The Evolutionarily Conserved Tetratrico Peptide Repeat Protein Pale Yellow Green7 Is Required for Photosystem I Accumulation in Arabidopsis and Copurifies with the Complex

Abstract

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; Yu et al., 1995). Differences between prokaryotic and eukaryotic PSI complexes become even more apparent when the roles of individual regulatory factors are considered. BtpA, for instance, a factor identified for prokaryote PSI biogenesis, is not present in eukaryotes (Bartsevich and Pakrasi, 1997). Here, we demonstrate that deletion of a homologous protein with regulatory function can have different consequences for PSI assembly in prokaryotes and eukaryotes. Inactivation of pale yellow green7 (Pyg7) in the higher plant Arabidopsis (Arabidopsis thaliana) completely abolishes photoautotrophic growth. In contrast, inactivation of Ycf37, the homologous gene in Synechocystis, still allows photoautotrophic growth of the cells, although they are severely impaired in photosynthetic activity and PSI accumulation (Wilde et al., 2001). This implies that the interplay between the regulatory factors and PSI complex formation has changed during evolution, presumably because the role of the regulatory factor became more specialized or stringent.

RESULTS

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.

Figure 1.

Wild-type (WT), pyg7-1, and pyg7-2 phenotypes of 18-d-old seedlings grown under high light (100 μmol m⁻² s⁻¹; A) or low light (5 μmol m⁻² s⁻¹; B).

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.

Figure 2.

The 77 K fluorescence emission spectra for wild-type (WT) and mutant pyg7-1 leaves of Arabidopsis. Spectra for wild-type leaves (solid line) and pyg7-1 leaves (dashed line) were recorded using homogenized leaf material. Chl was excited at 420 ± 10 nm. Data were normalized to the PSII emission peak at 682 nm. The figure is representative for spectra of at least five extracts from 18-d-old wild-type and pyg7-1 leaves of seedlings grown under low light (5 μmol m⁻² s⁻¹).

Figure 5.

Immunoblot analyses of thylakoid protein extracts from wild-type (WT) and pyg7-1 with antisera raised against the polypeptides indicated on the right side. Fifteen micrograms (WT, pyg7), 7.5 μg (WT/2), 5 μg (WT/4), or 2.5 μg (WT/8) of total membrane protein was loaded per lane. Plants were grown under high light (100 μmol m⁻² s⁻¹).

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 (F_v/F_m) describes the maximal efficiency of PSII photochemistry, which correlates with the number of functional reaction centers (Öquist et al., 1992). The reduced value of F_v/F_m in the mutant, 0.33 ± 0.08 versus 0.78 ± 0.03 in the wild type, may be caused by secondary effects because pyg7-1 does not show any detectable absorbance changes of P₇₀₀ at 810 nm. Because of the nonfunctional PSI, photosynthetic electron transport is blocked and PSII remains the major target of excessive light. This has also been reported for other PSI-specific mutants as well as mutants impaired in the cytochrome (Cyt) b₆/f complex and ATP synthase (Maiwald et al., 2003; Stöckel and Oelmüller, 2004). In several cases, this results in the down-regulation of PSII (compare with Stöckel and Oelmüller, 2004; P. Hein and R. Oelmüller, unpublished data). Taken together, these data indicate that mutation primarily affects electron transport through PSI.

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).

Figure 3.

Electron micrographs of chloroplasts from wild-type (WT) and pyg7-1 leaves. Plants were grown under low light (5 μmol m⁻² s⁻¹).

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).

Figure 4.

Northern-blot analysis for psaA/B, psaC, PsaD, PsaF, and PsaL as representative transcripts for PSI, psbA, PsbO for PSII, PetC (Rieske iron sulfur protein of the Cyt b₆/f complex), PetH (ferredoxin-NADPH-oxidoreductase), and large subunit of Rubisco with total RNA from 18-d-old wild-type (WT) and pyg7-1 seedlings. Lanes were loaded with 15 μg (WT; pyg7-1), 7.5 μg (WT/2), or 3.75 μg (WT/4) RNA per lane. Plants were grown under high light (100 μmol m⁻² s⁻¹).

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.

Figure 6.

