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J. Biol. Chem., Vol. 277, Issue 16, 14031-14039, April 19, 2002

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Lack of the Small Plastid-encoded PsbJ Polypeptide Results in a Defective Water-splitting Apparatus of Photosystem II, Reduced Photosystem I Levels, and Hypersensitivity to Light^*

Martin Hager, Marita Hermann, Klaus Biehler, Anja Krieger-Liszkay^§, and Ralph Bock^¶

From the  Institut für Biologie III, Universität Freiburg, Schänzlestraße 1, D-79104 Freiburg, Germany, the ^§ Institut für Biologie II, Universität Freiburg, Schänzlestraße 1, D-79104 Freiburg, Germany, and the ^¶ Westfälische Wilhelms-Universität Münster, Institut für Biochemie und Biotechnologie der Pflanzen, Hindenburgplatz 55, D-48143 Münster, Germany

Received for publication, December 18, 2002, and in revised form, January 28, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Photosystem II is a large pigment-protein complex catalyzing water oxidation and initiating electron transfer processes across the thylakoid membrane. In addition to large protein subunits, many of which bind redox cofactors, photosystem II particles contain a number of low molecular weight polypeptides whose function is only poorly defined. Here we have investigated the function of one of the smallest polypeptides in photosystem II, PsbJ. Using a reverse genetics approach, we have inactivated the psbJ gene in the tobacco chloroplast genome. We show that, although the PsbJ polypeptide is not principally required for functional photosynthetic electron transport, plants lacking PsbJ are unable to grow photoautotrophically. We provide evidence that this is due to the accumulation of incompletely assembled water-splitting complexes, which in turn causes drastically reduced photosynthetic performance and extreme hypersensitivity to light. Our results suggest a role of PsbJ for the stable assembly of the water-splitting complex of photosystem II and, in addition, support a control of photosystem I accumulation through photosystem II activity.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Photosystem II (PSII)¹ is a large cofactor-protein complex consisting of at least 17 protein subunits (Ref. 1; for review, see e.g. Ref. 2). The PSII reaction center is formed by a heterodimer of two pigment-binding proteins, D1 and D2, which, in photosynthetic eukaryotes, are encoded by the chloroplast psbA and psbD genes, respectively. The photochemical reaction carried out by the reaction center converts the energy of a photon into a separation of charge and, in this way, initiates electron flow. Around the reaction center, the outer parts of PSII are assembled, the inner and outer antennae funneling absorbed light energy to the catalytic core and the oxygen-evolving complex splitting water into protons, electrons, and dioxygen (reviewed in Refs. 3 and 4).

In addition to the well-studied large protein subunits, purified PSII particles contain a number of low molecular weight polypeptides, many of which are encoded by the plastid genome of photosynthetically active eukaryotes (5). Most of these small subunits do not bind redox cofactors and, hence, are unlikely to participate directly in electron transfer reactions. It is generally assumed that they rather function as photosystem-assembling or stabilizing factors. However, in many cases, molecular evidence supporting such a structural role is largely lacking.

The successful development of transformation technologies for Chlamydomonas (6) and tobacco chloroplasts (7) has paved the way to functional characterizations of plastid genome-encoded genes by reverse genetics. Linked to a selectable marker gene, mutant alleles can be introduced into plastids by chloroplast transformation, where they replace the endogenous wild-type allele by homologous recombination. During the past decade, reverse genetics has become a powerful tool in plastid functional genomics (reviewed in Ref. 8).

Here, we have taken a reverse genetics approach to define the function of one of the smallest polypeptides in PSII, PsbJ. Using chloroplast transformation, we have generated tobacco plants lacking the PsbJ polypeptide. Physiological and biochemical analyses revealed that the PsbJ-deficient mutant plants accumulate incompletely assembled oxygen-evolving complexes, have reduced levels of PSI and are extremely sensitive to light.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Plant Material and Growth Conditions-- Sterile tobacco plants (Nicotiana tabacum cv. Petit Havana) were grown on agar-solidified MS medium containing 30 g/liter sucrose (9). Homoplasmic transplastomic lines were rooted and propagated on the same medium. Photoautotrophic growth was tested on MS medium without sucrose. For protein isolation and physiological measurements, wild type and transformed plants were grown on sucrose-containing medium under low (2 µmol quanta m² s¹) and standard light (100 µmol quanta m² s¹), respectively. Leaves referred to as "young leaves" were harvested at the age of only a few days in order to avoid interference with photooxidative damage in the mutant chloroplasts. Leaves referred to as "mature leaves" were several weeks old and, in the case of the psbJ mutant, not yet photobleached.

Construction of a psbJ Plastid Transformation Vector-- The region of the tobacco plastid genome containing the psbE operon was isolated as a 2383-bp SalI/SpeI fragment corresponding to nucleotide positions 65,313-67,695 (according to Ref. 10). The fragment was cloned into a pBluescript KS vector (Stratagene, La Jolla, CA) cut with SalI and SpeI. To remove the HincII site from the remaining polylinker, the plasmid was linearized with SalI and the recessed ends were filled in using the Klenow fragment of DNA polymerase I followed by religation. The psbE operon was subsequently excised by digestion with HincII and ClaI (restriction sites correspond to nucleotide positions 66,341 and 67,167) and replaced by a similarly cut PCR product carrying the psbJ deletion. The deletion of the psbJ coding region was introduced into a PCR product by amplification with primers PT5' (5'-GACGATCTCACAAAGATGAA-3') and PJ1 (5'-TTTTGTTGACGACTCAATTCATTACCCCACTTCCCTCCA-3'). The 3' portion of oligonucleotide PJ1 binds to the sequence immediately upstream of the psbJ initiator codon and contains in its 5' portion the nucleotide sequence downstream of the psbJ termination codon up to the HincII site at 66,341. Oligonucleotide PT5' binds upstream of the ClaI site at 67,167. A chimeric aadA gene conferring resistance to aminoglycoside antibiotics (11) was cloned into the unique EcoRV site (position 66,054) to facilitate selection of chloroplast transformants. A plasmid clone carrying the aadA gene in the same orientation as the psbE operon yielded the final transformation vector ppsbJ (see Fig. 2).

