From the Institut für Biologie II, Biochemie
der Pflanzen,
Zellbiologie,
§§ Mikrobiologie, Universität Freiburg,
Schänzlestrasse 1, 79104 Freiburg, Germany, ** Service
de Bioénergétique, DBJC, CNRS URA 2096, Commissariat
à l'Energie Atomique Saclay, 91191 Gif-sur-Yvette, France, and
the ¶¶ Institut für Biochemie und Biotechnologie,
Universität Münster, Hindenburgplatz 55, 48143 Münster, Germany
Received for publication, December 17, 2002, and in revised form, February 4, 2003
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ABSTRACT |
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The function of cytochrome
b559 in photosystem II (PSII) was investigated
using a mutant created in tobacco in which the conserved phenylalanine at position 26 in the Photosystem II (PSII)1
is the membrane protein complex in thylakoid membranes that catalyzes
the light-induced oxidation of water and the reduction of
plastoquinone. The scaffold of the PSII reaction center is formed by
two protein subunits, D1 and D2, where the cofactors for photosynthetic
electron transport are located, and by cytochrome
b559. Cytochrome b559 is
located in close proximity to D1/D2 and is found in the smallest
experimentally obtainable PSII complex capable of carrying out
light-induced charge separation (1). This cytochrome, encoded by the
psbE and psbF genes, has a heterodimeric
structure consisting of one Different potential forms for cytochrome b559
are known: the high potential form, which is found in active
oxygen-evolving PSII; the intermediate form; and the low potential
form, which is found in PSII with an impaired oxygen-evolving complex
(for review, see Ref. 7). The different redox forms of cytochrome b559 are characterized by small changes of the
g-values in their EPR spectra, indicating very subtle changes in the
heme coordination (such as a small change in the relative orientation
of the histidine planes). There is an ongoing debate whether one
or two cytochrome b559 units are present per
PSII, although most recent publications (for reviews, see Refs. 2 and
7) and the x-ray structure suggest that there is only one (3).
Cytochrome b559 is an indispensable constituent
of PSII, but its function remains unclear. It is a redox-active
protein, and both photooxidation and photoreduction of its heme iron
have been observed. Because these reactions are very slow and
characterized by low quantum yields, it is generally assumed that the
cytochrome takes no active part in the primary electron transfer
reaction. A direct electron donation from cytochrome
b559 to P680+ bypassing the
physiological donor, the Mn-cluster, is observed only under prolonged
illumination at 77 K (8, 9). It has recently been shown that electron
donation from the cytochrome occurs via a redox-active carotene (10)
rather than via a redox-active chlorophyll as previously thought.
Several functions have been assigned to cytochrome
b559 in the literature, ranging from its
putative participation in the mechanism of water oxidation (11), a
function in the assembly of PSII (12-14), to a role in photoprotection
(for review, see Ref. 7). Morais et al. (13) investigated
cytochrome b559 mutants of Chlamydomonas
reinhardtii in which the heme-ligating histidine of the
In higher plants, the cytochrome b559 Plant Material--
Tobacco (Nicotiana tabacum
cv. Petit Havanna) plants were grown in soil at a light
intensity of 100 µmol quanta m Generation of Antibodies against the Conventional Electron Microscopy--
Specimens were prepared
for thin sectioning by fixing small pieces (1 mm2) of
tobacco leaves with 1.0% formaldehyde and 0.5% glutardialdehyde in
phosphate buffer (pH 7.0). For routine preparations, the samples were
dehydrated with ethanol and embedded in epoxy resin (20). Samples for
immunolabeling techniques were embedded in LR White (21). Thin sections
were performed with the ultramicrotome Ultracut E (Leica, Wetzlar,
Germany) and prepared on formvar-coated nickel grids. Routine sections
were stained with uranyl acetate (22) followed by lead citrate
(23).
Sections to be used for immunolabeling techniques were stained with
uranyl acetate only. Ultrathin sections were examined with a Philips
CM-10 electron microscope (FEI Company, Eindhoven, The Netherlands)
equipped with a Bioscan camera (model 792, Gatan, München,
Germany). The electron micrographs taken at an acceleration voltage of
60 and 80 kV were processed by a digital image system (Digital
Micrograph, Gatan, München, Germany).
