©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Mutant Strain of Chlamydomonas reinhardtii Lacking the Chloroplast Photosystem II psbI Gene Grows Photoautotrophically (*)

Pierre Künstner , Augustin Guardiola , Yuichiro Takahashi (1), Jean-David Rochaix

From the (1) Departments of Molecular and Plant Biology, University of Geneva, 30 quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland and the Faculty of Science, Department of Biology, Okayama University, Tsushima-naka, Okayama 700, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

The photosystem II (PSII)() 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.


EXPERIMENTAL PROCEDURES

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/ Fvalues were determined with the Plant Efficiency Analyzer of Hansatech Instruments.


RESULTS

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 msand completely blocked at 600 µE mson both HSM and TAP medium. As expected Fud7 and another PSII deficient strain, X, did not grow on HSM medium.


DISCUSSION

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.


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

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 ms. At least three independent measurements of fluorescence transients were performed.



FOOTNOTES

*
This work was supported by Grant 31.34014.92 from the Swiss National Fund and by a grant from the Human Frontier Science program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: PSII, photosystem II; HSM, high salt minimal; kb, kilobase(s).


ACKNOWLEDGEMENTS

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.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.