamydomonas size distribution alachor-treated 245/, 246/ of APM-treated 243/, 244/ during dark phase 234/ during light phase 235/ of metribuzin-treated 240/ Chlamydomonas reinhardii, herbicides that interfere with cell division, effect 241* use to identify modes of action .231-250 that interfere with photosynthetic processes, effect 239/ Chlorine addition to phenyl ring 84, 85 Chlorophyll bleaching 59 in presence of norflurazon or oxyfluorfen 116* changes under influence of oxyfluorfen 120/ contents of flax cotyledons 24/ effect of ioxynil 62/ incubated on monuron 61/, 62/ effect of DABCO 66* paraquat-treated, incubated in presence or absence of copper-penicillamine 72/ incubated on water 61/ effect of glyphosate 194/ of leaf sections of Conyza treated with or without paraquat .... 75/ level of paraquat-treated cotyledons, effect of monuron 69/ fluorescence 2 molecules in PS II antenna function 26 Chloroplast (s) of Chlorella pyrenoidosa, fluorescence parameters 4/ cup-shaped 233/ -directed protein synthesis, product 49-50 effects of glyphosate 198-199 experimental 81 gene product 49 with hydroxylamine pretreatment .. 6-7 isolation 39 from atrazine-resistant and atrazine-susceptible biotypes, lipid composition 106* pigment accumulation in wheat shoots, effect of substituted pyridazinones 99* pigment inhibition 99 thylakoid membranes, receptor site for triazine herbicides 37-55 trypsin-treated, photosynthesis inhibitors effect on reducing activity 10* Chloroplastic binding site 18 Chlorosis-inducing compounds 118 Chlorosis induction of necrosis 179

265 Chlorpropham (effects) 242, 257-260 on biosyntheses 258 on cell cycle 258 on respiration 258 Chlorella 254/, 256/, 259/ dark-adapted, luminescence 7 use to identify biochemical modes of herbicidal action 251-260 Chlorella pyrenoidosa, fluorescence parameters on chloroplasts 4/ Chromosomes 227/ abnormal configurations 219 3-CHPC, chemical name 81* CIPC (see Chlorpropham) 3-CIPC, chemical name 81* Club-shape appearance of trifluralintreated lateral root tips 216/ C M U structure 3* CNDO/s semiempirical quantum mechanical method 33 Colchicine 219,255 Common molecular target for inhibitors 2-9 Configuration of chloroplastic binding site 18 Copper-penicillamine, chlorophyll content of paraquat-treated flax cotyledons incubated in presence or absence 72/ Copper-zinc superoxide dismutase .... 58 Corn, DPE injury 136 Cotton seedling, ratio of linoleic to linolenic acid in polar lipids 104* Coulombic isopotential maps 33 Coulter counter with cell cycle analysis 232-238 Coulter counter, use to identify herbicidal modes of action 231-250 Covalent attachment site for azidoatrazine 46 CPTA effect on carotene content 157* Cyclase inhibitor 165 Cytochrome b-559 26 Cytochrome c-553 112/ D Dark phase, size distribution of Chlamydomonas cell during 234/ 3,4-DCIPC, chemical name 81* 2,3-DCIPC, chemical name 81* DCME (see N-Phenylurea) Deamination of phenylalanine 186 by P A L 187/ DABCO (see Diazobicyclooctane) Degradation of glyphosate 180 of lipids 125-127 of polypeptide 44