Immunolocalization of Cytochrome
b559--
Immunolabeling techniques were carried out on
ultrathin sections after embedding and sectioning (24). As primary
antibodies we used an anti- Morphometric Measurements--
Morphometric measurements were
performed on electron micrographs enlarged to a final magnification of
×62500. The grade of labeling intensity of cytochrome
b559 in wild-type and mutant chloroplasts was
compared by counting gold particles per area (1 µm2) on
longitudinally sectioned chloroplasts. 16 or more areas were counted on
different chloroplasts of the mutant and the wild type and compared
with control samples labeled with preimmune serum.
SDS-PAGE and Western Blot Analysis--
SDS gel electrophoresis
was carried out in 14% polyacrylamide gels according to the method
described by Laemmli (25). Western blotting was performed using a
Multiphor II Novablot unit (Amersham Biosciences). For detection, the
ECL system (Amersham Biosciences) was used according to the
manufacturer's protocol.
Preparation of Thylakoid Membranes and PSII
Particles--
Thylakoid membranes were prepared according to Ref. 26,
with the modification that the grinding medium additionally contained 0.1% (w/v) bovine serum albumin and 0.2% (w/v) ascorbate.
PSII-enriched membrane fragments (PSII particles) were prepared
essentially as described in Ref. 27. The activity of samples obtained
from wild-type plants was 325 µmol O2/(mg Chl * h)
(measured in the presence of 0.5 mM
p-phenylbenzoquinone). To determine contamination with
PSI 77-K fluorescence was examined, and the area of the emission band
at 730 nm was less than 5% for the wild type and ~50% for the mutant.
Activity Measurements--
Oxygen evolution activity was
measured using a Hansatech O2 electrode in a suspension
medium containing 0.3 M sucrose, 15 mM NaCl,
and 20 mM MES, pH 6.5. The chlorophyll concentration of the
samples was adjusted to 30 µg/ml. 0.5 mM methylviologen was used as electron acceptor and 1 µM nigericin as
uncoupler. PSII activity was measured by using 0.5 mM
phenyl-p-benzoquinone instead of methylviologen as electron
acceptor. PSI activity was measured as the rate of oxygen uptake using
30 µg of Chl/ml, 10 µM DCMU, 5 mM
ascorbate, 30 µM DCPIP, 0.5 mM
methylviologen, 1 mM NaN3, and 1 µM nigericin.
Chlorophyll Fluorescence and Thermoluminescence
Assays--
Chlorophyll fluorescence was measured in vivo
with a pulse-amplitude modulation fluorometer (PAM 101-3, Walz,
Effeltrich) as described in Ref. 28. The intensity of the measuring
analytic light (standard PAM 101 set) was sufficiently low (integral
intensity about 10
77-K fluorescence was measured using a FluoroMax-2 fluorometer (ISAII)
equipped with a Dewar cuvette. 40% glycerol was added prior to filling
the sample (5 µg Chl/ml) into a quartz capillary.
Thermoluminescence was recorded with a home-built apparatus described
in Ref. 29. Leaf segments (1 cm × 0.5 cm) were incubated for 5 min in the dark at 20 °C, then cooled within 1 min to 1 °C and
illuminated with a saturating single turnover flash. Light emission was
recorded during heating of the sample up to 70 °C with a heating
rate of 0.4 °C/min. Data analysis was performed according to Ref.
30.
Oxidation Reduction Spectra--
Difference absorption spectra
were recoded using an Aminco dual wavelength photometer. PSII
particles (120 µg of Chl/ml) were oxidized by adding 0.1 mM K3(Fe(CN)6). The
reduction was performed by first adding hydroquinone (0.3 mM), followed by ascorbate (0.6 mM), and
finally a few grains of dithionite.
EPR Spectroscopy--
EPR was performed using a Bruker ER-300
spectrometer, equipped with an Oxford ESR 9 cryostat, a
Hewlett-Packard 5350B frequency counter, and a Bruker 035-M
NMR gauss meter. Illumination at 200 K was performed in an
ethanol/solid CO2 bath in an unsilvered Dewar flask, using
a 300-watt projector lamp. 150 µl of material was transferred in a
fused-quartz sample holder. A typical chlorophyll concentration for the
measurements was 3.4 mg of Chl/ml.
Chloroplast Ultrastructure and Protein Composition of
PSII--
Studies of the ultrastructure of wild-type and mutant
chloroplasts by electron microscopy (Fig.
