Mark A Schöttler
email: schoettler@mpimp-golm.mpg.de

Claudia Flügel

Wolfram Thiele

Sandra Stegemann

Ralph Bock

Mark Aurel

The plastome-encoded PsaJ subunit is required for efficient photosystem I excitation, but not for plastocyanin oxidation in tobacco

Keywords: chlorophyll, CTAB -cetyltrimethylammoniumbromide, cytcytochrome, cyt photosynthesis, photosystem I, PsaJ, plastocyanin, Lhca

The functions of several small subunits of the large photosynthetic multiprotein complex photosystem I (PSI) are not yet understood. To elucidate the function of the small plastome-encoded PsaJ subunit, we have produced knock-out mutants by chloroplast transformation in tobacco (Nicotiana tabacum).

PsaJ binds 2 chlorophyll-a molecules and is localized at the periphery of PSI, close to both the Lhca2 and Lhca3 docking sites and the plastocyanin binding site. Tobacco psaJ knock-out lines do not display a visible phenotype. Despite a 25% reduction in the content of redox-active PSI, neither growth rate nor assimilation capacity are altered in the mutants. In vivo, redox equilibration of plastocyanin and PSI is as efficient as in the wild type, indicating that PsaJ is not required for fast plastocyanin oxidation. However, PsaJ is involved in PSI excitation: Altered 77 K chlorophyll-a fluorescence emission spectra and reduced accumulation of Lhca3 indicate that antenna binding and exciton transfer to the PSI reaction center are impaired in ∆psaJ mutants. Under limiting light intensities, growth of ∆psaJ plants is retarded and the electron transport chain is far more reduced than in the wild type, indicating that PSI excitation might limit electron flux at sub-saturating light intensities. In addition to defining in vivo functions of PsaJ, our data may also have implications for the interpretation of the crystal structure of PSI.

Introduction: Photosystem I (PSI) is a multisubunit protein complex in the photosynthetic membranes of higher plants, algae and cyanobacteria. It consists of 15 core subunits and 5 different antenna proteins in higher plants [1,2]. PSI catalyzes the final step of linear electron transport, the light-driven oxidation of plastocyanin (PC) and the reduction of ferredoxin. Despite the existence of a crystal structure of higher plant PSI [3], the exact functions of several subunits are still unknown. Best understood is the role of the three plastome-encoded subunits PsaA, PsaB and PsaC, which are essential for PSI assembly and function, because they bind all redox-active cofactors: PsaA and PsaB form the PSI reaction center heterodimer and bind the majority of the cofactors and antenna chlorophylls (chl.), whereas PsaC binds the cofactors F A and F B on the PSI acceptor side. In the absence of any of these subunits, functional PSI cannot accumulate in photosynthetic eukaryotes [START_REF] Takahashi | Directed chloroplast transformation in Chlamydomonas reinhardtii: insertional inactivation of the psaC gene encoding the iron sulfur protein destabilises photosystem I[END_REF][START_REF] Wostrikoff | Biogenesis of PSI involves a cascade of translational autoregulation in the chloroplast of Chlamydomonas[END_REF].

In higher plants, the function of the nuclear-encoded subunits has been elucidated in recent years using RNAi, antisense techniques and insertional mutagenesis in Arabidopsis thaliana (reviewed in [START_REF] Scheller | Role of subunits in eukaryotic photosystem I[END_REF][START_REF] Jensen | Molecular dissection ofphotosystem I in higher plants: topology, structure and function[END_REF]). This work has revealed non-essential functions of most nuclear-encoded PSI subunits, with the exception of PsaD, which is essential for PSI accumulation and is involved in the formation of the ferredoxin-binding site [START_REF] Ihnatowicz | Mutants of photosystem I subunit D of Arabidopsis thaliana: effects on photosynthesis, photosystem I stability and expression of nuclear genes for chloroplast functions[END_REF][START_REF] Haldrup | Arabidopsis thaliana plants lacking the PSI-D subunit of photosystem I suffer severe photoinhibition, have unstable photosystem I complexes, and altered redox homeostasis in the chloroplast stroma[END_REF]. All other knock-out mutants displayed much weaker phenotypes, often including a reduced PSI content due to destabilization of the complex and sometimes showing altered antenna binding and exciton transfer to the reaction center [START_REF] Ihalainen | Pigment organization and energy transfer dynamics in isolated photosystem I (PSI) complexes from Arabidopsis thaliana depleted of the PSI-G, PSI-K, PSI-L, or PSI-N subunit[END_REF][START_REF] Jensen | The PSI-O subunit of plant photosystem I is involved in balancing the excitation pressure between the two photosystems[END_REF][12].

In contrast, the functions of the plastome-encoded small PSI subunits, PsaI and PsaJ, has not yet been resolved in higher plants. In the unicellular green alga Chlamydomonas reinhardtii, PsaJ is required for the stabilization of the PC-binding site [13]. In the absence of PsaJ, a large fraction of photooxidized P 700 is not efficiently reduced by PC or cytochrome c 6 , although the PsaF subunit, which forms the actual binding site for both mobile redox carriers, is still present. This has suggested a role of PsaJ in adjusting the conformation of the PC binding site. These physiological data are circumstantially supported by structural data [3] and cross-linking studies [14] revealing a localization of the J-subunit adjacent to PsaF. Furthermore, in a Synechocystis ∆psaJ mutant, PsaF and PC binding were also affected [15]. In addition, as PsaJ is known to bind 2 chl.-a molecules in higher plants [16] and is also localized close to the binding sites of the Lhca2 and Lhca3 antenna proteins, an involvement in exciton transfer to the PSI reaction center has been proposed [16]. However, this hypothesis has not yet been addressed experimentally in any photosynthetic eukaryote.

To investigate both the possible role of PsaJ in the interaction between PC and PSI and the proposed function in light harvesting, we have constructed a psaJ knockout mutant in tobacco (Nicotiana tabacum L.). The availability of a workable chloroplast transformation technology for tobacco plants facilitates the construction of knock-out mutants for plastome-encoded genes and open reading frames [17,[START_REF] Hager | Targeted inactivation of the smallest plastid-genome encoded open reading frame reveals a novel and essential subunit of the cytochrome-b(6)complex[END_REF]. The wild-type (WT) copy of the gene of interest is replaced by homologous recombination with a knock-out allele generated by gene disruption with a selectable marker, most often the aadA gene conferring resistance to the aminoglycoside antibiotics spectinomycin and streptomycin [START_REF] Maliga | Plastid transformation in higher plants[END_REF][START_REF] Bock | Taming plastids for a green future[END_REF]. Analyzing a psaJ knockout transformant, we demonstrate here that the PsaJ protein is not required for efficient PC oxidation under physiological conditions, but that it is instead involved in exciton transfer to the PSI reaction center.

Experimental

Plant growth -WT tobacco (Nicotiana tabacum L. var. Petit Havana) and the ∆psaJ transformants were grown on soil in controlled environment chambers at approximately 600 µE m -2 s -1 light intensity at the uppermost leaves. The day length was set to 16 h, day temperature was 22° C, relative humidity 75%. Night temperature was 18°C, relative humidity was decreased to 70%. For low-light experiments, the growth conditions were as stated above, except that the light intensity was reduced to 20 µE m -2 s -1 .

Vector construction and chloroplast transformation -Plastid transformation vector p∆psaJ was derived from a cloned tobacco ptDNA fragment. A 1871 bp SpeI restriction fragment was subcloned into the unique SpeI site of vector pBluescript SK+ which had been dephosphorylated after linearization by treatment with calf intestinal phosphatase. The psaJ reading frame was disrupted by partial digestion with BglII and recovery of the linearized fragment followed by blunting of the overhanging ends by a fill-in reaction with the Klenow fragment of DNA polymerase I from E. coli (New England BioLabs). Subsequently, a chimeric aadA gene [START_REF] Svab | High frequency plastid transformation in tobacco by selection for a chimceric aadA gene[END_REF] was inserted as a blunt-end Ecl136II/DraI fragment into the blunted BglII site within the psaJ coding region. A clone was selected that contained the plastid selectable marker gene aadA in the same transcriptional orientation as the psaJ gene. This clone was designated p∆psaJ and used as chloroplast transformation vector.

Young leaves from sterile tobacco plants were bombarded with plasmid p∆psaJ-coated 0.6 µm gold particles using a biolistic gun (PDS1000He; BioRad). Primary spectinomycin-resistant lines were selected on RMOP regeneration medium containing 500 mg/l spectinomycin [START_REF] Svab | High frequency plastid transformation in tobacco by selection for a chimceric aadA gene[END_REF]. Spontaneous spectinomycin-resistant plants were eliminated by double selection on medium containing spectinomycin and streptomycin (500 mg/l each; [START_REF] Svab | High frequency plastid transformation in tobacco by selection for a chimceric aadA gene[END_REF][START_REF] Porra | Determination of accurate extinction coeffiicent and simultaneous equations for essaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy[END_REF]. Several independent transplastomic lines were subjected to three additional rounds of regeneration on RMOP/spectinomycin to enrich the transplastome and isolate homoplasmic psaJ knock-out lines.

Isolation of nucleic acids, polymerase chain reactions (PCR) and DNA gel blot analyses -

Total plant nucleic acids were isolated from leaf tissue samples by a cetyltrimethylammoniumbromide (CTAB)-based method. For gel blot experiments, DNA samples were digested with restriction enzymes, separated on 0.8 % agarose gels and blotted onto Hybond N nylon membranes (Amersham Biosciences, Little Chelfont, UK). For hybridization, α[ 32 P]dATP-labeled probes were generated by random priming (Multiprime DNA labeling kit, Amersham). A PCR product covering the entire psaJ coding region (generated with primers PpsaJf 5' GGTTTTTCAATGCGAGATCTA 3' and PpsaJr 5' CATGACAATAACTAGAATGAA 3') was used as probe for the restriction fragment length polymorphism (RFLP) analyses. Hybridizations were carried out at 65°C in Rapid Hybridization Buffer (Amersham) following the manufacturer's protocol. DNA samples were amplified with psaJspecific primers in an Eppendorf thermocycler using Taq DNA polymerase (Promega) and standard protocols (50 ng total genomic DNA in 50 µl reactions). The PCR program was 35 cycles of 45 s at 94°C, 90 s at 56°C and 90 s at 72°C with a 4 min extension of the first cycle at 94°C and a 6 min final extension at 72°C. PCR products were analyzed by electrophoretic separation in 1.5 % agarose gels.

Gas exchange measurements -Leaf assimilation capacity was determined in saturating light (800 µE m -2 s -1 , generated by a tungsten halogen lamp, Schott, Mainz, Germany) in a closed cuvette system with a Clark-type oxygen electrode (LD-2, Hansatech, Norfolk, GB). Leaf discs (10 cm 2 surface area) were measured in a CO 2 -enriched atmosphere (10% CO 2 ), to completely suppress photorespiration. Assimilation was measured until steady state. The chl. content of the leaf discs was determined after extraction in 80% acetone [START_REF] Porra | Determination of accurate extinction coeffiicent and simultaneous equations for essaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy[END_REF]. Assimilation rates were corrected for dark respiration, assuming comparable respiration rates in the light as in darkness [START_REF] Raghavendra | Beneficial interactions of mitochondrial metabolism with photosynthetic carbon assimilation[END_REF]. It was shown previously that the assimilation capacity determined under these conditions closely correlates with the maximum capacity of linear electron flux [START_REF] Schöttler | The role of plastocyanin in the adjustment of photosynthetic electron transport to the carbon metabolism in tobacco[END_REF].

