From the Departments of Molecular Biology and Plant
Biology, University of Geneva, 30 quai Ernest-Ansermet, CH1211 Geneva
4, Switzerland, ¶ Commissariat à l'Energie
Atomique/Cadarache, Département d'Ecophysiologic
Végétale et de Microbiologie, Laboratoire d'Ecophysiologie
de la Photosynthèse, Bâtiment 161, F-13108 Saint
Paul-lez-Durance, France, and
Department of Biochemistry and
Molecular Biology, Pennsylvania State University, University Park,
Pennsylvania 16802
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ABSTRACT |
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Certain Chlamydomonas reinhardtii
mutants deficient in photosystem I due to defects in psaA
mRNA maturation have been reported to be capable of CO2
fixation, H2 photoevolution, and photoautotrophic growth
(Greenbaum, E., Lee, J. W., Tevault, C. V., Blankinship, S. L., and Mets, L. J. (1995) Nature 376, 438-441 and Lee, J. W., Tevault, C. V., Owens, T. G.;
Greenbaum, E. (1996) Science 273, 364-367). We have
generated deletions of photosystem I core subunits in both wild type
and these mutant strains and have analyzed their abilities to grow
photoautotrophically, to fix CO2, and to photoevolve
O2 or H2 (using mass spectrometry) as well as
their photosystem I content (using immunological and spectroscopic
analyses). We find no instance of a strain that can perform
photosynthesis in the absence of photosystem I. The F8 strain harbored
a small amount of photosystem I, and it could fix CO2 and
grow slowly, but it lost these abilities after deletion of either
psaA or psaC; these activities could be
restored to the F8-psaA The proposal that two different light reactions are involved in
oxygenic photosynthesis arose from studies of an "enhancement effect" that was observed when two beams of different wavelengths were used to illuminate algae (3, 4). The discovery of cytochromes b and f led to the concept that they served as
intermediaries between the two photosystems, which operated in series
to achieve linear electron transfer from H2O to
NADP+ (5). The essence of this "Z-scheme" model is that
photosystem II (PS II)1
accomplishes the oxidation of water and the reduction of plastoquinone and cytochromes but is unable to reduce ferredoxin or
NADP+; photosystem I (PS I) is required for the reduction
of NADP+ and the oxidation of cytochrome f
through the mediation of the soluble protein plastocyanin. Although
other explanations have been proffered for the enhancement effect and
photosystem cooperativity (6), the Z-scheme is strongly supported by
experimental data (7) and is currently considered as the "central
dogma" of oxygenic photosynthesis (8, 9).
However, photosynthetic CO2 fixation and photoautotrophic
growth were recently reported (1, 2) in two PS I-deficient mutants, B4
and F8, both of which are nuclear mutants deficient in the
trans-splicing of the psaA mRNA (1, 10, 11).
Using a gas flow apparatus, Greenbaum et al. (1) were able
to measure an evolution of O2 coupled to CO2
fixation or to H2 evolution. The amount of CO2
fixed by these mutant strains was roughly equivalent to that measured
in wild-type (WT) cells. Although CO2 fixation in the PS
I-deficient strains initially appeared to be limited to anaerobic
conditions (1), photoautotrophic growth was later reported in the
presence of O2 (2). Several control experiments were
carried out by the authors to eliminate the possibility of trace
amounts of PS I. Irradiation with far red light ( The core of PS I is made up of the two largest subunits, PsaA and PsaB.
They bind all of the cofactors involved in intra-PS I electron
transport with the exception of the terminal iron-sulfur clusters,
which are bound by the extrinsic PsaC subunit. Methods to delete the
chloroplast genes psaA, psaB, and
psaC, have been described recently (12). Such mutants were
incapable of CO2 fixation or photoautotrophic growth, but
reintroduction of the deleted gene restored photoautotrophic growth
(13-15). Two hypotheses can be formulated to explain the discrepancy
between the results with deletion mutants and trans-splicing
mutants. On the one hand, the B4 and F8 strains could have harbored
undetected amounts of PS I that allowed synthesis of enough NADPH to
fix CO2 and grow photoautotrophically. On the other hand,
the B4 and F8 strains could have possessed an as yet unidentified
system that allowed PS I-independent reduction of NADP+. We
have undertaken an analysis of several different PS I-deficient mutants
(see Fig. 1) to examine critically the claim that PS I-deficient mutants can perform photosynthesis. We have used immunoblots to detect
PS I subunits as well as spectroscopic means to measure photooxidizable
P700, the primary electron donor of PS I (for reviews on PS
I, see Refs. 16 and 17). We have made use of real time mass
spectroscopy to measure the rates of O2 photoevolution, CO2 fixation, and H2 photoevolution. Our
results indicate very clearly that photosynthesis requires the presence
of active PS I.
