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Plant Physiol. (1998) 117: 525-532
The NAD(P)H Dehydrogenase in Barley Thylakoids Is
Photoactivatable and Uses NADPH as well as NADH1
Harald Bernhard Teicher and
Henrik Vibe Scheller*
Plant Biochemistry Laboratory, Department of Plant Biology, Royal
Veterinary and Agricultural University, 40 Thorvaldsensvej, DK-1871
Frederiksberg C, Denmark
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ABSTRACT |
An improved light-dependent assay was
used to characterize the NAD(P)H dehydrogenase (NDH) in thylakoids of
barley (Hordeum vulgare L.). The enzyme was sensitive to
rotenone, confirming the involvement of a complex I-type enzyme. NADPH
and NADH were equally good substrates for the dehydrogenase. Maximum
rates of activity were 10 to 19 µmol electrons mg 1
chlorophyll h1, corresponding to about 3% of linear
electron-transport rates, or to about 40% of ferredoxin-dependent
cyclic electron-transport rates. The NDH was activated by light
treatment. After photoactivation, a subsequent light-independent period
of about 1 h was required for maximum activation. The NDH could
also be activated by incubation of the thylakoids in low-ionic-strength
buffer. The kinetics, substrate specificity, and inhibitor profiles
were essentially the same for both induction strategies. The possible
involvement of ferredoxin:NADP+ oxidoreductase (FNR) in the
NDH activity could be excluded based on the lack of preference for
NADPH over NADH. Furthermore, thenoyltrifluoroacetone inhibited the
diaphorase activity of FNR but not the NDH activity. These results
also lead to the conclusion that direct reduction of plastoquinone by
FNR is negligible.
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INTRODUCTION |
The genomes of cyanobacteria and most plant chloroplasts contain
11 genes (ndhA-ndhK) with sequence similarity to
the subunits of NADH dehydrogenase (complex I) (Berger et al., 1993 ).
The ndh gene products form a complex that can be isolated
from thylakoid membranes (Funk and Steinmüller, 1995; Sazanov et
al., 1995; Quiles et al., 1996). In bacteria (other than cyanobacteria)
complex I consists of 14 subunits that are also conserved in
mitochondrial complex I (Friedrich et al., 1995). The cyanobacterial
and chloroplast NDH seem to lack homologs of the three essential
subunits that constitute the NADH-oxidation site in the bacterial and
mitochondrial complex (Grohman et al., 1996).
The function of NDH in chloroplasts is not understood, but a role in
cyclic electron transport and/or chlororespiration would seem to be
likely. A role in cyclic electron transport would imply electron
donation from stromal NADPH via the membrane-bound NDH complex to the
plastoquinone pool. Kubicki et al. (1996) showed that in sorghum the
ndh genes are preferentially expressed in bundle-sheath
chloroplasts, the apparent site of cyclic electron flow in
C4 species. Thus, this finding is in agreement
with a function of the NDH complex in cyclic electron transport.
NDH activity has been demonstrated in the thylakoid membranes of
several different species of plants, algae, and cyanobacteria (Mi et
al., 1992a, 1992b, 1994, 1995; Yu et al., 1993; Cuello et al., 1995;
Sazanov et al., 1995; Quiles et al., 1996; Seidel-Guyenot et al.,
1996). However, direct demonstration of an involvement in cyclic
electron transport in most cases has not been achieved. Mi et al.