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ix001

266

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

3-Dehydroquinate synthase 195 Diazobicyclooctane 63 effect on chlorophyll content of flax cotyledons incubated on monuron 66* Dibenzylamines, substituted, effect of dibenzylamine on carotene content 166* Dichlorination 84 Diethylalkylamines 154-158 Diethylaminoalkylbenzene(s) 158 effect on carotene content 156* 2-Diethylaminoethylesters of short chain aliphatic acids, effect on carotene content 161* 2-Diethylaminoethylbenzoates, effect of substitution on carotene content 160* Diethylaminoethylphenylether analogs 158-159 Diethylaminoethylphenylether effect on carotene content 157* Diethylaminoethylmethylphenylether, steric position of methyl group, effect on carotene content 164* 2-Diethylaminoethanol, effect of aryl and arylaliphatic esters on carotene content 162* Difunon structure 114 Dinitroaniline(s), site of action 20 structure 3* Dinitrophenol, interaction maps between a proton and 17/ Dinitrophenol structure 3* 2,4-Dinitrophenols, site of action 20 Dinoseb absorbance changes that show induction of passive swelling .. 90/ chemical name 81* derivative, interaction map 15 effect on rate of reduction of ferricyanide 92/ inhibition 87/ I-Dinoseb structure 3* Dipeptide, conformation 18 Dipeptide and photosynthesis inhibitors, intermolecular interaction between 19/ Diphenyl ether 119-122, 131 -carotenoid interactions in vitro .... 138 effects on phenolics 137 herbicides characterization 134-137 efflux of Rb from cucumber cotyledons in presence .... 133/ structure-activity relationship 132-134 light requirement and membrane disruption 134 86

+

Diphenyl ether (continued) injury to corn 136 injury, symptoms 140 involvement of carotenoids in lightactivating mechanisms 137-138 membrane injury for free radicals .. 144 model of proposed mechanism of action 148 reaction mechanism(s) 139-140 sites of action 20, 131-152 structure 3* Dipole moment of uracil 31 Diuron, chemical name 81* Diuron, dark grown wheat shoots 101* Division figure from trifluralin-treated cotton 227/ DNA content per cell, butamiphos effect.. 255 replication 214 synthesis 218 Dose-response curves 87/ DPE (see Diphenyl ether)

Electron chain carriers 112/ -deviator herbicides 73 microscopic studies of paraquattreated leaves 73 micrographs of cells from greened cucumber cotyledons pretreated with AFM 142/, 143/ cucumber cotyledons 140-141 polymorphic nucleus 226/ photosynthetic transport chain 2 -transfer chain 26 transfers within PS II 26-28 transport chain, electron acceptor sites .... 42/ divertors 63-73 inhibition, effect of side chain length 83 inhibition, photosynthetic 97-99 inhibitors 59, 80, 112 -withdrawing group effect on carotenoid-inducing ability 163 Electronic absorption spectra of plastoquinone 27 Electrophoresis, polyacrylamide gel .. 40 of thylakoid membrane polypeptides 45/ Electrostatic binding of herbicides .... 31 Energy, perturbation 14 Energy-transfer inhibitor 113 5-Enolpyruvylshikimate-3-phosphate synthase, glyphosate inhibition .. 197/

267

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ix001

INDEX

Enzyme (s) of aromatic amino acid synthesis, effects of glyphosate 193-198 inducer 165 of phenolic metabolism, effects of glyphosate 181/ of phenolic pathway in plants 181/ sites of glyphosate inhibition of aromatic amino acid synthesis 183/ Ethane formation in diphenyl ether and norflurazon-treated Scenedesmus 121* generation by flax cotyledons 72/ effect of ioxynil 65/ effect of monuron 64/ light-induced formation 120 Ethylene, light-induced formation .... 120

F

Fluorescence chlorophyll 2 induction measurement 126 parameters on chloroplasts of Chlorella pyrenoidosa, definitions 4/ parameters and inhibition with PS II inhibitors 5* transient 6 Fluorogram of pea chloroplast poly­ peptides tagged with radiolabeled azidoatrazine 48/ Fluorogram showing trypsin-sensitivity of polypeptide 47/, 51/, 53/ Fluorography 40, 44, 50 Fluridone structure 114 Free radical chain reactions 148 in DPE membrane injury 144 lipophilic reactions 140 mechanism 139 scavengers 136 Fresh weight increase, effect of glyphosate 194/ Fruit, citrus, recessive gene 171 Furfurylamine analogs effect on carotene content 168*

