Plant Physiol. (1992) 99, 979-986
0032-0889/92/99/0979/08/$01 .00/0
Received for publication November 19, 1991
Accepted February 3, 1992
Control of the Quantum Efficiencies of Photosystems I and 11,
Electron Flow, and Enzyme Activation following
Dark-to-Light Transitions in Pea Leaves
Relationship between NADP/NADPH Ratios and NADP-Malate
Dehydrogenase Activation State
Christine H. Foyer*, Maud Lelandais, and Jeremy Harbinson
Laboratoire du Metabolisme, Institut National de la Recherche Agronomique, Route de St-Cyr,
78026 Versailles Cedex, France (C.H.F., M.L.); and ATO/Agrotechnologie, Haagsteeg 6, Postbus 17,
6700 AA Wageningen, The Netherlands (J.H.)
ABSTRACT
whereas the electron transport system will only operate efficiently if NADP and ADP are plentiful; hence, the dilemma
that must be resolved by precise coordinate control (4, 8, 9).
Concepts of coordinate control of the electron transport
processes and the carbon reduction cycle have frequently
been derived from a knowledge of the discrete regulatory
properties of each individual reaction sequence. Only recently
has it been acknowledged that in vivo regulation is much
more complex and refined than might be expected from in
vitro measurements of individual components of the overall
process (4, 8). Physiological events may be reproduced in
reconstituted systems, but such systems frequently consist of
rather unnatural combinations of components at unphysiological concentrations. These disturb the balance of the native
control processes.
The function of photosynthetic control of electron transport
is to coordinate the synthesis of ATP and NADPH with the
rate at which these metabolites can be used in carbon metabolism, thus avoiding overreduction of the stroma as a result
of noncyclic electron flow (4). Several regulatory mechanisms, characterized in vivo and in vitro, may function to
minimize fluctuations in the [ATP]/[ADP][Pi] and [NADPH]
/[NADP] ratios. Under steady-state conditions, the rate-limiting step in electron transport resides between the photosystems at the level of plastoquinol oxidation by the Cyt b6/f
complex (7, 22). The rate of plastoquinol oxidation decreases
as the lumen pH decreases. This is a fundamental feature of
the restriction of electron transport (7, 22, 29). Measurements
in intact leaves, however, would suggest that the degree of
control in vivo exerted by this limitation on plastoquinol
oxidation is relatively constant with respect to irradiance in
air (4, 14), although not in the absence of CO2 (11). In
addition, it has been demonstrated that the acidification of
the thylakoid lumen also has direct effects on the quantum
efficiency of PSII (9, 30).
Although both feed-forward and feedback mechanisms are
well characterized in vitro (1, 2, 4, 8, 9, 16, 17), the relative
extent of the influence of each in vivo is unclear (4, 8).
Certainly, one reason for this is the failure to measure the
The quantum efficiencies of photosystems I and 11 (PSI and PSII),
[NADP]/[NADPH] ratios, and the activities of chloroplastic fructose-1,6-bisphosphatase and NADP-malate dehydrogenase were
measured in intact pea (Pisum sativum L.) leaves in air following
the transition from darkness to 750 microeinsteins per square meter
per second irradiance. PSII efficiency declined from a low value to
a minimum within the first 10 to 15 seconds of irradiance, after
which it increased progressively to the steady-state value. The
resistance of electron flow between the photosystems was high at
this time, but it was not the principal factor limiting electron flow.
Oxidation of P700 was restricted by acceptor side processes for
approximately the first 60 seconds of illumination. Once the acceptor side limitation was relieved, the oxidation state of P700 was
used to estimate the quantum efficiency of electron transport by
PSI. This was observed to increase progressively with time. The
quantum efficiencies of both photosystems increased in parallel,
consistent with a predominant role for noncyclic electron transport.
Fructose-1,6-bisphosphatase activity increased in an approximately
sigmoidal fashion with time of irradiance, paralleling the changes
in the quantum efficiencies of the photosystems. In contrast, the
activation of NADP-malate dehydrogenase did not show a lag
period but increased with time, reaching a maximum value at about
50 seconds of illumination, after which it declined. The NADP pool
was not extensively reduced during the first 10 seconds of illumination, but became so subsequently. It remained in the reduced
state until about 60 seconds of illumination and then became
relatively oxidized. The empirical relationship between NADPmalate dehydrogenase activity and the reduction state of the NADP
pool supports the suggestion that NADP-malate dehydrogenase
activity is a useful estimate of the reduction state of the stroma.
Precise coordinate regulation of the electron transport processes and carbon assimilation is an essential feature of photosynthesis. Coordinate regulation in vivo acts to reconcile
the conflicting requirements of these processes (4, 8, 9, 16).
The enzymes of the carbon reduction cycle require adequate
levels of ATP and NADPH to drive carbon assimilation,
979
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 1992 American Society of Plant Biologists. All rights reserved.
