(Received for publication, August 25, 1994; and in revised form, October 18, 1994)
From the
Photosystem II catalyzes the photooxidation of water to molecular oxygen, providing electrons to the photosynthetic electron transfer chain. The D1 and D2 chloroplast-encoded reaction center polypeptides bind cofactors essential for Photosystem II function. Transformation of the chloroplast genome of the eukaryotic green alga Chlamydomonas reinhardtii has allowed us to engineer site-directed mutants in which aspartate residue 170 of D1 is replaced by histidine (D170H), asparagine (D170N), threonine (D170T), or proline (D170P). Mutants D170T and D170P are completely deficient in oxygen evolution, but retain normal (D170T) or 50% (D170P) levels of Photosystem II reaction centers. D170H and D170N accumulate wild-type levels of PSII centers, yet evolve oxygen at rates approximately 45% and 15% those of control cells, respectively. Kinetic analysis of chlorophyll fluorescence in the mutants reveals a specific defect in electron donation to the reaction center. Measurements of oxygen flash yields in D170H show, however, that those reaction centers capable of evolving oxygen function normally. We conclude that aspartate residue 170 of the D1 polypeptide plays a critical role in the initial binding of manganese as the functional chloroplast oxygen-evolving complex is assembled.
In photosynthetic eukaryotes, the Photosystem II (PSII) reaction
center mediates the photooxidation of water to molecular oxygen,
providing electrons to the photosynthetic electron transfer chain and
releasing protons into the lumen of the chloroplast thylakoid membrane.
At least five nuclear-encoded and thirteen chloroplast-encoded PSII
polypeptide subunits have been identified (for reviews, see Erickson
and Rochaix(1992) and Ikeuchi(1992)). The function of PSII centers
active on both the donor and acceptor sides, as outlined in Fig. 1and described in the legend, requires the interaction of
the PSII polypeptides and their assembly with the lipid, metal, and
ionic cofactors of PSII. At the heart of PSII structure and function is
a heterodimer composed of two intrinsic membrane polypeptides, D1 and
D2 (Nanba and Satoh, 1987; Marder et al., 1987; Webber et
al., 1989; Tang et al., 1990). These two
chloroplast-encoded polypeptides provide ligands for the reaction
center chlorophyll P, pheophytin, and the primary and
secondary quinone acceptor molecules of PSII, Q
and
Q
, respectively (Trebst, 1987; Svensson et al.,
1990; Ruffle et al., 1991).
Figure 1:
Schematic diagram of electron transfer
events mediated by Photosystem II. Arrows indicate the linear
transfer of electrons from water (HO) to the second quinone
acceptor molecule (Q
). The D1 and D2 polypeptides of the
PSII reaction-center core provide ligands to redox active components on
both the donor and acceptor sides of the PSII reaction center
chlorophyll P
, as indicated. Absorption of light energy (h
) by the primary donor, P
, leads to
formation of the initial charge-separated state between P
and a pheophytin a molecule
(P
-Pheo
).
Charge separation is stabilized by further transfer of the electron
from pheophytin to the first and then second quinone acceptors, Q
and Q
, respectively. This latter
step is blocked by
the herbicide diuron (DCMU) which binds competitively with quinone at
the Q
-binding site of the D1 polypeptide.
P
is reduced by the secondary donor
Z (Y
), identified by Debus et al.(1988) and Metz et al.(1989) as tyrosine residue 161 of the D1 polypeptide
(D1Y161). The tyrosine radical is reduced, in turn, by the functional
oxygen-evolving complex (OEC) associated with the PSII core complex.
According to the model of Joliot and Kok, with each photooxidation of
the PSII reaction center, oxidizing equivalents are transferred one at
a time to the OEC, advancing the so-called S-states of the OEC. The
number associated with each S state (S
-S
)
indicates the number of oxidizing equivalents stored (Kok et
al., 1970; Joliot and Kok, 1975). Oxygen is released at the
transient formation of the S
state restoring the S
state. The cycle through the S-s
tates is thought to occur largely
through oxidation of a tetranuclear manganese cluster at the heart of
the OEC which supplies one electron for the reduction of
P
after each photooxidation of
chlorophyll and accumulates the 4 oxidizing eq needed to split water.
Current models predict that D1 and possibly D2 provide ligands to the
manganese cluster.
