Abnormal Regulation of Photosynthetic Electron Transport in a Chloroplast ycf9 Inactivation Mutant*

The ycf9 (orf62) gene of the plastid genome encodes a 6.6-kDa protein (ORF62) of thylakoid membranes. To elucidate the role of the ORF62 protein, the coding region of the gene was disrupted with an aadAcassette, yielding mutant plants that were nearly (more than 95%) homoplasmic for ycf9 inactivation. Theycf9 mutant had no altered phenotype under standard growth conditions, but its growth rate was severely reduced under suboptimal irradiances. On the other hand, it was less susceptible to photodamage than the wild type. ycf9 inactivation resulted in a clear reduction in protein amounts of CP26, the NAD(P)H dehydrogenase complex, and the plastid terminal oxidase. Furthermore, depletion of ORF62 led to a faster flow of electrons to photosystem I without a change in the maximum electron transfer capacity of photosystem II. Despite the reduction of CP26 in the mutant thylakoids, no differences in PSII oxygen evolution rates were evident even at low light intensities. On the other hand, the ycf9mutant presented deficiencies in the capacity for PSII-independent electron transport (ferredoxin-dependent cyclic electron transport and NAD(P)H dehydrogenase-mediated plastoquinone reduction). Altogether, it is shown that depletion of ORF62 leads to anomalies in the photosynthetic electron transfer chain and in the regulation of electron partitioning among the different routes of electron transport.

The ycf9 (orf62) gene of the plastid genome encodes a 6.6-kDa protein (ORF62) of thylakoid membranes. To elucidate the role of the ORF62 protein, the coding region of the gene was disrupted with an aadA cassette, yielding mutant plants that were nearly (more than 95%) homoplasmic for ycf9 inactivation. The ycf9 mutant had no altered phenotype under standard growth conditions, but its growth rate was severely reduced under suboptimal irradiances. On the other hand, it was less susceptible to photodamage than the wild type. ycf9 inactivation resulted in a clear reduction in protein amounts of CP26, the NAD(P)H dehydrogenase complex, and the plastid terminal oxidase. Furthermore, depletion of ORF62 led to a faster flow of electrons to photosystem I without a change in the maximum electron transfer capacity of photosystem II. Despite the reduction of CP26 in the mutant thylakoids, no differences in PSII oxygen evolution rates were evident even at low light intensities. On the other hand, the ycf9 mutant presented deficiencies in the capacity for PSII-independent electron transport (ferredoxin-dependent cyclic electron transport and NAD(P)H dehydrogenasemediated plastoquinone reduction). Altogether, it is shown that depletion of ORF62 leads to anomalies in the photosynthetic electron transfer chain and in the regulation of electron partitioning among the different routes of electron transport.
The plastid genome of a number of organisms has been sequenced to completion. Comparison of the plastome sequence among lower and higher plants has revealed the existence of highly conserved open reading frames (1), which have been named ycfs (hypothetical chloroplast frames (2)). Elucidation of the function of these genes has been enormously facilitated by the development of efficient plastid transformation techniques in the unicellular alga Chlamydomonas reinhardtii (3) and the higher plant tobacco (4 -5). This, together with the homologous recombination system of plastids, allows the targeted manipulation of the plastome. Indeed, the reverse genetics approach has been successfully used for unraveling the function of some of these conserved open reading frames (6 -9).
The ycf9 gene is co-transcribed with psbD and psbC, encoding the photosystem II (PSII) 1 proteins D2 and CP43, respectively (10 -12). ycf9, found in all green plant plastid genomes sequenced so far, as well as in the chromosome of Synechocystis sp. PCC 6803, is very highly conserved (13). It encodes a small 6.6-kDa hydrophobic protein (ORF62), which was recently reported to co-purify with the light harvesting complex of PSII (LHCII) (14).
Our earlier attempts to knockout the ycf9 gene in tobacco by biolistic chloroplast transformation yielded only largely heteroplasmic plants. Although the transformed plants did not show any apparent phenotypic differences compared with the wild type (WT) plants, it was suggested that the lack of homoplasmicity might reflect an important and maybe indispensable role of the protein (13). Further purification of the mutant lines to a 95% homoplasmic state was, however, achieved by further regeneration cycles of the heteroplasmic tissue in spectinomycin and ultimately through the formation of seeds. The ycf9 mutant plants have a phenotype indistinguishable from the WT under standard growth conditions, but their growth is severely impaired in lower light intensities. On the other hand, the ycf9 mutants are less susceptible to photodamage than the WT plants.
