The inhibition of photoreactions of chloroplasts by ioxynil

Vol.

30,

No.

1, 1968

THE

BIOCHEMICAL

INHIBITION

OF

November

results

have

of ioxynil

(4-hydroxy-3,5-diiodo

Paton

and

(1965a)

as the

following

the

throline

and

served

as the

Olsson

(1967)

inhibiting

cyclic

one

DCMU,

to ioxynil,

In this

1965).

donor

at

only

the

effect

found

at a 200-l

ascorbate-DCIP

was

leaves

-

Reactions

were

for

at

3 min.

run

BY IOXYNIL

of Science

much

less than

were

lux.

are:

to 10

prepared (19610)

in test-tubes

*The abbreviations used phenyl)-1,l -dimethylurea; diiodo-4-hydroxylbenzoate.

like

inside The

PMS, DCIP,

reaction

only

that

unlike

water

BDHB,

and water

which the

It also

inhibited while

cyclic the

by

and

DCMU,

served

as the

is structurally

quite

PMS-catalyzed (Avran

of chloroplasts with

Also,

Friend

photophosphorylation

photoreactions

NADP

O-phenon-

hand,

when

with

or ascorbate-DCIP

other

it inhibited

all

was

water

and

reinvesti-

as the electron

photophosphorylation

photoreduction

of NADP

but with

M ioxynil.

from but

the

photoreactions

-3

ioxynil,

either

NADP

to the

of chloroplasts.

0-phenanthroline

since

concentration, even

that

On

noncyclic

on various

1 Om6 AA.

by Avron

100,000

1965b).

DCMU,

regard

of concentration.

when

of interest

like

with

phosphorylation

range

found

and

completely

resistant

same

exactly

to act

higher

noncyclic

transport Smith,

literature

on photoreactions

they

electron

it is also

below

Chloroplasts

as described

at the

acted

of ioxynil

OOO-fold

- all

of plastoquinone

to inhibit

in the

to inhibit

and

ioxynil

found

concentrations

METHODS

(Paton

context

was

Shavit,

it was

the

that

photophosphorylation

gated,

PMS*

donor

cycling

When

with

photoreduction

similar

ioxynil

of plastoquinone

reported

donor.

appeared benzonitrile)

inhibited

electron

electron

recently

observed

photoreduction

the

COMMUNICATIONS

CHLOROPLASTS

Zippora Gromet-Elhanan Section, Weizrnann Institute Rehovoth, Israel

effect

as well

OF

RESEARCH

27, 1967

Controversial

Smith

BIOPHYSICAL

PHOTOREACTIONS

Biochemistry

Received

AND

lettuce

washed

once

a water

bath

mixtures

phenazine 2,6-dichloroph

(Lactuca

sativa

in a medium

maintained contained

var,romaine) without

at 20°, the

following

ascorbate. and

illuminated components

methosulphote; DCMU, 3-(3,4-dichloroenol indophenol, BDHB, n-butyl-3,5-

28

Vol.

30,

No.

in pmoles

ADP,

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1, 1968

in a total

5;

No,

volume

K phosphate,

chloroplasts

30-60

of ferricyanide

or NADP

and

DCMU.

The

pg

or

ferredoxin PMS

i Tris-HCI

IO (containing

containing

1.5 pmoles

of 3 ml

series

about

chlorophyll.

1 .O pmole

together

(pH

with

contained

0,l

7.8),

80;

4 x lo6

cpm

In addition,

of NADP 0.2

pmole

pmole

40;

MgCl2,

of P32)

and

once

the

and

saturating

DCIP,

20

of PMS,

NaCl,

reactions

and

when

-washed

contained

amounts

pmoles

8;

of ferredoxin,

ascorbate indicated

and

2x10m6M

20 pmales

of

ascorbate. Reduction reduction was

of ferricyanide

was

determined

Gromet-El

hanan AND

reduction

and

than

5x10m7M

and

toAvron

of Ben

Hayyim

by Avron

( 1960)

using

et the

al-*

and

Shavit

(1967)

and

modifications

NADP

(1963). ATP

formation

outlined

by

(1967b). -

loxynil

photophosphorylation

with

NADP

as the With

affected other Shavit,

found

water

electron

which

the

to be a very

was

(Avron, acceptor,

it was

giving

supposedly

inhibit

nor close

inhibitor

donor more

50%

as an electron

phosphorylation

potent

the electron

1961b)

ascorbate-DCIP

neither

inhibitors

was

when N-oxides

respectively.

many

accorcfjng

method

DISCUSSION

concentrations

(Avron

the

alkyl-hydroxy-quinoline

cyanide

with

by

as described

RESULTS

the

measured

was assayed

donar

NADP to the

(Figure

effective

inhibition ioxynil

reduction, oxygen

of photo1). with

Like ferri-

at 7x10w8M

and

at these

low

as is the

case

evolution

site

1965).

loxynil

concentration

- M

Figure 1 - Effect of Increasing Concentrations of loxynil on Chloroplost Open symbols represent photoreduction. Semiclosed and closed symbols phosphorylation. Control activity (expressed in pmoles/mg chlorophylI/hr) A,A to the following values : 0, 0 with ferricyanide 277 and 133; and 82; 0 , I wjth ascorbate-DCIP to NADP 41 and 64 respectively. vity with PMS ws 417 and with ascorbate-PMS 504.