Thylakoid membrane isolation and treatments with chaotropic agents -Thylakoid membranes were isolated as described [START_REF] Schöttler | The role of plastocyanin in the adjustment of photosynthetic electron transport to the carbon metabolism in tobacco[END_REF]. The chl. contents and a/b ratio were determined in 80% acetone [START_REF] Porra | Determination of accurate extinction coeffiicent and simultaneous equations for essaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy[END_REF].

For the in vitro stability measurements of PSI, thylakoid membranes equivalent to 200 µg chl. ml -1 were incubated in medium A (5 mM MgCl 2 , 30 mM KCl, 40 mM HEPES, pH 7.6) as a control or in medium A containing either 1 M NaCl, 2 M KI or 2 M NaBr. After 10 min of incubation in the chaotropic salt solution, thylakoids were sedimented by centrifugation (10.000 x g for 1 min) and resuspended in medium A.

Fluorometry -

The 77 K chl.-a fluorescence emission spectra were measured using a Jasco F6500 fluorometer with a red-sensitive photomultiplier. Samples equivalent to 10 µg chl. ml -1 were excited at 430 nm wavelength (10 nm bandwidth), and emission spectra were measured from 660 to 800 nm wavelength with 0.5 nm steps and a bandwidth of 1 nm. The spectra were corrected for the instrumental response of the photomultiplier. Room temperature chl.-a fluorescence of leaves was measured using a Dual-PAM instrument (Walz, Effeltrich, Germany). The redox state of Q A and of the PQ pool was calculated from (1-qL), a parameter more accurate than the commonly used (1-qP) [START_REF] Kramer | New fluorescence parameters for the determination of Q(A) redox state excitation energy fluxes[END_REF]. To determine the capacity for state transitions, leaves were excited using a red light LED (λ = 660 nm, 80 µE m -2 s -1 light intensity) and either blue light for predominant PSII excitation and PQ pool reduction (λ = 440 nm, 25 µE m -2 s -1 ) or far-red light (λ = 715 nm) for preferential excitation of PSI and PQ pool oxidation. Measurements were performed and analyzed according to [26].

Cyt-bf and PSII quantification -The cytochromes of the thylakoid membrane were determined in isolated thylakoids equivalent to 50 µg chl. ml -1 after destacking in a low-salt medium, to improve the optical properties of the sample [27]. The cytochromes were oxidized by addition of 1 mM ferricyanide and subsequently reduced by addition of 10 mM ascorbate and dithionite, resulting in reduction of cyt-f and the high potential form of cyt b 559 (HP, ascorbate -ferricyanide difference absorption spectrum) and reduction of cyt-b 6 and the low-potential form of cyt-b 559 (LP, dithioniteascorbate), respectively. At each redox potential, absorption spectra were recorded between 575 and 540 nm wavelengths with a spatial resolution of 0.2 nm using a Jasco V550 spectrophotometer (Jasco, Groß-Umstadt, Germany) with a head-on photomultiplier. The monochromator slid width was set to 1 nm. Difference absorption spectra were deconvoluted, using reference spectra and difference extinction coefficients for the cytochromes as described [27]. PSII contents were calculated from the sum of the cyt-b 559 HP and LP difference absorption signals [28].

PC and P 700 redox kinetics and quantification -Difference absorption signals of PC and P 700 were measured in the far-red range of the spectrum, essentially as described [START_REF] Kirchhoff | Plastocyanin redox kinetics in spinach chloroplasts: evidence for disequilibrium in the high potential chain[END_REF]. The contributions of PC and P 700 were deconvoluted by measuring difference absorption changes at 830-870 nm (predominantly arising from P 700 ) and 870-950 nm (predominantly arising from PC). Measurements were done using a novel instrument developed in cooperation with C. Klughammer and U. Schreiber (in preparation). This instrument allows the simultaneous determination of both difference absorption signals. Measurements were done on pre-illuminated intact leaves with fully activated Calvin cycle, so that a limitation of P 700 photooxidation by metabolic NADP + regeneration could be excluded. P 700 and PC were photooxidized using far-red light (715 nm wavelength), which selectively excites PSI. After 10 s of far-red light, a saturating pulse of red light (6000 µE m -2 s -1 , 200 ms duration) was applied and the far-red light was switched off, so that PC and P 700 could become fully reduced again after the end of the actinic light pulse. The reduction kinetics were fitted with a exponential function to determine the halftimes of PC and P 700 reduction.

For PSI quantification in isolated thylakoids, membranes equivalent to 50 µg chl. ml -1 were solubilized in medium A (see above) containing 0.2% (w/v) DDM, 100 µM methylviologen as artificial electron acceptor and 10 mM ascorbate as electron donor. P 700 , determined as described for measurements on intact leaves, was photooxidized by application of a saturating pulse of red light (6000 µE m -2 s -1 light intensity, 200 ms duration). For in vitro measurements of P 700 and PC redox kinetics, 1 µM PC, isolated as described [START_REF] Kirchhoff | Plastocyanin redox kinetics in spinach chloroplasts: evidence for disequilibrium in the high potential chain[END_REF], together with 10 mM ascorbate and 100 µM tetramethyl-1.4-phenylendiamine (TMPD) were used as donor system.

Protein electrophoresis and immunoblotting -Thylakoid proteins were separated by SDSpolyacrylamide gel electrophoresis using a Perfect Blue twin gel system (PeqLab GmbH, Erlangen, Germany). Proteins were transferred to a PVDF membrane (Hybond P, Amersham Biosciences, Little Chelfont, UK) using a semidry blot system (SEDEC-M, PeqLab) and a standard transfer buffer (25 mM Tris, 192 mM glycine, pH 8.3). Immunoblot detection was done using the ECL system (Amersham Biosciences) according to the instructions of the manufacturer. Antibodies against Lhca1-4 and against Lhcb proteins were purchased from Agrisera AB (Vännäs, Sweden). All immunoblot data were confirmed for three independently grown batches of WT and mutant plants.

Results

Construction of psaJ knock-out mutants and isolation of homoplasmic lines -To facilitate

the functional analysis of psaJ in higher plants, we constructed a psaJ knock-out by disrupting the reading frame with the selectable marker gene aadA (Fig. 1A). The psaJ knock-out allele was then introduced into the tobacco plastid genome by biolistic chloroplast transformation. Two homologous recombination events in the regions flanking the disrupted psaJ incorporate the knock-out allele into the plastid genome where it replaces the functional WT allele (Fig. 1A). Selection of bombarded leaf samples for resistance to spectinomycin conferred by the chimeric aadA marker gene yielded several transplastomic lines four of which were subjected to additional rounds of regeneration and selection to obtain homoplasmic tissue [START_REF] Svab | High frequency plastid transformation in tobacco by selection for a chimceric aadA gene[END_REF]. After three such rounds, plants were regenerated, rooted in sterile culture, subsequently transferred to soil and grown to maturity in the greenhouse. Seeds were obtained and analysis of the T1 generation by seed assays confirmed that the progeny was uniformly resistant to spectinomycin (data not shown). This suggested that the transformed chloroplast genome carrying the psaJ-disrupting aadA gene was present in a homoplasmic state and was inherited uniparentally maternally, as typical of a chloroplast-encoded trait [START_REF] Bock | Transgenic plastids in basic research and plant biotechnology[END_REF]. All plants from all independently generated transplastomic lines were phenotypically identical (see below) and two independent lines (Nt-∆psaJ#9

and Nt-∆psaJ#20) were selected for further analysis. To verify homoplasmy moleculary and to confirm correct integration of the selectable marker gene aadA into the psaJ reading frame, RFLP assays (Fig. 1B) were performed. These experiments ultimately verified that the WT allele had been replaced by the knock-out allele in all copies of the chloroplast genome (Fig. 1B and data not shown).

As all photosynthetic parameters (see below) of these two lines were highly similar, only average values for both lines will be presented.

Growth phenotype and functional organization of the photosynthetic apparatus in ∆ ∆ ∆ ∆psaJ

plants -When grown under standard conditions for tobacco plants (600 µE m -2 s -1 , 16 h daylength), ∆psaJ plants did not display any visible phenotype (Fig. 1C). Growth, leaf size, number of leaves and flowering time were identical with WT plants (Fig. 1C). As some phenotypes of photosynthesis mutants become more pronounced with increasing leaf age, we determined all photosynthetic parameters for leaves of different ages from fully grown plants at the onset of flowering. As the youngest leaf, leaf number four (from the top of the plant) was analyzed, which was already largely expanded. In addition, leaves number six, nine and eleven were measured, with leaf number eleven being the oldest leaf not yet displaying any visible symptoms of senescence, although assimilation capacity (Fig. 2A) and chl. content (Fig. 2B) were already severely reduced. In both WT and ∆psaJ plants, chl. contents were highest in the youngest leaves (525 mg chl. m -2 ), and decreased continuously with increasing leaf age to leaf number eleven, which contained only about 65% of the chl. content of leaf number four. No significant differences between the WT and ∆psaJ were observed (Fig. 2B).

Biochemical Journal Immediate Publication. Published on 9 Jan 2007 as manuscript BJ20061573

Copyright 2007 Biochemical Society However, interestingly, chl.-a/b ratios differed between WT and ∆psaJ plants (Fig. 2C): In ∆psaJ plants, the chl.-a/b ratio was slightly increased, the difference to the WT becoming more pronounced with increasing leaf age. Although such changes of the chl.-a/b ratio usually indicate alterations in the stoichiometries of the photosynthetic complexes, leaf assimilation capacities did not differ between the WT and the mutants (Fig. 2A): Again, the highest assimilation rates were obtained for the youngest leaves (about 600 µmoles electron pairs (EP) mg chl. -1 h -1 ), and declined to about half of the maximum assimilation capacity in the old leaves (275 µmoles EP mg chl. -1 h -1 ). These data already indicate that loss of PsaJ does not impair linear electron flux under saturating light intensities, suggesting that PsaJ is not essential for PSI assembly or function. To determine the molecular basis of the altered chl.-a/b ratios, we isolated thylakoids from the leaves used for the assimilation measurements. The contents of all redox-active proteins and protein complexes of the thylakoid membrane were determined by difference absorption spectroscopy and immunoblotting. The contents of PSII (Fig. 2D), cytochromebf-complex (cyt-bf) (Fig. 2E), and PSI (Fig. 2G), derived from difference absorption signals of cyt-b 559 (PSII), cyt-b 6 and cyt-f (cyt-bf), and P 700 (PSI), were determined in solubilized thylakoid membrane fragments. The PC contents, relative to P 700 , were determined in intact leaves, and absolute PC contents were calculated based on the P 700 quantification in solubilized thylakoids (Fig. 2F).

