W.P. Williams

Structural reorganisation of chloroplast thylakoid membranes in response to heat-stress

Abstract Chloroplasts isolated from broad bean (Vicia faba) show major structural reorganisations on heating to temperatures above 35°C. Exposure to increasing temperatures in the range 35–45°;C for 5 min, leads to a progressive destacking of the chloroplast membranes and the replacement of the normal granal arrangement by modified thylakoid attachment sites. An analysis of the size and packing densities of the freeze-fracture particles present in different membrane fracture-faces suggests that this rearrangement reflects the dissociation of the light-harvesting units of Photosystem II. The antennae complexes of Photosystem II appear to cluster together, maintaining regions of membrane adhesion, whilst excluding the core-complexes of Photosystem II and light-harvesting units of Photosystem I from these regions. If the chloroplasts are heated to higher temperatures, 45–55°C, phase-separated aggregates of non-bilayer-forming lipids are often observed. The release of these lipids from their normal constraints within the bilayer is consistent with the idea that they play a role in the packaging of the light-harvesting complexes within the thylakoid membrane.

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.

Comparison of ATP-induced and State 1/State 2-related changes in excitation energy distribution in Chlorella vulgaris

Abstract The addition of ATP to thylakoids isolated from Chlorella vulgaris is shown to lead to a quenching of fluorescence originating from Photosystem II and phosphorylation of chlorophyll a chlorophyll b light-harvesting protein (LHCP) directly analogous to that reported for higher-plant chloroplasts. The time courses of these two processes are shown to be identical. Parallel measurements of ATP-induced changes in the fluorescence properties of isolated algal thylakoids and light-driven (State 1 / State 2 changes) in whole cells strongly support the idea that LHCP phosphorylation plays an important role in State 2 adaptation under in vivo conditions.

Enhancement studies on algae and isolated chloroplasts. Part I. Variability of photosynthetic enhancement in Chlorella phyrenoidosa

Studies of the variability of enhancement in Chlorella pyrenoidosa confirm the existence of two types of variability: a very slow diurnal variation linked to the growth cycle and a much more rapid adaptive response to the immediate incident light conditions (State I-State II transitions). Measurements of the wavelength dependencies and relative contributions of these two types of variability suggest that they may be linked. A close examination of the enhancement signals associated with the State I-State II transition reveals that the transitions can take place in any one of three ways: by a change in Photosystem II efficiency alone, by a change in Photosystem I efficiency alone or by a simultaneous change in the efficiencies of both photo systems. Measurements of the rates of transition between State I, State II and the dark adapted state, Dark, suggest that the behaviour of State II and Dark are normally, but not always, identical. The transitions between the three states were found to be first order. For those samples exhibiting the same behaviour in Dark and State II, the rate of the State I-State II transition was found to be independent of the wavelength of Light II, suggesting that the return from State I to State II is essentially a dark process and that the driving force for the adaptive transition is the over-stimulation of Photosystem I. Finally, a model is proposed, involving an antagonistic control of the quantum yields of photochemistry of the two photosystems, that is capable of explaining the links between the two types of variability, their wavelength dependencies and the shapes of the individual enhancement signals.

Lipid-protein Interactions in the Thylakoid Membranes of Higher Plant Chloroplasts

The molecular organisation of the thylakoid membrane is usually described in terms of the fluid mosaic model. On this basis, the central matrix of the membrane is believed to consist of a fluid lipid bilayer. Membrane proteins are either attached to the surface of this bilayer by electrostatic forces or anchored within the bilayer by hydrophobic forces. A great deal of attention has been focussed on the spatial organisation of these protein components. Freeze-fracture studies have revealed the existence of large numbers of intra-membranous particles which can be characterised by their size, distribution between stromal and granal membranes, and the freeze-fracture faces with which they are associated (Staehelin et al. 1977; Arntzen 1978). Whilst there is still some debate regarding the origin of different groups of particles (Andersson, Anderson 1980), it is generally accepted that they reflect the presence of the chlorophyll-protein complexes of the two light-harvesting systems together with other intrinsic membrane proteins such as the cyt f / cyt b6 complex and the CF0 component of the coupling factor. Little or no attention, however, has been paid to the possible role of the membrane lipid fraction in thylakoid membrane organisation. This paper is directed at an examination of this problem.

Structural studies of plant membrane lipid dispersions subjected to oxidation in the presence of decomposing peroxychromate

Abstract Total membrane polar lipid extracts of broad bean ( Vicia faba ) leaves have been dispersed in phosphate buffer and oxidised in the presence of decomposing potassium peroxychromate. Changes in the organisation of the membrane structures formed by these lipids resulting from the oxidation of unsaturated fatty acvl residues were assessed by different biophysical methods. Fluorescence polarisation values of 1,6-diphenylhexatriene intercalated into oxidised dispersions indicated considerable restriction in probe motion. Temperature-dependence studies suggested that this restriction was not due to the removal of unsaturated residues by oxidation leaving predominantly gel-phase lipid. This view was confirmed by wide-angle. X-ray diffraction studies, which showed diffuse reflections in control and oxidised preparations centred around 0.46 nm as compared to a sharp reflection at about 0.41 nm in a hydrogenated sample, and electron microscopic studies. Freeze-fracture and negatively stained dispersions of unoxidised lipids show typical lamellar structures with inverted micelles of lipid sandwiched within the bilayer structure. Oxidation of the lipid destroys the bilayer arrangement and leads to a generally amorphous structure. These results are discussed in terms of the role of oxidation in senescing plant tissue.

Enhancement studies on algae and isolated chloroplasts. Part II. Enhancement of oxygen evolution in intact chloroplasts

Intact isolated chloroplasts were shown to exhibit a characteristic three-phase pattern of development of oxygen evolution activity. The first phase, Phase I, appeared to be an equilibration phase in which the isolated chloroplasts adapted to the conditions on the electrode surface. It was characterised by a rapidly increasing rate of oxygen evolution accompanied by decreasing enhancement signals. The second phase, Phase II, was an intermediate phase in which the rate of oxygen evolution was maximal and no enhancement was observed. In the last phase, Phase III, the rate of oxygen fell again, normal enhancement was still missing, but the samples appeared to undergo slow adaptive changes closely related to the State I-State II changes previously reported for whole cell systems. The concentrations of Mg2+ within the chloroplast were shown to play an important role in the control of the development of both the oxygen evolution and enhancement signals. It was shown how these signals could be explained in terms of a model that was consistent with that developed in Part I of this investigation to account for the variability of enhancement of the alga Chlorella pyrenoidosa.