Strains and Genetic Methods--
Our WT strain was isolated as
an mt+ segregant from a cross between two derivatives of
the 137c strain (18). The original deletions of psaA and
psaC were made in this strain (12). Isolates of the F8
strain (10) were kindly provided by Dr. E. Greenbaum (Oak Ridge
National Laboratory) and from the Chlamydomonas Stock Center (Duke
University). The FUD26 strain (19, 20) was obtained from Dr. J. Girard
Bascou (Institut de Biologie Physico-Chimique) and Dr. L. Mets
(University of Chicago). The B4 strain (1) was kindly provided by Dr.
L. Mets.
Bioballistic chloroplast transformations were performed with plasmids
designed to delete the psaA and psaC genes, and
homoplasmicity of these deletions was assessed by polymerase chain
reactions (12). The aadA cassettes used to delete
psaA or psaC were flanked by direct repeats;
homologous recombination between them removes the aadA
cassette, leaving one repeat behind (12). After several subcloning
steps on medium without antibiotic, spectinomycin-sensitive isolates
were obtained; loss of aadA was confirmed by specific polymerase chain reactions (12). All experiments herein used the
aadA-less strains to eliminate any differences due to
chloroplastic expression of the aminoglycoside adenyltransferase
enzyme. The third exon of psaA was reintroduced by
bombarding psaA
During characterization of one of the psaA Growth Conditions, Fluorescence Induction, and Immunoblot
Analysis--
Tris acetate/phosphate medium (TAP) and HSM for
heterotrophic and photoautotrophic growth, respectively, were prepared
as described (18). Chlamydomonas reinhardtii cultures were
maintained at 25 °C. In general, 100-ml TAP cultures were shaken at
180 rpm in 500-ml Erlenmeyer flasks under low illumination (~1 µmol
of photons m Spectroscopic Measurement of P700 Photooxidation and
Rereduction--
Cultures were harvested by centrifugation (8 min at
4000 rpm in a Sorvall GSA rotor at 4 °C) and washed with cold 50 mM Tris-HCl (pH 8.3). Aliquots corresponding to 7.5 × 107 cells and at least 100 µg of chlorophyll were
centrifuged, resuspended in 500 µl of 200 µg/ml Ficoll 400 in 50 mM Tris-HCl (pH 8.3), frozen on dry ice, and shipped from
Geneva to University Park. Control experiments demonstrated that
repeated freezing and thawing of Chlamydomonas cells in the
presence of Ficoll does not affect the photochemical activity of
P700 measured within 1 h after thawing.
The spectroscopic method is based upon techniques developed by
Harbinson and Woodward (24). The thawed sample suspensions, with the
addition of 10 mM sodium ascorbate and 4 µM
2,6-dichlorophenolindophenol, were placed in a 10 × 4-mm
quartz cuvette. The longer dimension of the cuvette was traversed by
the 832-nm probe beam from a 06GIC108 laser diode (Melles Griot,
Boulder, CO; 22-kHz sine-wave modulation; 20 milliwatts average power).
The beam was directed to a silicon photodiode detector shielded by a
red cut-off filter ( Mass Spectrometric Measurement of Gas Exchange--
Algal
cultures were harvested, and 1.5 ml of the suspension was placed in the
measuring chamber. Dissolved gases were directly introduced in the ion
source of the mass spectrometer (model MM 14-80, VG instruments,
Cheshire, United Kingdom) through a Teflon membrane. For O2
exchange measurements, the sample was sparged with N2 to
remove 16O2, and 18O2
(95% 18O isotope content; Euriso-Top; Saint Aubin, France)
was then introduced to reach an O2 concentration close to
the equilibrium with air. CO2 exchange measurements were
performed following the NaH13CO3 addition (0.3 mM final concentration; 99% 13C isotope
content; Euriso-Top) in the dark before recording CO2 exchange. Light was supplied by a fiber optic illuminator (Schott, Main, Germany), and neutral filters were used to vary light intensity. Unless specified, experiments shown here were performed at 300 µmol
of photons m
The hydrogenase enzyme is known to be inactivated by oxygen and to
require anaerobic conditions for its induction. Thus, for measurements
of H2 evolution, cultures were first subjected to anaerobiosis by sparging the sample with N2, closing the
chamber, and letting the algae consume O2 from the medium.
The O2 concentration was monitored by mass spectrometry
until it became undetectable (<0.2 µM), and the cultures
were incubated for a further 30 min. In companion experiments, glucose
(20 mM final concentration) and glucose oxidase (2 mg/ml
final concentration) were added to verify that O2 depletion
before illumination was complete and that hydrogenase induction and
activity were not affected by residual oxygen.
Wild Type--
To introduce the methods used in this work, we will
first describe characterization of the WT strain (see "Experimental
Procedures"), which can grow photoautotrophically as well as
heterotrophically on acetate-containing medium in the dark (Fig.