(1995), working with ndh mutants of Synechocystis
sp. PCC 6803, have presented the most clear evidence for an involvement of the NDH complex in cyclic electron transport. In contrast, ndh mutants of Synechococcus sp. PCC 7002 were
not deficient in cyclic electron transport (Yu et al., 1993). Cyclic
electron transport via NDH is most easily understood if the complex can
use NADPH, as shown by Mi et al. (1995). However, there is a lack of
consensus on the substrate specificity of the NDH complex. In barley
(Hordeum vulgare) and in pea, NADH-specific oxidation has
been reported (Cuello et al., 1995; Sazanov et al., 1995). Because the
thylakoid NDH activity in plants has been studied in only a few
instances, it is tempting to draw on studies in algae and
cyanobacteria. In the unicellular algae Chlamydomonas
reinhardtii and Pleurochloris meiringensis, which are
both species with a high rate of chlororespiration in the dark, the
dehydrogenase activity in the thylakoid membranes is most efficient
with NADH (Godde and Trebst, 1980; Seidel-Guyenot et al., 1996). In
cyanobacteria the situation is complex; whereas NADPH-specific activity
has been reported in Synechocystis sp. PCC 6803 (Mi et al.,
1995), different specificities have been reported for other species
(for review, see Schmetterer, 1994).
It is futile to look for a unifying principle covering all
oxygenic phototrophs. It seems likely that NDH is involved in both cyclic and respiratory pathways, and that its relative contribution to
different pathways may differ between species or even within a species
dependent on growth conditions. The issue of specificity is complicated
by the different assay conditions used, by mitochondrial contamination,
and by interference from NADPH oxidation by FNR. In most previous
studies the dehydrogenase activities have been assayed in the dark with
artificial acceptors such as ferricyanide or soluble quinones. Both
mitochondrial complex I and FNR will show high activity in such assays.
In this paper we have used a light-specific assay that eliminates the
interference from contaminating activities, and we clearly demonstrate
an NAD(P)H dehydrogenation that functions with equal efficiency with
both substrates.
A further unsolved question is which subunit(s) contains the
NAD(P)H-binding site of the NDH complex? The chloroplast genome has no
homolog of the NADH-binding flavoprotein of complex I, and evidence
against the presence of a nuclear-encoded chloroplast homolog has been
presented (Grohman et al., 1996). The genome of
Synechocystis sp. PCC 6803 contains open reading frames in the hydrogenase operon with some similarity to the NADH-oxidizing subunits in other bacteria (Appel and Schulz, 1996). Whether the gene
products are part of cyanobacterial NDH remains to be shown. Quiles et
al. (1996) reported the presence of a 53-kD NADH-oxidizing protein in
barley chloroplasts, and have suggested that this protein could be a
component of the NDH complex. The 53-kD protein was specific for NADH
rather than NADPH. Guedeney et al. (1996) showed that the flavoprotein
FNR binds to several polypeptides of the NDH in tobacco thylakoids, and
have suggested that FNR in thylakoids could be the functional
equivalent of the NADH-oxidizing domain in complex I. This could
explain the result of Mi et al. (1995), who provided evidence that the
Synechocystis sp. PCC 6803 mutant deficient in NDH was also
deficient in Fd-catalyzed cyclic electron transport. In contrast to
this result, cyclic electron transport of barley thylakoids could not
be inhibited by antibodies against FNR (Scheller, 1996).
In this study we present a number of decisive arguments against the
involvement of FNR in the NDH activity. We present evidence that
confirms and characterizes the presence of a bispecific, NDH-dependent
electron flow in barley, and address a number of conflicts that exist
from previous publications in this field. Finally, we report that
NAD(P)H dehydrogenation in isolated thylakoids of barley requires
induction for maximal rates of activity. Induction can be triggered by
brief illumination or by incubation of thylakoids in a
low-ionic-strength buffer.
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MATERIALS AND METHODS |
Isolation of Thylakoids
Seedlings of barley (Hordeum vulgare L. cv
Svalöf's Bonus) were grown at 20°C in vermiculite with a 12-h
photoperiod. Fluorescent tubes provided a PPFD of 80 µmol
m2 s1. After 7 d
the seedlings were harvested at the onset of the light period, and
leaves were homogenized in a buffer of 0.4 M Suc, 10 mM NaCl, 5 mM MgCl2, 10 mM Tricine (pH 7.5), and 10 mM sodium ascorbate
using a blender equipped with razor blades. The chloroplasts were
precipitated by centrifugation at 2000g for 10 min. The
chloroplasts were resuspended and lysed in 5 mM Tricine (pH
7.9) and the thylakoids were precipitated at 10,000g for 10 min. The thylakoid pellet was resuspended at a concentration of 0.5 to
2 mg Chl/mL in homogenization buffer with 20% glycerol and no
ascorbate. Thylakoid preparations used for the study of FNR and Cyt
c oxidase activity had a protein:Chl ratio of about 5:1. The
thylakoids were frozen in liquid N2 and stored at
 80°C. The thylakoids remained fully active in all of the pathways
investigated for at least 6 months.
