Plant Cell Physiol. 41(10): 1129–1135 (2000)
JSPP © 2000
Light-Induced Dynamic Changes of NADPH Fluorescence in Synechocystis
PCC 6803 and Its ndhB-Defective Mutant M55
Hualing Mi 1, Christof Klughammer and Ulrich Schreiber 2
Lehrstuhl Botanik I, Julius-von-Sachs Institut für Biowissenschaften, Universität Würzburg, Julius-von-Sachs Platz 2, D-97082 Würzburg,
Germany
;
Blue-green fluorescence emission of intact cells of Synechocystis PCC6803 and of its ndhB-defective mutant M55
was measured with a standard pulse-amplitude-modulation chlorophyll fluorometer equipped with a new type of
emitter-detector unit featuring pulse-modulated UV-A measuring light and a photomultiplier detector. A special illumination program of repetitive saturating light pulses with
intermittent dark periods (10 s light, 40 s dark) was applied to elicit dynamic fluorescence changes under conditions of quasi-stationary illumination. The observed effects
of artificial electron acceptors and inhibitors on the responses of wild-type and mutant M55 cells lead to the conclusion that changes of NAD(P)H fluorescence are measured. In control samples, a rapid phase of light-driven
NADP reduction is overlapped by a somewhat slower phase
of NADPH oxidation which is suppressed by iodoacetic acid
and, hence, appears to reflect NADPH oxidation by the
Calvin cycle. Mercury chloride transforms the light-driven
positive response into a negative one, suggesting that inhibition of NADP reduction at the acceptor side of PSI leads to
reduction of molecular oxygen, with the hydrogen peroxide
formed (via superoxide) causing rapid oxidation of NADPH.
The new fluorescence approach opens the way for new
insights into the complex interactions between photosynthetic and respiratory pathways in cyanobacteria.
Key words: Cyclic photosystem I — Hydrogen peroxide —
NADPH dehydrogenase — NADPH fluorescence — Respiration — Synechocystis PCC 6803.
Abbreviations: DBMIB, 2,5-dibromo-3-methyl-isopropyl-p-benzoquinone; FNR, ferredoxin-NADP+ oxidoreductase; IAA, iodoacetic
acid; LED, light emitting diode; NDH, NADPH dehydrogenase; PAM,
pulse-amplitude-modulation.
Introduction
Cyanobacteria are prokaryotic organisms which lack chloroplasts and mitochondria as separate organelles. They contain
two types of membrane systems, the Chl-containing thylakoid
membranes and the Chl-free cytoplasmic membranes. While
1
2
the latter display a purely respiratory chain, the thylakoid membranes are characterized by a unique assembly of electron
transport components which on one hand are involved in oxygenic photosynthesis and on the other hand carry out oxidative
phosphorylation, with some of the components being shared by
the two processes (Jones and Myers 1963, Aoki and Katoh
1982, Peschek and Schmetterer 1982, Sandmann and Malkin
1984). Dark-respiratory electron transport consumes O2 as well
as NADPH during the process of ATP-formation and plays an
important role in nitrogen fixation (Scherer et al. 1988). In the
thylakoid membranes, besides linear and respiratory electron
flow, also cyclic electron flow around PSI proceeds via the
same intersystem electron transport components. While cyclic
photophosphorylation provides part of the ATP for the Calvin
cycle (Myers 1987, Bendall and Manasse 1995) its most important role arises under energetically demanding growth conditions, such as, for example, upon cultivation in high salt medium (Fry et al. 1986, Jeanjean et al. 1993, Jeanjean et al. 1999)
or at low inorganic carbon levels for energizing the inorganic
carbon-concentrating process (Ogawa et al. 1985, Badger and
Price 1994).
NAD(P)H-dehydrogenase (NDH) in cyanobacteria has
been suggested to participate in the donation of electrons from
respiratory substrates to the photosynthetic electron transport
chain (Sandmann and Malkin 1983) and to be essential for
energization of inorganic carbon transport (Ogawa 1991). The
NDH is a multisubunit complex homologous to the mitochondrial complex I (Berger et al. 1991, Berger et al. 1993).
