Photosynthesis Research 34: 433-447, 1992.
O 1992 Kluwer Academic Publishers. Printed in the Netherlands.
Chloroplast energization and oxidation of P700/plastocyanin in
illuminated leaves at reduced levels of CO 2 or oxygen
Ulrich Heber, Spidola Neimanis, Katharina Siebke, Gerald Sch6nknecht & Eva Katona*
Julius-von-Sachs-Institut fiir Biowissenschaften der Universitiit Wiirzburg, Mittlerer Dallenbergweg 64,
D 8700 Wiirzburg, Germany
Received 26 February 1992; accepted in revised form 9 September 1992
Key words: chlorophyll fluorescence, cyclic electron transport, photorespiration, photosynthesis,
Photosystem II, proton gradient
Abstract
Chlorophyll fluorescence, light scattering, the electrochromic shift P515 and levels of some photosynthetic intermediates were measured in illuminated leaves. Oxygen and CO2 concentrations in the gas
phase were varied in order to obtain information on control of Photosystem II activity under conditions
such as produced by water stress, when stomatal closure restricts access of CO 2 to the photosynthetic
apparatus. Light scattering and energy-dependent fluorescence quenching indicated a high level of
chloroplast energization under high intensity illumination even when linear electron transport was
curtailed in CO2-free air or in 1% oxygen with 35/xl1-1 CO 2. Calculations of the phosphorylation
potential based on measurements of phosphoglycerate, dihydroxyacetone phosphate and NADP
revealed ratios of intrathylakoid to extrathylakoid proton concentrations, which were only somewhat
higher in air containing 35/xl 1-1 C O 2 than in CO2-free air or 1% oxygen/35/zl 1-~ CO 2. Anaerobic
conditions prevented appreciable chloroplast energization. Acceptor-limitation of electron flow resulted
in a high reduction level of the electron transport chain, which is characterized by decreased oxidation
of PT00, not only under anaerobic conditions, but also in air, when CO 2 was absent, and in 1% oxygen,
when the CO 2 concentration was reduced to 35/zll -I. Efficient control of electron transport was
indicated by the photoaccumulation of PT00+ at or close to the CO z compensation point in air. It is
proposed to require the interplay between photorespiratory and photosynthetic electron flows, electron
flow to oxygen and cyclic electron flow. The field-indicating electrochromic shift (P51s) measured as a
rapid absorption decrease on switching the light off followed closely the extent of photoaccumulation of
PT00+ in the light.
Abbreviations: F, F 0, F~, F M, F~-chlorophyll fluorescence levels; GA-glyceraldehyde; P515-field
indicating rapid absorption change peaking at 522 nm; Q A - primary quinone acceptor in Photosystem
II; Q N - non-photochemical quenching of chlorophyll fluorescence; Q q - photochemical quenching of
chlorophyll fluorescence
Introduction
In photosynthesis, two photosystems mediate the
* On leave from the Biophysics Department, University of
Medicine and Pharmacy 'Carol Davila', 8, Eroii Sanitari
Blvd., 76241 Bucharest, Romania.
endergonic oxidation of water and the formation
of what Arnon (1956) has termed assimilatory
power, i.e., the production of reductant in the
form of N A D P H and of phosphate energy in the
form of ATP. The photoreactions are essentially
irreversible. When light is abundant and electron
434
acceptors are in short supply as is frequently the
case under conditions of drought when leaves
close their stomata limiting access of CO 2, the
irreversibility of the photoreactions should be
expected to cause full reduction of reducible
components of the chloroplast electron transport
chain. Such reduction, which would lead to the
photoinactivation of electron transport, is never
observed in leaves under aerobic conditions.
Rather, electron carriers such as cytochrome f or
plastocyanine are frequently more oxidized in
the light in the absence than in the presence of
CO2. On the other hand, under anaerobic conditions considerable reduction is easily observed. It
has been concluded that oxygen can serve as an
efficient alternative electron acceptor when CO 2
is unavailable (Schreiber and Neubauer 1990).
However, oxygen reduction in the Mehler reaction is slow (Hosler and Yocum 1985, Wu et
al. 1991). Photorespiratory oxygen reduction and
the electron transport made possible in leaves by
the refixation of CO2, which is liberated during
photorespiratory carbohydrate oxidation, are
faster than the Mehler reaction is, but are still
far below the capacity of the electron transport
chain to transfer electrons to acceptors such as
CO 2. In consequence, a high oxidation level of
electron carriers in illuminated leaves with closed
stomata cannot be explained by an abundant
supply of electron acceptors. A main difference
between illumination of leaves or chlorplasts in
CO2-free air and in nitrogen, is that formation of
a large transthylakoid proton gradient is possible
only under aerobic conditions (Ziem-Hanck and
Heber 1980). The proton gradient not only
causes an increase in the chloroplast ATP/ADP
ratio and the phosphorylation potential, but is
also important in controlling Photosystem II activity (Weis and Berry 1987, Weis and Lechtenberg 1988, Genty et al. 1989, 1990b, Demmig
et al. 1989, Horton et al. 1990, Krause and Weis
1991) Acidification of the intrathylakoid space
during formation of the proton gradient can limit
electron donation to PS II from the water-splitting enzyme (Witt et al. 1986, Schreiber and
Neubauer 1987, 1989) thereby preventing a high
reduction level and subsequent photoinactivation
of the chloroplast electron transport chain. It is
the purpose of the present contribution to show
that reduction of electron carriers on the oxidiz-
ing side of Photosystem I occurs only at acceptor-limitations which exceed those likely to
occur in vivo even when the stomata of leaves
are tightly closed under extreme water stress. In
agreement with a recent proposal by Harbinson
and Foyer (1991), it is suggested that Photosystem I-dependent cyclic electron transport is
involved in the formation of a proton gradient
large enough to control Photosystem II when
electron flow to CO 2 is curtailed.
