Anthony P.R. Brain

Photoprotective Energy Dissipation Involves the Reorganization of Photosystem II Light-Harvesting Complexes in the Grana Membranes of Spinach Chloroplasts

The rapidly reversible macrostructural changes in higher-plant chloroplast thylakoid membrane organization accompanying photoprotective energy dissipation (qE) are studied using freeze-fracture electron and laser confocal microscopy. qE is shown to involve the aggregation of light-harvesting complexes and their segregation from photosystem II. Plants must regulate their use of absorbed light energy on a minute-by-minute basis to maximize the efficiency of photosynthesis and to protect photosystem II (PSII) reaction centers from photooxidative damage. The regulation of light harvesting involves the photoprotective dissipation of excess absorbed light energy in the light-harvesting antenna complexes (LHCs) as heat. Here, we report an investigation into the structural basis of light-harvesting regulation in intact spinach (Spinacia oleracea) chloroplasts using freeze-fracture electron microscopy, combined with laser confocal microscopy employing the fluorescence recovery after photobleaching technique. The results demonstrate that formation of the photoprotective state requires a structural reorganization of the photosynthetic membrane involving dissociation of LHCII from PSII and its aggregation. The structural changes are manifested by a reduced mobility of LHC antenna chlorophyll proteins. It is demonstrated that these changes occur rapidly and reversibly within 5 min of illumination and dark relaxation, are dependent on ΔpH, and are enhanced by the deepoxidation of violaxanthin to zeaxanthin.

Structural and functional changes associated with heat-induced phase-separations of non-bilayer lipids in chloroplast thylakoid membranes

The effect of hearing isolated bean chloroplasts on the structure of their thylakoid membranes has been examined by freeze‐fracture electron microscopy. A normal morphology of the membranes in which stacked grana can be observed is preserved up to 35°C. Incubation at 35–45°C causes complete destacking of the grana but no alteration in the distribution of the membrane‐associated particles between the exoplasmic and protoplasmic fracture faces. Heating above 45°C causes phase‐separation of non‐bilayer lipids into aggregates of cylindrical inverted micelles. Bleaching experiments show that destacking is associated with disruption of chlorophyll‐protein complexes of both photosystems I and II. The rate of electron transport through the photosystems is also perturbed. These results are discussed in terms of the role of non‐bilayer lipids in packaging the membrane proteins.

Light-harvesting antenna composition controls the macrostructure and dynamics of thylakoid membranes in Arabidopsis

We characterized a set of Arabidopsis mutants deficient in specific light-harvesting proteins, using freeze-fracture electron microscopy to probe the organization of complexes in the membrane and confocal fluorescence recovery after photobleaching to probe the dynamics of thylakoid membranes within intact chloroplasts. The same methods were used to characterize mutants lacking or over-expressing PsbS, a protein related to light-harvesting complexes that appears to play a role in regulation of photosynthetic light harvesting. We found that changes in the complement of light-harvesting complexes and PsbS have striking effects on the photosystem II macrostructure, and that these effects correlate with changes in the mobility of chlorophyll proteins within the thylakoid membrane. The mobility of chlorophyll proteins was found to correlate with the extent of photoprotective non-photochemical quenching, consistent with the idea that non-photochemical quenching involves extensive re-organization of complexes in the membrane. We suggest that a key feature of the physiological function of PsbS is to decrease the formation of ordered semi-crystalline arrays of photosystem II in the low-light state. Thus the presence of PsbS leads to an increase in the fluidity of the membrane, accelerating the re-organization of the photosystem II macrostructure that is necessary for induction of non-photochemical quenching.

The formation of non-bilayer structures in total polar lipid extracts of chloroplast membranes

Abstract The structural organisation of aqueous dispersions of total membrane lipid extracts of broad bean ( Vicia faba ) chloroplasts is dependent on pH and the presence of cations. In the absence of inorganic salts, sonicated dispersions of lipid extract in distilled water form smooth, single-shell vesicles approximately 30–50 nm in diameter. Reducing the pH of the dispersions, to neutralise the acidic lipids present in the extract, or the addition of low concentrations of metal cations, leads to the fusion of the vesicles and a partial phase-separation of the non-bilayer forming lipid monogalactosyldiacylglycerol to form spherical inverted micelles similar to those previously reported for binary mixtures of monogalactosyl and digalactosyldiacylglycerol (Biochim. Biophys. Acta 685, 297–306). Increasing concentrations of polyvalent, but not monovalent, cations lead to further structural rearrangements involving the formation of para-crystalline arrays of tubular and spherical inverted micelles. The factors determining the formation of these different structures, and their possible relevance to the structural organisation of the native chloroplast membrane, are discussed.

