First published online June 7, 2007; 10.1104/pp.107.100131
Plant Physiology 144:1924-1935 (2007)
© 2007 American Society of Plant Biologists
BIOENERGETICS AND PHOTOSYNTHESIS
Role of the Low-Molecular-Weight Subunits PetL, PetG, and PetN in Assembly, Stability, and Dimerization of the Cytochrome b6f Complex in Tobacco1,[C]
Serena Schwenkert,
Julia Legen2,
Tsuneaki Takami3,
Toshiharu Shikanai3,
Reinhold G. Herrmann and
Jörg Meurer*
Department of Biology I, Botany, Ludwig-Maximilians-University, 80638 Munich, Germany
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ABSTRACT
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The cytochrome b6f (Cyt b6f) complex in flowering plants contains nine conserved subunits, of which three, PetG, PetL, and PetN, are bitopic plastid-encoded low-molecular-weight proteins of largely unknown function. Homoplastomic knockout lines of the three genes have been generated in tobacco (Nicotiana tabacum ‘Petit Havana’) to analyze and compare their roles in assembly and stability of the complex. Deletion of petG or petN caused a bleached phenotype and loss of photosynthetic electron transport and photoautotrophy. Levels of all subunits that constitute the Cyt b6f complex were faintly detectable, indicating that both proteins are essential for the stability of the membrane complex. In contrast,  petL plants accumulate about 50% of other Cyt b6f subunits, appear green, and grow photoautotrophically. However, petL plants show increased light sensitivity as compared to wild type. Assembly studies revealed that PetL is primarily required for proper conformation of the Rieske protein, leading to stability and formation of dimeric Cyt b6f complexes. Unlike wild type, phosphorylation levels of the outer antenna of photosystem II (PSII) are significantly decreased under state II conditions, although the plastoquinone pool is largely reduced in petL, as revealed by measurements of PSI and PSII redox states. This confirms the sensory role of the Cyt b6f complex in activation of the corresponding kinase. The reduced light-harvesting complex II phosphorylation did not affect state transition and association of light-harvesting complex II to PSI under state II conditions. Ferredoxin-dependent plastoquinone reduction, which functions in cyclic electron transport around PSI in vivo, was not impaired in petL.
The cytochrome b6f (Cyt b6f) complex in cyanobacteria and plants resides in thylakoid membranes and links the electron transport between PSII and PSI. It functions as plastoquinol-plastocyanin oxidoreductase and mediates both linear and PSI cyclic electron flow, proton translocation across the membrane, as well as photosynthetic redox control of energy distribution between the two photosystems and gene expression (Allen, 2004 ; Cramer and Zhang, 2006). In flowering plants, the complex is composed of at least nine subunit species, forms a dimer, and is of dual genetic origin. Two subunits, PetC (Rieske FeS subunit) and PetM, are encoded by nuclear genes. The other subunits are encoded in plastid chromosomes. Of these, three genes encode large subunits PetA, PetB, and PetD, representing Cyt f, Cyt b6, and subunit IV, respectively. The remaining low-molecular-weight (LMW) subunits, PetG, PetL, and PetN, are hydrophobic and span the membrane once (de Vitry et al., 1996; Cramer et al., 2005). A further constituent subunit, PetO, which has only been described in Chlamydomonas, is reversibly phosphorylated upon state transition, but could not be detected in the crystal structure (Hamel et al., 2000). In contrast to other subunits analyzed, PetM is not essential for assembly or stability of the complex in cyanobacteria (Schneider et al., 2001, 2007). PetC possesses a highly mobile extrinsic domain and is assumed to contribute to the stability of the dimer by domain swapping (Kurisu et al., 2003). Mutants of PetC in Arabidopsis (Arabidopsis thaliana) and Chlamydomonas are able to assemble the complex (de Vitry et al., 1999; Maiwald et al., 2003); however, analyses of PetC mutants in maize (Zea mays; Miles, 1982), Oenothera (Stubbe and Herrmann, 1982), and Lemna (Bruce and Malkin, 1991) have shown that the FeS protein is essential for assembly of the complex in these organisms.
