Plant Cell Physiol. 38(11): 1207-1216(1997)
JSPP © 1997
Regulation of Steady-State Photosynthesis in Isolated Intact Chloroplasts
under Constant Light: Responses of Carbon Fluxes, Metabolite Pools and
Enzyme-Activation States to Changes of Electron Pressure
Simone Holfgrefe1, Jan E. Backhausen1, Camillo Kitzmann2 and Renate Scheibe1'3
1
2
Pflanzenphysiologie, Fachbereich Biologie/Chemie, Universitat Osnabriick, D-49069 Osnabruck, Germany
Lehrstuhl fur Pflanzenphysiologie, Humboldt-Universitdt, D-10099 Berlin, Germany
The reactions of isolated intact spinach chloroplasts at
saturating light and CO2 to changes in steady-state electron
flow were followed at the various stages of photosynthesis.
Alterations in the rate of electron flow were induced by the
addition of oxaloacetate (OAA), nitrite or methyl viologen
(MV). Two types of effect can be distinguished: (1) When a
small fraction of the electrons produced are accepted by
OAA or nitrite (up to 20% of the electrons produced in
the light), the activation state of the NADP+-dependent
malate dehydrogenase (NADP-MDH) was strongly decreased, whereas qP and the rate of O2-production were increased. qN, the stromal metabolite pools and the [14C]CO2-fixation rate were only marginally influenced. (2)
Higher amounts of nitrite or MV decreased O2 production
and strongly inhibited [14C]CO2 fixation. This treatment
further increased the ATP/ADP ratio, but had little effect
on the NADPH + H + /NADP + ratio. The stromal concentrations of 3PGA, DHAP and FBP, and the rates of 3PGA
and DHAP export were drastically changed. In particular,
the DHAP/3PGA ratio increased, and the rate of 3PGA export was decreased by minor changes in the rate of electron
flow. Addition of high amounts of nitrite or MV, but not
of OAA decreased the activation states of NADP-MDH
and fructose 1,6-bisphosphatase (FBPase), while the activation states of NADP+-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase
(PRK) remained unchanged under all conditions.
Redox-modulated chloroplast
oleracea — Thioredoxin.
enzymes
—
Spinacia
The ability of green plants to perform photosynthesis
in a wide range of environmental conditions is brought
about by several regulatory mechanisms. Electron supply
is frequently in excess to the amount required for CO2
fixation or photorespiration (Stitt 1986, Backhausen et al.
1994) and would lead to overreduction of the electron transport chain, if no other electron acceptors were present.
Electrons and/or ATP are also used to drive N- and S-assimilation, the synthesis of starch, protein and fatty acids,
and secondary metabolism. Additional electrons can also
be directed towards oxygen or into cyclic electron transport. Light/dark-modulation of chloroplast enzymes, mediated by the ferredoxin/thioredoxin system, is itself a reaction that consumes electrons. It is an important mechanism
which adapts metabolism in the chloroplast stroma in
order to prevent limitations caused by the lack of ATP or
by the restricted availability of electrons.
The light/dark-modulated Calvin-cycle enzymes,
GAPDH, FBPase, SBPase and PRK (Buchanan 1991), are
potentially rate-limiting regulatory enzymes. The same is
true for enzymes such as NADP-MDH being the regulated
enzyme of the malate valve (Scheibe 1990). The activity of
these regulatory enzymes is directly dependent upon the
Key words: CO2 fixation — Malate valve — Redox poising — rate of electron flow through the ferredoxin/thioredoxin
system that will shift the steady state in the redox cycles of
the target enzymes towards the reduced active enzyme
Abbreviations: ARC, anabolic reduction charge [NADPH +
HV(NADP+ +NADPH + H+)]; DHAP, dihydroxyacetone phos- forms (Scheibe 1991). In addition, intermediates of the
phate; EC, energy charge [(ATP+0.5 ADP)/(ATP+ADP +
Calvin cycle act as effectors for these redox cycles (Faske et
AMP)]; FBP, fructose 1,6-bisphosphate; FBPase, fructose 1,6-bis- al. 1995).