Central to PSII water oxidation is a cluster of 4 manganese ions which advances in oxidation state as
electrons are donated from it to the photooxidized PSII reaction center
chlorophyll, P. When 4 oxidizing eq
are accumulated in the manganese cluster, 1 molecule of oxygen is
released and the cycle of manganese oxidation begins again (see Fig. 1 legend). The minimal, purified chloroplast PSII particle
that retains maximal oxygen-evolving activity in vitro, in the
absence of elevated levels of Ca
or Cl
(reviewed in Critchley (1985) and Andersson and Åkerlund(1987)), consists of the D1
D2 heterodimer, several
additional intrinsic membrane polypeptides, and three extrinsic
membrane polypeptides referred to as the oxygen evolution enhancer
(OEE) (
)polypeptides, OEE1, OEE2, and OEE3, of approximately
33, 23, and 17 kDa, respectively. The OEE polypeptides are localized to
the lumenal side of PSII and participate with the intrinsic PSII
polypeptides in the formation of a fully activated chloroplast
oxygen-evolving complex (OEC) (reviewed in Andersson and
Åkerlund(1987), Ghanotakis and Yocum (1990), Debus(1992), and
Rutherford et al.(1992)). It is generally agreed that the OEC
active site, containing Ca
and 4 manganese atoms,
requires 20 to 24 coordinating ligands provided mainly by oxygen, but
with some nitrogen (DeRose et al., 1991). Histidine is now
known to be a ligand (Tang et al., 1994), and additional
coordination to manganese may be provided by carboxylate and amide
moieties and perhaps tyrosine. Most evidence suggests that it is the D1
and, possibly, D2 polypeptides which coordinate manganese (reviewed in
Diner et al.(1991), Debus (1992), and Vermaas and
Pakrasi(1992)). Aspartate residue 170 (Asp-170) of the D1 polypeptide
is found in the lumen only 9 amino acid residues away from D1 tyrosine
161 (Y
) which directly accepts electrons from manganese
(Debus et al., 1988; Metz et al., 1989). Asp-170 is
conserved in the predicted D1 sequences of all 38 species of
photosynthetic eukaryotes and cyanobacteria reviewed (Svensson et
al., 1991). The role of aspartate 170 of the D1 polypeptide was
recently examined in the cyanobacterium Synechocystis 6803 (Nixon and Diner, 1992; Boerner et al., 1992). These
authors found that substitutions at this site abolished or reduced
oxygen evolution in mutant cyanobacteria. Results of in vitro spectroscopic analysis of PSII particles isolated from mutant
cyanobacteria suggest that D1 aspartate 170 plays an important role in
the stable assembly of the manganese cluster of the cyanobacterial OEC.
Although photosynthesis in the prokaryotic cyanobacteria is similar in many respects to photosynthetic function in eukaryotic plants and algae, there are several features unique to the structure, function, regulation, and stability of the photosynthetic apparatus of the chloroplast. In eukaryotes, the photosystems are located in a thylakoid membrane within the chloroplast and, hence, totally isolated from the plasma membrane of the cell. Moreover, since the genes encoding polypeptides localized to the chloroplast thylakoid membrane are contained in two distinct genetic compartments as noted above, assembly of the chloroplast photosynthetic apparatus requires the coordinate expression of genes located in the nuclear genome and in the chloroplast genome, the latter of which is polyploid. This genetic complexity is in contrast to the prokaryotic cyanobacteria which lack intracellular organelles and which contain only one genome. Cyanobacteria also lack the highly ordered stacking of thylakoids into appressed and nonappressed membrane regions. There are substantial differences between cyanobacteria and chloroplast thylakoids with respect to the light-harvesting antennae polypeptides and pigments associated with the photosystems, the regulatory mechanisms affecting photosynthetic function (e.g. phosphorylation of the chloroplast reaction center polypeptides), and the ratio of PSII to PSI centers. There are also differences in PSII polypeptide composition. Cyanobacteria lack homologues for the OEE2 and OEE3 polypeptides (Stewart et al., 1985; Shen et al., 1992), and, although an OEE1 homologue exists, site-directed mutagenesis of the cyanobacterial OEE1 gene suggests that OEE1 is not essential for cyanobacterial PSII oxygen evolution in vivo (Bockholt et al., 1991; Mayes et al., 1991; Philbrick et al., 1991; Burnap et al., 1992). This is in marked contrast to the requirement for OEE1 and OEE2 in chloroplast oxygen evolution in vivo (Mayfield et al., 1987a, 1987b). Finally, differences in the interaction of PSII subunits are reflected by the fact that, in chloroplasts, loss of one subunit constituent has a more drastic consequence on the stability of the entire PSII complex (reviewed in Rochaix(1992)).
There has been until now little evidence as to whether specific changes in PSII polypeptide structure that affect PSII function in cyanobacteria would have a similar affect on the chloroplastic PSII. Given the above differences between photosynthesis in cyanobacteria and in the chloroplast of eukaryotes, we have undertaken studies to examine the relationship between PSII structure and donor-side function in a eukaryotic photosynthetic organism, C. reinhardtii. This unicellular green alga provides an excellent experimental system for a molecular and genetic approach to dissecting chloroplast PSII structure-function relationships (Rochaix and Erickson, 1988). DNA-mediated transformation of the nuclear (Debuchy et al., 1989; Fernandez et al., 1989; Mayfield and Kindle, 1990) and chloroplast (Boynton et al., 1988) genomes of this algae has been successful. Advantages of using Chlamydomonas to study PSII function include the much greater variable fluorescence compared to cyanobacteria, which allows for the rapid screening of algal colonies for mutant fluorescence phenotypes indicative of mutant PSII function, and the ability of Chlamydomonas to grow heterotrophically in the light or dark, which allows for easy maintenance of mutant strains deficient in photosynthesis. Here we report the site-directed mutagenesis of codon 170 of the chloroplast psbA gene encoding the D1 polypeptide of C. reinhardtii, the in vivo characterization of PSII function in algal transformants with amino acid substitutions at D1 residue 170, and our conclusions that residue 170 is critical for assembly and/or stability of manganese in the OEC of the chloroplast PSII.