Using an ndhB-gene-defective mutant (Ogawa 1991), an
NADPH-specific NDH functioning as mediator of respiratory
electron flow to the intersystem chain and of cyclic electron
flow was demonstrated in Synechocystis PCC 6803 (Mi et al.
1994, Mi et al. 1995). In cyanobacteria the oxidative pentose
phosphate cycle, which produces NADPH, is considered by far
the most efficient, if not only, substrate dehydrogenation pathway (Peschek 1999).
In view of the functional ‘bioenergetic duality’ inherent to
thylakoid membranes in cyanobacteria, it is not surprising that
despite considerable experimental efforts there are still many
open questions related to mechanistic details of photosynthetic
and respiratory electron transport in cyanobacteria (Peschek
1999). In order to investigate the delicate interplay between
these overlapping processes, it is essential that the membrane is
Present address: Shanghai Institute of Plant Physiology, The Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China
Corresponding author: E-mail, [email protected]; Fax, +49-931-888-6157.
1129
1130
NADPH fluorescence in Synechocystis PCC 6803
not disturbed, i.e. measurements should be carried out with intact cells. On the other hand, only few methods are available
for assessment of in vivo electron transport reactions. In
previous related work measurements of modulated Chl fluorescence and P700 absorbance played a major role (Mi et al.
1992a, Mi et al. 1992b, Mi et al. 1994, Yu et al. 1993, Badger
and Schreiber 1993) which could be carried out using the same
sample and the same basic measuring system [so-called pulseamplitude-modulation (PAM) fluorometer (Schreiber et al.
1986, Schreiber et al. 1988)].
In the present communication, we report on first measurements of NADPH fluorescence in suspensions of intact cells of
Synechocystis PCC 6803 and its ndhB-defective mutant, M55,
using a standard PAM fluorometer equipped with a new type of
emitter-detector unit. Such measurements have become possible with the recent advent of special light-emitting diodes
(LEDs) displaying a sufficiently high emission in the UV spectral range to excite NADPH fluorescence. In vivo NADPH fluorescence was already measured in early studies of algae and
photosynthetic bacteria by Duysens and Amesz (1957), Olson
et al. (1959), Olson and Amesz (1960) and recently also in intact chloroplasts and leaf segments of spinach (Cerovic et al.
1993). So far, no use of this method has been made to investigate the complex reactions within cyanobacteria, in which
NADPH plays a central role. In this first report, we will show
that light-induced changes in NADPH fluorescence display remarkable dynamics, which bear the potential of providing information on photosynthetic NADP reduction as well as dark
reduction associated with the reductive pentose phosphate cycle, NADPH oxidation linked to Calvin cycle activity, NDHmediated cyclic and respiratory electron transport as well as
NADPH oxidation by oxidative phosphorylation and by active
oxygen species.
Materials and Methods
Culture of cyanobacteria
Synechocystis PCC 6803 and its ndhB-defective mutant M55
(Ogawa 1991) were cultured in BG-11 medium (Allen 1968) at 27C
under fluorescent lamps at a light intensity of 60 mol m–2 s–1 photosynthetically active radiation. The suspensions were bubbled with air
enriched with 1.5% CO2. Cells were harvested and used for measurements during the logarithmic phase of growth.
Experimental set-up
NADPH fluorescence was measured with a PAM Chl fluorometer featuring a modified version of the emitter-detector-cuvette unit
ED-101PM (Heinz Walz GmbH, Effeltrich, Germany). The standard
650 nm measuring light LED was substituted by a blue LED (Nichia)
with an emission peak at 440 nm and a shoulder extending into the
UV-A. Wavelengths above 400 nm were eliminated by an optical filter
(2 mm UG 11, Schott). The resulting measuring light displayed a peak
around 380 nm. It was applied at maximal intensity, i.e. intensity setting 12 at the PAM-101 unit and 100 kHz modulation frequency. The
photomultiplier was protected by a combination of long-pass filter
(KV416, Schott), to eliminate the UV-measuring light and short-pass
filter (DT Cyan, Balzers), as well as blue-green glass filter (BG39,
Schott), to eliminate Chl fluorescence. It should be mentioned that after completion of the work presented here, a novel UV-A LED with an
emission peak at 375 nm has become commercially available (Nichia,
Type NSHU 590) with which the NADPH fluorescence measuring
system could be further improved. With this LED the measuring light
intensity is approximately four times higher, resulting in a 2-fold increase of the signal/noise ratio.