Materials and methods
Detached mature leaves of cabbage (Brassica
oleracea oleracea gemmifera), ivy (Hedera helix)
and beech (Fagus silvatica) grown in the Botanical Garden as well as of spinach (Spinacia
oleracea v. Polka 6510) and sunflower (Helianthus annuus L.) grown in the greenhouse were
used for the experiments. Part of the lamina of a
leaf was enclosed in a sandwich-type cuvette
while the petiole was kept in water. The gas flow
through the cuvette was 500 ml min-1. The composition of the gas could be varied while the
temperature was kept between 20 and 25 °C. Gas
mixtures were prepared using mass flow controllers (Tylan Corporation, Eching, Germany).
CO 2 and water contents in the gas stream were
determined using an infrared gas analyzer
(BINOS, Leybold Heraeus, Hanau, Germany).
The cuvette contained windows permitting illumination of the upper surface of the leaf with
short wavelength red light (filters K65 from
Balzers, Liechtenstein, and RG 630, or 610,
from Schott, Mainz, Germany, with half bandwidths ranging usually between 624 and 662 rim,
or between 600 and 662nm, respectively), a
broad band of red light (filters Calflex C from
Balzers and RG 630 from Schott, half bandwidth
from 625 to 755 nm) or far-red light (filters Calflex C from Balzers and RG 715 or RG 724 from
Schott with half bandwidths ranging from 709 to
773 (RG 715) or 715 to 757 nm (RG 724, thickness 2mm), or from 720 to 761nm (RG 724,
thickness 3 mm), respectively) or with two beams
containing red and far-red light. The system
allowed simultaneous measurements of CO 2 consumption, of changes in light transmission of the
435
leaf at a particular wavelength and of chlorophyll
fluorescence or P70o photooxydation. Apparent
changes in absorbance were measured at 522 nm
where the electrochromic signal (often termed as
P5~5) has its maximum and at 535 nm where the
light scattering signal peaks. Occasionally, the
light/dark changes in the electrochromic shift,
which gives information on the transthylakoid
electric field (Junge 1977), and in light scattering, which gives information on the transthylakoid pH gradient (K6ster and Heber 1982,
Bilger et al. 1988, Heber 1969), were determined
simultaneously at 535 nm where the P515 signal
can be distinguished from light-scattering
changes by its faster kinetics. The detecting device, a photomultiplier, was protected against
actinic light by two filters 9782 from Corning
Glass Works, Corning, New York, USA, and a
BG 18 filter from Schott, Mainz, Germany. The
measurement light was so weak as not to cause
detectable photosynthesis. Modulated chlorophyll fluorescence was measured with a PAM
chlorophyll fluorometer from Walz, Effeltrich,
Germany. Parameters of chlorophyll fluorescence (photochemical quenching of fluorescence
Qq, non-photochemical quenching QN, quantum
efficiency ~Ps n of electron flow through Photosystem II) were calculated from fluorescence
traces according to Schreiber et al. (1986), Bilger
et al. (1988) and Genty et al. (1989). For a more
detailed consideration of fluorescence parameters, see van Kooten and Snel (1990). P700 oxydation was monitored by measuring apparent
absorbance changes at 830nm using a PAM
fluorometer which was equipped with a proper
emitter-detector unit (Schreiber et al. 1988). Signals also contain a contribution by plastocyanin
which is oxidized and reduced together with P700
(Klughammer and Schreiber 1991). Measurements of dihydroxyacetone phosphate, phosphoglycerate and NADP were performed in leaf
extracts after freeze-clamping of leaves as described in Siebke et al. (1990) and Laisk et al.
(1991).
Results and discussion
Figure 1 shows a simultaneous recording of light
scattering at 535nm by a leaf of ivy and of
modulated chlorophyll fluorescence emitted from
the leaf under aerobic (A) and anaerobic conditions (B). CO 2 was absent from the gas stream
which was led over the leaf. The intensity of the
modulated exciting beam was very low and insufficient to cause measurable reduction of the secondary electron acceptor QA in the reaction
center of Photosystem II. Actinic illumination
was unmodulated and is not sensed by the fluorescence detector. The state of full oxidation of
QA is indicated by the fluorescence level F 0.
Three brief flashes of saturating actinic light
caused full reduction of QA which is indicated by
the transient fluorescence increase to the level
F~ in Fig. 1A. Simultaneously, there was a fast
transient increase ('spikes') in 535 nm absorption
by the leaf. As the difference spectrum showed a
maximum close to 522 nm, with a half bandwidth
of about 30 nm, the spikes are caused by the well
known electrochromic shift which indicates the
formation of the transthylakoid electric field P515
(Junge 1977). When actinic short wavelength red
light (100Wm -2) was turned on, fluorescence
first increased indicating reduction of QA and
then decreased while light scattering (with a
maximum at 535 nm) increased. The molecular
events which lead to increased light scattering
depend on the formation of a transthylakoid
proton gradient (Heber 1969). The secondary
decrease in fluorescence has first been observed
by Kautsky (Kautsky and Hirsch 1934) and is
termed Kautsky effect. It has been shown by
Krause (1973) to be caused, to variable extents,
by partial oxidation of QA and an increase in
nonphotochemical fluorescence quenching which
dominated the scene in Fig. 1A. In the presence
of oxygen, QA was more then 80% reduced by
the light. This is indicated by ( 1 - Q q ) = 0 . 8 ,
which is calculated from the height of the flashinduced fluorescence spikes during continuous
illumination and the extent of the rapid fluorescence decrease to the level F 0 seen after illumination was terminated (Schreiber et al. 1986).
