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1.
Homogeneous catalytic hydrogenation was used to modify the level of fatty acid unsaturation of thylakoid membranes in the pea chloroplast. Fluidity alteration has been monitored simultaneously using the spin-label probe, 16-doxyl stearate. Even in the case of 30% hydrogenation, no change in the reduction rate of flash-oxidized cytochrome f was observed, in contrast to the fact that the same decrease in the double-bond content of the thylakoid membrane resulted in a pronounced inhibition in the full-chain electron transport. We conclude that the rate of lateral diffusion of reduced plastoquinone is unaffected by the lowering of the fluidity of the thylakoid lipid matrix.  相似文献   

2.
The effect of alterations of lipid phase order of thylakoid membranes on the thermosensitivity of photosystem I (PS I) and photosystem II (PS II) was studied. Plant sterols stigmasterol and cholesterol were applied to decrease the fluidity in isolated membranes. After sterol treatment, a decrease of the temperature of 50 % inhibition of PSII activity was observed. Heat stress-induced stimulation of PSI-mediated electron transport rate was registered for control, but not for sterol-treated membranes. Effect of altered lipid order on oxygen evolving complex was evaluated by means of flash oxygen yields revealing changes in the stoichiometry of PSIIα and PSIIβ centers. The effect of sterol incorporation on the changes in the thermotropic behavior of the main pigment-protein complexes was studied by differential scanning calorimetry (DSC). DSC traces of control thylakoids in the temperature range 20–98 °C exhibited several irreversible endothermic transitions. Incorporation of cholesterol and stigmasterol results in superimposition of the transitions and only two main bands could be resolved. While high temperature band peaks at the same temperature after treatment with both sterols, the band that combines low temperature transitions shows different melting temperature (Tm): 70 °C for stigmasterol- and 65 °C for cholesterol-treated membranes. The data presented here emphasise the crucial role of lipid order for the response of thylakoids to high temperatures, mediated not only by changes in the fluidity of bulk lipid phase as result of sterol incorporation but also by changes in the thermotropic properties of pigment-protein complexes.Key words: Cholesterol, Fluidity, Heat stress, Oxygen flash yields, Thylakoid membrane, Stigmasterol  相似文献   

3.
R.C. Ford  J. Barber 《BBA》1983,722(2):341-348
The hydrophobic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene has been incorporated into the membranes of isolated thylakoids, separated granal and stromal lamellae and aqueous dispersions of extracted thylakoid galactolipids. Time-resolved fluorescence decays have been recorded on a nanosecond scale using single-photon counting in order to assess the motional properties of the probe. All the experimental systems used showed biphasic decay kinetics and the anisotropies of the decays have been interpreted in terms of a model for wobbling diffusion confined to a cone. The analysis has given information about dynamic and structural restraints of the lipid acyl chains. In the intact thylakoid membrane the degree of order of the fatty acid acyl chains is higher and their rate of motion slower than for isolated lipids. Even so, the dynamic and structural parameters indicate that the thylakoids can be considered as a relatively fluid membrane system when compared with many other biological membranes, a property which is probably required to facilitate efficient long-range diffusion of lipophilic mobile electron-transport components. It is suggested that the optimization of thylakoid fluidity is linked to regulation of the membrane protein/lipid ratio which is also likely to be responsible for the higher fluidity of stromal membranes relative to those of the grana.  相似文献   

4.
The effect of Mg2+-ions on the physical state of thylakoid membrane and kinetics of electron transport between two photosystems were studied. The rate of electron transport from photosystem 2 to P700+ and the activity of photosystem 2 were obtained from the kinetics of P700 redox transients induced by flashes of white light (t1/2 = 7 musec or 0.75 msec) fired simultaneously with the background continuous far-red light (707 nm). The spin-labeled stearic acids (I1.14 and I12.3) were used as indicators of Mg2+-induced structural changes. Addition of MgCl2 stimulates incorporation of spin-labels into the lipid region of the thylakoid membrane. It was found that Mg2+-ions modify the ESR spectrum of I12.3. The results evidence that the screening of charged groups on the thylakoid membrane surface induces structural changes in the lipid region of the membrane. We have concluded that these structural changes result in reorientation of lipid molecules in the thylakoid membrane. There is a correlation between Mg2+-induced structural changes and electron transport in chloroplasts. Addition of Mg2+-ions stimulates the photochemical activity of photosystem 2 by increasing the amount of active reaction centres and modifies the rate constant of electron transport from photosystem 2 to P700+. It has been demonstrated that ion regulation of electron transport in more effective in the oxidising side than in the reducing side of plastoquinone shuttle.  相似文献   