2A). Using specific antibodies against either PsaA (an
integral membrane core subunit) or PsaD (a stromal subunit involved in
docking ferredoxin to the acceptor side of PS I; Refs. 25 and 26), we
could easily visualize these polypeptides in membranes prepared from
the WT strain (Fig. 2A). The in vivo fluorescence
induction kinetics (Fig. 2B) are typical of normal algae
(27). The rise in fluorescence, which is correlated with reduced
QA in PS II, is due to the reduction of the plastoquinone
pool, while the subsequent drop has been interpreted as originating
from oxidation of the plastoquinone pool due to the combined action of
PS I and cytochrome b6f (28).
In order to measure PS I quantitatively, we monitored the kinetics and
extent of photooxidation and dark reduction of P700 by
following the increase of its absorbance at 832 nm (see "Experimental Procedures"). Photooxidation of P700 results in a broad
absorbance in the near-IR due to the loss of the ground state character
in oxidized chlorophyll and is monitored as an increase in absorbance at 832 nm. Optimization experiments indicated that the maximum amount
of P700+ could be observed within 1 s of
illumination with far-red light in the presence of sodium ascorbate and
2,6-dichlorophenolindophenol. The near-IR measuring beam is not able to
photooxidize P700 by itself, thus eliminating a possibility
of underestimating the amount of photochemically active
P700. The maximum level of P700 oxidation is
attained using far-red excitation, since this preferentially excites PS
I, thereby decreasing the rate of P700+
reduction in the light. In WT cells, P700 is oxidized
within 200 ms of illumination and is completely rereduced within 2 s after termination of excitation (Fig. 3).
We used continuous mass spectrometry to determine the in
vivo rates of O2 evolution, CO2 fixation,
and H2 photoevolution. We were able to distinguish
photosynthetic from respiratory activities by the inclusion of
[18O]O2 and
[13C]CO2 (25). The disappearance of
[13C]CO2 starting within 10 s after the
commencement of illumination is indicative of the activation of the
Calvin cycle enzymes, which then cooperate to convert gaseous
CO2 to carbohydrate (30). The WT cells removed
CO2 from the medium in a light-dependent manner
at a steady-state rate of 1.5 µmol min psaA
We found that the psaA and psaC deletion mutants
could not grow photoautotrophically and gave fluorescence induction
patterns typical of mutants lacking PS I (Fig.
2; results were identical for
psaB
We have previously shown that the psaB FUD26--
The FUD26 mutant was shown to be PS I-deficient due to
a 4-base pair deletion in the psaB gene, causing a
frameshift and truncation of the last 10 kDa of the PsaB polypeptide
(Ref. 19; see Fig. 1). Thus, this mutant should be incapable of
synthesizing active PS I. However, we found that the FUD26 isolate
provided to us was able to grow photoautotrophically (Fig.
2A) and had a fluorescence induction pattern consistent with
significant reoxidation of the plastoquinone pool (data not shown). We
detected the PsaA and PsaD polypeptides in significant amounts in
membranes from this strain (Fig. 2A). Moreover, we could
detect photooxidizable P700 at a level approximately 60%
that of WT (Fig. 3). This isolate was able to fix CO2 and
photoevolve H2 at rates 67 and 77% of WT, respectively
(Table I).
We were surprised by the presence of PS I in this strain, considering
the presence of a frameshift in the psaB gene. An isolate of
FUD26 obtained from Dr. Girard-Bascou showed no growth on minimal medium, similar to the original description of this mutant (20). We
hypothesize that the mutant has undergone some type of mutation event
that allows it to synthesize some full-length PsaB. We note that
Greenbaum et al. (35) estimated during their recent
experiments that this mutant had levels of PS I equivalent to 11-14%
of WT; it would thus appear that this mutant can revert to various
levels of PsaB expression. These results highlight the danger of using mutants in which the genetic information specifying the protein of
interest has not been completely obliterated.
F8--
The F8 mutant has a nuclear mutation that prevents
trans-splicing of the psaA mRNA (Refs. 10 and
11; see Fig. 1). The F8 strain was able to grow slowly on minimal
medium (Fig. 2A) and exhibited a fluorescence induction
pattern characteristic of mutants containing small amounts of PS I
(Fig. 2B; Refs. 15, 36). Membranes from this strain
contained small but detectable amounts of the PsaA and PsaD subunits.
Using densitometry, we estimated that these polypeptides were present
at roughly 10% of the WT amount when normalized to total membrane
protein (note that PsaA and PsaD were present at the same stoichiometry
as in the WT). Photooxidizable P700 in this strain
represented roughly 8% of the WT level (Fig. 3; Table I), indicating
that a small amount of active PS I was present in this strain. We
observed light-dependent CO2 fixation at
roughly 20% of the WT rate (Fig. 4; Table I) and H2
photoproduction at roughly 10% the WT rate (Fig. 5; Table I). An
isolate of F8 from the Chlamydomonas Stock Center was similar to the
one shown here in that it contained small amounts of PsaA and grew
slowly on minimal medium (data not shown); thus, this phenotype might
be a general phenomenon and not due to an isolated reversion event.