Linear and Cyclic Electron Transport
Linear electron flow in thylakoids was measured as rates of
O2 consumption, using a Clark-type
O2 electrode. The reaction medium contained (in a
total volume of 3 mL): 33 mM Tricine (pH 7.5), 83 mM NaCl, 10 mM MgCl2, 0.1 mM MeV, 6.7 mM NH4Cl, 0.3 mM NaN3, and thylakoids corresponding
to 60 µg of Chl. Fd-catalyzed cyclic electron transport was measured
as rates of P700+ reduction under anaerobic and
redox-poised conditions as described previously (Scheller, 1996).
Light-Dependent NAD(P)H Oxidation
Quantitation of light-dependent NAD(P)H oxidation was carried out
by a modification of the procedure of Mi et al. (1995). The reaction
mixture contained (in a total volume of 0.5 mL): 24 mM
Tris-HCl (pH 8.6), 48 mM NaCl, 9.6 mM
MgCl2, 200 µM NAD(P)H, 100 µM MeV, 10 µM DCMU, and thylakoids
corresponding to 4 to 8 µg of Chl. The oxidation of NAD(P)H at 25°C
was followed at 340 nm in a spectrophotometer (DW-2000, Aminco, Urbana,
IL) with a thermostated cuvette holder. The actinic light was supplied
by a halogen lamp (15 V, 150 W; model KL1500, Schott, Cologne, Germany) provided with fiber optics and passed through a 665-nm cut-on filter.
The photomultiplier tube was protected from the actinic light with a
340-nm narrow-bandwidth interference filter. The oxidation of NAD(P)H
was calculated using an extinction coefficient of 6.2 mM1 cm1.
Before spectrophotometric analysis, the reagents, except for NAD(P)H,
were mixed and the NDH activity was activated by illumination. The
sample was surrounded by a water-cooled jacket (25°C) and white light
was supplied by a halogen lamp (KL1500 Schott) provided with fiber
optics. Details of illumination times are given in ``Results''.
Alternatively, activation was accomplished by omitting
MgCl2 from the reaction mixture, and incubating
the sample for 1 h in the dark in the presence of 12 µM NADPH.
Light-Independent NAD(P)H Oxidation
The rate of light-independent NAD(P)H oxidation was measured
spectrophotometrically at 340 to 375 nm (using an extinction coefficient of 4.0 mM1
cm1) as the rate of NAD(P)H oxidation at 25°C
in an assay mixture containing (in a total volume of 0.5 mL): 20 mM Tris-HCl (pH 7.8), 8 mM
MgCl2, 200 µM NAD(P)H, and 120 µM duroquinone. The reaction also contained thylakoids
corresponding to 8 µg of Chl or 4 pmol of isolated barley FNR. The
reaction was started by the addition of duroquinone.
Chemicals and Various Assays
NADPH and NADH were obtained from Sigma (highest grade available)
or from Boehringer Mannheim. The use of high-grade pyridine nucleotides
is essential because lower-grade reagents may be contaminated with
inhibitors of NDH (Dalziel, 1963). All other reagents were of
analytical grade.
Chl was determined according to the method of Arnon (1949), and Cyt
c oxidase activity was measured according to the method of
Jesaitis et al. (1977). Protein concentration was determined by the
bicinchoninic acid procedure according to the method of Dunn (1989).
Barley FNR was isolated essentially according to the method of Serrano
and Rivas (1982). Free FNR and the proteins of the photosynthetic
membrane were resolved by SDS-PAGE using 8 to 25% polyacrylamide gels.