Plastid Transformation and Selection of Homoplasmic Transformed Tobacco Lines-- Young leaves from sterile tobacco plants were bombarded with plasmid ppsbJ-coated 1.1-µm tungsten particles using a biolistic gun (PDS1000He; Bio-Rad). Primary spectinomycin-resistant lines were selected on RMOP regeneration medium containing 500 mg/liter spectinomycin (7, 11). Plastid transformants were identified by PCR amplification according to standard protocols and using the primer pair P10 (5'-AACCTCCTATAGACTAGGC-3'; complementary to the psbA 3'-untranslated region of the chimeric aadA gene) and P11 (5'-AGCGAAATGTAGTGCTTACG-3'; derived from the 3' portion of the aadA coding region). Four independent transplastomic lines were subjected to four additional rounds of regeneration on RMOP/spectinomycin to obtain homoplasmic tissue.

Isolation of Nucleic Acids and Hybridization Procedures-- Total plant DNA was isolated by a rapid miniprep procedure (12). Total cellular RNA was extracted using the TRIzol reagent (Invitrogen). DNA samples were digested with Ecl136II/DraI, separated on 1.2% agarose gels, and blotted onto Hybond N nylon membranes (Amersham Biosciences). Total cellular RNA was electrophoresed on formaldehyde-containing 1.3% agarose gels and transferred onto Hybond N⁺ membranes. For hybridization, [-³²P]dATP-labeled probes were generated by random priming (Multiprime DNA labeling system; Amersham Biosciences). A radiolabeled PCR product covering part of the psbE operon (obtained by amplification with primer pair P7652 (5'-CCGAATGAGCTAAGAGAATCTT-3')/P7355 (5'-GACTATAGATCGAACCTATCC-3')) was used as probe for the restriction fragment length polymorphism analysis and for detection of psbE operon-specific transcripts. Hybridizations were carried out at 65-68 °C in rapid hybridization buffer (Amersham Biosciences). An NdeI restriction fragment corresponding to nucleotide positions 41,487-42,385 in the tobacco chloroplast genome (10) was used as a specific probe for detection of transcripts from the plastid psaA/B operon. To control for equal loading, blots were stripped and rehybridized to a cytoplasmic 18S rRNA probe (amplified with primer pair P5'18SNT (5'-GTATATTTAAGTTGTTGCAGT-3')/P3'18SNT (5'-AAACTTTGATTTCTCATAAGG-3')). Transcript quantitation was performed with a PhosphorImager using the Quantity One® software (Bio-Rad).

Immunoblot Analysis of Proteins and SDS-PAGE-- Thylakoid proteins from wild-type and mutant plants were isolated from total leaf material following published procedures (14). PSII-enriched membranes (BBY) were prepared according to a protocol originally developed for spinach (15). Following protein quantitation, equal amounts of thylakoid proteins were separated on Tricine-SDS-polyacrylamide gels (16) and stained with silver according to standard protocols (17). For Western blot analyses, electrophoretically separated proteins were transferred to Hybond-P polyvinylidene difluoride membranes (Amersham Biosciences) using the Trans-Blot Cell (Bio-Rad) and a standard transfer buffer (192 mM glycine, 25 mM Tris, pH 8.3). Immunoblot detection was performed using the enhanced chemiluminescence system (ECL; Amersham Biosciences).

Physiological Measurements-- Determination of PSII activity was performed on dark-adapted leaves from wild-type and mutant plants grown under low light (2 µmol quanta m² s¹) and standard light (100 µmol quanta m² s¹) conditions, respectively. PSII-dependent chlorophyll fluorescence was recorded at 650-nm wavelength with a pulsed amplitude modulation fluorimeter (Walz, Effeltrich, Germany; Ref. 18) under illumination of intact leaf tissue with white actinic light (flux density 50 µmol quanta m² s¹ and 100 µmol quanta m² s¹; pulse frequency of measuring light, 1.6 kHz). For complete reduction of Q_A, leaves were exposed to pulses of saturating light (1 s; flux density 6000 µmol quanta m² s¹). The redox state of the PSI reaction center chlorophyll P700 was monitored by following the changes in absorbance of dark-adapted leaves from wild-type and mutant plants at a 830-nm wavelength (19, 20). Absorbance measurements were performed using the pulsed amplitude modulation fluorimeter with a modified emitter/detector unit. Far red light with a peak wavelength of 730 nm was used to selectively excite PSI. To obtain complete rereduction of PSI, leaves were exposed to a strong white light pulse (5, 50, or 200 ms; 6000 µmol quanta m² s¹). Oxygen evolution of young leaves from plants grown at 2 µmol quanta m² s¹ was measured with a Clark O₂ electrode (Hansatech) at room temperature under saturating CO₂ levels to minimize competing O₂-consuming reactions. Oxygen evolution was monitored at flux densities of 30 µmol quanta m² s¹. Five independent measurements were performed to calculate average O₂ evolution values. Thermoluminescence was measured on leaf segments with a home-built apparatus. Thermoluminescence was excited with single turnover flashes at 0 °C. The flashes were spaced with a 1-s dark interval. Samples were then heated with a heating rate of 20 °C/min to 60 °C, and the light emission was recorded. Graphical and numerical data analyses were performed according to Ducruet and Miranda (21).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Targeted Inactivation of the Chloroplast psbJ Gene-- The psbJ gene is part of the plastid psbE operon, which comprises the four PSII genes psbE, psbF, psbL, and psbJ. This operon structure is conserved in all photosynthetically active multicellular plant species investigated to date but is not found in the unicellular green alga Chlamydomonas reinhardtii (22). The psbE operon is unique in that its tetracistronic primary transcript does not undergo processing into monocistronic or oligocistronic units (23). Consequently, translation must initiate efficiently on all four cistrons of the polycistronic mRNA. The resulting translation products are relatively small subunits of the PSII complex. The psbE and psbF genes specify the cytochrome b₅₅₉  and  subunits, which are essential for PSII assembly (24-26). The function of the other two polypeptides encoded by the psbE operon, PsbL and PsbJ, is much less clear. PsbL has recently been implicated in the stabilization of the PSII core complex and the dimeric form of PSII (27, 28). The PsbJ protein has been detected immunologically in thylakoid membranes of cyanobacteria (29, 30) and in purified PSII particles by MALDI-TOF mass spectrometry (1). Genetic analyses in cyanobacteria also support an association of PsbJ with PSII and, in addition, have established that, in Synechocystis, PsbJ is not essential for photoautotrophic growth (29).