1, A and B) showed
that the structural organization of the membrane was significantly
altered by the single amino acid exchange from phenyalanine to serine in the cytochrome b559
Fig. 1E shows a Western blot of thylakoid membrane proteins
from wild type and the cytochrome b559 mutant
stained with antibodies raised against the
Immunolocalization of cytochrome b559, using
secondary antibodies coupled to gold particles for detection, showed
that the staining of the wild-type chloroplasts was much higher than
for the mutant (Fig. 1, C and D). In the
wild-type chloroplast a clustering of gold particles can be seen at the
grana stacks, whereas in the mutant the less abundant gold particles
are more equally distributed. The gold labels, unequivocally
identifiable by their intense electron contrast and size (12-nm
diameter) were morphometrically evaluated. 92 ± 39 gold particles
were counted per µm2 for the wild type compared with
20 ± 13 particles for the mutant, whereas in controls with
preimmune serum less than 1 gold particle per µm2 was
counted. The mean values of wild-type and mutant chloroplasts are
significantly different at a 99.5% confidence level.
We then investigated the effect of the mutation on other
proteins of the photosynthetic machinery. Thylakoids and PSII-enriched membrane fragments (PSII particles) from wild type and the cytochrome b559 mutant were compared by Western blot
analyses for the abundance of CP47, FNR, OEC 33, and OEC 23. Fig.
2 shows that the amount of the
membrane-integral PSII protein CP 47 was considerably reduced in mutant
thylakoids, whereas there was no significant difference in PSII
particles, as expected. All other proteins examined remained largely
unchanged in thylakoids. On the other hand, in PSII particles a
signal for the 23-kDa protein was not detectable in the mutant, although it was present in thylakoid membranes. This indicates that
this luminal extrinsic protein was not attached to PSII. The extrinsic
OEC 33 behaved differently, being detectable in PSII particles of both
origins. Previous studies have shown that high concentrations of OEC 23 protein are present in the thylakoid lumen (31) and even in etioplasts
in the complete absence of PSII (14).
Characterization of the Mutant by 77 K Fluorescence--
Fig.
3 shows fluorescence emission spectra at
77 K recorded with thylakoid membranes from the wild type and the
mutant. The maxima at 684, 690, and 730 nm are generally attributed to
emissions originating predominantly from light-harvesting
complex and antenna pigments of PSII (684 and 690 nm) and PSI
(730 nm) (32). In the mutant, the ratio of emission at 684/730 nm
(ratio PSII/PSI) was lower compared with the wild type, indicating that
the amount of PSII was decreased relative to PSI. In addition, in the
mutant the first peak was slightly shifted by 2 nm to 682 nm.
Absorption spectra at room temperature of thylakoid membranes from
mature leaves showed no difference between the wild type and the
mutant. Likewise, the chl a/b ratio was unchanged, indicating that
there were no severe structural changes in the antenna system of PSII (data not shown).
To test whether the photosynthetic electron transport chain was
inhibited in the mutant, we measured the activity of PSII, PSI, and the
entire electron transport chain in the presence of an uncoupler (Table
I). PSII activity was reduced by 60% in
thylakoid membranes obtained from the mutant, whereas the PSI activity
remained unchanged compared with the wild type. The activity of the
total electron transport chain was reduced in the mutant by 75%.
Similarly, PSII-enriched membrane fragments isolated according to Ref.
27 showed a 75% lower activity than the wild type. This latter result is somewhat unexpected but could either be because of reduced PSII
stability during preparation or a certain degree of PSI impurity. Consequently, the loss in PSII activity may be overestimated.
Absorption Spectra of Cytochrome b559--
To test
whether the cytochrome b559 of the mutant
contained a heme group, we measured difference absorption spectra of
PSII-enriched membrane fragments between 535 and 590 nm. Fig.
4 shows the respective reduced minus
oxidized optical difference spectra of the wild type and the mutant
(Fig. 4, A and B). Ferricyanide was used as oxidant, whereas hydroquinone, ascorbate, and dithionite were subsequently added as reductants to estimate the potential form of
cytochrome b559. As shown in Fig. 4, an
absorption maximum was observed at 559 nm, indicative for cytochrome
b559 in both mutant and wild type. However, the
signal size was approximately five times smaller in the mutant on a
chlorophyll basis. In addition the Characterization of the Mutant PSII Particles by EPR
Spectroscopy--
To further characterize the electron transport in
PSII of the mutant, EPR spectra arising from radical species formed at
the donor and acceptor side of PSII upon 200 K illumination were
recorded (Table II). Upon illumination at
200 K, a normal multiline signal of the S2 state of the
Mn-containing water-oxidizing complex (33) was detected, showing that
the ability of the oxygen-evolving complex to advance from the dark
stable S1 state to S2 was not affected by the
mutation. TyrD+ could also be detected in both the mutant
and the wild type. 200 K illumination induced the
Fe-QA
In dark-adapted mutant samples, no oxidized high-potential cytochrome
b559 was seen by EPR following incubation with 5 mM potassium ferricyanide (an adequate oxidant for both the
cytochrome b559 and the non-heme iron).
