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Photosystem I Activity Is Increased in the Absence of the PSI-G Subunit

2002; Elsevier BV; Volume: 277; Issue: 4 Linguagem: Inglês

10.1074/jbc.m110448200

1083-351X

Poul Erik Jensen, Lisa Rosgaard, Jürgen Knoetzel, Henrik Vibe Scheller,

Mitochondrial Function and Pathology

PSI-G is a subunit of photosystem I in eukaryotes. The function of PSI-G was characterized inArabidopsis plants transformed with a psaGcDNA in antisense orientation. Several plants with significantly decreased PSI-G protein content were identified. Plants with reduced PSI-G content were indistinguishable from wild type when grown under optimal conditions, despite a 40% reduction of photosystem I. This decrease of photosystem I was correlated with a similar reduction in state transitions. Surprisingly, the reduced photosystem I content was compensated for by a more effective photosystem I because the light-dependent reduction of NADP+in vitro was 48% higher. Photosystem I antenna size determined from flash-induced P700 absorption changes did not reveal any significant effect on the size of the photosystem I antenna in the absence of PSI-G, whereas a 17% reduction was seen in the absence of PSI-K. However, nondenaturing green gels revealed that the interaction between photosystem I and the light-harvesting complex I was less stable in the absence of PSI-G. Thus, PSI-G plays a role in stabilizing the binding of the peripheral antenna. The increased activity in the absence of PSI-G suggests that PSI-G could have an important role in regulation of photosystem I. PSI-G is a subunit of photosystem I in eukaryotes. The function of PSI-G was characterized inArabidopsis plants transformed with a psaGcDNA in antisense orientation. Several plants with significantly decreased PSI-G protein content were identified. Plants with reduced PSI-G content were indistinguishable from wild type when grown under optimal conditions, despite a 40% reduction of photosystem I. This decrease of photosystem I was correlated with a similar reduction in state transitions. Surprisingly, the reduced photosystem I content was compensated for by a more effective photosystem I because the light-dependent reduction of NADP+in vitro was 48% higher. Photosystem I antenna size determined from flash-induced P700 absorption changes did not reveal any significant effect on the size of the photosystem I antenna in the absence of PSI-G, whereas a 17% reduction was seen in the absence of PSI-K. However, nondenaturing green gels revealed that the interaction between photosystem I and the light-harvesting complex I was less stable in the absence of PSI-G. Thus, PSI-G plays a role in stabilizing the binding of the peripheral antenna. The increased activity in the absence of PSI-G suggests that PSI-G could have an important role in regulation of photosystem I. photosystem light-harvesting complex chlorophyll high pressure liquid chromatography Photosystem (PS)1 I catalyzes the light-driven electron transfer from reduced plastocyanin to oxidized ferredoxin and is composed of a chlorophylla-binding core complex and a chlorophyll a- andb-binding peripheral antenna called LHCI. PSI from higher plants is a supramolecular complex consisting of at least 17 different polypeptides located in the nonappressed thylakoid membranes (1Scheller H.V. Jensen P.E. Haldrup A. Lunde C. Knoetzel J. Biochim. Biophys. Acta. 2001; 1507: 41-60Crossref PubMed Scopus (170) Google Scholar). The core of PSI consists of at least 13 different subunits (A−N). The PSI-A and PSI-B subunits are homologous and form a heterodimer, which binds the primary electron donor P700 (a chlorophyll dimer) and the electron acceptors A0 (a chlorophyll amolecule), A1 (a phylloquinone), and Fx (a [4Fe-4S] iron-sulfur cluster) (1Scheller H.V. Jensen P.E. Haldrup A. Lunde C. Knoetzel J. Biochim. Biophys. Acta. 2001; 1507: 41-60Crossref PubMed Scopus (170) Google Scholar, 2Golbeck J.H. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1992; 43: 293-324Crossref Scopus (191) Google Scholar, 3Jordan P. Fromme P. Witt H.T. Klukas O. Saenger W. Krauss N. Nature. 2001; 411: 909-917Crossref PubMed Scopus (2060) Google Scholar). The remaining cofactors, FA and FB (both [4Fe-4S] iron-sulfur clusters), are bound to PSI-C. The other subunits of PSI do not bind electron acceptors. In PSI of the cyanobacterium Synechococcus elongatus, the PSI-A/B dimer, together with some of the smaller membrane-intrinsic subunits, binds the 90 Chl a and 22 β-carotene molecules that constitutes the core antenna system (3Jordan P. Fromme P. Witt H.T. Klukas O. Saenger W. Krauss N. Nature. 