The psbJ gene of photosynthetic eukaryotes specifies a hydrophobic polypeptide of only 40 amino acids (theoretical molecular mass: 4.1 kDa) which is evolutionarily highly conserved (Fig. 1). In order to define the function of the PsbJ polypeptide in PSII of higher plants, we have constructed a chloroplast transformation vector carrying a psbJ null allele (Fig. 2). The psbJ coding region was deleted from a cloned fragment of the tobacco plastid DNA using PCR-based mutagenesis techniques. A chimeric selectable marker gene aadA was inserted into the intergenic spacer in between the psbE operon and the petA gene (Fig. 2). Earlier work had established that this spacer is a suitable site for the insertion of plastid transgenes (31, 32).

View larger version (26K): [in this window] [in a new window]   Fig. 1.   PsbJ is a highly conserved hydrophobic polypeptide. An alignment of PsbJ amino acid sequences from the cyanobacterium Synechocystis and selected plant species (for references and accession numbers, see Refs. 29 and 53) is shown. Residues identical in all sequences listed here are denoted by asterisks; residues conserved in at least six out of the seven species are marked by dots.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Construction of psbJ knockout plants. Physical maps of the region of the tobacco chloroplast genome containing the psbE operon and of the plastid-targeting fragment in the chloroplast transformation vector ppsbJ are shown. Genes above the line are transcribed from the left to the right; genes below the line are transcribed in the opposite direction. The aadA selectable marker gene is integrated into a unique EcoRV restriction site within the intergenic spacer in between the psbE operon and the petA gene. The arrows indicate possible sites of homologous recombination, resulting in different classes of transplastomic plants: recombination at sites 1 and 3 leads to incorporation of both the selectable marker gene and the psbJ deletion, whereas recombination at sites 2 and 3 produces transplastomic plants carrying the aadA but having a wild-type psbJ gene. Relevant restriction sites are indicated, and sites lost during vector construction due to ligation to heterologous ends are shown in parentheses. The binding site of the probe used for restriction fragment length polymorphism analyses and Northern blots (Figs. 3 and 4) is also marked.

Biolistic bombardment of sterile tobacco leaves with plasmid ppsbJ-coated tungsten particles was followed by selection of spectinomycin-resistant cell lines. Successful chloroplast transformation was verified by tests for double resistance on medium containing two aminoglycoside antibiotics, spectinomycin and streptomycin (11), and further confirmed by PCR assays using aadA gene-specific primers (32). Chloroplast transformants were purified to homoplasmy by passing them through additional regeneration cycles under antibiotic selection. Homoplasmy of the transplastomic lines (i.e. absence of any residual copies of the wild-type chloroplast genome) was confirmed by restriction fragment length polymorphism analysis (Fig. 3).

View larger version (35K): [in this window] [in a new window]   Fig. 3.   Southern blot analysis confirming successful plastid transformation, psbJ deletion, and homoplasmy of the transplastomic tobacco lines. Restriction digests with Ecl136II and DraI produce a 1.6-kb fragment for wild-type tobacco (Fig. 2). Plastid transformation with a vector carrying the aadA selectable marker gene but not the psbJ deletion results in a 2.75-kb fragment (control line E15). 1C, 1D, 3A, 3C, 4B, 4C, 8C, and 8D designate transplastomic lines produced by transformation with vector ppsbJ (Fig. 2). Lines 3A, 3C, 4B and 4C produce restriction fragments of identical size as the control line E15, suggesting that they carry the aadA but not the psbJ deletion and thus must originate from homologous recombination events at the sites labeled 2 and 3 in Fig. 2. Lines 1C, 1D, 8C, and 8D show hybridization to a slightly smaller band of 2.62 kb, indicating that they carry the 130-bp deletion in the psbJ gene as introduced by homologous recombination events at sites 1 and 3 in Fig. 2. Note that all transplastomic lines lack any hybridization signal corresponding to the wild-type plastid genome, demonstrating that they are homoplasmic.

Since there is no absolute linkage between the introduced psbJ deletion and the selectable marker gene aadA in our transformation vector (Fig. 2), two different types of chloroplast transformants are obtained: (i) transformants carrying only the aadA but not the psbJ deletion and (ii) transformants having incorporated both the aadA gene and the psbJ null allele by homologous recombination upstream of psbJ (Fig. 2). Southern blot analyses confirmed the isolation of transplastomic lines from each of the two types (Fig. 3). As expected, lines belonging to the first type were phenotypically indistinguishable from the wild type (as shown earlier; Ref. 31), whereas lines of the second type all exhibited the same mutant phenotype (subsequently referred to as psbJ plants; see below).

Since the psbJ gene is part of an operon that is transcribed as a tetracistronic mRNA, it was important to verify that the deletion introduced into psbJ did not affect transcription of the psbE operon or stability of its mRNAs. We therefore comparatively analyzed accumulation of psbE operon transcripts in wild-type and transplastomic tobacco lines (Fig. 4). No significant difference was found in transcript pattern and mRNA accumulation levels between the transplastomic lines only having the aadA marker and those additionally carrying the psbJ deletion indicating that the deletion in the psbJ coding region does not negatively affect synthesis or stability of psbE operon transcripts. Faithful expression of the engineered psbE operon in psbJ plants was subsequently also confirmed at the protein level by assaying accumulation of the cytochrome b₅₅₉ -subunit, the psbE gene product (see below).

