From the Developmental, Cellular, and Molecular Biology Group, Departments of Botany and Zoology, Duke University, Durham, North Carolina 27708-1000
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ABSTRACT |
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The QB binding site of the D1 reaction center protein, located within a stromal loop between transmembrane helices IV and V formed by residues Ile219 to Leu272, is essential for photosynthetic electron transport through photosystem II (PSII). We have examined the function of the highly conserved Ala251 D1 residue in this domain in chloroplast transformants of Chlamydomonas reinhardtii and found that Arg, Asp, Gln, Glu, and His substitutions are nonphotosynthetic, whereas Cys, Ser, Pro, Gly, Ile, Val, and Leu substitutions show various alterations in D1 turnover, photosynthesis, and photoautotrophic growth. The latter mutations reduce the rate of QA to QB electron transfer, but this is not necessarily rate-limiting for photoautotrophic growth. The Cys mutant divides and evolves O2 at wild type rates, although it has slightly higher rates of D1 synthesis and turnover and reduced electron transfer between QA and QB. O2 evolution, D1 synthesis, and accumulation in the Ser, Pro, and Gly mutants in high light is reduced, but photoautotrophic growth rate is not affected. In contrast, the Ile, Val, and Leu mutants are impaired in photoautotrophic growth and photosynthesis in both low and high light and have elevated rates of D1 synthesis and degradation, but D1 accumulation is normal. While rates of synthesis/degradation of the D1 protein are not necessarily correlated with alterations in specific parameters of PSII function in these mutants, bulkiness of the substituted amino acids is highly correlated with the dissociation constant for QB in the seven mutants examined. These observations imply that the Ala251 residue plays a key role in D1 protein.
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INTRODUCTION |
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Photosystem II (PSII)1 complexes of all oxygen-evolving organisms convert light energy into chemical free energy by transferring electrons from water to plastoquinone, associated with the release of molecular oxygen (1, 2). The D1 protein of the PSII reaction center turns over in a light-dependent manner more rapidly than any other chloroplast protein and is a primary target for photoinhibitory damage (3, 4). Based on homology with the crystal structure of the L protein from Rhodopseudomonas viridis (5), D1 is predicted to have five hydrophobic membrane-spanning helices (6). One of the stromally exposed regions of D1, extending from the C terminus of transmembrane helix IV through the N terminus of transmembrane helix V (IV-V loop), includes a stromal helix thought to lie parallel to the membrane surface (6). This loop, whose amino acid sequence is highly conserved among cyanobacteria, algae, and higher plants (7), is involved in binding both QB, the second stable quinone acceptor in PSII, and several classes of herbicides that inhibit photosynthetic electron transport at the QB docking site (8). Only a few of the many amino acid substitutions made in this region result in the loss of D1 function and photosynthetic capability (9, 10), suggesting that most positions can tolerate considerable variation in R group conformation or charge and still permit D1 function. However, particular residues may be necessary for optimal PSII activity or may provide functional advantages under certain environmental conditions (11, 12). Analysis of site-directed mutations in the region between Ile248 and Ala251 at one end of the parallel helix has led to the hypothesis that these residues may be buried in the thylakoid membrane (11), thus dividing the IV-V loop of D1 into two segments. One segment is postulated to be involved in QB binding and herbicide resistance (from the parallel helix to the helix V) and the other possibly in D1 degradation (from helix IV to the parallel helix).
Substitution of Val for Ala251 in the D1 protein in cyanobacteria and Chlamydomonas results in herbicide resistance and increased sensitivity to photoinhibition (13, 14), reduced photosynthetic yield associated with slower photoautotrophic growth (11, 15), and perturbation of the pattern of oxygen evolution in single turnover flashes (16). To examine further the role of the D1 Ala251 residue in PSII function and light sensitivity, we generated 12 of the 19 possible amino acid substitutions at this position in Chlamydomonas reinhardtii. Five of these substitutions (Arg, Asp, Gln, Glu, and His) were shown to lead to a nonphotosynthetic phenotype and to impaired D1 synthesis and accumulation (10). Seven (Cys, Gly, Ile, Leu, Pro, Ser, and Val), which retain photosynthetic function to various degrees, affect herbicide resistance and photoautotrophic growth rates to various degrees (17). In this paper, we examine D1 synthesis and turnover, photosynthetic rate, quantum efficiency, and electron transfer between QA and QB in these seven mutants. We also show that the bulkiness of the R group of the amino acid substituted in each of 12 Ala251 mutants is correlated with the dissociation constant (KO) for plastoquinone in the QB pocket, consistent with the hypothesis that the Ala251 residue of D1 is structurally involved in QB binding and is likely to be buried in the thylakoid membrane (11).
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MATERIALS AND METHODS |
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Strains and Culture Conditions--
Wild type and mutant strains
of C. reinhardtii designated "CC-" are available from
the Chlamydomonas Genetics Center at Duke University (Dr.