Surprisingly, even under prolonged 77 K illumination no signal of
oxidized cytochrome b559 could be detected,
although the amount of Car+/Chl+ formed was
significantly smaller than in the wild type. This indicates that some
cytochrome b559 was oxidized but invisible, perhaps because of a broadening of the signal.
Chlorophyll Fluorescence and Thermoluminescence Measurements in
Vivo--
To study the PSII activity in vivo, chlorophyll
fluorescence and thermoluminescence measurements were performed. Fig.
5 shows fluorescence curves measured on
dark-adapted leaves of wild-type (upper panel) and mutant
plants (lower panel). In the mutant, a high level of
F0 fluorescence was detected, and this causes the low variable fluorescence/maximum fluorescence
(Fv/Fm) ratio shown in
Table II. Upon switching on the actinic light (I = 300 µmol
quanta m
To investigate the reduction state of the donor and acceptor side of
PSII, we performed thermoluminescence measurements on leaf segments of
the mutant and the wild type. In thermoluminescence, the emitted light
originates from charge recombination of trapped charge pairs (for
review, see Ref. 35). The charge pairs involved can be identified by
their emission temperature, which strongly depends on the redox
potentials of the charge pairs involved. The B-band, which we
investigated here, arises from a recombination of the S2 or
S3 state of the oxygen-evolving complex with the semiquinone QB We have characterized a tobacco mutant carrying a single point
mutation in the plastid psbF gene, resulting in a
phenylalanine to serine change at position 26 in the It has been reported previously that cytochrome
b559 is essential for the assembly of stable
PSII reaction centers (12, 14). Null mutants for the The low amount of PSII exerts a dramatic effect on the ultrastructure
of the thylakoid membranes as can be seen in the electron microscopy
images (Fig. 1). Whereas thylakoid membranes of the wild type showed
the typical grana stack, grana formation and stacking were strongly
reduced in the mutant (Fig. 1). A number of previous studies have
suggested that grana stack formation is correlated with the amount of
PSII present in the thylakoid membrane (e.g. Ref. 37). It
has been proposed that the close appression of grana membranes arises
through recognition patterns and contact surfaces formed by LHCII
light-harvesting complex proteins that are associated with PSII (38).
It thus appears plausible that the low amount of PSII in the mutant and
the concomitantly reduced number of recognition sites for grana
stacking are responsible for the perturbed chloroplast ultrastructure.
Although PSII activity in thylakoid membranes of the PsbF mutant was
reduced, a certain quantity of active PSII was found (Table I) that
showed the characteristic EPR spectra (Table II). However, no EPR
signal for cytochrome b559 was seen. This
finding cannot be explained with a mutation-induced loss of the heme
group because difference absorption spectra showed a maximum at 559 nm,
indicating heme incorporation (Fig. 4). Mutants of Chlamydomonas reinhardtii, which carried mutations in the Sequence inspection of the Our determination of the distribution of high and low potential forms
of cytochrome b559 (Fig. 4) showed a lower
amount of high potential cytochrome b559 in the
mutant. However, the fluorescence (Fig. 5) and thermoluminescence
signals (Fig. 6) were altered in leaves of the mutant. Recordings of
fluorescence curves showed that the dark level of fluorescence
(Fo) was very high in the mutant, indicating a
largely reduced plastoquinone pool. In the light, photochemical and
non-photochemical quenching occurred, and the fluorescence declined to
a level comparable with that seen in wild-type leaves. Upon switching
of the actinic light, an increase of the dark-level fluorescence was
observed in the mutant, indicating that the same reduction level of the
PQ pool was reached as was observed during the dark adaptation prior to the measurement. In thermoluminescence analysis, a normal oscillation pattern of the B-band (S2,3QB Far-red illumination (intensity: 9 µmol quanta
m We therefore conclude that at least one physiological role of
cytochrome b559 is to keep the acceptor side of
PSII oxidized under conditions when forward electron flow does not
occur and the plastoquinone pool is reduced non-photochemically
(e.g. in the dark, in the presence of a high proton gradient
or a large concentration of reduction equivalents). It has been
proposed recently that cytochrome b559 in its
low potential form could function as a plastoquinone oxidase (17, 18).