980
FOYER ET AL.
extent of operation of these mechanisms simultaneously in
vivo. This is especially true of the induction phase of photosynthesis (3, 10, 27). The metabolic basis of photosynthetic
induction has been the subject of much investigation, but the
relative importance of each of the regulatory steps remains
debatable (3, 26), and the processes involved in the regulation
of electron flow are largely unexplored.
The transition from darkness to high irradiance may, at
first glance, appear to be a rather unnatural condition with
which to study the induction of photosynthesis. Recent work
by Kirschbaum and Pearcy (10, 20) has demonstrated, however, that this type of induction response is fundamental to
the utilization of brief saturating light flecks in understory
plants. In addition, it is one of the few situations in which
virtually total reduction of both the NADP pool and the
electron transport system can be observed. Large changes in
the rate of electron transport and energetic properties occur
rapidly. We have previously used simultaneous measurements of PSI and PSII activity and stromal enzyme activation
to study the processes involved in the coregulation of electron
flow, carbon assimilation, and the reduction state of the
stroma in steady-state conditions in intact pea leaves (4, 11,
13). Thus, it became pertinent to study the evolution of
control during the induction process to resolve several outstanding questions regarding the nature of photosynthetic
control of electron flow and the estimation of the reduction
state of the stroma during this transitional period. Concern
has been expressed with regard to the in vivo significance of
measured [NADPH]/[NADP] ratios in intact tissues (4, 8, 16).
Thus, it is advantageous to have an additional in situ indicator
of the reduction state of the stroma. Scheibe and Stitt (26)
have suggested that NADP-MDH' can be used as a physiological marker for the relative oxidation-reduction state
of the stroma (24-26). The precise relationship between the
activation state of this enzyme and the [NADP]/[NADPH]
ratio in vivo had not, however, been examined. Under steadystate conditions, the [NADP]/[NADPH] ratio is always relatively low (5, 19, 28) and the activity of NADP-MDH is
also found to be low (11, 13, 24-26). Thus, the induction
response of photosynthesis, in which both parameters vary
significantly, is an ideal system in which to study their
relationship.
The changing demands of carbon assimilation for NADP
and ATP in the induction period have enabled us to determine the sequence of regulatory interactions between electron
transport and carbon assimilation, including the sites of regulation of electron flow. In steady-state conditions in air, we
have shown that electron transport was largely noncycic
over a wide range of irradiances (13). In this study, we
demonstrate that the activity of both of the photosystems is
severely restricted during the initial stages of induction, but
that some PSII activity is still possible. We follow the progression of events leading to the steady-state situation of
predominant noncyclic electron flow where the limitation of
'Abbreviations: NADP-MDH, NADP-malate dehydrogenase;
4tpsi, relative quantum efficiency for electron transport by PSI; 4P5sl,
relative quantum efficiency for electron transport by PSII; FBPase,
fructose-1,6-bisphosphatase; P700+, the fraction of the P700 pool
that is oxidized.
Plant Physiol. Vol. 99, 1992
electron flow resides between the photosystems. We show
that the activation state of FBPase mirrors the changes in
PSII quantum efficiency. This observation clearly demonstrates the precise regulation between the capacity for noncyclic electron flow and the activation state of the enzymes
of the photosynthetic carbon reduction cycle.
MATERIALS AND METHODS
Plants
All measurements were made on pea plants (Pisum sativum
L. var Finale or Frisson). The varieties Finale and Frisson are
sister lines that are very similar in terms of growth, appearance, and field performance. These plants were grown hydroponically in a glasshouse at Versailles during the months
of May and June, 1990. Mature, healthy leaves were used for
all experiments.
Photometric Measurements
Chl fluorescence and light-induced absorbance changes
around 820 nm were measured as described previously (12,
13) with some modifications necessitated by the need to use
a leaf chamber suitable for fast sample freezing. Modulated
Chl fluorescence was excited by radiation from an array of
light-emitting diodes (Stanley, Tokyo, Japan, types H-2K and
H-3K, peak emission 660 nm) screened by a red filter (Walz,
Eiffeltrich, FRG). The modulation frequency was 1.2 kHz and
the intensity on the leaf surface was 0.8 umol mr2 s-1 PAR.
The modulated excitation for Chl fluorescence, the broadband radiation to saturate the reduction of the primary quinone acceptor of PSII pool, the intense far-red radiation to
oxidize P-700, the weak far-red radiation to oxidize the
fraction of the primary quinone acceptor pool of PSII that is
reduced in the dark, and the actinic radiation were all carried
to the leaf chamber via fiber optics. Only the 820 nm measuring beam was generated by an emitter close to the leaf
surface. Actinic light was provided by a quartz halogen lamp
filtered by NIR and Calflex dichroic mirrors and a metal film
neutral density filter (Balzers). The irradiance at the leaf
surface was 750 ztmol mr2 s-' PAR (measured by a quantum
sensor, Li-Cor). A silicon photodiode, screened by an RT-830
filter (3 mm thick, Hoya), was situated below the leaf and
was used to detect both Chl fluorescence and the 820-nm
measuring beam.