The relationship between QA reduction and (1 Qq) is non-linear. A comparison of F'0 with F 0
shows that F 0 was suppressed during illumination. While F[~ subsequently returned to the F 0
level, light scattering declined. Simultaneously,
flash-induced spikes in fluorescence and P515 increased. The recovery of the fluorescence spikes
436
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Fig. l, Chlorophyll fluorescence and light scattering of an ivy leaf before, during and after illumination with 100 W m -2 short
wavelength red actinic light under aerobic (A) and anaerobic (B) conditions. CO2 was absent from the gas stream which passed
over the leaf. Every 5 min the leaf received an 0.8 s flash of red light (500 W m-2). For explanation, see text.
occurred in more than one phase (not shown, but
see Demmig and Winter 1988). Only the first
one is shown. The recovery of F 0 and the return
of ~Ps u, the quantum efficiency of electron flow
through Photosystem If, from an initial value of
about 0.8 to values close to 0.7 within about
20 min after illumination (Table 1) are reliable
indicators that the leaf had suffered little photoinactivation during illumination (Bj6rkman 1987,
Baker and Horton 1987).
When the experiment was performed in nitrogen (Fig. 1B), different observations were made.
In this case, light scattering increased only transiently when the actinic illumination was turned
on after air was exchanged for nitrogen. It remained low during continued illumination, and
the flash-induced Psi5 signal was suppressed.
Superficially, the fluorescence response to illumination was reminiscent of the Kautsky effect, but
the causes underlying the slow secondary decline
in fluorescence seen in Fig. 1B are totally different. Fluorescence quenching under aerobic conditions requires formation of a large proton
gradient. Such a proton gradient was absent
under anaerobic conditions as shown by the
light-scattering response (compare with the lightscattering increase shown in fig. 1A). Decreased
light scattering is accompanied by decreased
ATP/ADP ratios (Kobayashi et al. 1982). The
reversed direction of the flash-induced spikes
indicates photoaccumulation of reduced pheophytine, the primary electron acceptor in the
reaction center of Photosystem II which is a
highly efficient quencher (Klimov et al. 1985).
No photoaccumulation of reduced pheophytin is
possible in the presence of oxygen. The observations show that in the light the electron transport
chain was reduced to such an extent that not
437
Table 1. Fluorescence parameters measured for an ivy leaf before, during and after a 1 h period of illumination with 100 W m 2
short wavelength red light in the presence of either CO2-free air or nitrogen. Data are from the experiment of Fig. 1.
q~Ps, = (FM -- F)/Fu is an indicator of the relative quantum efficiency of electron flow through Photosystem II, (1 - Qq) of the
relative reduction of QA (full reduction is 1.0) and QN = (FM-F~)/FM of the extent of non-photochemical fluorescence
quenching. Qq is photochemical fluorescence quenching. F ..... /Fo,initia I describes changes in the F o level. A light dependent
decrease in Fo is considered to indicate a regulated increase in radiationless dissipation of excitation energy, whereas increased Fo
levels indicate photoinactivation of the electron transport chain
CO2-free air
nitrogen
Before
illumin,
During
illumin,
20 min after
iUumin,
Before
illumin,
During
illumin,
20 min after
illumin.
qbPsit
0.77
0.06
0.67
0.74
-~
0.24
1 -- O 4
0
0.8
0
0
1.0
0
ON
0
0.78
0.33
0
_a
0.18
1.0
0.68
0.95
1.0
2.2
2.41
F o ~ne~
Fo.initial
No meaningful calculation possible.
even cyclic electron transport could occur which
according to A r n o n and Chain (1977, 1979) requires 'poising', i.e., a proper balance between
reduction and oxidation of electron carriers.
D a r k e n i n g produced some QA oxidation, and the
direction of the fluorescence spikes was accordingly reversed. Further oxidation was observed
on admission of oxygen. H o w e v e r , even in the
presence of 21% oxygen, the F 0 level of fluoresr¢
cence was much increased to F 0 (even in the
presence of far-red light, not shown) and saturating flashes produced much smaller fluorescence
spikes than observed in the aerobic experiment.
rp
T h e increase to F0, the very slow increase in the
F!
variable fluorescence of the spikes (FM-F0) and
low values of (FM-F0)/FM (Table 1) indicate
severe photoinhibition of the leaf after exposure
to light under anaerobic conditions (Baker and
H o r t o n 1987).
T h e experiment shown in Fig. 2 was perf o r m e d at an oxygen concentration of 1% with a
cabbage leaf (A) and of 0.2% with an ivy leaf
(B). C O 2 was absent. The purpose of reducing
the oxygen concentration was to eliminate
photorespiratory reactions and to facilitate partial reduction of the electron transport chain
already at low intensities of red light so as to
provide 'poising' conditions (Arnon and Chain
1979). Normally, such conditions require high
light intensities. Different oxygen concentrations
were chosen because of quantitative differences
in the response of the different plant species to
oxygen. Flashes to the fluorescence level F M
indicate full reduction of QA as in Fig. 1. Partial
reduction during illumination with 3 W m -2 short
wavelength red light is indicated by increased
fluorescence of the cabbage leaf. It is obscured
by F 0 quenching, an indication of increased
radiationless dissipation of excess light energy, in
the ivy leaf. Light scattering increased under
illumination with 3 W m -2 red light (R) particularly in the ivy leaf, less so in the cabbage leaf. It
is a consequence of the formation of a transthylakoid proton gradient. A saturating flash
given in the presence of red light increased fluorescence to a much lower level F " than the flash
given in the absence of illumination: Energization of the thylakoids had caused non-photochemical fluorescence quenching QN, an indication of increased radiationless dissipation of excitation energy. Superposition of far-red light (FR)
on top of the red light further increased light
scattering. It also decreased steady-state fluorescence and increased QN as shown by the deIt
creased flash-induced fluorescence level F M.