It has also previously been suggested that photoreduction of the
oxidized high potential form of cytochrome b559
occurs via electron donation from PQH2 (39). Oxidation of
PQH2 by cytochrome b559+
would result in delivery of an electron to oxygen and formation of
superoxide. Detoxification of superoxide could occur via the superoxide
dismutase activity of cytochrome b559 as
proposed by Ananyev et al. (40). It may also be possible
that the electron transfer leading to oxidation of PQH2 via
cytochrome b559 involves an alternative oxidase
(41) that ultimately reduces oxygen.
The high reduction level of the plastoquinone pool in the mutant may
either be a consequence of the thylakoid membrane's global structural
changes caused by a lower level of cytochrome
b559 or be because of a change of the redox
properties of cytochrome b559 perturbing the
putative plastoquinol oxidase function. The possibility of alterations
in the redox properties is supported by the lack of an EPR signal and
by the broadening of the -subunit (PsbF) was changed to
serine (Bock, R., Kössel, H., and Maliga, P. (1994) EMBO
J. 13, 4623-4628). The mutant grew photoautotrophically, but the amount of PSII was reduced and the ultrastructure of the chloroplast was dramatically altered. Very few grana stacks were formed in the
mutant. Although isolated PSII-enriched membrane fragments showed low
PSII activity, electron paramagnetic resonance indicated the presence
of functional PSII. Difference absorption spectra showed that the
cytochrome b559 contained heme. The
plastoquinone pool was largely reduced in dark-adapted leaves of the
mutant, based on chlorophyll fluorescence and thermoluminescence
measurements. We therefore propose that cytochrome
b559 plays an important role in PSII by keeping
the plastoquinone pool and thereby the acceptor side of PSII oxidized
in the dark. Structural alterations as induced by the single Phe
Ser point mutation in the transmembrane domain of PsbF evidently
inhibit this function.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
- and one
-subunit, both of which
form single transmembrane
-helices (2). The heme group is ligated by
two histidine residues, one located in the
- and one in the
-subunit, as revealed by x-ray crystallography (3). The heme is
bis(histidine) 6-fold-coordinated, as was demonstrated
spectroscopically (4, 5). The electron paramagnetic resonance (EPR)
spectrum of the oxidized heme iron is anisotropic, with gz ~3.00, gy
~2.20, and gx ~1.50, indicative of a low-spin species. These values
are consistent with two ligating histidine planes being perpendicular
(or nearly perpendicular) to each other (5). Likewise, the orientation
of the heme ring was determined to be perpendicular to the membrane
plane (6).
-subunit was replaced by different amino acids. In contrast to
earlier reports of cytochrome b559 knockout
mutants (e.g. 15) or mutations of the heme axial ligands
(16) that did not assemble PSII, these mutants assembled few but
functional oxygen-evolving PSII centers, although the cytochrome
b559 did not contain a heme group. This finding
seems to exclude a function of cytochrome b559
in photosynthetic water splitting, and it was proposed that cytochrome
b559 plays a protective role under
photoinhibitory illumination. Recently, it has also been suggested that
cytochrome b559 may function as a plastoquinol
oxidase (17, 18).
- and
-subunits are encoded by the psbE and psbF
genes in the plastid genome and are cotranscribed as part of a
tetracistronic transcription unit (psbE operon). Here we
describe the characterization of a cytochrome b559 mutant created in tobacco (Nicotiana
tabacum) in which the conserved phenylalanine at position 26 in
the
-subunit of cytochrome b559 was changed
to serine (19). In tobacco, the codon for phenylalanine is present at
the DNA level, whereas in spinach the codon for phenylalanine is
created post-transcriptionally from a genomically encoded UCU (serine)
codon by C-to-U RNA editing. The mutant was produced by inserting the
spinach editing site into the tobacco psbF gene via
chloroplast transformation (19). The resulting transplastomic tobacco
plants were incapable of editing the spinach site and hence contained a
serine instead of the conserved phenylalanine in position 26 of the
PsbF protein. This single amino acid change resulted in an altered
phenotype. The mutant tobacco plants grew photoautotrophically but
significantly slower than the wild type. The mutants had a light-green
leaf color and showed a high chlorophyll fluorescence phenotype. Here
we present a detailed analysis of this mutant at the levels of
chloroplast ultrastructure and photosynthetic electron transport.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2s
1.