We report here that inactivation of ycf9 in tobacco plants leads to an accelerated flux of electrons to photosystem I (PSI) without a parallel change in the maximum electron transfer capacity of PSII. Furthermore, depletion of ORF62 causes deficiencies in the PSII-independent routes of electron transfer (ferredoxin-dependent cyclic electron transfer and NAD(P)H dehydrogenase-mediated plastoquinone reduction; for a review see Ref. 15) and in the terminal oxidase (PTOX) (16 -17) pathway, which might be of importance for plant growth under suboptimal light conditions. Altogether, the results presented suggest a role for ORF62 in regulatory processes responsible for fine-tuning of photosynthetic electron transfer according to the prevailing environmental conditions.

Chloroplast Transformation of Tobacco and Genetic Analysis of ycf9
Inactivation Mutants-Inactivation of ycf9 in tobacco (Nicotiana tabacum var. Samsung) by chloroplast transformation was done as described before (13)  coding region. Plant regeneration was carried out on spectinomycin (500 g/ml), after which the plants were transferred to soil, and seeds were collected. Seeds were germinated on peat compost, and plants were grown at 25°C at a photosynthetic photon flux density of 150 mol photons m Ϫ2 s Ϫ1 with a 16-h light/8-h dark rhythm.
Total leaf DNA was isolated according to the hexadecyltrimethylammonium bromide extraction procedure of Rogers and Bendich (18), with slight modifications. Estimation of the proportion of transformed to non-transformed plastome copies was done by standard DNA gel blot analysis (19) by digesting DNA (8 g) with HincII and NcoI and using a DNA probe against the ycf9 coding region. The fragment used for the synthesis of the probe was amplified with the following pair of primers: forward, 5Ј-GAC TCT TGC TTT CCA ATT G-3Ј (annealing with plastome nucleotides 37596 -37614), and reverse, 5Ј-CAA GAG ATG AGA GAA TTA AG-3Ј (annealing with plastome nucleotides 37761-37781). For PCR analysis of WT and transformed plants primers flanking the region of aadA insertion were used: forward, 5Ј-TAC GAA TAA AGT GCG AAA GG-3Ј (annealing with plastome nucleotides 37425-37444), and reverse, 5Ј-CAT CAG GAG AAG CAA ATA CAA-3Ј (annealing with plastome nucleotides 37667-37687). The PCR program was as follows: 1 cycle (95°C 4 min), 30 cycles (95°C 30 s, 56°C 30 s, 72°C 2 min), and 1 cycle (72°C 10 min).
For all experiments mutant plants of the F1 generation were used. For some experiments (growth at low light, examination of the thylakoid polypeptide content, and phosphorylation status of PSII proteins) the number of individual mutant plants screened ranged from 10 to 20, in order to rule out segregation of the mutant phenotypes.
For examination of growth rates in lower light conditions, seeds were germinated as described above. After 3 weeks, WT and transformed plantlets of similar size were transferred to new pots, and the light intensity was reduced (20 mol photons m Ϫ2 s Ϫ1 ).
Protein Analysis and Enzyme Activity Assays-Thylakoid isolation, SDS-PAGE, and immunodetection were performed as described earlier (20). The gels were loaded on a chlorophyll (chl) basis (if not otherwise stated, 15 or 1 g of chl per well for studies of the NAD(P)H dehydrogenase (NDH) complex and PTOX or other thylakoid membrane complex proteins, respectively). For silver staining of polypeptides (21), thylakoid proteins were separated on Tricine/SDS-PAGE gels (22). NDH activity was solubilized from thylakoids as described (23). Thylakoids corresponding to 40 g of chl (ϳ180 g of protein) were solubilized with 2% Triton X-100 for 30 min on ice and centrifuged for 45 min at 105,000 ϫ g. The supernatant was diluted 2-fold with running buffer and ran in a linear 3-10% (2% bisacrylamide) native PAGE at 4°C (24). NDH activity was detected by incubating gel slices for 30 min at 30°C in darkness in 50 mM potassium phosphate, pH 8.0, 1 mM Na 2 EDTA, 0.2 mM NADH, and 0.5 mg/ml nitro blue tetrazolium.
For determination of ascorbate peroxidase (APX) activity, three leaf discs (3 cm diameter) were ground on liquid nitrogen and 3 ml of extraction buffer (0.1 M Tricine-KOH, pH 8.0, 1 mM dithiothreitol, 10 mM MgCl 2 , 50 mM KCl, 1 mM EDTA, 0.1% Triton X-100) were added to the resulting fine powder. The extract was filtered through Miracloth and was immediately assayed for peroxidase activity. 10, 20, or 30 l of filtered extract were added to 1 ml of reaction buffer (125 M ascorbate, 0.1 mM H 2 O 2 , 1 mM EDTA, 0.1 M Hepes-KOH, pH 7.8), and enzyme activity was recorded as changes in absorbance at 265 nm. Three leaves were assayed for both the WT and the mutant.