E. H. Harris, Box 91000, Duke University, Durham, NC 27708).
Stocks were maintained on plates of solid TAP or HSHAYA media (18)
under dim light (10 µmol m2 s
1). For
physiological, biochemical, and biophysical analysis, cells were grown
photoautotrophically in liquid minimal HS medium (18) bubbled with 5%
CO2 in air under low light (LL; 70 µmol m
2
s
1) or high light (HL; 600 µmol m
2
s
1) at 25 °C. Cells were harvested in early
exponential to midexponential growth (up to 2 × 106
cells ml
1), but sufficient doublings were allowed for
full adaptation to each environmental condition. In HL, these
conditions are saturating for maximal rates of photoautotrophic growth
of the wild type strain CC-125 (19).
In Vitro Site-directed Mutagenesis and Chloroplast Transformation of C. reinhardtii-- The A251A* (wild type control, CC-3394, GCT), and D1 mutants A251G* (CC-3393, GGT), A251I* (CC-3388, ATT), A251V* (CC-3387, GTT), A251R* (CC-3376, CGT), A251Q* (CC-3374, CAA), A251E* (CC-3377, GAA), A251H* (CC-3378, CAC), and A251D* (CC-3375, GAC) were obtained by site-directed mutagenesis of exon 4 of the chloroplast psbA gene followed by cotransformation as described previously (10, 17). Each amino acid alteration at position 251 is linked to a silent SspI RFLP marker (*) in the codon of residue 247 and all genotypes contained the selectable spectinomycin resistance marker in the 16 S rRNA (10).
Recovery of Same Site Suppressors--
Photosynthetically
competent same site suppressors arise spontaneously when cells of the
nonphotosynthetic A251R* transformant (10) are kept under conditions of
acetate starvation on plates in medium light (300 µmol
m2 s
1) or HL following 2-3 weeks of growth
on TAP plates (18). Under these conditions, the lawn of A251R* cells
bleaches, and the suppressors appear as isolated green colonies. DNA
from each of the photoautotrophically competent suppressors was
extracted, and the region of the chloroplast psbA gene
encoding the D1 loop between helices IV and V was amplified by
polymerase chain reaction and sequenced as described previously (10).
Four new amino acid substitutions at Ala251 were recovered:
A251C* (CC-3390, TGT), A251S* (CC-3392, AGT), A251P* (CC-3391, CCT),
and A251L* (CC-3389, CTT) in addition to A251G*, which was already
obtained by site-directed mutagenesis.
Immunoblot Analysis--
Protein isolation and solubilization
from cells grown in LL or HL, denaturing gel electrophoresis, and
immunodetection were done as described in Refs. 10, 20, and 21. The
antisera were used at the following dilutions: D1, 1:1000; tubulin
-subunit, 1:2000; PSII oxygen-evolving enhancing protein (OEE1),
1:500; and PSII light-harvesting chlorophyll
a/b-binding protein (LHCP), 1:4000. In three
determinations done on independently grown cultures, reproducibility of
protein accumulation in these mutants relative to the wild type control
was >95%.
In Vivo Pulse-chase Labeling of Chloroplast Proteins with [35S]Sulfate-- Pulse labeling of HL-grown cells was carried out as described in Ref. 19 with the following modifications. After a 15-min incubation with anisomycin (Pfizer) to inhibit cytoplasmic protein synthesis (final concentration, 140 µg/ml), 30 µl of carrier-free H235SO4 (NEN Life Science Products) was added to a final concentration of 125 µCi/ml. Aliquots (0.4 ml) of algal suspension were removed at 5, 8, 11, 14, and 17 min and injected into 6 ml of ice-cold acetone. The chase was initiated 5 min after the last time point in the pulse experiment by adding 0.995 µl of HS medium containing 200 mM Na2SO4, 50 mM NaHCO3, and lincomycin (500 µg/ml final concentration). After 5 min, the first aliquot (0.4 ml) was removed as the 0-min point of the chase. Subsequent samples were taken at 30, 60, 90, and 120 min. Protein solubilization, gel electrophoresis, and quantification of the labeled bands of interest, D1 and large subunit of ribulose-bisphosphate carboxylase oxygenase (LSU), were done as described in Ref. 10.