Pattern of ³⁵S-radiolabeled plastid-encoded membrane proteins from 18-d-old wild-type and pyg7-1 chloroplast proteins. Labeling occurred for 7.5 min and 60,000 cpm for wild-type (WT) and pyg7-1 proteins were loaded on a 15% SDS-polyacrymamide gel. The positions of PsaA/B (subunits A and B of PSI) and PsbA (D1 protein of PSII reaction center) are indicated. The proteins were identified by western-blot analyses. Plants were grown under high light (100 μmol m⁻² s⁻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 F_v/F_m 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) were chosen for high-resolution mapping of 1,338 F₂ individuals deriving from backcrosses to the ecotype Landsberg erecta. The two markers, CAT3 and F19G10-VII, enclose the mutant locus with 24 and four recombinations, respectively. Appearance of the pyg7 phenotype was examined by segregation analysis of the following progeny. The CAPS markers F19G10-VII and m235 could localize the mutation on the bacterial artificial chromosome clone T22J18 with four and seven recombination events, respectively. Database searches and sequence analyses of amplified DNA regions from pyg7-1 uncovered that the mutation is located in At1g22700. The gene contains five exons and four introns. The mutant sequence contains a single G-to-A exchange at position 621 of the coding region, which leads to a conversion of a Trp to a stop codon of the resulting protein in the mutant pyg7-1. Reverse transcription (RT)-PCR analyses uncovered that the pyg7 mRNA level is low in etiolated material and increases approximately 8-fold upon transfer of the seedlings to light (Fig. 7).

Figure 7.

Light-induced transcript accumulation of pyg7, hcf101 (14), and psaA from 12-d-old etiolated (dark) or light-grown (light, 100 μmol m⁻² s⁻¹) Arabidopsis seedlings or etiolated seedlings that were illuminated for 4, 10, 24, or 72 h. Amplification of actin cDNA was used as an internal control. Relative pyg7 mRNA levels were based on three independent experiments. Dark, 1.0 ± 0.1; 4-h light, 4.7 ± 0.2; 10-h light, 5.7 ± 0.3; 24-h light, 5.8 ± 0.4; 72-h light, 8.1 ± 0.5; continuous light, 8.0 ± 0.3.

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) and contains three tetratrico peptide repeat (TPR) motifs. An antiserum was raised against a Pyg7 peptide (see “Materials and Methods”). It detects a polypeptide with an apparent molecular mass of approximately 27 kD in the thylakoid membrane fraction of wild-type leaf extracts. No protein was detected in the mutant. Antisera against the reaction center protein PsaA of PSI and the reaction center subunit PsbA of PSII were used as a control (Fig. 8A). The antisera also detect a polypeptide of the expected molecular mass in protein extracts from wild-type Synechocystis cells, which was not present in extracts from the mutant Ycf37 (data not shown; see below). Immunoblot analyses of soluble and membrane fractions from Percoll-purified chloroplasts revealed that Pyg7 is associated with the thylakoid membranes of isolated chloroplasts (Fig. 8B). The localization of Pyg7 was further studied in a Suc gradient in which the two photosystems were separated after solubilization with Triton X-100 (see “Materials and Methods”). Figure 9 demonstrates that the highest amount of Pyg7 is found in fractions that contain the PSI reaction center (represented by PsaA and Lhca1). The very same distribution of Pyg7 in the Suc-gradient fractions was confirmed by mass spectrometry (MS).

Figure 8.

Immunoblot analyses of Pyg7 in comparison to Hcf101, PsbA, and PsaA. A, Thylakoid membrane extracts from 18-d-old wild-type and pyg7-1 seedlings grown in high light (100 μmol m⁻² s⁻¹) were analyzed with antibodies against Pyg7, PsbA, and PsaA; the soluble protein fractions were tested with Hcf101 antibodies (Stöckel and Oelmüller, 2004). B, Percoll-purified chloroplasts from 18-d-old wild-type seedlings (Chl) were separated into thylakoid (Thy) and stroma fractions (Str) and analyzed with antibodies against Pyg7, Hcf101, PsbA, and PsaA, respectively; 20 μg of protein were loaded per lane.

Figure 9.