Chlorophyll a Fluorescence Measurements-The ratio of variable to maximum fluorescence (F v /F max ) of intact leaves was measured with a pulse amplitude modulation fluorometer (PAM 101; Walz GmbH, Effeltrich, Germany). A non-actinic 1.6-kHz measuring beam alone was used to measure the initial F 0 , and maximum fluorescence, F max , was induced with a 2-s pulse of white light (5000 mol photons m Ϫ2 s Ϫ1 ). Each F v /F max measurement was preceded by a 1-h dark adaptation period. The fluorometer was controlled, and the data were digitized with the FIP software (QA-Data OY, Turku, Finland).
Fluorescence emission spectra were measured at 77 K with a diode array spectrophotometer (S2000, Ocean Optics, Dunedin, FL) equipped with a reflectance probe as described (26). Fluorescence was excited with light below 500 nm (defined with LS500S and LS700S filters, Corion Corp., Holliston, MA, placed in front of a slide projector), and the emission was recorded between 600 and 780 nm. A 100-l thylakoid sample (10 g of chl/ml in 0.1 M sorbitol, 10 mM Hepes, pH 7.4, 5 mM NaCl, and 10 mM MgCl 2 ) was used.
Measurement of PSII and Whole Electron Transfer Chain Activities-PSII and whole electron transfer chain activities of thylakoids were measured with a Hansatech oxygen electrode at 20°C. PSII activity was measured as oxygen evolution in a reaction mixture (1 ml) consisting of 5 mM NH 4 Cl, 0.33 M sorbitol, 40 mM Hepes-KOH, pH 7.6, 5 mM NaCl, 5 mM MgCl 2, 1 M glycine betaine, 1 mM KH 2 PO 4 , and thylakoids equivalent to 10 g of chl. 2,6-Dichloro-p-benzoquinone (0.25 mM) was used as an electron acceptor. Whole electron transfer chain activity was recorded as net oxygen consumption using methyl viologen (MV) as an electron acceptor in 1 ml of buffer consisting of 40 mM sodium phosphate, pH 7.4, 1 mM NaCl, 0.6 mM NaN 3 , 0.12 mM MV, 5 mM NH 4 Cl.
Determination of the Redox State of P700 -The redox state of P700 (reaction center chlorophyll of PSI) in isolated thylakoids was determined from the absorbance of P700 ϩ (oxidized P700) at 810 nm, using OD 860 as a reference. Absorbance changes were monitored using an ED-P700DW unit attached to the PAM 101 fluorometer. Measurements were done under anaerobic conditions in a temperature-regulated cuvette (25°C) containing 0.5 ml of buffer (50 mM Tricine, pH 7.5, 5 mM MgCl 2 , 6 mM glucose, 2 mM NH 4 Cl, 400 units/ml catalase, 50 M ferredoxin (Fd)) (27). The mixture was thoroughly flushed with nitrogen, and 2 units of glucose oxidase and thylakoids (25 g chl) were added. An initial illumination period (1000 mol photons m Ϫ2 s Ϫ1 ) of 30 s was applied to reduce Fd. After that, the samples were kept in darkness for 10 s, during which DCMU was added (final concentration of 10 M) through a small hole on the side of the cuvette. Thereafter, 30 cycles of actinic light (1.2 s) and darkness (8.8 s) were applied, and the average post-illumination change in the P700 ϩ signal of the 30 repetitions was resolved into the sum of two exponentials. The results reported consider only the fast component of the signal.
For determination of the amount of oxidizable P700, leaves were kept in darkness for 1 h on moist paper prior to measurements. Ten separate measurements were done from each leaf, and three leaves were used from both WT and transformed plants. The leaf was placed on a silver plate, and the light guide of the PAM-101 fluorometer was placed to a constant distance from the leaf (ϳ5 mm). The measurements consisted of a 12-s dark period (during which the system was calibrated with the aid of the ED-P700DW unit) followed by illumination with far-red LED (PAM-102-FR; Walz, GmbH, Effeltrich, Germany). The values were normalized by dividing with the chl content per leaf area, measured according to Ref. 28.