Western blotting was performed essentially according to the method of
Andersen et al. (1992) by electrophoretic transfer of thylakoid
proteins and free FNR to nitrocellulose membranes, incubation with
polyclonal rabbit antibodies raised against FNR isolated from barley,
and final incubation with secondary swine antibodies conjugated with
alkaline phosphatase.
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RESULTS |
Thylakoids Support Light-Dependent Oxidation of NADPH and NADH
To analyze the NDH activity without interference from
mitochondrial complex I or from the diaphorase activity of FNR, we used a light-dependent assay, in which NAD(P)H oxidation was driven by PSI
in the presence of MeV. The NAD(P)H oxidation was completely dependent
on light, and no background oxidation in the dark could be detected
(Fig. 1). Furthermore, no NAD(P)H
oxidation was observed in the absence of MeV (data not shown). In the
experiment shown in Figure 1, the rate of NADPH oxidation was 15 ± 2 µmol electrons mg1 Chl
h1 and the rate of NADH oxidation was 15 ± 3 µmol electrons mg1 Chl
h1. Whole-chain linear electron transport was
determined to be about 450 µmol electrons mg1
Chl h1, showing that the thylakoids had an
intact PSI and intersystem chain. Fd-catalyzed cyclic electron
transport in barley thylakoids in the presence of 25 µM
reduced Fd was determined by Scheller (1996) to be 36 µmol
electrons mg1 Chl h1,
i.e. 8% of whole-chain electron-transport rates or 2.5 times that of
the thylakoid NDH activity.
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| Figure 1.
The thylakoidal NDH activity in isolated
thylakoids of barley is NAD(P)H bispecific. The assay used in this
study for measuring the oxidation of NAD(P)H with MeV as the electron
acceptor is absolutely light dependent and thus specific for
thylakoidal NDH activity. The NDH was activated by illuminating the
thylakoids in the presence of the assay reagents for 45 min before
measurement.
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Even though the light-dependent assay eliminates the interference from
mitochondrial complex I, we resolved to quantitate the degree of
mitochondrial contamination. Cyt c oxidase activity was
measured on whole-leaf homogenate, on the supernatant after centrifugation of the homogenate at 2000g, and on isolated
thylakoid membranes. Expressed on the basis of total volume, the
whole-leaf homogenate was capable of oxidizing 8.2 µmol Cyt
c/min, the supernatant retained an activity of 5.8 µmol
Cyt c/min, and the thylakoid membranes had an activity of
0.5 µmol Cyt c/min, representing a contamination of the
thylakoid fraction by mitochondrial protein of 7%.
Rotenone is a classic inhibitor of mitochondrial complex I, acting on
the ubiquinone-reducing Fe-S centers (Trumpower, 1981). Inhibition of light-dependent NAD(P)H oxidation by rotenone (inhibitor concentration at 50% inhibition  150 µM)
confirms that light-dependent NAD(P)H oxidation is mediated by a
complex I-type enzyme, i.e. NDH (Fig. 2).
This experiment also confirmed the similarity with the NADH- and
NADPH-dependent activities. Antimycin A (20 µM) did not
inhibit the light-dependent oxidation of NADPH.
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| Figure 2.
Inhibition of light-dependent NADPH ( ) and NADH
( ) oxidation by rotenone in isolated thylakoids. The thylakoids were
activated by preillumination for 45 min. The activity of thylakoids in
the absence of rotenone was 19 µmol electrons mg1 Chl
h1 for the oxidation of NADPH and 20 µmol electrons
mg1 Chl h1 for the oxidation of NADH.
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FNR Is Not Involved in the Light-Dependent NADPH
Dehydrogenation
TTFA is an inhibitor of mitochondrial electron transport, acting
on succinate dehydrogenase (Trumpower, 1981). The light-dependent NADPH
dehydrogenase activity was not inhibited by TTFA (Fig.