Far-red light excites predominantly Photosystern I. Excitation of Photosystem II by the farred light was low. It was measured in a parallel
e x p e r i m e n t by measuring carbon assimilation of
the cabbage leaf in 1% oxygen containing
3 4 0 / x l l -~ C O 2. C O 2 uptake was 0.034nmol
cm - 2 S 1. The intensity of the short wavelength
438
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FR
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Fig. 2. Changes in 535 nm light scattering and absorbance and in modulated chlorophyll fluorescence produced in a cabbage leaf
(A) and in an ivy leaf (B) by a low intensity of short wavelength red light (3 W m -2) and a high intensity of far-red light (RG 9,
175 W m -z) given alone or in combination and by 0.8 s flashes of red light (500 W m 2). The oxygen concentration in the gas
phase was 1% in (A) and 0.2% in (B). CO 2 was absent. For explanation, see text.
red light was adjusted so as to give comparable
CO 2 uptake, or comparable PS II excitation by
the red beam.
The results of the experiment of Fig. 2 indicate
that increased thylakoid energization under farred light is a result of Photosystem I excitation
added to Photosystem II excitation. The effect of
Photosystem I excitation is also evident from the
residual energization remaining after R was
turned off. Both light scattering and non-photochemical fluorescence quenching were increased
in the presence of FR alone compared to scattering and fluorescence quenching in the presence
of R alone. Fluorescence returned after a transient flash-induced increase to a level distinctly
below steady-state fluorescence only in the presence of FR, not when R was present alone. Light
scattering was transiently increased (for more
than 30s) after a flash given on top of a FR
background. No comparable scattering increase
was observed when a flash was given on top of a
red background.
Table 2 shows that the quantum efficiency of
electron flow in Photosystem II was decreased
when FR was added to R. QA was more oxidized
in the presence than in the absence of the additional far-red beam. Apparently, far-red light
had decreased the electron pressure within the
electron transport chain. It had also increased
non-photochemical fluorescence quenching. We
conclude that coupled cyclic PSI-dependent
electron transport which is indicated by increased light scattering increases radiationless
dissipation of excitation energy.
Effective radiationless dissipation of light
energy should make charge separation in Photo-
439
Table 2. Fluorescence parameters measured for a cabbage leaf before, during and after illumination. The intensity of short
wavelength red light was 3 W m 2 and that of far-red light 175 W m 2 The data were calculated from the experiment shown in
Fig. 2A. For explanation of the fluorescence parameters, see legend to Table 1
qbps n
1 - Q,t
QN
Before
illumination
In red light
alone
In red plus
far-red light
In far-red
light alone
After
illumination
0.79
0
0
0.63
0.12
0.27
0.40
0.04
0.69
0.61
0.07
0.46
0.77
0
0.04
system I possible even if there is only limited
regeneration of electron acceptors. Figure 3
shows photooxidation of Pv0o in the reaction
center of Photosystem I and simultaneously oxidation of some plastocyanin which donates electrons to P700+ (Klughammer and Schreiber 1991)
on illumination of a predarkened spinach leaf
with short wavelength red light or with far-red in
air without CO 2 or with 600ppm CO 2. In the
following, plastocyanin oxidation will not be considered separately from the oxidation of P700
because it follows light-induced redox changes of
P700"
After several minutes of darkening to deacti-
K65+RG630
I'
RG715
t
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I
~,..~2,
I
21% 02
-b-+Q
o
13-
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Fig. 3. Changes in the redox state of PTo0in a spinach leaf
produced by short wavelength red light (K 65 and RG 630,
l l 0 W m 2) or far red light (RG 715, 175Wm -2) under
different atmospheric conditions. Only illumination with farred light caused almost full oxidation of PT~,0.
vate the photosynthetic apparatus, far-red light
caused fast initial photooxidation of 17700. This
was followed by a transient reductive phase
which is caused by reduction of electron carriers
by the PSII component of the far-red light
(Siebke et al. 1991). After the light activation of
Calvin cycle enzymes increased the availability of
electron acceptors, photooxidation of Pv00 increased to a maximum. Darkening caused slow
reduction o f PT00+ because the electron transport
chain was largely oxidized and electrons were in
short supply on the oxidizing side of Photosystem I. Under illumination with short wavelength red light, photooxidation of P700 followed
a pattern similar to that observed in far-red light
only when the experiment was performed in the
presence of CO 2. However, the extent of P~00
oxidation was reduced, and reduction of P700+ on
darkening was fast because the electron transport chain was more reduced when Photosystem
II excitation was strong. Different observations
were made when CO 2 was absent either from air
or from a gas mixture containing 1% oxygen in
nitrogen. In CO2-free air and after a prolonged
darkening period, appreciable oxidation of P700
could be observed only transiently even though
the presence of 21% oxygen facilitated electron
flow to oxygen in the Mehler reaction. In 1%
oxygen, the Mehler reaction is decreased. It is
not surprising, therefore, that what may be
termed over-reduction of the electron transport
chain prevented P700 photooxidation. The reversal of the direction of the trace seen in Fig. 3 on
illumination and darkening of the leaf in l%
oxygen compared to the other traces appears to
be caused by the photoaccumulation of reduced
ferredoxin in the light and by oxidation of the
reduced ferredoxin on darkening (Klughammer
and Schreiber 1991).