Significant differences between WT and ∆psaJ plants were obtained for the two photosystems: In WT plants, the PSI contents were about 2.3 mmoles PSI mol chl. -1 , independent of leaf age (Fig. 2G). In ∆psaJ knock-out plants, the PSI contents were significantly reduced already in leaf number four (approximately 80% of WT contents), and decreased further with increasing leaf age.

In the oldest leaves, only about 70% of the PSI contents in the WT were detected. Conversely, PSII accumulated to slightly higher amounts in the ∆psaJ mutant than in the WT (Fig. 2D). Whereas young WT leaves contained about 3.0 mmoles PSII mol chl. -1 , mutant leaves contained 3.7 mmoles PSII mol chl. -1 . The increase in PSII contents in ∆psaJ may reflect a redistribution of chl. in favor of PSII due to the reduced PSI content, so that the relative proportion of PSII increases. With increasing leaf age and decreasing leaf assimilation capacity, a reduction of PSII contents was observed in both the WT and the mutant. For PC and cyt-bf contents (Figs. 2E and2F), no significant differences between WT and ∆psaJ plants were detected. Both cyt-bf and PC accumulated to highest amounts in the youngest leaves, and decreased in parallel with assimilation capacity (Fig. 2A).

By and large, these spectroscopic quantifications were confirmed by immunoblot analyses using marker subunits of the photosynthetic complexes (Fig. 3). To test the dynamic range of the immunobiochemical detection reactions, a dilution series of the WT samples (100%, 50%, 20% and 10%) was used. Proteins from WT and ∆psaJ leaves number four, six, nine and eleven were semiquantitatively analyzed by direct comparison with the dilution series. As a diagnostic subunit of PSII, the essential D2 protein encoded by psbD was used. Due to high sequence homology with the slightly smaller D1 protein (PsbA), a double band is always observed with our anti-D2 antibody. No obvious differences between the WT and the ∆psaJ mutant were observed with this antibody. This is Copyright 2007 Biochemical Society unsurprising, because the spectroscopic measurements provide a much more precise quantification of complex abundance and the slight increase in PSII contents in the mutant (Fig. 2D) was far too small to result in a visual quantitative effect in the immunoblot. For the cyt-bf, the essential subunit cyt-f (PetA) was used as a diagnostic subunit [START_REF] Monde | Post-transcriptional defects of tobacco chloroplast mutants lacking the cytochrome-bf complex[END_REF]. Here, the about 50% decrease of cyt-bf content with increasing leaf age is clearly visible. For the PsaC protein, which binds the stromal iron-sulfur clusters of PSI and is essential for PSI accumulation [START_REF] Wostrikoff | Biogenesis of PSI involves a cascade of translational autoregulation in the chloroplast of Chlamydomonas[END_REF], the signals obtained in the mutants are slightly weaker than in the WT. However, the measured 20-30% lower PSI amounts in the mutant are at the borderline of detectability with immunoblotting techniques and the spectroscopic data provide a much more reliable dataset here. A strong leaf-age dependent decrease was observed for the marker subunit of the ATP synthase, AtpA, both in the WT and the mutant. The weak lower band results from a crossreaction of our antibody with the AtpB subunit, which shares some sequence similarity with AtpA. In essence, within the detection limits of immunoblots (which are only semiquantitative), the spectroscopic results were confirmed.

PSI stability and function -To determine if the reduced PSI contents in the ∆psaJ mutants (Fig. 2G) can be attributed to a reduced stability of PSI in the absence of its J-subunit, WT and mutant plants were subjected to stress treatments that potentially affect PSI. To assess PSI stability in vivo, tobacco plants were subjected to chilling stress. Plants were transferred to 4°C and 100 µE m -2 s -1 light intensity. These conditions have been described to selectively damage PSI, due to inhibition of the Mehler-Asada cycle and accumulation of reactive oxygen species on the PSI acceptor side. In Arabidopsis and barley, a few hours of cold stress were sufficient to result in the loss of up to 50% of PSI [32,[START_REF] Scheller | Photoinibition of photosystem I[END_REF]. Surprisingly, short term cold stress of up to 24 h had no effect on the amount of redoxactive PSI and on assimilation (data not shown). PSI damage was detectable only after several days of chilling stress, but no differences could be observed between the WT and the ∆psaJ mutant (not shown). Apparently, tobacco is far more tolerant to cold stress than Arabidopsis thaliana and barley.

Additional treatments that potentially destabilize PSI were performed in vitro using isolated thylakoids, which were subjected to repetitive cycles of freezing and thawing, to heat stress (10 min incubation at 70°C) or to incubation in chaotropic salt solutions. Afterwards, thylakoids were partly solubilized by addition of 0.2% (w/v) β-dodecyl-maltoside (DDM) to optimize the optical properties of the sample. Ascorbate was used as electron donor to keep P 700 reduced in darkness. Methylviologen functioned as electron acceptor during photooxidation. Up to 15 cycles of repetitive freezing in liquid nitrogen and thawing at 35°C had no significant effect on the amplitude of the P 700 difference absorption signal (Fig. 4A). In response to heat stress (10 min incubation at 70° C), a 20% reduction in the maximum amplitude of P 700 became apparent. However, no difference between the WT and the ∆psaJ mutant was observed. Also, 10 min incubation with different chaotropic reagents did not result in significantly different effects on the maximum oxidizable fraction of P 700 in the WT and the mutant.

Both 2 M KI and 2 M NaBr resulted in some reduction of the P 700 amplitude, the effect being slightly more pronounced in mutant thylakoids. Due to high standard deviations, these differences are not Copyright 2007 Biochemical Society statistically significant. Evidently, the J-subunit is not required to stabilize the intra-complex electron transfer from P 700 to the artificial acceptor methylviologen.

However, interestingly, solubilization of PSI with 0.2 % (w/v) DDM resulted in a clearly delayed P 700 reduction in the ∆psaJ mutant after the end of the saturating light pulse (not shown). To analyze this effect in more detail, and to exclude that different reduction kinetics of WT and mutant PSI might be due to different PC contents still associated with PSI after solubilization, P 700 redox kinetics of solubilized PSI particles were measured in the presence of 1 µM PC, with 10 mM ascorbate and 100 µM TMPD as electron donors to PC (Fig. 4B). In the WT, almost 85% of P 700 were reduced with a halftime of less than 8 ms by PC. Only a small fraction of approximately 15% of PSI displayed an extremely slow reduction (halftime > 500 ms). As the relative proportion of this fraction was independent of PC contents added to the sample, we conclude that this fraction represents PSI with damaged donor side, which cannot bind PC efficiently anymore. In the ∆psaJ mutant, the slow fraction was much larger with 50% of PSI being inefficiently reduced (Fig. 4B). Therefore, in the ∆psaJ mutant, the PSI donor side seems to be much more prone to damage or suffers from aberrant conformational changes, comparable to the situation in a Chlamydomonas psaJ mutant, where in vitro PSI redox kinetics also revealed a large fraction of P 700 not being efficiently reduced by PC or cytochrome c [13]. To test whether defects in PC-P 700 interaction are also observable in vivo, a detailed analysis of the redox equilibration between PC and P 700 was conducted on intact leaves.

Redox equilibration of PC and P 700 -

To analyze the in vivo redox equilibration of PC and P 700 , intact leaves were pre-illuminated to fully activate the Calvin cycle and avoid an acceptor side limitation of P 700 . Complete photooxidation of the "high-potential chain" comprising cyt-f, PC and P 700 and complete photoreduction of the plastoquinone (PQ) pool was achieved by applying an oversaturating light pulse (6000 µE m -2 s -1 , 200 ms duration). After the flash, both the over-saturating light pulse and the actinic illumination were switched off (time point 0 in Fig. 5A), and the reduction of PC and P 700 by electrons from the PQ pool was determined. In Fig. 5A, exemplary kinetics of PC and P 700 reduction measured in young leaves are shown. The fully oxidized states of PC and P 700 at the end of the light pulse were normalized to one, the completely reduced states were normalized to zero.

Reduction halftimes of P 700 of approximately 3.5 ms were obtained, without any significant differences between WT and the ∆psaJ mutant (Fig. 5A). With increasing leaf age, the reduction halftime of P 700 increased in both WT and mutant plants to about 10 ms (data not shown), which reflects the about 50% reduction in assimilation capacity of older leaves (cp. Fig. 2A). In accordance with the higher redox potential of P 700 , electrons accumulated in the PC pool only when the majority of P 700 was reduced. No significant differences between the WT and the ∆psaJ mutant were observed (Fig. 5A). Although the PC and P 700 reduction kinetics normally are limited by PQ re-oxidation at cytbf, a severe disturbance of the interaction between PC and P 700 as observed in the mutant in vitro (Fig. 4B), is expected to result in a visible delay of net P 700 reduction and an accelerated electron accumulation at PC. This was not observed, strongly arguing against a role of PsaJ in PC oxidation in higher plants. To test for subtle changes in the interaction between PC and P 700 , the reduction kinetics of both components were plotted against each other (Fig. 5B), and the apparent redox equilibration constants (k app ) were calculated [START_REF] Kirchhoff | Plastocyanin redox kinetics in spinach chloroplasts: evidence for disequilibrium in the high potential chain[END_REF]. K app values in the range of 5 to 12 were obtained. On average, both the WT and the ∆psaJ mutants had a k app of 7.5. Thus, neither redox kinetics nor redox equilibration between PC and P 700 are affected by PsaJ under physiological conditions in tobacco.

Antenna analysis -In addition to its close vicinity to the PC binding site, PsaJ is also part of the interface of PSI with the Lhca antenna proteins and has been hypothesized to be involved in exciton transfer from the antenna to the PSI reaction center [16]. To address the possible role of PsaJ in exciton transfer from the peripheral antenna to the PSI reaction center, light saturation curves of linear electron flux were measured. Under low light intensities, the quantum efficiency of PSII was found to be significantly lower in the ∆psaJ mutant than in the WT. Also, the PQ pool was considerably more reduced in the mutant, as concluded from significantly increased (1-qL) values (Fig. 6A). With increasing light intensity, this difference between the ∆psaJ mutant and the WT decreased progressively and eventually disappeared above 600 µE m -2 s -1 light intensity (Fig. 6A).

This suggests that exciton transfer to P 700 is less efficient in the mutant, so that PSI excitation limits linear electron flux and assimilation under low light intensities. Indeed, when plants were grown under low-light conditions (two weeks at 20 µE m -2 s -1 ), ∆psaJ transformants developed a clearly discernable mutant phenotype. Growth was retarded and the chl. content per leaf area was visibly reduced (Fig. 6B). When plants were transferred back to standard light intensities (600 µE m -2 s -1 ), these phenotypic differences disappeared.