5.
Plants, algae, and photosynthetic bacteria experience frequent changes in environment. The ability to survive depends on their capacity to acclimate to such changes. In particular, fluctuations in temperature affect the fluidity of cytoplasmic and thylakoid membranes. The molecular mechanisms responsible for the perception of changes in membrane fluidity have not been fully characterized. However, the understanding of the functions of the individual genes for fatty acid desaturases in cyanobacteria and plants led to the directed mutagenesis of such genes that altered the membrane fluidity of cytoplasmic and thylakoid membranes. Characterization of the photosynthetic properties of the transformed cyanobacteria and higher plants revealed that lipid unsaturation is essential for protection of the photosynthetic machinery against environmental stresses, such as strong light, salt stress, and high and low temperatures. The unsaturation of fatty acids enhances the repair of the damaged photosystem II complex under stress conditions. In this review, we summarize the knowledge on the mechanisms that regulate membrane fluidity, on putative sensors that perceive changes in membrane fluidity, on genes that are involved in acclimation to new sets of environmental conditions, and on the influence of membrane properties on photosynthetic functions.  相似文献   

6.
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.  相似文献   

7.
In chloroplasts of land plants, the thylakoid network is organized into appressed regions called grana stacks and loosely arranged parallel stroma thylakoids. Many factors determining such intricate structural arrangements have been identified so far, including various thylakoid-embedded proteins, and polar lipids that build the thylakoid matrix. Although carotenoids are important components of proteins and the lipid phase of chloroplast membranes, their role in determining the thylakoid network structure remains elusive. We studied 2D and 3D thylakoid network organization in carotenoid-deficient mutants (ccr1-1, lut5-1, szl1-1, and szl1-1npq1-2) of Arabidopsis (Arabidopsis thaliana) to reveal the structural role of carotenoids in the formation and dynamics of the internal chloroplast membrane system. The most significant structural aberrations took place in chloroplasts of the szl1-1 and szl1-1npq1-2 plants. Increased lutein/carotene ratio in these mutants impaired the formation of grana, resulting in a significant decrease in the number of thylakoids used to build a particular stack. Further, combined biochemical and biophysical analyses revealed that hampered grana folding was related to decreased thylakoid membrane fluidity and significant changes in the amount, organization, and phosphorylation status of photosystem (PS) II (PSII) supercomplexes in the szl1-1 and szl1-1npq1-2 plants. Such changes resulted from a synergistic effect of lutein overaccumulation in the lipid matrix and a decreased level of carotenes bound with PS core complexes. Moreover, more rigid membrane in the lutein overaccumulating plants led to binding of Rubisco to the thylakoid surface, additionally providing steric hindrance for the dynamic changes in the level of membrane folding.

Increases in lutein/carotenoid ratios lead to decreased thylakoid fluidity and hamper grana folding due to carotenoid-dependent changes in both photosynthetic complexes and lipid matrix organization.  相似文献   

8.
Spin-label ESR studies of lipid-protein interactions in thylakoid membranes   总被引:2,自引:0,他引:2  
G Li  P F Knowles  D J Murphy  I Nishida  D Marsh 《Biochemistry》1989,28(18):7446-7452
Lipid-protein interactions in thylakoid membranes, and in the subthylakoid membrane fractions containing either photosystem 1 or photosystem 2, have been studied by using spin-labeled analogues of the thylakoid membrane lipid components, monogalactosyldiacylglycerol, phosphatidylglycerol, and phosphatidylcholine. The electron spin resonance spectra of the spin-labeled lipids all consist of two components, one corresponding to the fluid lipid environment in the membranes and the other to the motionally restricted membrane lipids interacting directly with the integral membrane proteins. Spectral subtraction has been used to quantitate the fraction of the membrane lipids in contact with the membrane proteins and to determine the selectivity between the different lipid classes for the lipid-protein interaction. The fractions of motionally restricted lipid in the thylakoid membrane are 0.36, 0.39, and 0.53, for the spin-labeled monogalactosyldiacylglycerol, phosphatidylcholine, the phosphatidylglycerol, respectively. Spin-labeled monogalactosyldiacylglycerol exhibits very little preferential interaction over phosphatidylchline, which suggests that part of the role of monogalactosyldiacylglycerol in thylakoid membranes is structural, as is the case for phosphatidylcholine in mammalian membranes. Spin-labeled phosphatidylglycerol shows a preferential interaction over the corresponding monogalactosyldiacylglycerol and phosphatidylcholine analogues, in contrast to the common behavior of this lipid in mammalian systems. This pattern of lipid selectivity is preserved in both the photosystem 1 and photosystem 2 enriched subthylakoid membrane fractions.  相似文献   