The sample was contained in a 10 10 mm quartz cuvette, with
excitation and detection pathways at right angle, and the side opposite
the
detector
being
mirrored.
Saturating
actinic
light
(660 mol quanta m–2 s–1) was generated by an LED-array cone with
peak emission at 655 nm (High-Power-LED-Lamp, Walz). Chl concentration was 18 g ml–1. The suspension was kept at a constant temperature of 27C. The analog output signal of the PAM fluorometer
was fed into a digital storage oscilloscope with signal averaging
(Nicolet), from where the data were transferred to a PC for further
processing.
In the given set-up a blue-green fluorescence signal was also observed in the absence of cyanobacteria, with the cuvette being filled
with water. This background signal, which decreased with increasing
concentrations of cyanobacteria (as both the excitation light and the
fluorescence were absorbed) prevented a reliable quantitative assessment of absolute NADPH fluorescence levels. Therefore, the figures
refer to ‘blue-green fluorescence’ and no absolute units for NADPH
fluorescence yield are given. A more quantitative approach is possible
in experiments with isolated spinach chloroplasts, which allow reliable calibration of NADP content by comparing NADP reduction in
chloroplasts with and without envelope (C. Klughammer, H. Mi and
U. Schreiber, manuscript in preparation).
Results and Discussion
Phenomenology of light-induced changes of blue-green
fluorescence
In Fig. 1, typical induction transients of blue-green fluorescence emission in response to illumination by saturating red
light are shown, as measured with a standard PAM fluorometer
equipped with a modified emitter-detector unit (see Materials
and Methods). Fluorescence was excited by modulated UVlight peaking around 380 nm. The signal did not contain any
Chl fluorescence, as this was completely eliminated by the filter set in front of the photodetector. In Fig. 1A the responses of
wild-type cells of Synechocystis PCC 6803 and of its ndhB-defective mutant M55 upon illumination with a 10 s saturating
light pulse are compared. In both cases complex dark–light–
dark induction transients are observed. In the wild-type the response is dominated by a rapid fluorescence increase which is
followed by a dip and a slower rise phase. Upon light-off there
is a biphasic fluorescence decline leading to a level distinctly
below the initial dark level (undershoot) followed by a slow
rise back to the dark level. In the mutant M55 upon onset of illumination there is only a small, rapid fluorescence rise which
is followed by a dip phase leading below the initial level. Upon
light-off there is a pronounced further fluorescence decline (undershoot) before the signal slowly returns to its original level.
For an interpretation of these data, it is important to consider that the displayed traces are the averages of 10 single responses which were recorded after reaching a steady state dur-
NADPH fluorescence in Synechocystis PCC 6803
Fig. 1 Dark–light–dark induction transients of blue-green fluorescence of Synechocystis PCC6803 and its ndhB-defective mutant M55.
Actinic illumination (AL) by repetitive 10 s pulses of saturating red
light (660 mol quanta m–2 s–1) with 40 s dark intervals between consecutive illumination periods. Depicted transients are the averages of
10 single recordings. Suspensions of wild-type (WT) and mutant
(M55) cells were adjusted to the same Chl concentration (18 g ml–1).
(A) Comparison of wild-type cells (WT) and mutant cells (M55) in the
absence of artificial acceptors. (B) Comparison in the presence of
nitrite. KNO2 was added to final concentration of 5 mM at 3 min
before measurement.
ing repetitive illumination featuring 10 s light and 40 s dark
periods. Hence, under the given conditions the cells are in a defined state of pre-illumination before each illumination period.
The question arises whether the observed changes of bluegreen fluorescence are due to NADPH and, if this is the case,
what reactions can be ascribed to the various phases of the
complex induction kinetics.