PSI antenna organization and antenna function of plants grown under standard light conditions was further analyzed using Lhc isoform-specific antibodies (Fig. 7) and performing 77 K chl.-a fluorescence emission measurements (Fig. 8). Similar to the quantification of the photosynthetic complexes (Fig. 3), thylakoids isolated from leaves number four, six, nine and eleven were used for immunoblot analyses of antenna protein accumulation. For the Lhcb proteins, no significant differences between WT and ∆psaJ plants were observed (Fig. 7). In contrast, analysis of Lhca accumulation revealed a specific depletion of Lhca3 in ∆psaJ plants, whereas the other Lhca isoforms accumulated to approximately the same amounts as in WT thylakoids. Specific changes in the functional organization of the PSI antenna system are further supported by 77 K chl.-a fluorescence emission analysis (Fig. 8): 77 K fluorescence emission spectra of WT and ∆psaJ thylakoids were normalized to the PSII emission maximum at 686 nm wavelength, since no differences in PSII fluorescence emission properties were expected to occur in the ∆psaJ mutants. Indeed, the lowwavelength part of the fluorescence emission spectrum, which arises from PSII and LHCII fluorescence, was unaltered in ∆psaJ thylakoids compared to the WT. However, the far-red emission maximum of the PSI-LHCI system was clearly blue-shifted in the ∆psaJ mutant. Whereas for WT thylakoids, a typical emission peak at 733 nm was observed, in agreement with previously obtained 77 K emission spectra of tobacco [START_REF] Bondarava | Evidence that cytochrome b 559 mediates the oxidation of reduced plastoquinone in the dark[END_REF], the maximum emission of the ∆psaJ mutant was shifted to 728 nm Copyright 2007 Biochemical Society (Fig. 8). This blue-shift of chl.-a fluorescence emission was observed in all leaves of the mutant, independent of their ontogenetic state (not shown). Also, the overall fluorescence emission from the PSI-LHCI system relative to PSII was reduced in the mutant, which is in good agreement with the decreased PSI contents and the slight complementary increase in PSII contents (see Fig. 2).

To test whether the loss of PsaJ also interferes with the capacity of PSI to bind LHCII, state transitions were induced by blue light-enriched illumination of young leaves to reduce the PQ pool and induce antenna redistribution in favor of PSI (state 2). Subsequently, the PQ pool was reoxidized by far-red light-enriched illumination inducing return to state 1. When the amplitudes of state transitions were calculated [26], no significant differences between the WT (F T = 0.85, SD = 0.14) and the ∆psaJ mutant (F T = 0.72, SD = 0.16) were found, suggesting that PsaJ is not required for LHCII binding to PSI.

Discussion

Today, the overall function of higher plant PSI is quite well understood, mainly due to the availability of a crystal structure from pea [3] and the characterization of knock-out or antisense/RNAi mutants lacking specific nuclear-encoded subunits [START_REF] Scheller | Role of subunits in eukaryotic photosystem I[END_REF][START_REF] Jensen | Molecular dissection ofphotosystem I in higher plants: topology, structure and function[END_REF]. A notable exception is the limited knowledge about the role of the small plastome-encoded I-and J-subunits of PSI, whose functions have not yet been resolved in higher plants. This is because the generation of plants with transgenic chloroplasts is still significantly more challenging than nuclear transformation [START_REF] Maliga | Plastid transformation in higher plants[END_REF][START_REF] Bock | Taming plastids for a green future[END_REF]. The PsaJ subunit is particularly interesting, because, based on the available crystal structural data, a dual function has been proposed:

In view of its localization in close vicinity to both the LHCI and the PC binding sites, involvement in both PSI excitation and donor side reactions has been discussed. Furthermore, the plastid localization of the psaJ gene was considered to indicate an important function of this small subunit, as the other plastome-encoded PSI subunits, PsaA, PsaB and PsaC, have all been proven to be essential for PSI biogenesis in eukaryotes, whereas the vast majority of the nuclear-encoded subunits is not [START_REF] Jensen | Molecular dissection ofphotosystem I in higher plants: topology, structure and function[END_REF].

However, a comparable suggestion has recently been proven wrong for the cyt-bf, as deletion of the plastid-encoded PetL subunit does not result in a visible phenotype [START_REF] Fiebig | Rapid evolution of RNA editing sites in a small non-essential plastid gene[END_REF]. Therefore, the assumption that deletion of plastid-encoded subunits of photosynthetic complexes should result in stronger phenotypes than deletion of nuclear-encoded subunits is not necessarily correct.

The function of the J-subunit has been investigated previously in Chlamydomonas. Here, a role in the conformational stabilization of the PC-binding site has been identified [13]. Surprisingly, although the impaired PC binding by PSI should be expected to result in a growth phenotype, this was not observed [13]. PSI contents in the Chlamydomonas ∆psaJ mutants were normal, and the role of PsaJ in exciton transfer to the PSI reaction center was not addressed. In view of these unresolved issues and existing evidence that the functions of photosynthetic proteins can vary considerably between Chlamydomonas and higher plants [START_REF] Monde | Post-transcriptional defects of tobacco chloroplast mutants lacking the cytochrome-bf complex[END_REF], we addressed the functions of PsaJ in tobacco.

In this work, we have successfully generated homoplasmic knock-out transformants by disrupting the psaJ reading frame with the aadA antibiotic resistance marker. Our finding that, when grown at standard growth conditions, the knock-out lines do not display a visible phenotype, clearly demonstrates that PsaJ is not essential for PSI accumulation and function. The absence of a discernable phenotype is in agreement with the unaltered chl. contents per leaf area and the identical assimilation capacities of WT and knock-out plants (Fig. 2), and is reminiscent of the situation in Chlamydomonas. However, different from Chlamydomonas, loss of PsaJ resulted in a 20 to 30% reduction of PSI contents in tobacco which was slightly more pronounced in older leaves (Fig. 2).

More importantly, loss of PsaJ had no influence on in vivo PC-P 700 interactions and redox equilibration in tobacco (Fig. 5). Thus, PsaJ is obviously not required for the stabilization of the PC-binding site in vivo. However, in response to thylakoid solubilization, the reduction of PSI by PC was impaired in the mutant, whereas the same treatment had no effect on the P 700 reduction kinetics in the WT. This in vitro phenotype closely resembles that of Chlamydomonas PsaJ knock-out mutants, where the fast reduction of PSI by either PC or cyt-c 6 was almost completely abolished [13]. This may indicate that also in higher plants, PsaJ can help stabilizing the PC-binding site. However, this aspect of PsaJ function seems to become relevant only under non-physiological conditions in vitro. It is noteworthy in this respect, that the PC-P 700 interaction studies in Chlamydomonas reinhardtii were done using isolated PSI particles, so that, potentially, the observed effects could also have been caused by the in vitro conditions. Remarkably, no effect of the PsaJ deletion on overall growth of Chlamydomonas was reported [13], which might indicate that the observed strong effect on PC oxidation may occur only in vitro, as demonstrated here for tobacco.

Whereas the role of PsaJ for PSI stability and PC oxidation seems to be of only limited importance in higher plants, our data strongly suggest that its predominant function resides in PSI excitation. This conclusion is supported by several lines of evidence. First, the growth of the ∆psaJ mutant is retarded under low light conditions, whereas no phenotype is seen at higher growth light intensities (Figs. 1C and6B). Second, the PQ pool is more reduced in low light in the mutant than in the WT (Fig. 6A). Third, the 77 K chl.-a fluorescence emission spectrum of the PSI-LHCI complex is blue-shifted (Fig. 8). Finally, the accumulation of Lhca proteins is altered in the mutant (Fig. 7).

Together, these results support a scenario, in which PsaJ functions in LHCI binding and exciton transfer to the PSI reaction center, thereby enlarging the functional PSI antenna cross section. In the absence of PsaJ, PSI excitation seems to be less efficient, either due to uncoupling of LHCI from PSI or as a consequence of impaired exciton transfer from LHCI to the reaction center, for which the two chl.-a molecules bound to PsaJ could be required. The reduced efficiency of PSI excitation results in an imbalance between PSI and PSII excitation in low light and a more reduced redox state of the PQ pool at limiting light intensities. Surprisingly, the mutants are not capable of compensating for this imbalance in photosystem excitation, although their capacity for state transitions was found to be normal. Therefore, the PSI-oxidizing capacity seems to limit linear electron flux in low light. Under high light intensities, both the growth phenotype and the more reduced redox state of the mutant's PQ pool disappear, because now photosystem excitation is no longer limiting, but instead, linear electron flux itself becomes limiting for assimilation and growth.

The exact molecular basis of the altered antenna function in ∆psaJ plants is currently difficult to elucidate: Crystal structures indicate that PsaJ is most likely located in direct vicinity of Lhca2 [3,16]. However, due to the high homology between Lhca2 and Lhca3, the attribution of the protein structure closest to PsaJ to Lhca2 is not unequivocal [START_REF] Jolley | Structure of plant photosystem I revealed by theoretical modeling[END_REF]. Interestingly, our immunoblot analyses revealed that Lhca2 contents remain unaltered in the mutant, while Lhca3 levels are significantly reduced in the knock-out plants. Also, the 77 K chl.-a fluorescence emission data may be easier to reconcile with a predominant effect on Lhca3 accumulation and function than with an effect on Lhca2:

At least in vitro, the Lhca2 chl.-a fluorescence emission peaks at 701 nm wavelength [START_REF] Castelletti | Recombinant Lhca2 and Lhca3 subunits of the photosystem I antenna system[END_REF][START_REF] Croce | Origin of the 701nm fluorescence emission of the Lhca2 subunit of higher plant photosystem I[END_REF] and thus in the part of the fluorescence spectrum which is still very similar to the WT spectrum (Fig. 8). Only in the wavelength range above 700 nm, clear differences between ∆psaJ and WT thylakoids become apparent: Difference spectra of WT minus ∆psaJ mutant revealed a negative peak at 715 nm and a positive peak at 735 nm (not shown). The predominant contribution to in vitro 77 K fluorescence emission in this spectral range arises from Lhca3 and Lhca4 [START_REF] Castelletti | Recombinant Lhca2 and Lhca3 subunits of the photosystem I antenna system[END_REF][START_REF] Croce | Origin of the 701nm fluorescence emission of the Lhca2 subunit of higher plant photosystem I[END_REF]. Although in vitro emission from the Arabidopsis Lhca3 isoform is maximal at 725 nm [START_REF] Castelletti | Recombinant Lhca2 and Lhca3 subunits of the photosystem I antenna system[END_REF], in vivo data suggest that Lhca3 may emit predominantly in the 730 nm range, most likely due to interaction effects with the PSI reaction center and the other Lhca proteins. Significantly, Lhca3 antisense plants revealed a reduced and slightly blueshifted 77 K fluorescence emission, comparable to the effects observed in our ∆psaJ transformants [START_REF] Ganeteg | The properties of the chlorophyll a/b-binding proteins Lhca2 and Lhca3 studied in vivo using antisense inhibition[END_REF]. Most importantly, the difference spectra between the WT and the Lhca3 antisense plants [START_REF] Ganeteg | The properties of the chlorophyll a/b-binding proteins Lhca2 and Lhca3 studied in vivo using antisense inhibition[END_REF] are practically identical with those obtained here for WT and ∆psaJ plants. Thus, the observed 77 K spectra are in excellent agreement with the selective reduction of Lhca3 revealed by immunoblots.

