From the
The product of the chloroplast psbI gene is associated
with the photosystem II reaction center. To gain insights into the
function of this polypeptide, we have disrupted its gene in
Chlamydomonas reinhardtii with an aadA expression
cassette that confers resistance to spectinomycin through biolistic
transformation. The transformants are still able to grow
photoautotrophically in dim light, but not in high light, and they
remain photosensitive when grown on acetate containing medium. The
amounts of photosystem II complex and oxygen evolving activity are both
reduced to 10-20% of wild-type levels in these
psbI-deficient mutants. It appears that the PsbI polypeptide
plays a role in the stability of photosystem II and possibly also in
modulating electron transport or energy transfer in this complex.
The photosystem II (PSII)
Whereas in higher plants the psbI gene is
cotranscribed with psbK, in C. reinhardtii these two
genes are far apart on the chloroplast genome. This observation
confirms that the relative arrangement of most chloroplast genes
differs considerably between C. reinhardtii and plants. Since
psbI has the same orientation as atpA located
upstream, these two genes could be cotranscribed and the polycistronic
transcript rapidly processed to give rise to the mature 0.3-kb psbI transcript. Alternatively, psbI could be transcribed from
its own promoter. The PsbI protein of C. reinhardtii contains
one additional amino acid relative to plants but displays otherwise a
high amino acid sequence identity with its plant homologue
(Fig. 1). The protein contains a hydrophobic stretch, which most
likely represents a transmembrane domain. Together with the PsbA, PsbD,
PsbE, and PsbF products, the PsbI protein is part of the PSII reaction
center
(3, 4, 5) . It has been shown that in
C. reinhardtii and cyanobacteria loss of any of the first four
subunits leads to the complete inactivation of the PSII complex
(1, 26, 27) . In contrast to these results, this
study has revealed that the PSII complex is only partially inactivated
in the psbI-deficient transformants of C.
reinhardtii. In these cells both accumulation of the PSII core
subunits and PSII activity are reduced to the same level, 10-20%
of the wild-type values. These observations suggest that the PsbI
product is involved, at least partially, in the stability of the PSII
complex. We cannot rule out, however, that this subunit plays in
addition also a role in modulating electron transport or energy
transfer in this complex.
A and B refer to two sets of experiments. In each case three
independent measurements of O2 evolution were performed under an
illumination of 100 µE m
We thank N. Roggli for drawings and photography, Dr.
M. Sugiura for the tobacco psbI probe, and M.
Goldschmidt-Clermont and K. Redding for helpful comments.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
complex within
the thylakoid membrane of chloroplasts catalyzes the light-driven
reduction of plastoquinone with electrons from water (for review see
Refs. 1, 2). Although this complex includes over 20 polypeptides, it
has been possible to isolate a reaction center complex capable of
performing in vitro the primary electron transfer reactions of
PSII
(3, 4, 5) . This minimal complex contains
only five polypeptides, namely D1, D2, the
and
subunits of
cytochrome b559, and the PsbI polypeptide, which are all
encoded by the chloroplast genome
(3, 4, 5) .
The D1 and D2 proteins bind all the redox components of PSII required
to transfer electrons from the manganese cluster of the water splitting
complex to the plastoquinone pool
(1, 2) . These include
P680, the primary electron donor, pheophytin, the primary electron
acceptor, and QA and QB, the first and second quinone acceptors. D1
also provides the electron donor to P680, Z, and together with D2 is
thought to bind the manganese cluster involved in water oxidation. The
function of cytochrome b559 is not clear although it has been
proposed that it may catalyze a cyclic electron flow around PSII that
serves to protect the PSII reaction center against photodamage
(6, 7) . The sequence of the 4.8-kDa PsbI polypeptide
has been determined in several plants and cyanobacteria and found to be
highly conserved (for review see Ref. 8). Cross-linking studies
indicate that the N terminal part of the psbI gene product is
in close contact with the D2 protein and the
subunit of
cytochrome b559 on the stromal side of the thylakoid membrane
(9) . However, the function of the PsbI polypeptide within the
PSII reaction center complex is not yet known. To obtain insight into
the role of this polypeptide, we have inactivated the psbI gene of Chlamydomonas reinhardtii through directed
chloroplast gene disruption via biolistic transformation, and we have
examined the properties of the psbI-deficient transformants.
Strains and Growth Conditions
C. reinhardtii wild-type strain 137c and the chloroplast PSII mutant FuD7 in
which the psbA genes have been deleted
(10) were used.
The PSII mutant X was isolated during this study. The growth media Tris
acetate-phosphate (TAP) and high salt minimal (HSM) were prepared as
described
(11) . The transformants were either grown on TAP
plates or in TAP liquid medium containing 100 and 25 µg/ml
spectinomycin, respectively.