When a broad band of red light which in-
440
cluded considerable far-red light was used for
excitation instead of a short wavelength red
band, considerable photooxidation of PT00 was
observed in air even when the CO 2 concentration
was decreased to the CO 2 compensation point
(Fig. 4). However, after the leaf was fed through
the petiole with 60 mM glyceraldehyde for about
3 h, the extent of steady-state photooxidation of
PT00 decreased dramatically. By inhibiting phosphoribulokinase (Stokes and Walker 1972)
glyceraldehyde inhibits not only the Calvin cycle
but also photorespiratory reactions. Over-reduction in the presence of glyceraldehyde, but not in
its absence indicates that the main part of the
electron acceptors which make PT00 oxidation
possible in the presence of 21% oxygen is provided by photorespiratory reactions, not by the
Mehler reaction which is unaffected by glyceraldehyde. Thylakoid energization as indicated by
quenching of chlorophyll fluorescence is not prevented by glyceraldehyde (Wu et al. 1991). In
1% oxygen where photorespiratory reactions are
suppressed even in the absence of glyceraldehyde, steady-state photooxidation of PT00 was
similar with and without glyceraldehyde.
The role of photorespiration in providing electron acceptors for charge separation in Photosystem I is further explored in the experiment of
Fig. 5 which shows photooxidation of PT00 as a
function of incident light energy. Under photosynthetic conditions (340/zll -~ CO2, 21% oxygen), photooxidation of PT00 increased with light
intensity less steeply than under conditions
simulating performance of a leaf under drought
stress when stomata are closed. At a CO 2 compensation point of 35 ppm CO e, and in the presence of 21% oxygen, electron acceptors are
made available to the electron transport chain
behind closed stomata during the photorespiratory oxidation of carbohydrate and the refixation
of the evolved CO 2. In the presence of 35 ppm
CO 2 and 21% oxygen, even high light intensities
did not decrease PT00 photooxidation. In fact,
oxidation was increased compared to a situation
which permitted net CO 2 reduction although
CO 2 turnover is slow during photorespiration at
the CO 2 compensation point. Apparently, during
photorespiratory CO z turnover the activity of
Photosystem II is tightly controlled. However,
when at a constant CO z concentration of 35 ppm
AA
,!5s , l
I +GA,
[ PT00*
t
PT00 +
Spinach
~
34.0121
0,002
S
~
... -~'"
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Fig. 4. Illumination and darkening in a spinach leaf which
had been fed with the photosynthesis inhibitor glyceraldehyde (GA) through the petiole. A control leaf ( - G A ) was
left to stand in water. The gas atmosphere was either air
containing 3 5 / z l l -~ CO 2 or 1% oxygen in nitrogen with
3 5 / z l l i CO2.
I
I
I
400 Win-2
Fig. 5. PTo0photooxidation in spinach leaves as a function of
irradiance with a broad band of red light under different
atmospheric conditions. The first number assigned to a curve
denotes the CO 2 concentration i n / z l 1-1, the second number
the oxygen concentration in percent (example: 340/21 is air
with 3 4 0 / z l l ' CO2). A leaf fed with the photosynthesis
inhibitor glyceraldehyde (GA) produced the PTo0photooxidation shown in the G A curves. The insert shows a typical PT00
trace and illustrates the mode of evaluation of the data.
441
the oxygen concentration was decreased to 10, 5
and 1%, progressively higher reduction levels
were observed at high light intensities, whereas
low light intensities still permitted appreciable
photooxidation of PT00- Photorespiration is
known to decrease with decreasing oxygen concentration.
When CO 2 was completely eliminated from
the gas stream passing over the leaf, 21% oxygen
proved unable to maintain a high oxidation
status of Pvoo at high light intensities. Rather,
high light intensities decreased, by over-reduction, the PT00 oxidation observed at lower light
intensities. In contrast to this observation, Harbinson and Foyer (1991) have reported strong
PT00 photooxidation in CO2-free air at high light
intensities. In different experiments with leaves
of various plants, we have invariably observed
strong PT00 photooxidation in air when CO2 was
close to the CO 2 compensation level, but there
was considerable variety in PT00 photooxidation
in CO2-free air. In general, over-reduction was
indicated by decreased P700 photooxidation,
when the photosynthetic apparatus had been
deactivated by prolonged darkening, and when
high-intensity illumination was maintained for a
prolonged period of time (more than 15 min).
Figure 5 also shows photooxidation of PT00
when glyceraldehyde poisoning made oxidation
of NADPH in CO 2 assimilation and photorespiration impossible. As expected and shown already in Fig. 4, under these conditions PT00 oxidation was much decreased compared with oxidation in an unpoisoned leaf, when the light
intensity was high and the gas stream passing
over the leaf contained 35 ppm CO 2 and 21%
oxygen. Little difference was seen in a gas atmosphere containing 35 ppm CO 2 and 1% oxygen. Also, when electron pressure exerted by
Photosystem II was low at low light intensities,
comparable
PT00 photooxidation was possible
under different atmospheric conditions. At high
light intensities, photorespiratory CO 2 turnover
is needed to permit accumulation of PT00+.