phosphatase (EC 3.1.3.11); Fd, ferredoxin; FNR, ferredoxinUnder conditions where the redox pressure decreases
NADP+ reductase (EC 1.18.1.2); FTR, ferredoxin-thioredoxin
reductase; GAPDH, NADP+-dependent glyceraldehyde 3-phos- due to removal of electrons the rate of CO2 fixation will be
phate dehydrogenase (EC 1.2.1.13); MV, methyl viologen; determined by the most sensitive step. In order to elucidate
NADP-MDH, NADP+-dependent malate dehydrogenase (EC the critical steps that determine the observed rates of car1.1.1.82); OAA, oxaloacetate; 3PGA, 3-phosphoglycerate; Pi, bon assimilation, and to identify the responsible factors,
inorganic phosphate; PRK, phosphoribulokinase (EC. 2.7.1.19); various reagents that influence redox state and energy
qP, photochemical quenching of the variable chlorophyll fluorescence; qN, non-photochemical quenching of the variable chloro- charge of the chloroplast were added during steady-state
phyll fluorescence; RuBP, ribulose 1,5-bisphosphate; SBPase, CO2 fixation. The rates were varied by OAA, nitrite or MV
sedoheptulose 1,7-bisphosphatase (EC 3.1.3.37); Td, thioredoxin. as electron acceptors, added at various concentrations. All
3
Corresponding author.
of these components cause a removal of electrons without
1207
1208
Regulation of steady-state photosynthesis
ATP consumption, but there are large differences in their
mode of action. OAA is reduced to malate by NADPMDH under consumption of NADPH + H + . The rate of reduction is controlled by the activation state of the NADPMDH (Backhausen et al. 1994). In contrast, nitrite and MV
accept electrons from reduced ferredoxin. The reduction of
nitrite, being a physiological electron acceptor, is catalyzed
by nitrite reductase (Vega et al. 1980). Since no regulation
of this enzyme is known, the rate of nitrite reduction is
solely dependent upon the concentration of nitrite. However, at higher concentrations of nitrite its accumulation
decreases the stromal pH (Purczeld et al. 1978), leading
to artifactual results (Robinson 1988). MV is an artificial
electron acceptor which catalyzes electron transfer from
PSI to oxygen (Asada et al. 1990) and produces O2 radicals. Superoxide dismutase and the components of the
ascorbate-glutathione cycle serve to detoxify the active oxygen species (see Polle 1996). From the effects of the various
electron acceptors upon the enzyme-activation states and
on the metabolic situation inside the chloroplasts, regulatory and limiting steps within the Calvin cycle could be identified.
Material and Methods
Cultivation of spinach, chloroplast isolation, measurements
of O2 production, chlorophyll fluorescence and CO2 fixation—
Spinach (Spinacia oleracea L., var. US hybrid 424) was grown
hydroponically according to Walker (1988). Isolation of chloroplasts, incubation conditions, and determination of intactness, fluorescence parameters, rates of O 2 production and [l4C]COj fixation were as described previously (Backhausen et al. 1994). For all
experiments, only chloroplasts with an intactness of 92% and
above were used. After 2 min in the dark, the chloroplasts were illuminated with white light of 700 fiE m~2 s~'. The CO 2 concentration was saturating (5 mM NaHCO 3 at pH 7.6). The electron acceptors (OAA, nitrite and MV) were added after steady-state
photosynthesis was reached (as indicated by O 2 evolution and chlorophyll fluorescence). The composition of the medium was chosen
to be optimal for high rates of CO 2 fixation under control conditions. The supply of Pj (0.2 mM) was optimal, and did not become
limiting over the time period in which the samples were taken. All
experiments were performed at 15°C.
Measurements of metabolites—Samples for the measurements of DHAP, FBP and 3PGA were obtained by silicone-oil
centrifugation according to Heldt (1980). 3PGA, DHAP and FBP
were determined enzymatically (Stitt et al. 1989) to get stromal metabolite concentrations and the rates of export. The samples for
the determination of ATP, ADP and AMP were obtained by
silicone-oil centrifugation to concentrate the samples. Six samples
were pooled for each determination. ATP, ADP and AMP were
measured by a modified method of Hampp (1985) in a luminometric assay. The determination of NADP + and NADPH + H + was
performed by enzymatic cycling according to Slater and Sawyer
(1962) as described in Backhausen et al. (1994). From the measured concentrations of NADP + and NADPH + H + , the ARC was
calculated according to Andersen and von Meyenburg (1977). The
stroma volume of the chloroplasts was determined to be 27 p\ (mg
Chi)" 1 , using the method of Heldt (1980).