View larger version (79K): [in this window] [in a new window]   Fig. 4.   Northern blot analysis to detect transcripts from the plastid psbE operon in wild-type and transplastomic tobacco plants. A tetracistronic mRNA species of 1 kb accumulates in the wild type as well as in those transplastomic lines that carry the aadA gene downstream of the psbE operon but not the psbJ deletion (E15, 3A, 4B; see Figs. 2 and 3). The psbJ knockout lines 1C and 8C accumulate a 130-bp smaller mRNA species due to the deletion of the psbJ coding region. All transplastomic lines show an additional prominent RNA species of higher molecular weight (~2 kb), which is the result of read-through transcription initiating at the psbE operon promoter and terminating downstream of the aadA (31, 32). Reprobing of the blot with a 16 S rRNA-specific probe (not shown) confirmed that the accumulation levels of psbE operon transcripts do not differ significantly between the wild type and the various transplastomic lines.

Phenotype of Transplastomic Plants-- As established earlier for aadA transgenes inserted into the same genomic location (31, 32), transplastomic plants carrying the aadA selectable marker gene but not the psbJ deletion were phenotypically normal and indistinguishable from wild-type tobacco plants in all subsequent biochemical and physiological tests (see below). By contrast, homoplasmic psbJ knockout plants grown on synthetic medium under standard light conditions (100 µmol quanta m² s¹) displayed a clear mutant phenotype. While young leaves were almost normally green (Fig. 5), older leaves were completely white and showed strong symptoms of photobleaching. However, this phenotype was much less severe than that observed previously for tobacco photosynthesis null mutants (33, 34), suggesting that the lack of the PsbJ protein may reduce but does not completely abolish photosynthetic activity. The mutant phenotype was identical in all independently generated transplastomic lines and has remained stable during vegetative propagation, providing additional proof for the homoplasmy of the lines.

View larger version (73K): [in this window] [in a new window]   Fig. 5.   Phenotypic comparison of psbJ tobacco plants with E15 control plants. Plants were grown on sucrose-containing synthetic medium under standard (100 µmol quanta m2 s1) or extreme low light conditions (2 µmol quanta m2 s1). Bleaching of mature leaves in psbJ plants under 100 µmol quanta m2 s1 but not under 2 µmol quanta m2 s1 indicates hypersensitivity to light and accumulation of photooxidative damage over time.

When grown under extreme low light conditions (2 µmol quanta m² s¹), no bleaching of mature leaves occurred in psbJ plants (Fig. 5), confirming that the severe phenotype under standard light conditions is caused by the mutant's high sensitivity to light, which appears to result in the accumulation of photooxidative damage over time.

Photosynthetic Electron Transfer in psbJ Knockout Plants-- The highly light-sensitive phenotype of the psbJ lines was indicative of inefficient electron transfer reactions, potentially leading to the production of free radicals that in turn cause photooxidative damage to chloroplast membranes and proteins. In order to determine the efficiency of photosynthetic electron transport, we comparatively analyzed PSII and PSI functions in wild-type and psbJ plants. In view of the light-sensitive phenotype of the psbJ knockout plants and the strong dependence of photobleaching on leaf age, we included young and mature leaves from plants grown at 2 µmol quanta m² s¹ as well as similar leaf material from plants raised at 100 µmol quanta m² s¹ (Figs. 5-7). We define here "young" as the first (or at most the second, depending on the size of the youngest visible leaf) leaf from the tip of the plant and "mature" as a relatively expanded leaf (second, third, or fourth from the tip), which, in the case of the psbJ plants grown at 100 µmol quanta m² s¹ must not yet be photobleached.

We first measured PSII activity by chlorophyll fluorescence at room temperature. The minimum fluorescence F₀ was determined by exposure of dark-adapted leaves to measuring light of low intensity (Fig. 6). Subsequently, maximum fluorescence F_m was obtained by illumination with two saturating light pulses each resulting in complete reduction of the primary quinone-type acceptor of PSII, Q_A. High variable fluorescence (F_var = F_m  F₀) was detected for both young wild-type and young psbJ leaves (Fig. 6), strongly suggesting that psbJ plants synthesize functional PSII units capable of reducing the primary PSII acceptor Q_A. In contrast, mature leaves from psbJ plants grown at 100 µmol quanta m² s¹ showed almost no variable fluorescence. F_var was also drastically reduced in mature leaves from psbJ plants grown under extreme low light conditions (Fig. 6) mainly caused by a strong increase in minimum fluorescence, F₀. This may indicate a disproportion between the photon-capturing capacity of the PSII antenna and the transfer of absorbed light energy to the PSII reaction center resulting in dramatically enhanced chlorophyll fluorescence emission.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Fluorescence induction as a test for PSII activity. Leaf samples from dark-adapted wild-type and psbJ plants grown under the indicated light conditions were taken to measure PSII-dependent chlorophyll fluorescence. Leaves grown at 2 µmol quanta m2 s1 were exposed to actinic light of 50 µmol quanta m2 s1; leaves grown at 100 µmol quanta m2 s1 were exposed to actinic light of 100 µmol quanta m2 s1. PSII activity is clearly detectable in young leaves from psbJ plants, and strong photochemical quenching suggests intact transfer of electrons to downstream components of the electron transport chain. By contrast, in mature leaves from the psbJ mutant, the variable fluorescence (Fm  F0) is much lower than in the corresponding wild-type leaves and is close to zero in mature mutant leaves grown at 100 µmol quanta m2 s1.

The ratio (F_m  F₀)/F_m can serve as a measure of the maximum quantum yield of PSII-driven photochemistry. In young leaves from psbJ plants, this ratio (and hence the maximum photochemical capacity of PSII) was found to be only slightly reduced as compared with wild-type leaves, suggesting that the absence of the PsbJ protein does not dramatically impair the electron transfer reactions in PSII. By contrast, mature leaves from psbJ plants were severely affected (Fig. 6), indicating that the PsbJ protein is directly or indirectly involved in the protection of PSII from light-induced damage or destabilization.