3). TTFA also did not inhibit the
light-dependent NADH oxidation (data not shown). In contrast, TTFA
inhibited duroquinone-mediated diaphorase activity of isolated FNR, as
well as the light-independent, duroquinone-mediated NADPH oxidation by
thylakoids (Fig. 3). To address further the issue of the involvement of
FNR in the NDH activity we determined the amount of FNR in the
thylakoids. Immunoblots of thylakoids and isolated FNR incubated with
antibodies against FNR revealed that thylakoid fractions contained 5.8 nmol membrane-bound FNR/mg Chl (data not shown). In the
light-independent assay with duroquinone, the thylakoids oxidized NADPH
at a rate of 36 ± 7 µmol electrons mg1
Chl h1, whereas isolated FNR oxidized at a rate
of 91 ± 9 µmol electrons nmol1
h1. Based on an observation by Nielsen et al.
(1995) that free FNR is about 10 times more effective than bound FNR in
mediating the oxidation of NADPH by duroquinone, we can estimate that
the thylakoid-bound FNR should be able to mediate NADPH oxidation at a
rate of about 47 µmol electrons mg1 Chl
h1. Thus, the amount of FNR in the thylakoids
can account for all of the NADPH oxidation observed in the duroquinone
assay.
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| Figure 3.
TTFA as an inhibitor of NADPH oxidation by
isolated thylakoids or by FNR. The activity (± SD) was
determined as light-dependent NADPH oxidation by thylakoids in the
presence of MeV (), as NADPH oxidation in the dark by thylakoids in
the presence of duroquinone ( ), or as NADPH oxidation by isolated
FNR in the presence of duroquinone ( ). The light-dependent assay was
performed with thylakoids activated by preillumination for 45 min. The
activity of thylakoids in the absence of TTFA was 15 ± 2 µmol
electrons mg1 Chl h1 for the
light-dependent assay and 36 ± 7 µmol electrons
mg1 Chl h1 for the duroquinone-dependent
assay. The activity of FNR in the absence of TTFA was 91 ± 10 µmol electrons nmol1 FNR h1.
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Kinetic Properties of the NDH
Kinetic parameters were calculated for the light-activated
oxidation of NADPH by thylakoid preparations. The NDH activity did not
exhibit pure Michaelis-Menten kinetics (Fig.
4). The Eadie-Hofstee plot calculated
from the data in Figure 4 was hyperbolic rather than linear (Fig. 4,
inset), suggesting the presence of two kinetically different enzymes
capable of the independent oxidization of NADPH, a phenomenon
previously reported for thylakoidal NAD(P)H oxidation in the
chromophytic alga Pleurochloris meiringensis (Seidel-Guyenot et al., 1996). Enzyme kinetics can be approximated using hyperbolic regression as Vmax = 10 µmol electrons
mg1 Chl h1,
Km = 20 µM NADPH; or, using
the Lineweaver-Burk plot, as Vmax = 9 µmol electrons mg1 Chl
h1, Km = 13 µM NADPH. The experiment was carried out four times with
essentially the same results. Based on an observation by Sazanov et al.
(1995) that the stoichiometric ratio of NADH dehydrogenase to
photosynthetic reaction centers in spinach chloroplasts is about 1:100,
the turnover number (Kcat) of the NDH
characterized in this paper was calculated to be about 130 s1. The calculated
Kcat/Km ratio
of 107 M1
s1 indicates a relatively high kinetic
proficiency as well as a high substrate affinity, and suggests an
important role for this enzyme in mediating electron transport.
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| Figure 4.
Thylakoidal NDH activity (± SD) as a
function of NADPH concentration. The thylakoids were activated by
preillumination for 45 min. The curve shows a fit obtained by
hyperbolic regression. Inset, Eadie-Hofstee plot of the data.
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Photoactivation of NDH
The rate of NADPH oxidation observed in thylakoids was higher if
the thylakoids had been illuminated before measurement. Maximal rates
of activity were obtained approximately 30 to 60 min after the onset of
the illumination period. However, the light pulse required to achieve
maximal activation may be as short as 5 min, after which full
activation may be accomplished in the dark (Fig. 5). All of the results presented above
for the MeV-mediated oxidation of NAD(P)H were acquired after 45 min of
light induction.