F8: Deletion and Reintroduction of psaA and Deletion of
psaC--
The F8 mutant provided us the opportunity to distinguish
between the two hypotheses posed earlier by use of a genetic test. The
third exon of psaA was deleted from this strain (see Fig. 1
and "Experimental Procedures"). If it contained an activity able to
photoreduce ferredoxin or NADP+ in the absence of PS I,
then ablation of psaA should have little effect. However,
after deletion of psaA, the F8 strain lost the ability to
grow photoautotrophically and displayed a fluorescence induction curve
typical of a PS I-deficient mutant (Fig. 2). The small amount of PsaA
and PsaD detectable in F8 disappeared after deletion of psaA
(Fig. 2A), and photooxidizable P700 signals were no longer visible in the F8-psaA
We considered the possibility that transformation might cause an
unforeseen effect, besides the loss of PS I, that could explain the
lack of photosynthesis in the F8-psaA
Another unlikely possibility was that the DNA used to replace
psaA was somehow affecting the expression of other gene(s)
required for the hypothetical "PS I-independent photosynthesis"
process. However, we have also deleted the gene for PsaC in F8. These
genes are distant from each other in the chloroplast genome (18); additionally, the piece of DNA used to delete psaC was
different from that used to delete psaA and psaB
(12). As in our WT strain, the loss of PsaC in the F8 strain caused a
loss of photoautotrophic growth (Fig. 2A). We could no
longer detect PS I in the F8-psaC B4: Deletion of psaA and Reversion--
The B4 mutant is also
incapable of psaA mRNA trans-splicing (Ref.
1; see Fig. 1). The B4 mutant could not grow photoautotrophically (Fig.
2A) and had a fluorescence induction pattern typical of a PS
I-deficient mutant (Fig. 2B). We could not detect the PsaA or PsaD polypeptides in membranes (Fig. 2A), nor could we
observe photooxidizable P700 in whole cells of this mutant
(Fig. 3; Table I). Thus, we conclude that this strain has either
no PS I or very small amounts. Using mass spectrometry, we could not
detect light-dependent uptake of CO2 (Fig. 4)
or production of H2 (Fig. 5).
Since the B4 strain was incapable of CO2 fixation, we were
not surprised that deletion of psaA in this strain had no
effect. The B4-psaA
Given these facts, it is difficult to explain the reported fixation of
CO2 and photoevolution of H2 in the B4 strain
(1, 2). We hypothesize that the B4 mutant can phenotypically revert. We
isolated two photoautotrophic revertants from B4, and they both
expressed PsaA (13). Fortuitously, we managed to isolate a
PsaA-expressing photosynthetic revertant, B4-r2, from the transformant that gave rise to B4-psaA The Z-scheme of linear electron transport explains a large body of
data. Exceptions to the pathway of linear transport are well known and
include cyclic electron transport around PS I and cytochrome
b6f, injection of electrons into the
system by hydrogenase, the facultative usage of sulfide as an alternate
electron source in certain cyanobacteria (37-39), and the use of
oxygen as an alternate electron acceptor (13, 29). Be that as it may,
the thermodynamic underpinnings of the Z-scheme appear to rest on solid
ground. PS II is a strong oxidant (midpoint redox potential
(Em) of P680+ = +1100 mV)
that is capable of oxidizing water (Em = +820 mV)
but is a relatively poor reductant, able to reduce plastoquinone (Em = +100 mV) but not ferredoxin
(Em = However, the discovery that pheophytin (Em = The pioneering genetic and biophysical work of Levine and others (45,
46) in their analysis of various C. reinhardtii photosynthetic mutants provided the framework of the Z-scheme. Many
mutants specifically defective in PS I are available (e.g. Refs. 10 and 11). The psaA gene is split into 3 exons that are widely separated and located on different strands of the C. reinhardtii chloroplast genome (47). These exons are separately transcribed and then joined together in a process known as
"trans-splicing" (48), which requires many of the genes
necessary for PS I biosynthesis (11). The B4 and F8 mutants are
defective in the splicing of the first and second exons (1, 10, 11). In
general, PS I-deficient mutants have been reported to be
nonphotoautotrophic (18), but it was possible that this phenotype was
due to pleiotropic mutations that caused the coordinate but unrelated
loss of PS I and photosynthetic activity. A reverse genetics approach
would eliminate this possibility, as one could eliminate a gene for a
specific PS I subunit without any expectation as to the phenotypic consequences. The psaA, psaB, and psaC
genes have been deleted from the chloroplast genome in C. reinhardtii (12, 49), and these deletion mutants were
nonphotosynthetic. PS I subunit genes have also been deleted in two
cyanobacterial species with attendant loss of photoautotrophic growth
(50, 51). Thus, the "forward" and "reverse" genetic approaches
agree that PS I is required for photoautotrophic growth.
We have reported previously that the F8 mutant ceased to grow
photoautotrophically after deletion of psaA exon 3 and that the B4 mutant was nonphotoautotrophic (13). Here we extend that work.
