iwith each other. DCMU and atrazine can replace DNOC from its

i

I1Ulian J. Plant PhYliol., Vol. XXIX, No.4, pp. 335-344 (December.1986)

,.

with each other. DCMU and atrazine can replace DNOC from its binding site of the chloroplast membrane.

4. In the susceptible biotype tbe main TL band which is related to the secondary quinone electron acceptor Q. (Q.-band) apeared at +30·C. This band is totally absent in the resistant biotype.

5. In the resistant biotype a TL band at +IO·C can be observed Which is assigned to the primary acceptor Q. (QA-band).

INTRODUCTION

Differential response to triazine herbicides has been observed within certain

species for example, Ryan (1970) and Radosevich and Devilliers (1976)

Department of Botany, University of Chittagong. Chittagong, Bln gladesh.

TRIAZINE RESISTANCE IN ERIGERON CANADENSIS L. IV

INVESTIGATED BY THERMOLUMINESCENCE

MEASUREMENT

A. RASHID· AND S. DEMETBR

Biological Research Centre, Institute of Plant Physiology, Hungarian Academy of Sciences, H-6701 Szeged. Hungary.

SUMMARY

Thermoluminescence (TL) after continuous illumination was studied in untreated and herbicide treated (atrazine.2-chloro-4-etbyla­ mino-6-isopropylamino-s-triazine; DCMU, N-(3-4-dichlorophenyl)-N-N­ dimethyl-urea; DNOC. dinitro-o-cresol) chlosoplasts of triazine-suscepti­ ble and -resistant biotypes of Eri,eron canmieNlil L. The results obtained from the above experiment can be summarized in the following mannel'$ :

1. TL can be applied in the approximate determination of 16a value of photosytltem II (PS II) herbicides in the treated plants.

2. In the susceptible chloroplasts herbicide binding infiuences the redox state of the primary quinone electrone acceptor QA, which is reflected on the peak position of the main TL band.

336

reported in common grounsel

(Senecio vulgaris),

Radosevich (1977) reported in

redroot pigweed

(Amaranthus retroflexus)

and Machado

et al.

(1978) reported in

common lambsquarter

(Chenopodium album).

The difference is not associated

with uptake or metabolism of the inhibitor but of difforential chloroplast function

as related to the photosynthetic electro transport from

QA

to

QB

in the reducing

side of PS

(Pfister and Arntzen, 1979).

It

has been stated that TL provides

information about the redox state of the primary and the secondary acceptors of

PS II (Inoue and Shibata, 1978, 1979 and Droppa

et al.

1981). Therefore, the

present investigation was undertaken to characterizc the modifications occurring

in the redox state of the primary acceptor

QA

and the secondary acceptor

QB

due

to mutation induced alteration in the

QA-QB

herbicide binding complex of the

I

chloroplast membrane of

E. canadensis

by using TL measurement technique.

The variation in electron transfer rates from

QA

to

QB

was also one of the

objectives of this investigation.

MATERIALS AND METHODS

The supply of seedlings, the growing of seedlings and the isolation of

chloroplasts were described

elsewhere (Rashid and Demeter,

1985a).

Measurement of thermoluminescence :

The preparation of samples and the

technique of TL measurements have also been described previously (Rashid and

Demeter, 1985e). Samples were illuminatea with white light from a NARV A

halogen lamp of 650 W for 2 min at-60°C. The exciting light was passed through

a heat absorbing water filter (10 cm thickness) and a Balzer neutral density filter

giving an illumination intensity of 10 Wm-

The samples were heated in darkness

at a rate of :lO°C/min in order to get best resolution of the peaks (Sane

et al.

1977). Urea-type (atrazine and DCMU) and phenolic (DNOC) herbicides of

different concentrations were mixed with the samples before putting into the

sample holder.

RESULTS

TL originates from PS II and the bands of the glow curve can be related

to the charge recombination between positively charged donors and negatively

charged acceptors (Inoue and Shibata, 1978; Demeter

al.

1979). In our

experiment the main TL band has been found to appear at + 30°C during conti­

nuous heating from -60°C to + 80

337

et al.

1975)_ In the present experiment the relative intensity of the main TL

band of the glow curve was measured by using different concentrations of PS

II herbicides (atrazine, DCMU and DNOC) in isolated triazine-susceptible and

-resistant chloroplasts of

canadensis

The QB-band at 30"C has been found

to be replaced gradually by a new band at IOoC due to mixing of different con­

centrations of DCMU with the susceptible chloroplasts (Fig

1).