Taken together, these data support the suggestion that the Lhca protein localized in direct vicinity of PsaJ is not Lhca2, but rather Lhca3 [START_REF] Jolley | Structure of plant photosystem I revealed by theoretical modeling[END_REF].

Interestingly, an effect of PSI subunit deletion on the accumulation of antenna proteins has been reported also for other small subunits of the complex. For example, psaK antisense and T-DNA insertion mutants in Arabidopsis [12,[START_REF] Jensen | The PSI-K subunit of photosystem I is involved in the interaction between light-harvesting complex I and the photosystem I reaction center core[END_REF] display a reduced accumulation of Lhca3. However, in both PsaK mutants and Lhca3 antisense plants [START_REF] Ganeteg | The properties of the chlorophyll a/b-binding proteins Lhca2 and Lhca3 studied in vivo using antisense inhibition[END_REF], loss or reduced accumulation of Lhca3 results in a concomitant reduction in Lhca2 accumulation, which is most readily explained with a close physical interaction between Lhca2 and Lhca3 [START_REF] Jansson | Nearest-neighbor analysis of higher-plant photosystem I holocomplex[END_REF]. Our ∆psaJ mutant is unique in that this concomitant reduction in Lhca2 does not occur. The structural basis for this observation remains to be determined.

Another interesting question concerns the molecular cause of the reduced PSI contents in the ∆psaJ mutant. In theory, this could be either due to a slightly less efficient assembly of PSI or to an accelerated degradation. The latter effect could result from a subtle general destabilization of PSI subunit interactions in the absence of PsaJ. Alternatively, in the absence of its J-subunit, the PSI complex could be more accessible to proteolytic breakdown, for example, due to increased exposure of protease-susceptible domains at its surface. Our in vitro measurements of PSI stability in response to repetitive freezing and thawing, heat stress and treatments with chaotropic reagents (Fig. 4A) argue against a general destabilization of protein-protein interactions within the complex in the absence of PsaJ, as none of these treatments resulted in an accelerated loss of redox-active PSI. Although the PCbinding site may be bound slightly less tightly, our redox equilibration measurements indicate that this effect is not relevant under physiological conditions (Fig. 5). Therefore, a scenario, in which PSI without PsaJ is more prone to proteolytic degradation may seem more likely. However, as no PSIdegrading proteolytic activity has been characterized to date [42], it is currently not possible to test for an increased accessibility of PSI to native thylakoid proteases.

Finally, our finding that maximum assimilation rates in ∆psaJ are identical with those in the WT, despite of a 20% reduction in PSI accumulation, represents a potentially important observation. It implies that PSI, at least in WT plants, does not contribute to photosynthetic flux control. Instead, flux control is exerted at the level of the cyt-bf (Fig. 2E), in agreement with previous data [START_REF] Anderson | Cytochrome-bf complex: Dynamic molecular organization, function and acclimation[END_REF][START_REF] Anderson | Reduced levels of cytochrome bf complex in transgenic tobacco leads to a marked photochemical reduction of the plastoquinone pool, without significant change in acclimation to irradiance[END_REF].

Potentially, also PC could contribute (Fig. 2F), as its contents decrease in parallel with cyt-bf content and assimilation capacity [START_REF] Schöttler | The role of plastocyanin in the adjustment of photosynthetic electron transport to the carbon metabolism in tobacco[END_REF]. As the PsaJ mutant plants, despite a 20% reduction of PSI contents, can support the same maximum assimilation rates as the WT, it seems clear that plants possess an overcapacity of PSI. This overcapacity may be even greater than revealed by the ∆psaJ mutant, because cyt-bf accumulates to far lower levels than PSI (1.5 mmoles cyt-bf mol chl. -1 versus 2.3 mmoles PSI mol chl. -1 ). The halftime of PQ reoxidation, the rate-limiting reaction at the cyt-bf, is in the range of 2 to 3 ms [START_REF] Hope | Electron transfers amongst cytochrome f, plastocyanin and photosystem I: kinetics and mechanisms[END_REF], and therefore several times slower than PC oxidation by P 700 and electron transfer through the PSI complex to ferredoxin, which overall requires less than 1 ms. From these values, a several-fold potential overcapacity of PSI, relative to linear electron flux, can be expected, and therefore, the absence of any effect of the diminished PSI contents in ∆psaJ plants on overall assimilation is unsurprising. Currently, it is not understood why higher plants accumulate much higher PSI contents than theoretically necessary to support electron flux. One plausible explanation could be, that under certain environmental conditions, a high number of PSI complexes is beneficial to compensate for the low-affinity PC binding [46] and/or the inefficient unbinding of PC from PSI [46]. However, a rigorous analysis of the physiological functions of the PSI overcapacity will require modulation of PSI levels without altering PSI subunit composition.

Literature cited 1

Khrouchtchova, A., Hansson, M., Paakkarinen, V., Vainonen, J.P., Zhang, S., Jensen M NaBr). Afterwards, the maximum amplitude of photooxidizable P 700 was determined by difference absorption spectroscopy. None of the treatments resulted in selective destabilization of the mutant PSI complex. Heating to 70°C resulted in loss of about 25% of PSI in both WT and the ∆psaJ mutant, the other treatments had even smaller effects on the P 700 amplitude. However, in vitro P 700 redox kinetics of detergent-solubilized thylakoids reveal an impaired P 700 photoreduction in ∆psaJ mutants (B). The reduced state of P 700 was normalized to zero, the fully oxidized state was normalized to one. Reduced P 700 was photoxidized by a 200 ms light pulse (6000 µE m -2 s -1 ). After the end of the light pulse, the reduction kinetics of P 700 were analyzed. As PSI was disconnected from PSII due to detergent treatment, 1 µM PC and 100µM TMPD were added as artificial donor system. Analysis of redox kinetics revealed that the donor side of ∆psaJ mutants is highly vulnerable to thylakoid solubilization, because a fraction of slowly reduced P 700 , which is inaccessible to PC, became apparent after addition of 0.2 % DDM, whereas the PSI donor side function remained unaltered in WT thylakoids.

Figure 1 .

 1 Figure 1. Generation of psaJ knock-out plants. (A) Construction of a psaJ null allele by disruption with the aadA selection marker. Physical maps of the psaJ region in the WT plastid genome and the plastome in ∆psaJ plants are depicted. Genes above the line are transcribed from the left to the right, genes below the line are transcribed in the opposite direction. Relevant restriction sites used for cloning and RFLP analysis are indicated. Sites lost due to ligation to heterologous ends are shown in parentheses. (B) RFLP analysis to confirm homoplasmy and psaJ disruption by the chimeric aadA cassette. DNA samples were digested with the restriction enzyme SalI which produces a 5.5 kb fragment for the psaJ region in the WT (see part A). Disruption of the psaJ gene with the aadA selection marker results in a size increase of the probed restriction fragment by 1.1 kb. As expected, Southern blot analysis (probed with a radiolabeled PCR product covering the entire psaJ coding region) confirms the absence of the smaller 5.5 kb WT fragment from the transplastomic lines demonstrating that they are homoplasmic. (C) Phenotypic comparison of WT and ∆psaJ plants under standard growth conditions. The transplastomic knockout lines do not show any visible phenotype when grown in a controlled environment chamber under a light intensity of 600 µE m -2 s -1 .

Figure 2 .

 2 Figure 2. Assimilation capacities and chl. contents are unaltered, but the chl.-a/b ratio and photosystem accumulation are altered in ∆psaJ plants. The first fully expanded leaf (number four from the top) and leaves number six, nine and eleven were comparatively analyzed to determine possible phenotypic alterations arising during leaf ontogenesis. Data for the WT are presented as filled circles, data for the ∆psaJ mutant (PsaJ) are indicated as open circles. Leaf assimilation capacities of WT and ∆psaJ mutants are identical (A). No effect on the chl. content per leaf area is observed (B), but the chl.-a/b ratio is slightly elevated in the ∆psaJ mutant (C), indicating some changes in thylakoid complex stoichiometries. Thylakoids were isolated from the leaves measured to obtain the data sets displayed in Fig. 2D-G. Photosystem contents were determined from cyt-b 559 (PSII) and P 700 (PSI) difference absorption signals, respectively. Cyt-bf quantification was based on difference absorption signals of cyt-f and cyt-b 6 , PC was measured in the far-red range of the spectrum. In the ∆psaJ mutant, PSII contents (D) are significantly increased, probably as a consequence of reduced PSI accumulation (G). No differences between mutant and WT were observed for cyt-bf (E) and PC contents (F), which both correlate closely with assimilation capacities (A).

Figure 3 .

 3 Figure 3. Immunoblot analysis of marker proteins for the four multisubunit complexes of the photosynthetic light reactions. PsbD (D2-protein) is an essential component of the PSII reaction center. PetA (cyt-f) is essential for cyt-bf accumulation. PsaC is part of the PSI reaction center and required for biogenesis and stability of the PSI complex. AtpA is an essential component of the chloroplast ATP synthase. The first four lanes of each blot show undiluted thylakoids from WT leaves

Figure 4 .

 4 Figure 4. Overall stability of P 700 in response to stress treatments is unaltered in ∆psaJ thylakoids (A), but the PSI donor side is more prone to damage (B). Thylakoids isolated from young WT and ∆psaJ (PsaJ) leaves were subjected to 15 repetitive cycles of freezing in liquid nitrogen and thawing at 35°C, to heating to 70° C for 10 min, and to 10 min incubation with chaotropic salts (1 M NaCl, 2 M KI, 2

Figure 5 .