-Subunit of Cytochrome
b559--
Based on published sequences in the data bases,
two primers were designed to amplify the gene coding for the
-subunit of cytochrome b559 from
Narcissus pseudonarcissus L. Total RNA was isolated from
daffodil flowers using RNA-Easy kit (Qiagen, Hilden, Germany),
and a 280-bp cDNA fragment encoding the entire
-subunit of
cytochrome b559 was amplified by RT-PCR. The
fragment was then cloned into PCR-Blunt II-TOPO vector
(Invitrogen), yielding the plasmid pCYT, and the insert was
sequenced. The deduced amino acid sequence was highly homologous to
previously determined sequences of the cytochrome
b559
-subunit gene from other plants. To
express the cytochrome b559
-subunit as a GST
fusion in Escherichia coli cells, a
SmaI/EcoR I fragment containing the coding region
was excised from pCYT and cloned into pGEX-2T (Amersham Biosciences) to
yield pGST-cytochrome b599. Attempts to purify
the fusion protein by affinity chromatography using
glutathione-Sepharose failed. Therefore, total E. coli
protein extract was separated by SDS-PAGE, and the overexpressed
protein was excised from the gel and subsequently purified by
electroelution. The purified protein was finally used to immunize mice
and a rabbit.
-cytochrome b559
antiserum raised against the recombinant protein (see below), and 12 nm
Colloidal Gold-AffiniPure goat anti-rabbit IgG (H+L) and 12 nm
Colloidal Gold-AffiniPure goat anti-mouse IgG were employed as
secondary antibodies (Jackson ImmunoResearch Laboratories). In control
experiments preimmune serum was used instead of specific primary antibodies.
8 mol
quanta·m
2·s
1, frequency of modulated
light: 1.6 kHz) to prevent the reduction of PQ. As actinic white light,
a cold light source was used with an intensity of 300 µmol
quanta·m
2·s
1.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chain at position 26. In the mutant, the structure of thylakoid grana and stroma lamella was
disordered, whereas in the wild type the well ordered structure of
grana stacks separated by stroma lamella as well as starch granules was
nicely seen. The mutant had only small grana stacks, which were less well stacked than in the wild type and showed a much lower degree of
structural order. In addition, starch accumulation was much less
pronounced in the mutant, and starch granules (when present) were
significantly smaller than in the wild type.
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Fig. 1.
Electron microscopy and immunological
examination of cytochrome b559 in
wild-type and mutant tobacco leaves. A and
B, conventional electron microscopy of wild-type and
mutant leaves, respectively. C and D,
EM-immunolocalization of cytochrome b559 in
wild-type and mutant chloroplasts conducted with anti-PsbE antiserum.
E, Western blot with chloroplast proteins from wild-type
(left lane) and mutant (right lane) tobacco
leaves using the same antibodies. All samples were loaded on an equal
chlorophyll basis (15 µg/ml). Signal detection was performed with the
ECL system.
-subunit of cytochrome
b559. The alteration in the sequence of the
-subunit of cytochrome b559 led to a lower concentration of cytochrome b559 relative to the
chlorophyll concentration. Western blots conducted with a range of
chlorophyll concentrations per sample indicated a reduction of the
levels of PsbE to 10-30% of the wild type (data not shown).
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Fig. 2.
Immunoblots of thylakoid membranes and PSII
preparation from wild-type and mutant leaves. Blots were decorated
with antibodies specific for FNR, CP 47, OEC 33, and OEC 23. All
samples were loaded on an equal chlorophyll basis (15 µg/ml). Signal
detection was performed with the ECL system.
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Fig. 3.
Fluorescence emission spectra at 77 K. Thylakoid membranes from the wild type (dashed line) and the
cytochrome b559 mutant (solid line)
were measured with an excitation wavelength of 440 nm.
Activity of the photosynthetic electron transport chain in wild-type
and mutant thylakoid membranes
-absorption band was broadened
(FWHM 13 nm compared with 10 nm in the wild type). About 30% of the
oxidized cytochrome b559 was already reduced by
the addition of hydroquinone, indicating that a certain fraction was
present in the high potential form. Subsequent addition of ascorbate
reduced the signal to about 50% compared with the completely reduced
cytochrome b559. This indicates that cytochrome
b559 was present in all the known potential
forms.
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Fig. 4.
Difference absorption spectra from
PSII-enriched membrane fragments of wild-type (A) and
cytochrome b559 mutant
(B). The following spectra of the cytochrome
b559 heme are shown: hydroquinone-reduced minus
ferricyanide-oxidized (1), ascorbate-reduced minus
ferricyanide-oxidized (2), and dithionite-reduced minus
ferricyanide-oxidized (3). The samples contained 120 µg of
Chl/ml.
at g = 1.90 signal (34),
demonstrating that the acceptor side of PSII also functioned normally.