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| Figure 5.
Light activation of the thylakoidal NDH. The
thylakoids were illuminated in the presence of the assay reagents for
the time periods indicated under the open bars. After illumination, the samples were kept in the dark and rates of NADPH oxidation (± SD) were determined at different time points. The numbers
under the shaded bars indicate the total time from onset of the
experiment, i.e. the sum of the illumination and subsequent dark
periods.
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With the standard assay mixture containing 9.6 mM
MgCl2 and 48 mM NaCl, or with 240 mM NaCl, light was required for induction of activity (Fig.
6). Similar data were obtained when
CaCl2 was substituted for
MgCl2. However, after incubation for up to 60 min
in a low-salt assay medium (48 mM NaCl), light was not
required for induction of the activity.
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| Figure 6.
Induction of NDH activity. The assay mixtures
contained low concentrations of monovalent cations: 48 mM
Na+ ( ); or high concentrations of mono- and/or divalent
cations: 240 mM Na+ ( ), 48 mM
Na+/9.6 mM Ca2+ ( ), or 48 mM Na+/9.6 mM Mg2+
(). The thylakoids were incubated in the dark in the presence of the
respective assay reagents for 60 min, after which time the thylakoids
were illuminated for the specified times before measurement of NADPH
oxidation (± SD).
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DISCUSSION |
An assay has been used that specifically permits the measurement
of NAD(P)H oxidation via the thylakoid NDH in isolated thylakoids of
barley. The absolute light dependence of the assay (Fig. 1) excludes
interference from the mitochondrial NADH dehydrogenase. Using a series
of inhibitors it has been possible to determine that NAD(P)H is
oxidized via a rotenone-sensitive, complex I-type chloroplast NDH.
Rotenone inhibition was observed at higher concentrations (Fig. 2) than
that necessary to inhibit mitochondrial complex I (Slater, 1967;
Singer, 1979), but were in good agreement with previous reports of
rotenone inhibition of thylakoidal electron transport (Cuello et al.,
1995; Seidel-Guyenot et al., 1996).
In this study we demonstrate that electron transport via the
chloroplast NDH complex shows no NADPH/NADH substrate specificity (Figs. 1 and 2). The identical inhibition of both NADH and NADPH oxidative activities of NDH by rotenone indicates that both activities are components of the same enzyme (Fig. 2). The fact that NADPH is a
good substrate for the thylakoidal NDH strongly implies an involvement
of this complex in cyclic electron transport. The NADPH/NADH
bispecificity is an unusual (but not unique) property of an enzyme. The
best-documented case for a bispecific NAD(P)H-dependent enzyme is an
isoform of nitrate reductase (Campbell, 1996; Redinbaugh et al., 1996).
The very similar enzymatic properties with both substrates observed
with barley thylakoids, as reported in this paper, indicate that a
bispecific NDH complex carries out the oxidation of both substrates.
However, as the NAD(P)H-binding subunits of the NDH complex have still
not been identified, one should be cautioned against premature
conclusions. One possibility could be that the thylakoid NDH complex
exists in two different forms distinguished by a different set of
pyridine nucleotide-binding subunits. It can then be speculated that
the plant may alter the relative content of the two forms dependent on
the physiological requirements, e.g. for chlororespiration or cyclic
electron transport. Such a scheme could explain the contradictory
results obtained with different organisms.
NADH- and NADPH-oxidizing activities have been reported in chloroplasts
of barley (Cuello et al., 1995) and pea (Sazanov et al., 1995), as well
as in chloroplasts of P. meiringensis (Seidel-Guyenot et
al., 1996), and it has been suggested that the NDH complex is specific
for NADH, whereas the NADPH-oxidizing activity was mostly attributable
to the diaphorase activity of FNR (Cuello et al., 1995; Sazanov et al.,
1995). These workers have used a light-independent assay of thylakoidal
NDH activity with ferricyanide or soluble quinones as the electron
acceptor. Under these experimental conditions, FNR would be expected to
exhibit a very high NADPH-oxidation activity (Cuello et al., 1995;
Nielsen et al., 1995), as also demonstrated in the present
investigation (Fig. 3). Thus, NADPH oxidation by the NDH complex may
have been masked by FNR in the experiments by these workers.