High Light Treatment of Plants-For studies of light tolerance of PSII, detached leaves from 6-to 8-week-old plants were floated on a water bath and exposed to 1500 mol photons m Ϫ2 s Ϫ1 for 3 h. Small leaf discs (2 cm diameter) were cut from the leaves at specific time points for fluorescence measurements. For some experiments the leaves were detached, and the petioles were immersed either in water (control) or in 2 mM lincomycin for 16 h in darkness to inhibit plastid translation. The leaves were thereafter exposed to 850 mol photons m Ϫ2 s Ϫ1 for 1 h. F v /F max was determined prior to the beginning of the light treatment to ensure that the lincomycin treatment in darkness did not affect the photochemical efficiency of PSII.
Phosphorylation of PSII Core Polypeptides and LHCII-For determining the phosphorylation level of D1, D2, and CP43 according to changing light conditions, leaves were detached from the plants, the petioles were immersed in water, and the leaves were kept in darkness overnight to induce maximal dephosphorylation of the proteins (dark). Leaf disc samples (3 cm diameter) were thereafter transferred for 3 h to 50 (low light, LL), 150 (growth light, GL), or 850 mol photons m Ϫ2 s Ϫ1 (high light, HL). Thylakoid samples corresponding to 1 g of chl per lane were ran in SDS-PAGE, and thylakoid phosphoproteins were immunodetected with a rabbit polyclonal phosphothreonine antibody (New England Biolabs) as described (29).

RESULTS
Inactivation of the Chloroplast ycf9 Gene-Homoplasmicity for ycf9 inactivation was assayed by DNA gel blot analysis of the F1 generation of two independently transformed lines (G and F) regenerated at different stages of the selection process (G3, three regeneration cycles, F5 and F6, five and six regeneration cycles, respectively). As can be seen from Fig. 1B (ycf9 probe), no WT copies could be detected with DNA gel blot analysis in the mutant lines, suggesting that all of them had reached homoplasmicity. The presence of the aadA cassette in the mutant copies was further confirmed by using a probe against the aadA sequence (Fig. 1B, aadA probe). Importantly, this probe detected only one fragment, the expected 2650-base pair restriction fragment of the mutant plastid DNA (Fig. 1A), indicating that no additional mutations (e.g. in the nuclear genome) had been caused by random insertion of the aadA cassette.
Since the DNA gel blot technique might fail to detect very small amounts of non-transformed plastid DNA, we also performed PCR analysis of the WT and transformed plants (Fig.  1C). This analysis revealed the presence of a residual pool of WT plastid DNA copies in the mutants which, as judged from a dilution series of template DNA, constituted less than 5% of the total plastome population.
Chloroplast transformation of tobacco for insertional inactivation of ycf9 yielded in our previous work only largely heteroplasmic plants (50% of the plastid DNA molecules contained an insert) (13). Here, we obtained nearly homoplasmic (95%) ycf9 inactivation mutants by further regeneration cycles on spectinomycin and ultimately by the formation of seeds. Nevertheless, none of the mutant lines reached full homoplasmicity, a result that might still be indicative of an essential role of the ORF62 protein.
Growth of the ycf9 Mutants Under Different Light Conditions-ycf9 mutants (G3, F5, and F6) did not exhibit an altered phenotype under normal growth conditions (GL, 150 mol photons m Ϫ2 s Ϫ1 , Fig. 2A). However, when the light intensity in the growth chamber was lowered to 20 mol photons m Ϫ2 s Ϫ1 (LL), the growth of the mutants was clearly retarded as compared with the WT (Fig. 2B).
Thylakoid Protein Composition-Analysis of thylakoid polypeptide composition by silver staining of polyacrylamide gels revealed the absence of a small polypeptide in the ycf9 mutants (Fig. 3A), in line with a recent report by Ruf and co-workers (14) on a ycf9 inactivation mutant. These authors concluded that this protein band, which co-purified with isolated LHCII complexes, corresponded to ORF62, based on the specific absence of a 6.581-kDa mass peak (the theoretical molecular mass of ORF62) in the mutant LHCII fractions, as revealed by matrix-assisted laser desorption ionization/time of flight analysis.
Protein blot analysis showed that the amounts of major protein subunits of PSII, PSI, and their antenna, as well as the cytochrome bf complex, and the ATP synthase remained unchanged following inactivation of ycf9 (Fig. 3B). The ycf9 inactivation mutants, however, were specifically depleted of a 26-kDa protein (Fig. 3A), which corresponded to the CP26 component of the inner PSII antenna (Fig. 3B).
In addition, the mutants had clearly reduced amounts of the NdhH polypeptide, a subunit of the NDH complex (Fig. 3B). The NDH complex utilizes stromal NAD(P)H for reduction of plastoquinone (PQ) and therefore has been hypothesized to be involved in PSII-independent electron flow around PSI (30). Since to this point all the mutant lines exhibited the same properties, we chose one of them, F6, for most of the further experiments.