In addition, some photoaccumulation of ChlZ+
was observed. This indicates that the electron transfer reactions associated with PSII were not perturbed by the mutation. However, the
EPR signal sizes were significantly smaller in PSII particles from the
mutant as compared with the wild type when the samples were measured at
an equal chlorophyll basis. Here, one has to keep in mind that, because
of the altered PSII:PSI stoichiometry, the enrichment of PSII relative
to PSI was low in PSII particles from the mutant.
Fluorescence and EPR signals obtained with mutant and wild-type tobacco
plants
2s
1), first the
Fm was reached, but this was then quenched
during prolonged illumination. This quenching during illumination with actinic light was slower in the mutant than in the wild type, and a
longer illumination time was needed to reach the steady state. The
minimum fluorescence level finally reached was equivalent to the steady
state level in actinic light observed with wild-type plants (close to
the F0 level). After switching off the actinic light, the fluorescence increased to the fluorescence level observed prior to the illumination with actinic light. Lowering of the light
intensity of the measuring light beam did not change this behavior,
indicating that this high F0 level was not
caused by a too high intensity of the measuring light. Such elevated
F0 levels are most likely caused by dark
reduction of the plastoquinone pool, resulting in a reduced acceptor
side of PSII in the dark. In addition, illumination with far-red light
did not reduce the high dark-level fluorescence.
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Fig. 5.
Fluorescence measurements on leaf segments of
wild type (upper panel) and cytochrome
b559 mutant (lower
panel). Open arrow, onset/offset of the
measuring light. Closed arrows, onset/offset of actinic
light (white light at 300 µmol quanta
m 2s
1). Leaves were dark-adapted for at
least 30 min prior to the measurement. F0,
Fm levels are indicated for the curve from
wild-type plants.
(36). This recombination
yields a band at ~30 °C. The intensity of the B-band oscillated
with a period of four, which reflects the cycle of the S-states of the
Mn cluster. In general, in a dark-adapted sample 75% of the centers
are in the S1 state and QB is mainly oxidized.
In this case, the highest intensity of the B-band (maximum emission at 33 °C) is observed after the first flash, as can be seen clearly with wild-type leaves (Fig. 6, open circles). In contrast, almost no thermoluminescence signal
was detected in the mutant after the first flash, whereas after the second flash a large signal (B-band, maximum emission temperature at
35 °C) was observed (Fig. 6, filled circles). This B-band
oscillated with a periodicity of four, as expected, showing the highest
peak after two and six flashes, respectively. The signal on the fifth flash is again very small. This indicates a normal function of the
water-splitting complex and the forward electron transfer in PSII but a
shift in the oscillation pattern of the B-band by one flash.
View larger version (17K):
[in a new window]
Fig. 6.
Comparison of the flash dependence of the
integrated area of the B-band. Filled and
open circles represent the oscillation of the B-band in
mutant and wild-type leaf segments, respectively. Zero to eight flashes
were given at 1 °C after 5 min of dark adaptation of the leaf
segment at 20 °C.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit of
cytochrome b559. This mutation is interesting
because it does not directly influence the heme-ligating amino acid
residues (one His in the
-subunit and one His in the
-subunit)
but affects a highly hydrophobic stretch in the transmembrane helix in
the
-subunit. The mutation leads to a severe phenotype, and we have
shown here that it results in a reduced amount of the
-subunit of
the cytochrome (Fig. 1E), of CP47, and of the extrinsic
23-kDa protein associated with PSII (Fig. 2). Because the cytochrome
b559 is known to be essential for the PSII
assembly process (see below), it is not surprising that the mutant also
displayed a significant reduction in the amount of PSII centers, which
in turn causes ultrastructural alterations in the thylakoid membrane
(Fig. 1). In addition, the reduction state of the electron transport
chain was altered in the mutant as demonstrated by chlorophyll
fluorescence and thermoluminescence measurements (Figs. 5 and 6).
-subunit of
cytochrome b559 (12) or for both
- and
-subunits (15) are unable to assemble PSII. Mutants in which one of
the heme-ligating histidines was exchanged by a different amino acid
and in which the cytochrome no longer contained a heme group display
inefficient PSII assembly or even a complete failure in this respect
(13, 16). In the mutant investigated here, little PSII is
present (about 30% as seen by Western blots and immunolabeling),
indicating that a mutation not directly affecting the
heme-ligating histidine evidently also leads to perturbations of the
PSII assembly process.