Another source of NAD(P)H oxidation in our samples could be the
activity of mitochondrial enzymes. We have determined the contamination
of thylakoid preparations by mitochondrial proteins to be 7%. Using
the values of Rasmusson and Møller (1991) we can calculate that with
NADH as the substrate and O2 as the acceptor, the
mitochondrial enzymes could theoretically account for about 30 µmol
electrons mg1 Chl h1
under our conditions, whereas NADPH oxidation would be negligible. However, we were not able to detect any oxidation of NADPH or NADH in
the dark (Fig. 1), possibly because of the lack of Cyt c in
our samples. This allows us to conclude that the contribution of
mitochondrial NAD(P)H oxidation with O2 as the
electron acceptor in these samples is negligible.
The light-dependent, MeV-mediated oxidation of NADPH by barley
thylakoids does not have a component of FNR-mediated activity. The
NAD(P)H bispecificity of the thylakoidal NDH activity (Figs. 1 and 2)
is convincing evidence against the involvement of FNR. Further evidence
against the involvement of FNR is the insensitivity of MeV-mediated
oxidation of NADPH to TTFA (Fig. 3). In addition, the
Km value of 13 µM NADPH for
the light-dependent NADPH oxidation reported here differs significantly
from the Km value of 59 µM NADPH reported for the diaphorase activity of bound FNR (Nielsen et
al., 1995). FNR efficiently catalyzes reduction of soluble quinones,
and it has previously been speculated that FNR could directly mediate
the reduction of plastoquinone in the membrane (Hosler and Yocum, 1985;
Nielsen et al., 1995). However, no reduction by FNR of the
physiologically more-relevant quinones, i.e. decyl-plastoquinone, vitamin K1, or ubiquinone-10, could be detected (Nielsen et al., 1995).
If FNR could directly reduce plastoquinone, it should contribute to the
MeV-dependent NADPH oxidation. Because no component of FNR-dependent
activity was detectable, we conclude that direct reduction of membrane
quinones by FNR must be negligible. Contrary to the light-dependent NDH
activity, the light-independent NADPH oxidation activity appears to be
similar to FNR diaphorase activity. Accordingly, it is most likely that
light-independent NADPH oxidation in the presence of duroquinone is
predominantly attributable to FNR bound to the thylakoid membrane. In
summary, these results support the following three conclusions: (a) FNR
does not interfere with the light-dependent assay for NDH activity, (b)
FNR is not a component of the NDH complex in barley thylakoids, and (c)
electron transfer from NADPH via FNR directly to plastoquinone must be very limited if it occurs at all.
The concentration dependency of the NDH activity indicates the presence
of two enzymes (Fig. 4). The two activities appear to be saturated at
200 µM NADPH, i.e. the standard conditions for the
light-dependent assay. Under these conditions no inhibition of activity
by TTFA was observed (Fig. 3). Because TTFA strongly inhibits FNR, we
can conclude that FNR does not make any detectable contribution to the
activity shown in Figure 4. The indicated presence of a thylakoidal NDH
activity derived from two enzymes more likely should be interpreted as
the presence of a single enzyme in different states of activation, or
as activities reflecting a modification to a fraction of the NDH
complex pool.
The NDH-catalyzed NAD(P)H oxidation by light-induced thylakoid
membranes was determined to be 10 to 19 µmol electrons
mg1 Chl h1. The
variation in activities presented here may be attributable in part to
the fact that the mechanism and requirements for activation of NDH
activity are not yet fully understood. We believe that the rates
reported here represent true physiological activities, and that
previously published rates of NDH activity in plants have been
overestimated because of interference from contaminating activities.
