Photosynthesis Research 34: 449-464, 1992.
© 1992 Kluwer Academic Publishers. Printed in the Netherlands.
Photosystem I-dependent cyclic electron transport is important in
controlling Photosystem II activity in leaves under conditions of
water stress
Eva Katona*, Spidola Neimanis, Gerald Sch6nknecht & Ulrich Heber
Julius-von-Sachs-Institut fiir Biowissenschaften der Universitiit, Mittlerer Dallenbergweg 64, D-8700
Wiirzburg, Germany
Received 26 February 1992; accepted in revised form 9 September 1992
Key words: light scattering, photoinactivation, proton gradient, PT00 photooxidation, quenching of
chlorophyll fluorescence, redox poising
Abstract
Leaves of the C 3 plant Brassica oleracea were illuminated with red and/or far-red light of different
photon flux densities, with or without additional short pulses of high intensity red light, in air or in an
atmosphere containing reduced levels of CO 2 and/or oxygen. In the absence of CO 2, far-red light
increased light scattering, an indicator of the transthylakoid proton gradient, more than red light,
although the red and far-red beams were balanced so as to excite Photosystem II to a comparable
extent. On red background light, far-red supported a transthylakoid electrical field as indicated by the
electrochromic P515 signal. Reducing the oxygen content of the gas phase increased far-red induced light
scattering and caused a secondary decrease in the small light scattering signal induced by red light. CO 2
inhibited the light-induced scattering responses irrespective of the mode of excitation. Short pulses of
high intensity red light given to a background of red and/or far-red light induced appreciable additional
light scattering after the flashes only, when CO 2 levels were decreased to or below the CO 2
compensation point, and when far-red background light was present. While pulse-induced light
scattering increased, non-photochemical fluorescence quenching increased and F o fluorescence decreased indicating increased radiationless dissipation of excitation energy even when the quinone
acceptor QA in the reaction center of Photosystem II was largely oxidized. The observations indicate
that in the presence of proper redox poising of the chloroplast electron transport chain cyclic electron
transport supports a transthylakoid proton gradient which is capable of controlling Photosystem II
activity. The data are discussed in relation to protection of the photosynthetic apparatus against
photoinactivation.
Abbreviations: F, FM, F~, F~, Fo, F0-chlorophyll fluorescence levels; qbexc-quantum efficiency of
excitation energy capture by open Photosystem II; dOpsi~ - quantum efficiency of electron flow through
Photosystem II; P.~5 - field indicating rapid absorbance change peaking at 522 nm; PT00- primary donor
of Photosystem I; Q A - primary quinone acceptor in Photosystem II; Q N - non-photochemical fluorescence quenching; Q q - photochemical quenching of chlorophyll fluorescence
* On leave from the Biophysics Department, University of Medicine and Pharmacy 'Carol Davila', 8 Eroii Sanitari Blvd., 76241
Bucharest, Romania.
450
Introduction
Electron transport in photosynthesis is coupled
to the vectorial transfer of protons from the
chloroplast stroma into the intrathylakoid space.
The resulting transthylakoid proton gradient is
used for, and dissipated by, the synthesis of the
ATP required for carbon assimilation, photorespiratory carbohydrate breakdown and other
processes. Most workers agree that three protons
leave the thylakoids for each ATP synthesized,
but even H+/ATP values up to 4.5 have been
reported (Grfiber et al. 1987). Photosynthesis
can proceed rapidly at low chloroplast ratios of
ATP to ADP and of NADPH to NADP (Laisk
et al. 1991). However, a large proton gradient
has been shown to be important in controlling
Photosystem II activity and in facilitating radiationless energy dissipation under conditions
when excess light is absorbed by the chloroplast
pigment system (Bj6rkman 1987, Baker and
Horton 1987, Foyer et al. 1990, Genty et al.
1990, Krause and Weis 1991). Such control and
the accompanying dissipation of excess excitation
energy as heat are necessary to prevent full
reduction of the chloroplast electron transport
chain when stomata are closed under water
stress. Such reduction would lead to the photodestruction of the photosynthetic apparatus, an
effect, which can easily be demonstrated with
isolated chloroplasts (Heber et al. 1989).
In leaves, chloroplasts can reduce several electron acceptors. The proton/electron stoichiometry of linear electron transport is still not certainly known (Furbank et al. 1990). If Q-cycle activity at the level of the cytochrome b/f complex is
obligatory (Rich 1988), H+/e is 3. Auxiliary
reactions would not necessarily be required to
provide the ATP needed for carbon assimilation
or photorespiration of C3 plants, if the H+/ATP
ratio is 3. If Q-cycle activity is facultative as
several researchers assume (Moss and Bendall
1984, Ort 1986), such reactions are required.
The assimilation of nitrate consumes ATP only
when ammonia is incorporated into ketoglutarate, and a large proton gradient may be
formed during nitrate reduction. A proton gradient also builds up during the slow oxygen
reduction in the Mehler reaction and the accompanying reduction of peroxidatively produced
monodehydroascorbate (Asada and Takahashi
1987, Schreiber and Neubauer 1990). However,
Heber et al. (1978) and Furbank and Horton
(1987) have shown that, at high but not at low
light intensities, carbon assimilation of intact
chloroplasts or mesophyll protoplasts is inhibited
by low concentrations of antimycin A, which
inhibit cyclic, but do not inhibit linear electron
transport. A proton gradient is formed, and ATP
is synthesized during cyclic electron transport
around Photosystem I (Tagawa et al. 1963,
Arnon 1977, 1991, Arnon and Chain 1977, 1979,
Hosler and Yocum 1985). A necessary requirement for cyclic electron flow is proper 'redox
poising' which denotes a redox situation in which
electrons are not drained from the electron
transport chain whilst simultaneously full reduction of electron carriers is avoided.
It is questionable whether such redox poising
is possible when an oxidized chloroplast NADP
system traps electrons during carbon assimilation
of leaves (Laisk et al. 1991). On the other hand,
under excessive light, and when leaves close
their stomata under water stress and net photosynthesis is decreased together with nitrate reduction (Kaiser and F6rster 1989), increased
reduction of the chloroplast NADP system may
produce a redox situation which makes cyclic
electron flow possible. In preceding publications,
we have shown that under high intensity illumination, and in the complete absence of CO 2 from
air, excessive reduction of the chloroplast electron transport chain cannot be avoided in brightly illuminated leaves of C3 plants (Wu et al.
