Increased Synthesis of Photosystem II in Triticum vulgare
when Grown in the Presence of BAS 13-338
Salil Bose, R. M annar M annan
School o f Biological Sciences, Madurai Kamaraj University, M adurai 625021, India
and C. J. Arntzen
M SU-DOE Plant Research Laboratory, East Lansing M ichigan 48824, U SA
Z. Naturforsch. 39c, 5 1 0 -5 1 3 (1984); received D ecem ber 1, 1983
Photosynthesis, Electron Transport, Chlorophyll Fluorescence, Photosystem II, BAS 13-338
Addition o f BAS 13-338 (4-chloro-5-dim ethylam ino-2-phenyl-3(2H )-pyridazinone) to a sus­
pension o f chloroplast thylakoids caused an increase in the / level o f chlorophyll fluorescence
induction without affecting the F0 level and with a slight decrease in the Fmax level in a manner
similar to the addition o f D C M U to a thylakoid suspension. A ddition o f BAS 13-338 also
inhibited the rate o f Hill reaction H20 -» dichlorophenol indophenol with 50% inhibition
occurring at about 10 hm BAS 13-338. The inhibition was not reversed by diphenyl carbazide
used as an artificial electron donor to photosystem II. These results suggest that the site o f in h ib i­
tion by BAS 13-338 is between Q (next to the primary electron acceptor) and plastoquinone.
When the plants were grown in the presence o f sublethal dose o f BAS 13-338, the follow ing
changes were noted in the thylakoids o f the treated plants as com pared to the thylakoids isolated
from the control plants: The F0 and the norm alized variable fluorescence ( 4 F/ F0) levels
increased, chlorophyll a/ b ratio decreased, chlorophyll/P 700 ratio increased. Furthermore, the
rate o f photosystem II electron transport both under saturated intensity and the lim iting intensity
of illumination increased, and the ratio o f plastoquinone to Q decreased. These observations
have been interpreted as due to an increase in the ratio o f photosystem II to photosystem I in
plants grown in the presence o f BAS 13-338.
Introduction
Substituted pyridazinone com pounds have re ­
ceived considerable attention in recent years in the
study o f chloroplast developm ent and function ([ 1 ]
for review). Among various su b stitu ted p y rid a z i­
nones known to have interaction w ith p la n t system s,
4-chloro-5-dim ethylam ino-2-phenyl-3(2H )-pyridazinone or BAS 13-338 (also know n as SAN 9785) needs
special m ention because o f its m ax im u m ab ility in
increasing the ratio o f satu rated to u n sa tu ra te d fatty
acids with no or very little change in the pig m en t
content in the plants grow n in the presence o f
pyridazinone com pounds [2, 3]. T his p ro p erty o f
BAS 13-338 (hereafter m entioned as BAS for sim ­
plicity) offers a potential o f inducing h ea t resis­
tance in plants. However, before attem p tin g to test
Abbreviations: FeCN, potassium ferricyanide; D A D , diaminodurene; DCIP, dichlorophenol indophenol; D C M U
(diuron), 3-(3,4-dichlorophenyl)-l,l-dimethylurea; DBMIB,
2,5-dibromo-3-methyl-6-isopropyl-/?-benzoquinone;
PS,
photosystem (II or I); Q, quencher (= primary electron
acceptor o f PS II); PQ, plastoquinone; Chi, chlorophyll
{a + b).
Reprint requests to C. J. Arntzen.
0341-0382/84/0500-0510
$ 0 1 .3 0 /0
this potential, it is necessary to exam ine o th e r
changes, if any, induced in the structure and fu n c­
tion o f the photosynthetic ap p a ratu s by BAS. T his
com pound is also known as an in h ib ito r o f p h o to ­
system II electron transport, alth o u g h the site an d
the m ode o f action are not know n clearly [4, 5]. Both
o f these aspects are dealt w ith in this co m m u n ic a­
tion.