Prior to use, plants were kept in complete darkness for 20
min. Following the placement of the leaf on the leaf chamber,
the leaf was dark-adapted for a further 5 min. Imnmediately
prior to the commencement of actinic irradiation, the modulated measuring beam for Chl fluorescence was switched on,
and the actinic irradiation began. This lasted from 10 to 300
s, after which the leaf was quickly frozen (described below).
At intervals during the irradiation, an intense broad-band
actinic beam was briefly switched on. This had an intensity
(8000 ,umol m-2 s-' PAR) that appeared to saturate the yield
of Chl fluorescence produced by the modulated red measuring beam. From the change in the level of the yield of
modulated Chl fluorescence produced by the addition of this
intense radiation to the actinic background, the quantum
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 1992 American Society of Plant Biologists. All rights reserved.
COORDINATE REGULATION OF PHOTOSYNTHETIC PROCESSES
efficiency for electron trasport by PSII was calculated using
the technique of Genty et al. (6).
Changes in the oxidation state of P700 during photosynthetic induction were measured using 820 nm as a measuring
wavelength (14). The leaf was dark-adapted as described and
the actinic irradiation began. This was interrupted by a 1-s
dark period every 10 s to allow measurement of the fast
AA820 produced by dark reduction of oxidized P700. The
actinic irradiation was terminated after 400 s and an intense
far-red beam was used to oxidize all P700 and, therefore, to
calibrate the AA820 signal in terms of the total photooxidizable
P700 pool. The completeness of the P700 oxidation was
checked by adding a further weak far-red beam to the first
far-red beam once the AA820 produced by the latter had
stabilized. No further AA820 was produced by this addition,
even though the second far-red beam on its own was intense
enough to oxidize about 50% of the P700 pool in the leaf. A
linear compensation circuit was included in the AA820 signal
processing system to correct for a small nonlinearity that was
evident in the filter-diode-amplifier system. From the AA820
recorded during the period of actinic irradiation and the AA820
produced by the far-red irradiation, the quantum efficiency
for electron transport by PSI was calculated as described
previously (12, 29).
Leaf Freezing and Sample Preparation
The leaf chamber used in these experiments was designed
to allow rapid freezing of the irradiated portion of the leaf
without interruption of the irradiance. This was done by
forcing a chilled solid brass cutter through the leaf from
below using a pneumatic piston (Norgren Martonair). The
cutter and the leaf disc were then stopped by the top window
of the leaf chamber. This window was composed of a transparent acrylic ring, strong enough to stop the cutter, across
which was fixed a transparent self-adhesive plastic film. The
bulk of the leaf sample removed by the cutter was frozen
against this plastic film. During tests, a thermocouple fixed
to the upper side of the leaf was chilled from 20 to -5OC in
500 ms. Prior to use, the cutter was stored in liquid nitrogen.
The silicon photodiode used to detect Chl fluorescence and
the AA820 signals was situated below the leaf on a drop arm
that was released to swing away from the path of the freezing
block. This release was also affected by a pneumatic piston.
At the same time as the chilled cutter was freezing the leaf
from beneath, more liquid nitrogen was added from above
to ensure that the leaf disc remained frozen. This liquid
nitrogen filled a well bounded by the acrylic ring to the sides
and the plastic film on the base.
Two regimens of leaf freezing were followed. Leaves of
var Finale, which were used also for measurements of Chl
fluorescence and the AA820, were frozen and treated individually. Leaves of var Frisson, on which no photosynthetic
measurements were made, were frozen and pooled (eight
discs per time point) according to the duration of irradiance.
All discs were removed from the leaf chamber either attached
to the chilled cutter or to the upper leaf chamber (which
contained a reservoir of liquid N2) and transferred to liquid
nitrogen for grinding with frozen buffer (Finale) or to liquid
981
nitrogen for storage (Frisson) prior to grinding the pooled
samples with frozen buffer.
Enzyme and Metabolite Measurements
NADP-MDH and FBPase assays were performed as described previously (13). NADP and NADPH measurements
were made essentially as described by Maciejewska and
Kacperska (18). We calculate that with our average leaf
nucleotide [NADPH + NADP] pool size of 40 nmol mg-' Chl
and a rate of photosynthetic 02 evolution of 100 zmol 02
h-' mg-' Chl at 600 s of the induction period at this irradiance, the turnover time of the [NADPH + NADP] pool would
be on the order of 700 ms. This is a maximum value in this
study. The turnover time during the initial seconds of the
induction period would be less than 10% of this value.
