Carotenoid–Chlorophyll Coupling and Fluorescence Quenching Correlate with Protein Packing Density in Grana-Thylakoids

Abstract

ABSTRACT:The regulation of light-harvesting in photosynthesis under conditions of varying solar light irradiation is essential for the survival and fitness of plants and algae. It has been proposed that rearrangements of protein distribution in the stacked grana region of thylakoid membranes connected to changes in the electronic pigment-interaction play a key role for this regulation. In particular, carotenoid−chlorophyll interactions seem to be crucial for the downregulation of photosynthetic light-harvesting. So far, it has been difficult to determine the influence of the dense protein packing found in native photosynthetic membrane on these interactions. We investigated the changes of the electronic couplings between carotenoids and chlorophylls and the quenching in grana thylakoids of varying protein packing density by two-photon spectroscopy, conventional chlorophyll fluorometry, low-temperature fluorescence spectroscopy, and electron micrographs of freeze-fracture membranes. We observed an increasing carotenoid−chlorophyll coupling and fluorescence quenching with increasing packing density. Simultaneously, the antennas size and excitonic connectivity of Photosystem II increased with increasing quenching and carotenoid−chlorophyll coupling whereas isolated, decoupled LHCII trimers decreased. Two distinct quenching data regimes could be identified that show up at different protein packing densities. In the regime corresponding to higher protein packing densities, quenching is strongly correlated to carotenoid−chlorophyll interactions whereas in the second regime, a weak correlation is apparent with low protein packing densities. Native membranes are in the strong-coupling data regime. Consequently, PSII and LHCII in grana membranes of plants are already quenched by protein crowding. We concluded that this ensures efficient electronic connection of all pigment−protein complexes for intermolecular energy transfer to the reaction centers and allows simultaneously sensitive regulation of light harvesting by only small changes in the protein packaging. ■ INTRODUCTIONPhotosynthesis in higher plants and algae is initiated by the harvesting of sunlight through a series of pigment−protein complexes located in the thylakoid membranes of chloroplasts. Starting from these light harvesting complexes (LHCs), the absorbed energy is efficiently transferred from one complex to the other. These transfer processes end up in the photosystem reaction centers where the energy is converted into redox energy. However, plants are exposed to changing light intensities on a daily basis that can vary over several orders of magnitude. 1 Thus, a regulation mechanism is necessary that assures effective usage of sunlight energy under low-light conditions but avoids photo-oxidative damage to the reaction centers or other parts of the photosynthetic apparatus if the absorbed light exceeds its capacity. A main mechanism that evolved to minimize photo-oxidative damage in plants is called high-energy quenching (qE). qE enables the dissipation ...

PsbS‐dependent and ‐independent mechanisms regulate carotenoid‐chlorophyll energy coupling in grana thylakoids

“…As reported previously the Car → Chl energy transfer, ( y ‐axis), generally decreases with lower protein density and decreased quenching (increasing F OPE ). For example, in the first panel (a, data of samples deficient in PsbS, NPQ4), decreasing values with decreasing BBY membrane protein density are observed.…”

“…Thus, NPQ in nondiluted BBY increases in the order L17 > WT > npq4, that is, as expected NPQ is dependent on the PsbS level in grana membranes. As reported previously [29] the Car ? Chl energy transfer, U CarÀChl Coupling (y-axis), generally decreases with lower protein density and decreased quenching (increasing F OPE ).…”

“…Arabidopsis thaliana wild‐type, npq4 knockout, and PsbS overexpressor (L17) plants were grown for 7–8 weeks at about 100 μmol quanta·m −2 ·s −1 and 9 h daylight. BBY membranes were isolated as described elsewhere . Preparation of liposomes made of a thylakoid lipid mixture and their fusion with isolated BBY membranes to dilute the protein packing density in BBY membranes are described in Ref.…”

“…Holleboom et al . showed that a reduction in the protein density in isolated grana membranes by dilution with a lipid mixture mimicking the native composition leads to a decrease in the Car–Chl interactions, . This can be rationalized by larger distances of the LHCs to each other or to the PSII core by the decreased density of the packed membrane .…”

“…To explore the role of PSII/LHCII interactions for the PsbS‐dependent regulation of light harvesting and the Car–Chl interactions, , we investigated intact grana membrane from mutants deficient in PsbS (BBY‐NPQ4 ) and overexpressing PsbS (BBY‐L17 ) and compare them with WT (BBY‐WT) grana preparations. Similar as in Holleboom et al ., the protein density in each mutant was systematically varied by adding a lipid mixture that mimics the native lipid composition. The changes in quenching, as well as, Car–Chl interactions, , of samples of identical Chl to lipid ratio were then compared directly.…”