Primary photosynthetic processes: from supercomplex to leaf

This thesis describes fluorescence spectroscopy experiments on photosynthetic complexes that cover the primary photosynthetic processes, from the absorption of light by photosynthetic pigments to a charge separation (CS) in the reaction center (RC). Fluorescence spectroscopy is a useful tool in photosynthetic particles, because the latter are densely packed with fluorescence pigments like chlorophylls (Chl). The fluorescence of each pigment is affected by its environment and provide information about structure and dynamics of the photosynthetic complexes. In this thesis time-resolved fluorescence of Chl molecules is used for studying the ultrafast kinetics in membrane particles of photosystem II (PSII) (chapter 2, 3 and 4). In chapter 5 fluorescence lifetime imaging microscopy (FLIM) of is applied to study entire chloroplasts, either in the leaf or in isolated chloroplast form. The advantage of FLIM is that the interactions of the fluorescence pigments in both photosystems can be spatially resolved up to a resolution of 0.5 x 0.5 x 2 µm to indentify and quantify photosynthetic processes in their natural environment. Excitation energy transfer and charge separation in PSII membranes (chapter 2,3 and 4) In this thesis time-resolved fluorescence measurements of PSII containing membranes, the so called BBY particles, are performed in low-light conditions with open reaction centers. The BBY particles do not contain photosystem I (PSI) or stroma lamellae, but do support electron transfer and carry out oxygen evolution with high activity and are comparable with the grana in vivo. The fluorescence decay kinetics of the BBY particles are faster than observed in previous studies and also faster than observed for PSII in chloroplasts and thylakoid preparations. The average lifetime is 150 ps, which, together with previous annihilation experiments on light-harvesting complex II (LHCII) suggests that excitation migration from the antenna complexes contributes significantly to the overall charge separation time. This is in disagreement with the commonly applied exciton / radical-pair-equilibrium (ERPE) model that assumes that excitation energy diffusion through the antenna to the RC is much faster than the overall charge-separation time. A simple coarse-grained method is proposed, based on the supramolecular organization of PSII and LHCII in grana membranes (C2S2M2). The proposed modelling procedure for BBY particles is only approximate and many different combinations of excitation migration time and the charge separation time can explain the observed fluorescence kinetics. However it is clear that charge transfer should be rather fast and is accompanied with a large drop in free energy. In chapter 3, the fluorescence kinetics of BBY particles with open RCs are compared after preferential excitation at 420 and 484 nm, which causes a difference in the initial excited-state populations of the inner and outer antenna system. The fluorescence decay is somewhat slower upon preferential excitation of chlorophyll (Chl) b, which is exclusively present in the outer antenna. Using the coarse-grained model it was possible to fit the 420 and 484 nm results simultaneously with a two-step electron transfer model and four parameters: the hopping rate between the protein-pigment complexes, the CS rate, the drop in free energy upon primary charge separation and a secondary charge separation rate. The conclusion is that the average migration time contributes ~25% to the overall trapping time. The hopping time obtained in chapter 3 is significantly faster than might be expected based on studies on trimeric and aggregated LHCII and it is concluded that excitation energy transfer in PSII follows specific pathways that require an optimized organization of the antenna complexes with respect to each other. Analysis of the composition of the BBY particles indicates that the size of the light-harvesting system in PSII is smaller than commonly found for PSII in chloroplasts and explains why the fluorescence lifetimes are smaller for the BBY’s. In chapter 4, four different PSII supercomplex preparations were studied. The main difference between these supercomplexes concerns the size of the outer antenna. The average lifetime of the supercomplexes becomes longer upon increasing the antenna size. The results indicate that the rate constants obtained from the coarse-grained method for BBY preparations, which is based on the supercomplex composition C2S2M2, should be slightly faster (~10%) as predicted in chapter 3. The observation that the average lifetime of the supercomplexes is relatively slow compared to what one might expect based on the measurements on BBY particles, and this will require further future studies. Photosynthesis in plant leaves (Chapter 5) With the use of femtosecond two-photon excitation TPE at 860 nm it appears to be possible to measure fluorescence lifetimes throughout the entire leaves of Arabidopsis thaliana and Alocasia wentii. It turns out that the excitation intensity can be kept sufficiently low to avoid artifacts due to singlet-singlet and singlet-triplet annihilation, while the reaction centers can be kept in the open state during the measurements. The average fluorescence lifetimes obtained for individual chloroplasts of Arabidopsis thaliana and Alocasia wentii in the open and closed state, are approximately ~250 ps and ~1.5 ns, respectively. The maximum fluorescence state correspond to a state in which all reaction centers are closed. The kinetics are very similar to those obtained for chloroplasts in vitro with the FLIM setup and to in vivo results reported in literature. No variations between chloroplasts are observed when scanning throughout the leaves of Arabidopsis thaliana and Alocasia wentii. Within individual chloroplasts some variation is detected for the relative contributions of PSI and PSII to the fluorescence. The results open up the possibility to use FLIM for the in vivo study of the primary processes of photosynthesis at the level of single chloroplasts under all kinds of (stress) conditions. General conclusions This thesis gives new insight of the kinetic processes in PSII membranes. With the use of a coarse-grained method that provides an easy way to incorporate existing knowledge and models for individual complexes, valuable conclusions can be drawn about the excitation energy transfer and the CS which hopefully contributes to an improvement of the knowledge about PSII functioning. In general it was shown that a large drop in free energy is needed in PSII membranes for all simulations with the coarse-grained method. The presented results on the kinetics of chloroplasts obtained in vitro and in vitro are very similar and verify that conclusions drawn from isolated chloroplasts can be extrapolated to photosynthetic processes in their natural environment. <br/