 5 Figure5. Unaltered in vivo redox kinetics of PC and P 700 in ∆psaJ leaves. PC and P 700 redox kinetics were measured in intact WT and ∆psaJ leaves (leaves number four from the top). The reduced states of PC and P 700 were normalized to zero, the fully oxidized states of both components were normalized to one. The "high-potential chain" comprising cyt-f, PC and P 700 was fully oxidized by a strong lightpulse (200 ms duration, 6000 µE m -2 s -1 intensity), and the PSII side of the electron transport chain was concomitantly reduced. (A) Reduction kinetics. At time point 0, the saturating light pulse was switched off, and dark reduction of PC and P 700 by electrons from the PQ pool was traced. PC and P 700 reduction kinetics of WT and ∆psaJ plants are about identical, with average P 700 reduction halftimes of 3.5 ms. PC reduction was slower, as expected due to its lower redox potential relative to P 700 . P 700 signals of WT are given as filled circles, and PC signals are given as filled boxes. ∆psaJ signals are presented as open circles (P 700 ) and open boxes (PC), respectively. (B) To analyze the interaction of PC and P 700 quantitatively, the normalized PC (Y-axis) and P 700 (X-axis) reduction kinetics were

Figure 1 :

 1 Figure 1:

Biochemical Journal Immediate Publication. Published on 9 Jan 2007 as manuscript BJ20061573

Biochemical Journal Immediate Publication. Published on 9 Jan 2007 as manuscript BJ20061573

Biochemical Journal Immediate Publication. Published on 9 Jan 2007 as manuscript BJ20061573

  , P.E., Scheller, H.V., Vener, A.V., Aro, E.M. and Haldrup, A. (2005) A previously found thylakoid Pesaresi, P., Jahns, P., Leßnick, A., Tizzano, M., Schiavon, F., Salamini, F. and Leister, D. (2002) Single and double knockouts of the genes for photosystem I subunits G, K and H of Arabidopsis. Effect on photosystem composition, photosynthetic electron flow, and

	12	Varotto, C., state transitions. Plant Physiol. 129, 616-624
	13	membrane protein of 14kDa (TMP14) is a novel subunit of plant photosystem I and is Fischer, N., Boudreau, E., Hippler, M., Drepper, F., Haehnel, W. and Rochaix, J.D. (1999) A
		designated PSI-P. FEBS Lett. 579, 4808-4812 large fraction of PsaF is nonfunctional in photosystem I complexes lacking the PsaJ subunit.
	2	Ganeteg, U., Klimmek, F. and Jansson, S. (2005) Lhca5 -an LHC-type protein associated Biochemistry 38, 5546-5552
		with photosystem I. Plant Mol. Biol. 54, 641-651
		Xu, Q., Odom, W.R., Guikema, J.A., Chitnis, V.P. and Chitnis, P.R. (1994) Targeted deletion
		of PsaJ from the cyanobacterium Synechocystis SP PCC-6803 indicates structural interactions
		between the PsaJ and PsaF subunits of photosystem-I. Plant Mol. Biol. 26, 291-302
	16	Ben-Shem, A., Frolow, F. and Nelson, N. (2004) Light harvesting features revealed by the
		structure of plant photosystem I. Photosynth. Res. 81, 239-250

Biochemical Journal Immediate Publication. Published on 9 Jan 2007 as manuscript BJ20061573

Biochemical Journal Immediate Publication. Published on 9 Jan 2007 as manuscript BJ20061573 Copyright 2007 Biochemical Society

Chitnis, P.R. (1996) Photosystem I. Plant Physiol. 111, 661-669

Acknowledgements:

We thank Britta Hausmann for plant cultivation and Dr. Stefanie Hartmann for help with vector construction. This project was supported by a grant of the Deutsche Forschungsgemeinschaft to M.A.S. and R.B. (SFB 429, project A12) and by the Max Planck Society.

plotted against each other, and the apparent redox equilibration constants were calculated. For both WT and ∆psaJ, identical k app values in the range of 7.5 were obtained.


Taku Amo

Martin D Brand
email: martin.brand@mrc-dunn.cam.ac.uk

Were inefficient mitochondrial haplogroups selected during radiations of modern humans? -a test using modular kinetic analysis of coupling in mitochondria from cybrid cell lines

Keywords: mitochondrial DNA, human evolution, coupling efficiency, oxidative phosphorylation, cybrid, modular kinetic analysis ρ 0 , mtDNA-less, Δψ, mitochondrial membrane potential, TPMP, triphenylmethylphosphonium, FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone

We introduce a general test of the bioenergetic importance of mitochondrial DNA variants: modular kinetic analysis of oxidative phosphorylation in mitochondria from cybrid cells with constant nuclear DNA but different mitochondrial DNA. We apply this test to the hypothesis [Ruiz-Pesini, Mishmar, Brandon, Procaccio and Wallace (2004) Science 303, [223][224][225][226] that particular mitochondrial DNA haplogroups (specific combinations of polymorphisms) that cause lowered coupling efficiency, leading to generation of less ATP and more heat, were positively selected during radiations of modern humans into colder climates. Contrary to the predictions of this hypothesis, mitochondria from Arctic haplogroups had similar or even greater coupling efficiency than mitochondria from tropical haplogroups.

INTRODUCTION

Many mutations in mitochondrial DNA (mtDNA) lead to severe pathologies, such as LHON (Leber hereditary optic neuropathy) and MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes), although the penetrance of the pathological phenotype is often puzzling [START_REF] Taylor | Mitochondrial DNA mutations in human disease[END_REF][START_REF] Dimauro | Mitochondrial DNA and disease[END_REF]. The bioenergetic consequences of normal mtDNA variation are less well understood, although there are indications that such variation may affect bioenergetic function [START_REF] Ruiz-Pesini | Human mtDNA haplogroups associated with high or reduced spermatozoa motility[END_REF][START_REF] Montiel-Sosa | Differences of sperm motility in mitochondrial DNA haplogroup U sublineages[END_REF], disease susceptibility [START_REF] Baudouin | Mitochondrial DNA and survival after sepsis: a prospective study[END_REF][START_REF] Herrnstadt | An evolutionary perspective on pathogenic mtDNA mutations: haplogroup associations of clinical disorders[END_REF] and longevity [START_REF] Santoro | Mitochondrial DNA involvement in human longevity[END_REF]. Such analyses are difficult, however, because mitochondrial functions are mostly controlled by genes encoded in nuclear DNA, and it can be very difficult to be sure that small effects attributed to mtDNA variations are not confounded by different nuclear DNA backgrounds. In the present paper we use cybrids with different mtDNA complements but the same nuclear DNA background to overcome this problem [START_REF] King | Human cells lacking mtDNA: repopulation with exogenous mitochondria by complementation[END_REF]. We then assay bioenergetic function using the modular kinetic approach [START_REF] Brand | Top-down elasticity analysis and its application to energy metabolism in isolated mitochondria and intact cells[END_REF] to determine the effect of mtDNA sequence on mitochondrial bioenergetics. This novel application of modular kinetic analysis to cybrid mitochondria provides a powerful new tool, of general applicability, to analyse bioenergetic effects of differences in mtDNA in healthy human populations or in mitochondrial diseases.

Analysis of human mtDNA variation has identified specific combinations of polymorphisms that have been used systematically to classify mtDNAs into haplogroups, and to study the origin, radiations and evolution of human populations [10,11]. More recently, it has been proposed that these polymorphisms interacted with environmental factors, leading to positive selection of adaptive variants with decreased mitochondrial coupling efficiency and increased heat production that were beneficial for humans moving out of Africa into colder Eurasia and through the Arctic to the Americas [START_REF] Mishmar | Natural selection shaped regional mtDNA variation in humans[END_REF][START_REF] Ruiz-Pesini | Effects of purifying and adaptive selection on regional variation in human mtDNA[END_REF]. Specifically, haplogroups A, C, D and X, which are dominant in North-eastern Eurasian and native American populations, are predicted to have lower oxidative phosphorylation coupling efficiency than the other haplogroups, particularly L, which is dominant in African populations and B, which is present in central Asian and American populations and is thought not to have been cold-selected.

These haplogroups have specific polymorphisms in the mitochondrial genes MTATP6, MTCYB, MTND2 and MTND4, which code for components of the protein complexes of oxidative phosphorylation, so might cause slip or leak reactions and affect coupling efficiency.

The present study tests this hypothesis experimentally, to clarify whether and how oxidative phosphorylation coupling efficiency depends on genetic variants of mtDNA generated after modern humans migrated out of Africa.

EXPERIMENTAL

Subjects

Ethics approval was obtained from the Cambridge Research Ethics Committee. We recruited healthy volunteers by advertisement and obtained written informed consent from all participants. DNA was extracted from buccal swab samples by QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Mitochondrial haplogroups were determined by PCR-RFLP (restriction fragment length polymorphism) analysis according to criteria described in [11].

Entire mtDNAs were amplified in several overlapping fragments by PCR [START_REF] Torroni | Haplotype and phylogenetic analyses suggest that one European-specific mtDNA background plays a role in the expression Leber hereditary optic neuropathy by increasing the penetrance of the primary mutations 11778 and 14484[END_REF]. Each fragment was digested by restriction enzymes, and resolved on agarose gels. 15 ml of blood were taken from each of six volunteers who had Arctic (A, C and D) or tropical (L1, L2 and L3) haplogroups, for platelet preparation and cybrid construction.

Cells

A549.B2 ρ 0 (mtDNA-less) cells derived from human lung carcinoma A549 were cultivated in Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g/l glucose and 110 µg/ml sodium pyruvate, supplemented with 10% foetal bovine serum (FBS) and 50 µg/ml uridine. Platelets from the blood samples of volunteers were fused with A549.B2 ρ 0 cells as described elsewhere [START_REF] Chomyn | Platelet-mediated transformation of human mitochondrial DNAless cells[END_REF]. The resulting cybrid cell lines were routinely maintained in DMEM with 10% dialysed FBS. DNA was extracted from cultured cybrid cells as described previously [START_REF] Laird | Simplified mammalian DNA isolation procedure[END_REF] to confirm mitochondrial haplogroups by PCR-RFLP analysis. Cybrid B2.3243 cells, which have the same A549 nuclear background, but harbour a pathogenic mutation at np 3243 in the tRNA-leu(UUR) gene (MTTL1) [17,[START_REF] Dunbar | Different cellular backgrounds confer a marked advantage to either mutant or wild-type mitochondrial genomes[END_REF] were kindly donated by Ian Holt (MRC Dunn Human Nutrition Unit). The mutation was heteroplasmic: clone Z had a high proportion of wild type and clone X had a high proportion of mutant.

Mitochondria

Human cell mitochondria were prepared from cultured cybrids cells grown in DMEM with 10% FBS. Cells were allowed to grow up to approximately 90% confluence on 3 or 4 trays (500 cm 2 , Nunclon™). Following two washes with ice-cold STE buffer (comprising 0.25 M sucrose, 5 mM Tris-HCl (pH 7.4) and 2 mM EGTA), cells were scraped off the trays and harvested in STE by centrifugation at 480 g (10 min, 4ºC). Cell pellets were resuspended in 5 ml of STE supplemented with a protease-inhibitor cocktail (Calbiochem #539131) and homogenized using a glass homogenizer. After 8-fold dilution in STE and removal of cell debris (3 min centrifugation at 1100 g followed by filtration through muslin), mitochondria were pelleted by centrifugation at 12000 g (11 min, 4ºC). This procedure gave good quality mitochondria (respiratory control ratio with α-ketoglutarate + malate approximately 11) and yielded typically 5-8 mg of mitochondrial protein per 500 cm 2 tray. Protein concentration was determined by the biuret method with BSA as a standard.