2001; 411: 909-917Crossref PubMed Scopus (2060) Google Scholar). The subunits PSI-G, PSI-H, and PSI-N are unique to higher plants and algae, and their structure and exact position in the PSI complex are therefore not known. LHCI is also specific for higher plants and algae. This peripheral antenna is arranged around the core and is, at least in higher plants, composed of the products of four nuclear genes,Lhca1–4, with molecular masses of 20–24 kDa. LHCI binds about 100 chlorophyll a and b molecules and ∼20 xanthophyll molecules per P700 (4Lam E. Ortiz W. Malkin R. FEBS Lett. 1984; 168: 10-14Crossref Scopus (138) Google Scholar, 5Bassi R. Simpson D. Eur. J. Biochem. 1987; 163: 221-230Crossref PubMed Scopus (165) Google Scholar). Lhca1 and Lhca4 form heterodimers, whereas Lhca3 and Lhca2 may form homodimers (6Knoetzel J. Svendsen I. Simpson D.J. Eur. J. Biochem. 1992; 206: 209-215Crossref PubMed Scopus (72) Google Scholar, 7Bossmann B. Knoetzel J. Jansson S. Photosynth. Res. 1997; 52: 127-136Crossref Scopus (74) Google Scholar, 8Schmid V.H.R. Cammarata K.V. Bruns B.U. Schmidt G.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7667-7672Crossref PubMed Scopus (133) Google Scholar). The dimer complexes associate independently with the reaction center (9Jansson S. Andersen B. Scheller H.V. Plant Physiol. 1996; 12: 409-420Crossref Scopus (144) Google Scholar), and it has recently been shown that the complexes only bind to the core complex at the side of the PSI-F/J subunits (10Boekema E.J. Jensen P.E. Schlodde E. van Breemen J.F.L. van Roon H. Scheller H.V. Dekker J.P. Biochemistry. 2001; 40: 1029-1036Crossref PubMed Scopus (124) Google Scholar). PSI-G and PSI-K from eukaryotic phototrophs show significant sequence similarity (11Kjærulff S. Andersen B. Skovgaard Nielsen V. Lindberg Møller B. Okkels J.S. J. Biol. Chem. 1993; 268: 18912-18916Abstract Full Text PDF PubMed Google Scholar). A comparison of PSI-G and PSI-K fromArabidopsis displays ∼30% amino acid identity. In fact, the cyanobacterial PSI-K is equally similar to plant PSI-G and PSI-K. However, cyanobacterial PSI contains only one copy of PSI-K (3Jordan P. Fromme P. Witt H.T. Klukas O. Saenger W. Krauss N. Nature. 2001; 411: 909-917Crossref PubMed Scopus (2060) Google Scholar). Chemical cross-linking of plant PSI showed that PSI-G and PSI-K differed from all the other small PSI subunits by not forming cross-linking products with other small core subunits (9Jansson S. Andersen B. Scheller H.V. Plant Physiol. 1996; 12: 409-420Crossref Scopus (144) Google Scholar). This suggested that both subunits should be located away from the 2-fold symmetry axis. The location of PSI-K in cyanobacterial PSI agrees with this location (3Jordan P. Fromme P. Witt H.T. Klukas O. Saenger W. Krauss N. Nature. 2001; 411: 909-917Crossref PubMed Scopus (2060) Google Scholar). PSI-K has recently been shown to interact with Lhca2 and Lhca3 using plants in which the psaK gene was suppressed using antisense technology (12Jensen P.E. Gilpin M. Knoetzel J. Scheller H.V. J. Biol. Chem. 2000; 275: 24701-24708Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The role of PSI-G in plants and its location in the PSI complex are unknown, but a putative cross-linking product between PSI-G and Lhca2 has been reported (9Jansson S. Andersen B. Scheller H.V. Plant Physiol. 1996; 12: 409-420Crossref Scopus (144) Google Scholar). Thus, due to the homology between PSI-G and PSI-K and because light-harvesting chlorophyll a/b-binding proteins are only present in plants and green algae, a function of PSI-G in the interaction with LHCI is likely. To investigate the role of PSI-G, we transformed Arabidopsisplants with a psaG cDNA in antisense orientation under the control of a constitutive promoter. Transformants with no or very low levels of PSI-G protein were obtained, and the plants were analyzed at both the biochemical and leaf level. Arabidopsis thaliana (L.) Heyn cv. Columbia was used for all experiments. Plants were grown in peat in a controlled environment Arabidopsis Chamber (Percival AR-60L, Boone, IA) at a photosynthetic flux of 100–120 μmol photons m−2 s−1, 20 °C, and 70% relative humidity. The photoperiod was 12 h for plants used for transformation, whereas the photoperiod was 8 h for plants used for biochemical and physiological analysis to suppress the induction of flowering. A 550-bp fragment containing the entire coding region of PSI-G was amplified from a full-length cDNA clone (279G1T7; Arabidopsis Biological Resource Center DNA Stock Center, Columbus, OH) by PCR using primers based on a genomic sequence for the PsaG gene (GenBankTM accession number AC002328). The fragment was cloned in antisense orientation between the enhanced cauliflower mosaic virus 35S promoter and 35S terminator in the pPS48 vector (13Kay R. Shan A. Daly M. McPherson J. Science. 1987; 236: 1299-1302Crossref PubMed Scopus (734) Google Scholar,14Odell J.T. Nagy F. Chua N.-H. Nature. 