Figure 6 shows an experiment with a leaf of
H e l i a n t h u s a n n u u s . The rapid reduction of PT00+
and the rapid relaxation of the electrochromic
shift at 522nm (P515) were simultaneously recorded after a period of illumination. As in the
spinach experiment of Fig. 5, PT00 photooxida-
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Z'-'~minx,
~
35121[
O/21
0,001
35/1
I
0
0
;
2 0
I
I
I
400 Win-2
Fig. 6. P7oo photooxidation and the electrochromic shift P515
in a sunflower leaf as a function of irradiation with a broad
band of red light under different atmospheric conditions. The
insert shows a typical change in apparent absorbance of the
leaf at 515 nm and the mode of evaluation of the electrochromic shift as a fast absorbance change in the light/dark
transition. For explanation of the numbers, see legend to
Fig. 5.
tion was increased at high light intensities under
conditions of photorespiratory CO 2 turnover
(35 pm CO2, 21% oxygen) compared to a situation which permitted net CO z reduction
(340 ppm CO 2, 21% oxygen). A high reduction
level of electron carriers was indicated by decreased photooxidation of 17700at high light in the
absence of CO 2 at 21% oxygen and in the presence of 35 ppm CO 2, when the oxygen concentration was reduced to 1% oxygen. The electrochromic shift observed as a fast decrease of
absorbance on darkening (see insert to Fig. 6)
was small even at high light intensities during
photosynthesis (340 ppm CO2, 21% oxygen), but
large when stomatal closure was simulated
(35 ppm CO 2, 21% oxygen). At high light intensities, it was suppressed in CO2-free air and even
more so in the presence of 1% oxygen and
35 ppm CO 2. There was a similarity between the
general response of P700 photooxidation to differ-
442
ences in the composition of the gas atmosphere
and that of the electrochromic shift.
Figure 7 extends these observations to beech
leaves. Two experiments are shown. In one (Fig.
7A-C), PToo photooxidation, the electrochromic
shift and light scattering were simultaneously
recorded in a shade leaf. The electrochromic
shift has a large shoulder at 535 nm and can
therefore be measured at 535 nm together with
light scattering from which it can be distinguished by its difference spectrum and different
kinetics. In the second experiment with another
shade leaf (Fig. 7D), chlorophyll fluorescence
was recorded and the energy-dependent part of
non-photochemical fluorescence quenching was
calculated assuming that energy-dependent
quenching is relaxed after 6 rain darkening.
The response of PT00 to illumination was similar in principle in the shade leaf of beech (Fig.
7A) and in sun-adapted leaves of spinach (Fig. 5)
and sunflower (Fig. 6). The only noteworthy
differences were that PT00 photooxidation in
P?00 ÷
F o g u s , shade leaf
35/2.~..______--o
~A
--<~?;;;"
CO2-free air was not lower at high than at
intermediate light intensities and that less light
was required to photooxidize P70o in the shade
leaf of beech than in the sun-adapted leaves. In
agreement with the observations recorded for
spinach and sunflower, PToo photooxidation was
larger under conditions simulating stomatal closure (35 ppm CO2, 21% oxygen) than when CO 2
was either high or absent, or when the oxygen
concentration was reduced. As shown already
for sunflower, the electrochromic shift displayed
similarities to the response of PT00 to illumination. It was larger in the presence of 35ppm
CO2/21% oxygen than at other compositions of
the gas phase. Similarities between P515 and PT00÷
are due to a large extent to the formation of an
electric field across the core of the thylakoid
membrane when PToo+ accumulates. This field
decays when PT00+ is reduced on darkening.
Simultaneously, the decay of the transthylakoid
proton gradient creates a diffusion potential. Together, both phenomena may explain why Psz5
L a 53!
C
tight
scattering
0,(,07!
0,002
/
0,001
0,( 05(
/i
' .'"
0,C 02~
/
f6~o ,L
o'
0
Y
P515 ]
A A
i,'P"
o oo,
A
'
B
~
~
"''"
~
~
-
--
35/21
- - - -A ....
A
~
J
1
l
I
J
-o
0/21
......................
.
0
~
[
Wm-2
•
0121
...............
~ - . . . . . . . . . . . . . . . -3~;;27-"
_; :_:__-_---~
0,6
e'340/21
/
0,4
°,°°,li'
0
0
/
/
/
to
o t
0,2t
r
i
200
400
I
Wm-2
t
600
0
I
0
200
I
I
I
400 Win-2
I
600
Fig. 7. Changes in P700 photooxidation (A), in the electrochromic shift P5~5 (B), in 535 nm light scattering (C) and in the energy
dependent part (QE) of non-photochemical fluorescence quenching (D) of a shade leaf of Fagus silvatica as a function of
irradiance with a broad band of red lights. Inserts show typical traces for light-dependent changes in apparent absorbance close to
828 nm (PT0o, A) and at 535 nm (P515 shift, seen as a fast absorbance decrease on darkening at 535 nm, and light scattering (1.sc.),
seen as a slow decrease in apparent absorbance at 535 nm on darkening). For explanation of the numbers, see legend to Fig. 5.
443
and P700 display similar responses to changes in
light intensity and composition of the gas phase
in Figs. 6 and 7.
Figure 8 shows relative quantum yields of
electron flow through Photosystem II as calculated from fluorescence data (Genty et al. 1989).
In the sun plant Helianthus, quantum yields were
not much decreased under photorespiratory conditions compared with photosynthetic conditions.
A strong suppression of charge separation was
seen only under conditions of high reduction
levels of electron carriers (see also Fig. 6). In a
shade leaf of Fagus, control of Photosystem II
was more strongly expressed by decreased quantum yields under conditions of photorespiration
than in Helianthus.