Enzyme measurements—For the determination of the activation states of the redox-modulated enzymes, the extraction
method described for NADP-MDH in Backhausen et al. (1994)
was used. Full activation of GAPDH and activity assay were performed by the method of Baalmann et al. (1994). Full activation
and activity assays of FBPase and PRK (Faske et al. 1995) and of
NADP-MDH (Backhausen et al. 1994) were as described.
Results
Effects of electron acceptors on the photochemical and
non-photochemical fluorescence quenching—The addition
of electron acceptors to isolated intact chloroplasts during
CO2 fixation results in distinct changes in chlorophyll fluorescence indicating effects upon the redox state and energization of the thylakoids. Both, qN and qP show the typical
transients until steady-state photosynthesis is reached after
3-4 min of illumination (data not shown). The effectors
were added after 4 min, and in Fig. 1, the values obtained
after 6 min of illumination are shown. In all experiments,
an inverse relationship between qP and qN is observed.
Relatively strong increases in qP occur upon the additions
of weak electron acceptors such as 0.2 and 2 mM OAA,
and 0.2 mM nitrite (Fig. 1A). The new, stable qP values are
reached within 1-2 min, while the qN remains more or less
unchanged under these conditions (Fig. IB). In contrast,
with 2 mM nitrite or with MV, qP is as low as under control conditions (Fig. 1A), but qN increases significantly
(Fig. IB). With MV, however, qP and qN does not reach
stable values (data not shown). Namely qP decreases continuously, reaching a value of 0.16 after 10 min, while qN
increases to 110% of the controls.
0.3
I T I
0.25
A
h
0.2
B
0.7 h
T T T
T 1 1
0.6 H
0.5
Control
11.2 mM 2mM 0.2 mM 2 mM 2jiM
OAA OAA nitrite nitrite MV MV
Fig. 1 Photochemical (qP) and non-photochemical (qN) quenching of the variable chlorophyll fluorescence. Data were taken
2 min after the addition of the effectors (at 6 min of illumination).
Regulation of steady-state photosynthesis
0.4
0.2
control
0.2 mM 2mM
Q A A
QAA
0.2 roM 2 mM 2 fiM 10 )iM
n.tr.te
n.trlt^
M V
M V
Fig. 2 Redox state of the stromal NADPH + H + /NADP + system under the various conditions. Samples were taken under standard conditions after 6 min of illumination (2 min after the addition of the effectors) and subjected to silicone-oil centrifugation.
ARC=NADPH + H + / ( N A D P + + N A D P H + H + ) .
Effects on the energy carriers—The addition of the electron acceptors also affects the ARC (Fig. 2). In all cases, the
ARC is lower than in the controls indicating a relief of overreduction. With 0.2 mM OAA, the NADPH + H + concentration decreases slightly from 0.12 mM in the controls to
0.109 mM, while with \0fiM MV, which causes the largest
deviation from the controls, 0.092 mM NADPH + H + is determined.
For the ATP/ADP ratio, the addition of the electron
acceptors has more pronounced consequences. While weak
electron acceptors (OAA or 0.2 mM nitrite) result only in a
small increase to 105% of the control value (Fig. 3), higher
concentrations of nitrite and MV cause significant increases. Under these conditions, the concentration of ATP
increases from 0.29 mM in the controls to 0.48 mM, while
ADP decreases from 0.32 mM to 0.21 mM. It is interesting
to note that part of the change in the ATP/ADP ratio is
balanced by a decrease in the AMP concentration (data not
shown). In the controls, the AMP content is 0.09 mM, with
2.0
1.0
—
0.5
nn
Control
0.2 mM
2mM
0.2 mM
OAA
OAA
nitrite nitrite MV
2 mM
2pM
IOJIM
MV
Fig. 3 ATP/ADP ratios under the various conditions. Samples
were taken under standard conditions after 6 min of illumination
(2 min after the addition of the effectors) and measured after
silicone-oil centrifugation.
1209
OAA or 0.2 mM nitrite it decreases to 0.06 mM, and under
all other conditions, it is too low to be determined.
Effects on enzyme-activation states—The effects of
the additional electron acceptors on the activation states
of three enzymes of the Calvin cycle, FBPase, PRK and
GAPDH, and of the key enzyme of the malate valve,
NADP-MDH, were analyzed (Fig. 4). Apart from differring time courses of activation during induction of CO 2 assimilation under control conditions, their responses upon
the addition of electron acceptors are also different.