Fatty-acid composition of galactolipids from wheat shoots, effect of substituted pyridazinones 100* phosphatidylcholine of wheat roots 103* and phosphatidylethanolamine 103* polar lipids from light 101* G distribution in chloroplast lipids .... 106 polyunsaturated 144 Galactolipids, pyridazinone action 100-101, 100* peroxidation 121 Gases, hydrocarbon, origin 125 in thylakoids of blue-green algae, concentration 126* Gene ω-3 polyunsaturated, peroxidation .. 124/ derepression 171 FCCP induction 165 product, chloroplast 49 chemical name 81* Generation of singlet oxygen 58 effects Genetic mechanisms 49-50 on efflux of potassium and 136 permeability 93* Glutathione destruction Glyphosate (s) on reactions mediated by spinach absorption and translocation 180 thylakoids 83* analogs, metabolites and/or on reactions mediated by mung degradation products 177/ bean mitochondria 85* biochemical effects 175-206 induction of passive swelling 91* and physiological effects 180-201 inhibition of valinomycin-induced chelation studies 200 swelling 88* chemical structure 177/ Ferricyanide 31 degradation 180 Ferricyanide reduction discovery and development 176-179 by Hill reaction in chloroplasts, effect(s) (on) herbicide inhibition 99* anthocyanin content 194/ I for inhibition 83 fresh weight increase and rate 93 chlorophyll content 194/ effects of dinoseb on increasing .. 92/ anthraquinone production 196/ Flagella regeneration 232 chloroplast 198-199 herbicidal effect 237* enzymes of aromatic amino acid Flavins vs. carotenoids 139 synthesis .193-198 Fluidity of membranes, effect of enzymes of phenolic metabolism 184* inhibitory uncouplers on 86-89 50

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ix001

268

Glyphosate(s) (continued) effect(s) (on) (continued) membrane transport 200 PAL 186-192 physiological 180-201 respiration 200 shikimate accumulation 196/ soybean axis growth 191/ ultrastructural analysis 199-200 general characteristics and properties 178* half-life 179 inhibition of 5-enolpyruvylshikimate-3-phosphate synthase .... 197/ metabolic effects 200 nonbiochemical consideration ...179-180 specificity, effect on P A L 193 Glyphosine 176,198 Grapefruit and other citrus, recessive gene 171 Growth inhibition induced by herbicides 207-230 Green plant photosystems 24-26 H Haber-Weiss type reactions 68 Haemanthus katherinae endosperm cells, propham-treated 222/ Halogenated hydroxybenzonitriles, site of action 20 Herbicidal activity requirements 28 activity sites Ill mode of bleaching 111-130 addition assays 125-127 peroxidations 127 Herbicide (s) action (of) 23 mechanism 28-30 sites 112/ binding 24 assays 40 polypeptide, step-wise modification 46-49 proteins 30-31 site interactions 31-33 loss 41 modified by enzyme trypsin .40-43 with bleaching activity, chemical formulas 113/, 114/ cellular compartmentation 147 chemical names 236* effect on Chlamydomonas growth .. 237* Chlamydomonas reinhardii and Coulter counter 231-250 effect of herbicides that interfere with cell division on 241*