1985; 313: 810-812Crossref PubMed Scopus (1042) Google Scholar), and orientation of the insert was confirmed by sequencing. Subsequently, a fragment containing the enhanced 35S promoter followed by the antisense PsaG gene and the 35S terminator was excised with XbaI and ligated into the binary vector pPZP111 (15Hajdukiewicz P. Svab Z. Maliga P. Plant. Mol. Biol. 1994; 25: 989-994Crossref PubMed Scopus (1328) Google Scholar). The vector construct was transformed by electroporation (16Wen-Jun S. Forde B.G. Nucleic Acids Res. 1989; 178385Crossref PubMed Scopus (272) Google Scholar) intoAgrobacterium tumefaciens strain C58 (17Zambryski P. Joos H. Genetello C. Leemans J. Van Montagu M. Schell J. EMBO J. 1983; 2: 2143-2150Crossref PubMed Google Scholar).Arabidopsis plants were transformed by the floral dip method using Silwet l-77 (Lehle Seeds, Round Rock, TX) (18Clough S.J. Bent A.F. Plant J. 1998; 16: 735-743Crossref PubMed Google Scholar). Seeds harvested from transformed plants were germinated on MS medium (Sigma) containing 2% sucrose, 50 mg liter−1 kanamycin sulfate, and 0.8% agar for 2 weeks, and kanamycin-resistantArabidopsis plants were selected. Seedlings were then transplanted to peat. All biochemical and physiological experiments were performed with fully expanded rosette leaves harvested before bolting. Leaves from 8–10-week-old plants were used for isolation of thylakoids as described previously (19Haldrup A. Naver H. Scheller H.V. Plant J. 1999; 17: 689-698Crossref PubMed Scopus (112) Google Scholar). Total Chl and Chla/b ratio in thylakoids were determined in 80% acetone according to the method of Lichtenthaler (20Lichtenthaler H.K. Methods Enzymol. 1987; 148: 350-382Crossref Scopus (9261) Google Scholar). For preparation of PSI complexes, thylakoid membranes (1 mg Chl ml−1) were solubilized for 10 min with 1% dodecyl-β-d-maltoside (Sigma) at 0 °C. After centrifugation (5 min, 20,000 ×g), aliquots of the supernatant were applied on sucrose gradients. The sucrose gradients were prepared by freezing and subsequent thawing at 4 °C of 11 ml of 0.4 m sucrose, 20 mm Tricine-NaOH (pH 7.5), and 0.06% dodecyl-β-d-maltoside. The gradients were centrifuged for 20 h at 285,000 × g. The PSI band was collected with a syringe, and its protein composition was analyzed by fully denaturing SDS-PAGE. For pigment analysis, PSI-200 were extracted with 80% acetone under dim green light. The pigment composition was analyzed using a Dionex HPLC system with a Waters Spherisorb Analytical Column, ODS1 (250 × 4.6-mm inner diameter; 5-μm particle size). The mobile phase consisted of two solvents, A (acetonitrile/methanol/water (84:9:7)) and B (methanol/ethyl acetate (68:32)), both of which contained 0.1% triethylamin. The pigments were eluted with a linear gradient from 100% solvent A to 100% solvent B over 10 min, followed by an isocratic elution with 100% solvent B for 5 min, and a linear gradient of 100% solvent B to 100% solvent A in 1 min. The column was regenerated with 100% solvent A for 20 min before injection of the next sample. Injection volume was 80 μl, the flow rate was 1 ml min−1, and the eluate was monitored using a photodiode array detector in the range 290–595 nm. Pigments were identified by comparing retention times and absorption spectra with standard pigments (VKI, Hørsholm, Denmark). Quantification was performed by integration of the elution peaks at 445 nm using the program Chromeleon version 6 (Dionex, Sunnyvale, CA). Plants lacking the PSI-G subunit were identified by immunoblotting. Crude leaf extracts were prepared, and immunoblotting was carried out as described previously (12Jensen P.E. Gilpin M. Knoetzel J. Scheller H.V. J. Biol. Chem. 2000; 275: 24701-24708Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Isolated thylakoids were analyzed in similar immunoblotting procedures using antibodies as indicated in the figure legends. Primary antibodies were detected using a chemiluminescence detection system (ECL; Amersham Biosciences, Inc.) according to the manufacturer's instructions. NADP+ photoreduction activity of PSI was determined from the absorbance change at 340 nm as described by Naveret al. (21Naver H. Scott M.P. Golbeck J.H. Møller B.L. Scheller H.V. J. Biol. Chem. 1996; 271: 8996-9001Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) using thylakoids equivalent to 5 μg of Chl. Thylakoids were solubilized in 0.1%n-decyl-β-d-maltopyranoside before the measurement. Flash-induced P700 absorption change was measured at 834 nm, essentially as described previously (21Naver H. Scott M.P. Golbeck J.H. Møller B.L. Scheller H.V. J. Biol. Chem. 1996; 271: 8996-9001Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 22Tjus S.E. Møller B.L. Scheller H.V. Plant Physiol. 