Accelerated Publications Structural Characterization of a Complex of Photosystem I and Light-Harvesting Complex II of Arabidopsis thaliana†

Chloroplasts are central to the provision of energy for green plants. Their photosynthetic membrane consists of two major complexes converting sunlight: photosystem I (PSI) and photosystem II (PSII). The energy flow toward both photosystems is regulated by light-harvesting complex II (LHCII), which after phosphorylation can move from PSII to PSI in the so-called state 1 to state 2 transition and can move back to PSII after dephosphorylation. To investigate the changes of PSI and PSII during state transitions, we studied the structures and frequencies of all major membrane complexes from Arabidopsis thaliana chloroplasts at conditions favoring either state 1 or state 2. We solubilized thylakoid membranes with digitonin and analyzed the complete set of complexes immediately after solubilization by electron microscopy and image analysis. Classification indicated the presence of a PSI-LHCII supercomplex consisting of one PSI-LHCI complex and one LHCII trimer, which was more abundant in state 2 conditions. The presence of LHCII was confirmed by excitation spectra of the PSI emission of membranes in state 1 or state 2. The PSI-LHCII complex could be averaged with a resolution of 16 Å, showing that LHCII has a specific binding site at the PSI-A, -H, -L, and -K subunits. Oxygenic photosynthesis relies on a balanced system of light harvesting and the conversion of the light energy into chemical energy in the photosynthetic reaction centers of PSI1 and PSII. PSI and PSII have different absorption spectra. The light energy fluctuates in intensity and quality, and plants can adapt to the changing light conditions by directing the absorbed light to either PSI or PSII to keep the energy conversion efficient. The response of the photosynthetic apparatus to light fluctuations is called state transitions (13). State transitions occur in three main steps. Upon preferential excitation of PSII, the linear electron flow between PSI and PSII may become unbalanced, resulting in an over-reduction of the plastoquinone (PQ) pool and the † R.K., A.Z., and the groups of H.V.S., J.P.D., and E.J.B. were supported by the European Union, Grant HPRN-CT-2002-00248 (“PSICO network”); E.J.B was also supported by The Netherlands Organisation for Scientific Research, Council for Earth and Life Sciences. C.D.W. was supported by The Netherlands Foundation for Fundamental Research on Matter. * To whom correspondence should be addressed. Tel. +31 50 3634225; fax, +31 50 3634800; e-mail, [email protected]. ‡ University of Groningen. § The Royal Veterinary and Agricultural University. | Vrije Universiteit. 1 Abbreviations: LHCI, light-harvesting complex I; LHCII, lightharvesting complex II; PSI, photosystem I; PSII, photosystem II. © Copyright 2005 by the American Chemical Society Volume 44, Number 33 August 23, 2005 10.1021/bi051097a CCC: $30.25 © 2005 American Chemical Society Published on Web 07/28/2005 cytochrome b6f complex (4, 5). The first step is the initiation of a signal transduction, brought about by reduced state of the PQ pool and the cytochrome b6f complex, leading to the activation of kinases. The second step is the phosphorylation, by the activated kinases, of the mobile PSII antenna, the lightharvesting complex II (LHCII). In plants, the major LHCII proteins consist of three closely related chlorophyll a/bbinding proteins encoded by Lhcb1, Lhcb2, and Lhcb3 genes (6). However, it remains uncertain whether the mobile LHCII consists of a monomer or a trimer of lhcb proteins. Two kinase families are proposed to be involved in LHCII phosphorylation: TAK kinases (7, 8) and STT7 kinases in Chlamydomonas (9) and its homologue in Arabidopsis STN7 (10). The corresponding mutants of those kinases cannot perform phosphorylation of LHCII and state transitions. The phosphorylation of LHCII is thought to cause a conformational change, which allows LHCII to diffuse to PSI in the thylakoid membrane, constituting the last step in state transitions (1). The binding of phosphorylated LHCII to PSI brings the system in state 2 and allows a redistribution of the light energy between the two photosystems and a balancing of the linear electron flow. This association between PSI and LHCII is reversible. Preferential excitation of PSI leads to oxidation of the plastoquinol pool, a dephosphorylation of LHCII, and a release of LHCII from the PSI-LHCII complex (state 1). There are recent data indicating which PSI subunits play an essential role in the state transitions. Arabidopsis plants without the PSI-H and PSI-L subunits (11), as well as those without the PSI-O subunit (12), are highly deficient in state transitions. Chemical cross-linking using dithio-bis(succinimidylpropionate) followed by diagonal electrophoresis and immunoblotting