By spectroscopic observation of P700 photooxidation, we demonstrated that active PS I exists in all strains capable of photoautotrophic growth. We have eliminated the unlikely possibility that the loss of photosynthesis in psaA deletion mutants was
due to unrelated genetic damage by reintroducing psaA into
them and observing the restoration of CO2 fixation.
Photoautotrophic growth and CO2 fixation were also lost
after deletion of the psaC gene. This indicates that the
presence or absence of these capabilities correlates only
with the presence of PS I and not with the specific method used to
eliminate it. In this regard, the lack of H2 evolution in
PS I deletion mutants is of special note. H2 photoevolution was undetectable in mutants with deletions of psaA,
psaB, or psaC, no matter what their genetic
background. While CO2 fixation requires both NADPH and ATP,
H2 production requires only reduced ferredoxin and is thus
a more sensitive test for reduction of ferredoxin in vivo.
The lack of H2 photoevolution in PS I-deficient strains is
therefore a strong indication that ferredoxin is not photoreduced in
the absence of PS I.
Using the sensitive technique of mass spectrometry combined with
isotopic labeling, we have shown that PS I deletion mutants are
incapable of fixing inorganic carbon. However, we found that all PS
I-deficient cells were capable of sustained water photooxidation, although the rate could vary between strains (Table I). We have previously observed such O2 photoevolution in mutants
lacking PS I (13, 29), and it can represent a rate approximately
5-10% of that seen in WT cells. This activity reflects the ability of the chloroplast to reoxidize the plastoquinone pool independently of
the cytochrome b6f complex and PS I. Since the respiration rate was seen to increase by an approximately
equivalent amount, molecular oxygen is likely to serve as the ultimate
electron acceptor.
Because the kinetics of electron transfer from the primary donor to the
terminal acceptor are faster in PS I than in PS II, PS I should be able
to handle the electron flow from a larger quantity of PS II (52). In
fact, it has been proposed that the physical separation of the
photosystems is required for efficient linear electron flow due to the
much faster trapping kinetics of PS I (53). Whether or not the small
amount of PS I in "leaky PS I mutants" could account for the
observed amounts of photoevolved H2 is still under
discussion (35, 52). Although our work here does not directly address
this issue, we observed rates of H2 photoevolution that
seem compatible with the amount of detectable PS I (Table I). Under
normal conditions, the rate-limiting step of linear electron transport
is at the level of the cytochrome b6f
complex (54), and not at PS I. Therefore, one would expect that in
mutants with low levels of PS I, the relative CO2 uptake rates should be somewhat higher than the relative amounts of PS I. While we cannot absolutely exclude the possibility that PS I-independent and PS I-dependent electron transfer pathways
from H2O to ferredoxin could exist side-by-side in mutants
with low amounts of PS I, we cannot explain why this hypothetical PS
I-independent electron flow would not occur in mutants lacking PS I. Boichenko (55) has recently measured the action spectrum of
H2 photoevolution in F8 cells and found that it is
remarkably similar to that of PS I and significantly different from the
action spectrum of O2 evolution, which resembles that of PS
II. Our data are complementary to that of Boichenko, and we conclude
likewise that the observed CO2 fixation and H2
photoevolution activities in the B4 and F8 strains are due to PS I.
We find that the in vivo capabilities of
photoautotrophic growth, CO2 fixation, and H2
evolution require the presence of active PS I. We have found no case in
which a mutant lacking detectable PS I could perform these functions.
Thus, we disagree fundamentally with the conclusions arrived at by
Greenbaum, Lee, and colleagues (1, 2). Furthermore, our results
indicate that one must be very careful in the choice of mutant to
study; mutants defective in expression can be suppressed and small
deletions in genes can be repaired, but large deletions of entire open
reading frames encoding key structural components cannot revert or
result in leaky phenotypes.
mutant by reintroduction of
psaA. We observed limited O2
photoevolution in mutants lacking photosystem I; use of
18O2 indicated that this O2
evolution is coupled to O2 uptake (i.e. respiration) rather than CO2 fixation or H2
evolution. We conclude that the reported instances of CO2
fixation, H2 photoevolution, and photoautotrophic growth of
photosystem I-deficient mutants result from the presence of
unrecognized photosystem I.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
> 700 nm), which
excites PS I but not PS II, produced a small amount of H2
evolution in WT cells but not in the B4 mutant (1). Photobleaching experiments also failed to detect P700 in thylakoid
membranes from these mutants (2).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
mutants with plasmid pKR137 (15), which
carries psaA exon 3 with the psbD-aadA-rbcL
cassette (21) inserted at the EcoRI site approximately 300 base pairs downstream of psaA exon 3.
deletion
mutants of B4 (B4-psaA
-2), we isolated a photoautotrophic
revertant able to grow on minimal medium. This clone, renamed B4-r2,
was subsequently found to express PsaA and was not homoplasmic for psaA
. Further subcloning of B4-psaA
-2 on
antibiotic medium produced clones homoplasmic for psaA
.