Methods
The seeds o f Triticum vulgare (CV: P elisser
D urum wheat) were surface sterilized w ith 0.1%
m ercuric chloride and seedlings w ere raised in glass
petri plates (15 cm diam eter) over three layers o f
crude filter papers. Various concentrations o f BAS
were prepared and 2 0 ml o f a given solution w as
added once to a petri dish. T h ereafter, w atering was
done only w ith distilled water. T he seedlings w ere
grown at room tem perature u n d er w hite cool illu ­
m ination o f 4000 lux. C hloroplasts were iso lated
according to Brewer et al. [6 ], PS-II activity was
m easured spectrophotom etrically follow ing D a rr
et al. [7], C hl/P700 ratio was estim ated according to
Mullet et al. [8 ]. Analysis o f fluorescence in d u ctio n
was done according to Brewer et al. [6 ].
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S. Bose et al. • BAS 13-338 Effects on Photosystem II
511
Results
W hen BAS was added to a chloroplast thylakoid
suspension, the rate o f Hill reaction o f H 20 -*■ D C IP,
was inhibited in a m anner show n in Fig. 1. T he 50%
inhibition o f the rate occurred at ab o u t 10
BAS.
The rate was not restored upon add itio n o f diphenylcarbazide to the assay m edium . T he action o f
BAS as m easured by fluorescence induction kinetics
is shown in Fig. 2. A ddition o f BAS caused no
Fig. 3. Chlorophyll fluorescence induction curves in chlo­
roplast thylakoids isolated from plants grown in the pres­
ence o f various concentrations o f BAS 13-338. For assay
conditions see Methods.
BAS
1 3 -3 3 8 (juM)
Fig. 1. Inhibition o f the rate o f electron transport and o f
( A F - A I ) I A F as a function o f BAS 13-338 concentration.
Chloroplast thylakoids were isolated as described in M eth­
ods and BAS 13-338 was added to the assay m edium to a
given concentration prior to illumination. For assay condi­
tions see Methods. F0 is the ground level fluorescence
intensity, I is the intermediate level o f fluorescence as
determined in [6 ], Fmax is the maxim um fluorescence inten­
sity. A F = Fmax - F0 and AI = / - F0.
Fig. 2. Chlorophyll fluorescence induction curves in iso­
lated chloroplast thylakoids in the presence o f various con­
centrations o f BAS 13-338. The function ( AF —A I ) / A F
was estimated from induction curve as described in [6 ] and
plotted against the rate o f electron transport (inset) for a
given concentration o f BAS 13-338 (Fig. 1).
increase in the F0 level, b u t a m ark ed increase in th e
I level occurred. The F max level d id not increase,
rather, it decreased to som e extent w ith increasing
concentrations o f BAS. A sim ilar d ecrease w ith 5 (im
D CM U was observed in these ex p erim en ts as well
as by others [9]. W hen the increase in I level was
expressed as (A F - A / ) / A F and p lo tted as a func­
tion o f concentration o f BAS 13-338, th e g rap h
obtained was rem arkably sim ilar and o v erlap p in g
with the inhibition curve o f th e Hill reactio n
(Fig. 1). In fact, w hen the percent decrease o f
( AF—A I ) / A F was p lo tted as a fu nction o f percen t
rate o f Hill reaction, an alm ost p erfect co rrelatio n
was observed (Fig. 2). T his o b serv atio n is sim ilar to
the earlier observation rep o rted [6 ] th a t (A F - A I ) /
A F decreases linearly w ith the decrease in Hill reac­
tion when atrazine (a sym m etric triazin e h erb icid e)
was added to thylakoid suspension o f Amaranthus.
This sim ilarity betw een D C M U and BAS effects
im plies that the site o f in h ib itio n by BAS lies
between Q and plastoquinone. T hese observations
also show th at in h ib itio n o f (A F —A I ) / A F can be
m onitored as a m easure o f in h ib itio n o f Hill reac­
tion.
W hen plants w ere grow n in th e presence o f
various concentrations o f BAS and th e thy lak o id s
were isolated from leaves o f two w eek old seedlings
the chlorophyll fluorescence kinetics w ere re m a rk ­
ably different from th at o f the control th y lak o id s
(Fig. 3). First, the F0 value increased m ark ed ly by
100% at 200 jiM BAS treatm ent. T he v ariab le flu o ­
rescence A F increased m ore th an th e F0 as show n in
512
S. Bose et al. ■ BAS 13-338 Effects on Photosystem II
the norm alized variable fluorescence ( AF/ F0). T h e
value o f AF/ F q increased from 1.78 in the control to
2.35 at 50 (im BAS treatm ent and decreased to 1.94
at 200 jiM. Second, the value o f (A F — A I) A F also
increased from 0.68 in the control to 0.85 at 25 pM
BAS). As expected, a d d itio n o f 100 pM BAS to th e
control did not increase any o f these th ree p a ra m e ­
ters (last row in the table o f Fig. 3).