When wild-type leaves were exposed to continuous actinic light of low intensity, flashes of saturating white light superimposed on the actinic light resulted in a fluorescence rise, which approximately reached the initial value of F_m, and strong photochemical fluorescence quenching occurred (Fig. 6). By contrast, in young leaves from psbJ plants, the fluorescence rise induced by the saturating flashes clearly did not reach the initial F_m value, and mature leaves from plants grown at 100 µmol quanta m² s¹ even lacked almost any PSII photochemistry. These results lend further support to the idea that, in the absence of the PsbJ protein, the photosynthetic apparatus is highly light-sensitive.

In order to measure the efficiency of electron transfer to downstream components of the photosynthetic electron transport chain, we next set out to determine the activity of PSI. PSI function in wild-type and mutant plants was deduced from absorption measurements at 830 nm. Absorption changes at 830 nm correlate with the redox state of the PSI reaction center chlorophyll, P700. In the dark, P700 is present in its reduced form (19). Illumination of dark-adapted leaves with far red light selectively excites PSI, thereby converting P700 in its oxidized form (Fig. 7). This PSI-induced absorption shift is observed for the wild type as well as for all leaves from psbJ plants, including the mature leaves grown under normal light (100 µmol quanta m² s¹), which had almost no measurable PSII activity, confirming that the knockout of psbJ primarily affects PSII. However, in all leaf samples from the mutant, the intensity of the absorption change was significantly lower than in the equivalent wild-type sample, suggesting that psbJ plants may have fewer active PSI units than the wild type (Fig. 7).

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Test for PSI function by far red spectroscopy. Selective excitation of the PSI reaction center chlorophyll P700 results in a transition from its reduced to its oxidized state and hence reveals intact PSI photochemistry in both the wild type and the mutant. However, rereduction of P700 by white light pulses and, hence, by electrons released in PSII reveals remarkable differences. The 200-ms light pulse on mature mutant leaves grown at 2 µmol quanta m2 s1 as well as all pulses on mature mutant leaves grown at 100 µmol quanta m2 s1 cannot rereduce the PSI reaction center chlorophylls and instead result in an apparent overoxidation of the P700 pool. Note that plants grown at 100 µmol quanta m2 s1 generally possess more PSI than plants grown at 2 µmol quanta m2 s1, which is likely to reflect light-induced up-regulation of photosynthesis.

When short pulses of white light are superimposed onto the continuous far red light, electrons are released from PSII and transferred to PSI, where they lead to a rereduction of P700, which again can be monitored as an absorption change at 830 nm (Fig. 7). This PSII-dependent rereduction of P700 was observed for young mutant leaves but was significantly impaired in mature leaves from psbJ plants (Fig. 7).

Altogether, these physiological data suggest that, while psbJ plants are capable of synthesizing principally functional photosystems, (i) PSII function appears to be highly light-sensitive in a leaf age-dependent manner and (ii) PSI levels or activity may be reduced in the absence of the PsbJ polypeptide.

Net Photosynthetic Capacity of psbJ Plants-- Having established that the light reactions of photosynthesis still function to some extent in psbJ plants, we were interested to know whether or not the electron transfer capacity in the mutant was sufficient to sustain photoautotrophic growth. We therefore transferred cuttings from psbJ plants to sucrose-free synthetic medium. In this way, continued growth was made dependent on net photosynthetic carbon fixation. From our observations with the highly light-sensitive mutant phenotype (Fig. 5), we expected the light intensity to be the critical factor in these tests. We therefore assayed for photoautotrophic growth over a wide range of light intensities: 2, 10, 30, and 100 µmol quanta m² s¹. Under all of these light intensities, psbJ plants died after 2-4 weeks of incubation in the absence of an exogenously supplied organic carbon source (not shown), while all control plants survived and continued to grow. The phenotype at plant death was, however, slightly different. At 2 and 10 µmol quanta m² s¹, mutant plants showed severe wilting and turned brown, symptoms interpreted as death from carbon starvation. By contrast, plants under 30 and 100 µmol quanta m² s¹, while wilting similarly fast, turned white, showing strong evidence of photooxidation.

Low photosynthetic performance of psbJ plants was further supported by gas exchange measurements. While stomatal opening was not affected in the mutant, photosynthetic gas exchange was drastically reduced as compared with the wild type. Even in very young leaves from psbJ plants, the carbon dioxide-evolving processes exceeded the carbon dioxide-fixing processes; hence, no net carbon fixation occurs in psbJ knockout plants. Likewise, comparison of the oxygen evolution rates revealed that the photosynthetically most active leaves from psbJ plants produce only about one-tenth of the oxygen evolved by an average wild-type leaf (wild type: 2 µmol/m² s; psbJ plants: 0.2 µmol/m² s).

Accumulation of Thylakoid Proteins in psbJ Plants-- We next wanted to determine the molecular basis for the very low photosynthetic capacity in psbJ plants. To this end, we quantitatively assayed for the presence of the multiprotein complexes in the thylakoid membrane, which conduct the light reactions of photosynthesis. To get an overall impression of thylakoid protein accumulation levels in wild-type and psbJ plants, we first separated purified thylakoid proteins in high resolution SDS-polyacrylamide gels (16, 35) and stained the proteins with silver (Fig. 8). To facilitate unambiguous assignment of bands, we also loaded a PSI-deficient mutant (PSI; Ref. 33) and a PSII null mutant (PSII; produced by knockout of the psbD/C operon).² Unexpectedly, thylakoids from psbJ plants grown under low light conditions (2 µmol quanta m² s¹) contained wild-type levels of PSII proteins but had drastically reduced amounts of all PSI subunits (Fig. 8). The finding that a knockout of a small PSII subunit has a strong effect on PSI accumulation but no readily detectable effect on PSII accumulation was surprising. Subsequent analysis of thylakoid proteins from plants grown at standard light conditions (100 µmol quanta m² s¹) revealed that psbJ plants now also had significantly reduced PSII levels (Fig. 8), confirming that the lack of the PsbJ protein renders PSII light-sensitive and indicating that photoinduced protein degradation is indeed the cause of the highly light-sensitive phenotype of the psbJ mutant.