FBPase is inactive in the dark. Upon illumination, its
activation state in the controls increases steadily for
6-8 min to reach an activity of 55 ^mol (mg Chi)" 1 h ~ \ corresponding to 45% of the full activity. The addition of
0.2 mM OAA or of 0.2 mM nitrite (Fig. 4A, B) or of both
together (Fig. 4C) has minor effects on the activation state
of FBPase. Only the steady increase in the activation state
that occurs in the controls is slowed down or stopped. With
OAA, there is no difference between concentrations of
0.2 and 2 mM (Fig. 4A), but upon the addition of 2 mM
nitrite, a strong decrease of FBPase activity is observed
(Fig. 4B). The addition of MV leads to a similar decrease of
FBPase activity (Fig. 4D).
The activation state of the GAPDH in the light is regulated in a complex manner by dissociation of the reduced .
enzyme, which is mediated by its substrate l,3bisPGA
(Baalmann et al. 1995). Reduction of the enzyme does not
appear to require high electron pressure, and the time
course of the enzyme activation parallels the concentration
of l,3bisPGA in the stroma (Baalmann et al. 1994). In the
dark, an activity of 150-200 /nnol (mg Chi)" 1 h" 1 is determined in the standard assay (at 50 ftM 1,3bisPGA). Upon illumination, the apparent enzyme activity increases 5-fold
to about 700 ^mol (mg Chi)"1 h" 1 and remains stable. The
maximum activity for this enzyme is about 1,200 ftmol (mg
Chi)" 1 h" 1 . None of the effectors added has any significant
effect on the activation state of this enzyme. Only a small increase with OAA and nitrite added together, and a small
decrease with 4 ftM MV can be observed (Fig. 4E, F, G, H).
In the controls, the activation state of the PRK reaches
100% (equal to l,600^mol (mg Chi)" 1 h~') within a short
time and remains stable during the time of the experiment.
It is hardly influenced by any of the effectors (Fig. 41, J, K,
L).
In contrast, the activation state of NADP-MDH responds strongly to changes in the redox situation. The enzyme is essentially inactive in the dark, and upon illumination, a typical time course of activation is obtained. The
activity increases rapidly, reaching a maximum after 2 min
and then temporarily decreases. After 5 min, the activation
state is stabilized at 54% of the total activity (corresponding to 86//mol (mgChl)" 1 h" 1 ). The addition of all electron acceptors decreases the NADP-MDH activation state.
Upon addition of OAA, the activation state decreases to
Regulation of steady-state photosynthesis
1210
FBPasc
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NADP-CAPDH
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Fig. 4 Time courses of the activation states of FBPase (A-D), GAPDH (E-H), PRK (I-L) and NADP-MDH (M-P). Isolated intact
chloroplasts were illuminated and, as indicated by the arrows, after 4 min, 0.2 (•) or 2 mM OAA (•), 0.2 (A) or 2 mM (•) nitrite, or 2 (•)
or 4 iXM MV (•) were added. For the combined addition of 0.2 mM nitrite and 0.2 mM OAA (*), nitrite was added after 3 min, OAA was
added 1 min later, (o), untreated control. The activation states are shown as percent activity of the total capacity.
50% of the control value, but there is no difference between
0.2 and 2 mM OAA (Fig. 4M). Compared to 0.2 mM OAA
(Fig. 4M), a stronger decrease of the activation state is obtained with 0.2 mM nitrite, while 2 mM nitrite leads to a
further decrease to less than 5% of the control value within
1 min (Fig.4N). The combined addition of 0.2 mM OAA
and 0.2 mM nitrite also leads to a very effective decrease to
about 15% of the control (Fig. 4O). Similar effects are observed with MV (Fig. 4P), but here the activity decreases
steadily.
Effects on the levels of Calvin-cycle intermediates—
For a further analysis of the effects of the various electron
acceptors on the Calvin cycle, changes in the concentrations of 3PGA, DHAP and FBP were analyzed. In the
dark, stromal pool sizes of these intermediates are very
small. With the onset of illumination, their concentrations
increase in parallel with the rates of O2 evolution and CO2
fixation, and remain stable when steady-state photosynthe-
sis is reached (data not shown). In the controls, the stromal
concentration of 3PGA was found to be 2.8 mM, which is
20 times higher than that of DHAP or FBP (Fig. 5). Addition of electron acceptors decreases the 3PGA concentration in every case, and the decrease is the larger, the more
the redox state, as deduced from the activation state of the
NADP-MDH, is lowered (Fig. 5A). With OAA or 0.2 mM
nitrite, only a small decrease to 90% of the control can be
observed. Similar values are obtained upon the combined
addition of OAA and 0.2 mM nitrite (not shown). Again,
there is no difference between 0.2 mM and 2 mM OAA.