Importantly, immunodetection of the recently discovered PTOX (16 -17, 31-32) revealed a considerable reduction in the content of the enzyme in the thylakoids of the ycf9 mutant (Fig. 3B).
Electron Transfer Properties-To confirm the depletion of NDH complexes, the enzyme in its active state was solubilized with Triton X-100 from thylakoids of WT and F6, subjected to native PAGE, and the in-gel enzyme activity was determined. This assay showed a clear decline (3-fold) in NDH activity in the thylakoids of the mutant (Fig. 3C).
To investigate whether the other route of PSII-independent electron transfer (recycling of electrons from reduced Fd back to PQ) was likewise affected in the mutant, we determined P700 ϩ reduction rates in thylakoid samples in the presence of DCMU and reduced Fd (Table I and Fig. 4A). DCMU blocks the flow of electrons from PSII, and therefore P700 ϩ is mainly reduced through Fd-dependent cyclic electron flow around PSI (the NDH-mediated pathway does not function in isolated thylakoids in the absence of added NAD(P)H, and PSI recombination reactions do not interfere in the presence of Fd, see Ref. 27). Under such conditions the reduction rate of P700 ϩ was clearly slower in the mutant than in the WT (t1 ⁄2 0.34 and 0.23 s, respectively, Table I and Fig. 4A), pointing to a disturbance in the cyclic flow of electrons from Fd to PQ in the ycf9 mutant.
Thylakoids were also illuminated in the absence of DCMU, when PSII is the main source of electrons for reducing P700 ϩ . Under such conditions, the ycf9 mutant showed a faster P700 ϩ reduction than the WT (t1 ⁄2 0.09 s versus 0.16 s, Table I; Fig. 4B), suggesting an increased flow of electrons to P700 ϩ in the absence of ORF62.
Since a simple explanation for the faster P700 ϩ reduction of the mutant would be a decreased amount of functional PSI reaction centers (RC), the functionality of PSI was also examined. The amount of oxidizable P700 was determined by measuring the maximum change in OD 810 obtained by illuminating intact dark-adapted leaves with far-red light. This assay showed (WT, 0.25 Ϯ 0.01, F6, 0.23 Ϯ 0.01, arbitrary units) that there were no differences in the amount of functional PSI complexes between the WT and the mutant.
PSII oxygen evolution rates were measured from thylakoids of GL-and LL-grown plants under a range of light intensities (Table II). PSII oxygen evolution was similar in WT and F6, suggesting that the accelerated post-illumination reduction of P700 ϩ (Fig. 4B) was not caused by a more efficient PSII-dependent PQ reduction in the mutant. Importantly, no differences in PSII activities between the WT and F6 were observed either when the measurements were done with non-saturating

FIG. 4. Dark re-reduction of P700 ؉ in isolated thylakoids of WT and ycf9 inactivation mutant (F6) in anaerobic conditions in the presence of Fd.
A, re-reduction of P700 ϩ in WT and F6 in the presence of 10 M DCMU. B, re-reduction of P700 ϩ in WT and F6 in the absence of DCMU. P700 ϩ reduction was measured by monitoring changes in the absorbance at 810 nm in the dark after light-induced reduction of Fd. Each curve is an average of 30 repetitions. light intensities. Instead, the oxygen evolution rates of WT and F6 decreased in parallel upon lowering of light intensities, arguing against differences in energy transfer properties from the antenna to the PSII RC. Most importantly, despite the fact that the maximum electron transfer capacity of PSII was similar in F6 and WT, the rate of whole chain electron transfer was faster in the mutant (Table II), indicating an accelerated flow of electrons to PSI.
Size and Function of the LHCII Antenna-Recently, it was suggested that the ycf9 mutants are deficient in the transfer of energy to the PSII RC due to absence of the CP26 minor antenna protein (14). This suboptimal antenna function, in turn, was proposed to be the cause for reduced growth under limiting light conditions. We determined the amount of CP26 in the plants grown at 20 mol photons m Ϫ2 s Ϫ1 (LL) and found that, in contrast to the situation in growth light (150 mol photons m Ϫ2 s Ϫ1 , GL), the ycf9 mutants were able to accumulate CP26 under low light intensities (Fig. 5A), where the slow-growth phenotype becomes apparent (Fig. 2). Interestingly, the amount of CP26 was also increased in LL in the WT, in a similar manner as the phosphorylation level of LHCII polypeptides (Fig. 5A).