Photosynthetic O2 Evolution, Photochemical Quenching,
and Determination of Whole Chain Electron
Transport--
Photosynthetic O2 evolution and chlorophyll
a fluorescence were measured at 25 °C (19, 21) using a
Clark-type O2 electrode system (Hansatech Instruments,
Sheffield, UK) and a pulse-amplitude modulation fluorometer
(Heinz-Waltz PAM 101). Cells in early exponential growth (<0.1
A750) were resuspended at 0.165 A750 for HL-grown cells or 0.175 A750 for LL-grown cells and dark-adapted for 15 min. Initial Fv/Fm values were determined
immediately following dark adaptation (21), and quenching of
Fm during the 1-s 100-kHz flash did not appear to be a
problem as previously reported for the PAM method (22). Light response
curves of photosynthetic O2 evolution and chlorophyll
fluorescence were generated by increasing incident irradiance stepwise
from 10 to 900 µmol m2 s
1. Chlorophyll
content was determined according to Ref. 23. Photosynthetic data,
normalized for chlorophyll or biomass and plotted against incident
irradiance, were fitted by Marquardt nonlinear regression to the
equation of Smith/Talling (24) to determine net light-saturated O2 evolution (Pmax). Relative
apparent quantum yields (Ørel) were estimated by linear
regression from the initial slopes of the O2 evolution
curves plotted against absorbed irradiance (21). Maximum quantum yields
for O2 evolution (Ømax) were also calculated from the initial slopes of photosynthesis-irradiance curves, determined by nonlinear regression analysis, and the chlorophyll-specific optical
absorption cross-sections according to Ref. 25. Photochemical quenching
was calculated as described by Schreiber et al. (26). Values
reported for Pmax, Ørel, PAM
Fv/Fm, and chlorophyll are means ± S.E. of
at least three determinations done on independently grown cultures.
Values derived from fast repetition rate fluorometry and
thermoluminescence measurements (see below) represent the means of two
independent determinations.
Fast Repetition Rate Fluorometry (FRRF)
Measurements--
Chlorophyll a variable fluorescence was
measured using a FRR fluorometer (22, 27) that provides complex
information about the properties of PSII available from chlorophyll
fluorescence, i.e. photochemical yield, apparent PSII
cross-sections, connectivity between PSII units, and kinetics of
electron transfer from QA to
Q
. Aliquots of cells from algal cultures grown under
LL and HL conditions, at 1 µg of total chlorophyll/ml, were
dark-adapted for 2 min in a cylindrical cuvette of 5-mm inner diameter
and illuminated by blue light-emitting diodes (450 nm), which were
pulsed in flashlets of 1.5-µs duration. Flashlets can be repeated at
a variable rate, thus effectively providing either single-turnover
excitation (100 flashes given at 0.8 MHz) or the multiple turnovers of
PSII that are necessary to reduce fully the pool of secondary
plastoquinone acceptors (e.g. 3000 flashlets given at 10 kHz) (22).
Thermoluminescence Measurements-- Thermoluminescence (TL) and delayed luminescence (DL) were measured using a computerized laboratory-built apparatus (28). Five ml of algal suspension containing 5 µg of chlorophyll were gently filtered on Millipore HA 25 filters (25-mm diameter, 0.8-µm pore size) and placed on a temperature-regulated sample holder. Care was taken to keep the cells always covered by a layer of medium. Samples were placed in the dark for 180 s at 25 °C. For TL measurements, the sample holder was cooled to 3 °C, and, after temperature equilibration (60 s), the sample was excited by xenon flashes given at 1 Hz. The emitted luminescence signal was collected as a function of temperature during linear heating from 3 to 70 °C (at a rate of 0.5 °C/s), and the data were stored for further analysis. DL was measured at 25 °C, from 0.2 to 100 s after the end of excitation by xenon flashes. During the measurement, the temperature of the sample was kept constant within 0.4 °C.
Thermodynamics and Kinetics of the PSII Acceptor Side--
The
kinetic and thermodynamic characteristics of the acceptor side of PSII
were determined using the two-electron gate model (15). The
dissociation constant for plastoquinone in the QB pocket,
KO, was determined experimentally as the ratio of
amplitudes of the first two monoexponential components (one ~300 µs
and two ~2 ms in wild type) of QA
reoxidation in dark-adapted samples, following a single turnover (ST)
flash. The semiquinone equilibrium constant KE
was determined from fluorescence decay kinetics by calculating the
fraction of reduced QA
25 ms following
the ST flash (29). Thus, KE = 1/([QA
] 25 ms), and
Kapp was calculated as
Kapp = KE/(1 + KO). Alternatively, Kapp
was determined experimentally from analysis of the decay of DL in the
0.1-100-s time interval as the ratio of the number of centers
recombining from QB
(slow component,
1 = ~6 s) to the number of centers recombining from
QA
(fast component, 1 = ~0.7 s)
or as a ratio of the half-times of these two decay components (15).
Although each of these methods provided slightly different absolute
values of Kapp (Kapp was highest when determined from [QA
] 25 ms), the relative changes of Kapp between wild
type and each of the mutant strains were independent of the method
used. The remaining kinetic parameters of the two-electron gate model
(kon, koff,
kBA, kAB) were calculated
by solving the set of equations in Ref. 15.
Estimation of the Number of PSII Centers in the
Thylakoid--
The numbers of PSII reaction centers were determined
from O2 flash yields (30, 31). In wild type
(CC-125),2 chlorophyll a/O2 averages
2014 mol/mol for cells grown at 70 µmol quanta m2
s
1. Since each
O2 is generated from four sequential electron transfers through PSII, the numbers of PSII reaction centers can be calculated as
1 per 503 chlorophyll a (25). We extended this approach to all mutants by calculating the functional absorption cross-section of
PSII (from both the FRR fluorescence and O2 evolution
saturation profiles).