9.
Cholesteryl hemisuccinate has been incorporated into pea chloroplast thylakoids to investigate the relationship between fluidity and functioning of this membrane system. Levels of sterol which increased the apparent viscosity of the membrane, estimated by fluorescence polarization measurements using the lipophilic probe, 1,6-diphenyl-1,3,5 hexatriene, affected several photosynthetic processes. A decrease in fluidity was accompanied by an inhibition of dark limiting steps associated with electron transfer between photosystems two and one (PSII and PSI) as observed by the oxidation of the primary acceptor of PSII and by electron flow to ferricyanide. Also, treatment with cholesteryl hemisuccinate inhibited the saltinduced rise in chlorophyll fluorescence and changed the ionic conductivity of the membrane as judged by measurements of the decay of the lightinduced proton gradient. The results are discussed in terms of the effect of fluidity changes on the lateral diffusion of plastoquinone and chlorophyll protein complexes in the lipid matrix of the membrane.  相似文献   

10.
The effect of changes of membrane fluidity on the oxygen evolving capability of isolated thylakoids was investigated. Alteration of the lipid phase fluidity was achieved by incorporation of the plant sterol stigmasterol. Incorporation of stigmasterol in the lipid bilayer of thylakoid membranes results in rigidization of the hydrophobic phase of thylakoid membranes and decreases the degree of packing of the lipid head groups. These changes of lipid order are accompanied by a reduction of oxygen evolution, measured with 1,4-benzoquinone as an electron acceptor, and by a more pronounced inhibition of PSI-mediated electron transport. By analysis of the parameters of oxygen flash yields and oxygen burst under continuous illumination it was shown that after treatment with stigmasterol: 1.) the number of active oxygen-evolving centres decreased; 2.) the remaining active oxygen-evolving centres were not affected in respect to the oscillation pattern; 3.) the contribution of the slow oxygen-evolving centres in oxygen burst yield was increased. The effect of stigmasterol was compared with the well-studied effect of cholesterol. Results were discussed in terms of determining the role of lipid order for the organization and functioning of the photosynthetic machinery.  相似文献   

11.
Lipid-protein interactions in thylakoid membranes from lettuce, pea, tomato, and cucumber have been studied using spin-labeled analogues of the thylakoid membrane lipid components, monogalactosyl diglyceride and phosphatidylglycerol. The electron spin resonance spectra of the spin-labeled lipids all consist of two components, one corresponding to the fluid lipid environment in the membranes and the other to the motionally restricted lipids interacting with the integral membrane proteins. Comparison of the spectra from the same spin label in thylakoid membranes from different plants shows that the overall lipid fluidity in the membranes decreases with chilling sensitivity. Spectral subtraction has been used to quantitate the fraction of the membrane lipids in contact with integral membrane proteins. Thylakoid membranes of cucumber, a typical chilling-sensitive plant, have been found to have a higher proportion of motionally restricted lipids and a different lipid selectivity for lipid-protein interaction, as compared with those of pea, a typical chilling-resistant plant. This correlation with chilling sensitivity holds generally for the different plants studied. It seems likely that the chilling sensitivity in thylakoid membranes is not determined by lipid fluidity alone, but also by the lipid-protein interactions which could affect protein function in a more direct manner.  相似文献   

12.
Effects of various temperatures on the rates of electron transport between two photosystems, the light-induced uptake of protons, kinetics of proton efflux from the chloroplasts in the dark and photophosphorylation were studied in isolated chloroplasts. There are correlations between the physical state of thylakoid membrane and the rates of electron- and proton transport processes. The temperature dependence of "structural" parameter (fluidity of lipids in membrane) as well as the rates of electron- and proton transport processes reveal the breaks under the same temperatures. Stimulation of photophosphorylation by temperature increasing correlates with the heat activation of chloroplasts latent ATPase due to thermoinduced structural changes in the heat activation of chloroplasts latent ATPase due to thermoinduced structural changes in the protein part of CF0-CF1 complex. The rate of photophosphorylation also correlates with the physical state of membrane lipids. Thermoinduced "melting" of the thylakoid membrane inhibits the ATP formation because of a decrease in photosystem 2 photochemical activity and stimulation of membrane conductivity for protons.  相似文献   