Figure 2: Restriction map of the C. reinhardtii chloroplast psbA and rRNA genes and the mutations introduced at psbA codon 170. Upper: black boxes indicate the five exons of psbA (1-5) and the 16 S, 7 S, 3 S, 23 S, and 5 S rRNA genes. Hatched boxes indicate the four psbA introns and the intron in the 23 S rRNA gene. Bars above the restriction map show the relative locations of psbA DNA fragments cloned into recombinant plasmids pXb1.8, pRR, pRX, and pRRX. pCrBH4.8 contains a mutant 16 S rRNA gene which confers spectinomycin resistance to cells (Harris et al., 1989; Newman et al., 1990). Restriction sites for EcoRI (R), XhoI (X), XbaI (Xb), and BamHI (B) are indicated. Size of the bar is 1 kb. Lower: the wild-type DNA sequence of a portion of psbA exon 3 containing aspartate codon 170 is shown. The site-directed mutations indicated below the arrow were introduced into psbA in four different plasmid constructs. Indicated for each mutant codon is the corresponding amino acid residue substituted at position 170, the restriction site created by the mutation (far right), and the code used to label mutant constructs (far left).
Figure 3: Restriction fragment length polymorphism (RFLP) analysis of Chlamydomonas transformants. Analysis of DNA isolated from spectinomycin-resistant C. reinhardtii transformants shows the segregation of wild-type and mutant chloroplast DNA BsrI fragments containing a portion of psbA exon 3. DNA samples were prepared from 1.5-ml liquid cultures derived from 8 independent initial isolates (panel A) obtained after co-transformation of the WT2137 algal host with pCrBH4.8 and pRR-D170T plasmid DNAs (see Fig. 2) and from subsequent colony isolates (panel B) produced from the initial isolate shown in lane 3 of panel A. DNAs were subjected to BsrI restriction digestion and 1.5% agarose gel electrophoresis. Panels A and B show exposures obtained by autoradiography of Southern blots hybridized with a radiolabeled probe (261-bp fragment from pPXb261) indicated above the restriction map in panel C. A portion of psbA exon 3 is indicated by the black box. Restriction enzyme sites are given for BsrI (Bs), PstI (P), and XbaI (Xb). The BsrI site introduced by the D170T mutation in exon 3 is indicated with a star. Bars below the map show the DNA region contained in the wild-type (WT) and mutant (D170T) BsrI fragments of 303 bp and 240 bp, respectively.
Site-directed mutagenesis
targeted to codon 170 of psbA was performed using the
protocols described (Mutagene Kit, Bio-Rad) except that Bluescript
KS (Stratagene) was used as the phagemid vector, R408
(Stratagene) was used as the M13 helper phage, and kanamycin was not
added to superinfected cells. A synthetic 25-mer oligonucleotide
homologous to the region of psbA surrounding codon 170
(sequence given in Fig. 2) but mutant at the third position of
the codon (T) and degenerate at the first (C/A/T) and second (A/C)
positions, was used to prime second strand DNA synthesis.
Bombarded
plates were incubated 16 h in the dark, after which filters
containing cells were transferred to selective medium. TAP-SPEC agar
(165 µg of spectinomycin dihydrochloride/ml) selected for
spectinomycin-resistant cells. The spectinomycin-resistant control
transformant lines WTsr (from transformation of 137y with p228) and
FWTsr (from co-transformation of FuD7 with wild-type pRRX and p228)
were isolated. An additional control strain, FWT, was produced by
transformation of FuD7 with wild-type pRRX DNA followed by selection
for growth on minimal medium (HSM) in the light. Initial colonies were
picked into liquid medium and replated on selective medium, and
secondary colony isolates were obtained. Primary and secondary isolates
were subjected to colony hybridization and/or RFLP screening. All
incubations during recovery, selection, screening, and routine
maintenance of transformant cell lines were carried out in the dark.
Algal colonies grown on nylon membrane filters were screened by
colony hybridization using a modification of the method developed for
bacterial colonies (Grunstein and Hogness, 1975) as follows. Nylon
filters were placed colony side up, for 2 min each treatment, onto
0.75-ml puddles of the following solutions, in sequence: 10% SDS (3
0.75 ml); 0.5 M NaOH, 1.5 M NaCl (3
0.75 ml); and 0.5 M Tris-HCl, pH 8.0, 1.5 M NaCl (3
0.75 ml). After each puddle treatment, excess liquid was
removed from the filter using a Büchner funnel
connected to a vacuum line. Filters were processed and hybridized as
described above for Southern blot analysis.