Estimation of Carbon Assimilation-- Cell carbon and nitrogen were determined by filtering replicate aliquots of known cell densities on precombusted Gelman A/E glass fiber filters. The filters were rinsed with distilled water, dried, and combusted in a Perkin-Elmer 241 Elemental Analyzer. Carbon-specific growth rates were calculated as described by Falkowski et al. (32).
Estimation of Amino Acid Bulkiness-- In the absence of a robust, high resolution structural model for D1, molecular dynamic calculations of the predicted effects of amino acid substitutions at position 251 on QB binding are highly speculative. For this reason, we calculated the bulkiness of the substituted amino acids at position 251 as the volume of space enclosed by their van der Waals surface (33). These calculations do not account for changes in binding properties associated with side chain charge distributions or tertiary structural interactions between amino acid 251 and other residues in the binding domain.
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RESULTS |
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Isolation of Site-directed D1 Mutants and Recovery of Same Site Suppressors with Varying Levels of Photosynthetic Competence-- Three site-directed D1 mutants resulting from amino acid substitutions at residue Ala251 (A251G*, A251I*, and A251V*) retain some capacity for photoautotrophic growth. Four additional mutants at this position (A251C*, A251S*, A251P*, and A251L*) plus A251G*, were recovered from the nonphotosynthetic A251R* transformant (10) as spontaneous same site suppressors capable of photoautotrophic growth. Each of the codon changes in these suppressors required only a single base pair alteration, while a true Ala251 revertant of the A251R* strain, which was not obtained, would require two simultaneous base pair changes, a highly improbable event (Fig. 1). All 40 of the photoautotrophically competent suppressors isolated from A251R* showed single base pair substitutions in the Arg251 D1 codon (8 Cys, 9 Ser, 4 Pro, 15 Gly, 4 Leu) when the DNA fragment encoding the IV-V loop was sequenced. Hence, restored photoautotrophic capacity of the A251R* isolates probably cannot arise from intragenic suppressor mutations affecting residues other than amino acid 251 in the D1 protein or from extragenic suppressors.
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Levels of D1 and LHCP Proteins--
Levels of the D1 and LHCP
proteins of PSII have been examined in LL- and HL-grown cells by
immunoblotting (Fig. 2, A and
B), with the tubulin -subunit used as a control for equal
loading. Variations seen in the proportions of the 32- and 29-kDa D1
conformers might result from the specific amino acid substitutions
introduced. Therefore, both bands were summed to give the total amount
of D1 present, and these values were normalized to the amount of
-tubulin protein. When grown under LL, all mutants, with the exception of A251C*, showed a slight reduction (10-20%) in D1 level,
expressed as the sum of the 32- and 29-kDa conformers, compared with
wild type (Fig. 2B). In contrast, the D1 level under HL
growth varied considerably between mutants. Thus, although A251C* and
A251I* did not differ markedly from wild type, A251G*, A251S*, and
A251P* showed 16-28% reduction from the wild type levels, and A251L*
and A251V* appeared to contain elevated amounts of D1 (Fig.
2B). The 24-25-kDa D1 polypeptides, thought to represent N-terminal aborted translation products in the A251Q*, A251E*, A251H*,
and A251D* D1 mutants with nonphotosynthetic phenotypes (10), were
absent in all seven D1 mutants capable of photoautotrophic growth. The
level of D1 in wild type, A251C*, A251G*, A251S*, and A251P* under HL
was 57-59% of that in the same genotypes under LL. In contrast, under
HL A251I*, A251V*, and A251L* contained 68-80% of the LL amount of
D1.
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Synthesis and Degradation of the D1 Protein-- Wild type and the four mutants tested (A251C*, A251G*, A251L*, and A251P*) synthesized D1 at a substantially higher rate than LSU under HL during the 17-min labeling period and degraded D1 faster than LSU during the 2-h chase. Synthesis of D1 in A251C* and A251L* occurred at rates equal to or greater than wild type (Fig. 3), whereas D1 labeling in A251G* and A251P* was reduced by about one-third compared with wild type (Fig. 3 legend; 1.9 and 2.0 × 105 versus 3.0 × 105). Degradation of the newly synthesized D1 during the 2-h chase was elevated in A251G*, A251L*, and A251P* compared with wild type and A251C*. In the case of A251L*, the high D1 degradation rate was correlated with an increased D1 synthesis rate. A251P* had the lowest rate of D1 synthesis and the highest rate of D1 degradation of all of the mutants examined. Rates of LSU degradation were comparable with wild type in A251G* and A251P* (Fig. 3 legend) and lower than wild type in A251C* and A251L*. No labeling of 24-25-kDa bands typical of the aborted D1 translation products found in the nonphotosynthetic D1 mutants (10) was observed.
