DCMU-Induced Fluorescence Changes and Photodestruction
of Pigments Associated with an Inhibition of Photosystem I
Cyclic Electron Flow
Stuart M. Ridley
ICI Plant Protection Division, Jealott’s Hill Research Station, Bracknell, U.K.
Peter Horton
Department o f Biochemistry and ARC Research Group on Photosynthesis,
University o f Sheffield, U.K.
Z. Naturforsch. 3 9 c, 3 5 1 -3 5 3 (1984); received October 10, 1983
Diuron (DCM U), Photodestruction, Fluorescence, Photosystem I, Cyclic Electron Flow
Diuron (DCM U) induces the photodestruction o f pigm ents, w hich is the initial herbicidal
symptom. As a working hypothesis, it is proposed that this sym ptom can only be produced when
the herbicide dose is sufficiently high to inhibit not only photosystem II electron transport alm ost
completely, but also inhibit (through over oxidation) the natural cyclic electron flow associated
with photosystem I as well. Using freshly prepared chloroplasts, studies o f D C M U -induced
fluorescence changes, and dose responses for inhibition o f electron transport, have been com ­
pared with a dose response for the photodestruction o f pigm ents in chloroplasts during 24 h
illumination. Photodestruction o f pigm ents coincides with the inhibition o f cyclic flow.
Introduction
The m ajor initial sym ptom o f phytotoxicity in
plants treated with herbicides such as d iu ro n
(D C M U ), whose m echanism o f action is to block
chloroplast electron transport, is the d estru ctio n o f
existing chlorophyll, and the extent o f this d epends
on the intensity o f the light under w hich the plants
are grown. M uch m ore is now know n ab o u t the Q Bp rotein in the lam ellae w hich is involved in the
block, as other papers in this W orkshop report.
W hat is now needed is an understanding o f p ro ­
cesses leading to this lethal sym ptom , beyond the
tim e w hen D CM U binds to the Q B-p ro tein and up
until the m om ent when the process o f p h o to d e stru c­
tion actually starts. Few experim ents have ever been
done that follow the destruction o f chlo ro p last co m ­
ponents and processes in the tim e scale o f hours, b u t
attem pts to gain a greater understanding o f such
events have been m ade using isolated chloroplasts
Abbreviations: BSA, bovine serum albumine; D C M U
(diuron),
3-(3,4-dichlorophenyl)-l,l-dim ethylurea;
PS,
photosystem.
Reprint requests to S. M. Ridley.
0341-0382/84/0500-0351
$ 0 1 .3 0 /0
[1 —3], and individual leaves [4] illu m in ated and
analyzed over periods o f one o r m ore days. T his
paper provides su p p o rt for a w orking hypothesis
proposing th at natural cyclic electron flow associ­
ated with PSI m ust also be in h ib ited before D C M U
can induce the p h o to d estru ctio n o f pigm ents.
Materials and Methods
Intact chloroplasts from pea seedlings w ere iso lat­
ed by the m ethod o f W alker [5], and from m aize
mesophyll protoplasts [6 ] by the m eth o d o f D ay
et al. [7], E xperim ents on the p h o to d estru ctio n o f
pigm ents w ere carried o ut and m easu red as in [2 ]
b ut in a m edium ad d itio n ally containing 0.1% BSA.
Coupled noncyclic basal rate electron tran sp o rt was
recorded as 0 2 evolution as described by R idley [8 ].
The operation o f PS I cyclic electron flow was
recorded indirectly by m easuring [l4 C ]leucine in co r­
poration by a suspension o f chloroplasts [9] follow ­
ing 30 m in incubation (20 °C ) and illu m in atio n
(100 W /m 2) [2] in a m ed iu m (0.5 m l) containing
0.2 m KC1, 6 6 m M tricine (pH 8.3), 6 . 6 m M M gC l2,
5.72 |iM [l4 C]leucine, 1 (iM D C M U , and chloroplasts
(180 ng/m l). C hlorophyll fluorescence was m easu red
using an ap p aratu s described by H orton [10].