Respiratory flux and membrane potential

Oxygen consumption was measured at 37ºC using a Clark-type electrode (Rank Brothers, Cambridge, UK) calibrated with air-saturated medium comprising 0.115 M KCl, 10 mM KH 2 PO 4 , 3 mM Hepes (pH 7.2), 1 mM EGTA, 2 mM MgCl 2 and 0.3% (w/v) defatted BSA, assumed to contain 406 nmol atomic oxygen • ml -1 [START_REF] Reynafarje | O 2 solubility in aqueous media determined by a kinetic method[END_REF]. No correction was made for consumption of oxygen by the electrode, so mitochondrial oxygen consumption at low rates will have been slightly overestimated. Simultaneously with respiratory activity, Δψ (mitochondrial membrane potential) was measured using an electrode sensitive to the lipophilic cation TPMP + (triphenylmethylphosphonium) [START_REF] Brand | Measurement of mitochondrial protonmotive force[END_REF]. Mitochondria were incubated at 1.0 mg • ml -1 (for succinate respiration) or 1.5 mg • ml -1 (for α-ketoglutarate + malate respiration) in the presence of 80 ng • ml -1 nigericin to collapse ΔpH and 4 µM rotenone to inhibit complex I. The TPMP + -sensitive electrode was calibrated with sequential additions of TPMP + up to 2 µM, then 4 mM succinate or 3.2 mM α-ketoglutarate + 0.8 mM malate (with rotenone omitted) was added to initiate respiration. Experiments were terminated with 1.6 µM FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), allowing correction for any small baseline drift. Δψ values were calculated from the distribution of TPMP + across the mitochondrial inner membrane using a binding correction factor of 0.35 mg of protein • µl -1 .

Respiratory control ratios (the State 3 respiration rate with 0.8 mM ADP divided by the State 4 rate with oligomycin) with α-ketoglutarate + malate as substrate were determined in the absence of nigericin. Mitochondrial oxidative phosphorylation can be divided conceptually into three modules. These are (i) the reactions that produce Δψ, consisting of the substrate translocases, dehydrogenases and other enzymes and the components of the respiratory chain, here called 'substrate oxidation', (ii) the reactions that consume Δψ and synthesize, export and dephosphorylate ATP, consisting of the ATP synthase, the phosphate and adenine nucleotide translocases and any ATPases that may be present, called the 'phosphorylating system', and (iii) the reactions that consume Δψ without ATP synthesis, called the 'proton leak' [START_REF] Brand | The proton leak across the mitochondrial inner membrane[END_REF].

Modular kinetics

Oxygen consumption and Δψ were measured simultaneously using mitochondria incubated with 80 ng • ml -1 nigericin and 4 µM rotenone. Respiration was initiated by 4 mM succinate (or 3.2 mM α-ketoglutarate + 0.8 mM malate, with rotenone omitted). The kinetic behaviour of a 'Δψ-producer' can be established by specific modulation of a 'Δψ-consumer' and reciprocally, the kinetics of a consumer are revealed upon modulation of a producer [START_REF] Brand | Top-down elasticity analysis and its application to energy metabolism in isolated mitochondria and intact cells[END_REF]. To measure the kinetic response of proton leak to Δψ, the state 4 (non-phosphorylation) respiration of mitochondria in the presence of oligomycin (0.8 µg • ml -1 ; to prevent any residual ATP synthesis), which was used solely to drive the proton leak, was titrated with malonate (up to 0.5 mM). In a similar way, the state 4 respiration was titrated by FCCP (up to 0.8 µM) for measurement of the kinetic response of substrate oxidation to Δψ. State 3 (maximal rate of ATP synthesis) was obtained using an ADP regenerating system (100 µM ATP, 20 mM glucose, and 10 units • ml -1 hexokinase (from baker's yeast; Sigma)). Titration of state 3 respiration with malonate (up to 1.15 mM) allowed measurement of the kinetics of the Δψ-consumers (the sum of the phosphorylating system and proton leak).

Catalase assay

Cells were harvested by trypsinisation, washed with ice-cold PBS, and suspended in 50 mM potassium phosphate buffer (pH 7.0) containing 0.5% Triton X-100 and 0.1mM EDTA. The cells were disrupted by brief sonication, and the supernatant was obtained by centrifugation. Catalase activity of the supernatant was measured at 37ºC in triplicate as described elsewhere [START_REF] Amo | Unique presence of a manganese catalase in a hyperthermophilic archaeon, Pyrobaculum calidifontis VA1[END_REF]. One unit of catalase is defined as the activity required to decompose 1 µmol of hydrogen peroxide per min. Specific activity is expressed as units per mg protein, estimated using a BCA™ protein assay kit (Pierce, Rockford, IL, USA) with BSA as a standard. Values are given as mean ± SEM, with n being the number of independent mitochondrial preparations (Fig. 1) or cell clones (nine for Fig. 2 and Fig. 4; three for Fig. 3).

Statistics

To test for significant differences in modular kinetics between haplogroups, we interpolated the mean respiration rates of mitochondria from the individual preparations or clones at the lowest individual state 4 potential (for proton leak) or state 3 potential (for the phosphorylating system) or at any potential between the highest and lowest membrane potential (for substrate oxidation). The significance of differences between means was assessed by unpaired Student's t-test using Microsoft Excel X; p values < 0.05 were taken to be significant. There were no significant differences between Arctic and tropical haplogroups for any of the data in Fig. 2 or Fig. 4.

RESULTS

Generation of cybrid cell lines

To overcome effects of different nuclear backgrounds, cybrids were used to analyse the functional effects of mtDNA. Cybrid cell lines are constructed by repopulation of mtDNA-less (ρ 0 ) cells with exogenous mitochondria [START_REF] King | Human cells lacking mtDNA: repopulation with exogenous mitochondria by complementation[END_REF]. A549.B2 ρ 0 cells derived from human lung carcinoma A549 were used in the present study. To obtain different populations of mitochondria, we recruited healthy human volunteers, determined their mitochondrial haplogroups and selected six with Arctic (A, C or D) or tropical (L1, L2 or L3) haplogroups.

Platelets, which have no nuclei, were isolated from blood samples and fused with the ρ 0 cells. The resultant cybrids had mitochondrial DNA from the different donors, but their nuclear DNA was identical. After many generations of cybrid growth (diluting platelet-derived nuclear-encoded subunits), all nuclear-encoded mitochondrial protein subunits will be specified by the host cell, but all mitochondrial-encoded subunits will be specified by the donor mtDNA. Using cybrids made it possible to investigate the effects of mtDNA variation in cells with identical nuclear DNA.

Modular kinetic analysis of oxidative phosphorylation -validation of the method

Rather than arbitrarily measuring individual reactions to look for differences caused by mitochondrial DNA variants, we applied a systems approach: top-down elasticity analysis [START_REF] Brand | Top-down elasticity analysis and its application to energy metabolism in isolated mitochondria and intact cells[END_REF]. This is better described as modular kinetic analysis, since it analyses the kinetics of the whole of oxidative phosphorylation divided into a small number of modules (in this case three: substrate oxidation, proton leak and the phosphorylating system), connected by their common substrate or product, mitochondrial membrane potential, Δψ. Thus the analysis picks up any change in oxidative phosphorylation that is functionally important, and is unresponsive to any changes that have no functional consequences. Comparison of the kinetic responses of each of the three modules to Δψ obtained using mitochondria isolated from different cybrids reveals any effects of mitochondrial haplogroup on the kinetics of oxidative phosphorylation. The coupling efficiency of oxidative phosphorylation (the percentage of respiration rate at a given Δψ that is used for ATP synthesis) can then be calculated from the kinetic curves. Note that any slip reactions will appear as proton leak in this analysis [23].

To validate this experimental system, we examined two models: a known chemical inhibitor and a known pathogenic mutation. We first measured the kinetics of oxidative phosphorylation in the absence and presence of a low concentration of the complex III inhibitor, myxothiazol. Fig. 1A shows that only the kinetics of substrate oxidation were affected, as predicted, and that a change in kinetics giving rate changes of 10% was readily observed. In the second model system, we measured the kinetics of oxidative phosphorylation in mitochondria isolated from cybrids harbouring a pathogenic mutation at np 3243 in the tRNA-leu(UUR) gene (MTTL1) [17,[START_REF] Dunbar | Different cellular backgrounds confer a marked advantage to either mutant or wild-type mitochondrial genomes[END_REF]. This mutation decreases expression of all mitochondrially-encoded proteins, so is expected to decrease the activity of all the respiratory chain complexes (except complex II, whose subunits are all nuclear-encoded) and the ATP synthase. Fig. 1B shows that our assay system reported the expected difference in the kinetics of substrate oxidation. The kinetics of the phosphorylation system were not significantly different, despite the anticipated decrease in ATP synthase activity, presumably because of low control of the ATP synthase over the phosphorylation rate. As expected, the kinetics of proton leak were not changed. Thus our test system was able to identify small changes, less than 10%, in the kinetics of different modules of oxidative phosphorylation if they were present.

Modular kinetic analysis of oxidative phosphorylation -test of the hypothesis.

The electrochemical proton gradient across the mitochondrial inner membrane, formed as electrons are passed down the respiratory chain, is the primary energy source for cellular ATP synthesis. However, not all of the energy available in the redox reactions is Biochemical Journal Immediate Publication. Published on 13 Mar 2007 as manuscript BJ20061609 coupled to ATP synthesis. Some is consumed by proton slip reactions (fewer protons are pumped out of the matrix during electron flow) or proton leak reactions (protons are pumped normally but leak back through proton conductance pathways that bypass the ATP synthase).

As a result of these slip and leak reactions, heat is generated instead of ATP. These nonproductive proton slip and leak pathways are physiologically important. They account for a surprisingly high proportion of cellular and organismal respiration rate, estimated to be up to 20% [24,[START_REF] Brand | The efficiency and plasticity of mitochondrial energy transduction[END_REF], so differences in their capacities could plausibly play an adaptive role that could be selected during ancient human migrations.

To test the hypothesis, we measured the kinetics and coupling efficiency of oxidative phosphorylation using isolated mitochondria from cybrid cells harbouring Arctic or tropical haplogroups. Fig. 2A shows the averaged kinetics of the three modules of oxidative phosphorylation using succinate grouped by Arctic (closed symbols) or tropical (open symbols) mtDNA haplogroup (the results for the six individual cybrid lines are shown in Fig. 3).

There was a small difference, not statistically significant, between the Arctic and tropical haplogroups in the rate of the substrate oxidation module at any given Δψ (Fig. 2A, circles). This small difference was caused entirely by one of the three tropical haplogroup cybrids, L3, which had a lower maximum respiration rate than the other tropical haplogroup lines (Fig. 3A). We also measured the kinetics of substrate oxidation using α-ketoglutarate + malate as substrate instead of succinate to check for any effects of differences in the mitochondrial complex I genes. Once again, there was a small, but not significant, difference in the kinetics of substrate oxidation between the Arctic and tropical haplogroups (Fig. 2C) that was caused by lower rates only in the L3 cybrids (Fig. 3D). As there was otherwise no difference between Arctic and tropical haplogroups in the kinetics of substrate oxidation with either substrate, we assume this was an individual difference in the L3 line possibly caused by somatic mutations in mtDNA. We conclude that there is no general difference between Arctic and tropical mtDNA haplogroups in the overall kinetics of substrate oxidation.

Contrary to the hypothesis, the rate of the proton leak of mitochondria with the Arctic haplogroups was, if anything, slightly lower on average than that of the tropical ones at any value of Δψ (Fig. 2A, squares) (differences not statistically significant). For the individual haplogroups, the rate for tropical haplogroup L3 was relatively high and the rates for Arctic haplogroups C and D were relatively low (Fig. 3B), although any differences were small. This shows that mtDNA variations between the Arctic and tropical haplogroups had at most a Fig. 2B shows that on average, there was no difference in the rate of the phosphorylating system at any given value of Δψ between the Arctic and tropical haplogroups.