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Photosynthetic O2 Evolution and Whole Chain Electron Transport-- The relative apparent quantum yield (Ørel), maximum quantum yield (Ømax), and maximum rates of light-saturated photosynthetic O2 evolution (Pmax) of the aforementioned strains are compared in Table I. Mutants A251C*, A251G*, A251P*, and A251S* grown under LL have Pmax values somewhat higher than wild type, whereas Pmax values for A251I*, A251L*, and A251V* are lower than wild type. All mutants grown in HL except A251C* have lower Ørel and Ømax values than wild type, with A251I*, A251V*, and A251L* lowest of all. With the exception of the A251C* mutant, which has rates of O2 evolution equal to or higher than A251A*, all of the other photoautotrophic mutants appear to be impaired when grown under HL (Table I) and failed to acclimate to HL on a chlorophyll basis. Whereas wild type and A251C* show a 50% reduction in chlorophyll per unit biomass, and higher chlorophyll a/b ratios in HL, other mutants retain chlorophyll per unit biomass ratios and chlorophyll a/b ratios similar to those in LL.
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Photoautotrophic Growth and Carbon Accumulation--
LL and HL
photoautotrophic growth rates for the seven Ala251 mutants
with varying levels of photosynthetic competence have been described
elsewhere (17). Substitution of Cys, Gly, Pro, or Ser for Ala does not
affect the photoautotrophic growth rates under LL or HL, whereas Ile,
Val, and Leu substitutions lead to reduced growth rates under both
conditions (17). This slower growth is not correlated either with
decreases in whole chain (WC) or PSII
(
PSII) electron transfer (Table
II) or with the total carbon per cell and
the quantum requirement per carbon fixed (Table I). Therefore, the
rates of these processes per se in the mutants do not appear
to be limiting growth.
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Efficiency of Primary Photochemical Charge Separation-- The photochemical competence of each mutant grown under LL and HL was assessed as variable fluorescence measured as the single turnover of PSII using FRRF and by saturating, multiple turnover flashes with the PAM system. In general, the two Fv/Fm values obtained for each genotype grown under either LL or HL were in reasonable agreement (Table II), suggesting that potential quenching problems inherent to the PAM method (22) did not occur during the initial 1-s flash used for determination of dark-adapted Fm. The PAM Fv/Fm values for the wild type control A251A* were comparable with previously published values for wild type Chlamydomonas (19, 21). Mutants A251G*, A251P*, and A251S* showed a lower quantum yield of photochemistry (Ømax = Fv/Fm) than wild type and the other four mutants when grown under LL or HL and measured with either technique (Table II). Fv/Fm values obtained for the mutants A251G*, A251P*, and A251S* grown under HL approach those measured for the nonphotosynthetic Ala251 mutants that range from 0.13 to 0.46 (10).
The reduction in photochemical efficiency in HL suggests a loss of functional PSII reaction centers. Indeed, calculations show that A251G*, A251P*, and A251S* have ~2.5-fold fewer functional PSII reaction centers than wild type under HL, while A251I* and A251L* have 1.3-1.7-fold fewer than wild type (Table II). The lower concentration of PSII centers in HL-grown cells corresponds with the reduced probability of excitation transfer between PSII units (percentage of connectivity, Table II). With the exception of the A251C* mutant, which behaves like wild type, the connectivity is reduced 1.6-1.7-fold in A251S*, A251I*, and A251V* and is virtually absent in A251P*, A251G*, and A251L*. Furthermore, a decrease in the effective absorption cross-section of PSII (functional size of PSII antenna) of 13% can be seen in A251C*, A251I*, A251L*, and A251V* (Photochemical Fluorescence Quenching--
Photochemical
fluorescence quenching can be related to the level of reduction of
QA, the first stable acceptor of PSII, at a given
irradiance. The irradiance dependence of qP derived during the photosynthetic light response curves of selected mutants is shown
in Fig. 4. As observed for photosynthetic
O2 evolution, A251C* is the only mutant that has
qP values similar to wild type when grown under both LL and
HL. In contrast, A251G* (and A251P*, A251S*) and especially A251L* (as
well as A251I* and A251V*) exhibit a dramatic reduction in
qP, particularly when measured at low incident irradiances.
The low values of qP in dark-adapted mutants other than
A251C* and the A251A* wild type presumably reflect the higher
proportion of nonfunctional (photochemically incompetent) PSII centers
in these strains. The coefficient 1 qP is directly proportional to the average reduction state of QA. At
steady-state irradiance, qP is related to the cross-section
of PSII and the rate of oxidation of
QA
by the equation (1
qP)(I) = (
PSII × I)/(
PSII × I + 1/
PSII) for any given light intensity (I).
From Fig. 4 we can deduce that A251A* wild type and A251C* show much
lower reduction states of QA than any of the other mutants
at both growth irradiances. The more reduced QA pools in
these genotypes might potentiate photoinhibitory damage and lead to
loss of functional PSII reaction centers.
