Requirement for the H Phosphoprotein in Photosystem II of ... - NCBI

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and green algae requires most of the subunits of this com- plex. This has been demonstrated ...... Shochat S, Owens GC, Hubert P, Ohad I (1982) The dichloro-.

Plant Physiol. (1 997) 113: 1359-1 368

Requirement for the H Phosphoprotein in Photosystem I I of Chlamydomonas reinhardtii’ Elizabeth J. Summer, Volkmar H.R. Schmid, Brigitte U. Bruns, and Cregory W. Schmidt* Botany Department, University of Georgia, Athens, Georgia 30606-7271 sociation of light-harvesting complexes from PSII under conditions when excitation of PSI is limiting and the Cyt b,/f complexes are in a reduced state (Bennett, 1991). At least four subunits of PSII are also usually phosphorylated in a light-dependent manner (Bennett, 1991). These proteins include D1 and D2 of the reaction center, PSII-C (the core antenna chlorophyll-protein complex, CP43), and a 9-kD protein, respectively, encoded by the chloroplast psbA, psbD, psbC, and psbH genes. Phosphorylation of D1 has not been detected in C. reinhardtii or bryophytes (Ikeuchi et al., 1987a; Rintamaki et al., 199513). It is not clear whether phosphorylation of PSII core proteins corresponds to part of the mechanism for D1 turnover, initiation of state transitions, inactivation of PSII, or a combination of such events. There are similar sequence motifs for Thr phosphorylation of the light-harvesting proteins and PSII proteins (Mullet, 1983; Michel and Bennett, 1987; Dedner et al., 1988; Michel et al., 1988), but the identity of the enzyme(s) responsible for these modifications has not been established (Hind et al., 1995; Race et al., 1995; Sokolenko et al., 1995). For cyanobacterial PSII, only the PSII-H polypeptide has been reported to undergo phosphorylation (Race and Gounaris, 1993), even though there is no corresponding consensus sequence at the N termini of the proteins from these species (Abdel-Mawgoodand Dilley, 1990; Mayes and Barber, 1991). Nonetheless, universal phosphorylation of the PSII-H polypeptide would seem to reflect a fundamental physiological process: PSII-H was among the first chloroplast proteins shown to be phosphorylated and to such a high degree that it seems to undergo continua1 phosphorylation/ dephosphorylation in the light (Bennett, 1977). As a step toward further resolution of the requirements for small proteins in PSII biogenesis and function, we have examined the role of the PSII-H polypeptide in C. reinhardtii. Here, we show that deletion of the psbH gene leads to loss of proteins of the PSII core complex, but that turnover of these subunits is quite slow relative to that resulting from mutations affecting other subunits of the complex. We also demonstrate that formation of PSII complexes is impaired in the absence of PSII-H. This is in contrast to the situation in cyanobacteria, in which elimination of PSII-H does not affect assembly and stability of PSII, even though there is partia1 loss of electron transfer efficiency from the

To dissect the expression of the psbB gene cluster of the Chlamydomonas reinhardtii chloroplast genome and to assess the role of the photosystem II H-phosphoprotein (PSII-H) i n the biogenesis and/or stabilization of PSII, an aadA gene cassette conferring spectinomycin resistance was employed for mutagenesis. Disruption of the gene cluster has no effect on the abundance of transcripts of the upstream psbB/T locus. Likewise, interruption of psbB/T and psbH with a strong transcriptional terminator from the r b d gene does not influence transcript accumulation. Thus, psbB/T and psbH may be independently transcribed, and the latter gene seems to have its own promoter i n C.reinhardtii. In the absence of PSII-H, translation and thylakoid insertion of chloroplast PSll core proteins i s unaffected, but PSll proteins do not accumulate. Because the deletion mutant also exhibits PSll deficiency when dark-grown, the effect i s unrelated to photoinhibition. Turnover of proteins B and C of PSll and the polypeptides PSll protein A and PSll protein D i s faster than in wild-type cells but is much slower than that observed in other PSII-deficient mutants of C. reinhardtii, suggesting a peripheral location of PSII-H in PSII. The role of PSII-H on PSll assembly was examined by sucrose gradient fractionation of pulse-labeled thylakoids; the accumulation o f high-molecular-weight forms of PSll is severely impaired i n the psbH deletion mutant. Thus, a primary role o f PSII-H may be t o facilitate PSll assembly/stability through dimerization. PSII-H phosphorylation, which possibly occurs at two sites, may also be germane t o its role in regulating PSll structure, stabilization, or activity.

The biogenesis and structural integrity of PSII in plants and green algae requires most of the subunits of this complex. This has been demonstrated most clearly in nuclear mutants of Chlamydomonas reinhardtii with lesions in regulation of gene expression, as well as mutations that disrupt chloroplast structural genes (Mayfield et al., 1995). Especially at excessive light intensities, maintenance of the PSII complex is thought to be dependent upon high rates of synthesis of the D1 polypeptide in a manner that is coordinated with its rapid turnover (Barber, 1995). Recently, it has been proposed that phosphorylation of certain subunits of the PSII complex participates in activation of the proteolytic pathway (Giardi et al., 1994; Rintamaki et al., 1995a).However, phosphorylation of thylakoid proteins is best documented to promote state transitions through disThis work was supported by the National Science Foundation and the Department of Energy. V.H.R.S. was supported by a fellowship from the Deutsche Forschungsgemeinschaft. * Corresponding author; e-mail [email protected]; fax 1-706-542-1805.