Crystallographic studies have uncovered structural details of the complex in cyanobacteria and Chlamydomonas (Kurisu et al., 2003; Stroebel et al., 2003). Apart from the constituent subunits and the two cytochromes, Cyt b6 and Cyt f, the complex contains one chlorophyll a and one  -carotene molecule. An unexpected heme (c-type cytochrome) has recently been discovered, which may be involved in the cyclic electron transport around PSI (Kurisu et al., 2003; Stroebel et al., 2003; Cramer and Zhang, 2006). The LMWs are all located at the periphery of the complex, yet the two available crystal structures are not consistent in the exact assignments of PetG, PetL, and PetN. A knockout mutant of petG has been described in Chlamydomonas reinhardtii, demonstrating that the subunit is essential for either stability or assembly of the complex (Berthold et al., 1995). PetN, which represents the smallest open reading frame in the plastid genome, has a comparable stabilizing effect on the cytochrome complex, as judged from a tobacco (Nicotiana tabacum) knockout mutant (Hager et al., 1999). PetN and PetG are likewise essential in Synechocystis (Schneider et al., 2007). Disruption of petL has been reported to cause impaired photoautotrophic growth, reduced electron transfer, and reduced levels of the Cyt b6f complex in Chlamydomonas, but almost no effect in Synechocystis (Takahashi et al., 1996; Schneider et al., 2007). A corresponding knockout in tobacco indicated reduced stability of the complex, especially in aging leaves, although photoautotrophy was not affected (Fiebig et al., 2004; Schöttler et al., 2007). Although all three subunits are present in cyanobacteria and higher plants, the similarity between them in these two lineages is quite different (approximately 73%, approximately 52%, and approximately 29% for petG, petN, and petL, respectively), indicating a relatively high divergence of petL.
Several deletion mutants of petA, petB, and petD, in Chlamydomonas, Oenothera, and tobacco, have been described, which exert different effects in the assembly of the Cyt b6f complex (Herrmann et al., 1985; Kuras and Wollman, 1994; Monde et al., 2000). Two mechanisms have been suggested to explain the significantly reduced levels/stability of all subunits that constitute the complex in most of these mutants. Failure to assemble the complex could be due to faster degradation of improperly assembled proteins, which was verified for petB and petD by in vivo labeling experiments in Chlamydomonas (Kuras and Wollman, 1994). Alternatively, disruption of the hierarchical organization of expression of Cyt b6f complex subunits, designated as a controlled epistasy of synthesis (CES) process, may cause loss of assembly, as is the case for petA in Chlamydomonas. In this alga, each of the thylakoid membrane complexes contains at least one CES subunit, whose translation depends on the availability of its assembly partners (Choquet et al., 2001). It remains to be shown whether CES-like processes also exist in higher plants.
Besides linear and PSI cyclic electron flow and proton translocation, the Cyt b6f complex seems to be involved in redox signaling and state transition (Allen, 2004; Shikanai, 2007). For photosynthetic organisms, it is vital to modify and adjust their light-harvesting capacity depending on environment because high light causes photoinhibition, whereas low light limits photosynthesis. Therefore, regulatory mechanisms to prevent extensive damage include down-regulation of antenna size, nonphotochemical quenching, and redistribution of light-harvesting complex II (LHCII; state transition) to adapt and optimize photosynthetic efficiency. Redistribution of peripheral antenna proteins involves reversible phosphorylation and migration of LHCII proteins between the two photosystems, thus redistributing excitation energy between the photosystems (Aro and Ohad, 2003). Phosphorylation of thylakoid membrane proteins is mediated by two protein kinases, of which at least one (STN7) is controlled by the redox state of the plastoquinone (PQ) pool (Allen, 1992; Wollman, 2001; Bellafiore et al., 2005; Bonardi et al., 2005; Vainonen et al., 2005). It has been shown that the cytochrome complex senses the redox state of the PQ pool and is involved in signal transduction that activates LHCII kinase (Vener et al., 1998; Zito et al., 1999; Wollman, 2001).
To further elucidate the function of the LMW subunits of the Cyt b6f complex in higher plants, we have inactivated petG, petN, and petL in tobacco ‘Petit Havana’. PetG and PetN are essential for the stability of the entire Cyt b6f complex and for photoautotrophic growth. In contrast to this, PetL is dispensable for photoautotrophic growth, but crucial for accumulation of dimeric Cyt b6f complexes and photosynthetic redox regulation in tobacco.
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RESULTS
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Inactivation of petG, petN, and petL
Independent transformants were selected from each knockout type (petG, petL, and petN), compared, and one line of each was finally used for detailed study (Fig. 1, A–C
). Insertion of the aadA cassette into the respective genes and homoplastomy were verified by PCR analysis, sequence, Southern, northern, and western analysis (Swiatek et al., 2003; Fig. 1, D–F; data not shown). A transplastomic, spectinomycin resistance line containing the aadA insertion in a neutral site was used as wild-type control (Ohad et al., 2004). If not otherwise indicated, third to sixth leaves appearing from the meristem were used because transplastomic knockout lines in tobacco frequently exhibit secondary effects in older leaves (Ohad et al., 2004; Schwenkert et al., 2006; Schöttler et al. 2007; Umate et al., 2007).
Phenotypic Characterization of the Mutants
petG and petN deletion mutants lost their ability for photoautotrophic growth. They bleached when grown under normal tissue culture conditions (100 µmol photons m–2 s–1) and even under very low light (4 µmol photons m–2 s–1). Remarkably, their growth was severely retarded, petN stronger than petG, as compared to wild type and homoplastomic petA and petB mutants, also deficient in assembly of the cytochrome complex (data not shown). In contrast to petG and petN, petL did not show any photobleaching and is viable even when grown on soil. PetL protein does not accumulate in petL as judged from immunoblot analysis confirming homoplastomy of the knockout line (Fig. 1G).