1998; 116: 755-764Crossref PubMed Scopus (152) Google Scholar). The saturating actinic pulse (532 nm, 6 ns) was produced by a Nd:YAG laser (Quanta Ray model GCR-100; Spectra Physics, Mountain View, CA). Thylakoids (32 μg Chl ml−1) were dissolved in 300 μl of 20 mm Tricine (pH 7.5), 40 mm NaCl, 8 mm MgCl2, 0.1%n-decyl-β-d-maltopyranoside, 2 mmsodium ascorbate, and 20 μm 2,6-dichlorophenolindophenol. The solution was centrifuged for 50 s at 200 × gto remove starch grains before measurement. The sample (300 μl) was transferred to a cuvette with 1-cm path length. A diode laser provided the measuring beam, which was detected using a photodiode. A total of 16–64 flash-induced P700 absorption changes were collected and averaged for each sample. Photo-oxidizable P700 content was calculated from the absorption change at 834 nm using an extinction coefficient of 5000 m−1 cm−1 (23Matis P. Setif P. Isr. J. Chem. 1981; 21: 316-320Crossref Scopus (117) Google Scholar). Total P700 content was determined from the ferricyanide oxidized minus ascorbate reduced difference spectrum using an extinction coefficient of 64,000m−1 cm−1 at 700 nm. State transitions were measured with a pulse amplitude 101–103 fluorometer (Walz, Effeltrich, Germany) as outlined in Jensen et al. (12Jensen P.E. Gilpin M. Knoetzel J. Scheller H.V. J. Biol. Chem. 2000; 275: 24701-24708Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) and Lunde et al. (24Lunde C. Jensen P.E. Haldrup A. Knoetzel J. Scheller H.V. Nature. 2000; 408: 613-615Crossref PubMed Scopus (287) Google Scholar). The relative change in fluorescence was calculated as Fr = ((Fi′ − Fi) − (Fii′ − Fii))/(Fi′ − Fi). Fluorescence emission spectra at 77 K were recorded from 650 to 800 nm using an excitation wavelength of 435 nm and a bifurcated light guide connected to the spectrofluorometer. Intact leaves were dark-adapted for 30 min before measurement. Nondenaturing green gel electrophoresis was carried out as described by Knoetzel and Simpson (25Knoetzel J. Simpson D.J. Planta. 1991; 185: 111-123Crossref PubMed Scopus (53) Google Scholar), except that a Mini-Protean II electrophoresis cell (Bio-Rad, Hercules, CA) was used for the separation of pigment proteins. A total of 110 kanamycin-resistant plants derived from the original transformed plants were screened by immunoblotting analysis of total leaf protein extracts (data not shown). Approximately one-third of these had either no detectable PSI-G protein or significantly reduced amounts of PSI-G. The detection limit was about 3% of wild type levels of PSI-G. Under the growth conditions used, there was no obvious visible difference between plants lacking PSI-G and the wild type, and plants without PSI-G had a normal life cycle and produced seeds. Plants were screened by immunoblotting for the presence of PSI-G before further analysis. All experiments were performed with plants or thylakoids that had either no detectable PSI-G protein or <8% residual PSI-G protein. Several independent lines were analyzed to rule out any effects from position of the inserted transfer DNA. In thylakoids from plants with no detectable PSI-G or reduced amounts of PSI-G, the Chl a/b ratio was 2.76 ± 0.08 (±S.D.; n = 12), whereas in wild type plants, the ratio was 2.83 ± 0.09 (±S.D.; n = 6) (TableI). Although this difference is not significant, it suggests that plants without PSI-G have a lower PSI/PSII ratio or an increased peripheral antenna.Table IChlorophyll composition of thylakoids and PSI-200 from plants devoid of PSI-G or PSI-K compared to wild typeWild typeNo PSI-GNo PSI-KThylakoidsChla/b ratio2.83 ± 0.092.76 ± 0.083.20 ± 0.151-aValue is significantly different (p < 0.05) from the wild type value.PSI-200Chla/b ratio9.0 ± 0.29.1 ± 0.610.6 ± 0.31-aValue is significantly different (p < 0.05) from the wild type value.PSI-200Chl b11.1 ± 0.211.0 ± 0.89.4 ± 0.31-aValue is significantly different (p < 0.05) from the wild type value.The Chl a/b ratio in thylakoids was determined spectroscopically (20Lichtenthaler H.K. Methods Enzymol. 1987; 148: 350-382Crossref Scopus (9261) Google Scholar), and pigment composition of PSI-200 was determined using HPLC. Shown are the average ± S. D. (n = 3). The content of Chl b is expressed per 100 Chl a.1-a Value is significantly different (p < 0.05) from the wild type value. Open table in a new tab The Chl a/b ratio in thylakoids was determined spectroscopically (20Lichtenthaler H.K. Methods Enzymol. 1987; 148: 350-382Crossref Scopus (9261) Google Scholar), and pigment composition of PSI-200 was determined using HPLC. Shown are the average ± S. D. (n = 3). The content of Chl b is expressed per 100 Chl a. To analyze this further, the amount of P700 was determined using chemical oxidation. The number of chlorophylls per P700 reaction center was 666 ± 24 (±S.E.; n = 7) for wild type and 1109 ± 51 (±S.E.; n = 12) for thylakoids with reduced PSI-G content. The numbers are significantly different (p < 0.0001). The increased Chl/P700 ratio compared with the wild type corresponds to 40% less PSI in plants with reduced content of PSI-G. This result contrasts with the 17% decrease in the Chl/P700 ratio in plants without PSI-K (12Jensen P.E. Gilpin M. Knoetzel J. Scheller H.V. J. Biol. Chem. 2000; 275: 24701-24708Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Thus, the absence of PSI-G or PSI-K clearly leads to different compensatory responses. To visualize directly on the protein level that plants with reduced amounts of PSI-G have a lower content of the PSI