Applying two-photon excitation fluorescence lifetime imaging microscopy to study photosynthesis in plant leaves

This study investigates to which extent two-photon excitation (TPE) fluorescence lifetime imaging microscopy can be applied to study picosecond fluorescence kinetics of individual chloroplasts in leaves. Using femtosecond 860 nm excitation pulses, fluorescence lifetimes can be measured in leaves of Arabidopsis thaliana and Alocasia wentii under excitation-annihilation free conditions, both for the F0- and the Fm-state. The corresponding average lifetimes are ~250 ps and ~1.5 ns, respectively, similar to those of isolated chloroplasts. These values appear to be the same for chloroplasts in the top, middle, and bottom layer of the leaves. With the spatial resolution of ~500 nm in the focal (xy) plane and 2 μm in the z direction, it appears to be impossible to fully resolve the grana stacks and stroma lamellae, but variations in the fluorescence lifetimes, and thus of the composition on a pixel-to-pixel base can be observed

Multiscale photosynthetic exciton transfer

Photosynthetic light harvesting provides a natural blueprint for bioengineered and biomimetic solar energy and light detection technologies. Recent evidence suggests some individual light harvesting protein complexes (LHCs) and LHC subunits efficiently transfer excitons towards chemical reaction centers (RCs) via an interplay between excitonic quantum coherence, resonant protein vibrations, and thermal decoherence. The role of coherence in vivo is unclear however, where excitons are transferred through multi-LHC/RC aggregates over distances typically large compared with intra-LHC scales. Here we assess the possibility of long-range coherent transfer in a simple chromophore network with disordered site and transfer coupling energies. Through renormalization we find that, surprisingly, decoherence is diminished at larger scales, and long-range coherence is facilitated by chromophoric clustering. Conversely, static disorder in the site energies grows with length scale, forcing localization. Our results suggest sustained coherent exciton transfer may be possible over distances large compared with nearest-neighbour (n-n) chromophore separations, at physiological temperatures, in a clustered network with small static disorder. This may support findings suggesting long-range coherence in algal chloroplasts, and provides a framework for engineering large chromophore or quantum dot high-temperature exciton transfer networks.Comment: 9 pages, 6 figures. A significantly updated version is now published online by Nature Physics (2012

Digalactosyl-diacylglycerol-deficiency lowers the thermal stability of thylakoid membranes