View larger version (77K): [in this window] [in a new window]   Fig. 8.   Comparison of thylakoid membrane proteins from wild-type plants and psbJ plants. Thylakoids were purified from leaf material harvested from plants grown under low light conditions (2 µmol quanta m2 s1) or standard light conditions (100 µmol quanta m2 s1). Proteins were separated in 16.5% Tricine-SDS-polyacrylamide gels and stained with silver. To facilitate unambiguous assignment of bands, thylakoid proteins from a PSII-deficient mutant (PSII)2 and a PSI-deficient mutant (PSI; Ref. 33) were included. For rough evaluation of quantitative differences in protein accumulation, a 1:2 dilution of wild-type and psbJ thylakoids is also shown. PSI, selected protein bands belonging to photosystem I; PsaA/B, the two large reaction center proteins of PSI. Selected other thylakoid proteins belonging to PSII (PsbO, PSII), the light-harvesting antenna (CP29, CP26, LHCII), or the ATP synthase complex (AtpA/B) are also labeled. Note that the levels of all PS I proteins in psbJ plants are significantly reduced as compared with the wild type. M1 and M2, molecular weight markers.

Having confirmed at the protein level that, in psbJ plants, photooxidative damage occurs in a light exposure-dependent manner, we strictly separated young and mature leaves in all subsequent analyses of thylakoid proteins in order to distinguish between primary effects caused by the absence of the PsbJ polypeptide and secondary consequences such as photosystem degradation caused by light-induced damage. Using a collection of antibodies against subunits of all thylakoidal multiprotein complexes, we first analyzed protein accumulation in young leaves grown under low light conditions. These experiments confirmed that PSI subunits were drastically reduced in psbJ plants, whereas ATP synthase, cytochrome b₆f as well as most PSII subunits tested accumulated to wild-type levels (Fig. 9). However, a single PSII protein was virtually absent from thylakoids of the psbJ mutant: PsbP, the 23-kDa extrinsic protein of the water-splitting complex, also referred to as OEE2 (oxygen-evolving enhancer 2). Together with two other proteins, PsbO (33 kDa; OEE1) and PsbQ (16 kDa; OEE3), this extrinsic subcomplex is believed to stabilize the catalytic manganese cluster of the water-splitting complex and its ionic cofactors, chloride and calcium ions, which both are essential for efficient water splitting by PSII (36, 37).

View larger version (66K): [in this window] [in a new window]   Fig. 9.   Immunoblot analyses to quantitatively analyze thylakoid protein accumulation in psbJ plants and E15 control plants. Thylakoid proteins from young leaves grown under low light (2 µmol quanta m2 s1) or standard light (100 µmol quanta m2 s1) conditions were assayed with antibodies against individual subunits of thylakoidal multiprotein complexes. For quantitative evaluation, a dilution series for the control plants is shown. The immunoblot analyses confirm significant reduction of PSI subunits (PsaC, PsaF) in psbJ plants (see Fig. 8) while revealing essentially unchanged levels of the ATP synthase subunit AtpB, cytochrome f (PetA), a key component of the cytochrome b6f complex, and most PSII subunits. The only protein that is undetectable in the psbJ mutant is the 23-kDa extrinsic protein of the water-splitting complex (PsbP). The absence of PsbP protein was confirmed for both thylakoid preparations and PSII particle isolations from mutant and control plants. Note also that the antenna protein CP26 is reduced in psbJ plants, and, at 100 µmol quanta m2 s1, also PsbE and PsbO are present at slightly lower levels than in the wild type.

Besides a virtually complete absence of PsbP, the light-harvesting antenna protein CP26 was found to be reduced in the psbJ mutant (Fig. 9). However, the reduced CP26 levels are unlikely to contribute to the low PSII activity and the severe light-sensitive phenotype of psbJ plants, since it has been shown that tobacco and Arabidopsis plants lacking CP26 grow photoautotrophically and moreover are phenotypically entirely normal under standard growth conditions (35, 38). Hence, reduced CP26 accumulation is likely to be a side effect of the low PSII performance caused by the defective water-splitting apparatus in the absence of PsbJ.

When identical immunoblot analyses were performed with thylakoids from young leaves grown under standard light conditions, two other PSII proteins were found to be present at reduced levels: PsbO, the manganese-stabilizing protein of the water-splitting complex, and PsbE, the -subunit of cytochrome b₅₅₉ (Fig. 9). This may indicate that the cytochrome b₅₅₉ is one of the first targets of the light-induced destabilization of PSII as caused by the defective water-splitting apparatus in the psbJ mutant.

Immunoblot analyses with thylakoids from mature leaves revealed that now all PSII subunits were drastically reduced, including the two reaction center proteins D1 and D2 (PsbA and PsbD) as well as the inner antenna proteins CP43 and CP47 (Fig. 10). This observation confirms that light-induced PSII degradation occurs in psbJ plants and that photooxidative damage accumulates over time.

View larger version (80K): [in this window] [in a new window]   Fig. 10.   Analysis of thylakoids from mature leaves indicates massive photooxidative damage-induced protein degradation in psbJ plants. Under both low light (2 µmol quanta m2 s1) and standard light (100 µmol quanta m2 s1) conditions, protein levels of all PSII subunits tested (CP43, CP47, PsbA, PsbE, PsbO) were found to be drastically reduced in mature leaves from psbJ plants, suggesting that photooxidative damage accumulates over time, which in turn results in accelerated degradation of thylakoid proteins. As compared with the young leaves analyzed in Fig. 9, reduced PSII activity is now also evident at the protein level, resulting in an even more dramatic down-regulation of PSI levels; PsaF accumulation is reduced to less than 25% of the levels in the E15 control line.