Herbicide(s) (continued) Chlamydomonas reinhardii (continued) effect of herbicides that interfere with photosynthetic processes on 239* use to identify herbicidal action modes 231-250 Chlorella use to identify biochemical modes of action 251-260 diphenyl ether, characterization .134-137 use to disrupt membrane structure 209 DPE efflux of Rb from cucumber cotyledons in presence .... 133/ light requirement and membrane disruption 134 site(s) of action 131-152 electron deviator 73 -glycoside complexes 136 glyphosate 176 growth inhibition, induced 207-230 growth inhibitor 210/ incorporation of radioactivity in presence 117* -induced peroxidations 119-125 inhibition of cell enlargement and elongation 212, 213 inhibition of ferricyanide reduction by Hill reaction in chloroplasts 99* interaction with cellular and liposome membranes 79-96 interference with cell division ...238-242 interference with photosynthesis .... 238 effect on lipid composition of plant membranes 97-109 photosynthetic deviator 75/ photosynthetic inhibitor 75/ role of light and oxygen in action 57-77 selected effects on efflux of potassium and permeability 93* induction of passive swelling 91* inhibition of valinomycininduced swelling 88* reactions mediated by mung bean mitochondria 85* reactions mediated by spinach thylakoids 83* selectivity and resistance 30 structure-activity relationships .132-134 triazine, receptor site in chloroplast thylakoid membranes 37-55 Hill reaction in chloroplasts, herbicide inhibition of ferricyanide reduction 99* Hydrocarbon gases, origin 125 86

+

INDEX

269

Inhibitor (continued) photosynthetic (continued) herbicides, role of light and oxygen in action 57-77 with a simulated peptidic target .18-20 -triggered luminescence in Chlorella 8/ of PS II-catalyzed electron trans­ port, interaction 14 Inhibitory uncouplers (effect of) 30, 80 fluidity 86-89 permeability 86-89 swelling responses 86-89 Inhibitory herbicides 28 Interaction, intermolecular, between dipeptide and photosynthesis inhibitors 19/ I Interactions map(s) 14—18 I for inhibition of coupled phos­ dinoseb derivative 15 phorylation 83 nitrofen 15 I for inhibition of ferricyanide between a proton reduction 83 and a dinitrophenol 17/ Incorporation of C-labeled pre­ and nitrofen 17/ cursors into α-amino levulinic a triazinone 17/ acid 199* Intermolecular interaction between di­ Inducers of /^-carotenes 159-162 peptide and photosynthesis Induction of *ram-carotenes, struc­ inhibitors 19/ tural features 163-165 Internode cell enlargement in Chara .. 211/ Inhibition β-Ionone stimulatory effect on caro­ by alachlor 242-249 tene production 153 of carotene biosynthesis 113, 116 Isopotential curves 14-18 of cell division 218-219 Isopropyl m-chlorocarbanilates (see of cell enlargement 209-213 Chlorpropham) chloroplast pigment 99 Isotropic cell(s) 215/ by dinoseb 87/ enlargement 214, 216/ of electron transport, effect of Ioxynil 139 side chain length 83 chemical name 81* growth, induced by herbicides ...207-230 effect on chlorophyll content of flax photosynthetic electron transport .97-99 cotyledons 62/ of PS II, binding sites associated .23-35 effect on pigment content of flax Inhibition of valinomycin-induced cotyledons 65/ swelling by selected herbicides .. 88* and ethane generation 65/ Inhibitor structure 3* chemical families 2-9 electron transport 59, 82 Κ energy-transfer 113 photosynthesis K permeability 86 effect on reducing activity of trypsin-treated chloroplasts 10* L interferences with chloroplastic membrane protein 10-14 Lenacil structure 3* intermolecular interaction be­ LHC (see Light-harvesting complex) tween dipeptide 19/ Light photosynthetic -activating mechanism(s) for binding of C-atrazine in DPE 137-140 presence 12/, 13/, 19/ -dependent quenching, inhibition .. 82 -harvesting complex 50 comparison between half-effect values of luminescence -induced formation of ethane and data and p l 9* ethylene 120

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ix001

Hydrocarbon production by spheroplasts in presence of Cu(II) 126* Hydrogen bonding, triazine molecule 15 Hydrogen peroxide generation 58 Hydrophobic environment 31 Hydroxyl free radical(s) 58, 73 Hydroxybenzonitrile structure 3* Hydroxybenzonitriles, halogenated, site of action 20 Hydroxyphenolics, P A L activity during glyphosate treatment 188/ Hydroxylamine conversion to nitrite 68 nitrite formation 70/ pretreatment of chloroplasts with .. 6-7 Hydroxyurea 218 50

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BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