1991, Heber et al. 1992). However, full photosynthetic control is shown by extensive oxidation
of PT00 in the reaction center of Photosystem I, if
photorespiratory CO 2 turnover is facilitated at a
CO 2 concentration close to the CO 2 compensation point.
In this communication, we wish to present
evidence of thylakoid energization by cyclic electron transport in leaves of C3 plants, when linear
electron flow is restricted by the availability of
electron acceptors. Using a different approach,
Harbinson and Foyer (1991) have also recently
concluded that cyclic electron transport supports
a large transthylakoid photon gradient when access of CO 2 to the photosynthetic apparatus is
reduced. Although our conditions of demon-
451
stration are not identical with photorespiratory
conditions of brightly illuminated water-stressed
leaves, extrapolation of the observations made
when linear electron transport is restricted by
acceptor availability at low flux densities of absorbed light lead to the conclusion that thylakoid
energization of leaves whose stomata are closed
is supported by Photosystem I-dependent cyclic
electron flow.
Materials and methods
Detached mature leaves of cabbage (Brassica
oleracea var. oleracea) grown in a greenhouse
were used for the experiments. Leaves were cut
under water, and petioles were kept in water
during the experiments. Part of the lamina of a
leaf was enclosed in a sandwich-type cuvette
which could be aerated. The composition of the
gas atmosphere could be varied using mass flow
controllers (Tylan Corporation, Eching, Germany). Water contents of the gas stream leaving
the cuvette, and CO 2 contents of the gas streams
entering and leaving the cuvette were determined using an infrared gas analyzer (BINOS,
Leybold-Heraeus, Hanau, Germany). The relative humidity of the gas stream entering the
cuvette was about 50%. The gas flow through
the cuvette was 500 ml min -1 and the temperature between 20 and 25 °C. The upper surface of
the enclosed part of leaf lamina could be illuminated through a window in the cuvette with a
beam of either red or far-red light or with the
two beams simultaneously. The red beam (filters
RG 630 from Schott, Mainz, Germany, and K65
from Balzers, Liechtenstein) had a half-bandwidth ranging from 624 to 662nm. It excited
both Photosystems I and II. The far-red beam
(filters RG 610 and RG 715 or RG 724 from
Schott and Calflex C or Calflex X from Balzers,
as indicated in the legends to the figures) had a
half-bandwidth ranging from about 714 to
766nm or from 725 to 752nm. It should be
noted that filters of the same designation used in
the previous publication (Heber et al. 1992) had
slightly different half bandwidths. The far-red
beam excited predominantly Photosystem I
(Melis et al. 1987). A third beam (filters RG 630
from Schott and Calflex C from Balzers) with a
half-bandwidth ranging from 625 to 755 nm was
given in the form of l s high intensity
( > 5 0 0 W m -2) pulses. An extremely weak
(0.008 W m -2) monochromatic measuring beam
recorded transmission of the leaf at various
wavelengths in the green part of the spectrum. It
was used to detect electrochromic absorption
changes, which give information on the transthylakoid electric field and peak at 522nm
(Junge and Witt 1968, Junge 1977) and changes
in light scattering, which give information on the
transthylakoid pH gradient and peak at about
535 nm (Heber 1969, Krause 1973, K6ster and
Heber 1982, Bilger et al. 1988). Filter arrangements to protect the recording photomultiplier
against actinic light were as reported previously
(Heber et al. 1992). The electrochromic signal
could also be measured at 535 nm by virtue of its
fast kinetics which distinguished it from the slow
light scattering changes. Modulated chlorophyll
fluorescence was measured by the PAM fluorometer of Walz (Effeltrich, Germany) as described by Schreiber (Schreiber 1986, Schreiber
et al. 1986). Fluorescence intensity levels were
designated and the photochemical quenching parameter (Qq) was calculated from fluorescence
traces according to van Kooten and Snel (1990);
see also Schreiber et al. (1986). The non-photochemical quenching parameter (Q~) was defined
and calculated according to Bilger et al. (1988)
as relative decrease in the maximum fluorescence
level. Quantum efficiencies for excitation capture
by (riPe×c) and electron transport through PS lI
(~ps H) were calculated according to Genty et al.
(1989, 1990). P700 photooxidation was monitored
measuring optical absorbance changes at about
830 nm by a PAM fluorometer which was equipped with a proper emitter-detector unit
(Schreiber et al. 1988).
Results and discussion
Figure 1 shows changes in the apparent absorbance at 535 nm of a cabbage leaf which was kept
in the dark (with only the dim green measuring
beam on) or illuminated either with a beam of
far-red light or a beam of red light, or with the
two beams superimposed on one another. The
oxygen concentration was varied from 1 to 21%,
452
i
Fig. 1. Changes in apparent absorbance at 535 nm of a cabbage leaf produced by illumination with 4.2 W m --z (R1) or 8.4 W m -z
(R2) red light, or by 100 W m -~ (FR) far-red light, or by a combination of R 1 and FR. The red light had a half-bandwidth ranging
from 624 to 662 nm, while the far-red light had one ranging from 725 to 752 nm. R1 and FR were chosen so as to produce a
comparable rate of CO 2 assimilation in an atmosphere containing 1% 02 and 500 ~11 -~ CO 2. (A) CO s was not added, and the
oxygen content of the gas phase was varied between 21 and I%. (B) 35/zl 1-' CO 2 was present in the gas phase whose oxygen
concentration was varied as in (A). For further explanation, see text.