RESULTS
44si as a Function of Time of Irradiance
The changes in tpsn, measured folowing the transition
from darkness to light, were complex (Fig. 1). In the first
period of irradiance, 4psjl declined from a value of about 0.08
at 5 s to 0.05 at about 15 s. It is of interest to note that (ps,,
was not zero at the initial point of measurement, and that in
the initial period of irradiance IPSHl actually decreased. However, it did not fall to zero (Fig. 1). After approximately 25 s
of irradiance, tPs5l increased in a sigmoidal fashion as the
duration of irradiance increased until a steady-state 4PSII was
reached. Maximum 4P6i was between 0.75 and 0.80 in these
leaves.
Figure 2 shows the changes in the oxidation state of P700
and in tps, with time of irradiance following the dark-to0.3
a0.
U
c
.IU 0.2
0
E
E 0.1
0
0
0
0
50
100
150
200
250
300
Duration of irradiance (s)
Figure 1. The relationship between 4psi, and the duration of irradiance at 750 Mmol m-2 s-1 PAR. Prior to irradiance, the leaves were
dark-adapted for 20 to 30 min. Illumination commenced at time 0.
Each data point represents the mean of at least three samples.
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 1992 American Society of Plant Biologists. All rights reserved.
0.6
100
m
75
6
0
0
0.24
Xo
(L
0.0
50
1-u
Plant Physiol. Vol. 99, 1992
FOYER ET AL.
982
-__r.
-u
ci)
@c
measurements made on pooled leaf samples of the variety
Frisson.
In dark-adapted leaves of both varieties, the activity of
FBPase was between 20 and 30 ,umol h-1 mg-' Chl. With the
commencement of irradiance, FBPase activity rapidly increased after a short lag period, reflecting the activation of
the chloroplast isoenzyme (Fig. 4).
%I*
.
-v
a
0
25
o.o
0
0
80
160
240
320
400
time (s)
Figure 2. The relationship between the relative oxidation of P700
(as a percentage) (M), the 4sPs (A), and the duration of irradiance for
an individual pea leaf (var Finale) irradiated at 750 ,mol m-2 s-'
following a dark incubation period of 20 min. The values of 4psi are
only shown after 70 s because prior to this time P700 oxidation was
restricted by a shortage of electron acceptors; under these conditions, the degree of oxidation of P700 cannot be used to calculate
1PS,1.
light transition in a single pea leaf. The typical response of
P700 following the transition from darkness to irradiance can
be seen. Maximum P700 oxidation occurred after about 70 s
irradiance. The P700+ pool then progressively declined to a
steady-state value at about 350 s. A significant feature of the
changes in P700 is the biphasic rise and fall in the degree of
oxidation. The precise detail of changes in P700 oxidation
varied somewhat from leaf to leaf, and thus the biphasic
nature of the changes in P700+ can be obscured by averaging.
The biphasic pattern of P700 oxidation was, however, an
intrinsic feature of the induction process and is a reflection
of changes in the factors that determine the oxidation state
of P700. After the P700+ pool had reached its maximum
value and began to decline, it was possible to obtain valid
estimates of bps, from the P700+ measurement.
The quantum efficiencies of the photosystems increased in
parallel with increasing time of irradiance (Fig. 3). The values
of 4ps, were high at the onset of irradiance and appeared to
be independent of tpsjl. The value obtained for 4psi at 20 s
is erroneous, however, because the P700 oxidation state was
restricted on the acceptor side. This initial value is included
only for completeness. Thus, the nature of the relationship
between (bps and 4psjj cannot be precisely determined at this
time. The point where a parallel increase in the quantum
efficiency of both photosystems appeared to begin coincided
with the time of maximum P700 oxidation.
Relationship between FBPase Activity and Duration of
Irradiance
are of two
obtained from
individual leaf samples of the variety Finale; others were
The
enzyme
data for FBPase and NADP-MDH
types: the majority of the data points
were
Relationship between NADP-MDH Activity and the
Duration of Irradiance
The activation of NADP-MDH was very similar in leaves
of both varieties Finale and Frisson (Fig. 5). The dark activity
of NADP-MDH was very low but increased rapidly following
the onset of irradiance. Maximum activation was reached at
about 50 s of irradiance. After 80 s, the activity declined once
more (Fig. 5). Following this decline, NADP-MDH activity
remained relatively low, as is generally observed in steadystate conditions (11, 13, 24-26).
Relationships between the Redox State of the NADP Pool
and NADP-MDH Activity
The reduction state of the NADP pool, expressed as the
ratio [NADPH]/[NADP + NADPH], was measured following
the transition from darkness to constant irradiance (Fig. 6).
This ratio was low in the dark (0.35). Following the commencement of irradiance, the degree of reduction did not
change greatly for the first 10 s in these experiments. Between
10 and 20 s, the NADP pool became substantially reduced.