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270

Light (continued) and oxygen role in action of photo­ synthetic inhibitor herbicides 57-77 phase, size distribution of Chlamydomonas during 235/ requirement and membrane disrup­ tion, DPE herbicides 134 role in action of photosynthetic inhibitor herbicides 57-77 Linoleic to linolenic acid ratio in polar lipids of cotton seedlings .. 104* Linoleic reactions of lipoxygenase 144 α-Linolenic acid, peroxidation 124 α-Linolenic acid, mechanism for degradation 123 Linolenic acid content 102, 107 Linolenic to linoleic acid, BASF 13 338 application effect on ratio .. 105/ Lipid (s) analyses 140-141 composition 101* of chloroplasts isolated from atrazine-resistant and atrazine-susceptible biotypes .... 106* of plant membranes, effects of herbicides 97-109 and triazine resistance 104-108 of cotton seedlings, linoleic to linolenic acid ratio 104* degradation 125-127 free radicals 59 hydroperoxides 59 peroxidation reactions, consequences 63 peroxide decomposition products 141-144 Lipophilic free radical reactions 140 free radical scavenger 150 -hydrophilic properties of bio­ regulators 163 Liposomes 82 Lipoxygenase reactions with linoleic acid 144 Luminescence, chemical-triggered 7-9 Luminescence of dark-adapted Chlorella 7 Lycopene accumulation 154

M Malonyl dialdehyde precursors 141 MDA (see Malonyl dialdehyde) Mechanism for degradation of α-linolenic acid 123 Mechanism of herbicidal action 28-30 Membrane(s) cell injury 140-147 chloroplast thylakoid, receptor site for triazine herbicides 37-55

Membrane(s) (continued) degradation 119 disruption, DPE herbicides 134 interaction of herbicides with cellular and liposome 79-96 permeability to ions 89-94 permeability measured as potassium leakage from paraquat-treated flax cotyledons 74/ transport, glyphosate effect 200 Metabolic effects of glyphosate ...178*, 200 Metabolic pathway affected by herbicides 237* Metabolism, effects of glyphosate on enzymes of phenolic 184* Metabolism of herbicide 28 Metribuzin 238 -treated Chlamydomonas, size distribution 240/ structure 3* MGDG to total phospholipids, ratio .. 108* Microtubules 222/ organizing center 221 Mitochondria, experimental 81, 82 Mitochondria responses 84-86 Mitosis 214 butamiphos action 255, 257 Mitotic disruption mechanism 221 index 218 inhibitors 218 Mode of action of *rû«5-carotenoid bioregulators ... 165, 171 of herbicidal bleaching 111-130 in wheat shoots 101-103 Model of proposed mechanism of DPE action 148 Molecular weight of herbicide-binding proteins 24 Molecular weight of polypeptide in PS II 31 Monuron, effect on carbon dioxide fixation 60* chlorophyll content 61/, 62/ chlorophyll level of paraquattreated 69/ of DABCO on chlorophyll content of flax cotyledons incubated with 66* formation of nitrite from hydroxylamine 71* on pigment content and ethane generation 64/ Multinucleate cell occurrence 223-224 Multipole spindle apparatus 222/ Ν +

NADP reduction Necrosis

24 179

INDEX

271

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Neurosporene 158 Nitrene formation 44 Nitrite formation from hydroxylamine 68, 70/ Nitro group, effect behavior of compounds toward proton 15 Nitrofen 11,113,119 addition to isolated spinach chloroplasts 122 interaction map(s) 15 between a proton 17/ (diphenyl ether) structure 3* Nitrophenols 30 p-Nitrophenylethers 113, 119 Noncompetitive inhibition 43 Norflurazon inhibitory site 117 Norflurazon structure 114 Ο

