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First published online May 5, 2006; 10.1104/pp.106.078147 Plant Physiology 141:870-878 (2006) © 2006 American Society of Plant Biologists The Evolutionarily Conserved Tetratrico Peptide Repeat Protein Pale Yellow Green7 Is Required for Photosystem I Accumulation in Arabidopsis and Copurifies with the Complex1Institut für Allgemeine Botanik und Pflanzenphysiologie, Friedrich Schiller University, 07747 Jena, Germany
Pale yellow green7-1 (pyg7-1) is a photosystem I (PSI)-deficient Arabidopsis (Arabidopsis thaliana) mutant. PSI subunits are synthesized in the mutant, but do not assemble into a stable complex. In contrast, light-harvesting antenna proteins of both photosystems accumulate in the mutant. Deletion of Pyg7 results in severely reduced growth rates, alterations in leaf coloration, and plastid ultrastructure. Pyg7 was isolated by map-based cloning and encodes a tetratrico peptide repeat protein with homology to Ycf37 from Synechocystis. The protein is localized in the chloroplast associated with thylakoid membranes and copurifies with PSI. An independent pyg7 T-DNA insertion line, pyg7-2, exhibits the same phenotype. pyg7 gene expression is light regulated. Comparison of the roles of Ycf37 in cyanobacteria and Pyg7 in higher plants suggests that the ancient protein has altered its function during evolution. Whereas the cyanobacterial protein mediates more efficient PSI accumulation, the higher plant protein is absolutely required for complex assembly or maintenance.
Plants are of fundamental importance to maintain life on earth because they supply oxygen and energy during photosynthesis. The basic processes of light capturing are organized into two multiprotein complexes, PSI and PSII. Both complexes are present in prokaryotic and eukaryotic organisms and composed of approximately the same proteins and cofactors. Ultimately, light energy is converted into a proton gradient across the photosynthetic membrane, which gives rise to the synthesis of ATP, and drives electron flow through the thylakoid membrane. Among the two photosynthetic complexes, PSI is the most conserved multiprotein complex in all photosynthetic organisms (Pakrasi, 1995  ; Chitnis, 2001; Scheller et al., 2001). The molecular mechanism of PSI biogenesis is still enigmatic, although possible scenarios have been proposed in numerous reviews (Pakrasi, 1995; Schwabe and Kruip, 2000; Chitnis, 2001; Scheller et al., 2001). In recent years, several regulatory factors for PSI biosynthesis have been identified that are conserved in prokaryotic and eukaryotic organisms (Wilde et al., 1995, 2001; Bartsevich and Pakrasi, 1997; Boudreau et al., 1997; Ruf et al., 1997; Mann et al., 2000; Naver et al., 2001; Shen et al., 2002a, 2002b; Lezhneva et al., 2004; Stöckel and Oelmüller, 2004). This implies a strong conservation in the organization, biogenesis, structure, and dynamics of prokaryotic and eukaryotic PSI. The initial step in PSI biogenesis is the formation of the reaction center consisting of the heterodimer PsaA/B. The formation of this complex is thought to depend on proper and on-time delivery of all associated cofactors including iron, sulfur, heme, etc.
Although many structural and regulatory proteins for PSI are evolutionarily conserved and already present in cyanobacteria, a more detailed analysis uncovered that some of these compounds also differ in function. For instance, whereas PsaC stabilizes the reaction center of eukaryotic PSI and is absolutely required for its function, the cyanobacterial complex is also functional without PsaC (Takahashi et al., 1991
The recessive pyg7-1 mutant was generated by ethyl methanesulfonate mutagenesis. When grown in soil, homozygous mutant seedlings are lethal. After germination, they developed pyg cotyledons, but no primary leaves. The mutant can only be propagated heterotrophically. Even though grown on Suc-supplemented media, pyg7-1 plants reveal a significantly reduced growth rate and display yellowish leaf pigmentation. Furthermore, the leaves are thinner and almost transparent compared to the wild type (Fig. 1 ). Under UV light, the pyg7-1 mutant exhibits a high-chlorophyll (Chl) fluorescence phenotype. Chl content of the mutant grown under high-light conditions (100 µmol m–2 s–1) was decreased by 95% ± 1% and under low-light conditions (5 µE m–2 s–1) by 91% ± 1%, indicating a minor light-dependent photosensitivity of the mutant. Of the two pyg7 alleles (pyg7-1 and pyg7-2) available (see Fig. 1 and below), pyg7-1 was used for detailed analyses.