Thus, in an assay measuring electron transport from NAD(P)H to
potassium ferricyanide in barley (Cuello et al., 1995), the rate of
NADPH oxidation can be calculated to be about 400 µmol electrons
mg1 Chl h1. However, as
these workers point out, the specificity of the assay is such that much
of the high rate with NADPH may be ascribed to the diaphorase activity
of FNR. The rate of NADH oxidation in the same study can be calculated
as 125 µmol electrons mg1 Chl
h1. Ferricyanide, however, is a much more
efficient acceptor of electrons than the more natural acceptor,
plastoquinone-1 (Godde, 1982). When this factor is taken into account,
the rate of NADH oxidation found by Cuello et al. (1995) is comparable
to the rate presented here.
The NDH activity reported here corresponds to about 3% of linear
electron- transport rates. Fd-dependent cyclic electron transport corresponds to about 5% of linear electron transport, depending on the
exact redox conditions (Scheller, 1996). Combined activities of cyclic
electron transport of less than 10% of linear electron transport agree
with cyclic electron-transport rates determined in microorganisms in
vivo (Maxwell and Biggins, 1976; Myers, 1987; Yu et al., 1993; Ravenel
et al., 1994).
The activation of NADPH-dependent electron flow through the thylakoidal
NADPH dehydrogenase is shown to be light dependent (Fig. 5). The
initial light-dependent step in the activation process may be related
to the generation of ion gradients or oxidation of a redox mediator.
Apparently, this initial step triggers a slower activating step. In the
isolated thylakoid membranes, activation by phosphorylation or by
thioredoxin cannot take place. One possibility would be activation by
dephosphorylation. Redinbaugh et al. (1996) have reported activation by
dephosphorylation of nitrate reductase, another bispecific
pyridine-nucleotide dehydrogenase.
When the ionic strength is low, light is not required to trigger the
activation, whereas rates of NDH activity are generally lower for
ionically activated than for photoactivated samples (Fig. 6). NDH is
localized in the grana margins and stroma lamellae (Steffánsson,
1996). The mechanism of ionic activation could be related to a
destacking of the thylakoid membranes and exposure of previously masked
pools of NDH. Because the activities observed upon light treatment or
destacking are not additive effects, it is tempting to speculate that
light activation leads to a lateral displacement of the NDH complex
toward the stroma lamellae. Present investigations are aimed at
determining the mechanisms of activation.
A model of the different pathways of cyclic electron transport is shown
in Figure 7. Fd-catalyzed cyclic electron
transport may be mediated in an antimycin-sensitive reaction by a low
potential Cyt b559 (Miyake et al., 1995).
The antimycin-insensitive Fd:quinone reductase has not been identified.
Possibly, the antimycin-insensitive Fd:quinone reductase and NDH are
identical. Ongoing experiments with inhibitors of NDH and Fd:quinone
reductase should allow us to decide between these possibilities.
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| Figure 7.
Model of cyclic electron transport in thylakoid
membranes of barley, showing the site of action of inhibitors and
artificial acceptors (italics) used in this study. A, B, C, D, and E,
Subunits of PSI; DQ, duroquinone; FQR, antimycin-insensitive Fd:quinone reductase; and Pc, plastocyanin.
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FOOTNOTES |
1
This work was supported in part by grants from
the Center of Plant Biotechnology, the Danish Natural Science Research
Council, and the Danish Agricultural and Veterinary Research Council.
*
Corresponding author; e-mail scheller{at}biobase.dk; fax
45-35-28-33-33.
Received November 26, 1997;
accepted February 25, 1998.
| |
ABBREVIATIONS |
Abbreviations:
Chl, chlorophyll.
FNR, Fd:NADP+
oxidoreductase.
MeV, methyl viologen.
NDH, NAD(P)H dehydrogenase.
TTFA, thenoyltrifluoroacetone.
| |
ACKNOWLEDGMENT |
Prof. Birger Lindberg Møller is thanked for many valuable
discussions and for his support throughout this study.
| |
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Abstract
Introduction