Figure 9:
Oxygen yields from Chlamydomonas cells following saturating light flashes. Panel A,
relative yields of oxygen were measured following each of a series of
saturating light flashes (533 ms apart) given to dark-adapted whole
cells, as described under ``Experimental Procedures.'' The
FWTsr (control) and D170H
(transformant) cells
were dark-adapted for 10 min prior to illumination. Panel B,
lifetime of the S
state for these cells under the same
conditions as for A. The cells were dark-adapted for 5 min and
then given a single saturating light flash. Following this flash and at
the time intervals indicated, a series of flashes were given 533 ms
apart and the relative amplitude of oxygen was measured on the third
overall flash. The relative concentration of the S
state is
plotted as the difference, between the third overall flash yield for
delay time (t) and the yield of the third overall flash for a
delay time of 5 min, divided by the steady state oxygen flash
yield.
To facilitate chloroplast transformation of two different algal hosts, the mutant XbaI fragment from each mutant pXb1.8 was transferred into the larger plasmid constructs, pRR and pRRX (see Fig. 2). The resulting mutant pRR constructs (pRR-D170H, pRR-D170N, pRR-D170T, pRR-D170P) were used in co-transformation experiments in which the wild-type photosynthetic C. reinhardtii strains 2137 or 137y served as algal hosts. The mutant pRRX constructs (pRRX-D170H, pRRX-D170N, pRRX-D170T, pRRX-D170P) were used for all co-transformation experiments in which FuD7 was the algal host strain.
When the psbA deletion mutant FuD7 was used as the algal host for co-transformation, spectinomycin-resistant transformant colonies were screened by colony hybridization. Subsequent Southern blot analysis of DNA isolated from secondary isolates of original colonies which showed positive hybridization signals (data not shown) confirmed the presence of two copies of psbA per chloroplast genome. We were unable to detect any FuD7 host DNA molecules in these transformants using a polymerase chain reaction DNA amplification assay with the sensitivity to detect one copy of the FuD7 chloroplast DNA molecule in 2000, (data not shown) providing further evidence that the mutant lines are homoplasmic.
Table 1summarizes some of the mutant and control algal transformant lines isolated, the algal strains which served as host, and the transforming DNAs used. Three of the mutant transformants (D170N, D170T, D170P) are obligate heterotrophs. The D170H transformant grows very slowly on minimal medium.
To verify that the algal co-transformant lines contained the desired site-directed mutations, total RNA was isolated from transformant algal cells and used as a template for avian myeloblastosis virus reverse transcriptase in dideoxy DNA sequence reactions. All algal transformants showed the presence of the predicted mutations at codon 170 (Fig. 4), and no other differences were observed between the mutant and wild-type RNA templates. Finally, the mutant phenotype (poor or no growth on minimal medium) could be rescued by transformation of mutants with wild-type pXb1.8 DNA.
Figure 4: DNA sequence analysis using RNA templates isolated from mutant Chlamydomonas transformants. Total RNA isolated from wild-type (WT) and transformant cell lines served as a template for dideoxy sequence analysis using synthetic oligonucleotide primers complementary to psbA RNA as described under ``Experimental Procedures.'' An autoradiogram of an 8% acrylamide gel shows the region corresponding to DNA fragments 53-62 nucleotides in length. The sequence generated at wild-type (WT) codon 170 is shown to the left of the gel next to the complimentary sequence for the aspartate (Asp-170) GAC codon. Listed below the WT and each of the transformant sequences is the amino acid residue encoded at position 170, the codon sequence (5` to 3`), and the actual DNA sequence (3` to 5`) seen on the autoradiogram, respectively. AGCT above the WT sequence denotes the dideoxy sequencing reactions loaded in each of four consecutive wells, respectively.
Figure 5:
Kinetics of chlorophyll fluorescence
emission from Chlamydomonas cells. Chlorophyll fluorescence
induction transients were measured in situ from C.
reinhardtii cells on algal plates dark-adapted for 10 min prior to
assay, as described under ``Experimental Procedures.'' The
relative amounts of PSII variable fluorescence in the FuD7 algal host
and in isolates D170H, D170N
,
D170T
, and D170P
of transformant cell lines are
shown here. Initial levels of fluorescence were normalized to 0.2 for
each strain. In the absence of normalization, the FuD7 host strain
(which lacks PSII) displays a high level of initial fluorescence,
approximately 5 times the absolute initial fluorescence of wild-type
cells. Panel A, full time scale is 1.43 s; panel B,
enlargement of the first 0.68 s of induction of selected algal strains
as marked.
Figure 6:
Quantification of PSII in mutant Chlamydomonas transformants. A
[C]diuron herbicide binding assay (Vermaas et al., 1990) was used to quantitate PSII in whole cells.
Assay of the number of diuron binding sites, assessed on a per
chlorophyll basis, provides an accurate quantification of assembled
PSII reaction centers. Assays were performed in duplicate, such that
each point represents the mean of a pair of measurements for bound
[
C]diuron plotted against the mean of a pair of
measurements for free [
C]diuron, obtained after
cells were incubated with labeled diuron at seven initial
concentrations (ranging from 5-100 nM). The double
reciprocal plot of 1/bound (mg of chlorophyll per nmol of
[
C]diuron bound) versus 1/free
(1/µM [
C]diuron free) for the
nonaveraged data was used for extrapolation of chlorophylls/binding
site at the y-intercept and -1/K
at
the x-intercept. The results are summarized in Table 1.