We attempted to isolate photoautotrophic revertants of
B4-psaA
-2 in three separate trials by spreading cells on
high salt minimal agar medium (HSM; 9 × 105 cells
cm
2) and illuminating them (30-60 µmol of photons
m
2 s
1) either aerobically or anaerobically.
For some of these experiments, we UV-irradiated the HSM plates (8 mJ
cm
2 of 260-nm radiation) immediately after plating the
cells and held them in the dark for 24-48 h before illumination in an
attempt to increase the reversion frequency. In total, 3.7 × 109 cells were tested (1.7 × 109 cells
without UV and 2.0 × 109 cells with UV treatment),
and no photoautotrophic revertants were isolated. However, B4 appears
to revert at a low rate, and we have succeeded in isolating only two
photoautotrophic revertants from it.
2 s
1). For growth tests, 12 µl of log phase cultures were spotted onto agar media. TAP plates
were kept under low light (<1 µmol of photons m
2
s
1). HSM plates were subjected to anaerobiosis using the
Biomérieux Generbag Anaer system (Marcy L'Etoile, France)
according to the manufacturer's instructions and were strongly
illuminated (300 µmol of photons m
2 s
1).
Fluorescence induction kinetics were measured using a laboratory-built video fluorometer (22, 23) on cultures grown upon TAP plates under low
light. Immunoblot analysis of total cellular membranes using the
anti-PsaA and anti-PsaD antibodies was performed as described
previously (15).
> 760 nm) and was measured at the time
constant of 3 ms. The actinic light from a 300-watt tungsten lamp was
filtered using a hot mirror and a red cut-off filter to provide a broad
band peaking at 715 nm with a full-width half-maximum of 40 nm and an
intensity of 60 µmol of photons m
2 s
1,
incident on the sample at a right angle to the probe beam. The actinic
light was controlled with a shutter (Vincent Associates, Rochester, NY;
2-ms full opening/closing time). The absorbance changes
(
A) were analyzed using IgorPro (WaveMetrics, Lake
Oswego, OR), and their amplitudes were calculated as
0.434*
I/I, where I is the intensity
of probe beam, and
I is the actinic light-induced change.
Due to variations in sample densities (in the range of 1.6-7 × 107 cells ml
1), the
A signals
were normalized to cell density.
2 s
1 incident light. All gas
exchange measurements were performed at 25 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
1 mg of
chlorophyll
1 when illuminated with 300 µmol of photons
m
2 s
1 (Fig. 4; Table
I). When the photon flux was reduced
100-fold, CO2 uptake occurred at 1.2% of this rate (Fig.
4). The O2 evolution rate was equivalent to this value
(Table I), as expected; for each molecule of O2 produced,
one molecule of CO2 is fixed. The respiration rate, as
measured by uptake of 18O2, was not greatly
affected by the light (Table I). Under anaerobic conditions, the
chloroplast enzyme hydrogenase is induced and can serve as an alternate
acceptor of electrons from ferredoxin (31-33). After removal of
O2, production of H2 in WT cells was observed
immediately upon illumination (Fig. 5).
Summary of characteristics of the strains under study
and psaC
--
In order to examine mutants that should
contain no PS I, we made use of mutants with deletions in the
chloroplast genes psaA, psaB, or psaC
(Ref. 12; see Fig. 1). These deletions
remove the entire coding sequence of psaA exon 3 (which
encodes the last 661 amino acid residues of the 751-residue PsaA
polypeptide), 96% of the psaB gene, or all of the
psaC gene. Thus, such deletion mutants should be incapable
of reversion.
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Fig. 1.
Graphic depiction of the strains used in this
study. Each oval represents one strain; the
upper circle is the nucleus, and the
lower shape is the chloroplast. Within the
chloroplast, the oval to the left is the
chloroplast genome, and the double oval at the
bottom represents the thylakoid membrane. WT, the
mature psaA mRNA is assembled from three separate
transcripts arising from the chloroplast genome. Translation of this
message gives rise to the PsaA polypeptide (represented by the
serpentine form), which forms half the core of PS
I. psaA , the third exon of psaA has been
deleted in this strain by chloroplast transformation. Thus, there will
be no PsaA polypeptide. FUD26, the chloroplastic
psaB gene has a four-base pair deletion (*) in the FUD26
mutant, which results in a frameshift and a truncated gene product.
B4, F8, these mutants each have a mutation (X) in
the nuclear genome that blocks trans-splicing of the
psaA mRNA. They have very little or no mature
psaA mRNA, and consequently very little or no PsaA
polypeptide. Gray forms and shadowed
letters symbolize this indeterminate quantity.
F8-psaA
, the third exon of psaA has been
deleted in F8 in the same way as the psaA
mutant.