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Ability of the Reaction Center to Transfer Electrons from
QA to QB and Stability of
QB in the D1 Binding
Pocket--
The effect of each amino acid substitution on the rate of
electron transfer from QA to the plastoquinone pool and the
stability of QB
in the quinone binding
pocket of the D1 protein can be assessed from a combination of variable
fluorescence and luminescence measurements. The ratio of the maximum
fluorescence yield induced by a multiple turnover flash (MTF) to that
observed following a single turnover flash (STF) is indicative of the
rate of reduction of secondary plastoquinone acceptors in PSII
(i.e. QB and the mobile plastoquinone pool) by
QA (Table III). MTF/STF
ratios ranged from 1.45 in the A251A* wild type control to 1.10 in
A251L*, which appears to be most impaired in transferring electrons
from QA to QB. Among the photosynthetically
competent mutants, the correlations between MTF/STF ratios and the
percentage of PSII centers containing reduced QA
measured 25 ms following a
saturating flash (Table III) fall into two groups. The A251A* wild type
and mutants unimpaired in photosynthetic O2 evolution show
a range of MTF/STF from 1.19 to 1.45 with only 11-21% in
[QA
] 25 ms, whereas the impaired
mutants (A251I*, A251V*, and A251L*) show values of 24-42%. A251V*,
which is strongly reduced in Pmax like A251I*
and A251L*, shows a smaller reduction in MTF/STF and [QA
] 25 ms than the other two
mutants. The proportion of the PSII reaction centers with
QA
in the nonphotosynthetic mutants
(10) is especially high (89-96%), which suggests that almost no
electrons are transferred to QB in these mutants at
physiologically significant rates.
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Apparent Equilibrium Constant Kinetics and Thermodynamics of
Electron Transfer between QA and
QB--
With the exception of A251P* and A251S*, all other
mutants show significantly reduced apparent redox equilibrium constants for the electron resonance between QA and QB
(Kapp) as calculated according to the
"two-electron gate" model (15). Lower values of
Kapp are indicative of lower overall rates of
reduction of QB by
QA
(Tables II and III). Moreover, the
relationship between qP at LL (Fig. 4) for each of the
mutants reflects the overall equilibrium rate constant
Kapp (Table III). In the nonphotosynthetic
mutants (10), Kapp approaches 0, confirming that
electron transfer to secondary quinones in these strains is blocked at
the primary quinone acceptor QA.
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DISCUSSION |
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We have used a combination of biophysical, biochemical, and physiological assays to characterize the effects of seven amino acid substitutions at the Ala251 residue of the D1 protein that result in varying levels of photoautotrophic competence. These seven mutants and the five nonphotosynthetic Ala251 D1 mutants we characterized previously (10) can be grouped into four phenotypic classes with respect to D1 synthesis and steady state level, photosynthetic capacity, photoautotrophic growth rate, and herbicide resistance (Fig. 6): 1) similar to wild type (A251C*), 2) impaired in photosynthesis but not in photoautotrophic growth (A251G*, A251P*, and A251S*), 3) markedly impaired in photoautotrophic growth and photosynthesis and herbicide-resistant (A251I*, A251L*, and A251V*), and 4) not photosynthetically competent (A251R*, A251D*, A251Q*, A251E* and A251H*). The effects of the individual amino acid substitutions on D1 synthesis/accumulation, growth, and carbon accumulation, O2 evolution, composition of the PSII reaction centers, and QA to QB electron transfer will be considered below.
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The Arg, Asp, Gln, Glu, and His Substitutions--
Previously, we
showed that these five amino acid substitutions at the
Ala251 D1 residue led to a nonphotosynthetic phenotype,
markedly impaired D1 synthesis and accumulation, and severely affected
primary photochemical charge separation efficiency (10). Here, we
report that electron transfer between QA and QB
is almost completely blocked in these five mutants (Table III). No
binding of QB in its niche on the D1 protein could be
detected in these strains. Electrons that can be extracted from water
oxidation are blocked at the primary quinone acceptor QA,
which has been suggested to lead to a doubly reduced
QA2 (36). This could increase the
possibility for recombination of the radical pair
P680+/Phe
and generate
highly toxic 1O2 via the
3P680 state (37). The conformation of the IV-V
loop of the D1 protein in these mutants is presumably structurally
altered compared with wild type (10).
Importance of the Ala251 Residue Deduced from Suppressor Analysis of the Nonphotosynthetic A251R* Mutant-- Among about 40 isolates recovered after acetate starvation of the nonphotosynthetic A251R* mutant and sequenced, only same site suppressors were obtained. This strongly suggests that other mutations in the D1 protein or in other chloroplast proteins are unable to compensate for the structural modification caused by insertion of a bulky, charged amino acid at position 251. These results reinforce the hypothesis that the amino acid residue present at position 251 plays a critical role in conformation and function of the IV-V loop (10, 16, 17, 35).