29

Photoreactions. represent photocorresponds with NADP 69 Control

acti-

Vol. 30, No. 1, 1968

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The phosphorylation was inhibited 10m4M.

accompanying

when the concentration

However,

which

In contrast

was not inhibited

phosphorylation

in this system was resistant

and Margulies,

here with

phosphorylation tion,

without

include

affecting

@ al.,

Elhanan, ioxynil,

the electron (Trebst,

1966;

Gromet-Elhanan,

1967a, b) and phenol all these compounds

blocked

as cyclic

from ascorbate-DCIP P-trifluoromethyl

19670, b); Dio-9

(Neumann

A similar

1962).

transport

and photo-

to NADP.

These

benzimidazoles

1967).

and cyclic

to

photophosphoryla-

and phlorizln

and Drechsler,

noncyclic

of the

to exert effects similar

of electron

donor as well

various

1963);

to inhibit

phosphorylation

1960; Wessels,

inhibition

transport

‘1).

donor (Fig. 1). The omission of

were reported

namely,

with water OS the electron

salycilaldoxime

(Buchel

ioxynil,

even

cyclic

of inhibition

of the PMS system to ioxynil.

In the last few years many compounds those reported

(Figue

required

wos observed when the PMS-catalyzed

(Jagendorf

at

to ioxynil

the PMS-catalyzed

the pattern

as the electron

to NADP

S@!/c inhibition

at the concentrqtions

the sensitivity

effect of ascorbate by DCMU

(19650)

but rather followed

somewhat

giving

al I types of photophosphorylation

by ioxynil

with ascorbate-DCIP

increased

wos inhibited

of NADP

totally bbcked

noncycl ic photophosphorylation,

protective

was raised,

with the results of Poton and Smith

phosphorylation

flow from ascrobate-DCIP

of ioxynil

the photoreduction

at concentrations

ascorbate

the electron

However,

(Grometunlike

photophosphorylation

at

the same range of concentrations. The effects of the above mentioned explained

by proposing

compounds,

II ond the second near photoreaction

reduction

with ascorbate-DCIP

implies chain electron

transport

phospharylation lation

ascorbate-DCIP chain which in the resistant

on the electron

transport

cannot

and NADP,

for the cyclic

system would leading

indicate

can be sites : one of the photo-

donor to all these compounds

be located

but is rather

is exclusive

chain

I. The resistance

as the electron

that the second site of inhibition between

OS ioxynil,

that they act in the same manner at two different

near photoreaction of NADP

as well

on the electron

located

transport

on that part of the

system. The absence of

that there is no site of phosphory-

from ascorbate-DCIP

to NADP.

ACKNOWLEDGEMENTS I would like to thank Dr. B. J. Heywood of May and Baker Ltd., Dagenham, Essex for a gift of ioxynil. I wish to thank Miss 0. Cohen far technical assistance.

30

Vol.

30, No.

1, 1968

BIOCHEMICAL

AND

BIGPHYSICAL

RESEARCH

COMMUNICATIONS

REFERENCES Avron, M. (1960). Biochem. Biophys. Acta c, 257. Avron, M. (1961a). Anal. Biochem. z, 535. Avron, M. (1961b). Biochem. J. 78, 735. Avron, M. and Shavit, N. (1963rAnoI. Biochem. 5, 549. Avron, M. and Shavit, N. (1965). Biochim. Biophys. Acta 109, 317. Ben Hayyim, G., Gromet-Elhanan, Z. and Avron, M., in preparation. Buchel, K. H., Draber, W., Trebst, A. and Pistorius, E. (1966). Zeit. Naturforsch. 21b, 243. Friend, J. and Olsson, R.n67). Nature 214, 942. Gromet-Elhanan, Z. (1967a). Abstracts, 7mnternationaI Congress of Biochemistry, Tokyo, H-43. Gromet -El hanan, Z. (1967b). Arch. Biochem. Biophys. in press. Jagendorf, A. T. and Margulies, M. (1960). Arch. Biochem. Bi ophys. 90, Neumann, J. and Drechsler, Z. (1967). Plant Physiol. 42, 573. Paton, D. and Smith, J. E. (1965a). Weed Res. 2, 75.Paton, D. and Smith, J. E. (1965b). Nature 207, 1211. Trebst, A. (1963). Zeit. Naturforsch. 18b, 817. Wessels, J. S. C. (1962). Biochim. BioFys. Acta 65, 561. -

31

184.