Abstract
Light-Harvesting Complex II (LHCII) is a chlorophyll-protein antenna complex that efficiently absorbs solar energy and transfers electronic excited states to photosystems I and II. Under excess light intensity LHCII can adopt a photoprotective state in which excitation energy is safely dissipated as heat, a process known as Non-Photochemical Quenching (NPQ). In vivo NPQ is triggered by combinatorial factors including transmembrane ΔpH, PsbS protein and LHCII-bound zeaxanthin, leading to dramatically shortened LHCII fluorescence lifetimes. In vitro, LHCII in detergent solution or in proteoliposomes can reversibly adopt an NPQ-like state, via manipulation of detergent/protein ratio, lipid/protein ratio, pH or pressure. Previous spectroscopic investigations revealed changes in exciton dynamics and protein conformation that accompany quenching, however, LHCII-LHCII interactions have not been extensively studied. Here, we correlated fluorescence lifetime imaging microscopy (FLIM) and atomic force microscopy (AFM) of trimeric LHCII adsorbed to mica substrates and manipulated the environment to cause varying degrees of quenching. AFM showed that LHCII self-assembled onto mica forming 2D-aggregates (25–150 nm width). FLIM determined that LHCII in these aggregates were in a quenched state, with much lower fluorescence lifetimes (~0.25 ns) compared to free LHCII in solution (2.2–3.9 ns). LHCII-LHCII interactions were disrupted by thylakoid lipids or phospholipids, leading to intermediate fluorescent lifetimes (0.6–0.9 ns). To our knowledge, this is the first in vitro correlation of nanoscale membrane imaging with LHCII quenching. Our findings suggest that lipids could play a key role in modulating the extent of LHCII-LHCII interactions within the thylakoid membrane and so the propensity for NPQ activation.
Highlights
Photosynthetic light harvesting occurs at the thylakoid membranes of chloroplasts of plants, algae and cyanobacteria [1]
Light-Harvesting Complex II (LHCII) trimers were optically characterized in a dilute, stable form in detergent solution in standard cuvette-based absorption and fluorescence spectrometers: an absorbance spectrum with Chl a Qy maximum at 675 nm (Fig. 1D), an emission maximum at 681 nm (Fig. 1D) and a fluorescence decay curve fitted to an amplitude-weighted fluorescence lifetime of τ = 3.8–4.0 ns (Fig. 1E)
10.5 ± 2.2 13.5 ± 2.1 a Denotes concentration of LHCII solution incubated with mica surface for 20 min followed by washing surface with copious buffer to remove unbound protein. b Incubated with dioleoyl phosphocholine (DOPC) lipid vesicles for 20 min, following LHCII adsorption to mica. c Mean of at least 4 atomic force microscopy (AFM) images taken of different regions of the substrate, standard deviation (S.D.) shown. d Mean of well-resolved LHCII-LHCII pairs, S.D. shown (n = 49, 51, respectively)
Summary
Photosynthetic light harvesting occurs at the thylakoid membranes of chloroplasts of plants, algae and cyanobacteria [1]. NPQ is triggered under excess light conditions by the interplay of three factors: the transmembrane ΔpH [10], the PsbS protein [11] and the enzymatic de-epoxidation of the LHCII bound xanthophyll violaxanthin to zeaxanthin [12]. Together these factors bring about a change in BBA-Bioenergetics1859(2018)1075–1085 conformation of LHCII, and possibly the minor LHC antenna complexes CP24, 26 and 29 [13,14,15], that leads to formation of dissipative energy transfer pathways correlated to a dramatic shortening of the Chl excited state lifetime [16, 17]. The dynamics and efficiency of NPQ formation and relaxation have been shown to be crucial to plant fitness in fluctuating light environments as experienced in nature [18,19,20]
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