In vitro ferredoxin-dependent desaturation of fatty acids in cyanobacterial thylakoid membranes.

Thylakoid membranes isolated from the cyanobacterium Synechocystis sp. strain PCC6803 were capable of desaturating the acyl groups in monogalactosyl diacylglycerol. This desaturation reaction required the reduced form of ferredoxin.     

Degradation of proteins from thylakoid membranes in senescing wheat leaves at high temperature

This investigation determined whether thylakoid proteins would be degraded more rapidly or not in senescing wheat (Triticum aestivum L. em. Thell.) leaves concurrently exposed to high temperatures. Excised leaves were pulse-labelled with [³⁵S]-methionine for a 12 h period, and then incubated at 22,32 or 42°C for 0, 1, 2, or 3 d, before extracting a thylakoid enriched membrane sample. After electrophoretic separation, two prominent [³⁵S]-labelled protein bands were chosen for further analyses. Band A contained the D-1 thylakoid protein and band B contained thylakoid proteins of the light harvesting complex (LHCII) associated with photosystem II (PSII). Total protein, [³⁵S]-labelled protein, band A protein, and band B protein within the thylakoid enriched membrane samples were measured. Unlabelled thylakoid enriched membrane samples, extracted from leaves given similar treatments, were used to measure uncoupled whole-chain and photosystem II (PSII) electron transport and chlorophyll fluorescence. Accentuated decline in whole-chain and PSII electron transport, increasing F_o values, and decreasing F_max values were a result of high temperature injury in leaves treated at 42°C. None of the thylakoid enriched membrane protein fractions were degraded more rapidly in high-temperature treated leaves. Degradation of the total [³⁵S]-labelled membrane proteins and band B was inhibited by the 42°C treatment. The results indicate that high temperature stress may disrupt some aspects of normal senescence.     

Resistance to low temperature photoinhibition is not associated with isolated thylakoid membranes of winter rye

In vivo measurements of chlorophyll a fluorescence indicate that cold-hardened winter rye (Secale cereale L. cv Musketeer) develops a resistance to low temperature-induced photoinhibition compared with nonhardened rye. After 7.2 hours at 5°C and 1550 micromoles per square meter per second, the ratio of variable fluorescence/maximum fluorescence was depressed by only 23% in cold-hardened rye compared with 46% in nonhardened rye. We have tested the hypothesis that the principal site of this resistance to photoinhibition resides at the level of rye thylakoid membranes. Thylakoids were isolated from cold-hardened and nonhardened rye and exposed to high irradiance (1000-2600 micromoles per square meter per second) at either 5 or 20°C. The photoinhibitory response measured by room temperature fluorescence induction, photosystem II electron transport, photoacoustic spectroscopy, or [¹⁴C]atrazine binding indicates that the differential resistance to low temperature-induced photoinhibition in vivo is not observed in isolated thylakoids. Similar results were obtained whether isolated rye thylakoids were photoinhibited or thylakoids were isolated from rye leaves preexposed to a photoinhibitory treatment. Thus, we conclude that increased resistance to low temperature-induced photoinhibition is not a property of thylakoid membranes but is associated with a higher level of cellular organization.     

Two novel intrinsic annexins accumulate in wheat membranes in response to low temperature

Four immunologically related proteins that belong to the annexin family were identified in cold acclimated wheat (Triticum aestivum). Two soluble forms with molecular masses of 34 and 36 kDa were found to bind phospholipid membranes in a calcium-dependent manner. These two forms are similar to the previously reported doublet in several plant species. The other two forms, with molecular masses of 39 and 22.5 kDa, were found associated with the microsomal fraction. Biochemical analysis showed that both forms are intrinsic membrane proteins and their association with the membrane is calcium independent. This is, to our knowledge, the first report of the presence of these annexin forms in plants. Membrane purification by two phase partitioning demonstrated that the p39 form is localized to the plasma membrane. Immunoblot analysis showed that the protein level of both p39 and p22.5 increases gradually reaching a maximum level after one day of low temperature exposure. The protein accumulation was similar in both hardy and less hardy cultivars, suggesting that the accumulation is not correlated with freezing tolerance. The results are discussed with respect to the possible role of these new intrinsic membrane annexins in low temperature signal transduction pathway.     

The increase in unsaturation of fatty acids of phosphatidylglycerol in thylakoid membrane enhanced salt tolerance in tomato

Overexpression of chloroplastic glycerol-3-phosphate acyltransferase gene (LeGPAT) in tomato increased cis-unsaturated fatty acid content in phosphatidylglycerol (PG) of thylakoid membrane. By contrast, suppressing the expression of LeGPAT decreased the content of cis-unsaturated fatty acid in PG. Under salt stress, sense transgenic plants exhibited higher activities of chloroplastic antioxidant enzymes, lower content of reactive oxygen species (ROS) and less ion leakage compared with the wild type (WT) plants. The net photosynthetic rate (P _N) and the maximal photochemical efficiency (F_v/F_m) of photosystem II (PSII) decreased more slightly in sense lines but more markedly in the antisense ones, compared to WT. D1 protein, located in the reactive center of the PSII, is the primary target of photodamage and has the highest turnover rate in the chloroplast. Under salt stress, compared with WT, the content of D1 protein decreased slightly in sense lines and significantly in the antisense ones. In the presence of streptomycin (SM), the net degradation of the damaged D1 protein was faster in sense lines than in other plants. These results suggested that, under salt-stress conditions, increasing cis-unsaturated fatty acids in PG by overexpression of LeGPAT can alleviate PSII photoinhibition by accelerating the repair of D1 protein and improving the activity of antioxidant enzymes in chloroplasts.     