, data not shown), in that the fluorescence rose to a high level and remained constant (34). Photoautotrophic growth can be
restored to these strains by reintroduction of the deleted gene (14,
15). We could detect no PsaA or PsaD polypeptides in membranes from the
psaA
strain (Fig. 2A). As has been observed previously
(12, 34), the psaC
mutant could accumulate some PsaA
polypeptide at roughly 5-10% the WT value (Fig. 2A).
However, membranes from this mutant contained no detectable PsaD
polypeptide, indicating that the stromal side of PS I is significantly
perturbed due to the lack of PsaC. No photooxidizable P700
could be detected in either mutant (Fig.
3; Table I).
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Fig. 2.
A, growth tests and immunoblot analysis
of the various mutants. The indicated cultures were grown to log phase
in acetate-containing medium under low illumination and then spotted
onto either acetate or minimal agar. The acetate plates were kept under
low light (<1 µmol of photons m 2 s
1),
while the minimal plates were subjected to anaerobic conditions and
illuminated (300 µmol of photons m
2 s
1).
Membrane proteins (75 µg/lane) prepared from the cultures were
immunoblotted using a combination of anti-PsaA and anti-PsaD
antibodies. The leftmost lane is a 5-fold
dilution of the WT extract into the psaA
extract (thus,
20% of WT). Two independent transformants each are shown of the
psaA and psaC deletion mutants of F8. B4-r2 is a
photoautotrophic derivative of B4. Note the presence of a
cross-reacting protein that is present in all extracts and that
migrates slightly faster than PsaA. B, in vivo
fluorescence induction kinetics. Log phase TAP cultures of the various
mutants were illuminated with blue light (75 µmol of photons
m
2 s
1), and the red chlorophyll
fluorescence was measured using a laboratory-built video fluorometer.
Strains shown are WT (
), psaA
(
), F8 (
),
F8-psaA
(
), F8-psaA
+psaA
(
), B4 (
), and B4-r2 (
).
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Fig. 3.
Spectroscopic detection of P700
in whole cells by absorbance changes at 832 nm. The curves show
the photooxidation and rereduction of P700 in whole cells
as actinic light is turned on (upward arrow) and
off (downward arrow). Each trace represents an
average of 32 light on/off cycles. The amplitudes have been normalized
to cell density.
mutant cannot fix
CO2 (13). The psaA
and psaC
mutants were also incapable of CO2 fixation (Fig.
4; Table I), and no significant
H2 photoevolution could be detected in the
psaA
mutant (Fig. 5; Table
I). Significant O2 photoevolution does occur in these
mutants (Table I); however, in this case, the electrons appear to be
directed toward respiration rather than CO2 fixation or
H2 evolution. Note that the light respiration rates in
these PS I-deficient mutants are essentially the sum of the dark
respiration and O2 evolution rates (Table I). Previous
experiments (13) with the psaB
mutant under varying light
showed that it could evolve O2 at a maximal rate about 4% that of the maximal WT rate but that this evolved O2 was
almost quantitatively matched by an increase in the respiration rate (i.e. there is no net O2 evolution).
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Fig. 4.
CO2 exchange measured during
dark/light transitions in different mutants using mass spectrometry and
13C-enriched CO2. The light (300 µmol of
photons m 2 s
1, unless otherwise indicated)
was turned on when denoted by the vertical dotted
line. Chlorophyll concentrations were 20 µg
ml
1. A, WT (
,
), psaA
(
), and psaC
(
) mutants. WT CO2
consumption in low light (3 µmol of photons m
2
s
1) was 1.2% of the consumption rate at high light
fluence (300 µmol of photons m
2 s
1),
which allowed saturating oxygen evolution in all mutants and about 75%
of the saturating oxygen evolution activity in WT. B,
CO2 exchange during dark/light transitions in the B4 (
),
F8 (
), F8-psaA
(
), and
F8-psaA
+psaA (
) strains.
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Fig. 5.
Photoproduction of H2 during
dark/light transitions measured by mass spectrometry. Cultures
were first subjected to anaerobic conditions in order to induce
hydrogenase. The light (300 µmol of photons m 2
s
1) was turned on when indicated by the arrow.
Chlorophyll concentrations were 20 µg ml
1.
A, WT (
) and psaA
(
) strains.
B, F8 (
), F8-psaA
(
), and
F8-psaA
+psaA (
) strains. C, B4
(
) and B4-r2 (
) strains.
strains (Fig. 3).
Along with the loss of PS I upon deletion of psaA, F8 also
lost the ability to fix CO2 (Fig. 4) and photoevolve
H2 (Fig. 5).
mutants. The
particle bombardment technique used to introduce DNA into the
chloroplast might also insert DNA into the nucleus, thus creating
random mutations. However, it seems unlikely that secondary nuclear
mutations could explain the loss of photosynthesis, since we have
examined several independent transformants. To further exclude this
possibility, we reintroduced psaA exon 3 into two
independently derived F8-psaA
strains and selected
transformants by virtue of a co-integrated antibiotic resistance gene
(see "Experimental Procedures"). We found that this restored to all
of the transformants (referred to as
"F8-psaA
+psaA") both the slow
photoautotrophic growth and the fluorescence induction curve seen
previously with the original F8 mutant (data not shown; Fig. 2 shows
results from one representative transformant from each of the two
F8-psaA
strains). The levels of PsaA and PsaD
polypeptides (Fig. 2A) as well as photooxidizable P700 (Fig. 3) were restored to their original low levels
after reintroduction of psaA. Moreover, the CO2
fixation and H2 evolution activities returned to the same
levels seen in F8 (Figs. 4 and 5; Table I).
transformants, either
immunologically (Fig. 2A) or spectroscopically (Fig. 3).