It remained in the reduced state for 40 to 100 s and then
became more oxidized. The relationship between the redox
0.8
max. *PS2
0.6
690s
0. 4
0.2
30<
20s
.
.
.
0.00.4
0.2
0.0
0.6
0.8
1.0
#PS1
Figure 3. The relationship between 4Psi and 4psIl during induction.
The maximum dark-adapted value of 4pSn was 0.76. The data were
collected from a dark-adapted pea leaf subjected to 750 ,umol m-2
51 irradiance. The first record was made at 20 s and the last at
690 s.
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 1992 American Society of Plant Biologists. All rights reserved.
983
COORDINATE REGULATION OF PHOTOSYNTHETIC PROCESSES
1.00
I
0-
wI
z
C.
E 80
T
0.80
0
100
_80
+
0.60
z
0.40
-
0.20
E 60
z
0
* 40
0
60
120
240
300
time (s)
0
0)
XL 20
m
180
i .
50
100
I
0
I
150
I
I
200
I
250
I
300
Duration of irradiance (s)
Figure 4. The relationship between the activity of FBPase and the
duration of irradiance at 750 ,mol m-2 s-' following dark adaptation
of 20 to 30 min. Irradiance commenced at time 0. Maximum
activities were 80 to 100 Mmol h-1 mg-' Chl. Points (U) are the
means of between three and eight samples obtained from leaves
of var Finale. Other points (A) represent the activity of five pooled
samples of var Frisson.
T
u 70
Figure 6. The relationship between the ratio of NADPH and the
sum of NADP and NADPH, and the duration of irradiance at 750
m-2 s-1 PAR for pea leaves in air. These data were obtained
Drmol
from pooled leaf samples from var Frisson.
state of the NADP pool and the activation state of NADPMDH, which were both measured in fractions of the pooled
extracts of six leaves, was approximately hyperbolic (Fig. 7).
DISCUSSION
During photosynthetic induction, the efficiencies of the
photosystems are severely limited by the regulated capacity
of certain components of the electron transport chain and the
photosynthetic carbon reduction cycle. This was evidenced
by the low efficiency of PSII, the absence of oxidized P700,
the reduced state of the NADP system, and the changes in
E 60
1.0
c 50
0
E 40
o 0.8 <
A
> 30
A
A
f
.
~~~~+
.
AC
20
u
.
z
*
0
0
0.6 _~~~~~~~~~~~~
!
z
a 10
%
U0,
0.4-
U
0o
0
z
0
50
100
150
200
250
300
Duration of irradiance (s)
Figure 5. The relationship between NADP-MDH activity and the
duration of irradiance at 750 /Amol m-2 s-' following dark adaptation
for 20 to 30 min. Points (U) are the means of between three and
eight samples of var Finale. Other points (A) were obtained from
five pooled samples of var Frisson. Maximum activity, obtained by
incubating samples with DTT, was 108 ± 5.3 umol h-1 mg-1 Chi.
z
0
I
0
.
30
60
NADP-MDH activity (pmol h-1
90
(mg
Chl-)1)
Figure 7. The relationship between the activity of NADP-MDH
activity and the ratio between NADPH and the sum of NADPH and
NADP. These measurements were made on pooled leaf samples of
pea var Frisson irradiated for different times in air at an irradiance
of 750 umol m-2 s-1 following a dark adaptation of 20 to 30 min.
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 1992 American Society of Plant Biologists. All rights reserved.
984
FOYER ET AL.
activation states of NADP-MDH and FBPase. In the initial
seconds following the onset of illumination, (bps,, fell, P700
was largely unoxidized, and the NADP pool was relatively
oxidized. A block in electron transport during this brief initial
stage of photosynthetic induction has been described previously and is suggested to reside at the level of Fd-NADP
reductase (23). During the earliest stages of photosynthetic
induction, the restriction of photosynthetic electron transport
lies after PSI. This is the reason why P700 oxidation is
restricted (13). As this restriction was alleviated and P700
oxidation increased (Fig. 2), the limitation of electron transport moved to one between PSII and PSI (Fig. 3). During the
first 20 s of irradiance, the minimal efficiency of PSII and
lack of oxidized P700, coupled with the oxidized condition
of the NADP pool and NADP-MDH, are consistent with a
limitation after P700 but before NADP. This phase is often
preceded by a brief burst of electron transport activity, but
no burst was evident in these experiments. tPSn declined
initially, however, and the existence of a transient peak of
electron transport before our first measurement at 5 s is
possible.
The limitation of electron flow between PSI and NADP+
is short-lived (10-20 s). The NADP pool then became extensively reduced for up to 100 s after the onset of illumination.