Figure 7:
Relaxation of the chlorophyll fluorescence
yield in mutant Chlamydomonas cells following saturating light
flashes. The kinetics of relaxation of the chlorophyll fluorescence
yield in whole cells was monitored following each of a series of
saturating light flashes given 600 ms apart. C. reinhardtii control cells (panel A) and transformants (panels
B-E) were suspended in TAP growth medium at a cell concentration
close to 0.5 A
The most likely
interpretation of the data is that, in dark-adapted D170T cells, for
example, only a single electron is available on the donor side for
reduction of P The rate of charge
stabilization on the donor side of PSII in transformants can be
inferred from experiments that monitor the rate of charge recombination
between Q
Figure 8:
Charge recombination between the PSII
donor and acceptor sides in mutant Chlamydomonas cells. The
kinetics of relaxation of the chlorophyll fluorescence yield in whole
cells in the presence of 40 µM DCMU was monitored
following a single saturating flash given to control
(FWTsr
C. reinhardtii is an ideal organism for studies
involving the genetic engineering of chloroplast-encoded proteins which
function in photosynthesis. This unicellular eukaryote contains a
single large chloroplast providing a relatively large target for
biolistic transformation, is able to grow heterotrophically on acetate
allowing for the maintenance of strains defective in photosynthetic
function, and has a well-characterized genetic system. Since the first
report of biolistic chloroplast transformation (Boynton et
al., 1988), this technique has been used to address many questions
related to chloroplast function (reviewed in Boynton and Gillham
(1993)), including studies focused on PSII structure and function
(Przibilla et al., 1991; Roffey et al., 1991, 1994;
Heiss and Johanningmeier, 1992; Lers et al., 1992; Schrader
and Johanningmeier, 1992; Monod et al., 1994; Takahashi et
al., 1994). The work we report here explores the role of the D1
polypeptide in activating chloroplast PSII donor-side function.
Several lines
of evidence led us to the conclusion that the differences observed in
the PSII phenotype of the mutant transformant cell lines are due to the
specific mutations introduced at psbA codon 170 and not to any
other factors. First, extensive molecular characterization of each
different psbA plasmid construct used for transformation ruled
out the possibility that other mutation(s) had been introduced
inadvertently into the mutagenized region (pXb1.8) of psbA.
Second, DNA sequence analysis using RNA templates isolated from each
algal transformant cell line confirmed the presence of the
site-directed mutations at psbA codon 170 and revealed no
differences in any other region of the transcript analyzed. Third, all
mutant cell lines, which grew very poorly or not at all on minimal
medium, could be rescued to wild-type photosynthetic phenotype by
transformation with a wild-type pXb1.8 construct, indicating that any
mutations responsible for the altered photosynthetic phenotype were
localized to the region of D1 contained in pXb1.8 (see Fig. 2).
Fourth, the phenotypes of independent isolates of transformants bearing
the same psbA mutation were similar (Table 1).
The dramatic
reduction in variable fluorescence in the mutants (Fig. 5) is
correlated with loss of donor-side function (Table 1); the
greater the reduction in variable fluorescence emitted by an algal
mutant, the greater the reduction in rates of oxygen evolution. In
situ monitoring of algal colonies for the kinetics of chlorophyll
fluorescence induction should provide a rapid and effective method, in
future experiments, for identifying mutants with specific defects in
donor-side function.
When
histidine or asparagine are substituted at residue 170 in D1, the
resulting algal mutants (D170H, D170N) are partially inhibited in
oxygen evolution and the kinetics of fluorescence relaxation are rather
more complex. We attribute this phenotype to a heterogeneous population
of PSII centers in these mutants, some with a functioning OEC and
others without. While absolute rates of oxygen evolution in the D170N
mutant were too low to allow a detailed investigation of
oxygen-evolving PSII centers, active centers in the D170H transformant,
which evolves oxygen at rates 30-60% those of wild-type cells,
were analyzed using a Joliot type oxygen electrode. Such analysis
revealed little perturbation in function of the OEC, with the S-states
cycling relatively normally (Fig. 9A). The S It is not yet clear whether aspartate 170 of the D1
polypeptide provides a ligand to manganese atoms in the mature
tetranuclear manganese cluster of the activated OEC, whether this
residue provides a ligand to manganese only during initial assembly of
the manganese cluster, or whether aspartate 170 never provides a direct
ligand to manganese, but rather has a critical effect on the
conformation of the manganese binding site. The fact that different
amino acid substitutions at residue 170 allow for wild-type OEC
function in cyanobacterial mutants (Nixon and Diner, 1992; Boerner et al., 1992) suggests that residue 170 may not provide a
ligand to manganese in the mature OEC. Relative differences in the
overall rates of oxygen evolution by mutants in vivo appear to
reflect differences in the relative effectiveness of the amino acid
residue at position 170 to provide an initial ligand to manganese. In
our algal mutants, histidine was more effective than asparagine (Table 1); in cyanobacteria, glutamate is more effective than
histidine (Nixon and Diner, 1992). X-ray and magnetic resonance
spectroscopy suggests that oxygen and some nitrogen atoms provide
ligands to the manganese cluster, although evidence for chloride has
been reported also (for reviews, see Diner et al.(1991) and
Debus(1992)). Analysis of the D170H mutants, using magnetic resonance
techniques to assay possible increased nitrogen ligation, may provide a
means of resolving the role of D1 residue 170 in the mature OEC.