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352
S. M. Ridley and P. Horton • D C M U Inhibition o f Photosystem I Cyclic Electron Flow
Results and Discussion
The photodestruction o f pigm ents induced by
D C M U has been sim ulated in vitro by incu b atin g
isolated pea chloroplasts in the light for p erio d s o f
about 24 h and m onitoring pigm ent levels at in te r­
vals [2, 3]. A ttem pts have been m ad e here to estab ­
lish a relationship betw een the extent o f p ig m en t
photodestruction induced by D C M U , and the d e ­
gree to which D C M U can im m ed iately in h ib it
electron flow. This has involved a study o f the
effects o f varying doses o f the h erb ic id e on th ree
different processes recorded as far as possible u n d er
sim ilar conditions (100 W /m 2 illu m in atio n , 200 jam
Chi) using intact or freshly ru p tu re d chloroplasts
(Fig. 1):
(a) chlorophyll and carotenoid levels in chloroplasts
following 24 h illum inated incubation;
(b) coupled noncyclic basal rate electron tran sp o rt,
m easured im m ediately in freshly p re p a re d c h lo ro ­
plasts as 0 2 evolution in the presence o f ferricyanide;
(c) cyclic electron transport, the o p era tio n o f w hich
was recorded indirectly by the lig h t-d e p en d e n t in ­
corporation of [I4 C]leucine into proteins by a sus­
pension of freshly prepared intact chloroplasts co n ­
taining 1 [am D CM U . W hen PS II is alm ost blocked
N o n - c y c lic e le c tr o n
u C-Leu
in c o r p o r a t io n ( C y c l i c f lo w )
Fig. 1. Effects o f D C M U doses on chlorophyll and carote­
noid photodestruction in isolated chloroplasts (200 jam Chi)
during 24 h illumination (as a % o f illum inated controls),
and on noncyclic and cyclic electron flow in freshly pre­
pared chloroplasts. Controls rates (average o f duplicates):
coupled noncyclic flow, 48 (amol 0 2 ev o lv e d /h /m g Chi;
[l4C]leu incorporation in the presence o f 1 (am D C M U (an
indicator of the operation o f natural cyclic flow),
7.0 nm ol/h/m g Chi. The rate is the same without D C M U
and comparable with rates in [9] and [11], but the ATP
then comes from noncyclic phosphorylation.
by D C M U , ATP for protein synthesis com es from
cyclic rather than noncyclic p h o sp h o ry latio n [ 1 1 ],
and so we believe this to be a reasonable in d icato r
of the operation o f natural cyclic electron flow
itself. The D C M U inhibitions (Fig. 1) are reversed
by the addition o f ATP, so the m echanism o f p ro ­
tein synthesis is not being directly in h ib ited , only
the production o f ATP. It is ap p a ren t (Fig. 1) th a t
pigm ent photodestruction after 24 h closely follows
the progressive in h ib itio n in fresh chloroplasts o f
cyclic electron transport rath er th an the progressive
inhibition of noncyclic electron flow. I 50 values o f
0.36 |iM and 4.3 fiM for the in h ib itio n o f coupled
noncyclic and cyclic electron flow, respectively, have
been obtained. C oncentrations o f D C M U , th a t in ­
hibit noncyclic electron transport up to ab o u t 98%
in fresh chloroplasts, fail to induce any p ig m en t
photodestruction w hen chloroplasts are illu m in ated
for 24 h. At this level o f in h ib itio n the C h i;D C M U
ratio is about 2 0 0 : 1 , w hich is sim ilar to th at n eeded
to optim ize fluorescence quenching th ro u g h cyclic
electron flow by providing the correct redox ‘p o is­
ing’ of the electron carriers com m on to both cyclic
and noncyclic electron transport [12], H igher doses
o f D C M U induce a progressive destruction o f p ig ­
ments in chloroplasts during 24 h illu m in atio n , and
also inhibit fluorescence quenching [ 1 2 ] and
[ 14C]leucine incorporation (cyclic flow) in fresh
chloroplasts, presum ably by causing the cyclic ca r­
riers to becom e over-oxidized as noncyclic flow b e ­
comes com pletely inhibited.
The poising effect o f D C M U [12] has been inves­
tigated further by fluorescence using m aize m esophyll chloroplasts incubated w ith pyruvate (w hich
enhances cyclic flow [13]). Since fluorescence yield
is principally determ ined both by the redox state o f
the acceptor side o f PS II (and hence the rate o f
noncyclic electron flow), and the tran sth y lak o id pH
gradient, it can be used to probe the m etabolic state
o f photosynthetic m aterial [10]. F ig u re 2A shows
that fluorescence quenching (due to p roton g rad ien t
form ation) is stim ulated by a low concentration o f
D CM U , but reversed by a high concentration.