Reduction of the NADP pool was accompanied by an increase
in the activation state of the thiol-modulated enzymes
NADP-MDH and FBPase. These two enzymes are regulated
in a slightly different manner (1, 2, 17). NADP-MDH activity
is believed to be modulated solely by the flux of electrons
from the thioredoxin system and the [NADPH]/[NADP]
ratio (2, 24-26). In contrast, the activation of FBPase requires
both reducing equivalents and the substrate, fructose-1,6bisphosphate, to stabilize the activation state of the enzyme.
There is a short but clearly defined lag phase in the activation
of FBPase. This is not apparent in the activation of NADPMDH. The high [NADPH]/[NADP] ratio would favor rapid
NADP-MDH activation. Reduced Fd would be relatively
plentiful and available for thioredoxin reduction and enzyme
activation. FBPase activation is slower than that of NADPMDH, presumably because of the lack of fructose-1,6-bisphosphate.
As the thiol-modulated enzymes became activated, the
level of oxidized P700 rapidly increased, as did 4s6i. The
quantum efficiencies of the photosystems were thus directly
limited by the capacity of the enzymes that require activation
during the initial minutes of illumination. It is interesting to
note the close correlation that exists between the activity of
PSII (Fig. 1) and the activation state of FBPase (Fig. 5). This
is consistent with precise coordination between the activity
of the photosynthetic carbon reduction cycle and the flux of
electrons through PSII. During steady-state photosynthesis
at high light intensities, the rate of electron transport is
considered to be limited by the activation state of enzymes
of the photosynthetic carbon reduction cycle (16). This simple
model is complicated, however, by the absence of change in
the degree of photosynthetic control of electron transport
with increasing irradiance (4, 14). Changes in the degree of
photosynthetic control would be expected if the photosynthetic carbon reduction cycle was to become increasingly
limiting as irradiance increased. It is clear that under steady-
Plant Physiol. Vol. 99, 1992
state conditions, the activity of the electron transport chain
is balanced with the demands of the photosynthetic carbon
reduction cycle so as to avoid overreduction of the PSI
acceptor pool and the associated hazards of oxygen radical
generation.
During the early stages of photosynthetic induction, P700
oxidation is clearly limited by the absence of electron acceptors. The increase in the AA820 signal during the first period
(Fig. 2) is due largely to the relief of this restriction. During
the initial phase of induction, it is not evident how the P700+related AA820 signal can be used to calculate the quantum
yield of PSI. The method employed in this work requires that
the oxidation state of P700 is determined by the relative rates
of excitation of PSI and of electron donation from reduced
electron transfer components between the photosystems.
This condition is met once the AA820 reaches its maximum
and then begins to decline. Once this stage is passed, the
efficiencies of both photosystems change in parallel (Fig. 3).
The typical relationship between 4psi and 4psi, in pea leaves,
subjected to a range of irradiances (11, 13), is mirrored during
the latter stages of induction. It implies a predominant role
for linear electron flow as thylakoid electron flow increases
during photosynthetic induction in these leaves.
In the earlier phase, when P700 oxidation is limited on the
acceptor side, it is not possible, using these techniques, to
determine whether or not cyclic electron flow around PSI is
occurring. The removal of the restriction on the acceptor side
of PSI occurs in parallel with a large increase in (Pps, and, by
implication, the rate of noncyclic electron flow. It is important
to note that (Ps,, never declines to zero and there is always
some electron flow through PSII. This flow could be supported by 02 reduction after PSI (19) or by other substrates
(e.g. thioredoxin- or Fd-linked reduction of other substrates).
It has been demonstrated (14, 15), that during the increase in
the P700+ pool, there are only small changes in the half-time
for P700+ reduction. Thus, the change in tPSHl, which is
essentially the quantum yield for noncyclic electron flow, is
not due to changes in the capacity for electron flow between
the two photosystems to any great extent but rather to the
relief of the limitation of electron transport after PSI. Changes
in both (Pps, and (Pps, are related due to changes in the
capacity for electron flow between the photosystems (12-14).
There are, therefore, two distinct phases in the regulation or
limitation of electron flow in leaves during photosynthetic
induction. In the first phase, the resistance for electron flow
between the photosystems, although high, is not limiting. In
the second phase, the limitation lies between the two photosystems, and the increasing rate of electron transport is
determined by the decreasing resistance for electron flow
between the two photosystems.
The overreduction of the NADP pool during the initial
minutes of induction may be necessary to force the activation
of the enzymes of the photosynthetic carbon reduction cycle.
The slow activation of FBPase and NADP-MDH arises from
the hysteretic nature of the enzymes and their activation.
Once the thiol-modulated enzymes of the photosynthetic
carbon reduction cycle become active, the [NADPH]/[NADP]
ratio falls, as does the activity of NADP-MDH, which has
been reported previously (27, 28). Light activation of ribulose1,5-bisphosphate carboxylase can be an important factor
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 1992 American Society of Plant Biologists. All rights reserved.