Electron Transport Is Retarded in petL and Abolished in petG and petN
Chlorophyll a fluorescence measurements showed that no photochemical quenching occurred in petG and petN at any light intensity, indicating that electron transport is completely blocked. The maximal PSII yield, Fv/Fm, is significantly decreased in petG and petN, but apparently unchanged in petL (wild type 0.81 ± 0.01; petG 0.54 ±0.03; petN 0.58 ± 0.02; and petL 0.78 ± 0.02). This indicates increased light sensitivity as a secondary effect of the petG and petN mutations. At lower light intensities, photochemical quenching in petL is comparable to wild type, but, at increased light intensities, above 500 µmol photons m–2 s–1, the photosynthetic yield is significantly reduced in petL plants (Fig. 2A
), whereas nonphotochemical quenching remains largely unchanged (data not shown). This indicates that, especially at higher light intensities, the PQ pool remains relatively reduced and the electrons are not transferred efficiently from PSII to PSI, presumably due to reduced activity of the cytochrome complex.
PSI yield in terms of the light-induced redox state of P700+ was measured as absorbance changes at 830 nm. Although PSI is functionally not affected in petL, PSI yield is significantly impaired in the mutant, indicating that the electron flow from the Cyt b6f complex to PSI is impaired in the mutant (Fig. 2B).
Accumulation of Cyt b6f Complex Subunits in the Mutants
Immunoblot analysis demonstrated that subunits of the Cyt b6f complex, Cyt f, Cyt b6, and subunit IV, are faintly detectable in both petG and petN. This demonstrates that both LMW subunits are essential for proper assembly of the Cyt b6f complex in higher plants, although they are located peripherally in the complex (Stroebel et al., 2003; Fig. 3
). In petL, subunits of the cytochrome complex accumulate to levels of approximately 50% of the wild type. PSII and PSI components are slightly affected in petG and petN as illustrated by immunoblot analysis with antibodies raised against subunits CP43 and PsaF of these photosystems. Remarkably, levels of ATP synthase subunit  are significantly up-regulated in petN and petG, but unchanged in petL as compared to wild type.
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Figure 3. Immunoblot analysis of wild type, petG, petL, and petN thylakoid membrane proteins. Proteins were separated by SDS-PAGE on a 15% polyacrylamide gel, electroblotted onto polyvinylidene difluoride membranes, and probed with antisera against Cyt f, Cyt b6, and subunit IV of the cytochrome complex, and representative subunits of PSI (PsaF), PSII (CP43), of the ATP synthase - and -subunits. Five micrograms of chlorophyll (100% of wild type and mutant) were loaded per lane.
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Assembly of the Cyt b6f Complex in petL
To investigate the Cyt b6f complex in petL, isolated mutant and wild-type thylakoids were solubilized with 1% -dodecylmaltoside (-DM). The membrane assemblies separated by nondenaturing blue-native (BN) gel electrophoresis in the first dimension were subjected to SDS-PAGE in the second dimension. The components of thylakoid membrane complexes are designated according to serological and mass spectrometric identification (Granvogl et al., 2006; Schwenkert et al., 2006). The analysis revealed that predominantly dimers of the Cyt b6f complex accumulate in the wild type, but surprisingly only the monomeric form of the complex is detectable in silver-stained gels of petL (Fig. 4
). Immunoblot analysis uncovered that the Rieske FeS protein is detached from the monomeric form of the complex in mutant and wild type (Fig. 6B). Assembly and amounts of all other thylakoid membrane complexes are not affected by the mutation.
To clarify whether the dimer in petL is absent due to instability during the solubilization process, milder lysis conditions for solubilization of thylakoid membranes were used. Thylakoid membranes were partially solubilized with 0.2% -DM and the lysates were separated by Suc density centrifugation. Under these conditions, preferentially the LHCII and PSII monomer, ATP synthase, as well as the dimeric and monomeric Cyt b6f complex were released from the membrane, whereas other higher order assemblies tend to remain in the pelleted residual membrane fraction (Fig. 5A
). Even under these mild conditions of solubilization and separation, dimeric cytochrome complexes could not be detected in petL. In contrast to BN-PAGE using 1% -DM, the Rieske FeS protein is not released from the monomer in wild type, but is found detached in petL and detected in the upper part of the gradient containing free proteins (Fig. 5B). This indicates that PetL is involved in stabilizing the Rieske FeS protein.
To investigate further whether loss of PetL prevents dimerization or causes instability of the complex, in vivo radiolabeling experiments were performed. For this, rapidly expanding young leaves of wild-type and mutant plants were incubated in 35S-Met-containing medium. After 40 min of incubation, BN-SDS-PAGE showed predominantly the assembled dimeric form of the Cyt b6f complex and only minor amounts of the monomer in wild type. Again, in petL exclusively, the monomeric form of the Cyt b6f complex could be detected, suggesting that dimer assembly is impaired in the mutant (Fig. 6A
).
