Relationship between Thylakoid Membrane Fluidity and the Functioning of Pea Chloroplasts

Abstract

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 lppboilic 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 chlorophyli 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 dfffusion of plastoquinone and chlorophyli protein complexes in the lipid matrix of the membrane.There is increasing evidence that the fluidity of the chloroplast thylakoid membrane may play an important role in controlling the light reactions of photosynthesis (4, 24). The fluidity of biological membranes is determined mainly by the length and the degree of saturation of alkyl chains in fatty acids of the constituent lipids. Thylakoid membranes of higher plants contain galactolipids with highly unsaturated fatty acids and a small portion of phospho-and sulfolipids (1,5,15). Thus, a relatively low viscosity may be expected for the thylakoid membrane at room temperature (2). However, the membrane fluidity can be changed by varying the temperature of the chloroplast suspension and a great deal of evidence has accumulated suggesting effects of temperature on function ofchloroplasts, including electron transport, fluorescence changes, and other related reactions (6,9,13,14,16,19,20,22,29,30,33,34 thylakoid lipid phase on the functioning of various photosynthetic processes such as electron transport, ionic conductivity of the membrane, and cation-induced Chl fluorescence changes. Although this sterol occurs only at low levels in higher plants its effect on the physical properties of biological membranes in general is well documented. Thus, we have simply used this compound, added as the hemisuccinate, in an attempt to artificialy manipulate the fluidity of the thylakoid membrane. MATERIALS AND METHODSPea chloroplasts were prepared according to Nakatani and Barber (21), and then osmotically disrupted, washed once, and suspended in 0.33 M sorbitol and 1 mm Tris-HCl solution (pH 7.5). Cholesteryl hemisuccinate was added to thylakoid membranes by the method of Shinitzky et al. (28); 3.5% (w/w) of polyvinyl pyrrolidone (PVP, Mr 40,000, Sigma) was incubated with 25 mm Tris-HCl buffer (pH 7.5) for at least 30 min and cholesteryl hemisuccinate (Tris-salt), dissolved in a small amount of tetrahydrofuran, was added to give the required concentration. The cholestery...

Response of isolated thylakoid membranes with altered fluidity to short term heat stress

“…After incubation, the membranes were washed two times in order to remove the non-incorporated sterol and resuspended in a medium appropriate for further measurements. Alternatively, the method of Yamamoto et al (1981) to incorporate cholesterol by the use of polyvinyl pyrrolidone as a mediator was checked. Fluorescence polarization (P) of 1,6-diphenyl-1, 3, 5-hexatriene (DPH) indicates that with both methods, the level of cholesterol incorporation is approximately the same.…”

Effect of Membrane Fluidity on Photosynthetic Oxygen Production Reactions

“…Several studies on this topic that use thylakoid membranes with artificially manipulated lipid phase (incorporation of cholesterol or cholesteryl hemisuccinate) or lipid mutants (genetically altered membrane fluidity) discuss the importance of fluidity of the lipid matrix on functional characteristics of the photosynthetic apparatus, located in thylakoid memAbbreviations: 1,4 BQ, 1,4-benzoquinone; chl, chlorophyll; DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea; DCPIP, 2,6-dichlorophenolindophenol; DPH, 1,6-diphenyl-1,3,5-hexatriene; DPPC, dipalmitoylphosphatidylcholine; MES, 2-(morpholino)ethanesulfonic acid; MV, methyl viologen; PSI (II), photosystem I (II); TMA-DPH, trimethylammonium-diphenyl-DPH; Tricine, N-[tris(hydroxymethyl) (Ford and Barber, 1983;Siegenthaler and Tremolieres, 1998;Yamamoto et al, 1981).…”

Changes in the Cationic Regulation of Structure and Function in Thylakoids Isolated from Wheat Seedlings Treated with BASF 13.338

“…The addition of Mg2' to the low-salt suspension of control chloroplasts induced an increase in the yield ofChl a fluorescence as reported for several other systems by a number of workers (4,18). However, the Mg2+-induced increase in the room temperature Chl a fluorescence was either absent or significantly diminished in thylakoid membranes isolated from plants grown in the presence of BASF 13.338.…”

“…Barber et al (4) suggested that the cation-induced increase in Chl a fluorescence can be used as an intrinsic probe for monitoring changes in the fluidity of the thylakoid membranes. Direct experimental evidence in support of this suggestion is derived from the correlation between cation-induced fluorescence changes and polarization of diphenyl hexatriene fluorescence in aging chloroplasts (4) as well as in thylakoid membranes infused with cholesteryl hemisuccinate (4,18).…”