Possible differences in the efficiency of energy transfer from the LHCII antenna to PSII were examined by measuring fluorescence induction kinetics in the presence of DCMU in WT and F6 thylakoids from plants grown in both GL and LL. It must be noted that in the presence of DCMU the kinetics of fluorescence rise is determined by antenna size and not by other components downstream of PSII. As can be seen in Fig.  5B, no differences were found in the kinetics of fluorescence rise between WT and F6 thylakoids. The faster fluorescence rise in both WT and F6 thylakoids from LL-grown plants as compared with GL-grown plants indicates that an acclimation to LL by an increase in the PSII antenna size occurs similarly in the WT and the ycf9 mutant. 77 K fluorescence emission spectra from isolated thylakoids of WT and F6 (Fig. 6) revealed no differences in the positions or relative heights of the PSII (CP43 and CP47 at 685 and 695 nm, respectively) or PSI (735 nm) emission peaks. Thus, the mutant did not appear to differ from the WT in the amounts of the photosystems and their antennas. The difference in the relative heights of PSII and PSI peaks between GL and LL plants was obvious for both the WT and F6 (Fig. 6) and reflects acclimation of the photosynthetic machinery to the prevailing growth irradiance, shade and low light plants having a lower PSII/PSI ratio (33).
High Light Tolerance and Phosphorylation of Thylakoid Proteins-Susceptibility of the mutant to photoinhibition of PSII was investigated by recording changes in F v /F max (variable/maximum fluorescence) during exposure of plants to high light. As can be seen in Fig. 7A, photoinhibition of WT tobacco proceeded more rapidly than that of F6. In order to rule out factors involved in the PSII repair cycle (e.g. D1 repair rate), plastid translation was inhibited with lincomy-cin during a 16-h dark adaptation period, and the control (in water) and the lincomycin-treated plants were subsequently exposed to high light (Fig. 7B). The difference in F v /F max values between the WT and F6 was also evident in the lincomycin-treated plants, indicating that the slower photoinhibition kinetics of the mutant was not due to a more efficient PSII repair machinery.
Next, the phosphorylation of PSII core polypeptides was examined, the level of which has been shown to respond to the redox state of the PQ pool (34 -35). For this purpose, darkadapted leaf discs of both WT and mutant plants were exposed to different light intensities, and changes in the phosphorylation level of PSII core polypeptides were determined. Fig. 8 shows that phosphorylation of PSII core polypeptides increased  with increasing light intensities in the ycf9 mutant, indicating that the kinase was active and responded to oscillations in the PQ redox state in a similar manner as in the WT. However, in F6 the phosphorylation level of these proteins was consistently lower than in the WT, suggesting a greater oxidation of PQ in the mutant under all light conditions studied. Ascorbate Peroxidase Activity-Results from measurements of the rate of P700 ϩ reduction ( Fig. 4B and Table I) and whole chain electron transfer (Table II) indicated a faster flux of electrons to PSI. This was not transduced, however, into a more rapid carbon fixation, as can be deduced from the similar growth rate of the WT and F6 under standard conditions ( Fig.  2A). It is known that in the presence of excess reductant (reduced Fd), the electron transport system may divert electrons to molecular oxygen instead of NADP ϩ (Mehler reaction) (36), at a rate proportional to the concentration of reduced Fd (37). Diversion of electrons to oxygen leads to a rapid formation of superoxide radicals, which have to be metabolized by the detoxification system of chloroplasts. In this system superoxide dismutase and APX work in chain to ultimately convert superoxide into water and monodehydroascorbate (38).
We determined APX activities from total leaf extracts and found that ascorbate degradation was faster in the mutant than in the WT (5.3 Ϯ 0.5 and 3.9 Ϯ 0.1 mmol of ascorbate oxidized h Ϫ1 (mg chl) Ϫ1 , respectively). Chloroplast APX constitutes around 80% of the total APX content of the cell (39), and therefore measurement of APX activity from total leaf extracts mainly reflects the activity of the chloroplastic enzyme.

Depletion of ORF62 Leads to Anomalies in Photosynthetic
Electron Transfer-Comparison of the electron transfer properties of thylakoids of WT and the ycf9 inactivation mutant revealed a higher rate of whole chain electron transfer in the latter (Table II). This was not accompanied, however, by an increase in the electron transfer capacity of PSII, which was similar in WT and F6 under a wide range of measuring light intensities (Table II). Based on this and the finding that the amount of functional PSI RCs remained unchanged in the mutant, we conclude that the function of some intermediate component of the electron transfer chain between PSII and PSI is modified upon severe depletion of ORF62. In line with this conclusion is the finding that the post-illumination reduction of P700 ϩ was faster in the thylakoids of the mutant in the absence of DCMU (Fig. 4B). It is important to emphasize that these results are not caused by artifacts generated during measurements in vitro, since a similar difference with respect to the WT has been obtained for cyanobacterial ycf9 inactivation mutants using intact cells instead of isolated thylakoids in the experiments (unpublished results).
