Abstract
Mobile light-harvesting complex II (LHCII) is implicated in the regulation of excitation energy distribution between Photosystem I (PSI) and Photosystem II (PSII) during state transitions. To investigate how LHCII interacts with PSI during state transitions, PSI was isolated from Arabidopsis thaliana plants treated with PSII or PSI light. The PSI preparations were made using digitonin. Chemical cross-linking using dithio-bis(succinimidylpropionate) followed by diagonal electrophoresis and immunoblotting showed that the docking site of LHCII (Lhcb1) on PSI is comprised of the PSI-H, -L, and -I subunits. This was confirmed by the lack of energy transfer from LHCII to PSI in the digitonin-PSI isolated from plants lacking PSI-H and -L. Digitonin-PSI was purified further to obtain an LHCII.PSI complex, and two to three times more LHCII was associated with PSI in the wild type in State 2 than in State 1. Lhcb1 was also associated with PSI from plants lacking PSI-K, but PSI from PSI-H, -L, or -O mutants contained only about 30% of Lhcb1 compared with the wild type. Surprisingly, a significant fraction of the LHCII bound to PSI in State 2 was not phosphorylated. Cross-linking prior to sucrose gradient purification resulted in copurification of phosphorylated LHCII in the wild type, but not with PSI from the PSI-H, -L, and -O mutants. The data suggest that migration of LHCII during state transitions cannot be explained sufficiently by different affinity of phosphorylated and unphosphorylated LHCII for PSI but is likely to involve structural changes in thylakoid organization.
Highlights
In oxygenic photosynthesis two photosystems, PSI1 and Photosystem II (PSII), work in series to convert light energy into chemical energy
The light intensity passing through the filters was about 50 –70 mol photons mϪ2 sϪ1, The plants were dark-adapted for 30 min before measuring the maximum fluorescence signal (Fm), and subsequently the leaves were exposed to orange light (L2, which favors PSII) for 20 min (Fig. 1)
The majority of light-harvesting complex II (LHCII) trimers are bound to PSII [15], but a fraction of mobile LHCII is able to dissociate from PSII
Summary
Plant Materials—Arabidopsis thaliana (L.) Heyhn ecotype Col-0 was used for all experiments. The leaf was exposed to orange light (which favors PSII, L2) for about 20 min, and the maximum fluorescence yield in State 2 (Fm2) was determined. The fiber with the leaf was transferred rapidly to the red light (which favors PSI, L1), and after 20 min the maximum fluorescence yield (Fm1) was measured. Diagonal Electrophoresis—The digitonin-PSI preparations (treated with PSII light) were diluted to 0.3 mg of Chl/ml in 20 mM Tricine, pH 7.5, 0.3% (w/v) DM, and 10 mM NaF. Digitonin-PSI preparations (15 g of Chl) were diluted to a total volume of 500 l in 50 mM Tricine buffer, pH 7.5, 0.01% (w/v) digitonin, and 0.5 mM ascorbate and 0.05 mM methyl viologen were added prior to measurements. The half-life (t1⁄2) was calculated after fitting of exponential curves to the absorption curves
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