It has been reported that amount and thus stability of the cytochrome complex in petL plants could depend on leaf age (Schöttler et al., 2007). Immunoblot analysis of the second dimension performed with leaves of different age probing with antisera elicited against subunits of the cytochrome complex detected monomer and dimer in the wild type, but the dimer was below the limit of detection in petL leaves of middle age (Fig. 6B). However, in the first two leaves appearing from the meristem, traces of dimer were detectable in the mutant (Fig. 6C), indicating that PetL is not primarily required for dimerization, but rather for stability of the dimer. Taken together, both assembly and stability of the complex appear to be more strongly affected, particularly with increasing leaf age. The Rieske protein is not detectable in the monomer both in the wild type and petL, implying that it is lost during solubilization with 1% -DM.
Because the data presented above indicate that an unstable association of the Rieske protein might lead to monomerization in petL, a stable Arabidopsis petC transposon insertion line, which lacks the Rieske protein (Maiwald et al., 2003), was analyzed by BN- and SDS-PAGE. In contrast to other Rieske mutants, lack of the Rieske protein does not prevent accumulation of other Cyt b6f subunits in Arabidopsis. Interestingly, the second dimension, immunoblotted and probed with an antiserum against Cyt f, demonstrated that this mutant is well able to accumulate dimeric Cyt b6f complexes (Fig. 7
).
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Figure 7. Arabidopsis wild-type and petC plants were analyzed by BN-SDS-PAGE. Immunoblot analysis of the second dimension with antisera against Cyt f demonstrates that dimeric and monomeric complexes accumulate at normal levels in the mutant.
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petL Is Defective in Phosphorylation of the LHCII
The phosphorylation state of PSII reaction center proteins and LHCII was investigated by illumination with far-red light of 728 nm (state I) and with increasing light intensities of 650 nm (state II) for 30 min. Thylakoids were then rapidly isolated and immunoblot analysis was performed with anti-phospho-Thr antibodies. In both the wild type and the petL mutant, the PSII reaction center proteins CP43, D1, and D2 were phosphorylated upon illumination. Furthermore, LHCII proteins were more phosphorylated with increasing light intensities. This process appears to be impaired in petL because phosphorylation is reduced to approximately 50% of wild-type levels (Fig. 8A
).
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Figure 8. Phosphorylation patterns and formation of PSI-LHCII supercomplexes. A, Wild-type and petL plants were illuminated with far-red light of 728 nm (state I) or treated with increasing light intensities of 10, 20, and 40 µmol photons m–2 s–1 at 650 nm (state II). Thylakoids were isolated and immunoblot analysis was performed using a phospho-Thr-specific antibody (New England Biolabs). Equal amounts of chlorophyll (5 µg) were loaded per lane. Immunoblot analysis with an antiserum specific for Cyt b559 was performed as a loading control. B, Kinase activation was induced in darkness by addition of reduced duroquinol to isolated thylakoids. Five micrograms of chlorophyll were loaded per lane and equal loading was checked by Coomassie Brilliant Blue (CBB). C, BN-PAGE was performed with thylakoids isolated from plants incubated under state I and state II conditions (40 µmol m–2 s–1). Thylakoids were solubilized with 1.5% digitonin, allowing the detection of PSI-LHCII supercomplex under state II conditions in wild type as shown by immunoblot analysis of the BN-PAGE gel with antisera against LHCBI (bottom). Equal amounts of chlorophyll (100 µg) were loaded per lane. D, Second-dimension SDS-PAGE and immunoblot analysis with phospho-Thr antibodies were performed with the BN gel shown in C. Under state II conditions, an additional complex is detectable, representing an association of LHCII-P with PSI (indicated by arrows). E, Wild type under state I and state II conditions was probed with a PsaC antiserum showing the additional LHCII-P-PSI complex appearing only under state II conditions. [See online article for color version of this figure.]
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To show that hindered LHCII phosphorylation is primarily due to reduced levels of the cytochrome complex and not because of the reduction state of the PQ pool or other secondary effects of the petL mutation, the PQ pool was reduced in darkness by adding duroquinol to isolated wild-type and petL thylakoids. Immunoblot analysis showed that phosphorylation of D1 and D2 proteins was equally activated in both wild type and mutant, but phosphorylation of LHCII remained significantly reduced in petL (Fig. 8B).
Association of the PSI-LHCII supercomplex under state II conditions can be observed in BN gels after solubilization of thylakoids with digitonin (Fig. 8C). In the second dimension of the BN gel, the phosphorylated LCHII can be detected in the free LHCII trimer and PSII supercomplexes, as well as associated with PSI as confirmed by electrophoretic mobility and the use of a specific PsaF antiserum (Fig. 8E). Although there is less LHCII phosphorylation in petL, the PSI-LHCII supercomplex is formed (Fig. 8, C and D). Upon analysis of the BN-SDS-PAGE second dimension with phospho-Thr antibodies, it appeared that equal amounts of PSI-LHCII are present in wild type and mutant, whereas there is less phosphorylated trimeric LHCII in the mutant (Fig. 8D).