With 2 mM nitrite or 10 fiM MV, the 3PGA concentration
decreases to 0.75 or 0.45 mM, respectively, which is only
30% or 15% of the control value (Fig. 5A).
The changes in the stromal concentrations of FBP and
DHAP are in the opposite direction. FBP and [DHAP +
GAP] are in equilibrium by the aldolase and triose phosphate isomerase reactions, but according to Edwards and
Regulation of steady-state photosynthesis
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0.2 mM 2mM (12 mM 2 mM 2fiM
Cunt nil
OAA OAA nitrite nitrite MV MV
Fig. 5 Stromal concentrations of 3PGA (A), DHAP (B) and
FBP (C), and the ratio of DHAP/3PGA (D). Samples were taken
after 6 rain of illumination (2 min after the addition of the effectors) and subjected to silicone-oil centrifugation. For the calculation of their concentrations, a stroma volume of 27 /A (mg Chi)" 1
was used.
Fig. 6 Product-formation rates under the various conditions.
All data were obtained after 6 min of illumination. The rate of O2
evolution (A) was measured polarographically. The rate of CO2
fixation (B) was determined by incorporation of [I4C]CO2 into
acid-stable products. The export rates of 3PGA (C) and DHAP
(D) were calculated after enzymatic determination of these compounds in the supernatants after silicone-oil centrifugation.
Walker (1983), the equilibrium position is strongly influenced by the concentrations of FBP and [DHAP+GAP].
Therefore, the effects on the stromal DHAP concentration
(Fig. 5B) are different from the changes in the FBP pool
(Fig. 5C). With 0.2 mM OAA and/or 0.2 mM nitrite, the
DHAP and FBP concentrations increase to 130-135% of
the control value. When the FBPase-activation state is decreased by the addition of 2 mM nitrite or 10 fiM MV
(Fig.4B, D), there is a strong increase in the FBP pool
(Fig. 5C). Values of about 300% of the controls are reached, but the DHAP concentration increases only to 170% of
the control. The effects of the external electron acceptors
become clearer when the DHAP/3PGA ratio is regarded
(Fig. 5D). It is lowest in the controls (0.05), nearly doubled
with weak electron acceptors (0.11), and increases about
tenfold with 2 mM nitrite (0.43) or 10 ^M MV (0.68).
Rates of product formation—The product formation
during photosynthesis was analyzed by various methods.
1212
Regulation of steady-state photosynthesis
First, the total rate of carbon fixation was measured both
by determining the incorporation of [I4C]CO2 into acidstable products and by enzymatic determination of the
total 3PGA and DHAP. Under the conditions used
(0.2 mM Pj), only a small part of the [I4C]CO2 is incorporated into starch (5-8%; data not shown). Since it is known
that the fixed carbon can be exported as DHAP and 3PGA,
their concentrations in the medium were measured after
silicone-oil centrifugation and were used to calculate their
export rates.
In parallel experiments, the rates of O2 evolution and
[14C]CO2 fixation were followed. After 3 min of illumination, an O2-evolution rate of 92^mol (mg Chi)" 1 h" 1 and a
[l4C]CO2-fixation rate of 98/miol (mg Chi)" 1 h" 1 can be observed. Upon the addition of 0.2 mM OAA or nitrite, the
rate of [14C]CO2 fixation is not largely influenced (Fig. 6B),
while that of O2 evolution is increased (Fig. 6A). The export rate for DHAP remains nearly constant (Fig. 6C), and
that of 3PGA decreases only slightly (Fig. 6D). Under conditions that lead to a lowered activation state of FBPase,
the COyfixation rate decreases to 34% (2 mM nitrite) or
16% (10 fiM MV) of the control, but the rates of O2 evolution (Fig. 6A) are much higher than those of CO2 fixation
(Fig. 6B). It is interesting to see that the rate of DHAP export is still at about 60% of the control (Fig. 6C), but
3PGA export strongly decreases (2 mM nitrite) or ceases
completely (lOfxM MV) (Fig.6D).