Considering the accelerated whole chain electron transfer in F6, the next question is how the faster linear electron flow is utilized in the mutant. In chloroplasts there are three main sinks for PSI electrons as follows: (i) the Calvin-Benson cycle, (ii) cyclic electron flow around PSI, and (iii) the Mehler reaction. Similar growth rates of WT and F6 in normal light ( Fig.  2A) indicate that the accelerated flow of electrons to PSI is not transduced into a faster carbon fixation in the Calvin-Benson cycle. Neither are electrons recycled back from Fd to PQ, as can be deduced from the deficiency in Fd-dependent cyclic electron transfer in F6 (Fig. 4A). Another possible route for the return of electrons to PQ is the NDH complex. This complex reduces PQ using stromal NAD(P)H as a substrate (40) and has been hypothesized to be involved both in the electron flow around PSI in the light and in a putative respiratory chain in the chloroplast ("chlororespiration," see Refs. 17, 30, 41, and 42) resembling the one occurring in cyanobacteria. However, the reduction in NdhH protein levels (Fig. 3B) and the decline in NDH activity (Fig. 3C) of the mutant argue against this alternative.
Instead, by taking into account the increased APX activity of the mutant as an indicator of increased O 2 reduction through the Mehler reaction, it seems that electrons leak from the system and react with O 2 to form superoxide. This harmful species is then metabolized by the concerted action of superoxide dismutase and APX (Mehler-peroxidase reaction; see Ref. 38). Stimulation of APX activity has been reported in maize leaves, in which excess reductant was produced due to restrictions in CO 2 assimilation upon chilling (43).
Considering all the different steps of photosynthetic electron transport affected in the ycf9 mutant, one putative component whose function might be modified upon depletion of ORF62 is the cytochrome bf complex. Oxidation of plastoquinol by the cytochrome bf complex is considered to be the rate-limiting step in photosynthetic electron transport (44 -45), and thus, it might be hypothesized that this control step is altered in plants nearly deprived of ORF62. Furthermore, it has been reported that conditions favoring cyclic electron flow induce migration of the cytochrome bf complex from the grana to the stroma-exposed membranes (46), thereby raising the possibility that this migration could be involved in the triggering of cyclic electron flow. The deficiencies in cyclic electron transport of the ycf9 mutant might then be related to a modification of the function or migration of the cytochrome bf complex. Finally, the cytochrome bf complex is distributed over the grana appressions and the stroma-exposed thylakoids (47), and accordingly, it is conceivable that ORF62 could interact with the cytochrome bf complex in the grana. Based on results by Ruf et al. (14), ORF62 co-purifies with the LHCII antenna, and therefore it is expected to be located mainly in the grana stacks. We have previously reported the localization of ORF62 in the stromaexposed membranes on the basis of the specific immunodetection of a 6.5-kDa protein in this thylakoid fraction (13). However, testing of the antiserum with the homoplasmic mutant plants revealed that our earlier conclusion on the location of ORF62 did not hold.
Another alternative is that ORF62 interacts with a component independent of the cytochrome bf complex. In line with this is the effect of ycf9 inactivation on the NDH complex (Fig.  3, B and C), cyclic electron flow (Fig. 4B, Table I) and PTOX (Fig. 3B), all affecting directly the PQ pool. The presence in thylakoid membranes of a terminal oxidase (PTOX) that catalyzes oxidation of plastoquinol and reduction of oxygen to water was recently reported (16 -17), but so far the role of this enzyme in photosynthesis is unknown. It is considered that electron flow from plastoquinol to O 2 is marginal in normal light conditions (17), and indeed decreased PTOX amounts (Fig. 3B) did not affect the growth of the ycf9 mutant under optimal light conditions (Fig. 2). However, one could argue that PTOX might have a still unknown role, which might affect the overall photosynthetic rate and be of importance for acclimation to suboptimal light intensities.
ycf9 Mutants Tolerate High Light Better Than the Wild Type-A faster flow of electrons to PSI without changes in the maximum electron transfer capacity of PSII can be expected to result in oxidation of the PQ pool. This, in combination with a stimulation of the scavenger system of thylakoids, might be beneficial under high light stress (48 -49). Our photoinhibition experiments showed that indeed the ycf9 mutant was less susceptible to photoinhibition than the WT (Fig. 7A). Importantly, this capacity to cope better with higher irradiances was not due to a more efficient PSII repair machinery since the differences in high light tolerance remained after pretreatment of leaves with lincomycin (Fig. 7B). In addition to the photoinhibition experiments, the lower phosphorylation level of PSII proteins in the ycf9 mutant (Fig. 8) is also in good agreement with a considerable oxidation of PQ, since the level of PSII phosphorylation is known to respond to PQ reduction (34,35). Notably, PSII phosphorylation in the mutant was lower also in darkness (Fig. 8). In the dark there is predicted to be no pumping of electrons through the NDH complex in the mutant, and thus PQ remains fully oxidized. In the case of the WT, however, one could expect that the observed residual PSII phosphorylation results from NDH activity.