Octanol-water partition coefficient .... 163 Oleosomes 140-141 Onium compounds, bioregulation of pigment biosynthesis 153-173 Ortho substitution 84 Osmotic concentration, cell elongation 212 properties of membranes 86 swelling 82 Oxidative phosphorylation, uncouplers 84 Oxyfluorfen effect on carotenoid content 120/ effect on chlorophyll content 120/ structure 114 Oxygen, role in action of photo­ synthetic inhibitor herbicides 57-77 Ρ

P680 26,112/ P700 112/ P A L (see Phenylalanine) ammonia-lyase) Paraquat 123,238 ability to promote formation of nitrite from hydroxylamine .... 71* reduction 63 structure 114 -treated cotyledons, carbon dioxide uptake and evolution 67/ -treated cotyledons, effect of copper-penicillamine on chlorophyll content 72/ -treated cotyledons, effect of monuron on chlorophyll level 69/ Particle number 247/ Partition coefficient, 1-octanol-water 154 PCILO method 18 PCP (see Pentachlorophenol)

Peel damage 163 Pentachlorophenol 236* Permeability effects of FCCP, selected herbi­ cides, and carbanilates 93* effect of inhibitory uncouplers 86-89 membrane, to ions 89-94 Peroxidation of α-linolenic acid 124* herbicide-induced 119-125 of polyunsaturated fatty acid 121 as secondary herbicidal effects 127 Perturbation energy 14 Phenol-type herbicides 43 Phenolics effects on DPE 137 metabolism disruption 180 metabolism, effect of glyphosate .... 189* pathway in plants 181/ L-Phenylalanine 193 Phenylalanine ammonia-lyase 137 activity during glyphosate treatment 188/ deamination 1186, 187/ effect of AOPP activity 191/ effects of glyphosate 186-192, 187/ specificity of glyphosate's effect .... 193 inhibitors 189, 190/ N-Phenylcarbamate(s), site of action .. 20 ΛΓ-Phenylcarbamate, structure 3* Phenylpyridazinones 112 2-Phenylpyridazinones, structureactivity relationship, bleaching .. 118 N-Phenylurea(s) interaction maps between a proton 16/ site of action 20 structure 3* -type inhibitors 2 radiolabeled competition studies 11-14 site of action 11 Pheophytin 26 Phosphatidylcholine, fatty-acid composition 103* Phosphatidylethanolamine, fatty acid composition 103* Phospho-2-oxo-3-deoxyheptonacetaldolase 195 Phospholipid composition for wheat roots 103* Phospholipids, total ratio of MGDG .. 108* Af-Phosphonomethyl glycine (see Glyphosate) Phosphorothioamidate herbicide 252 Phosphorylation, coupled effect of side chain length 83, 85 Phosphorylation, coupled, I for inhibition 83 Photoaffinity labels 31,39 Photoaffinity probe 44 Photochemical quenching capacity ...2, 4-6 50