Plasmid Constructions
The larger 2.6-kb
PstI- EcoRI fragment of the chloroplast DNA fragment
R7 of C. reinhardtii (12) was cloned in the Bluescribe
plasmid. The second NsiI site was removed after partial
digestion with NsiI, blunting, and religation giving rise to
the pBSR7pt2.6 plasmid (see Fig. 2). The plasmid pUC-atpX-aad
containing the atpA- aadA cassette
(13) was
cut with EcoRV and SmaI, and the excised cassette was
inserted into pBSR7pt2.6 after cutting with ScaI and
NsiI and blunting, thus removing the psbI gene
(Fig. 2). The orientation of the cassette was determined by
digestion with PstI
(13) . The plasmid with aadA in the same orientation as atpA was called pBS
R7pt2.6aad1 (Fig. 2). The psbD- aadA cassette
was excised from the cg12 plasmid
(25) with BamHI and
blunted, and ClaI (partial digestion) and inserted into
pBSR7pt2.6aad1 which had been cut with SpeI and blunted, and
with ClaI (partial digestion). Recombinant DNA plasmids for
transformation were prepared using standard methods
(14) .
Figure 2:
Map of the chloroplast genome region
containing psbI and strategy used for directed psbI disruption. The region partly covers EcoRI fragments R7
and R15. Transcription of psbI occurs in the same direction as
that of atpA. The site of insertion of the chloroplast
expression cassette aadA containing either the 5`- atpA or 5`- psbD region is indicated. Regions on the
chloroplast genome corresponding to the 5`- atpA and
3`- rbcL segments of the cassette are shown. Arrows indicate the direction of transcription. Restriction sites for
EcoRI ( R), PstI ( P), NsiI
( N), and SacI ( S) are marked. The
NsiI site in parenthesis was removed for the
replacement of the psbI gene with the aadA cassette
( cf. ``Experimental
Procedures'').
Chloroplast Transformation in C.
reinhardtii
Chloroplast transformation in C. reinhardtii wild-type cells was performed as described previously
(15) selecting for resistance to spectinomycin. Five
transformants named 7-2, 10-1, 10-2, 14-1, and
14-2 were characterized.
Isolation of Nucleic Acids
Total DNA and RNA were
isolated as described
(10) . The 0.24-9.5-kb RNA ladder
from Life Technologies, Inc. was used for determining the size of
transcripts.
Western Analysis
Antibody against the D1 protein
was a gift from L. McIntosh. Total cell proteins were separated by
electrophoresis, electroblotted on nitrocellulose membranes, reacted
with antibodies, and visualized by the ECL (enhanced chemiluminescence)
method (Amersham) as described by the manufacturer.
Measurement of PSII Activities
These measurements
were performed as described
(15, 16) . Fluorescence
transients and F/ F
values were determined with the Plant Efficiency Analyzer of
Hansatech Instruments.
Characterization of the psbI Gene of C.
reinhardtii
The chloroplast DNA region of C. reinhardtii comprising psbI was sequenced earlier
(17) and
the gene identified recently
(18) . We localized this gene
independently on the chloroplast EcoRI fragment R7 using a
psbI probe from tobacco for DNA hybridization (data not
shown). Comparison of the amino acid sequence of the PsbI polypeptide
of C. reinhardtii reveals significant sequence identities with
its homologue from higher plants (76%), cyanobacteria (65%), and
Euglena gracilis (57%) (Fig. 1). The C. reinhardtii PsbI protein contains 37 amino acids, 1 residue more than in
plants. A highly conserved segment of 21 predominantly hydrophobic
amino acids could represent a transmembrane domain. Another conserved
feature is the presence of several charged residues near the carboxyl
terminus.
Directed Deletion of the psbI Gene
The physical
map of the chloroplast DNA region of C. reinhardtii comprising
psbI is shown in Fig. 2. A plasmid containing the 2.6-kb
PstI- EcoRI fragment was digested with ScaI
and NsiI to remove the psbI gene. The deleted region
was replaced with the aadA expression cassette conferring
resistance to spectinomycin in which the coding region of aadA is flanked by a chloroplast promoter and 5`-untranslated region
(from either atpA or psbD) and the 3` downstream
region of rbcL (Fig. 2; 13, 25). The constructs were
introduced into the chloroplast genome through biolistic transformation
selecting for resistance to spectinomycin. Analysis of transformants
obtained with the cassette containing the atpA 5` region in
the same orientation as atpA revealed a 2-kb deletion
comprising the atpA gene (data not shown) presumably due to
recombination between the two 5` atpA regions located 1 kb
apart (Fig. 2). Since insertion of this cassette in the opposite
orientation could have resulted in a deletion between the two
3`- rbcL regions, we used a cassette containing the psbD promoter and 5`-untranslated region and chose transformants with
the orientation shown in Fig. 2. The DNA from five transformants
was isolated, digested with EcoRI, and hybridized with an R7
probe (Fig. 3). It can be seen that while the probe hybridizes as
expected to the 3.6-kb R7 fragment in wild-type and in the photosystem
II mutant Fud7 ( lane F), it hybridizes to a 5.1-kb fragment in
all five transformants examined as predicted from the plasmid used for
transformation (Fig. 2). The aadA probe hybridizes to
the same fragment of the transformants, but not to the DNA of
untransformed strains (Fig. 3). Since the chloroplast genome is
polyploid we tested the transformants for homoplasmicity. No signal
corresponding to the wild-type R7 fragment was detectable in the
transformants under conditions where a single copy of fragment
R7/chloroplast was detected (data not shown).