Reduced Growth of ycf9 Mutants in Low Light Is Not Due to Deficiencies in Antenna Function-The ycf9 inactivation mutants presented a WT-like phenotype under standard growth conditions ( Fig. 2A). However, a clear difference in size became apparent when the plants were grown under low light intensities (Fig. 2B). Recently, it was proposed that the reduced growth rate of the tobacco ycf9 mutants in LL was due to inefficient energy transfer to the PSII RC, caused by the lack of the CP26 antenna protein (14). Our results, however, do not support this view, although the CP26 protein amounts are indeed strongly reduced in the mutant under normal light conditions (Fig. 3, A and B). In contrast, under LL, where the slow-growth phenotype becomes apparent, CP26 does clearly accumulate in the mutant (Figs. 2 and 5A). Moreover, the similar kinetics of fluorescence induction (Fig. 5B) rules out the possibility of deficiencies in energy transduction to the RC. Further evidence comes from experiments with CP26 antisense Arabidopsis plants (50). These plants do not exhibit reduced growth in LL and have wild type PSII activities under a wide range of measuring light intensities. Moreover, ycf9 mutants of Synechocystis likewise show slower growth rates than the WT under low irradiances, 2 despite the fact that the light-harvesting system of cyanobacteria, the phycobilisome antenna, differs considerably from its eukaryotic counterpart and has no subunits homologous to CP26.
Adaptation of plants to low light intensities is accompanied by rearrangements in the photosynthetic machinery, which result in a reduction of the PSII/PSI ratio (33). This is considered to set the photosynthetic apparatus for cyclic electron flow in order to adjust the ATP/NADPH ratio (51)(52). 77 K fluorescence emission spectra showed that the ycf9 mutant was able to adjust the PSII/PSI ratio in LL in a similar manner as the WT (Fig. 6). Nevertheless, its growth in LL was clearly retarded compared with the WT (Fig. 2). Cyanobacterial psaE (essential for Fd-dependent cyclic electron flow) and ndhF (NDH subunit) mutants (53)(54) have been reported to exhibit decreased growth under low light conditions. This has been interpreted as a failure to produce sufficient ATP in such conditions due to deficiencies in either of the two PSII-independent electron transfer pathways. It is therefore conceivable that the defects detected in both the NDH-mediated (Fig. 3, B and C) and the Fd-dependent electron transfer routes (Fig. 4A, Table I) in the ycf9 mutant are at least partly responsible for the impairment of growth under limiting light conditions. ORF62, a Possible Role in the Regulation of Photosynthetic Electron Transfer-Photosynthetic organisms have a capacity to regulate photosynthetic electron transfer in order to respond efficiently to changes in environmental conditions. Adjustments in the function of the photosynthetic machinery are required to coordinate the synthesis of ATP and NADPH with the rate of their consumption, and thereby to optimize the use of excitation energy and to avoid damage to the photosystems (for reviews see Refs. 33, 55, and 56). Optimization of the use of limited energy and adjustment of the synthesis of metabolites to their rate of use in the Calvin cycle is achieved in part by a redistribution of energy between PSII-dependent and PSIIindependent electron transfer routes (15,57,58). Despite intense study, it is not known how the balance between the activities of the photosystems is established and what is the molecular mechanism that triggers the shift between the different modes of electron transfer.
On the basis of our results, ORF62 is involved in this finetuning of photosynthesis. In plants deprived of ORF62, the partitioning between PSII-dependent and PSII-independent electron flow is impaired. Furthermore, without affecting the maximum electron transfer capacity of PSII, depletion of ORF62 leads to a faster flow of electrons to PSI, probably resulting in the stimulation of O 2 reduction in the Mehler reaction. It is tempting to propose a model in which ORF62 could be part of an "electron gate" which, according to the physiological needs of the cell, would divert electrons to the different electron transport pathways. Accurate assignment of ORF62 to a specific thylakoid protein complex will be essential for development of hypothesis of how this might be accomplished at the molecular level. Obviously, further work is needed for unraveling the complex regulatory networks that coordinate the function of the thylakoid protein complexes and the exact molecular role of ORF62 in these processes.