Atrazine and

..

_..

,;1

"

..

.40 O.lD -40 0 ·40 -40 0 '40 -40 0 .40

Temperature (-C) _{Temp,uotur. ro}

Fig. 1: Gradual disappearance of Pig. 2: Comparison of the effect the Qs-band at+30·C and of atrazine, DCM and concomitant appearance DNOC on the peak posi­ of \theQ.cband aHI0·C tions of the glow bands in the glow curye of TL of TL in susceptible (a, due to the treatment of b, c and d) and resistant susceptible chloroplasts (m, n. 0 and p) chloro­

with DCMU. plasts.

338

fGund to be arGund 80 nM (Fig 3). Interestingly, no. QB-band at

+

30

e was

G~erved

in the TL Gf resistant chlGrGplasts even in untreated cGntrGI sample.

SENSITIVE

-

...

•

-

c:

toI

-

c:

t­

toI

>

-g

toI

a::

80

60

40

20

10-8

I

_so

-80nM

10-

DCMU(M~

Fig. 3: Estimtion of Ito concentration of DCMU in the susceptible chloro­ plasts by plotting the amplitude of the Qb-band as a function of

increasing concentrations of DCMU.

A band at

+

lOoe (QA-band) was fGund Gnly in this case. That band remained

unchanged (Fig 2) at high concentrations of atrazine, DeMU and DNOe 50 p.M,

20 p.M and 250 I'M respectively).

Since DeMU and DNOC induced TL bands are well separated frGm each

Gther therefGre, we tried to follGW the displacement of these herbicides in the

chlGrGplast membranes. Fig 4 shGWS the concentration dependence Gf the dis­

Temperatur. rcl

Fig. 4 : Displacement of bound DNOC from the susceptible chloroplast tbylakoids by DCMU as monitored by TL. After S min incubation of thechJoroplasts with 200 uM DNOC, different cancentrations of DCMU were added after 2min. a} no DCMU added b} 7S nM DCMU c) 200nM

DISCUSSION

Measuremeut of oxygen evolution (Rashid and Demeter, 1985a), flash­

induced SIS nmabsorption change (Rashid and Demeter, 1985c) and fluorescence

induction (Rashid, 1983) could be applied in the determination of Iso concentra­

tion ofPS IT herbicides (atrazine. DCMU and DNOC) of

Erigeron canadensis

L.

However, these techniques could not give any data about the changes occuring

at the acceptor side of PS II accompanying with the appearance of herbicide

resistance in this species. Therefore, we tried to introduce a new method, TL

for the investigation of the mechanism of herbicide resistance in plants. It has

been stated that TL provides information about the redox states of tbe primary

and the secondary acceptors of PS II (Inoue and Shibata, 1979; Droppa

et al.

1981). Thus TL seems to

be

an useful technique for studying the mechanism

and sites of action of herbicides that inhibit the electron transport of PS II at

level the of secondary acceptor QB.

In the current experiment, the

at + 30°C has been found to

be

340

/

herbicides blocked the electron transport QA to QB (Trebst and Draber, 1979)

and as a results electrons accumulated on QA (Velthuys and Amesz, 1974).

Charge recombination of the reduced primary acceptor QA- with the donor side

of PS II generated the QA-band. Plotting the amplitude of the

as a

function of DCMU concentrations the

value was found to be 80 nm (Fig 3).

This finding is in good correlation with our data obtained from oxygen evolution

(Rashid and Demeter 1985a) and

SIS

nm absorption change measurements

(Rashid and Demeter, 1985c).

The differences in the peak positions of the

may

explained

in the following ways:

The structural modifications of the proteinaceous

components of QA and QB due to binding of DCMU, atrazine and DNOC

changes the mutual orientation and separation of QA and P680, so that probabi­

lity of the reverse electron transport from QA to P680 might also change.

(2) TL originates from charge recombination occurring between positively

charged donors and negatively charged acceptors (Arnold, 1966: Lurie and

Bertsch, 1974; Ichikawa et al. 1975; Inoue and Shibata, 1979; Demeter et

01.