Taken together, the immunoblot analyses establish that the lack of PsbJ causes a defect in the water-splitting apparatus. This finding explains the light-sensitive phenotype of psbJ plants, confirms the results from our physiological measurements, and also explains why mutant plants grown under low light conditions are capable of conducting photosystem-dependent electron transport but evolve very little oxygen and cannot grow photoautotrophically.

In order to confirm that the loss of PsbP is the primary defect causing the mutant phenotype of the psbJ knockout, we measured flash-induced patterns of thermoluminescence in wild-type and psbJ plants. In thermoluminescence measurements, the emitted light originates from charge recombinations of trapped charge pairs (for a review, see Ref. 39). The charge pairs involved can be identified by their emission temperatures, which strongly depend on the redox potentials of the charge pairs. The most important thermoluminescence band for investigating the electron transfer within PS II is the B-band. Recombination of the S₂ or S₃ state of the oxygen-evolving complex at the donor side of PS II with the semiquinone Q yields the B-band at ~30 °C (40). Fig. 11 shows the oscillation of the B-band measured on dark-adapted leaf sections of the psbJ mutant and the wild type. In the wild type, a thermoluminescence curve is observed, which consists of two bands: the B-band and the afterglow band (21, 41). We focus here on the changes in the intensity of the B-band at 24 °C in dependence on the number of exciting single turnover flashes. As expected, the highest intensity of the B-band is observed after the first flash and the intensity of this band oscillates with a period of 4. In the mutant, the intensity of the B-band is lowered, while that of the afterglow band is comparable with the wild type. Additionally, the maximum temperature of the B-band decreased by about 5 K compared with wild type. The most striking observation is that the B-band shows no oscillation in dependence on the number of excitation flashes. It thus seems that the cycle of the S-states is perturbed after the formation of S₂ (Fig. 11). The S₂ state can clearly be formed, but subsequently, no longer positive charge equivalents can be accumulated. As for water oxidation, a complete S-cycle formation (S₀-S₄) is required, it is no longer surprising that the psbJ mutant evolves very little oxygen. These data confirm very recent thermoluminescence studies on psbJ-deficient cyanobacterial and tobacco mutants, which revealed that while the cyanobacterial mutant shows almost wild type-like signal oscillation, the tobacco mutant largely lacks detectable oscillation (30).

View larger version (13K): [in this window] [in a new window]   Fig. 11.   Thermoluminescence measurements of dark-adapted leaves from the wild type and the psbJ mutant. While the wild type (filled circles) shows the typical oscillation cycle, reflecting the different oxidation states of the manganese cluster (S0-S4), the PsbP-deficient oxygen-evolving complexes in the psbJ mutant (open circles) are largely locked in state 2 (S2) and show a much less pronounced oscillation of oxidation states.

Down-regulation of Photosystem I Is Not Transcriptional-- Having established that the low PSII capacity in psbJ plants results in drastically reduced PSI levels, we were interested to learn whether this PSII-mediated regulation of PSI was exerted through transcriptional or post-transcriptional control mechanisms. We therefore comparatively analyzed mRNA accumulation of the two large PSI reaction center polypeptides PsaA and PsaB. These analyses revealed that the transcripts from these genes accumulate to identical levels in the wild type and in psbJ plants (data not shown). This suggests that the observed down-regulation of PSI levels in psbJ plants occurs neither transcriptionally nor at the level of mRNA stability but rather indicates that PSII dysfunction (as in the psbJ mutant) may result in a post-transcriptional down-regulation of PSI levels.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Absence of the PsbJ Polypeptide Results in a Defective Water-splitting Complex of PSII-- The oxidation of water molecules in PSII is catalyzed by the luminal manganese cluster (believed to contain four manganese ions) and a redox-active tyrosine residue of the D1 protein, referred to as Tyr^z. In addition, at least three membrane-extrinsic proteins, PsbO, PsbP, and PsbQ, are important for maintenance of photosynthetic oxygen evolution in plants. These extrinsic proteins are assumed to be present in a 1:1:1 stoichiometry per PSII complex and have been implicated in stabilizing the manganese cluster and its obligatory cofactors Ca²⁺ and Cl.

In the course of this work, we have shown that correct assembly of this extrinsic protein subcomplex of PSII is dependent on the presence of a small hydrophobic polypeptide, PsbJ. In the absence of PsbJ, the water-splitting complex of PSII lacks the PsbP subunit, which results in a drastically reduced photosynthetic oxygen evolution rate. Similar physiological data have been obtained previously for chloride ion-depleted PSII-enriched membrane fragments (42). Since maintaining the local concentration of chloride ions is believed to be a major function of the PsbP protein, we propose that the PSII complexes in the psbJ mutant are functionally equivalent to chloride ion-depleted PSII particles and that, in both cases, the water-splitting system is inhibited at the step of the S₂ to S₃ transition.

A recent study investigates photosynthetic electron flow in PsbJ-deficient mutants of Synechocystis PCC6803 and Nicotiana tabacum (30). The tobacco psbJ knockout reported in this study was constructed in a different manner. The aadA gene was inserted directly into the psbJ reading frame, whereas we avoided disruption of the psbE operon structure and placed the aadA in the downstream intergenic spacer (Fig. 2). However, similar physiological data were obtained; oxygen evolution was found to be reduced, and the oscillation of thermoluminescence signals with the number of exciting flashes turned out to be impaired. In addition, the lifetime of the reduced primary quinone acceptor Q was found to be increased more than 100-fold (and hence plastoquinone reduction was drastically slower than in the wild type (30)). The high chlorophyll fluorescence observed by the authors of this study in their fluorescence induction kinetics may be attributable to higher light intensities during plant growth (10 µmol quanta m² s¹ as compared with 2 µmol quanta m² s¹ in our study) and/or to leaf age, which we have shown to be critical parameters for avoiding interference with photooxidative damage (Figs. 5-10). Furthermore, in the present work, we provide new molecular and biochemical data that may explain many of the physiological observations made with PsbJ-deficient plants. Our finding that the knockout of psbJ is associated with the loss of the extrinsic PsbP protein of the water-splitting complex provides a biochemical explanation for the observed low oxygen evolution capacity of the psbJ mutants. In addition, our molecular analyses have revealed an interesting regulatory connection between photosystem II activity and photosystem I synthesis (see below).