Nitrite is known to be reduced by ferredoxin at the acceptor side of PSI, thus competing with NADP for electrons. In the
dark, nitrite causes slow oxidation of NADPH via ferredoxinNADP+ oxidoreductase (FNR) and ferredoxin. Fig. 1B shows
that in the presence of nitrite the kinetic response of M55 becomes more similar to that of wild-type cells, as the amplitude
of the initial positive transient is distinctly increased. Nitrite
has practically no effect on the wild-type induction kinetics.
Very similar results as with nitrite were also obtained with hydrogen peroxide (H2O2) (data not shown) which is also known
to oxidize NADPH in the dark. Both nitrite and H2O2 were
found to cause a drop of the blue-green fluorescence dark level
(not shown). In wild-type cells the H2O2-induced quenching
was 25% of the maximal light-induced fluorescence increase,
whereas it was 80% in M55 cells. If it is assumed that H2O2 induces complete NADPH oxidation, these findings would suggest that approximately 25 and 80% of total NADP are reduced in the dark in wild-type and M55 cells, respectively. As
the kinetic responses of M55 cells become more similar to that
of wild-type cells when NADPH is preoxidized, it appears that
a major cause for the difference in kinetic responses is the dif-
1131
ferent reduction levels of NADPH in darkness in suspensions
of the two types of cells.
As a consequence of the steady state conditions reached
during repetitive illumination (see above), the net signal
change associated with a single illumination period (10 s light,
40 s dark) is zero. Therefore, assuming that the signal does reflect NADPH, there is the same amount of NADP reduction as
NADPH oxidation. It also has to be realized that during illumination reducing equivalents are not only stored in NADPH, but
also in form of reduced carbon (e.g. glucose-6 phosphate).
While it may be assumed that the former is rapidly oxidized by
the Calvin cycle after light-off to the extent that ATP is available, the latter will be more slowly broken down in the reductive pentose phosphate cycle with parallel reduction of NADP.
Considering the complexity of the in vivo reactions involving NADP(H), at the present state of information an explanation of the various transients of the induction kinetics can be
only tentative. However, based on our knowledge of photosynthetic reactions in wild-type and M55 the following interpretations appear reasonable:
First rapid rise: light driven reduction of NADP;
Dip phase: oxidation of NADP via Calvin cycle activity, which
sets in as soon as the ribulosebisphosphate carboxylase is activated and ATP becomes available;
Secondary rise phase: accumulation of NADPH as its oxidation in the Calvin cycle becomes limited, possibly going along
with a limitation in ATP-supply;
Stationary phase: matched rates of light-driven NADP reduction and NADPH oxidation via the Calvin cycle; electron storage in reduced carbon;
Rapid decay phase: dark-oxidation of NADPH via the Calvin
cycle or alternative acceptor systems, as, for example, active
oxygen species;
Slow decay phase: NADPH oxidation during oxidative phosphorylation;
Slow rise phase: dark-reduction of NADP associated with reductive pentose phosphate cycle.
These tentative interpretations shall serve as working hypothesis for the experiments presented in the following sections.
Inhibition of light-driven NADP reduction
Both in wild-type and M55 cells, the presence of methyl
viologen largely suppressed the light-induced increase of bluegreen fluorescence (data not shown). The PSII inhibitor
DCMU caused distinctly different effects on the responses of
wild-type and M55 cells. As shown in Fig. 2, in the presence of
10 mM DCMU almost no light-induced transients were observed in the mutant cells, whereas there was still a pronounced rapid response in wild-type cells. This observation
agrees with our previous findings that the wild-type displays a
high capacity of cyclic electron flow around PSI via the NDH
which assures reduction of the intersystem electron chain even
when electron donation by PSII is prevented by DCMU (Mi et
1132
NADPH fluorescence in Synechocystis PCC 6803
Fig. 2 Dark–light–dark induction transients of blue-green fluorescence of wild-type and M55 cells in the presence of DCMU. DCMU
was added 2 min before measurements at a concentration of 10 mM.