Wild-type and pyg7-1 plants grown under axenic conditions were analyzed for P700 absorbance changes, 77 K emission, and Chl a fluorescence. The mutant pyg7-1 did not show any detectable absorbance changes of P700 at 810 nm, indicating that PSI is not functional (data not shown). The 77 K fluorescence spectra were recorded to examine the distribution of excitation energy in the mutant pyg7-1 and the wild type. The emission band at 731.1 ± 0.5 nm in the wild type, characteristic for a functional PSI, was shifted to 727.8 ± 0.8 nm in the mutant (Fig. 2 ). This blue shift indicates that the amount of the various Lhca components relative to each other is changed. Western analyses revealed that the Lhca1, 2, and 4 proteins are only slightly reduced in the mutant, whereas Lhca3 is reduced by more than 80% (see Fig. 5B). These data are consistent with the idea that the impairment in pyg7-1 is likely to be caused by lesions in those PSI proteins that are directly involved in electron transfer. The smaller blue shift in pyg7-1 compared to other PSI-deficient mutants (Haldrup et al., 2000 ; Lezhneva et al., 2004; Stöckel and Oelmüller, 2004) might be caused by the severe reduction of Lhca3 antenna protein of PSI.
No significant shift in the emission bands at 682.4 ± 1.2 nm was observed, indicating that the residual amount of PSII is functional in the mutant.
No photochemical quenching (qP) could be measured in pyg7-1 (data not shown). The Chl a ratio of variable to maximal fluorescence parameter (Fv/Fm) describes the maximal efficiency of PSII photochemistry, which correlates with the number of functional reaction centers (Öquist et al., 1992 Lack of PSI in pyg7-1 is accompanied by changes in the plastid ultrastructure (Fig. 3 ). In low light, only a few lamellae and no assimilatory starch can be detected in pyg7-1. In high light, the membrane structure in the mutant plastids is less organized (data not shown).
Northern analysis of pyg7-1 plants using representative probes for nuclear and plastid-encoded PSI, PSII, and Cyt b6/f complex subunits, as well as the large subunit of Rubisco revealed that expression and transcript accumulation are not affected in the mutant (Fig. 4 ). Immunoblot analyses of thylakoid proteins uncovered that none of the PSI subunits (PsaA, PsaC, PsaD, PsaF, PsaL, and PsaH) were detectable in pyg7-1 (Fig. 5A) . This was independent of the light intensity in which the seedlings were grown (data not shown). The significant decrease in the level of D1, the reaction center protein of PSII, in the mutant may be caused by enhanced destabilization due to increased photoinhibition in pyg7-1 plants in higher light intensities. Subunits PsbS and PsbO of PSII, subunit IV, and Cyt b6 of the Cyt b6/f complex, as well as AtpB of ATP synthase, accumulate in comparable amounts in wild type and pyg7-1 (Fig. 5A).
In organello labeling experiments of wild-type and pyg7-1 chloroplast proteins with 35S-Met (Fig. 6 ) demonstrate that all major thylakoid proteins are synthesized in the mutant. This indicates that accelerated degradation, rather than a block in protein synthesis, is responsible for the absence of PSI reaction center polypeptides in pyg7-1.