with o r w i t h o u t 35/~11-1 C O 2 simultaneously
present. T h e intensities of the different b e a m s
were balanced in a separate experiment so as to
p r o d u c e identical p h o t o s y n t h e t i c C O 2 u p t a k e in
an a t m o s p h e r e containing 1% oxygen and
5 0 0 / z l 1-1 C O z. In this situation, photorespiration is suppressed. In the separate assimilation
e x p e r i m e n t 100 W m -2 far-red light 2produced as
m u c h C O 2 u p t a k e as 4 . 2 W m red light
(0.047 nmol cm -2 s - l ) . This was taken to reflect
the extent o f P h o t o s y s t e m II excitation by the
far-red b e a m which served to excite p r e d o m i n antly P h o t o s y s t e m I. T h e p u r p o s e of the experim e n t o f Fig. 1 was to obtain information on light
scattering, L i g h t - d e p e n d e n t changes in the scattering o f 535 n m light are k n o w n to indicate
c h a n g e s in the transthylakoid p r o t o n gradient
( K r a u s e 1973, K6ster and H e b e r 1982, Bilger et
al. 1988, but see H e b e r et al. 1986 and Brugnoli
a n d B j 6 r k m a n 1992 for precautions necessary in
the interpretation o f 535 n m signals).
I n Fig. 1A, experiments were p e r f o r m e d in the
a b s e n c e o f C O 2, in Fig. 1B with 35/~11 - I C O 2
p r e s e n t in the gas stream. This C O 2 concentration is close to the C O 2 c o m p e n s a t i o n point
which c o r r e s p o n d s to the intercellular C O 2 conc e n t r a t i o n in air, if water stress enforces complete stomatal closure ( C o m i c and Briantais
1991). A t the C O 2 c o m p e n s a t i o n point, p h o t o respiratory and respiratory C O 2 p r o d u c t i o n are
as fast as the p h o t o s y n t h e t i c refixation of evolved
C O z, but b o t h are much slower than light-satu-
453
was reversible on darkening. The difference
spectrum of the faster part of the signal is broad
covering both the range of the electrochromic
shift P515 (Junge 1977) and of light scattering
(Heber 1969). Figure 2 shows a similar difference spectrum (A2) measured in air with
500/~11-1 CO 2 in order to minimize the acceptor
limitations introduced in the experiment of Fig.
1. Insert I A in Fig. 2 demonstrates that the
kinetics of the signal seen at 522 nm follow close-
rated assimilatory electron flow in air. In the
absence of C O 2 from the gas stream entering the
cuvette, a considerable part of the respiratory
and photorespiratory CO 2 will escape from the
leaf so that light-dependent linear electron flow
is decreased even when compared with electron
flow at the CO 2 compensation point.
Figure
1A
shows that
far-red
light
( 1 0 0 W m -2) given alone in CO2-free air produced an increase in 535 nm absorption which
mm
50
m
L,O
.
30
A830
38
20
10
,5,525
3
1
-
-lo
-20
I
500
J
J
510
z
I
520
z
I
530
I
,
540
,
J
550
I
nm
Fig. 2. Difference spectra of far-red or flash-induced changes in apparent absorbance of a cabbage leaf. Ordinate in m m pen
deflection in the recordings (but see sensitivity bar in the figure). T h e insets show original traces close to 525 nm. T i m e scales and
recording sensitivities are identical in the three insets. Conditions: (A) T h e gas phase was air with 500 pAl -~ C O 2 to minimize
acceptor limitations. The intensity of the far-red beam ( R G 724) was 44 W m 2. Bars in A522 of the inset I A show that absorbance
changes were m e a s u r e d shortly after the dark/light and before the light/dark transition (A 1 and A2). Only A 2 is shown as a
light/dark difference spectrum. T h e dark/light difference spectrum A~ was similar. T h e inset I A also shows P700/plastocyanin
oxidation and reduction as AA830 in m e a s u r e m e n t s m a d e simultaneously with the A522 m e a s u r e m e n t s . (B) The gas phase was 21%
oxygen with 30 pJ l ~ C O 2 to introduce acceptor limitation. Red background light was low (2.8 W m 2) to permit linear electron
flow in spite of acceptor limitation. Far-red light ( R G 724, 90 W m 2) was turned on and off on top of the red as indicated in the
inset I B. T h e difference spectrum is shown under B. (C) The gas phase was 2% oxygen and C O 2 was absent to produce strong
acceptor limitation. Far-red light ( R G 724, 44 W m 2) was given as background. 1 s flashes of red (ca. 500 W m - 2 ) were given
every minute. T h e inset I c shows flash-induced changes in absorbance. Bars denote where m e a s u r e m e n t s were made. C 1 is the
m a x i m u m increase, C 2 the m a x i m u m decrease. C 3 was m e a s u r e d 8 and C 4 28 s after the flash. Only the difference spectra C1, C 2
a n d C 4 are shown. C 3 was similar to C 4.
454
ly the kinetics of the far-red induced oxidation
and of the slow dark reduction of P700 and plastocyanin.
The difference spectrum of the slow part of
the 535 nm signal in Fig. 1A which was almost
absent in 21% oxygen, but large in 1% oxygen,
had a peak at 535 nm. It is very similar to the
difference spectrum C 4 in Fig. 2, which is identified as a light scattering increase (Heber 1969).
Red light (4.2 W m -2) given alone after a dark
period in Fig. 1A produced no appreciable
change in absorption. Doubling of the red light
(to 8 . 4 W m - 2 ) , however, decreased transmission of 535 nm light. The difference spectrum of
this signal was very similar to that of the slow
light scattering increase caused by far-red light in
the presence of reduced oxygen levels, but there
was also a small component distinguishable by
fast kinetics in the signal which extends the
difference spectrum into the 520nm region of
the P515 signal.
When in Fig. 1A the red beam ( 4 . 2 W m 2)
and the far-red beam (100 W m -2) were
superimposed on one another, 535 nm transmission decreased far more than seen by doubling
the red light. The difference spectrum was
broad. It contained a slow light scattering component and a faster P515 component. The light
scattering component could be almost eliminated
by adding some CO 2 to 21% oxygen (Fig. 1B).
An almost pure far-red-dependent P515 signal
remained as shown in a different experiment in
the difference spectrum of Fig. 2B.