Suc density gradient centrifugation of 2% Triton X-100-solubilized PSII and PSI complexes. Prior to loading on a 5% to 28% linear Suc gradient, the two sedimented photosystems (see “Materials and Methods”) were adjusted to 1.5 mg/mL Chl and solubilized by 2% Triton for 50 min. Suc gradients were centrifugated for 26 h at 39,000 rpm and fractionated from top to bottom into 15 equal fractions. Equal volumes of each fraction were loaded on a SDS-polyacrylamide gel and investigated by immunoblot analyses with antibodies against PsaA, Lhca1, Pyg7, and PsbA.

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).

DISCUSSION

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 F_v/F_m is considerably reduced in the mutant. The increase in Q_A 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) and for hcf101, another PSI-deficient mutant (Lezhneva et al., 2004; Stöckel and Oelmüller, 2004). The fluorescence data are consistent with the observation that the Lhca1, 2, and 4 proteins are still detectable in the mutant, although they cannot be associated with PSI. Interestingly, Lhca3, which is located adjacent to PsaK (Jansson et al., 1996; Ben-Shem et al., 2003), is severely reduced in the mutant. Finally, loss of PSI in pyg7-1 has strong effects on the organization of the thylakoid membrane (Fig. 3). Stacking of grana thylakoids is comparable to wild type, whereas stroma thylakoids are barely detectable. Like other photosynthetic mutants, pyg7-1 does not accumulate assimilatory starch (Fig. 3; Haldrup et al., 2000; Amann et al., 2004; Lezhneva et al., 2004; Stöckel and Oelmüller, 2004).

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), also contains three TPR motifs. Boudreau et al. (1997) have shown that ycf3 is only loosely associated with PSI. Thus, the role of the TRP sequences for PSI association needs to be analyzed in more detail.

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). Inactivation of the higher plant Pyg7 results in complete loss of PSI. A similar evolutionary shift in function has been observed for another protein pair involved in PSI accumulation, Hcf101 from higher plants (Lezhneva et al., 2004; Stöckel and Oelmüller, 2004) and Slr0067 from Synechocystis (T. Stöckel, unpublished data). This indicates that proteins that have accessory functions in cyanobacteria develop into crucial regulators in higher plant chloroplasts. The ycf37/pyg7 genes provide an interesting system to investigate this hypothesis because homologous genes are also present in the cyanelle of Cyanophora paradoxa, the plastids of Cyanidium caldarium, Porphyra purpurea, and Guilliardia theka. In the green algae Chlamydomonas rheinhardtii and higher plants, the gene was transferred to the nucleus.

MATERIALS AND METHODS

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) supplemented with 1.35% (w/v) Suc. To ensure synchronized germination, the plates were kept in darkness at 4°C for the first 48 h. Pyg7-1 is an ethyl methanesulfonate-induced mutant. The T-DNA insertion line N807379, named pyg7-2, was obtained from the Nottingham Arabidopsis Stock Centre (Sessions et al., 2002). Segregation and sequencing analyses using the primer pairs 5′-TGTTACATAACCGGGTTGCAG-3′ and 5′-TGTTTCTGCTTGAGGTTTAGATTG-3′ confirmed that the pyg phenotype of the mutant is caused by a T-DNA insertion in exon 3 (363 nucleotides downstream of the ATG codon) of At1g22700.

PAM101/PDA-100 and P₇₀₀ Absorbance Measurements

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 F_m and the F_v/F_m 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 F_m′ of illuminated leaves was determined by the application of saturating flashes every 30 s until a stable fluorescence level (F_t) 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 = (F_m′ − F_t)/( F_m′ − F₀). The quantum yield of PSII (ΦPSII) was calculated as ΦPSII = (F_m′ − F_t)/F_m′ and the nonphotochemical quenching parameter (NPQ) as NPQ = (F_m − F_m′)/F_m′.

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 (ΔA_810max) between the reduced and the oxidized states of P₇₀₀ was used to estimate the photochemical capacity of PSI (Barth and Krause, 2002).

77 K Measurements

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.

Photoinhibitory Measurements

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 F_v/F_m 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).

Antisera and Immunoblot Analyses

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).