Although PsaA and PsbA are synthesized in the mutant (Fig. 6), PsaA (and other PSI subunits) were never detectable in western studies. In contrast, PsbA accumulates in a light intensity-dependent manner. Thus, the decrease in the steady-state PsbA protein level under high light in pyg7-1 may be caused by photoinhibition. Because the Fv/Fm values increase in the mutant during recovery after photodamage (data not shown), PsbA synthesis appears to be normal in the mutant.
The gene pyg7 has been mapped on chromosome 1 using simple sequence-length polymorphism (SSLP) and cleaved-amplified polymorphic sequence (CAPS) markers (see "Materials and Methods"). Finally, CAPS markers CAT3, F19G10-VII, and the SSLP marker CIW12 (Lukowitz et al., 2000
The open reading frame of pyg7 encodes a polypeptide of 296 amino acids with a predicted molecular mass of 33.7 kD. Analysis of the N-terminal sequence using the prediction programs ChloroP (Emanuelsson et al., 1999 ) and TargetP (Nielsen et al., 1997; Emanuelsson et al., 2000) revealed a putative plastid-directing transit sequence of 61 amino acids. Thus, the predicted mature protein possesses an estimated molecular mass of 26.8 kD.
Analysis of the primary sequence of Pyg7 revealed high homology to the cyanobacterial Ycf37 from Synechocystis (Wilde et al., 2001
To confirm that the mutated gene is responsible for the observed phenotype, we analyzed an independent knockout line, N807379 (see "Materials and Methods"). Chl a fluorescence induction data of pyg7-2 resembled that of pyg7-1. PCR and sequence analyses with the homozygous knockout plants confirmed the information available in the databases. No PSI activity, PsaA, and Pyg7 proteins were detected in pyg7-2 (data not shown; see Fig. 1).
Several regulatory proteins involved in PSI biogenesis of higher plants, Chlamydomonas and Synechocystis, have been isolated and characterized (Wilde et al., 1995 , 2001; Bartsevich and Pakrasi, 1997; Boudreau et al., 1997; Ruf et al., 1997; Mann et al., 2000; Naver et al., 2001; Shen et al., 2002a, 2002b; Lezhneva et al., 2004; Stöckel and Oelmüller, 2004). Pyg7, a TPR protein, is a novel membrane-bound regulator of PSI, which represents the homolog of the cyanobacterial Ycf37 (Wilde et al., 2001). Immunological studies with antisera raised against a conserved Pyg7 polypeptide from Arabidopsis revealed that the TPR proteins Pyg7 and Ycf37 do not accumulate in the higher plant and cyanobacterial mutants. Both organisms are impaired in PSI activity, but differ substantially in that Ycf37-deficient Synechocystis cells can grow photoautotrophically and accumulate a functional PSI complex, whereas the higher plant mutant is lethal and lacks PSI completely.