The increase in the signals seen in Fig. 1 when
red and far-red beams were superimposed cannot be due to Photosystem II excitation as shown
by a comparison with the signals brought about
by the doubling of the red beam. It must be
attributed to Photosystem I excitation by the
far-red light. Far-red light can increase the transthylakoid proton gradient by supporting coupled
cyclic electron transport. We conclude that the
experiment shows cyclic electron transport in a
C3 leaf.
However, even the low concentration of
35 kdl 1 CO 2 suppressed light scattering in the
presence of 21% oxygen (Fig. 1B), probably in
part by disrupting the cyclic pathway, as during
the interplay of assimilatory carbon reduction
and photorespiratory carbon oxidation NADPH
is consumed. NADP drains electrons from the
cyclic electron transport chain. Light scattering
may also be decreased by decreasing the transthylakoid proton gradient owing to increased
ATP consumption. It should be noticed that the
light scattering signal can be almost completely
quenched by decreasing the transthylakoid proton gradient to levels which are still able to
support appreciable carbon assimilation (Heber
1969, Dietz et al. 1984).
When photorespiratory carbohydrate oxidation was decreased by decreasing the oxygen
concentration, light scattering increased both in
the absence of CO 2 (Fig. 1A) and in its presence
(Fig. 1B) indicating not only decreased ATP
consumption but also increased cyclic electron
transport as shown by a comparison of the light
doubling experiments (8.4 W m -2 red light versus 4.2 W m-2 red plus 100 W m -2 far-red light).
In the presence of only 1% oxygen and in the
absence of CO: (Fig. 1A) excitation of Photosystem II by 8 . 4 W m - : red light (but not by
4.2 W m -z) was sufficient to reduce the electron
transport chain to such an extent that cyclic
electron flow (which is possible in principle also
under illumination with red light, but which requires some oxidized plastoquinone) was impeded. This is shown by a secondary suppression
of light scattering (i.e. the transthylakoid proton
gradient) after the initial light-dependent increase. It should be noted that additional Photosystem I excitation by the far-red beam not only
prevented such inhibition in the same experiment but also stimulated light scattering more
than at the higher oxygen concentrations. Obviously, Photosystem I is capable of controlling
Photosystem II so that excessive reduction of the
electron transport system, which leads to an
inhibition of thylakoid energization, is prevented.
The experiment in Fig. 1 is a demonstration of
'poising' in leaves. In experiments with thylakoids, Arnon has demonstrated a requirement of
cyclic electron transport for a balanced redox
situation of the electron transport chain which
was described as poising (Arnon et al. 1958).
Cyclic electron transport requires oxidized electron carriers between the two photosystems and
reduced carriers on the reducing side of Photosystem I. Neither full reduction of the chloro-
455
plast electron transport chain (Ziem-Hanck and
Heber 1980) nor excessive oxidation of the chloroplast N A D P system (Laisk et al. 1991) will
permit cyclic electron flow to occur.
Figure 3 shows light scattering changes of a
cabbage leaf in an atmosphere containing 15%
oxygen and either no CO 2 (A) or 500 ppm CO 2
(B). Light used for continuous illumination was
either red to excite both Photosystems II and I
or far-red to excite largely Photosystem I, or a
combination of both. From a third light source,
high intensity red flashes lasting 1 s were given
every 40 s both in the dark and during continuous illumination with red or far-red or red plus
A
~' 15% 0 2 , - CO2
off
~}
far-red light. For red background light, three
different intensities were used. When the far-red
light was given, it had a constant intensity of
100 W m -2. The intensity of the red flashes was
about 600 W m
Flashes given in the dark produced fast transient absorption increases with a difference spectrum very similar to that shown in Fig. 2 (C~).
They were caused by the electrochromic shift
P5~5 and indicated the rapid formation and
breakdown of a transthylakoid electrical field.
Continuous far-red light increased absorption
rapidly, and the difference spectrum was similar
to that shown in Fig. 2 (A2). The absorption
B
15% 0 2 , 500#1 1"1CO2
off
" off
6
~ off
IaA=O.
~
0TJ
-
~
~11[l ~ FR 0
R ~
¢30~
R
dark
FR+R
dark
dark
(30j j
~
,
~
5 rain
~}
•
(10)
.
FR ~ R ~
dark
{10~
[I
i
t , FR ~ R ~
¢2,5~
Oil
FR
On
On
FR + R
i
!
i dark
on
on
on
Fig. 3. Changes in apparent absorbance at 535 nm of a cabbage leaf produced by 100 W m 2 far-red light (FR) and three different
intensities of red light (R: 2.5, 10 and 30 W m 2) or combinations of red and far-red light. 1 s flashes of a broad band of red light
(600 W m 2 half-bandwidth from 625 to 755 nm) were given every 40 s. Half-bandwidths of red and far-red lights were as in Fig.
1, Arrows indicate onset and termination of illumination. (A) CO 2 was not added to the gas phase which contained 15% oxygen.
(B) 500 tzl 1-1 CO 2 was present in the gas phase which contained 15% oxygen. For explanation, see text.
456
changes produced by the flashes relaxed in the
presence of far-red light with biphasic kinetics
(not clearly seen because of low time resolution).
When continuous red light replaced the far-red,
apparent absorption at 535 nm remained low at
the lowest intensity (2.5 W m-2), but increased
progressively in Fig. 3A, but not in Fig. 3B, as
the intensity of the red light was increased to 10
and 30 W m 2. Since this increase, which was
seen only in the absence, but not in the presence
of CO2, had a difference spectrum which peaked
at 535 nm, it was mainly caused by increased
light scattering (Heber 1969) and indicated formation of an appreciable transthylakoid proton
gradient.
The combination of red and far-red light had
in the absence of CO2, but not in its presence,
large effects on the light-dependent changes in
apparent absorbance particularly at the lower
intensities of red light (Fig. 3A). As in Fig. 1,
the changes in apparent absorption caused by
red plus far-red light were larger than expected
on the basis of additive effects. Both P5~5 and
light scattering increased. On darkening, the fast
relaxation of P5~5 permits to distinguish the
electrochromic change from the more slowly relaxing light scattering change. It can be seen that
light scattering changes were appreciable in the
absence of CO 2 and largely absent in its presence
(except for a small and transient light scattering
signal seen when red light of 30 W m 2 was turned
on). In the presence of CO2, photosynthetic
energy consumption had suppressed the light
scattering changes.