PetL Is Not Essential for Ferredoxin-Dependent PQ Reduction
In higher plants, the principal pathway of cyclic electron transport around PSI is sensitive to antimycin A and depends on the function of a small thylakoid protein, PGR5 (Munekage et al., 2002). Activity of PGR5-dependent PSI cyclic electron transport can be detected as ferredoxin (Fd)-dependent PQ reduction in ruptured chloroplasts. It has long been discussed whether this electron transport occurs through the Cyt b6f complex (Kurisu et al., 2003; Stroebel et al., 2003; Okegawa et al., 2005; Cramer and Zhang, 2006). To test this possibility and to evaluate the effect of monomerization of the cytochrome complex on electron transport, the activity of Fd-dependent PQ reduction was measured in petL (Fig. 9
). Although addition of NADPH to ruptured chloroplasts did not change the reduction level of PQ, subsequent addition of Fd reduced PQ in both wild type and petL, as monitored by increased chlorophyll fluorescence (Fig. 9). Both reduction rate and the final level of reduction were almost identical in wild type and petL, indicating that PetL is not essential for PGR5-dependent PSI cyclic electron transport. Addition of antimycin A drastically impaired PQ reduction in both mutant and wild type. The remaining PQ reduction activity depends on the chloroplast NDH complex rather than PGR5 (Munekage et al., 2004). We conclude that PGR5-dependent PQ reduction via Fd does not require dimerization of the Cyt b6f complex.
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Figure 9. Fd-dependent PQ reduction activity in ruptured chloroplasts. Chloroplasts were isolated from wild-type and petL leaves and osmotically shocked in hypotonic buffer (10 µg of chlorophyll/mL). Electron donation to PQ was monitored as increase in chlorophyll fluorescence by addition of 0.25 mM NADPH and 5 µM Fd under weak illumination (1 µmol photons m–2 s–1). To inhibit the PGR5-dependent PQ reduction, 10 µM antimycin A (AA) was added prior to analysis. The fluorescence level was standardized by Fm. [See online article for color version of this figure.]
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DISCUSSION
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To clarify and compare the roles of the plastid-encoded LMW subunits PetG, PetL, and PetN in assembly and stability of the Cyt b6f complex, transplastomic knockout lines were generated in tobacco. Whereas petL grew photoautotrophically, growth of petN and petG was retarded even when cultivated on Suc-supplemented medium in comparison to wild type. The latter mutants lack the cytochrome complex almost completely.
petG and petN Lack the Cyt b6f Complex Almost Completely
petG and petN accumulate only traces of other subunits of the Cyt b6f complex, suggesting that they represent constituent subunits that are required for early stages of complex assembly. Although they reside only peripherally in the complex, their absence causes assembly problems or leads to rapid degradation of the assembly. Deficiencies in petN were less pronounced, indicating that PetG functions in the nucleation process of assembly, whereas PetN represents a later addition. It is surprising that these small subunits exert such a crucial role on the assembly and/or stability of the cytochrome complex, specifically because they are not located at a central position. Either this is due to rapid degradation of the translation products, to a CES-like process involved in the coordination of protein stoichiometry of the cytochrome complex, or to CES-independent assembly deficiencies. This pattern is reminiscent of the essential role of the LMW subunits PsbE, PsbF, PsbL, and PsbJ on the assembly/stability of PSII and photoautotrophic growth in tobacco (Swiatek et al., 2003; Ohad et al., 2004). Other LMW subunits of PSII, like PsbH, PsbI, PsbK, PsbM, and PsbZ, are dispensable for assembly of at least monomeric complexes, but exert crucial roles on light trapping and regulation of the redox components involved in primary photochemical reactions (Swiatek et al. 2001; Minagawa and Takahashi, 2004; Shi and Schröder, 2004; Schwenkert et al., 2006; Umate et al., 2007).
Severe light sensitivity of petN and petG could account for reduced levels of PSI and especially PSII proteins as has been reported earlier for mutants deficient in photosynthesis (Barkan et al., 1986). This could lead to rapid degradation and impaired synthesis of photosynthetic proteins. Higher levels detected for ATP synthase subunits in petN and petG compared to the wild type could be explained as compensation either from a physiological or from a structural point of view (Fig. 3) to compensate for impaired ATP production, to maintain membrane structure, or, rather, to maintain a high protein-lipid ratio. Interestingly, the same effect has been observed for a Cyt b6f complex mutant in Oenothera and Arabidopsis (data not shown), and a reverse effect has been noted in mutants lacking ATP synthase but accumulating higher levels of subunits of the cytochrome complex (Dal Bosco et al., 2004).