subunits and also to analyze changes in the amounts of antenna proteins, immunoblotting analysis of thylakoid proteins was performed (Fig.1). The lanes on the gel were loaded with proteins corresponding to equal amounts of chlorophyll. Generally, the thylakoids with reduced amounts of PSI-G have ∼21–40% less of most PSI subunits (Fig. 1A). This number is in good agreement with the 40% lower P700 content observed in the same thylakoids. A trace of PSI-G protein corresponding to ∼8% of wild type is evident in the lane with thylakoids from the antisense plants. The immunoblot analysis with the PSII antibodies (D1, PSII-W, and PSII-S) shows that the amount of PSII is similar to the amounts found in wild type or slightly increased. The PSI antenna proteins Lhca1 and Lhca4 are reduced by ∼5–10%, whereas the Lhca2 and Lhca3 proteins are reduced slightly more. The PSII antenna proteins Lhcb1–4 are either unaffected or increased (Fig. 1B). As already shown in Fig. 1, the thylakoid preparation used contains residual PSI-G protein. In the different preparations of thylakoids and plants used throughout this study, the residual PSI-G content was estimated to be either below the detection limit (3% or less) or 8% of wild type level. If PSI-G affects the stability of the PSI complex, then one would expect an enrichment of PSI containing PSI-G during preparation of PSI-200 particles. To analyze this, thylakoids containing 8% PSI-G were solubilized using dodecyl-β-d-maltoside, and the resulting complexes were separated by sucrose gradient centrifugation. The PSI-200 enriched fraction was analyzed by SDS gel electrophoresis and immunoblotting (Fig. 2), and the chlorophyll composition was analyzed by HPLC (Table I). From Fig. 2, it is clear that an enrichment of PSI-200 particles containing PSI-G is not taking place because the residual PSI-G protein in the particles corresponds to the amount of PSI-G in the thylakoids. The chlorophyll analysis also clearly shows that PSI-200 particles with a peripheral antenna very similar to wild type are purified. Thus, under the solubilization conditions used in this experiment, the PSI complexes without PSI-G are stable with respect to antenna proteins and pigments. To analyze whether PSI-G has any effect on electron transfer, NADP+ photoreduction was determined using thylakoids purified from plants without PSI-G and wild type plants. With thylakoids from wild type plants, an activity of 33.1 ± 2.3 μmol NADPH s−1 (μmol P700)−1 (±S.E.;n = 9) was determined, and with thylakoids devoid of PSI-G, an activity of 49.1 ± 6.2 (±S.E.; n = 9) was determined. These values are significantly different (p < 0.02). Hence, the activity is 48% higher in the absence of PSI-G. The relatively high standard deviation on the measurements performed on thylakoids devoid of PSI-G is related to the varying amounts of residual PSI-G protein in the preparations used. To analyze the role of PSI-G in antenna function, fluorescence emission at 77 K after excitation at 435 nm was recorded. The fluorescence emission spectra using thylakoids of both wild type and plants devoid of PSI-G are shown in Fig. 3. The spectra revealed a consistent 1-nm blue-shift from 734 nm to 733 nm in plants lacking PSI-G, which could suggest that the interaction between LHCI and the PSI core is affected in the absence of PSI-G. The 1-nm blue-shift was also seen when measurements were performed on intact leaves but was not seen when measurements were performed on purified PSI-200 (results not shown). To investigate the antenna function further, the PSI antenna size was estimated by determining P700 absorption changes as a function of flash intensity. The excitation laser flash was initially set at saturating intensity to ensure quantitative excitation of all P700, and the intensity was then successively lowered with the use of neutral gray filters. At each flash intensity, the amplitude of P700 absorption change was monitored (Fig. 4). We have previously demonstrated that plants devoid of PSI-K have less Lhca2 and Lhca3 and are impaired in energy transfer from the peripheral antenna to the core (12Jensen P.E. Gilpin M. Knoetzel J. Scheller H.V. J. Biol. Chem. 2000; 275: 24701-24708Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). It was therefore of interest to estimate the actual antenna size in thylakoid samples from plants devoid of PSI-K as well (Fig. 4). To estimate the relative antenna cross-section, the light response curves were fitted to a single hit Poisson distribution (A(I) = Asat × (1 − exp(−kI)), where A(I) is the signal at the given flash intensity, Asat is the maximum signal with a saturating flash, I is the flash intensity, and k is the relative antenna cross-section). With this analysis, it was shown that the cross-section of PSI in the absence of PSI-K was 17.6 ± 10.7% (±S.D.; n = 8) smaller than that in the wild type (p = 0.001). In good agreement with this reduction in functional antenna size, the content