S
o
1
1=
o
u
C
ft
10
Fig. 7 Time courses of the stromal 3PGA concentration (A) and
of [I4C]CO2 fixation (B) upon addition of NH4C1 and/or MV. Isolated intact chloroplasts were illuminated, and after 4 min, as indicated by the arrow, 1 mM NH4C1 (•) or 10 /*M MV (a) were added separately or together (v). (o), untreated control.
Effects of partial uncoupling—High rates of electron
flow will increase the zJpH across the thylakoid membrane
and can cause a down-regulation of the photosynthetic
electron transport (Heber and Walker 1992). We used the
combined application of 10 fiM MV and 1 mM NH4C1 to
estimate the extent to which this feedback-inhibition contributes to the observed effects on the CO 2 fixation and on
the changes in the stromal metabolite contents. The addition of 1 mM NH4CI alone decreases qN from 0.65 down to
0.4, but is not inhibitory for photosynthesis under control
conditions (Fig. 7B). The combination of 1 mM NH4C1
with 10 fiM MV causes a decrease of the CO2 fixation but is
less effective than MV alone (Fig. 7B). The same is true for
the stromal 3PGA (Fig. 7A) and DHAP concentrations
(not shown). The activation state of NADP-MDH and the
ARC, however, remain low. After 7 min of illumination,
ARC values of 0.36 or 0.34 are determined with or without
NH4CI, respectively, compared to 0.48 in the untreated controls (data not shown).
Discussion
In this work, we analyzed the response of isolated intact spinach chloroplasts to changes in the rate of electron
flow. These were introduced by the addition of different
electron acceptors at saturating light and CO2 after steadystate photosynthesis was reached. It was our intention to
follow the events caused by these treatments in the various
steps of photosynthesis, starting with the electron-transport chain (O2 evolution and chlorophyll fluorescence), the
reactions in the stroma (energy carriers, enzyme-activation
states and Calvin-cycle intermediates), and the rates of product formation. From this analysis, we hoped to identify
underlying regulatory patterns at the various stages of photosynthesis.
Under control conditions, the rate of O2 evolution
nearly equals the rate of [14C]CO2 fixation, both reaching
about 100fimol (mgChl)" 1 h~'. This meets the expected
stoichiometry, but a significant part of the fixed carbon exits from the chloroplast as 3PGA. In this case, electrons are
only required to produce 3PGA from [14C]CO2, but no
NADPH + H + is used for its further reduction into trioseP. On the other hand, electrons are continuously consumed
by the ferredoxin-thioredoxin system and by the Mehler
reaction. The ferredoxin-thioredoxin system requires a constant electron flow for the redox cycle to maintain the activation states of the different target enzymes (Scheibe 1991).
In the absence of OAA, the Mehler reaction is used to poise
the stromal ATP/NADPH + H + ratio by removing excess
electrons. Mass-spectrometric analyses have shown that O2
is reduced at detectable rates (5-15% of the total electron
flow) during steady-state photosynthesis (Marsho et al.
1979, Badger 1985). On the other hand, from studies with
antimycin A it is clear that the cyclic electron flow operates
Regulation of steady-state photosynthesis
at high rates in isolated chloroplasts (Woo 1983, Backhausen et al. 1994), but since electrons are cycled this does
not increase the rate of O2 evolution. It has been shown
that both reactions occur in a concerted manner (Steiger
and Beck 1981).
The addition of the various electron acceptors clearly
redirects the electron fluxes. The effects on photosynthesis
caused by the different treatments can be divided into two
groups: (1) controlled or moderate consumption of electrons that can be coped with, and (2) uncontrolled electron
removal that leads to inhibition of CO2 fixation. As an example for the first case, 0.2 or 2 mM OAA or 0.2 mM
nitrite increase the rate of O2 evolution and qP, but qN, the
stromal metabolite concentrations and the rates of product
formation are affected only marginally. Of the activation
states of the redox-modulated enzymes, only that of
NADP-MDH decreases significantly, but there is hardly
any interference with the Calvin cycle.
In contrast, uncontrolled electron removal caused by
2 mM nitrite or by MV has a significant influence upon photosynthesis. These electron acceptors lead to a decrease of
the stromal 3PGA concentration and of the rate of 3PGA
export, to an increased qN and ATP/ADP ratio, and to
lowered activation states of both FBPase and NADPMDH. It can be concluded that these inhibitions arise from
excessive consumption of electrons, but in addition, further interactions of MV and nitrite with the chloroplast metabolism must be considered. With MV, due to active O2
species, proteins might be damaged (Kaiser 1976, 1979,
Charles and Halliwell 1980). Nitrite can accumulate in the
stroma and decrease its pH value (Purczeld et al. 1978).