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

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272

Photochemistry, photosystem II 26-28 Pigment electron transfers within 26-28 bleaching 113 Photooxidation 102 content of flax cotyledons Photorespiration 58 and ethane generation 65/ Photosynthesis effect of monuron 64/ glyphosates effects 198 effect of ioxynil 65/ inhibition, chloroplast 99 herbicides interfering with 238 molecule 26 inhibitors effect on reducing activity of trypsin-treated Plant death 73 181/ chloroplasts 10* Plants, phenolic pathway Plastocyanin 112/ inhibitors interferences with a Plastoquinone(s) 26, 112/ chloroplastic membrane chemical structure 25/ protein 10-14 electronic absorption spectra 27 Photosynthetic in PS II complex 24 efficiency 108 Polyacrylamide gel electrophoresis electron transport and fluorography 40 chain 2 inhibition 97-99 Polyacrylamide gel electrophoresis of thylakoid membrane with PS II inhibitors 5* polypeptides 45/ deviator herbicides 75/ Polymorphic nucleus 225/ inhibitor(s) electron micrograph 226/ binding of C-atrazine in presence 12/, 13/, 19/ -3 Polyunsaturated fatty acid perooxidation 124/ comparison between half-effect values of luminescence data Polypeptide(s) degradation 44 and ρΙ · 9* polyacrylamide gel electrophoresis herbicides 75/ of thylakoid 45/ role of light and oxygen step-wise modification of herbicidein action 57-77 binding 46-49 with a simulated peptidic target 18-20 tagged with radiolabeled azidoatra-triggered luminescence in zine, fluorogram 48/ Chlorella 8/ Polyunsaturated fatty acid(s) 132, 144 processes, interference by in thylakoids of blue-algae, herbicides 239* concentration 126* reactions 40 Potassium, effects of FCCP, selected Photosystem II herbicides, and carbanilates on binding sites associated with efflux 93* inhibition 23-35 182 diameter 26 Prephenate dehydrogenase 81* inhibitor (s) 1-21 Propanil, chemical name Propanil structure 3* -catalyzed electron transport 221 interaction with 14 Propham -treated Haemanthus katherinae with chlorophyll 5* endosperms cells 222/ with fluorescence parameters .... 5* Prophase division 220/ with photosynthetic electron transport 5* Protein(s) binding 112/ photochemistry and electron herbicide-binding 30-31 transfer within 26-28 molecular weight 24 primary electron acceptor, indirect synthesis effect of chlorpropham .... 258 measurements of effects 2-6 triazine receptor 49-50 redox activity 113 Proteolysis 46 secondary electron acceptor, B, direct interferences 6-7 PS II (see Photosystem II) Photosystems, green plant 24-26 PUFA (see Polyunsaturated fatty acid) 3* Physiological effects, glyphosate ... 181-201 Pyramin (pyridazinone) structure Phytoene synthetase 117 Pyridazinone(s) action on galactolipids 100-101, 100* Phytotoxic symptoms 59 differential sensitivity 103-104 Phytotoxicity of glyphosate 179-180 effect on chloroplast pigment Pi-pi complex 31 accumulation 99* between uracil and a salt model ... 32/ 14

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INDEX

273

Pyridazinone(s) (continued) inhibition site of action structure substituted

97 20 3t 98/

Quantum mechanical method, CNDO/s semiempirical 33 Quenching, photochemical, capacity .2,4-6 Quenching singlet oxygen 58, 116

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ix001

R Radioactive amino acid incorporation 39 Radioactivity in presence of herbicides 117/ Radiolabeled photoaffinity triazine probe 46 Radiolabeled substances from cucumber cotyledons, effect of A F M on efflux 135/ Rb from cucumber cotyledons, efflux in presence of DPE herbicides 133/ Rb from cucumber cotyledons treated with A F M in α-tocopherol 146/ Reaction mechanism(s) of DPE ...139-140 Recessive gene in grapefruit and other citrus 171 Receptor herbicide 43-46 protein Β complex 20 protein, triazine 49-50 site for triazine herbicides in chloroplast thylakoid membranes 37-55 Redox activity, PS II 113 Redox potential, effect of herbicide binding 29 Reduction of ferricyanide, effects of dinoseb 92/ Reduction of NADP 24 Resistance mechanism(s) 134-137 trait 108 triazine, and lipid composition 104-108 Respiration, chlorpropham effect 260 Respiration effects of glyphosate 200 Respiratory uncoupler 232 Riboflavin 139 Rieske-type protein 112/ R N A synthesis inhibition 218 Root development, lateral, in peas 216/ lateral, zone of elongation 215/ tip from trifluralin-treated cotton 225/ 86

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Root (continued) tip from trifluralin-treated plant R U 21731 structure