13.
Triazine treatment of thylakoid membranes isolated from the primary leaves ofZea mays L. showed an 80% inhibition of photosystem (PS) 2 activity. No detectable change of PS 1 activity was found. The inhibited membranes showed a selective reduction of the most unsaturated linolenic acid (C18∶3) biosynthesis by about 15% coupled with a corresponding increase in stearic (C18∶0), oleic (C18∶1) and linoleic (C18∶2) acids. Thus the inhibition of electron transfer of PS 2 induced by triazine treatment was followed by lipid peroxidation and changes in the thylakoid membrane fluidity.  相似文献   

14.
Lipids in dimeric photosystem II complexes prepared from two species of cyanobacteria, Thermosynechococcus vulcanus and Synechocystis sp. PCC6803, and two higher plants, spinach and rice, were analyzed to determine how many lipid molecules and what class of lipids are present in the photosystem II complexes. It was estimated that 27, 20, 8, and 7 lipid molecules per monomer are bound to the dimeric photosystem II complexes of T. vulcanus, Synechocystis, spinach, and rice, respectively. In each of the organisms, the lipid composition of the photosystem II complexes was quite different from that of the thylakoid membranes used for preparation of the complexes. The content of phosphatidylglycerol in the photosystem II complexes of each organism was much higher than that in the thylakoid membranes. Phospholipase A2 treatment of the photosystem II complexes of Synechocystis that degraded phosphatidylglycerol resulted in impairment of QB-mediated but not QA-mediated electron transport. These findings suggest that phosphatidylglycerol plays important roles in the electron transport at the QB-binding site in photosystem II complexes.  相似文献   

15.
The lipid fluidity of thylakoid membrane regions separated by Yeda press and sonication methods has been investigated using diphenylhexatriene fluorescence polarization measurements and rotational correlation times derived from the ESR spectra of the spin-labels 5-doxyldecane and 12-doxylstearate. According to both techniques, stromal lamellae vesicles with essentially only Photosystem I activity were more fluid than the granal membranes. The differences in lipid fluidity between the two fractions were interpreted in terms of the ratio of the amounts of protein compared to lipid in the membranes. Stromal lamellae fractions contained lower protein/lipid ratios compared with the granal membranes.  相似文献   

16.
Quantitative analysis of time-resolved anisotropy measurements of DPH or TMA-DPH in lipid vesicles yields more than one mathematically correct solution. The solutions differ with respect to the average orientation and to the reorientational dynamics of the probe molecules in the bilayer. This leads to quite opposite results regarding the effects of cholesterol on membrane fluidity. One solution predicts an increase in fluidity, the other a decrease. Angle-resolved fluorescence depolarization (AFD) measurements of probes in oriented lipid bilayers enable determination of the average orientation of the probes in the bilayer and, if the fluorescence decay function is known, of the reorientational dynamics. Analysis of AFD measurements of DPH and TMA-DPH show that increasing unsaturation leads to a decrease in molecular order and a decrease in reorientational dynamics (= fluidity) of the probes. At temperatures above the phase transition of the lipids, the addition of cholesterol causes an increase in molecular order and an increase in reorientational dynamics (= fluidity). The plant sterol stigmaterol, which is structurally closely related to cholesterol, has different effects than cholesterol. The effects vary with the structure of the surrounding lipids. The membrane fluidity concept as it was originally proposed by Chapman attempts to describe the structural and dynamic properties of lipids in a membrane using one single parameter indicated as 'membrane fluidity'. Our results show that it is necessary to distinguish between structural parameters describing molecular order and motion parameters describing molecular dynamics, thus supporting a similar suggestion by Seelig and Seelig. In order to be useful, the membrane fluidity concept has to be limited to the parameters describing molecular dynamics.  相似文献   

17.
Sulphoquinovosyldiacylglycerol is a negatively charged lipid which exists in the thylakoid membrane. It is proposed that a large proportion of this acidic lipid does not form a part of the bulk lipid matrix but is closely associated with protein complexes where it is tightly bound and participates in either optimising catalytic activities, or maintaining the complexes in a functional conformation. Experimental evidence for this proposal is emerging from studies with isolated photosystem 2, and coupling factor complexes.Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement.  相似文献   

