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, 132 (3), 265-276

Light-induced Formation of Dimeric LHCII

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Light-induced Formation of Dimeric LHCII

Ewa Janik et al. Photosynth Res.

Abstract

It emerges from numerous experiments that LHCII, the major photosynthetic antenna complex of plants, can appear not only in the trimeric or monomeric states but also as a dimer. We address the problem whether the dimeric form of the complex is just a simple intermediate element of the trimer-monomer transformation or if it can also be a physiologically relevant molecular organization form? Dimers of LHCII were analyzed with application of native electrophoresis, time-resolved fluorescence spectroscopy, and fluorescence correlation spectroscopy. The results reveal the appearance of two types of LHCII dimers: one formed by the dissociation of one monomer from the trimeric structure and the other formed by association of monomers into a distinctively different molecular organizational form, characterized by a high rate of chlorophyll excitation quenching. The hypothetical structure of such an energy quencher is proposed. The high light-induced LHCII dimerization is discussed as a potential element of the photoprotective response in plants.

Keywords: Dimer; Fluorescence quenching; LHCII complex; Photoprotection; Spinacia oleracea.

Figures

Fig. 1
Fig. 1
Electrophoretic analysis of LHCII oligomeric forms in LHCII preparations isolated from Spinacia oleracea leaves. Spinach leaves were dark-adapted (LHCII) or pre-illuminated with light intensity of 1200 µmol photons m−2 s−1 (LHCII-HL) before LHCII isolation. LHCII was suspended in 0.1% DM. Figure shows exemplary results of the electrophoretic gel analyses with band assignment (FP free pigments fraction)
Fig. 2
Fig. 2
Electrophoretic analysis of LHCII oligomeric forms in LHCII samples illuminated with different light intensities. LHCII was suspended in 0.1% DM. Electrophoresis was conducted under constant illumination starting from the sample loading. Each slot was illuminated with different light intensity (from 11 µmol photons m−2 s−1 to 1240 µmol photons m−2 s−1). This was carried out by gel illumination in an intensity gradient of a LED light source (as it is schematically presented in Fig. S1). Light intensity in each slot was precisely measured using a photometer before commencing each experiment. Upper panel shows exemplary results of the electrophoretic gel analyses with band assignment (FP free pigments fraction). Lower panel presents the quantity analysis of LHCII trimers, dimers, and monomers in the samples. Relative intensities of the bands corresponding to different LHCII forms were quantified by scans analyzed with ImageJ2x software. Experiment was repeated ten times. Similar, exemplary effect of different light intensities on the LHCII trimers and monomers mixture is presented as Supplementary data (Fig. S2)
Fig. 3
Fig. 3
Normalized autocorrelation curves of different LHCII forms. Monomers (a) and trimers (b) were suspended in 0.05 and 0.03% DM solution, respectively. Dark-adapted LHCII trimers and monomers mixture in 0.1% DM solution (c) were subjected to illumination with blue light intensity of 100 µmol photons m−2 s−1 for 30 min (d). Diffusion coefficient values [as mean values ± SD (n = 8 biological replicates)] of the LHCII forms detected during FCS measurements are presented in the figure
Fig. 4
Fig. 4
Amplitude-weighted average lifetime <τ> values in relation to amplitude of lifetime components measured from LHCII trimer, dimer, and monomer. Values of average lifetime were calculated on the basis of the fluorescence decay kinetics fitted with three components characterized by the following lifetimes: τ 1 = 3.7 ns, τ 2 = 1.8 ns and τ 3 = 0.3 ns. Average lifetimes for the LHCII trimers and monomers are presented as mean values. Error bars indicate standard deviation (n = 10 biological replicates). Fluorescence intensity decays were analyzed by deconvolution with the instrument response function and analyzed as a sum of exponential terms. The quality of the fit was judged from the χ2 value (~1). Excitation and detection was at 470 and 680 nm, respectively. Fluorescence decay curves were recorded from LHCII complexes located in polyacrylamide gel separated by means of the non-denaturing gel electrophoresis
Fig. 5
Fig. 5
77 K Chl a fluorescence emission spectra of the LHCII dimers. Dimers were induced by low (18 µmol photons m−2 s−1) or high (1200 µmol photons m−2 s−1) light intensity. The spectra were normalized in the maximum. Spectra were measured from the LHCII complexes located in polyacrylamide gel
Fig. 6
Fig. 6
Room temperature CD spectra in the red region of different LHCII forms. The spectra were measured from the LHCII complexes separated by means of the non-denaturing gel electrophoresis. During separation the LHCII trimers and monomers mixture was illuminated with high light intensity (HL, 1200 µmol photons m−2 s−1). The spectra were registered from the complexes located in polyacrylamide gel. Spectra are normalized in the negative maximum at 650 nm
Fig. 7
Fig. 7
Proposed graphical presentation of high light-induced dimer structure created using YASARA (Krieger et al. 2002). a Top view (from the stromal side), b bottom view (from the lumenal side), c, d side views from the membrane plane perspectives. For clarity of presentation, interacting Chl a and b are marked red. Distance between Mg2+ ions in marked chlorophylls is equal to 0.906 nm. The illustration is based on the crystal structure of LHCII from PDB (ID: 2BHW). blue Chl a, green Chl b, yellow violaxanthin, magenta neoxanthin, orange lutein

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