Nevertheless, our results to date show that substitutions at D1 residue
170 affect the ability of chloroplast PSII centers to evolve oxygen and
clearly show that this residue is critical for assembly and/or
stability of the manganese cluster of the chloroplast OEC.
The 50% reduction in the level of PSII
observed when aspartate 170 is altered to proline suggests that, as
well as leading to a complete loss of oxygen evolution, the mutation
also may perturb the steady state balance between assembly and turnover
of the PSII reaction-center complex. The D1 polypeptide displays a
rapid, light-dependent turnover (Mattoo et al., 1984; Ohad et al., 1984) which has been the subject of intense study in
recent years. Current models suggest that D1 is periodically damaged,
is consequently removed from the PSII complex, and is replaced with a
newly synthesized D1 restoring activity to the complex (for reviews,
see Mattoo et al. (1989), Guenther and Melis(1990), and
Prásil et
al.(1992)). As damage is thought to result largely from the very
strong oxidants generated during normal function of the PSII complex,
attempts to improve photosynthetic efficiency are currently focused on
the D1 turnover process itself (Barber and Andersson, 1992). The use of
site-directed mutagenesis to manipulate chloroplast PSII electron
transfer will aid in dissecting the molecular events of in vivo PSII activation and photodamage and should provide important
insights as to how plants modulate photosynthetic function as light
levels change. and dark-adapted for 10 min.
The amplitudes of the relaxation curves were normalized to what they
would be if the cell concentrations were exactly 0.5 A
for all cell suspensions. The earliest time point for each
trace is at 50 µs following the actinic flash. A,
FWTsr
; B, D170H
; C,
D170N
; D, D170P
; E,
D170T
. The relative, normalized variable fluorescence (F - F
/F
. It is assumed this
electron comes from the tyrosine residue 161 (Y
) of D1,
resulting in formation of the radical species
Y
. In wild-type cells,
P
is reduced rapidly by an electron
from Y
which is rereduced in turn by the OEC, between
actinic flashes. In such cells, the high fluorescence state is
repeatedly observed 50 µs after each flash.
Q
is still dissipated by electron
transfer to Q
or by charge recombination with the donor
side over longer time scales, resulting in the characteristic
relaxation of the fluorescence yield of chlorophyll. However, in the
D170T and D170P mutants, the oxygen-evolving complex which normally
provides electrons for the reduction of Y
is apparently absent or unable to function, leading to formation
of
Y
P
PheoQ
after the second flash. P
is known
to quench chlorophyll fluorescence (Butler et al., 1973)
despite the presence of Q
. Hence, the
extent to which fluorescence quenching appears with each successive
actinic flash is a reflection of the inactivity of the OEC. The greater
the reduction in ability of a mutant to evolve oxygen, the more marked
is the fluorescence quenching, again consistent with a functional
defect in the tertiary electron donor (the OEC).
and the donor side. In this
assay, algal cells in suspension are treated with the inhibitor DCMU to
block oxidation of Q
by Q
(see Fig. 1). After a single saturating light flash in the
presence of DCMU, the rate of relaxation of the fluorescence yield of
chlorophyll is, to a first approximation (ignoring energy transfer
between PSII centers), the rate of reoxidation of
Q
by charge recombination with the
donor side. The rate of charge recombination depends on the
concentrations of Q
and
P
; the higher the concentration of
P
, the faster the charge
recombination (Nixon and Diner, 1992). Fig. 8shows the results
of such studies. In the ``wild-type'' control line
(FWTsr
), where the OEC is intact and the concentration of
P
is determined by the equilibrium
S
Y
P
⇔
S
Y
P
(refer to
Fig. 1legend), the fluorescence yield decays relatively slowly
in a biphasic manner. Charge recombination in the D170H transformant is
very similar to that seen in control cells. However, in the algal
transformants D170N, D170P, and D170T, the rate constant for decay of
the fast phase is approximately 3 to 4 times that observed for control
cells. These results, consistent with our observations that the D170N,
D170T, and D170P transformants are drastically reduced in their ability
to evolve oxygen (Table 1) and exhibit a fluorescence quenching
at 50 µs indicative of an increased concentration of
P
after two consecutive actinic
flashes (Fig. 7), further strengthen our conclusion that the
D170N, D170T, and D170P mutants are defective in PSII donor-side
function.
) and transformant (D170H
,
D170N
, D170P
, D170T
)
cell lines.