The bottom trace was recorded 2 min after 10 mM DBMIB was added
to a wild-type sample containing 10 mM DCMU. Conditions were the
same as for Fig. 1.
Fig. 3 Effect of iodoacetic acid on induction transients of blue-green
fluorescence in wild-type and mutant cells. IAA was added 2 min
before measurements at a concentration of 5 mM. Other conditions
were the same as for Fig. 1.
al. 1992a, Mi et al. 1992b). In the presence of DCMU, the
wild-type signal declines during illumination (compare with
control in Fig. 1). This reflects the exhaustion of NADPH,
which is partially oxidized by the Calvin cycle and thus withdrawn from cyclic flow around PSI. In M55 cells, which are
deficient in NDH, there is no dark reduction of the intersystem
electron transport chain and no NDH-mediated cyclic electron
flow. Hence the mutant cells are lacking a donor for PSI in the
presence of DCMU which, therefore, will prevent NADP reduction.
When, in addition to DCMU, the plastoquinone antagonist 2,5-dibromo-3-methyl-isopropyl-p-benzoquinone (DBMIB), which inhibits electron transport at the cyt b/f complex,
is also added, the response of wild-type cells is also largely
suppressed (bottom trace in Fig. 2).
Taken together, these data leave little doubt that the observed light-induced changes of blue-green fluorescence indeed
do reflect changes in NADPH fluorescence.
cence signal below the base line.
These findings agree with the assumption that the major
effect of IAA stems from inhibition of Calvin cycle enzymes
and, hence, that the IAA-induced difference in NADPH formation during a light pulse can be taken as a measure of NADPH
consumption by the Calvin cycle. Apparently, in wild-type
cells the rate of the Calvin cycle is sufficiently high to prevent
full NADP reduction even upon onset of saturating illumination (large effect of IAA on amplitude of rapid positive transient). This is possible only if there is a correspondingly high
ATP supply. On the other hand, in M55 cells, full NADP reduction is obtained (no effect of IAA on amplitude of rapid positive transient). This suggests that in M55 cells, which lack
NDH-mediated cyclic photophosphorylation, the Calvin cycle
is limited by ATP supply. It may be further noted that even in
the presence of IAA, when no NADPH is oxidized in the
Calvin cycle, upon light-off there is a rapid decline of fluorescence yield, which may reflect NADPH oxidation by active oxygen species (see below).
Effect of iodoacetic acid (IAA)
IAA is known to inactivate enzymes by cross linking with
cysteine residues. It acts as an inhibitor of Calvin cycle activity (Marsho et al. 1979). Fig. 3 shows that in the presence of
IAA the amplitude of rapid NADP reduction is approximately
doubled in wild-type but not in M55 cells. In both types of cells
the dip phase is suppressed. Furthermore, particularly in the
wild-type cells, there is a distinct suppression of the transient
post-illumination drop (undershoot) of the blue-green fluores-
Effect of mercury chloride (HgCl2)
HgCl2 is known to inhibit numerous enzyme reactions. It
was also found to affect NDH-mediated cyclic electron flow
around PSI (Mi et al. 1992a, Mi et al. 1992b). As shown in Fig.
4, the relatively low concentration of 20 mM HgCl2 has a dramatic effect on the apparent changes in NADPH upon illumination by a pulse of saturating light, in the wild-type as well as in
mutant M55 cells. Notably, the initial positive responses of the
controls are transformed by HgCl2 into negative transients, thus
NADPH fluorescence in Synechocystis PCC 6803
Fig. 4 Effect of HgCl2 on induction transients of blue-green fluorescence in wild-type and mutant cells. HgCl2 was added at a concentration of 20 mM Ca. 2 min before measurements. Other conditions were
the same as for Fig. 1.
suggesting that NADPH is oxidized in a rapid light-driven
reaction.