In Organello Radiolabeling of Proteins

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). For labeling, 10⁷ chloroplasts were used. The chloroplasts were preincubated in reaction buffer containing a 5 μm amino acid mixture without Met and 10 μm Mg-ATP for 10 min under 50 μmol m⁻² s⁻¹ at 23°C prior to adding 5 μCi ³⁵S-Met. The reaction was stopped by adding 20 volumes of ice-cold lysis buffer (7 mm magnesium acetate, 118 mm potassium acetate, and 46 mm HEPES-KOH, pH 7.6) after 7.5 min. After centrifugation at 10,000 rpm (SS34; Sorvall), total membranes were washed twice with lysis buffer and resuspended in a HEPES-sorbitol buffer (330 mm sorbitol, 50 mm HEPES-KOH, pH 8.0). The radioactive incorporation of Met was measured using a scintillation counter (Beckman).

Isolation of Chloroplasts, Soluble Plastid Proteins, Thylakoid Membranes, and PSI

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-β-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).

Positional Cloning of pyg7-1

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), and CAPS markers PhyB and AG (www.arabidopsis.org), were used. For high-resolution mapping, genomic DNA from single leaves of 1,338 individual F₂ plants was isolated. SSLP markers, nga248, SO392 (Bell and Ecker, 1994), CIW12 (Lukowitz et al., 2000), m235, and CAPS markers CAT3 (www.arabidopsis.org), as well as the newly developed marker F₁9G10-VII (5′-AGTTGGTCCTCGAGCTCTCC-3′ and 5′-GCTGCTTAAGAATGCGCAGC-3′), were used for fine-mapping procedures.

RT-PCR and Northern-Blot Analyses

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 the RT reactions, 1 μg mRNA, 0.5 μg oligo(dT) primers, as well as 2 units of Moloney murine leukemia virus reverse transcriptase were used to synthesize cDNA. In subsequent PCR reactions, under standard conditions, gene-specific primers for pyg7, hcf101, psaA, as well as for actin2, with 25 and 23 cycles of replication, respectively, were used. The resulting PCR fragments were separated on 1% agarose gels. Genomic DNA and a PCR reaction without a template served as a control.

For northern analysis, total RNA from 18-d-old wild-type and mutant plants was isolated according to Heim et al. (1993). Northern-blot analyses were performed as previously described (Heim et al., 1993). The following primer pairs were used to produce the appropriate probes: 5′-GATGGCGATGTCAAGTGG-3′ and 5′-GCTTCATCTATATCCGCGTG-3′ for PetC; 5′-AATCTCCTCTGTATCCCC-3′ and 5′-TTTCCTTGCCAACAGGTC-3′ for PetH; 5′-AGGCTTCCACAGTTTTGGTTT-3′ and 5′-CCCAAACATCTGACTGCATTT-3′ for psaA; 5′-GGATGTACTCAATGTGTCCG-3′ and 5′-AGCTAGACCCATACTTCGAG-3′ for psaC; 5′-ATGGCAACTCAAGCCG-3′ and 5′-CTCTTCCTGGATTCGCTTTC-3′ for PsaD; 5′-ATTCATTGCTGCTCCTCC-3′ and 5′-GCCCGAATCTGTAACCTTC-3′ for psbA; 5′-CTGCTTCGAGCCTACTTCCTT-3′ and 5′-GCAGTGTTCTTCACGTTCTCC-3′ for PsbO; 5′-GCGTATGTAGCTTATCCC-3′ and 5′-TCCCCCTGTTAAGTAGTC-3′ for rbcL; and 5′-AACCCCGACTTATGGAAG-3′ and 5′-TAAGACCAGGAGCGTATC-3′ for the 18S rRNA gene. For hybridization, the probes were labeled with ³²P-CTP by random priming.

Electron Microscopy

Electron micrographs were performed as previously described (Kusnetsov et al., 1994).

MS

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). Peptide analysis by coupling liquid chromatography with electrospray ionization-MS and tandem MS (MS/MS) was described previously (Stauber et al., 2003; Sherameti et al., 2004).

Protein Identification

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). The similarity between the measured MS/MS spectrum and the theoretical MS/MS spectrum, reported as the cross-correlation factor (X_corr), was equal or above 1.5, 2.5, and 3.5 for singly, doubly, or triply charged precursor ions, respectively. To identify corresponding loci, identified protein sequences were subjected to a BLAST search at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) and FASTA searches by using the Arabidopsis Genome Initiative protein database at The Arabidopsis Information Resource (http://www.arabidopsis.org). Identification of conserved domains and signal peptides was performed by using SMART (Ponting et al., 1999).

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number NP_850950.