Bilayer and non-bilayer transformations in aqueous dispersions of mixed sn-3-galactosyldiacylglycerols isolated from chloroplasts A freeze-fracture study

Abstract A number of different particle and ‘particle-like’ structures are observed in freeze-fracture replicas prepared from aqueous dispersions of mixtures of mono- and digalactosyldiacylglycerol. The smallest of these structures (10–12 nm in diameter) corresponding to inverted lipid micelles sandwiched within lipid bilayers are often organised into extensive planar arrays. A number of larger ‘particle-like’ features are also observed in replicas of this type. An analysis of the relationship between these structures suggests that they reflect responses to stresses associated with a temperature-dependent incorporation of the lipids of the inverted micelles into the lamellar structure.

The phase behaviour of 1,2-diacyl-3-monogalactosyl-sn-glycerol derivatives

Abstract Monogalactosyldiacylglycerol was isolated from the blue-green alga Anacystis nidulans . Part of this lipid, which is rich in the 1–16:1/2–16:0 derivative, was hydrogenated to yield a lipid fraction rich in the 1–16:0/2–16:0 derivative. The phase behaviour of the two fractions were studied using differential scanning calorimetry, wide-angle X-ray diffraction and freeze-fracture electron microscopy. Both fractions exhibited complex polymorphic behaviour. Two distinct gel phases were identified; a stable form (MGDG 1 ) and a metastable form (MGDG II ). The transition temperatures for the two forms were 345 K and 325 K for the 1–16:0/2–16:0 fraction and 311 K and 279 K for the 1–16:1/2–16:0 fraction, respectively. The corresponding enthalpy values were 59.3 and 24.5 kJ · mol −1 and 51.4 and 11.5 kJ · mol −1 . Inverted hexagonal (H 11 ) phases were seen at higher temperatures. The transition to the H 11 phase appears to occur directly from MGDG 1 gel phase, but may involve the formation of a lamellar liquid -crystalline phase existing between the melting points of the two gel phases in the case of the transitions from the MGDG 11 gel phase.

Phase separations in membranes of Anacystis nidulans grown at different temperatures

Freeze fracture electron microscopy studies were performed on samples of Anacystis nidulans quenched from different temperatures. Membrane lipid phase separations were observed to take place over the ranges 15--30 degrees C, 5--25 degrees C and -5--15 degrees C for cultures grown at 38, 28 and 18 degrees C, respectively. Differential scanning calorimetry heating curves showed endotherms which coincided with these temperature ranges. Variations of phase separation temperatures with growth temperature, and hysteresis effects in the calorimetric measurements, were related to changes in the fatty acid composition of membrane lipids.

Low pH and phospholipase A2 treatment induce the phase-separation of non-bilayer lipids within pea chloroplast membranes

Membrane lipid Hexagonal phase Freeze‐fracture Chloroplast

Changes in thylakoid membrane thickness associated with the reorganization of photosystem II light harvesting complexes during photoprotective energy dissipation

Using freeze-fracture electron microscopy we have recently shown that non-photochemical quenching (NPQ), a mechanism of photoprotective energy dissipation in higher plant chloroplasts, involves a reorganization of the pigment-protein complexes within the stacked grana thylakoids1. Photosystem II light harvesting complexes (LHCII) are reorganized in response to the amplitude of the light driven transmembrane proton gradient (ΔpH) leading to their dissociation from photosystem II reaction centers and their aggregation within the membrane1. This reorganization of the PSII-LHCII macrostructure was found to be enhanced by the formation of zeaxanthin and was associated with changes in the mobility of the pigment-protein complexes therein1. We suspected that the structural changes we observed were linked to the ΔpH-induced changes in thylakoid membrane thickness that were first observed by Murikami and Packer2,3. Here using thin-section electron microscopy we show that the changes in thylakoid membrane thickness do not correlate with ΔpH per se but rather the amplitude of NPQ and is thus affected by the de-epoxidation of the LHCII bound xanthophyll violaxanthin to zeaxanthin. We thus suggest that the change in thylakoid membrane thickness occurring during NPQ reflects the conformational change within LHCII proteins brought about by their protonation and aggregation within the membrane

The effect of chemical modification of Saccharomyces cerevisiae on electrophoretic mobility, cell-wall structure and amphotericin B uptake

Saccharomyces cerevisiae NCYC 239 suspended in solutions of NaCl showed two distinct plateaus in plots of electrophoretic mobility vs. pH, corresponding to pKa values of approx. 2 and 5. This is in contrast to cells suspended in buffer where only a single pKa (4) can be determined. Modification of cells with KI/I2 or nitrous acid led to altered electrophoretic mobility, indicating the presence of sulphydryl and amino groups, respectively, in the yeast cell surface, whereas uranyl nitrate modification had little effect, suggesting phosphate groups to be absent. Electron micrographs showed visible effects of KI/I2 and nitrous acid modification on cell membrane structure, and in these modified cells amphotericin B uptake was rapid. It is suggested that diffusion through the cell wall is the rate-limiting step for amphotericin B uptake. An activation energy of 20 kJ X mol-1 was determined for uptake of amphotericin B by unmodified cells.