Especially the reductive activation of FBPase is known to
be sensitive to even small decreases in the pH, because low
pH values decrease the affinity of reduced Td towards FBPase (Soulie et al. 1985) and the reactivity of the reduced Td
(Scheibe et al. 1986). A general problem is that high rates
of electron flow without concomitant ATP consumption
will increase the ApH to values which may inhibit PSII activity (Heber and Walker 1992, Krieger and Weis 1993),
and thus decrease the rate of electron flow. The control experiments with partial uncoupling in the presence of 10 ^M
MV show that this zlpH-induced inhibition in fact contributes to the restrictions in CO2 fixation. But even after
decreasing the ApH, the ARC and the NADP-MDH activation state remained low, indicating that, apart from the
additional effects that may be caused, the electron flow
towards NADP + and Td is strongly restricted. From the
differences between the rates of O2 production and of
DHAP formation it can be taken that about 40-50% of the
electrons generated in the electron transport chain are
diverted towards MV (10 ^M).
Another possibility is that a shortfall of NADPH +
H + restricts the operation of the Calvin cycle. However,
when the electron transport is completely uncoupled, and
1213
osmotically shocked chloroplasts are supplied with ferricyanide as an electron acceptor, the rate of O2 production
is about threefold higher (data not shown) compared with
intact chloroplasts with CO2 as final acceptor. Thus,
the electron-transport capacity of the thylakoids is sufficient, and the chloroplasts possess the capacity to provide
NADPH + H + with high rates (Giersch et al. 1980). Our
measurements of the stromal NADPH + H + and NADP +
concentrations show that even in the presence of 10 fiM
MV or 2 mM nitrite, the changes in the pool sizes of the
pyridine dinucleotides are only small, and occur in a range
so that their function neither as electron donor for NADPMDH or GAPDH nor as electron acceptor for FNR is influenced.
However, the activation states of FBPase and NADPMDH exhibit the largest changes when the rate of electron
flow is varied. The NADP-MDH responds with a high sensitivity because NADP + inhibits its Td-mediated reductive
activation (Scheibe and Jacquot 1983). Since additional
electron acceptors will decrease both, ARC and redox state
of Td, even small changes have a strong impact on the activation state of NADP-MDH. Consequently, the electron
flow towards OAA is self-regulated, and only depends on
the activation state of NADP-MDH, but (in contrast to
nitrite) not on the added concentration of OAA.
From the three redox-modulated enzymes of the Calvin cycle investigated here, only the activation state of
the FBPase changed significantly in the presence of additional electron acceptors. FBPase is reduced by Td, and
Mg2+-FBP acts as a positive effector for the reductive activation (Wolosiuk et al. 1980). FBP shifts the equilibrium
between inactive and active enzyme in the redox cycle
towards the active form (Scheibe 1990, Faske et al. 1995),
and thus should facilitate reductive activation. But a comparison of the stromal FBP concentration with the FBPaseactivation states (Fig. 8A) indicates that there is an inverse
relationship. This apparent contradiction can be explained
by the hypothesis that activation is largely dependent upon
electron pressure. Under control conditions, a steady increase of FBPase activity with time was observed. This
appears to be caused by the high electron pressure (as indicated by qP) in the absence of additional electron acceptors in isolated chloroplasts, thus leading to a high activation state. Addition of electron acceptors decreases the
FBPase-activation state, lowers the stromal concentration
of 3PGA (Fig. 8B), and results in a build-up of FBP
(Fig. 8C). As a further consequence, this also increases the
energy charge (Fig. 8D). This relationship indicates the predominant role of the FBPase in the redox-mediated control
of CO2 fixation and demonstrates the connection between
redox state and energy charge.
Finally, the activation states of PRK and GAPDH are
apparently of minor importance in controlling the Calvin
cycle under the investigated conditions. In the case of
Regulation of steady-state photosynthesis
1214
20
40
60
80
100
FBPase activity (% of control)
100
200
300
400
Stromal FBP (% of control)
<
a
_ o
E o
£
0.6
0.8
1.0
EC(% of control)
Fig. 8 Relationships between various parameters ui cnloroplast
metabolism. The correlations between the activation state of the
FBPase and the stromal FBP (A) and 3PGA concentrations (B), respectively, between the stromal FBP and the stromal 3PGA concentrations (C), and between the stromal 3PGA concentration and
the EC (D) are shown. The data from the Figs. 1, 4 and 5 were
used and recalculated as % of the control. The values obtained
after lOmin of illumination under control conditions were taken
as 100%.