PetL Stabilizes Rieske Conformation, Leading to Dimerization of the Cyt b6f Complex
Structural alterations of the cytochrome complex in petL lead to impaired electron flow, as shown by fluorescence and spectroscopic analyses in vivo. PSII quantum yield is reduced at higher light intensities and PSI is far more oxidized in the mutant. The outlined data demonstrate that PetL is either involved in stability or dimerization of the cytochrome complex as has been discussed previously (Takahashi et al., 1996; Breyton et al., 1997). Formation of the dimer is severely impaired and only traces could be found in the two youngest leaves in petL (Fig. 6C). The PetL-deficient dimer is highly unstable and below the limit of detection in mature leaves. Even under mild solubilization conditions and separation in Suc density gradients, the dimeric form of the complex could not be detected in the mutant. In vivo labeling experiments using comparable conditions of solubilization suggest, as well, that dimer formation is affected. In young petL plants, only monomeric complexes are labeled (Fig. 6A). The lower amount of cytochrome complex in petL could be due to faster degradation of the monomeric form of the complex, which accumulates in petL. Nevertheless, photoautotrophic growth was not impaired in tobacco, but considerably in Chlamydomonas (Takahashi et al., 1996).
The Rieske FeS protein is composed of a hydrophilic region and a single C-terminal membrane-spanning hydrophobic stretch. It acts as a link between two monomers, with the membrane domain spanning one monomer and the extrinsic domain stabilizing the other monomer of the complex (Kurisu et al., 2003). It is evident that loss of the Rieske protein from monomeric complexes in petL must be a secondary effect of the mutation because the Rieske protein is essential for PQ oxidation; otherwise, plants would not be viable. The Rieske protein is lost from the monomeric complex in petL even during mild solubilization conditions. This and the fact that it remains attached to the complex under the same conditions in the wild type indicate that PetL is directly involved in the attachment of the Rieske protein to the complex. Applying somewhat harsher solubilization conditions, as is the case in BN gels, the Rieske protein is also missing in the monomeric wild-type cytochrome complex, which could be caused by simultaneous loss of both PetL and the Rieske protein (Breyton et al., 1997).
The recently released crystal structures (Kurisu et al., 2003; Stroebel et al., 2003) assign the PetL protein to the periphery of the complex opposite the dimerization axis. This positioning does not support the idea that the protein is directly involved in the dimerization process. Yet, it is likely that lack of PetL destabilizes the attachment of the Rieske protein, either via conformational changes or its association with the Cyt b6f complex leading to destabilization of the dimer.
We have demonstrated that an Arabidopsis petC mutant has the ability to form a dimeric Cyt b6f complex (Fig. 7) and another petC mutant has been described from Chlamydomonas with a similar phenotype (de Vitry et al., 1999). This suggests that loss of the Rieske protein from the complex is a consequence of monomerization rather than its cause. Therefore, it is evident that loss of PetL leads to a conformational change instead of loss of the Rieske protein, thus preventing dimerization or leading to decreased stability of the dimer.
The Cyt b6f Complex Is Involved in State Transition
Binding of plastoquinol at the quinol oxidation site of the cytochrome complex is directly involved in the activation of LHCII phosphorylating kinases and thus in state transition (Vener et al., 1997; Zito et al., 1999). A transgenic line expressing fusion between petL and petD, which has been described from C. reinhardtii, is not impaired in reducing the PQ pool but is unable to perform state transition (Zito et al., 2002). This work reinforces the direct involvement of the cytochrome complex in the activation of the LHCII kinase in tobacco. LHCII phosphorylation is significantly reduced upon both state II conditions and reduction of the PQ pool in darkness. As phosphorylation in general is not affected, it appears that the reduced level of LHCII phosphorylation is caused by decreased amounts of the cytochrome complex and/or loss of dimerization.
The fact that formation of PSI-LHCII supercomplexes is not affected in petL is consistent with the finding that state transition is not impaired (data not shown; Schöttler et al., 2007). Still, how can the PSI-LCHII supercomplex be formed if there is less phosphorylation? This is explained by the fact that association of the phosphorylated LHCII with the photosystems is a competitive and dynamic process (Wollman, 2001; Rochaix, 2007). Presumably, a limited amount of LHCII-P can be bound to PSI, leading to accumulation of a mobile LHCII-P trimer pool of different size depending on the LHCII phosphorylation status. Indeed, levels of LHCII-P trimer are significantly reduced in the mutant as compared to the wild type (Fig. 8D), although it is evident that accumulation of the trimer is identical (Fig. 8C). Similarly, a Rieske knock-down is not affected in state transition despite a greatly photochemically reduced PQ pool (Anderson et al., 1997).