The Cys Substitution--
For most of the parameters analyzed, the
A251C* mutant appears indistinguishable from the wild type A251A*
control (this paper and Ref. 17). However, detailed analysis of the
electron flow between water and the plastoquinone pool in the thylakoid
membranes indicated a noticeable reduction in the rate of
QA reoxidation and a destabilized
S2QB
(S3QB
) state, which can be
attributed to a shift of the apparent equilibrium constant
(Kapp) between
QA
QB and QA
QB
(Table III). Although the Cys
substitution has some impact on the electron transfer between
QA and QB, this mutant is able to grow and
evolve O2 as well as wild type under LL and HL (Table I),
photoacclimates properly during growth in HL by down-regulating its
LHCP and chlorophyll levels (Fig. 2B and Table I), and does not show resistance to PSII herbicides (17). Based on the MTF/STF ratio
in A251C* (Table III), the ability of this mutant to reduce fully its
plastoquinone pool, as well as its overall rate of electron transport
from water to the terminal acceptors, is similar to wild type. Cysteine
possesses a reversibly reducible SH group, which might help mediate
electron flow between QA and QB. This could
explain why the overall photosynthetic output and biomass production in
this mutant are indistinguishable from those of wild type.
The Gly, Pro, and Ser Substitutions-- The A251G*, A251S*, and to a lesser extent A251P* mutants are not only markedly impaired in the electron transfer between QA and QB but also in efficiency of primary photochemical charge separation (Tables II and III). These defects on the acceptor and donor sides of D1 lead to an overall reduction of the photosynthetic function (Table I) and to an increased PSII excitation pressure (Table II and Fig. 4). The mutants also fail to down-regulate their chlorophyll content normally when grown in HL (Table I) and have been shown to contain a larger functional antenna per PSII reaction center and lower probability of excitation energy transfer between the reaction centers than wild type (Table II). This observation is corroborated by immunoblot analysis showing that LHCP accumulation was also not down-regulated to the level of the wild type control under HL (Fig. 2).
The redox state of the plastoquinone pool in the thylakoid membrane has been shown to regulate expression of the nuclear cab gene encoding LHCP by molecular signaling (34, 38). Reduced plastoquinone was postulated to repress cab gene transcription via a signaling pathway involving a phosphorylated factor coupled to the redox state of plastoquinone through a chloroplast kinase. Substitutions of Gly, Pro, and Ser for Ala251 slow the electron transfer rate between QA and QB (Table III) and therefore result in a mainly oxidized quinone pool in the membrane, which might prevent redox repression of cab gene transcription in HL. Because the mutant cells would no longer sense the difference in redox state under HL, cab gene expression would continue at almost the same level as in LL, resulting in accumulation of LHCP-bound chlorophyll in the antennae. If a reduction in the number of chlorophyll molecules harvesting light ameliorates the potentially destructive redox chemistry occurring in chloroplasts exposed to bright light, the question arises as to why the A251G*, A251P*, and A251S* mutants with their large antennae grow as well as wild type under HL (17). Decreases in PSII centers per cell seen in these three mutants (Table II) might be a consequence of photodamage or might reflect another adaptation of the cell to the HL environment. Photoinhibition has been defined as a light-dependent decrease in maximum photosynthetic efficiency (39), which results in damage and degradation of the D1 protein. For example, in A251G* and A251P*, D1 synthesis in short term pulse-chase experiments is reduced by about one-third, whereas D1 turnover is increased by 27 and 73%, respectively, compared with wild type (Fig. 3). These mutants also tend to accumulate less D1 than wild type (Fig. 2). Alternatively, photoinhibition may be considered a mechanism that matches overall photosynthetic electron flow to the photon flux rate through the down-regulation of PSII photochemistry (40-42). In fact, the decrease in the number of PSII reaction centers per cell observed in A251G*, A251P*, and A251S* (Table II) might reflect an adjustment of the photosynthetic electron transport system to high PSII excitation pressure. The 3.3-h photoautotrophic doubling times obtained with wild type C. reinhardtii, A251C* and the A251G*, A251P*, and A251S* mutants in microtiter plates under optimum conditions (saturating light and CO2) (17) are the shortest known for this alga. Hence, our observation that the growth rate of the latter three mutants is not photosensitive under these conditions suggests that photosynthetic electron transfer from QA to the plastoquinone pool is not rate-limiting for overall photosynthetic electron flow at light saturation. The calculated time for whole chain electron transport (The Ile, Leu, and Val Substitutions-- Replacement of Val for Ala251, the only substitution at this position previously characterized in cyanobacteria and in C. reinhardtii, has been reported to result in resistance to certain PSII herbicides and increased the sensitivity to photoinhibition (13, 14). The cyanobacterial mutation was subsequently shown to affect the apparent equilibrium constant on the acceptor side (11) and the properties of the oxygen-evolving complex on the donor side (16). The phenotype of the new A251V* mutant of C. reinhardtii characterized in this paper is similar to that of the two aforementioned Val mutants. In addition, we created and characterized Ile and Leu substitutions that fall into the same phenotypic category (this paper and Ref. 17). Based on herbicide resistance, we find the A251I* and A251V* mutants to be virtually identical, while the A251L* mutant results in a more extreme herbicide resistant and photosynthetic defective phenotype.