Short red flashes produced very different effects in the absence and in the presence of CO 2.
Positive and rapidly reversible spikes were
caused by a transient increase in P515 (difference
spectrum C 1 in Fig. 2). Large negative spikes
seen only in the absence of CO 2 (Fig. 3A) had a
broad difference spectrum which distinguished
this signal from P5~5 and light scattering changes
(Fig. 2 C2). Its response to changes in the composition of the gas phase suggest that it is related
to a transient photoaccumulation of reduced
ferredoxin. The negative spikes were followed by
a slow light scattering increase (identified by the
difference spectrum shown in Fig. 2 C4) which
was maximal 10 to 20 s after a flash had been
given. Transiently increased thylakoid energiza-
tion indicated by these scattering increases was
particularly pronounced in the presence of a low
intensity red background light. It was less conspicuous, when the background scattering approached light saturation.
The flash-induced secondary increase in thylakoid energization observed in the absence of
CO 2 was not only completely missing in the
presence of CO 2 but actually replaced by a small
flash-induced decrease in apparent absorbance
suggesting flash-induced additional photosynthetic energy consumption.
Upon darkening, the system slowly reverted to
the state observed before illumination.
Figure 4 shows simultaneous recordings of
modulated chlorophyll fluorescence and apparent absorbance changes at 535 nm of a cabbage
leaf. In Fig. 4A, the oxygen concentration was
10% and CO z was absent. The leaf was flashed
every 40 s with high intensity red light (1 s) either
in darkness or in the presence of continuous
far-red or of red light. In some cases extra
flashes were given outside this routine to probe
for the state of leaf energization. As in the
experiment of Fig. 1, the intensities of the red
and far-red beams had been balanced in a separate experiment so as to cause comparable carbon assimilation in an atmosphere containing
500 t~ll -~ CO 2 and 1% oxygen.
Flashes given in darkness caused a transient
increase in the P5~5 signal (upper traces in Figs.
4A and B) and increased fluorescence transiently
from the minimum level F 0 indicating full oxidation of the primary quinone acceptor QA of
Photosystem II to a maximum level F M which
indicates its full reduction. The initial ratio (F MF0)/FM = ~cxc was close to 0.67 in Fig. 4A indicating some loss of the quantum efficiency of
excitation energy capture in Photosystem II
owing to the previous history of the leaf (Genty
et al. 1989). The maximum relative quantum
efficiency is close to 0.8 (Bj6rkman and Demmig
1987).
When far-red background light (134Wm 2)
was given, apparent 535 nm absorption increased
initially fast owing to the formation of a transmembrane field and then slowly owing to increased light scattering. Although the light scattering change indicated increased thylakoid energization, no energy-dependent fluorescence
457
A
m~
R
I
AA =0.~
~L
W
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Fig. 4. Simultaneous recording of 535 nm absorbance changes (upper trace) and of modulated chlorophyll fluorescence (lower
trace) of a cabbage leaf which was illuminated with far-red (134 W m -2, FR) or red (9.6 W m 2, R) background light. It also
received brief (1 s) pulses of high intensity red light (600 W m-2). Half-bandwidths of red light and of flashes were as in Fig. 2.
The far-red light had a half-bandwidth ranging from 714 to 766 nm. The intensities of the red and far-red beams were balanced so
as to produce comparable CO 2 assimilation in 1% oxygen containing 500/xl 1-~ CO 2. CO2 uptake in this situation was 0.1 nmol
m 2 s ~. Arrows indicate onset and termination of illumination. (A) The gas atmosphere contained 10% oxygen. CO 2 was absent.
The leaf was flashed every 40 s. Additional flashes were given as indicated, o.a. = optical artifact. (B) Before the experiment, the
leaf was illuminated for 1 h in a nitrogen atmosphere to produce some photoinhibition. The gas atmosphere was CO2-free air, and
the leaf was flashed every min. For further explanation, see text.
q u e n c h i n g was i n d i c a t e d in Fig. 4 A by a decrease
of m a x i m u m fluorescence levels ( F ~ ) u n d e r farr e d i l l u m i n a t i o n . This is surprising, b e c a u s e by
i n c r e a s i n g n o n - r a d i a t i v e e n e r g y dissipation, such
e n e r g i z a t i o n s h o u l d have d e c r e a s e d F ~ . H o w e v e r , a state t r a n s i t i o n i n d u c e d by far-red light
m a y h a v e i n c r e a s e d F ~ ( H o r t o n et al. 1990)
o b s c u r i n g the d e c r e a s e e x p e c t e d from t h y l a k o i d
energization.
B r i e f light pulses given o n top of the far-red
b a c k g r o u n d light had c o n s i d e r a b l e effects. W h i l e
light s c a t t e r i n g i n c r e a s e d slowly after the flashes,
c h l o r o p h y l l fluorescence d e c r e a s e d to a n d e v e n
s o m e w h a t b e l o w the F 0 level in Fig. 4A. W h e n a
s e c o n d flash was given a few s e c o n d s after the
first flash while light scattering h a d r e a c h e d its
m a x i m u m , the m a x i m u m fluorescence level F ~
c o r r e s p o n d i n g to full QA r e d u c t i o n was m u c h
d e c r e a s e d s h o w i n g that c o n s i d e r a b l e thylakoid
e n e r g i z a t i o n h a d d e v e l o p e d after the first flash.
B r i e f d a r k e n i n g after a flash d e c r e a s e d fluoresc e n c e slightly b e l o w its initial m i n i m u m level
458
indicative of full oxidation of QA at the beginning of the experiment, demonstrating quenching of the 'dark'-level fluorescence.
When the far-red background light was turned
off, 535 nm absorbance decreased in two kinetic
phases. The first phase was fast and indicated
relaxation of an electrochromic component
(P515) and the second was slow. It showed relaxation of the light scattering change. Surprisingly,
F M decreased somewhat in the dark, probably in
a reversal of the state transition. The transient
fluorescence decline to a very low level seen
after a flash in the presence of far-red light was
absent in the dark, and flashing a second time
shortly after another flash did not produce a
decrease in the maximum fluorescence level as
observed under far-red background illumination.