The kinetic curves of the six individual haplogroups almost overlapped (Fig. 3C). We conclude that there is no difference between Arctic and tropical mtDNA haplogroups in the overall kinetics of the phosphorylating system.

From the kinetic curves shown in Figs 2 and3, we can analyse the coupling efficiency of oxidative phosphorylation. The classic measure is the respiratory control ratio: respiration at maximal rate of ATP synthesis (state 3) divided by respiration with no ATP synthesis (state 4) (Fig. 4A). We also independently measured the respiratory control ratio using αketoglutarate + malate as substrates (Fig. 4D and4E). Fig. 4A and4E show that there were small differences in average respiratory control ratio; mitochondria from the Arctic haplogroups were slightly better coupled than those from the tropical haplogroups. However, these differences were not statistically significant, and they were in the wrong direction to support the hypothesis even if haplogroup L3, which has lower respiration rate, was omitted (data not shown).

A more precise measure of coupling efficiency is given by the percentage of respiration that is coupled to ATP synthesis, calculated as shown in Fig. 2A at any chosen membrane potential. If this percentage is lower, then a greater proportion of the redox energy is diverted to heat production. Fig. 4B shows that at the value of Δψ achieved in state 3 (maximal rate of ATP synthesis), mitochondria from the Arctic haplogroups were slightly better coupled than those from the tropical haplogroups. Again, this difference was not statistically significant, and it was in the wrong direction to support the hypothesis.

Mitochondria in cells normally operate at less than maximal rates. Fig. 4C examines the coupling efficiency across the whole range of rates of ATP synthesis and values of Δψ from state 3 to state 4 (where ATP synthesis is minimal). As expected [START_REF] Brand | Control of the effective P/O ratio of oxidative phosphorylation in liver mitochondria and hepatocytes[END_REF], coupling efficiency decreased as mitochondria moved from state 3 towards state 4. Once again, there was a small difference, but not statistically significant, between the Arctic and tropical haplogroups, and again it was in the wrong direction to support the hypothesis.

Catalase activity

Differences in respiration rates of mouse cybrid cells harbouring different mtDNA sequences can be masked by compensatory changes in mitochondrial density due to a retrograde response that correlates with three-fold changes in catalase activity [27]. However, such compensatory changes would not mask changes in the modular kinetics of oxidative phosphorylation in mitochondria isolated from cybrids, and did not prevent the expected results in our pathogenic mutant model (Fig 1B). Nonetheless, to test whether catalaseassociated compensation occurred in our human cybrids, we measured catalase activity to see if depended on mitochondrial haplogroup. Catalase specific activity did not differ between arctic (15.4 ± 1.0 units/mg protein, n = 9) and tropical (14.6 ± 1.1 units/mg protein, n = 9) cybrid cells (p = 0.6), suggesting that this retrograde response was not a factor in our experiments.

Discussion

We conclude that the hypothesis that mitochondrial haplogroups that lower the coupling efficiency of oxidative phosphorylation were positively selected during radiations of modern humans [START_REF] Mishmar | Natural selection shaped regional mtDNA variation in humans[END_REF][START_REF] Ruiz-Pesini | Effects of purifying and adaptive selection on regional variation in human mtDNA[END_REF] is not supported by our experimental test using modular kinetic analysis of cybrid mitochondria. Recent mitochondrial genetic studies have also cast doubt on the hypothesis [START_REF] Elson | Comparative genomics and the evolution of human mitochondrial DNA: assessing the effects of selection[END_REF]29].

The strengths of our approach to test this hypothesis are (i) for the first time, we used a direct empirical measurement, rather than inference from genetic and physiological traits, (ii) we separated the effects of mitochondrial haplogroup from any confounding effects of nuclear DNA background and (iii) we used a simple in vitro system with the powerful modular kinetic approach where the relevant variables could be tightly controlled and manipulated. However, our approach also has some weaknesses. (i) A small effect of mitochondrial haplogroups operating over many generations might still be too small to be detected by biochemical experiments. We estimate that our experiments had the statistical power to identify a 7.3% difference in coupling efficiency between the two haplogroups. (ii)

We measured the coupling efficiency of oxidative phosphorylation using isolated mitochondria in simple defined media, but effects of mitochondrial haplogroups might only emerge at cellular or higher levels (for example, they might involve mitochondrialcytoplasmic feedback loops or retrograde signalling to nuclear genes, or might be tissue-specific). (iii) Interaction between nuclear and mitochondrial genomes may affect oxidative phosphorylation [30], so the effect of mitochondrial haplogroup might depend on nuclear background. A549, the parental line of the A549.B2 ρ 0 cells used here, has mtDNA of European haplogroup H, which is proposed to be relatively cold-adapted [START_REF] Mishmar | Natural selection shaped regional mtDNA variation in humans[END_REF][START_REF] Ruiz-Pesini | Effects of purifying and adaptive selection on regional variation in human mtDNA[END_REF]. Perhaps mtDNA of tropical haplogroups does not complement A549 nuclear DNA optimally and causes slightly lower coupling efficiency, masking evolutionarily relevant efficiency changes in human populations.

The novel method introduced in this study, modular kinetic analysis using cybrids, has provided a powerful empirical test of a specific hypothesis of altered mitochondrial coupling efficiency in human populations carrying different mitochondrial haplogroups. However, it is also a more general method that can be used to investigate the bioenergetic consequences of other differences in mitochondrial haplogroups and haplotypes in humans or other species. As shown in Fig. 1B, it is also a powerful method to investigate in a precise and controlled way the bioenergetic phenotypes of hereditary or sporadic pathological mitochondrial DNA mutations that are thought to cause many mitochondrial diseases.

Figure legends

Biochemical

  Journal Immediate Publication. Published on 13 Mar 2007 as manuscript BJ20061609 © 2007 Biochemical Society

Biochemical

  Journal Immediate Publication. Published on 13 Mar 2007 as manuscript BJ20061609 © 2007 Biochemical Society modest effect on mitochondrial proton leak, but in the wrong direction to support the hypothesis.

Fig. 1 .

 1 Fig. 1. Modular kinetic analysis -validation of the method. Modular kinetic analysis of the kinetic responses of the three modules of oxidative phosphorylation (substrate oxidation, circles; proton leak, squares; Δψ-consumers (sum of phosphorylating system and proton leak), triangles) to membrane potential, Δψ, in mitochondria isolated from cybrids, using 4 mM succinate as substrate. (A) Kinetic responses in mitochondria isolated from A, D, L1 and L2 cybrids in the absence (filled symbols) and presence of 25 nM of the complex III inhibitor, myxothiazol (open symbols). Data are means ± SEM (n = 4 independent mitochondrial preparations). The kinetics of substrate oxidation were significantly different at membrane potentials above 98 mV. (B) Kinetic responses in mitochondria isolated from cybrids harbouring a pathogenic mutation at np 3243 in the tRNA-leu(UUR) gene (MTTL1). Filled symbols, clone Z (high proportion of wild type); open symbols, clone X (high proportion of mutant). Data are means ± SEM (n = 3 independent mitochondrial preparations). The kinetics of substrate oxidation were significantly different at all membrane potentials.

Fig. 2 .

 2 Fig. 2. Modular kinetic analysis -averaged values. Modular kinetic analysis of the kinetic responses of the three modules of oxidative phosphorylation to membrane potential, Δψ, in mitochondria isolated from cybrids. Filled symbols, Arctic haplogroups (A, C and D); open symbols, tropical haplogroups (L1, L2 and L3). (A) Succinate as substrate. Comparison of the kinetic responses of substrate oxidation (circles; Δψ titrated with uncoupler, FCCP), proton leak (squares; Δψ titrated with inhibitor, malonate), and Δψ-consumers (sum of phosphorylating system and proton leak, triangles; Δψ titrated with malonate). Circled are state 3 (condition with maximal ATP synthesis) and state 4 (condition with no ATP synthesis). The coupling efficiency of oxidative phosphorylation (percentage of respiration used for ATP synthesis at a given Δψ) was calculated from the kinetic curves: the inset bar graph shows the example of Arctic haplogroups, at the Δψ of state 3. (B) Kinetic response of the phosphorylating system to Δψ, calculated from (A) by subtracting respiration driving proton leak from respiration driving the Δψ-consumers at each Δψ. (C) Kinetic response of substrate oxidation using 3.2 mM α-ketoglutarate + 0.8 mM

Fig. 3 .

 3 Fig. 3. Modular kinetic analysis -full dataset. Modular kinetic analysis in mitochondria isolated from cybrids, using succinate as substrate, of (A) substrate oxidation, (B) proton leak and (C) the phosphorylating system. (D) Modular kinetic analysis of substrate oxidation using α-ketoglutarate + malate as substrate. Filled symbols, Arctic haplogroups (circles, haplogroup A; squares, haplogroup C; triangles, haplogroup D); open symbols, tropical haplogroups (circles, haplogroup L1; squares, haplogroup L2; triangles, haplogroup L3). Data are means ± SEM (n = 3 cell clones; 3 or 4 (A, B and C) or 1 (D) repeats using independently prepared mitochondria for each clone). The kinetic response of the phosphorylating system to Δψ was calculated by subtracting respiration driving proton leak from respiration driving the Δψ-consumers at each value of Δψ.

Fig. 4 .

 4 Fig. 4. Coupling efficiency of oxidative phosphorylation in mitochondria isolated from cybrids. Filled bars, Arctic haplogroups (A, C and D); open bars, tropical haplogroups (L1, L2 and L3). (A) Respiratory control ratio using succinate as substrate (State 3 respiration rate/State 4 respiration rate). (B) Coupling efficiency using succinate as substrate at the Δψ of state 3. (C) Coupling efficiency using succinate as substrate at selected Δψ values between state 3 and state 4. Values at 145 mV or less were significantly different from values at 155 mV. (D) Respiration rates with α-ketoglutarate + malate as substrate. (E) Respiratory control ratio using α-ketoglutarate + malate as substrate. Data are means ± SEM (n = 9 cell clonesthree different clones for each of the three constituent haplogroups; 3 or 4 (A, B and C) or 1 (D and E) independent mitochondrial preparations per clone). Values in (A, B and C) were calculated from data in Fig. 2A.

  Figure 4

Biochemical Journal Immediate Publication. Published on 13 Mar 2007 as manuscript BJ20061609 © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 13 Mar 2007 as manuscript BJ20061609 © 2007 Biochemical Society

© 2007 Biochemical Society

Acknowledgements

We thank Valerie Church, Julie Buckingham and Helen Boysen for excellent technical assistance, and Ian Holt (MRC Dunn Human Nutrition Unit) for advice and A549.B2 ρ 0 and B2.3243 cells. Supported by the Medical Research Council and partly by the Uehara Memorial Foundation (T.A.). Biochemical Journal Immediate Publication. Published on 13 Mar 2007 as manuscript BJ20061609