The cells were suspended in the same media and at the same cell
concentrations as in Fig. 7. All curves were normalized to the
same initial value at the first time point.
Oxygen-evolving PSII Complexes in the D170H Transformant
Are Relatively Unperturbed
Since the D170H transformants evolve
oxygen at 30-60% of the wild-type rate but accumulate wild-type
quantities of PSII reaction centers, it was important to determine
whether only some of these reaction centers were normally active or
whether all reaction centers were active but showed perturbed function
of the OEC. Use of the Joliot type oxygen electrode, in which a single
layer of dark-adapted cells is sedimented onto the electrode surface,
allows for the observation of oscillations in oxygen evolution produced
after each flash in a series of consecutive saturating flashes as the
active oxygen-evolving complexes cycle through the S-states in a
synchronized manner (Fig. 9A). In light-grown wild-type
cells, the majority of PSII centers relax to the S state
after a short dark incubation and thus the peak of oxygen evolution
occurs on the third flash as the S
state is advanced to the
S
state, resulting in release of oxygen and return to the
S
state (see Fig. 1legend). In D170H transformant
cells, peak oxygen evolution also occurred on the third flash. An
estimation of the half-life time of the S
state was made
based on measurements obtained using the Joliot electrode as described
under ``Experimental Procedures'' and Fig. 9B legend. The results of such studies (Fig. 9B) show
that the S
half-life time in the D170H mutant (10 s) is
roughly comparable to that of the control cells (14 s). Thus, the PSII
centers of D170H which are active in oxygen evolution appear to
function quite normally.
Site-directed Mutations at psbA Codon 170 Result in the PSII
Phenotype of Homoplasmic Transformant Cell Lines
The single
chloroplast of a C. reinhardtii cell contains 80-100
copies of the circular chloroplast DNA molecule (Harris, 1989), and
each DNA molecule contains two identical copies of the D1 gene, psbA, which is located in the inverted repeat (Erickson et
al., 1984). Integration of the transforming DNA into one or more
chloroplast DNA molecule(s) of the host cell via homologous
recombination (Newman et al., 1990) followed by segregation of
the newly formed recombinant chloroplast DNA molecules during colony
formation results in co-transformed cell lines which contain both the
selectable marker and the unselected mutant psbA genes.
Approximately 25% of our initial spectinomycin-resistant colonies were
co-transformants which contained mutant copies of psbA, and
all secondary colony isolates were homoplasmic for psbA (Fig. 3), regardless of whether a psbA deletion mutant
host or wild-type algal host was used. Although independent integration
events could account for the presence of two mutant psbA
copies per chloroplast DNA molecule in transformants, it is likely that
gene conversion and/or inter/intramolecular recombination maintains
homogeneity of gene copies within the chloroplast-inverted repeat
(Palmer, 1983; Erickson et al., 1984; Blowers et al.,
1989). Treatment of algal host cells with 5-fluoro-2`-deoxyuridine
(FdUrd) prior to bombardment reduces the number of copies of the
chloroplast DNA molecule and increases the number of transformants
recovered (Newman et al., 1990; Kindle et al., 1991).
However, FdUrd is known to be mutagenic (Wurtz et al., 1979).
For this reason, we do not treat cells with FdUrd and still are able to
recover drug resistance transformants at a frequency of
10
to 10
.
Finally, the fact that the PSII phenotypes of the D170T
(FuD7 host) and D170T
(wild-type host) cell lines
were indistinguishable with respect to oxygen evolution, herbicide
binding, and PSII quantitation ( Fig. 6and Table 1) as
well as fluorescence measurements (Erickson et al., 1992)
suggests that the nuclear genome of the host strain makes no
significant contribution to the differences seen in PSII function in
these mutants and strongly supports our conclusion that the alteration
observed in PSII phenotype of a mutant is due solely to the amino acid
substitution in the D1 polypeptide. We also demonstrated that the
presence of the spectinomycin resistance marker present in all
co-transformants had no effect on our assays of PSII function. Although
this result was expected since initial characterization of the C.
reinhardtii spectinomycin-resistant mutant spr-u-1-6-2, which contained the same 16 S rDNA mutation
we used as a selectable marker, showed the mutation did not
significantly alter photosynthesis or chloroplast protein synthesis
(Chua and Gillham, 1977; see also Boynton and Gillham(1993)), we
constructed and analyzed the ``control'' transformant algal
strains WTsr, FWTsr, and FWT (see ``Experimental
Procedures'').
Algal Transformants Accumulate Normal Levels of PSII
Reaction Centers, but Are Defective in PSII Donor-side
Function
It is known that the D1 polypeptide is essential for
assembly and/or stability of chloroplast PSII (Bennoun et al.,
1986), as is the D2 polypeptide (Erickson et al., 1986). In
cyanobacterial systems, introduction of a wide range of single amino
acid substitutions into the D1 or D2 polypeptides has resulted in the
depletion or complete loss of PSII centers (for reviews, see Debus
(1992), Nixon et al. (1992a), and Vermaas and Pakrasi (1992)).