Red background light corresponding to the
far-red beam in its ability to excite Photosystem
II produced only a very small P515 signal (Fig.
4A). Light scattering increased slowly while F~
levels decreased indicating increased non-radiative energy dissipation (which was not seen
under a far-red background), but flashing did not
induce much additional light scattering. QA was
somewhat reduced under red illumination as
seen by the steady-state fluorescence level F
which was clearly above F 0. After QA was fully
reduced in a flash, it was only slowly reoxidized
as seen by the slow return of fluorescence to its
steady-state level. A second flash placed shortly
after a first flash failed to indicate increased
energization by a decrease in the maximum fluorescence level or by light scattering. QA occurred
slightly more reduced.
The data recorded in Fig. 4A show clearly that
under the conditions of the experiment flashinduced additional thylakoid energization can
only be produced in the presence of far-red light
which is known to excite Photosystem I. It is
neither observed as a post-illumination event in
the dark nor in the presence of red background
light whose intensity has been matched to that of
the far-red light so as to produce comparable
Photosystem II excitation.
The experiment shown in Fig. 4B is similar to
that of Fig. 4A with the exceptions that the
cabbage leaf had been illuminated before the
experiment for 1 h under anaerobic conditions in
order to cause partial photoinhibition of Photo-
system II and that the gas atmosphere was C O 2f r e e air. Apparently, this treatment not only
produced some increase in the level of F 0 (not
shown), but interfered also with the ability of
reduced QA to reoxidize after a brief flash. This
is indicated by the slow relaxation of fluorescence from the maximum level F M to its 'dark'
level F 0. In contrast to the experiment of Fig. 4A
where far-red background illumination failed to
quench flash-induced F~ fluorescence although it
increased thylakoid energization as indicated by
increased light scattering, far-red light decreased
F~ in the experiment of Fig. 4B by increasing
non-radiative energy dissipation. After the flash,
fluorescence decreased (much more clearly than
in the experiment of Fig. 4A) below the F 0 level
while light scattering increased transiently as it
had done in the experiment of Fig. 4A. The
observations made after the optical responses
induced by far-red light had relaxed in the dark
were similar to those made in the experiment of
Fig. 4A.
To investigate in more detail the state of leaf
energization after a 1 s high intensity red flash,
additional 1 s flashes were given different times
after the first flash had induced a slow transient
scattering response in a leaf which was illuminated with far-red background light (Fig. 5).
Apparent absorbance at 535 nm and modulated
chlorophyll fluorescence were recorded simultaneously. The gas phase contained 5% O 2 and no
CO 2. The relative quantum efficiency of excitation capture ~exc of the leaf had decreased from
about 0.8 to 0.55 as a consequence of previous
anaerobic pretreatment in the light which had
also caused some increase in the F 0 level of
fluorescence. This increased level is given as a
straight line in Fig. 5A. QA remained fairly
oxidized under the far-red background light as
shown by a comparison of F, F 0 and F~ levels.
By fully reducing QA, the first flash produced a
rise in fluorescence to the maximum level F~.
( 1 - Qq) = (F-F0)/(F~-F0) was 0.12. Disregarding some nonlinearity between (1 - Qq) and
QA reduction, this shows that about 12% of
Photosystem II reaction centers were closed by
the far-red background light. It should be noted
that no F 0 quenching was observed in the presence of background FR alone. Non-photochemical quenching in the presence of background
459
A
B
I
10
20
'5
•w•
FM'
c
qJ
o
,7
30 s
a" F O'
30 s
1
, ~J
?
!
D
0.5
\
',,o"
"o, QN'
/ 'V-°° "-,
~_ __~. . . . . ix--."_0._.- - z ~ ' -
_O
I
1
10
20
I
30 s
Fig. 5. Flash-induced changes in 535 nm light scattering of a cabbage leaf (upper traces), of chlorophyll fluorescence lower left
trace) and of fluorescence parameters (lower right: Flash-induced suppression of 'dark' fluorescence F, = 1 to the level F[~,
flash-induced non-photochemical fluorescence quenching Q; = (F~-F~)/Fd; flash-induced changes in the redox state of QA as
indicated by changes in the fluorescence parameter ( 1 - Q 0 ' (Fully reduced QA corresponds to (1 Qq) = t.) The flash (1 s,
600Wm 2) had a half-bandwidth ranging from 625 to 755nm. It was given on far-red background light (134Wm 2,
half-bandwidth: 714-766 nm). Subsequent flashes were given after various times to probe for fluorescence changes induced by the
first flash. An evaluation of these changes is compared with light scattering in (B) and an example of the procedure is shown in
(A). The gas atmosphere contained 5% 0 2. CO 2 was absent. Before the experiment, the leaf was subject to photoinhibitory
treatment as in the experiment of Fig 4B.
f a r - r e d was
QN = ( F M - F ~ ) / F M = 0 - 1 6 .
The
q u a n t u m efficiency of e l e c t r o n flow t h r o u g h
P h o t o s y s t e m II was q%s 11 = ( F ~ - F ) / F ~ = 0.50.
A f t e r t h e flash, t h e a p p a r e n t a b s o r p t i o n at
535nm increased and fluorescence decreased
b e l o w t h e F 0 level as it did in the e x p e r i m e n t s
d e s c r i b e d in Fig. 4. T h e ' d a r k ' - l e v e l of the chlor o p h y l l f l u o r e s c e n c e a f t e r t h e flash, F~, was det e r m i n e d b y switching off t h e b a c k g r o u n d f a r - r e d
light for s e v e r a l s e c o n d s at d i f f e r e n t t i m e s a f t e r
t h e first flash (not shown). A s e c o n d flash ind u c e d a d d i t i o n a l light s c a t t e r i n g a n d c a u s e d fluor e s c e n c e to rise to F ~ (Fig. 5A). A r e l a t i v e
m e a s u r e of QA r e d u c t i o n is then 1 - Qq = ( F -
F ~ ; ) / ( F ~ - F ( ; ) a n d a m e a s u r e of t h e f l a s h - i n d u c e d
i n c r e m e n t in n o n - p h o t o c h e m i c a l f l u o r e s c e n c e
q u e n c h i n g in O ; = ( F ~ - F d ) / F ~ .
