Abstract
Under excess illumination, the Photosystem II light-harvesting antenna of higher plants has the ability to switch into an efficient photoprotective mode, allowing safe dissipation of excitation energy into heat. In this study, we show induction of the energy dissipation state, monitored by chlorophyll fluorescence quenching, in the isolated major light-harvesting complex (LHCII) incorporated into a solid gel system. Removal of detergent caused strong fluorescence quenching, which was totally reversible. Singlet-singlet annihilation and gel electrophoresis experiments suggested that the quenched complexes were in the trimeric not aggregated state. Both the formation and recovery of this quenching state were inhibited by a cross-linker, implying involvement of conformational changes. Absorption and CD measurements performed on the samples in the quenched state revealed specific alterations in the spectral bands assigned to the red forms of chlorophyll a, neoxanthin, and lutein 1 molecules. The majority of these alterations were similar to those observed during LHCII aggregation. This suggests that not the aggregation process as such but rather an intrinsic conformational transition in the complex is responsible for establishment of quenching. 77 K fluorescence measurements showed red-shifted chlorophyll a fluorescence in the 690-705 nm region, previously observed in aggregated LHCII. The fact that all spectral changes associated with the dissipative mode observed in the gel were different from those of the partially denatured complex strongly argues against the involvement of protein denaturation in the observed quenching. The implications of these findings for proposed mechanisms of energy dissipation in the Photosystem II antenna are discussed.
Highlights
Photosynthetic light harvesting is a controlled process, well integrated into the light phase of photosynthesis
It has been previously demonstrated that the removal of detergent, acidification, and addition of dibucaine or zeaxanthin all enhance the transition of LHCII into the quenched state with varying degrees of efficiency, in all cases the nature of the quenching state is the same [2]
The results obtained in this study provide important insights into the role of protein conformational changes and aggregation in the formation of excitation quenchers in LHCII
Summary
LHCII trimers were prepared from dark-adapted spinach leaves by an isoelectric focusing procedure as described [12]. Oligomeric LHCII was obtained by incubation of the trimers with 50 mg of SM-2 absorbent (Bio-Rad), allowing a 10-fold decrease in the fluorescence yield. Denaturation of LHCII complexes was achieved by incubation of samples at 70 °C for 5 min. Trimers or monomers of LHCII were introduced into a 6% polyacrylamide gel by the following procedure. Samples were diluted in a solution containing 20 mM HEPES (pH 7.8) and 0.03% n-dodecyl -D-maltoside, mixed with a solution of acrylamide/bisacrylamide (37.5:1), and polymerized with 0.05% ammonium persulfate and 0.03% TEMED for ϳ20 min using a Bio-Rad Mini-PROTEAN system.
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