W hen the area above the fluorescence in d u ctio n
curves in the presence o f 5 pM D C M U was analyzed
in thylakoids isolated from the control and the
treated plants, the relative ratio o f PQ to Q was
found to be less in the thylakoids o f treated plants
(Table I). Since the am ount o f PQ p er chlorophyll
did not change in treated sam ples (d ata not show n),
results indicated a 36% increase in the am o u n t o f Q
in the thylakoids o f treated plants. S tudies in th e
Hill reactions showed th a t the rate o f electron tra n s­
port in the thylakoids o f treated plants was h ig h er
than the control rate. T able II show s th a t in case o f
H 20 —> D CIP Hill reaction ab o u t 30 —40% increase
in the light-saturated rate in the lig h t-lim ited rate
occurred in thylakoids o f plants trea ted w ith 50 pM
BAS. A sim ilar increase in the rate was observ ed
with DAD (+ F eC N ), and D A D ( + F e C N ) +
DBMIB m ediated Hill reactions. T ab le I also show s
that the chlorophyll a / b ratio in these thylako id s
decreased m arkedly; the degree o f decrease d e p e n d ­
ed on the concentration o f BAS in the grow th
medium. The chlorophyll content in le a f (area or
fresh weight
(< 10%).
basis)
decreased
only
m arg in ally
Discussion
A decrease in the Chi a / b ratio and an in crease in
the chlorophyll fluorescence yield are gen erally
regarded as a consequence o f an increase in th e u n it
size of PS II and com m only referred to as sh ad eplant characteristics. An increase in the C h l/P 7 0 0
ratio is an obvious consequence o f the increase in
the unit size o f PS II. It should be noted, how ever,
that all of these observations can altern ativ ely be
interpreted in term s o f an increase in the n u m b e r o f
P S II units. F urther observations, nam ely, an in ­
crease in the rate the PS-II electron tran sp o rt m e a ­
sured under high light intensity and an increase in
Q /P Q ratio exclude the form er p ossibility and
support the latter. In the case o f an increase in the
unit size alone, the Q /P Q ratio should not change and
the light-saturated rate (on chlorophyll basis) o f
PS II should decrease. A nother o b serv atio n (to be
reported elsewhere) that the / 5 0 value D C M U in ­
hibition o f PS-II Hill reaction is increased (by ab o u t
40%) in the treated plants is an o th er evidence
supporting this conclusion. Since the ch lo ro p h y ll
content is not affected on leaf area (or fresh w eight)
basis, the increase in the n u m b er o f PS II u n its has
occurred not only at the thylakoid level b u t also in
the leaf as a whole.
Acknowledgements
Table I. Effect o f BAS 13-338 treatment on chlorophyll
content, Chi a !Chi b ratio and P Q /Q ratio.
Treatment
Control
50 |iM
1 0 0 hm
Total Chi
[m g/m m 2]
Chi a/
Chi 6 -ratio
2.80
2.67
2.53
3.26
2.55
2.32
PQ
Q
1 0 .8
—
8 .0
Chi
P700
263
424
519
This work was partially su p p o rted by a D e p a rt­
ment o f Science and Technology grant to S. B.
[11 (8)/80 SERC]. One o f the au th o rs (R. M.) w ould
like to thank the U niversity G rants C o m m issio n for
a research fellowship. We thank Beth K lingel for
reading, correcting and skillfully typing this m a n u ­
script. The authors are grateful to BASF co m p an y
for supply with pure BAS 13-338.
Table II. Effect o f BAS 13-338 treatment on PS-II electron transport rates with different electron
acceptors. The rates are expressed as nm oles o f acceptor red uced /m g Chi x hr.
Treatment
Control
50 |iM
loo hm
H20 -» DCIP
Saturating
light intensity
Limiting
light intensity
229
300
255
40
55
50
H20 -» D A D
F eC N
H 20 -> D A D
DBMIB + F eC N
256
355
387
1 0 0
69
108
S. Bose et al. • BAS 13-338 Effects on Photosystem II
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