Dimerization of the Cyt b6f Complex Is Not Essential for PSI Cyclic Electron Transport
In petL, Fd-dependent PQ reduction, which functions in PSI cyclic electron transport in vivo, was not impaired (Fig. 9). This is consistent with the fact that induction of nonphotochemical quenching was not affected in the mutant. We obtained similar results with the Arabidopsis mutant pgr3-2 (Yamazaki et al., 2004), in which levels of PetL and probably also PetG are reduced (data not shown). Previously, we have characterized electron transport in the Arabidopsis mutant pgr1, which has a conditional defect in the activity of the cytochrome complex (Okegawa et al., 2005). Due to an amino acid alteration in the Rieske subunit, the Cyt b6f complex is hypersensitive to lumenal acidification in pgr1 (Munekage et al., 2001; Jahns et al., 2002). We did not find any evidence suggesting that Fd-dependent PQ reduction is affected by the pgr1 defect (Okegawa et al., 2005). None of the results suggests that Fd-dependent PQ reduction in the PGR5-dependent PSI cyclic pathway involves the Cyt b6f complex, although we still cannot rigorously rule out this possibility.
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MATERIALS AND METHODS
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Strategies for Generation of Knockout Constructs and Plastid Transformation
In a transplastomic approach, the petG, petL, and petN genes of tobacco (Nicotiana tabacum ‘Petit Havana’) have been inactivated by replacing all (petG, petN) or most (petL) of the genes with the chimeric amino glycoside 3' adenyl transferase (aadA) cassette conferring resistance to spectinomycin (Koop et al., 1996). The individual constructs were inserted into the pBluescript KS II+ plasmid (Stratagene) and the constructs were introduced into tobacco leaf chloroplasts applying biolistic transformation (Boynton et al., 1988; Svab et al., 1990; Svab and Maliga, 1993). The transformed material was regenerated, selected, and cultured as described recently (Schwenkert et al., 2006).
The BstYI fragment of the tobacco chloroplast genome (position 67,607–69,574 bp) was obtained from clones described earlier (Shinozaki et al., 1986) and cloned into the BamHI site of pBluescript. The Bsu15I-PacI fragment (position 68,531–68,689) spanning the petG region was replaced by the aadA cassette, thus removing the entire petG gene. The HindIII-BspHI fragment (position 28,555–30,968) containing petN was cloned into the pBluescript EcoR32I-Ecl136II sites. The gene was removed completely by replacing the aadA cassette with the internal MunI fragment (position 29,259–29,668). The SalI-SmaI fragment (position 67,607–69,500) was cloned into the corresponding sites of pBluescript. The N-terminal part of petL (position 68,259–68,355) was cut off with NdeI and DdeI, and exchanged with a terminatorless aadA cassette excised with SmaI and SphI. Thus, 19 of the 31 amino acids coding for petL were removed (Fig. 1).
Growth Conditions
Tobacco plants were grown under standard light conditions (100 µmol m–2 s–1) or under lower light regimes (approximately 4–10 µmol photons m–2 s–1) generally at 25°C and 16-h-light/8-h-dark cycles (Osram L85W/25 universal white fluorescent lamps). Plants from photoautotrophic mutant lines were grown on soil under greenhouse conditions. If not otherwise indicated, all analyses were carried out with young leaves of 6- to 8-week-old plants, grown in vitro and under greenhouse conditions (day 26°C, night 20°C), respectively. Tobacco lines carrying the aadA cassette in a neutral insertion site and referred to as RV plants served as wild-type control (Ohad et al., 2004).
Arabidopsis (Arabidopsis thaliana) wild-type (ecotype Landsberg erecta) and mutant plants were grown on Murashige and Skoog medium containing 1.4% Suc in a growth chamber at 30 µmol m–2 s–1, 16-h-light/8-h-dark cycles, and at 22°C.
DNA Gel-Blot Analysis
For Southern analysis, total wild-type and mutant DNA (5 µg/lane) was restricted with appropriate enzymes and the resulting fragments were separated electrophoretically in an agarose gel. DNA transfer from the gel onto nitrocellulose membranes was performed by capillary blotting as described by Sambrook et al. (1989). Hybridization was carried out with a radiolabeled, single-stranded RNA probe generated by in vitro transcription using PCR products generated with oligonucleotides containing a T7 promoter at their 5' ends. The following oligonucleotides were used as templates: 5'-GATAATACGACTCACTATAGGGCACACAATTTAAGTAGATGCG-3' and 5'-GATAATACGACTCACTATAGGGTTAATTAATCAAAGGTCCAA-3' for petG and 5'-GATCCAAAACTTGAGATAATGG-3' and 5'-TTTGTAGCATTTTGGCGACA-3' for petN.
RNA Gel-Blot Analysis
RNA gel-blot analysis was performed as described (Lezhneva and Meurer, 2004). The petL probe was amplified by PCR using oligonucleotides 5'-GGATGGATAGATGTTACAGATGATG-3' and 5'-GTCATTGAGATCATGTCAATTCGGATTA-3'.