18.
We have used three doxyl stearic acid spin labels to study the transverse hetero-geneity in lipid fluidity in thylakoids, photosystem II (PS II) preparations, and thylakoid galactolipid vesicles. This comparative study shows that spin labels incorporated into the membrane of the PS II preparation experience far more immobilization than do the same spin labels incorporated into either thylakoids or vesicles prepared from the polar lipids extracted from thylakoids. The spin label immobilization found in the PS II preparation is manifest even near the center of the bilayer, where lipid mobility is normally at its maximum. Analysis of the lipid content of the PS II preparation, relative to chlorophyll, suggests that the PS II preparation may be lipid depleted. This lipid depletion could explain the results presented. However, electron microscopy [Dunahay et al. (1984) Biochim. Biophys. Acta 764:179–193] has not indicated that major delipidation has occurred, and so it remains possible that the immobilization found in the PS II preparation is due primarily to the normal (but close) juxtaposition of adjacent PS II complexes and the cooperative immobilization of their surrounding lipids. Based on the results presented, we conclude that highly mobile lipids are not required for oxygen evolution, the primary photochemistry or the secondary reduction of exogenously added quinones. Unfortunately, the relationship between the plastoquinone pool and the fluidity of the membrane in the PS II preparation remains ambiguous.Abbreviations PS II photosystem II - SDSA 5-doxylstearic acid - 12DSA 12-doxylstearic acid - 16DSA 16-doxylstearic acid - 7N14 2-heptyl-2-hexyl-5,5-dimethyloxazolidine-N-oxyl - chromium oxalate potassium trioxalatochromiate - EPR electron paramagnetic resonance - Chl chlorophyll - MGDG monogalactosyldiacylglycerol - DGDG digalactosyldiacylglycerol  相似文献   

19.
A variant of fluorescence recovery after photobleaching allows us to observe the diffusion of photosynthetic complexes in cyanobacterial thylakoid membranes in vivo. The unicellular cyanobacterium Synechococcus sp. PCC7942 is a wonderful model organism for fluorescence recovery after photobleaching, because it has a favorable membrane geometry and is well characterized and transformable. In Synechococcus 7942 (as in other cyanobacteria) we find that photosystem II is immobile, but phycobilisomes diffuse rapidly on the membrane surface. The diffusion coefficient is 3 x 10(-10) cm(2) s(-1) at 30 degrees C. This shows that the association of phycobilisomes with reaction centers is dynamic; there are no stable phycobilisome-reaction center complexes in vivo. We report the effects of mutations that change the phycobilisome size and membrane lipid composition. 1) In a mutant with no phycobilisome rods, the phycobilisomes remain mobile with a slightly faster diffusion coefficient. This confirms that the diffusion we observe is of intact phycobilisomes rather than detached rod elements. The faster diffusion coefficient in the mutant indicates that the rate of diffusion is partly determined by the phycobilisome size. 2) The temperature dependence of the phycobilisome diffusion coefficient indicates that the phycobilisomes have no integral membrane domain. It is likely that association with the membrane is mediated by multiple weak interactions with lipid head groups. 3) Changing the lipid composition of the thylakoid membrane has a dramatic effect on phycobilisome mobility. The results cannot be explained in terms of changes in the fluidity of the membrane; they suggest that lipids play a role in controlling phycobilisome-reaction center interaction.  相似文献   

20.
The effects of lipid peroxidation (LPO) on the physical state (fluidity) of the rat brain synaptosomal lipid bilayer matrix and the annular lipid domains were investigated using the fluorescent probe pyrene. The parameters of pyrene fluorescence intensity alpha = IE/IM were measured at excitation wavelengths 280 nm and 340 nm (alpha 280 and alpha 340), reflecting fluidity of lipid bilayer matrix and annular lipids, respectively. LPO induction was shown to result in changes of fluidity of both the bilayer and annular lipids. Upon reducing formation of LPO products by carnosine, fluidity changes of both the lipid bilayer matrix and annular lipids were diminished. Conformational changes of the annular lipid domain by LPO may therefore be considered as a possible cause of the functional changes in the receptor mediated responses and of the inactivation of membrane-bound enzymes by oxidative stress.  相似文献   

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