We investigated the effects of digalactosyl-diacylglycerol (DGDG) on the organization and thermal stability of thylakoid membranes, using wild-type Arabidopsis thaliana and the DGDG-deficient mutant, dgd1. Circular-dichroism measurements reveal that DGDG-deficiency hampers the formation of the chirally organized macrodomains containing the main chlorophyll a/b light-harvesting complexes. The mutation also brings about changes in the overall chlorophyll fluorescence lifetimes, measured in whole leaves as well as in isolated thylakoids. As shown by time-resolved measurements, using the lipophylic fluorescence probe Merocyanine 540 (MC540), the altered lipid composition affects the packing of lipids in the thylakoid membranes but, as revealed by flash-induced electrochromic absorbance changes, the membranes retain their ability for energization. Thermal stability measurements revealed more significant differences. The disassembly of the chiral macrodomains around 55°C, the thermal destabilization of photosystem I complex at 61°C as detected by green gel electrophoresis, as well as the sharp drop in the overall chlorophyll fluorescence lifetime above 45°C (values for the wild type—WT) occur at 4–7°C lower temperatures in dgd1. Similar differences are revealed in the temperature dependence of the lipid packing and the membrane permeability: at elevated temperatures MC540 appears to be extruded from the dgd1 membrane bilayer around 35°C, whereas in WT, it remains lipid-bound up to 45°C and dgd1 and WT membranes become leaky around 35 and 45°C, respectively. It is concluded that DGDG plays important roles in the overall organization of thylakoid membranes especially at elevated temperatures

Excitation energy transfer in native and unstacked thylakoid membranes studied by low temperature and ultrafast fluorescence spectroscopy

In this work, the transfer of excitation energy was studied in native and cation-depletion induced, unstacked thylakoid membranes of spinach by steady-state and time-resolved fluorescence spectroscopy. Fluorescence emission spectra at 5 K show an increase in photosystem I (PSI) emission upon unstacking, which suggests an increase of its antenna size. Fluorescence excitation measurements at 77 K indicate that the increase of PSI emission upon unstacking is caused both by a direct spillover from the photosystem II (PSII) core antenna and by a functional association of light-harvesting complex II (LHCII) to PSI, which is most likely caused by the formation of LHCII-LHCI-PSI supercomplexes. Time-resolved fluorescence measurements, both at room temperature and at 77 K, reveal differences in the fluorescence decay kinetics of stacked and unstacked membranes. Energy transfer between LHCII and PSI is observed to take place within 25 ps at room temperature and within 38 ps at 77 K, consistent with the formation of LHCII-LHCI-PSI supercomplexes. At the 150-160 ps timescale, both energy transfer from LHCII to PSI as well as spillover from the core antenna of PSII to PSI is shown to occur at 77 K. At room temperature the spillover and energy transfer to PSI is less clear at the 150 ps timescale, because these processes compete with charge separation in the PSII reaction center, which also takes place at a timescale of about 150 ps. © 2007 Springer Science+Business Media B.V

Molecular basis of photoprotection and control of photosynthetic light-harvesting

In order to maximize their use of light energy in photosynthesis, plants have molecules that act as light-harvesting antennae, which collect light quanta and deliver them to the reaction centres, where energy conversion into a chemical form takes place. The functioning of the antenna responds to the extreme changes in the intensity of sunlight encountered in nature1, 2, 3. In shade, light is efficiently harvested in photosynthesis. However, in full sunlight, much of the energy absorbed is not needed and there are vitally important switches to specific antenna states, which safely dissipate the excess energy as heat2, 3. This is essential for plant survival4, because it provides protection against the potential photo-damage of the photosynthetic membrane5. But whereas the features that establish high photosynthetic efficiency have been highlighted6, almost nothing is known about the molecular nature of the dissipative states. Recently, the atomic structure of the major plant light-harvesting antenna protein, LHCII, has been determined by X-ray crystallography7. Here we demonstrate that this is the structure of a dissipative state of LHCII. We present a spectroscopic analysis of this crystal form, and identify the specific changes in configuration of its pigment population that give LHCII the intrinsic capability to regulate energy flow. This provides a molecular basis for understanding the control of photosynthetic light-harvestin