Chl a fluorescence measurements demonstrate that Fv/Fm is considerably reduced in the mutant. The increase in QA reduction further indicates that the electron flow downstream of PSII is blocked. Determination of absorbance changes of P700 at 810 nm revealed that PSI in pyg7-1 is not functional. Western analysis confirmed that essential subunits of the reaction center are either absent or severely reduced in pyg7-1 (Fig. 5) and the corresponding knockout line, pyg7-2. Furthermore, the blue shift in the 77 K fluorescence emission spectrum of the mutant (Fig. 2) is in agreement with the notion that the transfer of excitation energy from Lhca1/Lhca4 to the PSI reaction center P700 is impaired. A similar blue shift was also reported for plants lacking PsaF (Haldrup et al., 2000
Inactivation of pyg7 leads to PSI deficiency and to the inability of the mutant to grow photoautotrophically. Because transcripts for representative plastid and nuclear-encoded subunits of the PSI reaction center are present in wild-type amounts in the mutant, it is unlikely that Pyg7 plays a role in transcription and/or transcript accumulation (Fig. 4). Furthermore, organello labeling experiments of thylakoid proteins revealed that major subunits of the PSI reaction center are synthesized in pyg7-1 (Fig. 6). Because we could isolate only a limited number of plastids from homozygote mutant seedlings, pulse-chase experiments gave no reasonable results. However, we could demonstrate that the radiolabel disappears much more rapidly from PsaA in the pyg7-1 plastids compared to wild-type plastids after transfer of the isolated organelles to radioactive-free medium (data not shown). The prediction of a plastid transit peptide suggests that Pyg7 is a chloroplast protein. This was confirmed by cell fractionation and immunoblot analyses as well as MS analyses of the thylakoid subfraction (see Fig. 8). Pyg7 is a membrane-bound protein and associated with PSI, further supporting the idea that the protein affects the stable accumulation of the PSI complex rather than being involved in transcriptional or posttranscriptional processes (see Figs. 4, 5, and 8). Western analyses and MS analyses demonstrate that Pyg7 is present in substantial amounts in purified PSI fractions (see Fig. 9). Pyg7 might be required for the assembly and/or stabilization of the complex. The protein can affect the stability of the PSI core subunits, the availability and/or binding of any of the cofactors to these subunits, or the assembly of the subunits or cofactors into a functional PSI complex. The presence of three TPR motifs in the C-terminal part of the protein suggests that this region might be involved in protein-protein interactions with other PSI subunits. Three TPR motifs are already present in the cyanobacterial Ycf37 protein and they exhibit the highest degree of sequence conservation to the eukaryotic protein. It remains to be determined whether the cyanobacterial Ycf37 is also associated with the PSI reaction center. Ycf3, another regulator of PSI that interacts with PsaA and PsaD in Chlamydomonas (Naver et al., 2001
Pyg7 is of ancient phylogenetic origin and its homolog, Ycf37, from Synechocystis is already required for proper PSI accumulation. However, there is a remarkable difference in the function of both proteins: Cyanobacteria can still grow photoautotrophically in the absence of Ycf37; also, the reduced PSI-to-PSII ratio and the higher phycobilin-to-Chl ratio suggest a function of Ycf37 in PSI stability or assembly (Wilde et al., 2001
Growth Conditions and Plant Material
Arabidopsis (Arabidopsis thaliana) seedlings were grown in growth chambers under continuous white light and a light intensity of 5 and 100 µmol m–2 s–1 at 22°C, respectively. For physiological experiments, seeds were sterilized with 33% (v/v) bleach and 0.08% N-laurylsarcrosinate, washed four times with 1 mL of sterile distilled water, and placed on petri dishes with solidified one-half-strength Murashige and Skoog medium (Murashige and Skoog, 1962
In vivo Chl a measurements were performed with 18-d-old Arabidopsis seedlings using the pulse amplitude-modulated fluorometer PAM101, equipped with a PAM data acquisition system (PDA-100; Walz). Prior to measurements, the fiber optic of the emitter/detector unit (101-ED) was positioned closely to the upper surface of the plants, which were dark adapted for 7 min before the minimal fluorescence F0 was recorded. A saturating white-light pulse of 6,000 µmol m–2 s–1 for 600 ms was used to determine the maximal fluorescence Fm and the Fv/Fm ratio. After 1 min, actinic red light (650 nm, 40 µmol m–2 s–1) emitted from a photodiode (102 L) was turned on and the fluorescence parameter Fm' of illuminated leaves was determined by the application of saturating flashes every 30 s until a stable fluorescence level (Ft) was reached. Subsequently, the actinic light was switched off to determine the minimal fluorescence F0' in the light-adapted state. The fluorescence quenching parameter qP was calculated as qP = (Fm' – Ft)/( Fm' – F0). The quantum yield of PSII (
The light-induced in vivo absorbance changes of P700 at 810 nm were measured using the PAM101/PDA-100 fluorometer connected to a dual-wavelength emitter/detector unit (ED P700DW). Saturating far-red light (730 nm, 15 W m–2) emitted by a far-red diode (102-FR) for 1 min was applied to oxidize P700. After 30 s of far-red light, a strong white-light pulse of 6,000 µmol m–2 s–1 was applied for 400 ms. The maximal signal difference (
Fluorescence spectra at 77 K were recorded using a FluoroMax-2 fluorometer (Jobin Ivon). The excitation was set at 420 ± 10 nm and the spectra were measured over 650–750 nm to reveal fluorescence emitted from PSII and PSI. Leaf tissue of 18-d-old plant material was homogenized in 2 mL of reaction buffer (50 mM MES-NaOH, pH 6.0, 10 mM MgCl2, 5 mM CaCl2, and 25% glycerol) and immediately used for recording spectra.