It is thus apparent that even small alterations to the primary
structure of one of these two core PSII polypeptides can lead to
destabilization of PSII, limiting the usefulness of such mutants in
structure-function studies. Of particular interest for our studies was
the question of whether our site-directed algal mutants contained PSII.
Since all the transformants reported in this study assemble substantial
levels of mutant PSII centers, the mutant PSII phenotype is due not to
a lack of PSII reaction centers, but rather to a specific functional
defect in the PSII centers present in the mutants.A Role for D1 Aspartate Residue 170 in Assembly of a
Functional Chloroplast Oxygen-evolving Complex
The C.
reinhardtii mutants with amino acid substitutions at D1 residue
170 are partially or totally inhibited in oxygen evolution. Two types
of fluorescence relaxation measurements ( Fig. 7and Fig. 8) allowed us to further assess electron donation to the
PSII reaction center. Mutants in which D1 aspartate 170 is replaced by
threonine (D170T) or proline (D170P) evolve no oxygen and display a
progressively increased quenching of fluorescence yield observed at 50
µs following each in a series of consecutive saturating flashes (Fig. 7, D and E), as well as an accelerated
rate of charge-recombination between the acceptor and donor sides of
PSII (Fig. 8). Such results provide strong evidence for a loss
of function of the OEC which acts as the tertiary donor to PSII. The
catalytic site of the OEC is a cluster of 4 manganese ions which is
assembled in a multistep process (see Tamura and Cheniae(1987),
reviewed in Debus(1992)). Because light is required for oxidation of
the first Mn atom bound at a high affinity site, as
well as for oxidation of a second and subsequent ligated
Mn
, the term photoactivation has been applied to the
process of assembly of the manganese cluster (see MiyaoTokutomi and
Inoue(1992) for a recent discussion of photoactivation). Our results
imply an inability of mutants to assemble a functional OEC and suggest
that they may be defective in OEC manganese binding. In the green algal
mutant Scenedesmus LF1 (Metz et al., 1980, 1986;
Diner et al., 1988) and in cyanobacterial mutants with a
modified C terminus (Nixon et al., 1992b), all of which lack a
functional OEC but which contain aspartate at residue 170 of D1, the
fluorescence yield also drops off at each successive flash, but much
more gradually. This is presumably because Mn
bound
at aspartate 170 of D1 can provide electrons for the reduction of
Z
. Analysis of PSII core complexes depleted for
manganese and isolated from Synechocystis sp. PCC 6803 mutants
with amino acid substitutions at position 170 of D1 revealed an inverse
relationship between the K
for the oxidation of
exogenous Mn
and the ability of the mutants to
assemble active manganese clusters, providing further evidence for a
role of this residue in the binding of manganese necessary for assembly
of the oxygen-evolving complex (Nixon and Diner, 1992; Diner and Nixon,
1992). The apparent K
of
1 µM measured by these authors for the manganese oxidation site in
wild-type cells was similar to the value observed for the site of
photooxidation of the first manganese assembled during activation of
the PSII OEC in vitro (Blubaugh and Cheniae, 1990), leading to
the suggestion that aspartate 170 of D1 binds the manganese necessary
for the first stage in assembly of the tetranuclear cluster.
state decayed only slightly faster than in control cells, as
judged by measurements of oxygen flash yields (Fig. 9B)
and by the rate of charge recombination in the presence of DCMU (Fig. 8). Moreover, the fact that neither
S
Q
recombination (
Fig. 9B) nor
S
Q
recombination rates (Fig. 8) are greatly perturbed in the D170H transformant
compared to the control cells indicates that the D1 substitution in
D170H has no significant effect on the equilibrium constant for the
reaction Q
Q
⇔
Q
Q
and suggests that PSII
acceptor-side function is apparently unaltered in this mutant. Similar
results have been obtained using a cyanobacterial mutant containing a
histidine substitution at D1 residue 170 (Nixon and Diner, 1992). We
suggest that the replacement of aspartate by histidine and, possibly
asparagine, may lower the probability of successful completion of the
earliest stage in OEC assembly, without affecting the performance of
centers in which assembly of the manganese cluster is actually
completed.
What Happens to PSII Centers Lacking an Active
OEC?
Following exposure to low intensity light in
vitro, PSII membrane fragments which lack the manganese cluster of
the OEC accumulate pigment cations, and suffer subsequent damage to the
PSII reaction center (Blubaugh et al., 1991) in a process
known as low-light photoinhibition. Quenching of variable fluorescence
accompanies low-light photoinhibition and such quenching may persist
even after dark incubation due to the stable nature of some quenching
pigment cations and the inability of damaged reaction centers to
rapidly reduce the oxidized primary donor
P. In our flash train experiments (Fig. 7), the initial fluorescence levels reached at the first
flash are lower for all the Asp-170 mutants than for the control cells
and are particularly low for D170T and D170P. This depression in
initial fluorescence yield may result from a slowing of
P
reduction, and/or from
accumulation of long-lived quenching species following growth of the
mutants in low light.