1979). The peak position ofTL band is determined by the redox span between

the donors and the acceptors molecules participating in the recombination (Vass

et al. 1981). The herbicide induced change in the redox state of QA is reflected

in shifts in the peak positions of the

The two different peak positions

(around

+

lOoC for DCMU and atrazine and -lOoC for DNOC) obtained in

our TL measurement can be supported by a classification done by Trebst and

Draber (1979), who classified urea-type and phenOlic herbicides into two different

groups on the basis of their chemical structure. We therefore, assume that

herbicides belonging to two different groups have different effects on the state of

QA which

is

evident from our results. Boger

01. (1981) proposed binding sites

for urea-type and phenolic herbicides. Our results can also be supported by the

work of Pfister et al. (1981), who reported a 32 kDaIton polypeptide for binding

urea-type herbicides and Oettmeier and Masson (1980), who reported a 41

kDalton polypeptide for binding phenolic herbicides on the reducing side of

PS

n.

Since DCMU and DNOC induced TL bands are well separated from each

other, therefore, we tried to follow the displacement of these herbicides in the

susceptible chloroplast membrane. The results obtained from· the displacement

experiment suggest that DCMU, atrazine and DNOC have a common site of

action in chloroplast membrane. Our displacement experiment can be supported

by the work of Reimer et

01. (1979) and Trebst (1979). who demonstrated that

bromonitrothymol (BNT), a phenolic herbicide has replaced the bound radio­

actively labelled metribuzin (C

JL

3,

..

I

J

TRIAZINE RESISTANCE 'IN ERIGERON CANADENSIS

341

concentration range of their

value. Oettmeier

et al.

(1982) showed that the

ureas interfere noncompetitively but the phenolic herbicides interfere competi­

tively with the specific binding sites of the phenolic herbicide. dinoseb.

The apparant contradictory results can be resolved

explaining that

there might have different but overlapping binding areas for DCMU, atrazine

and DNOC. The binding of one herbicide may influence the binding of the

other. Our interpretation can be supported

a model proposed by Trebst

(1981).

In the above mentioned TL experiments it has been noticed that the' 'QB­

'band appeared at

+

30°C in the case of susceptible biotype, Due to the treat­

ment of DCMU, atrazine and DNOC, the electron transport is blocked between

QA to QB and consequently the QB-band has been found to be replaced by the

QA-band at

+

10DC,

+

8°C and -IODC respectively in susceptible chloroplasts.

Surprisingly, the QB-band did not appear at all at +30°C even in untreated

control sample of resistant chloroplasts. A band at + IODC (QA-band) has been

fonnd to appear in the glow curve of TL in this case. These observation suggest

that following light excitation of the resistant chloroplasts, the primary acceptor

pool remains in reduced state for a long period while in the susceptible biotype

Q-A is quickly reoxidized by the secondary acceptor QB.

A mechanism which can explain our result would

be

that in the resistant

chloroplasts, the semiquinone QB- is stabilized (i.e. mid-point potential of

QB/QB- is raised) so that the center would be predominantly in QB- state after

dark adaptation. The more positive potential of Qs- explains why the singly

reduced secondary acceptor QB- does not take part in charge recombination

reaction and consequently the lack of the QB-band in the TL spectrnm.

It

has been shown by Bowes

et al.

(1980) that in succeptible chloroplasts

the rate of the reaction QA-Qs-+QAQS- is faster than the reaction QA-QB--+

QAQB

According to the redox theory of qui nones (Clarke, 1960), a more

positive redox potential of QB- results in an equivalent lowering of the redox

potential of QB

- .

Thus in resistant chloroplasts the QA-Qs- -+ QAQB2-

reaction

342 A. RASHID AND S. DEMETER

biotype chloroplasts showed a much higher "intermediate"

level in the

resistant chloroplasts than in the susceptible types. This implies that the rate

of reoxidation of the primary electron acceptor by the secondary electron accep­

tor pool is slower in the resistant biotype than in the susceptible one.

In the susceptible chloroplasts the QA-band is shifted upon herbicide

addition indicating that not only the state of QB but that of QA also is influenced

by herbicide binding to the chloroplast membrane. Opposite to this, in resistant

chloroplasts the QA-band remained unchanged even at high concentrations of

DCMU, atrazine and DNOC. This observation suggests that in the resistant

chloroplasts the

complex has changed due to the mutation of chloroplast

DNA (Pfister and Arntzen,

and herbicide binding at the level of the

secondary acceptor does not exert any effect on the redox state of the primary

acceptor.

ACKNOWLEDGEMENT

The first author gratefully acknowledges the Hungarian Academy of

Sciences and UNESCO for providing him financial support for tbis research

work.

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