Mutant and wild-type Arabidopsis seedlings were illuminated with a photon flux density of 1,800 µmol m–2 s–1 for 1.5 h before transfer to 20 µmol m–2 s–1 for subsequent recovery. Photoinhibition was assayed by calculating the Fv/Fm as a measure of the maximal photochemical efficiency of PSII. The room temperature Chl fluorescence of 15-min dark-adapted plants was performed using the Fluorocam (Photon Systems Instruments).
The Anti-Pyg7 antibodies were raised against the N-NKVARPRRDALKDRVK-C peptide (Eurogentec). For immunoblot analyses, a dilution of 1:500 was used. The PsbA, PsbS, Lhcb1, 2, 5, and 6 antibodies were obtained from Agrisera. All other antibodies have been described previously (Stöckel and Oelmüller, 2004
Translational active chloroplasts from 18-d-old mutant and wild-type seedlings were purified on a Percoll gradient and resuspended in reaction buffer (330 mM sorbitol, 50 mM HEPES-KOH, pH 8.0, 10 mM dithiothreitol, and 100 µg/mL phenylmethylsulfonyl fluoride) according to van Wijk et al. (1995)
Chloroplasts for immunolocalization analyses were isolated from 18-d-old plants. The chloroplast-enriched fraction was purified on a Percoll gradient. Intact chloroplasts were washed twice with isolation medium (0.3 M sorbitol, 5 mM MgCl2, 5 mM EGTA, 5 mM Na2EDTA, 20 mM HEPES-KOH, pH 8.0, and 10 mM NaHCO3) and disrupted in breaking buffer (50 mM HEPES-KOH, pH 8.0, 10 mM MgCl2). The stromal and membrane fractions were separated by centrifugation at 10,000 rpm (SS34; Sorvall) for 20 min. The soluble proteins from the supernatant were precipitated with trichloroacetic acid and resuspended in 100 mM Na2CO3, 10% (w/v) Suc, and 50 mM dithiothreitol. The membrane proteins in the pellet were resuspended in breaking buffer.