of Chlb, which is only found in the peripheral antenna, is 15–16% lower in the absence of PSI-K. In contrast with these differences in antenna composition and function in the absence of PSI-K, the P700 light saturation curve for thylakoids devoid of PSI-G is essentially identical to that of the wild type (Fig. 4), and the Chlb content of PSI-200 particles devoid of PSI-G also does not differ from that of the wild type (Table I). Therefore, we conclude that PSI-G has no direct effect on the PSI antenna. Furthermore, PSI-G and PSI-K have very different effects on PSI, despite their homology.Figure 4Light-saturation curves for P700 oxidation in thylakoids devoid of PSI-G or PSI-K compared with wild type. The curves were acquired by flashing cuvettes containing mildly solubilized thylakoids with laser light of varying intensity and measuring the resulting P700 oxidation. The absorbances are plotted relative to saturation (ΔAsat = 100). Each point is the average obtained with seven to eight thylakoid samples. For clarity, the error bars have been omitted, but the standard errors were less than 2% for all points.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Pigment·protein complexes were solubilized from wild type, PSI-G-less, and PSI-K-less thylakoid membranes using octylglucoside and separated by nondenaturing green gel electrophoresis. This separation revealed seven major pigment·protein complexes in the wild type (Fig.5). PSI including LHCI is kept in its intact form in the green band called CPI* (PSI reaction center with light-harvesting complexes), whereas the PSII-associated antenna proteins are separated into trimeric LHCII, CP47, CP43, and LHC monomers consisting of CP29, CP26, CP24, and monomeric LHCII. Plants devoid of PSI-G had reduced amounts of PSI complexes in a green band that migrates with the same eletrophoretic mobility as wild type CPI*. In addition, a PSI band with significantly higher eletrophoretic mobility appears, indicating that PSI complexes devoid of PSI-G have lost light-harvesting proteins, probably due to their instability during electrophoresis in the absence of PSI-G. The behavior of PSI devoid of PSI-K is quite different from that of PSI devoid of PSI-G (Fig. 5). Apparently, the absence of PSI-G causes greater instability of the complex during electrophoresis in this system than the absence of PSI-K. State 1-state 2 transitions are a dynamic mechanism that enables plants to respond rapidly to changes in illumination and involve the dissociation of a mobile pool of the LHCII from PSII and concomitant association of this LHCII with PSI. State transitions are detected as differential changes in fluorescence at room temperature from PSII in leaves that are exposed to alternating PSII and PSI light, i.e. blue light or blue light together with far-red light (Fig. 6). Expressed as relative fluorescence changes (Fr), plants devoid of PSI-G have only 45% of the state transitions observed in the wild type plants. Thus, the capacity for redistribution of absorbed excitation energy between the two photosystems is significantly reduced in plants without PSI-G. We have successfully produced Arabidopsis plants with highly reduced or no detectable PSI-G protein using the antisense technique and thereby obtained an important source for investigating the role of the PSI-G protein in vivo as well as in vitro. PSI-G has been suggested to be positioned on the periphery of the PSI core and has therefore been suggested to interact with the peripheral antenna (9Jansson S. Andersen B. Scheller H.V. Plant Physiol. 1996; 12: 409-420Crossref Scopus (144) Google Scholar). However, the data presented here clearly demonstrate that PSI-G is not required for function of the PSI antenna. The antenna size measurements obtained using flash-induced P700 oxidation showed that the functional size of the peripheral antenna was unaffected in the absence of PSI-G, whereas in the absence of PSI-K, a clear reduction in antenna size was measured. This is further supported by the pigment analysis of PSI-200 particles, in which a clear decrease in Chl b content was seen in the absence of PSI-K, and no change was seen in the absence of PSI-G. However, this is somewhat in contrast to the separation of the protein·pigments complexes by mildly denaturing gel electrophoresis (green gels), in which samples from plants devoid of PSI-G revealed reduced amounts of PSI·LHCI complexes (CPI*) and the appearance of a new green band with higher electrophoretic mobility at the same time. The appearance of this second PSI band indicates that Lhca proteins are lost from the PSI holocomplex, most probably due to a destabilization of the structural organization of the antenna in the absence of PSI-G. A similar but not as pronounced effect is observed in samples from plants without PSI-K. Although the absence of PSI-G caused a destabilization of PSI under the mildly denaturing conditions of the green gels, there is no indication that PSI was destabilized in vivo or during the preparation of PSI with nonionic detergents. In leaves and thylakoids from plants lacking PSI-G, the low-temperature fluorescence emission spectra revealed a 1-nm blue-shift in the far-red emission peak from 734 nm to 733 nm. The fluorescence emission peak at 734 nm in plants is thought to arise from the Lhca1/Lhca4 dimer (8Schmid V.H.R. Cammarata K.V. Bruns B.U. Schmidt G.