showed that the docking site of LHCII on PSI is comprised of the PSI-H, -L, and -I subunits (13). Thus, despite ample evidence for a functional interaction between LHCII and PSI, direct evidence for the existence of a physical complex between the two and the precise docking site of LHCII on PSI has not been demonstrated. Another open question is the origin of LHCII which associates to PSI. LHCII is mainly organized as trimers, and the usual ratio of LHCII trimers to dimeric PSII core complexes is about 8:1. In Arabidopsis, most PSII core complexes bind only 2-4 LHCII trimers, which implies a pool of nonbound or very loosely bound LHCII (14). Thus, the LHCII which associates to PSI can originate either from the nonbound LHCII pool or from PSII-LHCII supercomplexes. A further question deals with the association state of the LHCII proteins binding to PSI. Binding of phosphorylated LHCII monomers to PSI has been suggested (1, 8), but more recent work shows that phosphorylation is not a prerequisite for binding to PSI (13). Structural studies usually require purified proteins. The purification of a PSI-LHCII supercomplex after digitonin solubilization of Arabidopsis membranes was recently described (13). However, these PSI-LHCII supercomplexes appeared to be too fragile to obtain in sufficient quantity for structural analysis. Single-particle electron microscopy is an effective method for both the sorting and averaging of molecular projections of large membrane proteins. We have previously shown that this technique can be used to analyze the composition of PSII-LHCII supercomplexes in partially solubilized grana membranes immediately after solubilization and without any purification (15). Hence, we have performed an analysis of all possible PSI and PSII projections from Arabidopsis thylakoid membranes of chloroplasts, brought either in state 1 or state 2 before they were solubilized with digitonin. Analysis of the set of single-particle projections reveals for the first time a supercomplex consisting of one PSI complex and one trimeric LHCII, which is more abundant in state 2. The projection map could be resolved at 16 Å resolution, which reveals the precise position of LHCII. MATERIALS AND METHODS Plant Material and Growth Conditions. Arabidopsis thaliana (L.) Heynh. ecotype Columbia was used for all experiments. Plants were grown in compost 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 8 h to suppress the induction of flowering. Induction of State Transitions. Four-to-six week-old plants were used for light treatment. An orange filter (Rosco, 105 orange, Teadan Aps, Stenløse, Denmark) adjusted with a gray filter (209 neutral density, LEE Filters, Andover, U.K.) to 70 μmol photons m-2 s-1 was used to induce state 2 in plants, and a red filter (HT 027 medium red, LEE Filters) adjusted with gray filters to 50 μmol photons m-2 s-1 was used to induce state 1. The filters were mounted in a controlled environment chamber equipped with 400-watt Powertone HPI-T Plus lamp (Philips). Plants were exposed to state 1 or state 2 light for 1 h; leaves were harvested under state 1 or state 2 light and immediately frozen in liquid nitrogen. We have previously shown that this light treatment induces state transitions efficiently (13). Isolation of Thylakoid Membranes. Leaves from lighttreated plants were used for isolation of thylakoids as described previously (16). Total chlorophyll and chlorophyll a/b ratio in thylakoids were determined in 80% acetone according to ref 17. The 77 K fluorescence emission spectra were recorded of the thylakoid preparations from state 1 and state 2 to verify that the light treatment induced state transitions in the plants. Solubilization of Thylakoid Membranes. Thylakoid membranes were resuspended in 20 mM Bis-Tris (pH 6.5) with 5 mM MgCl2 at a final concentration of 0.5 mg of Chl/mL and solubilized with digitonin at a final concentration of 0.5% (w/v) for 30 min at 4 °C with stirring according to ref 13, followed by centrifugation in an Eppendorf table centrifuge for 15-20 min. The entire solubilized sample was directly used for preparation of specimen for electron microscopy without any purification step. Fluorescence Measurements. For the low-temperature, steady-state fluorescence measurements, the membranes were diluted in a buffer containing 20 mM Bis-Tris, pH 6.5, 20 mM NaCl, 5 mM MgCl2, and 66% (v/v) glycerol as a cryoprotectant. The final optical density of the membranes was less than 0.1 cm-1 at 680 nm. Fluorescence emission spectra were measured with a 0.5 m imaging spectrograph (Chromex 500IS) and a CCD camera (Chromex Chromcam I). The spectral resolution was 0.15 nm. For broadband excitation, a tungsten halogen lamp (Oriel) was used with an interference filter transmitting at 420 nm, bandwidth 15 nm. A helium bath cryostat (Utreks, 10936 Biochemistry, Vol. 44, No. 33, 2005 Accelerated Publications