Separation of PSI and PSII occurred by Suc gradient centrifugation. A crude thylakoid membrane preparation was obtained from isolated plastids. Plastids were resuspended in 10 mM Tris-HCl, pH 8.0, 5 mM KCl, 3 mM MgCl2, 2 mM MnCl2, and stirred on ice for 2 h. The Chl concentration was adjusted to 1.05 mg/mL, and the photosynthetic complexes were partially solubilized at 4°C for 50 min in the presence of 400 mM (NH4)2SO4, 30 mM octyl-
A segregating F2 progeny was generated by crosses of male pollen donor plants of heterozygous lines of pyg7-1 in Columbia background with female recipient plants of Landsberg erecta ecotype, followed by selfing of the resulting F1 plants. To assign the mutant locus to one of the Arabidopsis chromosomes, 30 F2 plants homozygous for the mutant pyg7 locus as well as a combination of SSLPs, nga248, nga280, nga111, nga168, nga162, nga8, nga6, nga76, nga151 (Bell and Ecker, 1994
RNA for greening experiments was isolated from 7-d-old etiolated seedlings, 7-d-old etiolated seedlings illuminated for 4, 10, 24, or 72 h, as well as green seedlings illuminated for 7 d with continuous white light of 100 µmol m–2 s–1 using TRIzol Reagent (Invitrogen) according to the manufacturer's instructions. RT-PCR reactions were performed with the RT-PCR kit (RevertAid first-strand cDNA synthesis kit; Fermentas) using the following primer pairs 5'- AGGCTTCCACAGTTTTGGTTT-3' and 5'-CCCAAACATCTGACTGCATTT-3' for psaA; 5'-CAAGACTCTCCTCACAGAAC-3' and 5'-CTCTGAACCAAGAACCGTTG-3' for pyg7; 5'-GCTGATGTCTATGGTCCAAGTCTACC-3' and 5'-CAATTACCGCTGCTGTCAATGGCGC-3' for hcf101. Actin2 (5'-GGTAACATTGTGCTCAGTGGTGG-3' and 5'-CTCGGCCTTGGAGATCCACATC-3') was used as a control (Robinson et al., 1999
For northern analysis, total RNA from 18-d-old wild-type and mutant plants was isolated according to Heim et al. (1993)
Electron micrographs were performed as previously described (Kusnetsov et al., 1994
Aliquots of the eluted protein fractions and excised protein bands from SDS gels were used for MS. Trypsin digestion of protein mixtures, in-gel trypsin digestion of excised protein bands, and elution of the peptides from the gel matrix were performed according to Sherameti et al. (2004)
The measured MS/MS spectra were matched with the amino acid sequences of tryptic peptides from the Arabidopsis database in FASTA format. Raw MS/MS data were analyzed by Finnigan Sequest/Turbo Sequest software (revision 3.0; ThermoQuest). The parameters for the analysis by the Sequest algorithm were set according to Stauber et al. (2003) Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number NP_850950.
We wish to thank Dr. M. Hippler for PsaA and PsaD antibodies, Dr. R.B. Klösgen for PsbO antibodies, Dr. W. Fischer for electron microscopy, and Dr. A. Wilde for the Synechocystis Ycf37 strain. We also thank H. Becker for skillful technical assistance. The SAIL insertion line N807379 was obtained from the Nottingham Arabidopsis Stock Centre. The nucleotide sequence of pyg7 is identical to that of At1g22700 deposited in the EMBL database. Received January 30, 2006; returned for revision March 27, 2006; accepted March 27, 2006.
1 This work was supported by Friedrich Schiller University, Jena, Germany.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Ralf Oelmüller (b7oera{at}hotmail.com). Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.106.078147. * Corresponding author; e-mail b7oera{at}hotmail.com; fax 49–3641–949–232.
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ABSTRACT
RESULTS
PSII) was calculated as 
A810max) between the reduced and the oxidized states of P700 was used to estimate the photochemical capacity of PSI (Barth and Krause, 2002
-D-glucopyranoside, and 0.45% sodium cholate. The photosynthetic complexes were collected by high-speed centrifugation (49,000 rpm, 2 h, 4°C; Ti45 rotor; Beckman) and the pellet was resuspended in 10 mM Tris HCl, pH 8.0, 3 mM MgCl2, 2 mM MnCl2, and 2% Triton X-100. After adjustment of the Chl concentration to 1.5 mg/mL, the two photosystems were solubilized by stirring on ice for 50 min. The suspension was clarified by centrifugation (20,000 rpm, 20 min; Sorvall) and 500-µl aliquots were loaded onto a linear Suc gradient (10 mL, 5%–28%; centrifugation 26 h, 39,000 rpm; SW40 rotor; Beckman). The gradient was fractionated and aliquots of the fractions were used for MS or SDS-PAGE (Schägger and von Jagow, 1987
-subunit of Arabidopsis: effects on photosynthesis, thylakoid protein composition, and nuclear chloroplast gene expression. Plant Physiol 133: 191–202