215/ 3t

S

S-2846 (see Butamiphos) Salt bridge 31 Salt model, pi-pi complex between uracil and 32/ Scendesmus, ethane formation in diphenyl ether-treated 121* Scendesmus, ethane formation in norflurazon-treated 121* SchhTs base-type reactions 148 Seedling growth inhibition 180 Sensitivity, differential, to pyridazinones 103-104 Shikimate accumulation 195 effect on glyphosate 196/ Shikimate conversion to anthranilate .. 195 Silicomolybdic acid 7 Singlet oxygen quenchers 116 Sites of herbicidal action 112/, 130 diphenyl ether 131-152 Size distribution of Chlamydomonas alachlor-treated 245/, 246/ of APM-treated 243/, 244/ during light and dark phase ...234/, 235/ metribuzin-treated 240/ Soybean growth 189 effect of AOPP and glyphosate 191/ Specificity of glyphosate's effect on PAL 193 Spin polarization pattern 27 Spin-trapping experiments 139, 144 Spindle apparatus, multipole 222/ Stability aspects of glyphosate 178* State 4 respiration stimulation 85 Steric effect of methyl group on carotene content 164* Structural features for induction of *ra/is-carotenes 163-165 Structure-activity relationship for bleaching of 2-phenylpyridazinones 118 Structure-activity relationships for DPE herbicides 132-134 Styrene 332 Superoxide 139 anion 123 formation 58, 68 importance in action of herbicides .. 68 Swelling passive, induction by carbanilates 91* dinoseb 90/ FCCP 91* selected herbicides 91* responses of membranes, effect of inhibitory uncouplers 86-89

274

BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

Τ Trypsin (continued) -mediated loss of atrazine affinity .. 41 T B A R M (see Thiobarbituric sensitivity of polypeptide, acid-reacting materials) fluorogram 47/ Thiobarbituric acid-reacting -treated chloroplasts, photosyn­ materials 141 thesis inhibitors effect 10/ Thylakoid -treated membranes, C-atrazine membranes, chloroplast, receptor binding 41 sites for triazine herbicides ...37-55 Turgor pressure, cell elongation 212 membrane polypeptides, poly­ Type 1 reactions 59 acrylamide gel electrophoresis 45/ Type 2 reaction, singlet oxygen 58 protein 49 Tocopherols 136 U α-Tocopherol 26 efflux of Rb from cucumber 147-148 cotyledons treated with 146/ Ultrastructural analysis of glyphosate effects 199-200 protect against A F M injury 145 178/ Topographic probe 40 Uptake of glyphosate Toxicity, glyphosate 178/ Uracil dipole moment 31 Translocation, glyphosate 180 and salt model, pi-pi complex Trialkylamines, effect on carotene between 32/ content 155/ site of action 20 Triazine(s) 112 structure 3/ binding site 39, 43-46 112 herbicides 30 Ureas in chloroplast thylakoid mem­ branes, receptor site 37-55 V interaction maps between a proton and 16/, 17/ Valinomycin 86, 87 resistance, lipid composition 104-108 -induced swelling, inhibition by molecule, hydrogen bonding 15 selected herbicides 88/ probe, radiolabeled photoaffinity .. 46 receptor protein 49-50 W 5-Triazines, sites of action 20 Triazinones 112 (metribuzin) structure 3/ Wall-loosening factor, cell elongation 212 site of action 20 Water conductivity potential, cell elongation 212 Triethylamine bioregulators 171 Water-octanol partition coefficient .... 163 Trifluralin (dinitroaniline) 11,242 formation and inhibition 221 Wheat roots fatty acid composition of phos­ structure 3/ phatidylcholine 103/ -treated fatty acid composition of phosChara plant, apex 216/ phatidylethanolamine 103/ cotton lateral root 220/, 227/ grown in diuron 101/ lateral root tips, club shape phospholipid 103/ appearance 216/ pyridazinones effect on chloroplast lateral root tip from pigment accumulation 99/ pea plant 215/ pyridazinones effect on fatty acid Triplet chlorophyll generation 58 composition 100/ Triplet state(s) 29 separation of modes of action ...101-103 of radical pair (P-580 Pheo) 27 Trypsin digestion 10 Ζ of PS II particles 30, 31 herbicide binding sites modified by 40-43 Zone of elongation in lateral root 215/ 14

Publication Date: February 11, 1982 | doi: 10.1021/bk-1982-0181.ix001

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