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7667-7672Crossref PubMed Scopus (133) Google Scholar), and apparently binding of the dimer to the reaction center core rather than heterodimerization gives rise to the far-red fluorescence (26Knoetzel J. Bossmann B. Grimme L.H. FEBS Lett. 1998; 436: 339-342Crossref PubMed Scopus (39) Google Scholar,27Ihalainen J.A. Gobets B. Sznee K. Brazzoli M. Croce R. Bassi R. van Grondelle R. Korppi-Tommola J.E.I. Dekker J.P. Biochemstry. 2000; 39: 8625-8631Crossref PubMed Scopus (61) Google Scholar). However, when any of the four Lhca proteins are completely missing or unable to interact with PSI, a large blue-shift of about 7 nm is seen (28Zhang H. Goodman H.M. Jansson S. Plant Physiol. 1997; 115: 1525-1531Crossref PubMed Scopus (49) Google Scholar, 29Haldrup A. Simpson D.J. Scheller H.V. J. Biol. Chem. 2000; 275: 31211-31218Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 30Ganateg U. Strand Å. Gustafsson P. Jansson S. Plant Physiol. 2001; 127: 150-158Crossref PubMed Scopus (81) Google Scholar). Thus, a blue-shift in the long-wavelength fluorescence emission should indicate that the interaction between the Lhca complexes and the PSI core is perturbed. However, no blue-shift was found in the purified PSI-200 particles devoid of PSI-G. This suggests that the blue-shift observed in thylakoids and intact leaves lacking PSI-G is due to fluorescence from Lhca complexes that are not bound to the core. The immunoblotting analysis indicates a 20–40% decrease in the amounts of most PSI core subunits. A similar reduction in the amounts of the Lhca2 and Lhca3 subunits in the absence of PSI-G was also observed. However, the Lhca1 and Lhca4 proteins were only reduced by 10%. Thus, Lhca1·Lhca4 complexes that are not connected with a PSI core are present, and this will result in fluorescence emission that is more blue-shifted than that seen when all the LHCI complexes are connected. It is known that deficiency in PSI does not necessarily lead to a similar deficiency in LHCI because the barley mutant viridis-zb63, which contains less than 5% of the PSI core proteins, still accumulates wild type amounts of the LHCI complexes (31Nielsen V.S. Scheller H.V. Møller B.L. Physiol. Plant. 1996; 98: 637-644Crossref Google Scholar). In the case of PSI-K, a clear reduction in the amounts of Lhca2 and Lhca3 was shown by immunoblot analysis, and this correlated with a 2-nm blue-shift, although there was no significant change in the amounts of Lhca1/Lhca4 (12Jensen P.E. Gilpin M. Knoetzel J. Scheller H.V. J. Biol. Chem. 2000; 275: 24701-24708Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). This can be explained by the fact that the Lhca complexes are located on one side of the PSI core (10Boekema E.J. Jensen P.E. Schlodde E. van Breemen J.F.L. van Roon H. Scheller H.V. Dekker J.P. Biochemistry. 2001; 40: 1029-1036Crossref PubMed Scopus (124) Google Scholar) and are thereby in contact with each other. Thus, a change in the binding environment of one of the antenna complexes is likely to affect the fluorescence properties of the other. In the absence of PSI-G, the situation is more subtle because the more severe destabilization during green gel electrophoresis suggests an interaction, whereas functional antenna size, Chl b content, and the low-temperature fluorescence data do not support a role for PSI-G in direct contact with the peripheral antenna. The data presented demonstrate that PSI-G is not required for attachment of the Lhca complexes to the core. The amount of PSI was reduced by 40% in the absence of PSI-G. This was independently verified by the state transition measurements, in which plants with reduced PSI-G content had ∼55% less state 1-state 2 transition than did the wild type. Thus, the energy distribution to PSI at the expense of PSII is significantly reduced in these plants. PSI is required for state transitions (32Delosme R. Olive J. Wollman F.A. Biochim. Biophys. Acta. 1996; 1273: 150-158Crossref Scopus (164) Google Scholar); hence, the lower amount of PSI in the absence of PSI-G explains the decreased capacity for redistribution of absorbed energy. The absence of PSI-G has a positive effect on electron transport because the in vitro NADP+ photoreduction was stimulated by 48% in thylakoids with reduced amounts of PSI-G. Removal of PSI-N, PSI-H, or PSI-L results in a less efficient PSI, and the plants compensate for this by increasing the amount of PSI, whereby a normal phenotype is maintained at least under optimal growth conditions (19Haldrup A. Naver H. Scheller H.V. Plant J. 1999; 17: 689-698Crossref PubMed Scopus (112) Google Scholar, 24Lunde C. Jensen P.E. Haldrup A. Knoetzel J. Scheller H.V. Nature. 