Abbreviations: ACA, aminocaproic acid; BAM, benzamidine; D1, PSII protein A; D2, PSII protein D; PSII-B, PSII-C, and PSII-H, PSII proteins B, C, and H; TAP, Tris-acetate phosphate. 1359

Summer et al.


Q, to QB quinone sites (Mayes et al., 1993). Our work

entailing disruption of the psbH gene by replacement of this sequence with that for a gene for antibiotic resistance also contributes to an understanding of the expression of an evolutionarily conserved cluster of chloroplast genes for severa1 PSII proteins. Evidence is provided for the expression of upstream psbB and psbT genes in a manner that is independent of psbH transcription. Thus, the formation of a psbB-psbT-psbH mRNA precursor is not requisite for the generation of the mature transcripts. Moreover, interruption of the psbB gene cluster with an antibiotic-resistance cassette, in either a forward or reverse transcriptional orientation, has no effect on the transcription of psbH. Thus in Cklamydomonas, it appears that this gene possesses its own promoter.


Cklamydomonas reinkardtii strain 137 (mating type +) was used for a11 experiments. Cells were grown in TAP or in minimal media in liquid or 1.3% agar. As required, plates and liquid media were supplemented with ampicillin (100 pg mL-') or spectinomycin (100 pg mL-'). DNA Constructs

Plasmid recombination into the psbB / T-N-H gene cluster of the C. reinkardtii chloroplast genome is illustrated in Figure 1.R19, a large ctDNA EcoRI fragment, was the source for 51-3, a EcoRIlPstI subclone in pBluescript SK(-). The 3.6-kb insert of 51-3 contains psbN, psbH, and part of the trnE coding region (Johnsonand Schmidt, 1993).To confer spectinomycin and streptomycin resistance to the constructs, the 1.9-kb aadA cassette was utilized (Goldschmidt-Clermont, 1991). The cassette, originating from pUC-atpX-AAD, contains aadA behind the promoter and a 5' untranslated sequence of atpA and is followed by the 3' termination sequence of the C. reinkardtii chloroplast rbcL gene. The




I 1 kb













1 1







- Figure 1. Mutation sites in the psbB/r, psbN, and psbH gene cluster. Drawn to scale are maps of the C. reinhardtiichloroplast genome and sites for insertional and deletion mutagenesis with the aadA cassette. The limits of the R19 EcoRl fragment and the 51-3 subfragment are also indicated. Also shown are restriction sites for Pstl (P), EcoRl (E), Hindlll (H), Bsm I ( B ) , Nhel (N),and Msc I (M).The bars at the bottom of the figure indicate the respective positions of subclones used for generating the probes for the northern analyses of psbB, psbN, and psbH in Figure 3.

Plant Physiol. Vol. 11 3 , 1997

cassette was excised with EcoRIl NotI, blunt-ended with the Klenow fragment, and cloned into the EcoRV site of pBluescript KS(-) to produce the plasmid pBSaadA. Plasmid p(aadA)iNH was constructed by ligating the SmaI/ EcoRVexcised aadA sequence into the blunt-ended BsmI site (position 2265) of 51-3. Plasmid p(aadA)AH contains the aadA cassette but lacks the entire psbH gene. To create p(aadA)AH, the 5' chloroplast sequences and the entire aadA cassette was excised from p(aadA)iNH by digestion with SmaI and partial digestion with HindIII, filling in the ends using the Klenow fragment and ligating into a NkeI/ EcoRI subclone of 51-3. Plasmid p(aadA)UN was created by ligating the aadA cassette into the HpaI site at position 1394 of 51-3, whereas p(aadA)iHEwas produced by insertion at the MscI site at position 3404 of 51-3. A11 plasmids were transformed by heat shock into XLIBlue Esckerickia coli competent cells and selected on Luria broth plates containing ampicillin and spectinomycin (100 pg mL-', each). Gene orientations were confirmed by restriction mapping and partial sequencing (Sequenase version 2.0, United States Biochemical). Plasmid DNA for cloning, sequencing, and transformation was isolated by standard procedures. Alga1 Transformation