Antimycin A also reverses the D C M U -in d u ced
quenching, indicating the involvem ent o f cyclic flow
in the quenching (Fig. 2B). The responses to the
yield o f chlorophyll fluorescence titrated w ith
DCM U in high light reveal an I 50 for in h ib itio n o f
cyclic flow o f 3.0 hm [14], which is close to the I 50 o f
4.3 |a m for the in h ib itio n o f cyclic flow recorded by
S. M. Ridley and P. Horton • D C M U Inhibition o f Photosystem I C yclic Electron Flow
0 2yM DCMU
0 lu M DCMU
I
10pM DCMU
Antimycin (2 p M )
t
Fig. 2. DCM U-induced quenching o f chlorophyll fluores­
cence in intact maize mesophyll chloroplasts, and inh ib i­
tion o f quenching by (A) 10 |!M D C M U , and (B) 2 m M
antim ycinA. Pyruvate (10 m M ) was present as a substrate
[13], and 50 ng Chl/m l were used at a light intensity o f
180 W /m 2.
m easuring the incorporation o f [l4 C]leucine in high
light (Fig. 1). In a prelim inary experim ent (d ata not
shown), chloroplasts (200 jim Chi) were in cu b ated
and illum inated for 24 h w ith varying doses o f
an tim y cin A (up to 2 0 |im) in an attem p t to in h ib it
cyclic electron transfer to a variable degree. T his
induced a photodestruction o f pigm ents th a t b e ­
cam e greater w ith increasing doses o f antim ycin,
thus adding w eight to the general argum ent o f p ig ­
m ent photodestruction being related to in h ib itio n o f
[1] C. E. Stanger and A. P. Appleby, W eed Sei. 20,
3 5 7 -3 6 3 (1972).
[2] S. M. Ridley, Plant Physiol. 59, 724—732 (1977).
[3] S. M. Ridley, Carotenoid Chemistry and Biochem istry
(G. Britton and T. W. G oodwin, eds.), p. 353 —369,
Pergamon Press, Oxford 1982.
[4] K. E. Pallett and A. D. Dodge, J. Exp. Bot. 31, 1051 to
1066 (1980).
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353
cyclic flow, although the possibility o f ad d itio n a l
actions o f an tim y c in A [15] co n trib u tin g to the
chlorosis cannot yet be ruled out.
These experim ents p ro v id e fu rth e r evidence th at
the degree o f D C M U -related in h ib itio n o f flu o res­
cence quenching in freshly p rep ared intact ch lo ro ­
plasts can be equated to th e extent to w hich
D C M U -induced pigm ent d estru ctio n occurs follow ­
ing 24 h illum ination o f isolated chloroplasts; the
hypothesis being th a t b o th o f these are in d u ced by
the inhibition o f cyclic electron flow by D C M U .
Previous experim ents have led to the suggestion
that when P 700 activity is d estroyed directly (w hich
w ould cause a block in cyclic flow) the d egree o f
D C M U -induced pigm ent p h o to d estru ctio n is g rea t­
ly enhanced [3], and th a t th e loss o f pigm ents
induced by D C M U can be p revented by restoring
cyclic electron flow artificially [2, 3]. All o f these
findings are in general ag reem en t w ith th e w orking
hypothesis th at in h ib itio n o f PS I cyclic electron
flow by D C M U (through o v er-o x id atio n o f electron
carriers w hen noncyclic flow is blocked) is necessary
for inducing the prim ary h erb icid al sym ptom o f
pigm ent photodestruction.
Acknowledgments
We wish to thank R onald Sw art at J e a lo tt’s Hill
for the [ 14C]leucine in co rp o ratio n d ata in Fig. 1, and
Paul Fem yhough in the A R C U n it at S heffield for
the data in Fig. 2.
[10] P. Horton, Proc. R. Soc. Lond. B 217, 4 0 5 - 4 1 6
(1983).
[11] J. M. Ramirez, F. F. del Campo, and D. I. A m on,
Proc. Nat. Acad. Sei. 5 9 , 606 —612 (1968).
[12] J. D. Mills, R. F. Slovacek, and G. Hind, Biochim.
Biophys. Acta 504, 298 —309 (1978).
[13] D. Crowther, R. C. Leegood, D. A. Walker, and
G. Hind, Biochim. Biophys. Acta 722, 127—136
(1983).
[14] T. Brearly and P. Horton, Proc. 6th Int. Congr. Photo­
synthesis, 1983, in press.
[15] D. S. Bendall, Biochim. Biophys. Acta 683, 1 1 9 - 151
(1982).