PRK, the redox change of the enzyme is responsible for the
change of activity (Porter et al. 1987), and association/dissociation of the enzyme might also be involved (Porter
1990). ATP is known to interfere with both activation and
inactivation. Since reduction and oxidation rates of the enzyme are inhibited in a similar way, no change in the PRK
activation state will occur (Faske et al. 1995). Furthermore,
there is good evidence that the enzyme activity in the chloroplast stroma is in large excess, and the flux through this enzyme in vivo is exclusively regulated by metabolite effects
upon the catalysis, as caused by 6-P-gluconate, 3PGA,
ATP or Pi (Gardemann et al. 1982, 1983). In transgenic
tobacco plants, the activity of PRK could be decreased far
below wild-type level without any effect on photosynthesis
or metabolite levels (Gray et al. 1995).
For GAPDH, regulation of its activation state is different from that of the other redox-modulated enzymes. Reduction of this enzyme is completed even at low light intensities, and changes in its activation state are then depending
on the concentration of l,3bisPGA which leads to dissociation and increase of activity (Baalmann et al. 1994). 1,3bisPGA is in equilibrium with the ATP/ADP ratio and the
3PGA concentration, but in the stroma, the relationship
between these two is strictly inverse. At high ATP/
ADP ratios, only little 3PGA is found, and therefore, the
stromal l,3bisPGA does not change significantly upon
changes of these parameters. Such inverse relationship between the ATP/ADP ratio and the 3PGA content without
any effect upon the GAPDH-activation state has been also
observed upon changing the Pj concentration in the incubation medium of isolated chloroplasts (Baalmann et al.
1994). In spinach leaves, when fed with low concentrations
of MV, again the ATP/ADP ratio and qN increased, while
3PGA decreased (Neuhaus and Stitt 1989).
At first sight, the significance of GAPDH activation is
difficult to see. Even the enzyme activity measured in the
dark (150-200/rniol (mg Chi)" 1 h"1) seems to be sufficient
to account for the reduction of the 3PGA produced by CO2
fixation in the light. But this does not reflect the situation in
vivo, because the assay for the determination of the enzyme activity is performed under saturating NADPH + H +
(200 fiM) and 1.3bisPGA (50^M) concentrations. The Km
value for NADPH + H + of the activated enzyme is 60 fiM
(Baalmann et al. 1995), and thus NADPH + H+ is saturating in the stroma, but l,3bisPGA is not. The stromal concentration of 1,3bisPGA can be calculated from the concentrations of ATP, ADP and 3PGA (Bucher 1963). Under
control conditions, l,3bisPGA is at 0.6ftM, which is far
below the Km value (20 fM) of the activated enzyme
(Baalmann et al. 1995).
In isolated chloroplasts, GAPDH activation states of
only 50-60% were measured. This raises the question why
the enzyme is not activated to a higher degree, since the
data indicate a limitation of the CO2 assimilation at this
Regulation of steady-state photosynthesis
step, being responsible for the high stromal 3PGA concentration under control conditions. Further, the rate of
3PGA export correlates quite well with the stromal concentration of 3PGA, as was earlier described by others (Flugge
et al. 1980). However, with isolated chloroplasts, full activation is only achieved, when ATP and 3PGA are exogenously added in the light (Baalmann et al. 1994). We conclude that in vivo larger pool sizes of stromal ATP and
3PGA are needed for full activation of GAPDH. But even
under conditions which should allow its full activation,
there is evidence that the capacity of the chloroplasts to reduce 3PGA into triose-P is not in excess (Fridlyand et al.
1997). In transgenic (antisense-gap A) tobacco plants, even
small decreases in the amount of enzyme affected the rate
of RuBP regeneration at saturating CO2 (Price et al. 1995).
The authors wish to thank K. Jager (nee Meyer) for growing
the spinach, and Dr. Paul W. Quick, Sheffield, for linguistic help.
This work was supported by the Graduiertenforderung des
Landes Niedersachsen (GradFog; J.E.B.) and by the Deutsche
Forschungsgemeinschaft (SFB 171-C15; R.S., and Pe 486; C.K.).
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(Received February 10, 1997; Accepted September 2, 1997)