Light scattering exhibited little similarity to
either P700 oxidation or the P5~5 signal. As expected from its relationship to the transthylakoid
proton gradient, it was suppressed at low light
intensities when air levels of CO 2 were available.
In this situation, light energy is limiting photosynthesis and thylakoid energization is decreased
Netionthus
0,6
0,~
4
~-~__
340121
~.0.~ O,2
i
I
i
i
i
i
Fagus, shade leaf
o,6
~0,4
(t)
Q- 0,2
~
'
"
"
-.-.
_ 3_4_0_/2~
I
200
400
Win-2
600
Fig. 8. Relative quantum yield dPpsn of electron flow through
Photosystem II as a function of irradiance in a sunflower and
a beech leaf. Values were calculated from fluorescence data
according to Genty et al. (1989). For explanation of numbers, see legend to Fig. 5.
by photosynthetic energy consumption. Photorespiratory CO 2 turnover is slow. It consumes
less energy than net photosynthesis. Therefore,
light scattering is increased compared to light
scattering in the presence of high CO 2 levels. In
the absence of CO 2, or when the oxygen concentration is decreased to eliminate photorespiratory reactions, energy consumption is lower than
during photorespiratory CO 2 turnover. Light
scattering therefore rises steeply with light intensity. At high light intensities, light scattering was
lower than at intermediate intensities. To a large
part, this is a consequence of the mode of data
evaluation which is shown in the inset to Fig. 6.
The fast decrease in absorbance seen when the
light was turned off was attributed to Pst5. The
following increase shown in the inset before light
scattering slowly relaxed was not observed at low
light intensities. It increased with light intensity
and is neglected in the evaluation of the data
because of uncertainties of extrapolation.
What is important to note is that at intermediate and high intensities of actinic illumination light scattering was not much dependent on
the composition of the gas atmosphere, although
the extent of P700 photooxidation was strongly
affected by oxygen and CO 2. Over a broad range
of light intensities, light scattering was not lower
in CO2-free air or in 1% oxygen plus 35 pJl
CO 2 than in air containing either 340 or 35 txl 1 J
C 0 2 " P7oo oxidation, on the other hand, was
larger in air containing 340 or 35/xll t than in
C O r f r e e air or in 1% oxygen where reduced
oxidation was observed at intermediate and high
light intensities compared to low light. Apparently, control of Photosystem II by the transthylakoid proton gradient is insufficient to prevent over-reduction when linear electron flow is
curtailed by lack of electron acceptors.
Energy dependent fluorescence quenching QE
has also been proposed to be a useful indicator
of the state of the proton gradient (Krause 1973,
Briantais et al. 1980, Bilger et al. 1988). It can
be calculated from that part of the non-photochemical fluorescence quenching which is reversible after illumination within a time span of
6 min although the relaxation of the proton gradient is known to be much faster than this (Oja
et al. 1986). As should be expected from the
different fluxes and energy requirements of
444
photosynthesis, photorespiratory C O 2 t u r n o v e r
and photorespiratory carbohydrate oxidation,
O E w a s lowest under rate-limiting illumination,
when air levels of CO 2 were present, and highest
in the absence of CO 2. Very similar observations
were made with light scattering. As also seen
with light scattering, Q E w a s similarly high at
high light intensities in air without CO 2 or with
35 or 340ppm CO 2. Once again it must be
concluded from this and the strong dependence
of P700 photooxidation on the composition of the
gas phase that the proton gradient cannot control
Photosystem II to an extent sufficient to prevent
flooding of the electron transport chain with
electrons, when linear electron flow is decreased
much below the fluxes sustained by the interplay
between photorespiratory CO 2 release and the
refixation of the released CO 2.
Unfortunately, light scattering and fluorescence changes are only indirectly related to
changes of the proton gradient. They give no
direct information on its magnitude. Attempts
were therefore made to obtain information on
the proton gradient using a totally different approach. The assimilatory force F A is defined as
the product of phosphorylation potential and
redox state of the chloroplast NADP system
(ATP)
(NADPH)
F A - (ADP)(P~) (NADP +)
(1)
It can be calculated from measurements of phosphoglycerate and dihydroxyacetone phosphate at
the leaf level (Heber et al. 1987, Dietz and
Heber 1989). Problems and limitations of this
method have been discussed in detail (Siebke et
al. 1990) and need not be reiterated here. If F n
is known, additional information on the redox
state of the chloroplast NADP system permits
calculation of the chloroplast phosphorylation
potential. Information on the chloroplast NADP
system can be obtained by NADP measurements. In the cytosol, the N A D P H / N A D P ratio
is high in leaves both in the dark and in the light
(Heber and Santarius 1965). Measured NADP is
therefore largely of chloroplast origin not only in
the dark but also in the light. With chloroplastic
NADP and total pool sizes (NADP + NADPH)
known, chloroplast N A D P H / N A D P ratios can
be calculated (Laisk et al. 1991). Under the
simplifying assumption that phosphorylation
potentials are close to equilibrium with the proton motive force (Grfiber 1990), and that the
membrane potential component of the proton
motive force can be neglected in this consideration, the following relation will hold:
AGATP = AGo,ATP + RT ln(ATP)/((ADP)(Pi) )
= 3(RT ln(H + ) J ( H + )o)
(2)
It can be seen that it is possible to calculate
proton gradients from the standard free energy
of ATP hydrolysis and known phosphorylation
potentials. The H+/ATP ratio of ATP synthesis
at the thylakoid membrane is assumed to be 3
(Junge et al. 1970, Portis and McCarty 1976).