COORDINATE REGULATION OF PHOTOSYNTHETIC PROCESSES
contributing to the induction process (22). This enzyme has
its own unique mechanisms for the regulation of the activation state (21). Pea leaves do not contain carboxyarabinitol1-phosphate, as discussed previously (13), but we cannot
eliminate the possibility that activation of ribulose-1,5-bisphosphate carboxylase contributes to the phase where activation of enzymes is a major limitation. The measurements
of the activities of NADP-MDH and the [NADPHJ/[NADP]
ratios presented here show that the relationship between
these was approximately hyperbolic during induction and
qualitatively similar to that reported by Scheibe (25). A
detailed interpretation of data of this kind is complicated by
several factors. First, the measurement of the reduction state
of the NADP pool is complicated by binding of NADP or
NADPH to chloroplast proteins (8). Second, there are fluxes
of reducing equivalents throughout the stroma. As a consequence, the relationships between the redox state of stromal
components may be significantly influenced by kinetic constraints in addition to thermodynamic considerations. These
kinetic limitations are poorly understood. Nonetheless, it is
clear that the activity of NADP-MDH is, at least qualitatively,
a good indicator of the stromal reduction state.
CONCLUSIONS
PSI electron flow was restricted on its acceptor side during
approximately the first 60 s of irradiance following a darkto-light transition. During this phase, some PSI will become
photosynthetically inefficient but not oxidized. Precise interpretation of the AA820 data, therefore, is not possible and the
AA820 measurement cannot be useful to probe 4ips, in this
situation. Thus, it is not possible to determine whether cyclic
electron flow or noncycic electron flow is occurring at this
time. Biphasic induction kinetics in the AA820 signal were
observed as the acceptor side became progressively oxidized.
Once the restriction of electron flow on the acceptor side was
alleviated, P700 oxidation was determined by donor side
processes and $ps, could be determined accurately.
The quantum efficiency of PSII was not zero during the
first seconds of irradiance. It had a very low value at the
beginning of irradiance and subsequently fell even lower
during the first 10 to 20 s of irradiance before rising progressively to its steady-state level.
Once the restriction of electron flow on the acceptor side
of PSI has been removed and reliable estimates of (ps, can
be made, it is clear that both 4~ps, and 4Ppsn are modulated in
parallel. This would be expected in a system where noncycic
electron flow is predominant. The change from a limitation
on the PSI acceptor side to one on the donor side, in constant
irradiance, is important. It marks a change in the site of
limitation of thylakoid electron transport from that after PSI
to restriction between PSII and PSI. As long as P700 oxidation
is limited on its acceptor side, precise photosynthetic control
of electron transport at the level of the plastoquinol oxidation
is not possible. The parallel increase in Ips, and tpsii is a
reflection of the change to a limitation of electron flow
between the photosystems.
The increase in FBPase with increasing irradiance paralleled exactly the changes in (Pps, and, hence, the quantum
efficiency of noncycic electron flow. This is a further dem-
985
onstration of the coordination of the capacities of thylakoid
and stromal reactions participating in photosynthesis.
Changes in the activation state of NADP-MDH mirrored
changes in the [NADPH]/[NADP] ratio such that the relationship was hyperbolic, saturating only at the highest
NADP-MDH activities and extreme [NADPH]/[NADP] ratios. The activation state of NADP-MDH is, therefore, a
valuable tool with which to estimate the reduction state of
stromal components.
LITERATURE CITED
1. Crawford NA, Droux M, Kosower NS, Buchanan BB (1989)
Evidence for function of the ferredoxin/thioredoxin system in
the reductive activation of target enzymes of isolated intact
chloroplasts. Arch Biochem Biophys 271: 223-239
2. Cseke C, Buchanan BB (1986) Regulation of the formation and
utilisation of photosynthate in leaves. Biochim Biophys Acta
853: 43-63
3. Edwards G, Walker DA (1983) Induction. In C3, C4: Mechanisms and Cellular and Environmental Regulation of Photosynthesis. Blackwell Science Publications, Oxford, p 156-200
4. Foyer CH, Furbank R, Harbinson J, Horton P (1990) The
mechanisms contributing to photosynthetic control of electron
transport by carbon assimilation in leaves. Photosynth Res 25:
83-100
5. Fredeen AL, Raab T, Rao IM, Terry N (1990) Effects of phosphorus nutrition on photosynthesis in Glycine max (L.) Merr.