Preparation of Thylakoid Membranes, SDS-PAGE, and Immunoblot Analysis
Membrane proteins were isolated essentially as described (Umate et al., 2007). Proteins separated by SDS-Tris-Gly-PAGE (15% acrylamide; Laemmli, 1970) were transferred to polyvinylidene difluoride membranes (GE Healthcare), incubated with monospecific polyclonal antisera (Lezhneva et al., 2004), and visualized by the enhanced chemiluminescence technique (GE Healthcare).
Suc Density Gradient Centrifugation
Isolated thylakoid membranes (final chlorophyll concentration 1 mg/mL) were partially solubilized with 0.2% -DM in 20 mM BisTris, 5 mM NaCl, and 5 mM MgCl2 for 45 min at 4°C. After centrifugation at 18,000g, 4°C for 12 min, the solubilized fraction was layered onto 5% to 30% Suc gradients containing 0.06% -DM. Gradients were run for 18 h in a Beckman SW40Ti rotor at 200,000g (4°C) and fractionated from top to bottom into 38 aliquots of 300 µL each, using an ISCO 640 gradient fractionator. The separation pattern of partially solubilized protein complexes in the gradients was verified by serology.
BN-PAGE
BN-PAGE analysis was performed as described earlier (Schwenkert et al., 2006). Thylakoids were isolated and solubilized with 1% -DM for 10 min or with 1.5% digitonin for detection of PSI-LHCII-P supercomplexes (Zhang and Scheller, 2004) for 1 h, followed by centrifugation at 20,000g for 1 h at 4°C. The solubilized proteins were separated on a 4% to 12% polyacrylamide gradient gel. Excised gel lanes were denatured and analyzed on a 15% polyacrylamide gel containing 4 M urea. The gels were subsequently silver stained (Blum et al., 1987) or used for immunoblotting. Protein spots were assigned as described previously (Granvogl et al., 2006). For in vivo labeling experiments, primary leaves of 2-week-old mutant and wild-type seedlings were incubated for 40 min in 35S-Met at a final concentration of 0.7 µCi µL–1 (GE Healthcare) as described (Amann et al., 2004).
Analysis of Phosphorylated Thylakoid Membrane Proteins
For phosphorylation experiments, all buffers were supplemented with 10 mM NaF. Wild-type and mutant plants were treated with light at 650 (PSII light) or 728 (PSI light) nm. To activate the protein kinase in isolated thylakoids in darkness, reduced duroquinol (1 mM) was added and the assays were incubated for 20 min at room temperature (Allen et al., 1989). The extent of phosphorylation was detected with phospho-Thr antibodies (New England Biolabs).
Chlorophyll a Fluorescence Induction Kinetics
Chlorophyll a fluorescence induction kinetics of wild-type and mutant leaves was measured using a pulse-modulated fluorimeter (PAM101; Walz). Leaves, dark adapted for at least 15 min, were used to analyze minimal (F0) and maximal (Fm) fluorescence yields, the latter being determined by application of a saturating light pulse (1-s duration, 4,500 µmol photons m–2 s–1). The potential maximum quantum yield of PSII was measured as (Fm – F0)/Fm = Fv/Fm. (Schreiber et al., 1998).
PSI Activity
PSI yield was measured on leaves as absorption changes at 820 nm induced by saturating pulses and far-red light (12 W m–2 as measured with a YSI Kettering model 65 A radiometer) in the absence or presence of actinic light (650 nm, 20 and 250 µmol m–2 s–1) using the PSI attachment of PAM101 (Walz; Klughammer and Schreiber, 1994).
In Vitro Assay of PSI Cyclic Electron Transport Activity
Chloroplast isolation and assay of Fd-dependent PQ reduction in ruptured chloroplasts were performed as described (Munekage et al., 2002).
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ACKNOWLEDGMENTS
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We would like to thank Martina Reymers and Gisela Nagy for excellent technical assistance. We are grateful to Vera Bonardi for helpful suggestions. Further, we would like to express our gratitude to Dario Leister for critical reading of the manuscript and providing the petC mutant. Jean-David Rochaix is acknowledged for supplying PetL antibodies.
Received March 23, 2007;
accepted June 2, 2007;
published June 7, 2007.
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FOOTNOTES
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1 This work was supported by the German Science Foundation (DFG; grant SFB TR1 to J.M. and R.G.H.). 
2 Present address: Institute of Biology, Department of Biology, Chemistry and Pharmacy, Freie Universität Berlin, Koenigin-Luise-Str. 12-16, 14195 Berlin, Germany.
3 Present address: Graduate School of Agriculture, Kyushu University, Fukuoka 812–8581, Japan.
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: Jörg Meurer (joerg.meurer{at}lrz.uni-muenchen.de).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
www.plantphysiol.org/cgi/doi/10.1104/pp.107.100131
* Corresponding author; e-mail joerg.meurer{at}lrz.uni-muenchen.de; fax 49–89–1782274.
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