Calculations shown in Table 3 suffer from the
uncertainties brought about by the assumptions
made. Also, we have not considered binding of
N A D P H to ribulose bisphosphate carboxylase
(Ashton 1982). If we had considered it, calculated phosphorylation potentials and calculated
ApH values would have been somewhat higher
than shown in Table 3. Nevertheless, as the main
purpose is to compare ApH values at different
compositions of the gas phase, the generally
similar ApH values calculated for spinach leaves
which had been illuminated in 21 or 1% oxygen,
with either 340 ppm, 35 ppm or no CO 2 present,
appear to be meaningful. The results are in fair
agreement with the conclusion from the biophysical measurements of light scattering and
fluorescence (Fig. 7) that energization as expressed by ApH is similar under high intensity
illumination at the very different atmospheric
conditions. Nevertheless, the calculated ratios of
the intrathylakoid proton concentration to the
stromal proton concentration ((CH)i/(CH) o in
Table 3) also reveal interesting detail. If stromal
proton concentrations are considered comparable under the different conditions of gas phase
composition, the highest intrathylakoid p r o t o n
concentration was calculated for photorespiratory conditions (21%, 0 2, 35/zl1-1 CO2), the
lowest for conditions in which over-reduction
was observed (CO2-free air, 1% oxygen with
35/xl 1-1 CO2, see Fig. 3). The calculated proton
concentration for photosynthetic flux was between these extremes.
445
Table 3. M e a s u r e m e n t s of dihydroxyacetone phosphate and phosphoglycerate and of N A D P in leaves which had been
illuminated (300 W m 2 red light) in air or in l % oxygen with different concentrations of C O 2 were used to calculate F A and
chloroplast N A D P H / N A D P ratios (see text). From both, chloroplast phosphorylation potentials were calculated u n d e r
simplifying assumptions: that the H ÷/ATP-ratio of the thylakoid A T P synthetase is 3, that the bulk m e m b r a n e potential can be
neglected for A T P synthesis and that the chloroplast phosphorylation potential is close to equilibrium with the transthylakoid
proton gradient
Phosphorylation potential,
calculated from calculated
F A and calculated
N A D P H / N A D P ratios
AGATP, kJ mol ~
calculated ApH
(c~)~/(c~+~),,
21% 02,
340/xll iCO 2
21% 02,
35~tll ~COn
21% 0 2
noCO 2inair
1% 0 2,
35/xll 'CO,
n=4
n-5
n=5
n=5
692 ± 203
804 -+ 222
503 ± 259
399 ± 177
48.1 ± 0.75
2.81 ± 0.045
645 ± 65
48.5 ± 0.7
2.83 ± (I.04
676 ± 65
47.4 ± 1.93
2.76 ± 0.08
575 ± 106
46.8 ~- 1.2
2.73 + 0.07
537 + 87
Conclusions
Electron transport is needed for the formation of
the proton motive force which is the energy
potential for chloroplast ATP synthesis. When
stomata close under water stress, linear electron
transport must decrease because of decreased
availability of CO 2. A dramatically decreased
proton gradient is indicated by light scattering
under anaerobic conditions (Fig. 1), but not in
the presence of oxygen. In fact, thylakoid energization is increased under rate-limiting light when
CO 2 and oxygen are decreased below air levels
(Fig. 7). Oxygen is reduced to H20 2 in a reaction which does not consume ATP. The H20 2
formed oxidizes ascorbate which in turn is reduced by NADPH in another reaction that does
not consume ATP (Asada and Takahashi 1987).
Both reactions will contribute to the proton motive force (Schreiber and Neubauer 1990). However, it appears that, by itself, oxygen reduction
in the Mehler reaction is incapable of preventing
over-reduction of the electron transport chain
(Fig. 4). In contrast, effective control of Photosystem II was observed when photorespiratory
CO 2 turnover was possible (Figs. 4-7). Both
photorespiration and the refixation of evolved
CO 2 consume ATP and NADPH. The photorespiratory ratio of NADPH oxidation to ATP
consumption is only slightly higher than the
photosynthetic ratio. In low light, photosynthesis
is highly energy-efficient in the absence of much
light scattering (Heber 1969, Dietz et al. 1984).
It is difficult to see, therefore, how photorespira-
tion could, by sustaining some limited linear
electron flow, could contribute to the high light
scattering level observed under high intensity
illumination. Rather, it appears that the limited
linear flow is important in preventing over-reduction of the electron transport chain. Its very
limitation, on the other hand, should result in an
increased NADPH/NADP ratio, a condition
that permits cyclic electron flow by directing
electrons from the reducing side of Photosystem
I back into the electron transport chain. This
increases thylakoid energization. Figure 2 and
Table 2 show that Photosystem I-supported cyclic electron flow increases radiationless dissipation of excess excitation energy. Table 3 shows
that the ratio of the intrathylakoid to the extrathylakoid proton concentration is higher
under conditions of photorespiratory CO 2 turnover than when such turnover is restricted. We
conclude that cyclic electron transport and linear
electron transport to oxygen cooperate to form a
proton motive force which is large enough for an
efficient control of Photosystem II, a prerequisite
for photoprotection of the photosynthetic apparatus.
Acknowledgments
This work was supported within the Sonderforschungsbereich 251 of the University of
W6rzburg and by the Stiftung Volkswagenwerk.
E.K. gratefully acknowledges support by the
Gottfried-Wilhelm-Leibniz-Program
of
the
446
Deutsche Forschungsgemeinschaft and by the
Ministry of Education and Science, Romania,
Grant 91CH.
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