Planta 181: 399-405
6. Genty BE, Briantais JM, Baker NR (1989) The relationship
between the quantum yield of photosynthetic electron transport and photochemical quenching of chlorophyll fluorescence. Biochem Biophys Acta 990: 87-92
7. Haehnel W (1984) Photosynthetic electron transport in higher
plants. Annu Rev Plant Physiol 35: 659-693
8. Heber U, Schreiber U, Siebke K, Dietz KJ (1990) Relationships
between light-driven electron transport, carbon reduction and
carbon oxidation in photosynthesis. In I Zelitch, ed, Perspectives in Biochemical and Genetic Regulation of Photosynthesis.
Alan R Liss, New York, pp 17-37
9. Horton P (1989) Interactions between electron transport and
carbon assimilation: regulation of light-harvesting and photochemistry. In WR Briggs, ed, Photosynthesis, Plant Biology
Series, Vol 8. Alan R Liss, New York, pp 393-406
10. Kirschbaum MUF, Pearcy RW (1988) Gas exchange analysis of
the fast phase of photosynthetic induction in Alocasia macrorrhiza. Plant Physiol 87: 818-821
11. Harbinson J, Foyer CH (1991) Relationships between the efficiencies of photosystems I and II and stromal redox state in
C02-free air. Evidence for cyclic electron flow in vivo. Plant
Physiol 97: 41-49
12. Harbinson J, Genty B, Baker NR (1989) Relationship between
the quantum efficiencies of photosystems I and II in pea leaves.
Plant Physiol 90: 1029-1034
13. Harbinson J, Genty B, Foyer CH (1990) Relationship between
photosynthetic electron transport and stromal enzyme activity
in pea leaves. Toward an understanding of the nature of
photosynthetic control. Plant Physiol 94: 545-553
14. Harbinson J, Hedley CL (1989) The kinetics of P-700+ reduction
in leaves: a novel in situ probe of thylakoid functioning. Plant
Cell Environ 12: 357-369
15. Harbinson J, Woodward FI (1987) The use of light induced
absorbance changes at 820 nm to monitor the oxidation state
of P-700 in leaves. Plant Cell Environ 9: 131-140
16. Heber U, Neimanis S, Dietz KJ, Vill J (1986) Assimilatory
power as a driving force in photosynthesis. Biochim Biophys
Acta 852: 144-155
17. Leegood RC (1990) Enzymes of the Calvin cycle. In PJ Lea, ed,
Methods in Plant Biochem. Vol 3. Academic Press, London,
pp 15-37
18. Maciejewska U, Kacperska A (1987) Changes in the level of
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 1992 American Society of Plant Biologists. All rights reserved.
986
19.
20.
21.
22.
23.
24.
FOYER ET AL.
oxidised and reduced pyridine nucleotides during cold acclimation of winter rape plants. Physiol Plant 69: 687-691
Marsho TV, Behrens PN, Radmer KJ (1979) Photosynthetic
oxygen reduction in isolated intact chloroplasts and cells from
spinach. Plant Physiol 64: 656-659
Pearcy RW (1990) Sunflecks and photosynthesis in plant canopies. Annu Rev Plant Physiol Plant Mol Biol 41: 421-453
Portis AR Jr (1990) Rubisco activase. Biochim Biophys Acta
1015: 15-28
Rich P (1982) A physiochemical model of quinone-cytochrome
6-c complex electron transfer. In BL Trumpower, ed, Functions
of Quinones in Energy Consuming Systems, Academic Press,
New York, pp 73-83
Satoh U (1981) Fluorescence induction and activity of ferredoxin-NADP reductase in Bryopsis chloroplasts. Biochim Biophys Acta 638: 327-333
Scheibe R (1987) NADP+ malate dehydrogenase in C3 plants.
Regulation and role of a light-activated enzyme. Physiol Plant
71: 393-400
Plant Physiol. Vol. 99, 1992
25. Scheibe R (1990) Light/dark modulation: regulation of chloroplast metabolism in a new light. Bot Acta 103: 327-334
26. Scheibe R, Stitt M (1988) Comparison of NADP-malate dehydrogenase activation, QA reduction and 02 evolution in spinach leaves. Plant Physiol Biochem 26: 473-482
27. Siebke K, Laisk A, Neimanis S, Heber U (1991) Regulation of
chloroplast metabolism in leaves: evidence that NADP-dependent glyceraldehyde phosphate dehydrogenase, but not
ferredoxin-NADP reductase, controls electron flow to phosphoglycerate in the dark to light transition. Planta 185:
337-343
28. Takahama U, Shimizu-Takahama M, Heber U (1981) The
redox state of the NADP system in illuminated chloroplasts.
Biochim Biophys Acta 637: 530-539
29. Weis E, Ball JT, Berry J (1987) Photosynthetic control of electron
transport in leaves of Phaseolus vulgaris: evidence for regulation of photosystem II by the proton gradient. In J Biggins, ed,
Progress in Photosynthesis Research, Vol II. Martinus Nijhoff,
Dordrecht, The Netherlands, pp 553-556
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 1992 American Society of Plant Biologists. All rights reserved.