-subunit of
cytochrome b559, did not contain a heme group
and showed no such absorption maximum at 559 nm (13). The lack of
detectable EPR signals for cytochrome b559 in
PSII particle preparations from the mutant could be explained by a
broadening of the spectra because of a structural change, such as an
increase of the anisotropy caused by H-bonds to the imidazole group of
the heme-ligating histidines. The difference absorption spectra also
showed a broadening of the
-band of the cytochrome
b559 spectrum.
- and
-subunits of cytochrome
b559 reveals that the site of mutation is
located in a region with extremely hydrophobic amino acid residues
(Phe-Phe-Leu in the
-subunit and Leu-Phe-Ile in the
-subunit).
The recently determined crystallographic data for PSII (3) suggest that
this region provides the contact site of the two helices. The change
from phenylalanine to serine in the mutant could have two effects. The
introduction of a small polar amino acid residue might alter the
contact site between the
- and
-subunits, leading to a structural change and increased flexibility in the orientation of the two helices.
Alternatively, the introduction of a much smaller side group might lead
to a closer contact at this site, resulting in an opening of the
helices toward the stroma.
recombination) was observed. Most interestingly, however, the oscillation pattern was shifted by one flash. This shift can be explained either by the presence of QB
in the
dark within the majority of the reaction centers or by a change in the
stability of the S-states of the Mn cluster. Normally, in a
dark-adapted sample, 75% of the Mn cluster of the PSII centers are in
the S1 state and 25% in the S0 state. If the
acceptor side of PSII is oxidized, a single flash results in the
formation of S2QB
, yielding the
highest thermoluminescence emission. If the S1 state were
less stable in the mutant, the Mn cluster could be in the
S0 state in the dark. This in turn would lead to a shift of
the oscillation pattern of the B-band by one flash, resulting in the
maximum TL emission on the second flash. However, a lower S-state is
excluded by our EPR data (Table II); 200 K illumination, which is
equivalent to one flash, was sufficient to give a multiline signal
(S2 state) in the PSII preparation from mutant leaves, indicating that the Mn cluster was predominately in the S1
state in the dark. The high fluorescence state in the dark and the
shifted oscillation of the B-band are therefore indicative of a reduced acceptor side in the dark. If
S1QB
and
S0QB
are present in the dark, the
flash-dependence of the thermoluminescence emission as shown in Fig. 6
is readily explained.
2s
1) did not decrease the high dark level
of fluorescence. Therefore, it can be excluded that the alteration of
the PSII:PSI stoichiometry and increased activity of cyclic electron
flow around PSI were responsible for the high reduction state of the
plastoquinone pool in the mutant.
-band of the absorption spectrum.
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ACKNOWLEDGEMENTS |
---|
We thank Katharina Kienzler for technical assistance, Peter Faller for critical reading, and Randall Cassada for correcting the English version of the manuscript. Anti-FNR antibodies were kindly provided by R. J. Berzborn, Ruhr-Universität Bochum, and anti-OEC 33 and anti-OEC 23 by C. Jansson, Stockholm University.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
¶ Supported by grants from the Deutsche Forschungsgemeinschaft (Graduiertenkolleg "Molekulare Mechanismen der Pflanzendifferenzierung" (to N. B.) and "Biochemie der Enzyme" (to L. P.).
Supported by a Marie Curie individual fellowship
(MCFI- 2000-00611).
To whom correspondence should be addressed. Tel.:
49-761-203-2698; Fax: 49-761-203-2601; E-mail:
anja.liszkay@biologie.uni-freiburg.de.
Published, JBC Papers in Press, February 4, 2003, DOI 10.1074/jbc.M212842200
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ABBREVIATIONS |
---|
The abbreviations used are: PS, photosystem; P680, primary electron donor in PSII; QA, primary quinone electron acceptor in PSII; QB, secondary quinone electron acceptor in PSII; Chl, chlorophyll; CP, chlorophyll-binding protein; D1, D2, subunits of PSII; DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea; DCPIP, 2,6-dichloroindophenol; EPR, electron paramagnetic resonance; Fo, dark-level fluorescence; Fm, maximum fluorescence; Fv, variable fluorescence; FNR, ferredoxin-NADP+ oxidoreductase; MES, 2-(N-morpholino)ethanesulfonic acid; PQ, plastoquinone; S-states, oxidation states of the Mn cluster; TL, thermoluminescence; OEC, oxygen evolving complex.
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