Transformation of C. reinhardtii chloroplasts with a particle gun transformation system (Biolistic PDS-1000/ He, Bio-Rad) was essentially as described by Boynton and Gillham (1993).Five micrograms of plasmid DNA was ethanolprecipitated onto 12 mg of tungsten M-17 beads (Bio-Rad) and resuspended in 40 pL of 100% ethanol, and 5 pL was spread on carrier discs for each transformation event. Cells were grown to 106 cells mL-' in TAP plus 0.5 mM 5-fluorodeoxyuridine (Sigma) to reduce ctDNA levels, and resuspended to a density of 10' cells mL-'. Aliquots of 0.5 mL were added to 0.5 mL of molten 0.2% agar cooled to 42"C, poured in the middle of a TAP / ampicillin plate, and then placed in darkness at room temperature for 2 h. Following bombardment using 1100-psi rupture discs, the plates were incubated in darkness for 1h before the surface agar was loosened with 4 mL of minimal medium for replating on TAP plates containing 100 pg mL-' spectinomycin. After 2 weeks, colonies were restreaked on fresh antibiotic-containing plates and then individual colonies were selected for growth in liquid TAP-spectinomycin media for further analyses. When the loss of photosynthetic competence and high chlorophyll fluorescence became clear for heteroplasmic isolates of aadAAH mutants, selection for metronidazole resistance was used to isolate homoplasmic mutants (Schmidt et al., 1977). The plating/ selection protocol was repeated until homoplasmicity for the chloroplast gene disruption, as determined by Southern-blot analysis, was obtained. Nucleic Acid Purification and Analyses

DNA for Southern-blot analyses and PCR was isolated from alga1 cells by the protocol described in Harris (1989), except the aqueous phase of phenol/ chloroform extracts

PSll Phosphoprotein H and PSll Biogenesis

was adjusted to 0.7 M NaCl before the addition of 0.1 volume of 10% cetyltrimethyl ammonium bromide in 0.7 M NaC1. After extraction of the solution with 1 volume of chloroform, the DNA was isopropanol-precipitated, resuspended in H,O, and used for Southern-blot analysis. DNA for use in PCR reactions was further purified using DNA purification resin (Wizard Maxipreps, Promega) following the manufacturer’s recommendation for plasmid DNA. RNA was purified by lysis of harvested cells with SDS in the presence of proteinase K, followed by chloroform/ phenol extraction and then isopropanol, ethanol, and lithium chloride precipitation, as described previously (Johnson and Schmidt, 1993). The RNA (5 pg per lane) was separated on denaturing formaldehyde-containing agarose gels. Northern blotting to nylon membranes and hybridization with probes of the indicated chloroplast gene sequences were performed as previously described (Johnson and Schmidt, 1993).Probes for northern and Southern analysis were made by nick translation (Promega) of plasmid DNA in the presence of [a-32P]ATPand used at 106 cpm mL-’ of hybridization buffer. The clones used for probes of psbA, psbD, and psbC transcripts have been described previously (Jensen et al., 1986). The clones used as probes of psbB/T, psbN, and psbH are indicated in Figure 1. Determination of the aadA Cassette lntegration Site

The presence of the aadA cassette in the proper chloroplast genomic fragment was demonstrated by Southernblot analysis. Five micrograms of total DNA was digested to completion with EcoRI, separated on 0.7% agarose gel, and transferred to a nylon membrane following standard protocols. The membrane was probed with nick-translated 51-3, stripped, and reprobed with pBSaadA. More precise determination of the insertion site of the aadA cassette was further accomplished by PCR amplification of the respective flanking sequences. PCR reactions were done following standard protocols. DNA (0.1 pg) was amplified in a reaction containing 1 mM of each primer, 3 mM MgCI,, buffer (Perkin-Elmer), 1 mM dNTP, and 1 pL of TAQ (Perkin-Elmer) for 40 cycles at denature, anneal, and extend temperatures of 92,40, and 72”C, and cycle times of 1, 2, and 2 min, respectively. To PCR amplify sequences encompassing the 3’ integration sites, the primer sequences were 5‘-GATGACGCCAACTACCTC, corresponding to positions 448-431 of aadA (as given in GenBank accession no. X02340), and 5’-CCCGCCACTGTCATC,corresponding to positions 2855-2841 of 51-3 (as given in GenBank accession no. L13303). The resulting products were 1.27 and 0.8 kb, respectively, for DNA from mutants generated from constructs p(aadA)iNH and p(aadA)AH. The 0.8-kb PCR product was sequenced directly using the above 51-3 primer and Sequenase (version 2.0, United States Biochemical). For the 5’ integration sites, a primer with the sequence 5’-CAGTTGGAAGAATTTGTCCof aadA (positions 1131-1148 of GenBank accession no. X02340) and 5’CTGCCTAGGCAAGTAAAC of 51-3 (positions 1100-1118 of GenBank accession no. L13303) was used. DNA from both mutants yielded 1.7-kb products, which were then cloned into pBluescript SK(-) and digested with EcoRI and


SmaI by filling in the ends with the Klenow fragment. Resulting clones were sequenced with T3 primer. Fluorescence Measurements, Oxygen Evolution, and Growth Curves

Fluorescence was measured at 27°C with cells in liquid cultures using a pulse-amplitude-modulationfluorometer (PAM 101, 103, Walz, Effeltrich, Germany). Cells were first dark-adapted for 5 min. Initial fluorescence was measured with weak (

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