A251I* and A251L*, and to a lesser extent A251V*, are severely impaired in the QAProperties of the Amino Acid at Position 251 in Relation to D1 Structure and Function-- In cyanobacteria, amino acids 248-251 of the IV-V loop of the D1 protein are thought to be buried in the thylakoid membrane, dividing the loop into two functional domains (11, 16). Our grouping of the 12 Ala251 mutants of C. reinhardtii into four phenotypic classes in terms of their effects on photosynthetic efficiency, photoautotrophic growth, and herbicide resistance (Fig. 6) relative to the nature of the substituted R group is consistent with the present structural models of the IV-V loop. Insertion of a charged or polar and relatively bulky amino acid at this position (e.g. Arg, Asp, Gln, Glu, or His) would be predicted to destroy the conformation of the D1 IV-V loop because of interactions of the R groups of these amino acids with polar heads of the lipid bilayer of the thylakoid (10). This is consistent with the total blockage of the electron flow between QA and QB and the nonphotosynthetic phenotype observed in these five mutants.
Substitution of Ala251 with an uncharged amino acid having a bulky side chain (Fig. 5, e.g. Ile, Leu, or Val) does not disrupt D1 function completely but impairs photosynthetic electron transfer to various extents that appear to correlate with the position of the methyl group on the R group. Leu, whose methyl group is attached at C-3, has a more severe phenotype than Ile and Val, which have methyl groups attached at C-2. In contrast, substitution of small uncharged amino acids for Ala251 (e.g. Cys, Gly, Pro, or Ser) has no effect on the mutants' photoautotrophic growth or herbicide sensitivity (17) and photosynthetic O2 evolution, but all four mutations affect the function of the QB binding niche to varying degrees. In the case of the Gly, Pro, and Ser substitutions, the acceptor side of PSII is affected. The effect of the Ser and Pro substitutions may be related to the polarizable hydroxyl group and five-ring structure, respectively, in the R groups of these two amino acids. That Gly, which has a hydrogen attached to the carbon backbone instead of the methyl group in the normal Ala residue, perturbs function suggests that steric interactions on the side chain are critical for plastoquinone binding. Interestingly, substitution of Cys with a sulfhydryl group, results in the fewest biophysical, biochemical, and physical consequences of the 12 mutations examined. The fact that Cys can exist in either reduced or oxidized form might help mediate proton and/or electron transfer to plastoquinone in the QB pocket. In the case of the seven Ala251 mutants that retain the capacity for photoautotrophic growth, one has difficulty in correlating growth rate, carbon accumulation, and photosynthetic efficiency under LL and HL with the capacity for electron transfer between QA and QB. Furthermore, no correlation has been found between QB ![]() |
ACKNOWLEDGEMENTS |
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We thank Anita M. Johnson for help with
chloroplast transformation experiments, Dr. P.B. Heifetz for advice
regarding the O2 evolution and PAM fluorescence
measurements, and Drs. A. Sorokine and Z. Kolber for discussions and
suggestions regarding the FRRF and thermoluminesence measurements. Drs.
P. B. Heifetz, S. P. Mayfield, G. Piperno, and B. D. Kohorn kindly provided the D1, OEE1, tubulin -subunit, and LHCP
antibodies, respectively.
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FOOTNOTES |
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* This work was supported by Department of Energy Grant DE-FG05-89ER14005 (to J. E. B., N. W. G., and C. B. O.).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.
Present address: Dept. de Botanique, Faculté de Pharmacie,
Université Paris-Sud, 5 rue Jean-Baptiste Clément, 92296 Chatenay-Malabry, Cedex, France.
§ Present address: Environmental Biophysics and Molecular Biology Program, Brookhaven National Laboratory, Upton, NY 11973. Dr. Prásil is now at the Institute of Microbiology, MBU AVCR, 379 81, Trebon, Czech Republic.
¶ Supported by the Department of Energy under contract DE-AC02-76CH00016.
Present address: Dept. of Plant Genetics, Weizmann Institute
of Science, Rehovot 76100, Israel.
** Supported by the Willstatter and Forschheimer Centers.
Present address: Research School of Biological Sciences,
Australian National University, Canberra 2601, Australia.
§§ To whom correspondence and reprint requests should be addressed: DCMB Group, Box 91000, Duke University, Durham, North Carolina 27708-1000. Tel.: 919-613-8157; Fax: 919-613-8177; E-mail: jboynton{at}acpub.duke.edu.
1 The abbreviations used are: PSII, photosystem II; LL, low light; HL, high light; LHCP, light-harvesting chlorophyll a/b-binding protein; LSU, large subunit of ribulose-bisphosphate carboxylase oxygenase; TL, thermoluminescence; DL, delayed luminescence; ST, single turnover; FRR, fast repetition rate; FRRF, FRR fluorometry, MTF, multiple turnover flash; STF, single turnover flash; LDS, lithiumdodecyl sulfate.
2 B. Förster, unpublished observations.
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