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.
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| Format: | Doctoral thesis biblioteca |
| Language: | English |
| Subjects: | chloroplasts, fluorescence, fluorescence microscopy, membranes, photosynthesis, photosystem ii, plants, spectroscopy, chloroplasten, fluorescentie, fluorescentiemicroscopie, fotosynthese, fotosysteem ii, membranen, planten, spectroscopie, |
| Online Access: | https://research.wur.nl/en/publications/primary-photosynthetic-processes-from-supercomplex-to-leaf |
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