2000; 408: 613-615Crossref PubMed Scopus (287) Google Scholar, 33Naver H. Haldrup A. Scheller H.V. J. Biol. Chem. 1999; 274: 10784-10789Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). This is explained by a mechanism in which the amounts of the two photosystems will adjust individually to maintain optimal electron transport. Plants with a reduced amount of PSI-G have 40% less PSI but display a normal phenotype under our growth conditions. This can be explained if PSI in the absence of PSI-G is more efficient and is supported by the higher in vitro PSI activity measured in the absence of PSI-G. Under light-limited conditions, it would not be possible to significantly increase the efficiency of PSI electron transport. However, under high irradiance levels when PSII photoprotective thermal dissipation is engaged, PSI will be absorbing many more photons than it is receiving electrons from PSII. Thus, PSI will have to deal with this excess excitation energy. One way is cyclic electron flow around PSI, but the capacity of this pathway is modest in comparison with the excess photon load when zeaxanthin/ΔpH-dependent energy dissipation is fully engaged in PSII (34Ort D.R. Plant Physiol. 2001; 125: 29-32Crossref PubMed Scopus (285) Google Scholar). The oxidized primary donor of PSI, P700+, is a strong quencher of excited states in the PSI antenna and can accumulate when PSI photochemistry outpaces PSII. We suggest that PSI-G plays an important role in this process. PSI-G is present in one copy per P700, and it is assumed that PSI-G is always attached to PSI. If PSI-G has a regulatory role, it will have to be able to execute this function while attached to the core complex. One hypothesis is that PSI-G changes conformation in response to changes in light quantity/quality or changes in pH and that this conformational change results in increased electron transfer to ferredoxin. Under normal conditions, PSI-G will maintain the electron transfer rate through PSI at a level that is below the potential maximum rate, but the amounts of the two photosystems will have been adjusted to maintain optimal electron transport. In high light or under other photoinhibitory conditions, PSI-G changes conformation and thereby allows higher electron transfer rates via more efficient electron transfer to ferredoxin. This will lead to overoxidation of P700, and P700+ will be the prevailing species. P700+ will stay oxidized until an electron arrives from PSII and will act as an efficient quencher of excitation energy in PSI. Thus, the role of PSI-G could be to shift PSI back and forth between a normal working mode and a quenching mode. This means that the PSI-G antisense plants will be in constant quenching mode. If this hypothesis is correct, then PSI without PSI-G should be better protected against PSI photoinhibition but would be more poorly adapted to variations in light intensity. Another way that PSI-G could affect PSI activity would be to uncouple at least part of the peripheral antenna, whereby excess excitation energy is prevented from reaching the core antenna and reaction center. In this way, PSI-G could perhaps be thought to cause a nonphotochemical de-excitation of pigments. However, no difference in PSI cross-section was found under almost the same conditions of solubilization at which an increased NADP+ reduction was seen. Hence, we think that PSI-G most likely acts on electron transport rather than on light harvesting. The mechanism behind the conformational change or uncoupling in response to light or pH changes is not known. This will require detailed knowledge about putative interactions with pigments (carotenoids and/or Chl a) and the location of PSI-G in the PSI complex. In conclusion, PSI-G and PSI-K are integral membrane proteins of 11 and 9 kDa, respectively. The two eukaryotic subunits are equally similar to the cyanobacterial PSI-K subunit, and they have clearly evolved from the same ancestral protein. However, the functions of the two proteins in higher plants are quite different. PSI-K has a role in interaction with Lhca2 and Lhca3, whereas PSI-G does not have an important role in interaction with the peripheral antenna. PSI-G is not necessary for attachment of the light-harvesting complexes to the core and is probably not in direct contact with LHCI. More importantly, PSI-G seems to regulate PSI electron transport, and this could be important for photoprotection of PSI. Future investigations will focus on this aspect of PSI-G with a dynamic role in PSI and on the molecular mechanism by which PSI-G exerts its proposed function. We are grateful to Prof. Birger Lindberg Møller for invaluable discussions and support. We thank Drs. Wolfgang Schröder, Krishna Niyogi, Torill Hundal, and Stefan Jansson for the kind gift of antibodies.