Figure 3:
DNA analysis of the
psbI-defective transformants. 5 µg of DNA from wild-type
( WT), Fud7 ( F), and five transformants (7-2,
10-1, 10-2, 14-1, 14-2, corresponding to
lanes 1-5) was digested with EcoRI, separated
on 0.8% agarose gels and blotted. The blots were hybridized with the
EcoRI fragment R7 ( upper) and an aadA probe
( lower). Open and dark wedges correspond to
fragments of 5.1 and 3.6 kb, respectively.
Hybridization of a
psbI probe to total RNA from wild-type and the Fud7 mutant
revealed a O.3-kb transcript that was absent as expected in all
transformants examined (Fig. 4).
Loss of psbI Leads to Reduced Photosystem II Activity and
Reduced Accumulation of the PSII Complex
PSII activity of the
transformants was determined by measuring oxygen evolution and
fluorescence transients. As shown in Table I, all transformants evolved
oxygen at 10-20% of wild-type levels. It can also be seen that
the ratio between variable fluorescence and maximum fluorescence,
F/ F
, is
significantly reduced in the transformants. To test whether the
diminished PSII activity of the transformants was due to reduced
accumulation of active PSII complex, cell proteins were fractionated by
polyacrylamide gel electrophoresis, blotted, and reacted with an
antibody against the D1 reaction center protein. Fig. 5 reveals that
the amount of D1 protein accumulated in the transformants ranges
between 10 and 20% as compared to wild-type. As expected the D1 protein
is completely absent in the Fud7 mutant in which the psbA genes have been deleted
(10) . It has been shown previously
that the amount of D1 protein provides a measure of the amounts of the
other PSII core subunits and that in the absence of D1 the PSII complex
is unstable
(10, 26) . In contrast, the PsaF protein
from PSI accumulates to the same level as in wild-type (Fig. 5)
indicating that absence of the PsbI product affects specifically the
accumulation of PSII subunits.
Figure 5:
Immunoblot analysis. Total cell proteins
were separated by polyacrylamide gel electrophoresis and blotted onto
nitrocellulose filters. The blot was incubated with antibodies raised
against D1 ( PsbA protein) and PsaF protein from PSI. The lanes
are labeled as in Fig. 3. Dilutions of wild-type proteins (1, 2, 5, 10,
20, and 30%) were included for quantitation of the blots.
Loss of the PsbI Polypeptide Leads to Increased
Photosensitivity
Wild-type and the five transformants were grown
on TAP (acetate) or HSM (minimal) plates and incubated at increasing
light intensities (20, 60, 600 µE ms
). As shown in Fig. 6, the transformants grew
under low light on HSM plates. Growth of these cells was markedly
diminished at 60 µE m
s
and
completely blocked at 600 µE m
s
on both HSM and TAP medium. As expected Fud7 and another PSII
deficient strain, X, did not grow on HSM medium.
Figure 1:
Sequence comparison of the PsbI
polypeptide. a, C. reinhardtii; b, barley
(19); c, rice (20); d, tobacco (21); e,
liverwort (22); f, Synechoccus sp. PCC6301 (23); g,
douglas-fir (C. H. Tsai and S. H. Strauss, EMBL/Genbank/DDBJ data
banks); h, E. gracilis. *, identical
residues; dots indicate residues identical in C.
reinhardtii and plants.
The psbI-deficient transformants
are still able to grow in minimal medium under low light. In contrast,
psbK-deficient mutants of C. reinhardtii are unable
to do so, and they accumulate less than 10% of PSII complex
(16) . Since it has been shown that in cyanobacteria loss of
psbK does not affect phototrophic growth and PSII function
(28) it is likely that in C. reinhardtii the PsbK
protein is also not involved in the photochemistry of PSII and that
this subunit, as the PsbI product, plays a role in PSII stability.
These observations suggest that the threshold level of PSII in C.
reinhardtii for growth on minimal medium is around 10% of
wild-type level. The PsbI subunit also appears to play some role in
light sensitivity, as the psbI-deficient transformants are
significantly more sensitive to high light than wild-type when grown
photoautotrophically or mixotrophically on acetate medium. Absence of
another small hydrophobic protein associated with PSII, the Ycf8
product, also leads to impaired cell growth in high light
(29) .
Like the Ycf8 protein, the PsbI product could be required for
maintaining normal PSII activity under stressfull high light
conditions.
Table:
Photosynthetic activity of the psbI::aadA
mutants
s
.
At least three independent measurements of fluorescence transients were
performed.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.