Additionally, neither CO2 fixation nor H2
photoevolution could be detected in the F8-psaC
derivatives (Table I). All of these results are entirely consistent
with the hypothesis that the photosynthetic activities occurring in F8
are dependent upon the small amount of PS I that it accumulates.
strain appeared identical to the
original B4 strain; it was unable to grow photoautotrophically (Fig.
2A), it had no PsaA polypeptide (Fig. 2A; note
that detection of some PsaD in this experiment is not reproducible and
may represent some stromal PsaD polypeptide contaminating the membrane
preparations), nor could it fix CO2 or photoevolve
H2 (Table I). The kinetics of the small photoinduced signal
at 832 nm are much faster in this mutant than in the other strains
displaying photoinduced P700 signals, indicating that this
signal is not related to P700.
before all copies of the
psaA gene had been removed (see "Experimental
Procedures"). The B4-r2 strain was essentially the same as WT; it
expressed PsaA at a high level (Fig. 2A), showed a normal
fluorescence induction pattern (Fig. 2B), contained
photooxidizable P700 (Fig. 3) at roughly 40% of the WT
level (Table I), and could fix CO2 and evolve
H2 at normal rates (Fig. 5; Table I). However, after the
deletion had become homoplasmic, giving rise to the
B4-psaA
strain, we could not obtain such photosynthetic
revertants (see "Experimental Procedures"). Taken together, it
would seem that the only way for B4 to revert to a photosynthetically
competent state is to express PsaA. We have performed the complementary
experiment by transformation with a construct that replaces
psaA exon 3 with a "prespliced" psaA gene,
thus bypassing the trans-splicing defect in B4. Introduction of this gene into either B4 or B4-psaA
confers
photoautotrophic growth to the
transformants.2 Thus,
photosynthesis in B4 also correlates with the presence of PS I.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
430 mV) or NADP+
(Em =
320 mV). PS I is a strong reductant, whose
iron-sulfur clusters (Em =
580 mV) are capable of
reducing ferredoxin but whose oxidized primary donor
(Em = +450 mV) is incapable of oxidizing water.
610
mV) is the primary electron acceptor within PS II (40, 41) made
feasible the idea that PS II could reduce ferredoxin or
NADP+. This coupled with the fact that PS II and PS I are
differentially localized to granal and stromal thylakoid membranes,
respectively (42), prompted the proposal that PS II in the granal
membrane could reduce ferredoxin, while PS I in the stromal membrane
was involved in cyclic transport for the generation of ATP. Albertsson et al. (43) observed reduction of NADP+ coupled
with H2O oxidation in vesicles enriched in PS II, and Arnon
and Barber (44) presented evidence that isolated PS II reaction centers
could reduce ferredoxin. The involvement of residual PS I in these
reactions was suggested by the fact that both required plastocyanin, a
well known PS I electron donor and that the first could be blocked by a
cytochrome b6f inhibitor. While PS
I-dependent reactions can be studied in the absence of PS
II due to the existence of specific inhibitors of PS II and the ability
to independently excite PS I using far-red illumination, the study of
PS II-dependent reactions in the absence of PS I poses more
difficulties. The use of genetic mutants provides a novel way to
overcome this problem.
CONCLUSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank N. Roggli and D. Hughes for assistance in preparation of figures. Our thinking has been stimulated by interesting discussions with A. W. Rutherford, E. Greenbaum, J. W. Lee, L. J. Mets, T. G. Owens, and A. Melis.
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FOOTNOTES |
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* This work was supported in part by Swiss National Fund Grant 3100-050885.97.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.
§ Recipient of a National Science Foundation Plant Biology Fellowship (Grant DBI-9740162). To whom correspondence should be addressed. Present address: Depts. of Chemistry and Biological Sciences, 120 Lloyd Hall, 6th Ave., The University of Alabama, Tuscaloosa, AL 35487-0336. Fax: 205-348-9104; E-mail: Kevin.Redding{at}mail.ua.edu.
** Recipient of National Science Foundation Grant MCB-972366.
2 C. Rivier, K. Redding, and J.-D. Rochaix, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: PS I and II, photosystem I and II, respectively; P700, primary donor of PS I; TAP, Tris acetate/phosphate medium; HSM, high salt minimal agar medium; WT, wild type (or wild-type).
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REFERENCES |
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