As our knowledge on the complex in vivo reactions in cyanobacteria is still incomplete, any attempt to definitely explain the effect of HgCl2 would be premature. However, as the
effect is also observed in the mutant M55, it can be stated that
the HgCl2-effect is not related to the function of NDH, i.e. at
least of that type of NDH which is mutated in M55. The following observations appear relevant for a tentative explanation: the rapid negative transient in the presence of HgCl2 is
substantially slowed down after removal of molecular oxygen
and also by addition of KCN (Fig. 5). The data agree with the
working hypothesis that HgCl2 blocks NADP reduction, possibly by affecting FNR-activity, thus stimulating O2-reduction.
The rapid negative transient could reflect oxidation of NADPH
by the H2O2 which originates from superoxide formation at the
acceptor side of PSI by the Mehler reaction and superoxide dismutase activity (Mehler 1951, Asada and Takahashi 1987).
While superoxide dismutase is known to be inhibited by KCN,
superoxide may also dismutate in an uncatalyzed reaction, although at a much lower rate. This may explain why the negative transient is not completely suppressed by KCN. Furthermore, it may be assumed that superoxide can also interact
directly with NADPH and thus cause its oxidation.
Conclusions
The presented data allow a number of conclusions. Intact
cells of cyanobacteria display blue-green fluorescence, which
can be monitored by a standard PAM fluorometer in conjunc-
1133
Fig. 5 Effects of O2-removal and cyanide on rapid light-induced
quenching of blue-green fluorescence in the presence of HgCl2. Wildtype cells in the presence of 20 mM HgCl2. O2 was removed with the
help of the glucose–glucose oxidase trap (3 mM glucose, 30 U ml–1
glucose oxidase). KCN concentration was 1 mM. Other conditions
were the same as for Fig. 1.
tion with a new emitter-detector unit: This fluorescence is characterized by dynamic light-induced changes which appear likely to reflect changes in NAD(P)H. In principle, NADH and
NADPH display very similar fluorescence properties and,
hence, cannot be distinguished by this method.
The conclusion that NAD(P)H fluorescence changes are
involved is supported by the fact that the rapid positive transient can be affected in a predictable manner by alternative
photosynthetic electron acceptors, like methyl viologen, nitrite
and H2O2, and can be suppressed by electron transport inhibitors, in wild-type as well as mutant M55 cells of Synechocystis
PCC6803.
The PSII inhibitor DCMU inhibits the light-induced transients in the ndhB-defective mutant M55, but not in wild-type
cells, supporting previous suggestions of NDH-mediated cyclic electron transport around PSI. Almost complete inhibition
in wild-type cells can be obtained by adding DBMIB as well as
DCMU.
Using a particular illumination program featuring repetitive pulses of saturating light with intermittent dark periods
(10 s light, 40 s dark) complex responses of various light and
dark reactions involving NAD(P)H can be obtained under quasi-stationary illumination, which provide information on various reactions involving NADP reduction and NADPH oxidation in the light and in the dark.
A rapid negative response which normally is overlapping
the initial rise phase is inhibited by IAA and, hence, appears
likely to reflect NADPH oxidation by the Calvin cycle.
HgCl2 causes a remarkable transformation of the light-in-
1134
NADPH fluorescence in Synechocystis PCC 6803
duced rapid positive transient into a negative transient which is
affected by O2-depletion and KCN. The latter effects argue for
a possible involvement of the H2O2 which is formed via superoxide at the acceptor side of PSI.
In view of the complexity of the in vivo system, these observations are still incomplete and the drawn conclusions
should be considered tentative only. However, there can be
hardly any doubt that the new fluorescence approach does allow very detailed insights into the dynamic interplay between
light and dark reactions of cyanobacteria under in vivo conditions. This should open the way for more detailed investigations and eventually to a deeper understanding of the interactions between photosynthetic and respiratory electron transport
chains, in which NADPH undoubtedly plays a central role.
Ackowledgements
We thank Prof. T. Ogawa (Bioscience Center, Nagoya University,
Japan) for the gift of the M55 strain. H.M. was supported by a fellowship of the Max-Planck-Gesellschaft and a travel grant from the Chinese Academy of Science (97XD14023 and KJ982-J1-625). Financial
support by the Deutsche Forschungsgemeinschaft (SFB 176) is gratefully acknowledged.
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(Received December 17, 1999; Accepted July 25, 2000)