F i g u r e 5B
s h o w s the d e p e n d e n c e of t h e s e f l u o r e s c e n c e s p a r a m e t e r s on the t i m e i n t e r v a l r b e t w e e n two
flashes. It can be s e e n that an i n c r e a s e in light
s c a t t e r i n g is a c c o m p a n i e d by an i n c r e a s e in nonphotochemical fluorescence quenching. Nonphotochemical fluorescence quenching declined
while light s c a t t e r i n g d e c l i n e d . C h a n g e s in F 0
w e r e , as e x p e c t e d , a n t i p a r a l l e l to c h a n g e s in
n o n - p h o t o c h e m i c a l fluorescence q u e n c h i n g and
to light s c a t t e r i n g (see also Fig. 4 A a n d B). It is
particularly remarkable that non-radiative ener-
460
gy dissipation in Photosystem II increased while
reduction of QA decreased. Since the increased
energization of the thylakoids indicated by increased light scattering is a Photosystem I effect
(Figs. 1, 3 and 4), the data show clearly that
Photosystem I can control Photosystem II not by
preventing reoxidation of photoreduced QA by a
large proton gradient but rather by controlling
reduction of QA.
In Fig. 6, 535 nm absorbance changes of a
cabbage leaf and photooxidation of PT00 as indicated by absorbance changes in the 830 nm region were simultaneously recorded. As should be
expected, the far-red background light (FR1) not
only produced a Pst5 signal (upper trace, seen as
fast increase in absorbance at 535 nm on illumination and as a fast decrease on darkening) and
slower light scattering changes, but also considerable photooxidation of PT00 (lower trace). By
exciting Photosystem II and transporting electrons from water into the intersystem chain between the two photosystems, the flashes resulted
in a partial reduction of PT00÷. As PT00 was slowly
reoxidized, light scattering increased. It decreased again when PT00 oxidation reached a
steady state. It is important to note that this
FR1
steady state did not represent full oxidation.
W h e n after darkening and illumination with
short wavelength red light and a second darkening period a far-red b e a m (FR2) was turned on
which not only had a lower intensity than the
far-red b e a m used initially ( R G 715), but was
also shifted further into the far-red, photooxidation of PT00 was increased by 10% c o m p a r e d with
the oxidation observed initially. As with F R I ,
flashes reduced photooxidized PT00- However,
reoxidation after the flashes was faster than in
the presence of FR 1 background, although the
flashes had produced comparable water oxidation by PS II, and not only Photosystem II but
also Photosystem I excitation was reduced by a
shift towards the far-red. Apparently, poising
was altered and electrons escaped via a linear
electron transport pathway instead being forced
into the cyclic pathway. This is also shown by
decreased light scattering and the absence of
extra scattering after the flashes. In the presence
of short wavelength red light, with PS II excitation comparable to that caused by FR1, the
flashes failed to produce appreciable changes in
the redox state of P700" m small flash-induced
oxidation was transient. As in the experiment of
dark
R
dark
FR2
off
[
e~
J
"<|
/ I
/
O~
/
\
A A830nm = 0.005
1 min
/ A A535am = 0.01
/
~n
off
off
o!
Fig. 6. Simultaneous recording of apparent absorbance at 535 nm (upper trace) and of absorbance at 830 nm (lower trace) of a
cabbage leaf, which was first illuminated with 134 W m -2 far-red light (FR1), then after a period of darkening with 9.6 W m -2 red
light (R) and after another period of darkening with 100 W m 2 far-red light (FR2). FR2 (half-bandwidth from 725 to 752 nm)
contained less light capable of exciting Photosystem II than F R 1 (half-bandwidth from 714 to 766 nm). The intensities of FRI and
R were balanced so as to produce comparable Photosystem II excitation. This was achieved by adjusting the intensity of R so as
to give the same rate of CO 2uptake in 1% oxygen with 500/xl I J CO2 as in FR 1(0.11 nmol cm -2 s-l). The gas atmosphere of the
flashing experiment contained 5% oxygen. CO2 was absent. The apparent time lag between the traces is caused by slight
displacement of the pens used for recording. For explanation, see text.
461
Fig. 4, much less extra light scattering was observed after the flashes than after flashing on top
of a FR~ background.
The observations suggest that the extra energization of the thylakoid system which controls
Photosystem II and is induced in the presence of
high intensity far-red background light by short
pulses of high intensity red light is caused by
cyclic electron flow around Photosystem I. The
light pulses serve to improve 'poising' of the
electron transport chain, i.e., to improve the
redox environment for coupled cyclic electron
transport which can support a transthylakoid
proton gradient.
Conclusions
The experiments shown in Figs. 1-6 were intended to examine proposals made previously
(Heber et al. 1990, Harbinson and Foyer 1991,
Wu et al. 1991) that photorespiratory CO 2 turnover behind closed stomata protects waterstressed leaves against photodestruction of the
chloroplast electron transport chain not only by
relieving excessive reduction of the electron
transport chain but also by making cyclic electron transport possible which, together with
linear electron transport to oxygen, can support
a transthylakoid proton gradient large enough
for controlling Photosystem II.
Nevertheless, the experiments of Figs. 1-6
were neither performed with water-stressed
leaves nor under conditions which simulate in all
respects gas exchange behind fully closed
stomata. Such conditions (high intensity illumination at the CO 2 compensation point in 21%
oxygen) were met in the experiments of the
preceding publication (Heber et al. 1992).