Remarkably, the phenotypic effects of psbJ inactivation are much more dramatic in tobacco than in cyanobacteria (29, 30). Our finding that tobacco psbJ deletion mutants are affected in water splitting and lack the extrinsic PsbP protein may explain this difference. The oxygen-evolving complexes of cyanobacteria and plants show remarkable structural differences, the most significant being that the cyanobacterial complex lacks the extrinsic oxygen evolution-enhancing PsbP and PsbQ proteins. Thus, the lack of the PsbJ polypeptide is unlikely to have similarly dramatic consequences for cyanobacterial water splitting as it has for oxygen evolution in plant PSII.

How does the PsbJ protein mediate association of PsbP with PSII? Two possibilities can be envisaged: (i) PsbJ provides a docking site for PsbP binding, or (ii) PsbJ changes the conformation of PSII and, in this way, allows for stable binding of PsbP. In the former case, PsbJ would interact directly with PsbP; in the latter case, a direct physical interaction would not necessarily be involved. Thus far, our attempts to identify protein-protein interaction partners of PsbJ have remained unsuccessful,³ and further experimentation is required to define the exact position of PsbJ in relation to the PSII oxygen-evolving complex. It is noteworthy in this respect that the recently resolved x-ray structure of PSII crystals active in water splitting has revealed the presence of an unassigned helix in proximity to the extrinsic proteins and the manganese cluster of the water-splitting complex (1). However, which of the unassigned helices in PSII corresponds to PsbJ currently remains an open question.

psbJ Tobacco Plants Are Incapable of Photoautotrophic Growth and Exhibit Hypersensitivity to Light-- In the absence of efficient water splitting, the electron transfer reactions in PSII are highly sensitive to light-induced inhibition, commonly referred to as donor-side photoinhibition. Photoinhibition is elicited by the failure to reduce PSII electron-transferring cofactors with electrons generated by oxidation of water molecules. This, in turn, results in the accumulation of highly oxidizing compounds, such as P680+ and oxidized Tyr^z, (for a review, see e.g. Ref. 44), which can severely damage surrounding macromolecules. Inhibition of electron transfer in PSII is ultimately followed by degradation of the PSII reaction center protein D1. Our finding that psbJ plants are impaired in photosynthetic water splitting, which makes them highly susceptible to irreversible donor-side photoinhibition, explains both the incapability of mutant plants to grow photoautotrophically and the accumulation of massive photooxidative damage leading to rapid photobleaching of mutant leaves at higher light intensities (Fig. 5). This is in line with the earlier finding that a mutant of Chlamydomonas (FUD 39), which lacks the extrinsic 23-kDa protein of PSII (PsbP; Ref. 43), shows chloride ion deficiency in PSII and is highly susceptible to photodamage (37, 45).

Interestingly, it has been shown recently that cytochrome b₅₅₉ also undergoes light-induced degradation during photoinhibition of PSII (46). When analyzing early events in light-induced PSII degradation in psbJ knockout plants (Fig. 9), we noticed that the -subunit of cytochrome b₅₅₉ (PsbE) was found to be reduced before a reduction of the reaction center proteins could be detected, possibly suggesting that, upon donor side photoinhibition, degradation of cytochrome b₅₅₉ may precede D1 degradation. Being the first higher plant mutant affected in water splitting, the psbJ mutant provides an excellent model for further studying the molecular events during donor side photoinhibition.

Reduced PSII Activity in psbJ Knockout Plants Leads to Down-regulation of PSI-- An interesting aspect of the psbJ knockout plants was their significantly reduced PSI levels. At first sight, it may seem surprising that the lack of a small PSII protein subunit affects PSI. However, a growing body of evidence supports the idea that the expression of photosynthesis-related genes is influenced by redox signals originating from photosynthesis itself (Refs. 47 and 50; for a recent review, see e.g. Ref. 51). Our comparative analyses of protein and mRNA accumulation in the psbJ mutant and several other photosynthetic mutants (Figs. 8-10 and data not shown) provide genetic support for PSI accumulation being controlled by PSII activity. At present, we cannot yet exactly define the level at which this regulation occurs. Translational regulation of gene expression in photosynthesis is well established (47, 50, 52) and has been suggested to employ redox signals generated in photosynthetic electron transport. Thus, it seems possible that, in psbJ plants, low PSII activity generates a redox signal that negatively regulates translation of PSI genes. Experiments are in progress that test this hypothesis and are hoped to provide insights into how photosystem stoichiometry is regulated.

	ACKNOWLEDGEMENTS

We are grateful to Dr. Pal Maliga for providing the chimeric aadA gene and Drs. Udo Johanningmeier, Alice Barkan, Ralf Oelmüller, Ralf-Bernd Klösgen, and Roberto Bassi for antisera against several thylakoid proteins. We thank Natascha Bondareva for help with PSII preparations, Katharina Kienzler for help with thermoluminescence measurements, and Dr. Michael Hippler for stimulating discussion.

	FOOTNOTES

^* This work was supported by Deutsche Forschungsgemeinschaft Grant BO 1482/1-4, a grant from Graduiertenkolleg "Molekulare Mechanismen pflanzlicher Differenzierung"), and a grant from the State of Baden-Württemberg (Landesforschungsschwerpunkt) (to R. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 49-251-8324790; Fax: 49-251-8328371; E-mail: rbock@uni-muenster.de.

Published, JBC Papers in Press, February 4, 2002, DOI 10.1074/jbc.M112053200

² M. Hager and R. Bock, unpublished results.

³ D. Karcher and R. Bock, unpublished.

	ABBREVIATIONS

The abbreviations used are: PSII, photosystem II; PSI, photosystem I; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES