Distribution of phenmedipham and other biscarbamates inside leaf fragments of Chenopodium album and Spinacia oleracea plants

Chemosphere, Vol.18, Nos.9/10, Printed in Great Britain

DISTRIBUTION

pp

OF PHENMEDIPHAH CHENOPODIUH

2077-2082,

1989

0045-6535/89 $3.00 Pergamon Press plc

AND OTHER BISCARBAMATES ALBUM AND SPINACIA

INSIDE LEAF FRAGMENTS

OLERACEA

+

OF

PLANTS

by S. Mona (II). J.M. Ducruet(2),

F. Nurit(1).

P. Ravanel(3)

and M. Tissut (I

(I) Laboratoire de Physiologie cellulaire v@g~tale, Universit~ Joseph Fourier BP 53×, 38041 Grenoble c~dex. France. (2) Laboratoire de Biophysique. Centre d'Etudes Nucl~aires de Saclay. 9 1 1 9 1 G i f sur Yvette c~dex. France. (3) Laboratoire de Pharmacoonosie. Universit@ Joseph Fourier. Domaine de La Merci, 38700, La Tronche, France

The

target of the herbicide

PS II level I. Furthermore. phenylureas

for

phenmedipham under

binding

are

conditions

phenmedipham

we have previously on

the

QB

bound

to purified

where

electron

as

other

part

present

shown

by

competition

is associated

study

thylakoids,

and the amount

of the mesophyll

cuticule

the

thylakoids

DI

with

II

It

the amount

conditions which exclude

remains

that

I mg

is clearly

2

One

bound

the herbicide

Among

to the QB that

the

of

the

purpose

of phenmedipham associated

nmoles

chlorophyll,

is suggested

phospholipids.

when

and

to

at the

competes with

is fully inhibited.

diuron.

thylakoidal

tissues

PS

(1.5 nmole)

of this product which foliar

chloroplasts

polypeptide

corresponding

through

experiments the

nside

shown 2 that phenmedipham

the ratio between

cells and

out under experimental

on

a fraction

with

is to measure

site

transfer

these 2 nmoles mg -I chlorophyll, site,

is located

bound

to other

treatment

to

parts

is carried

the limiting step represented

by the

penetration.

Very

little

herbicides

in

information

the

represents

a first

inhibitor

generally

out for this work. was obtained

apoplastic attempt applied First,

and

to foliage penetration

this problem

of

This

of

study

without

Two methods were cuticular

(0.5 cm x I cm) by vacuum

the stomatal

2077

mesophyll.

purposes.

of phenmedipham

whatever

the

the distribution

in the case of a photosynthesis

for agronomic

allowed

of phenmedipham

concerning

parts

of standard size

This treatment

solution

available,

symplastic

to clarify

in leaf fragments

(6.2 Pa pressure). the aqueous

is presently

chambers

its concentration.

set

limitation treatment

to be filled up with

.OO

2078

Secondly, we carried out a determination

of the

inhibition by phenmedipham

of the

photosynthesis rate in leaf fragments, with particular care to establish the minimum amount of product

introduced

complete.

conditions

These

correspond

to

the

inside the of

appearance

leaf,

almost of

complete

toxic

fluorescence changes and photodestruction of P S ]

for which

inhibition

inhibition

symptoms

in

percentage was

of

photosynthesis

=

diuron-induced

plants

of pigments associated with an inhibition

cyclic electron flow3.

Photosynthesis oxygen emission

inhibition was measured by two methods

on PS

obtained

by

and

:

a. leaf fluorescence has been used to detect photosynthesis acting

: leaf fluorescence

114,5.

The

short-term

quantitative fluorescence

estimation

inhibition by herbicides

of such

measurements

an

on whole

inhibition leaves

or

can on

be

leaf

fragments, by determination of the FI/FP ratio, following Cadahia et al.5; b. photosynthesis polarographic

intensity

and

measurements

of

inhibitory

effects

light-dependent

02

were

carried

evolution,

out as

through

previously

described6. The treated

two methods were for

half

biscarbamate

for

hour

in vacuo,

derivatives (table

as surfactant water

an

tested comparatively

30

(figure to

60

I). The

with

on leaf fragments

phenmedipham

but

of standard

also

with

I), with or without

addition of 0.3% of

low pressure exposure

of leaf fragments

minutes,

did

not

induce

significant

changes

photosynthetic rate.

TABLE !

CHEMICALSTRUCTURES~ D COMMONI~ES OF THE DIFFERENTBIBCARBAMATEDERIVATIVES STUDIED.

~NH-COO-RI

R!

R2

R3

Name

CH3 CIt3 CH3 CH3 C2H5

H H C1 fl H

CH3 C1 C1 CF3 H

Phenmed i ph 3-CI 3,4-di C1 3-CF3 De~ediph~

four

size, other

isophorone immersed of

in

their

2079

Figure

I - Percentage of photosynthesis

(abscissa)

or fluorescence

inhibition determined by oxygen emission

induction (ordinate),

for different phenylcarbamates

at I0 or I00 HH, in the presence or absence of isophorone.

Chenopodi.um album

leaves. B:

I0 pH without

O:

I00 pH without

I : desmedipham

isophorone, isophorone,

; 2 : 3-Cl

A:

id. with

isophorone

~:

id. with

isophorone

; 3 : 3,4-diCl

; 4 : phenmedipham

Treatments with I00 Ftl herbicide and isophorone

induced a total

except fop compound 2. Average of 4 measurements,

'°°[ %

" T

; 5 : 3-CF 3 inhibition,

bars z~ ~.

[[ 2

5

//

,,/,

/,

(9 0t'-"

/

/

/

/

(1)

0 u) 5 O 'L_

0

1

I 5O

oxygen

i 100 %

2080

Figure

I shows that

measurement, isophorone

with

100%

inhibition

one exception which

0.3%.

The

inhibition

is the 3-Cl

percentage

generally higher than through 02 emission rates.

This

can

be explained

(II0 O) was obtained by both methods

by

derivative,

measured

intensity

the fact

that

in the presence

through

fluorescence

of of was

particularly for low inhibition

fluorescence

I level

is directly

related to the proportion of herbicide-blocked centers, hence to the real inhibition of

PS

II

electron

photosynthetic

electron

conditions of medium compare

transfer,

whereas

transport,

to strong

the percentage

in

oxygen which

evolution

PS

II

is

not

light and low proportion

inhibitions

(0 and

reflects a

limiting

100% excluded)

obtained

+ 15 (at a level of 5%). The same experiment was repeated with Standard

leaf

fragments

of

Chen opodium

album

and

of

close

to

100%

through

fluorescence

measurement,

step

and

in

If we

by each method, interval equal

to

14C phenmedipham

90

Spinacia

floated in vacuo for one hour. At this stage, photosynthesis be

overall

of blocked centers.

the average difference between them was 39% with a 95% confidence

~M.

the

oleracea

were

inhibition was shown to to

90%

by

02

evolution

determination. The treated fragments were rapidly washed with water and ethanol 30% and

ground

labelled

in 98%

ethanol.

phenmedi pham

After

was

centrifugation,

dissolved

in

determination of the phenmedipham content scintillation

counting.

Under

these

it was

the

shown

supernatant.

that

95"/. of

After

the

dilution,

n the leaf fragments was achieved through

conditions,

we

could

compare

the

amount

of

phenmedipham found in isolated thylakoids and class A chloroplasts 2 to the amount of the herbicide remaining in leaf fragments. TABLE 2

~,MOONT OF PHENMEDIPI~M (nmoles) GIVING A 100% INHIBITION OF THE PHOTOSYNTHETIC ELECTRON TRanSFER PER MG CHLOROPHYL IN PURIFIED THYLAKOIDS I PURIFIED CLASS A CHLOROPLA~S AND LEAF FRAi3MENTS.

Thylakoids

Class A 0.5 x I cm leaf Chloroplasts fragments

Thyl.I/ f Class A

Class A j J

leaf

Thyl.j Jleaf

Spinacia 01eracea

2.0 ~ 0.4

4.8 ~ 0.3

15.9 ~ I

42X.

30%

I~

Chenopodium album

2.1 L 0.2

4.8 L 0.3

18.0 L 1.3

44%

2T~

11%

2081

The compared

increase

to thylakoids

herbicide

is

bound

phospholipids 2. A

]n

part

herbicide

can

plasmalemma, the

walls

0.3%

of

although

probably

This might

explain

I. This

was

effect was

found

to

fragments

that

would

solutions

increases

isolated

chloroplasts

effect

on

the amount

induces

ratio

between

the amounts

reaches

ratio between

close

as

between

volume,

the the

through

solution vacuum

the true concentration

action,

the

of herbicide

isophorone,

in

the

transfer

leading

from

Under

our

This

the same

means

leaf

not

but

part

of

the

the free

in the aqueous

phase

Ii00

that

the

in

ratio

fragments, as

This difference from

In

and 90 ~M.

of magnitude

result

from

the

and foliar

order

stomatal

phenmedipham

0.3 ~M

inhibition.

does

by

We

penetration

conditions,

respectively 300.

the

leaf fragments.

filled

2.

Chenopodium)

to a further

space

In

herbicide

chloroplasts.

isolated

to

which

to

were

inside

barrier

bound

therefore

and

droplets.

for

apoplastic

process

part

chlorophyll

needed for a 100%

penetration

apoplastic lipid

increase

easier

is not of

(mitochondria,

the same as for table

in class A chloroplasts

to I/3

the

14C phenmedipham.

to the inside of the

fragments is

exactly

In

to

lipophilic

hydrophilic

with

membrane

mg-!

is added.

Ii00

the concentrations

much

the

in leaf two

a

solution

of phenmedipham

a value

surprising

equilibrium

these

nmoles

to the cells,

that

inside

the

is bound

the

the

effect of

two-fold

2.5

isophorone

and

a

!

mean

when

in

In

important

were

of

membranes

also demonstrated

result

from the external

cell

the powerful

conditions

the

elsewhere.

concentration

as

lipophilic

150 , 75Z of

with

located

concentrate

(35

0.3%

are

membrane...).

an

the

of phenmedipham

different

certainly

or from the xylem vessels

terms,

Hence,

can

parts

nuclear

at

of this

associated

part

the major

in the

reticulum,

isophorone

of the product,

limited

found

is

one

other

fragment,

the experimental

leaf

that

chambers

not

be

it w~s without

suggest

which

leaf

three

of part

thylaRoid,

25%

when

constitute

movement.

this last case,

content

the

to

In the and

fragment,

phenmedipham

in figure

Isophorone

two

certainly

cell

is seen

between

leaf

protein

bound to class A chloroplasts

by the binding

envelope.

QB

endoplasmic

phenmedipham

The

the

the

of

leaf,

However,

other

to

chloroplasts,

symplastic

of

can be explained

to the chloroplast

product

class

in the amount of phenmedipham

a

the is

diffusion

introduction

of

apoplastic

space.

inside

the leaf

a

is

2082

much lower

than 90 ~M, since

it has been depleted by partition

into the membrane

and binding to the QB site. Taking this hypothesis as true, the

volume

of

the

phenmedipham

it becomes possible

solution

•

which

was

to evaluate approximately

introduced

inside

the

leaf

q

through vacuum treatment

:

¥=

[c] q C v

q =

: amount of product inside the leaf. At Ii00, q is 15.9 nmoles per chlorophyll in spinach. : concentration of the solution in which leaf fragments were soaked. the Ii00, it is 90 ~M in spinach. : volume of this solution introduced inside the fragments.

m9 At

In the case of our phenmedipham treatments of spinach leaves,

[C~ was 90 ~M,

0.52

relation

nmoles/fragment

containing

0.033

mg

chlorophyll.

The

gives

therefore a value of 5.8 ~l for v, which represents approximatly 32% of the total volume of the leaf. Such a value to

vacuum

is in agreement with the weight infiltration

(data

not

shown).

increase of the fragments As

a

whole,

it

is

in reponse

interesting

to

underline that for phenmedipham, when one part reaches the QB site, 7.6 others are distributed presumably

throughout

the leaf, among which

associated with

the lipophilic

amount of an herbicide within of action

is an

can be found the fraction of product

space. The

relation

between

the

total

the leaf and its fraction really bound to the site

important parameter

to explain the activity of a PS 11 herbicide

on whole plants in terms of their inhibitory power on isolated chloroplasts.

REFERENCES

I. A. Trebst, E. Ristorius, G. Boroschewski

and H. Schultz -

Z. Naturforsch.,

1968, 23b, 342-348. 2. P. Ravanel, M. Tissut, F. Nurit et S. Mona - Weed Res., submitted. 3. S.M. Ridley and P. Horton - Z. Naturforsch.,

1984, 394, 351-353.

4. C.D. Miles and D.J. Daniel - Plant Sci. Letters, 1973, l, 2375. E. Cadahia E., J.M. Ducruet and P. Gaillardon - Chemosphere,

1982, l.._!,445-450.

6. O. Belbachir, M. Matringe, M. Tissut et D. Chevallier - Pest. Biochem. and Physiol., (Received

1980, 14, 303-308.

in UK

1 March

1989)