Rather, in the present work, CO 2 was decreased
or removed from the gas atmosphere and the
oxygen concentration was decreased so as to
slow photorespiratory carbohydrate oxidation
down. The reason for such manipulation was to
increase acceptor-side limitations to electron
flow. Figure 3 shows that it is more difficult to
demonstrate thylakoid energization by far-red
stimulated cyclic electron flow when thylakoid
energization is high (i.e. light scattering is large)
than when it is low. For this reason the acceptor-
side limitation of electron flow was increased by
partial or full exclusion of CO 2 from the gas
stream and by a partial reduction of the oxygen
concentration. In this situation, even low excitation of Photosystem II resulted in sufficient electron accumulation at the reducing side of Photosystem I to make cyclic electron transport easily
observable as increased light scattering in the
presence of increased excitation of Photosystem
I by far-red light (Fig. 1).
When the acceptor side of the electron transport chain is oxidized, as it is in the presence of
air levels of CO 2 under rate-limiting light, cyclic
electron flow cannot occur, because its redox
requirements are not met (Fig. 3). If electrons
are trapped by oxidized NADP they are unavailable for cyclic transport. In fact, Heber et al.
(1978) and Furbank and Horton (1988) have
observed inhibition of carbon assimilation by
antimycin A, an inhibitor of cyclic electron transport, only under high-intensity illumination, not
under rateqimiting light. In C 3 plants, linear
electron transport and Q-cycle activity may be
capable of satisfying the proton transfer requirements of assimilatory ATP synthesis under some
conditions, particularly at low light intensities
(Moss and Bendall 1984, Ort 1986). In Fig. 3,
the synergistic effect of red and far-red light on
light scattering, which is observed in the absence
of CO 2 and interpreted to indicate thylakoid
energization by cyclic electron flow, is conspicuously absent in the presence of CO 2.
On the other hand, cyclic electron transport
becomes inefficient if electron carriers between
the photosystems are reduced and thus unable to
accept electrons from the cyclic pathway (Fig.
1A, 1% oxygen, light R2). Under nitrogen, intact chloroplasts are unable to initiate carbon
reduction unless they are primed by a pulse of
oxygen (Ziem-Hanck and Heber 1980). Poising
conditions must be established and maintained to
enable cyclic electron flow to occur (Arnon 1977,
Arnon and Chain 1979). This is possible under
aerobic conditions even under high intensity illumination by the interplay of photorespiratory
CO 2 release and the refixation of the released
CO 2 which drains electrons from the electron
transport chain thereby avoiding excessive electron accumulation between the photosystems
and simultaneously providing poising conditions
462
(Wu et al. 1991, Heber et al. 1992). This situation occurs when stomata are closed under water
stress preventing access of external CO 2 to the
photosynthetic apparatus. Under high intensity
illumination, and with CO 2 at the compensation
point, the accumulation of Pv00+ (Harbinson and
Foyer 1991, Heber et al. 1992) shows that electron carriers such as plastocyanin and cytochrome f are largely oxidized. There is a large
redox gradient between reduced ferredoxin and
plastoquinone which facilitates electron donation
during cyclic electron transport around Photosystem I even if the plastoquinone pool is much
reduced (Moss and Bendall 1984).
Flashing the leaf with saturating red light
pulses (1 s) initiated light scattering changes in
the absence, but not in the presence of CO 2 (Fig.
3). The increased transthylakoid proton gradient
indicated by the scattering changes persisted for
many seconds after the flash. It was very small or
absent, when red light was given alone, but
appreciable or even considerable in the presence
of additional far-red light. In the presence of
CO 2, flashing the leaf failed to increase light
scattering because electrons were rapidly drained
from the electron transport chain by the availability of oxidized electron acceptors on the reducing side of Photosystem I. In its absence,
electron drainage was slow as shown by the slow
oxidation of P700 in Fig. 6.
The observations recorded in Figs. 4 and 5
show that cyclic electron transport can control
Photosystem II activity even when the primary
quinone acceptor QA of Photosystem II is not
much reduced. In far-red background light scattering at 535 nm transiently increased after the
red flash. Probing the state of Photosystem II by
fluorescence analysis revealed in addition to increased non-photochemical fluorescence quenching a decreased quantum efficiency of charge
separation in and of electron transport through
Photosystem II.
It is obvious that the relationship between the
redox situation of the electron transport chain
and cyclic transport which has first been outlined
by Arnon and his colleagues for a thylakoid
membrane system supplemented with ferredoxin
(Arnon et al. 1958), and which is demonstrated
here for leaves, can also explain observations
made with C4 plants. Mesophyil chloroplasts
require a poised electron transport system to be
able to produce ATP during coupled cyclic electron flow. Part of the phosphoglycerate produced
in the bundle sheath chloroplasts is known to
move into the mesophyll to be reduced in mesophyll chloroplasts. This reaction serves purposes
of poising. In malate-type C 4 plants such as
Maize, bundle sheath chloroplasts which oxidize
malate thereby generating N A D P H in a dark
reaction possess a reduced capacity of Photosystem II (Edwards and Walker 1983). This
avoids over-reduction which would inhibit the
cyclic electron flow needed for ATP synthesis
(cf. Furbank et al. 1990).
However, there is an important distinction
between C 3 and C a plants in their different ATP
requirements of carbon assimilation. In C 3
plants, a major role of cyclic electron flow appears to be in photoprotection, because assimilatory ATP requirements may he largely satisfied
by proton transport during linear electron flow to
acceptors as different as CO2, nitrite and oxygen
and by Q-cycle activity during linear flux. Photoprotection, on the other hand, requires control
of Photosystem II activity which in turn needs a
high level of thylakoid energization. We propose
that a balance between photosynthetic and
photorespiratory electron flows provides the
backstage for proper poising which brings cyclic
electron flow into play when high thylakoid energization is needed to prevent damage to the
photosynthetic apparatus. In C 4 plants, an important additional role of cyclic electron flow is
to facilitate the extra ATP synthesis needed for
their more specialized photosynthetic process
(Furbank et al. 1990).
Acknowledgements
This work was supported within the Sonderforschungsbereich 251 of the University of
W/irzburg and by the Stiftung Volkswagenwerk.
E.K. acknowledges support by the GottfriedWilhelm-Leibniz-Program of the Deutsche
Forschungsgemeinschaft and by the Ministry of
Education and Science, Romania, grant 91CH.
We are grateful to Drs Wolfgang Bilger and
Ulrich Schreiber for helpful discussions.
463
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