31P-NMR observation of the temperature and glycerol induced non-lamellar phase formation in wheat thylakoid membranes

³¹P-NMR spectra at 162 MHz were used to monitor phase changes of wheat thylakoid membranes as a function of temperature. At room temperature the³¹P-NMR line was a superposition of anisotropic component characteristic of phospholipid lamellar phase and isotropic line due to inorganic phosphorus or small membrane vesicles arising as an effect of preparation. For temperatures higher than +35 °C an increase of the isotropic component occurs, which is irreversible as the sample is cooled. For the temperatures between +55 °C and +60 °C the presence of the hexagonal phase cylinders is suggested, as monitored by phosphorus lineshape. However, the addition of glycerol stimulates a formation of the isotropic phase. The effect of reconstitution of freeze-dried thylakoid membranes by addition of water or water-glycerol medium to the sample was examined. As lyophilizate was gradually diluted, the increase of isotropic line component was observed. For thylakoid membranes suspended in D₂O at the highest dilution examined, the line contribution due to small membrane fragments is not greater than 50%, but in presence of glycerol, this contribution could reach 70%. This suggests that the presence of glycerol increases the formation of the small membrane particles as the thylakoid membrane is reconstituted from lyophilizate. The wheat thylakoid membranes reconstituted from lyophilizate show, in comparison to native membranes, the increased contribution of small membrane vesicles. Moreover, the³¹P -NMR spectra suggest the appearance of the hexagonal phase cylinders even at +50 °C.Abbreviations DGDG digalactosyldiacylglycerol - DLPC dilinoleoyl phosphatidylcholine - DLPE dilinoleoyl phosphatidylethanolamine - EDTA ethylenediamine-tetraacetic acid - MGDG monogalactosyldiacylglycerol - NMR nuclear magnetic resonance - PC phosphatidylcholine - PG phosphatidylglycerol - PSII photosystem II - TGDG trigalactosyldiacylglycerol - Tris Tris-(hydroxymethyl)-aminomethan - S/N signal to noise ratio     

Effects of high temperature and low pH on photosystem 2 photochemistry in spinach thylakoid membranes

The effects of temperature (25–45 °C) and pH (7.5–5.5) on photosystem (PS) 2 was studied in spinach (Spinacia oleracea L.) thylakoid membranes using chlorophyll a fluorescence induction kinetics. In high temperature and low pH treated thylakoid membranes a decline in the variable to maximum fluorescence ratio (F_v/F_m) and PS 2 electron transport rate were observed. More stacking in thylakoid membranes, studied by digitonin fractionation method, was observed at low pH, while the degree of unstacking increased under high temperature conditions. We conclude that the change in pH does not significantly affect the donor/acceptor side of PS 2 while high temperature does. Fluorescence emission spectra at 77 K indicated that low pH is associated with energy redistribution between the two photosystems while high temperature induced changes do not involve energy re-distribution. We suggest that both, high temperature and low pH, show an inhibitory effect on PS 2 but their mechanisms of action are different.     

Comparative physiological and proteomic response to abrupt low temperature stress between two winter wheat cultivars differing in low temperature tolerance

Abrupt temperature reduction in winter wheat at either autumn seedling stage prior to vernalisation or early spring crown stage can cause severe crop damage and reduce production. Many studies have reported the physiological and molecular mechanisms underlying cold acclimation in winter wheat by comparing it with spring wheat. However, processes associated with abrupt temperature reduction in autumn seedling stage prior to vernalisation in winter wheat are less understood. In this study, physiological and molecular responses of winter wheat seedlings to abrupt low temperature (LT) stress were characterised in the relatively LT‐tolerant winter wheat cultivar Shixin 828 by comparing it with the relatively LT‐sensitive cultivar Shiluan 02‐1 using a combination of physiological, proteomics and biochemical approaches. Shixin 828 was tolerant to abrupt LT stress, while Shiluan 02‐1 exhibited high levels of reactive oxygen species (ROS) and leaf cell death. Significant increases in relative abundance of antioxidant‐related proteins were found in Shixin 828 leaves, which correlate with observed higher antioxidant enzyme activity in Shixin 828 compared to Shiluan 02‐1. Proteomics analysis also indicated that carbohydrate metabolism‐related proteins were more abundant in Shiluan 02‐1, correlating with observed accumulation of soluble sugars in Shiluan 02‐1 leaves. Amino acid analysis revealed a strong response to LT stress in wheat leaves. A negative effect of exogenous sucrose on LT tolerance was also found. This study indicates that high ROS scavenging capacity and high abundance of photosynthesis‐related proteins might play a role in winter wheat response to abrupt LT stress. In contrast, excess accumulation of soluble sugars might be disadvantageous for LT tolerance in the wheat cultivar Shiluan 02‐1.     

Structural-functional state of thylakoid membranes of wheat genotypes under water stress

Plants were grown in field conditions in the wide area under normal water supply and severe water deficit. Two wheat (Triticum aestivum L.) genotypes contrasting by architectonics and differing in drought-resistance were used: Giymatli-2/17, short stature, with broad and drooping leaves, drought-sensitive, and Azamatli-95, short stature, with vertically oriented small leaves, drought-tolerant). It was found out that Giymatli-2/17 was characterized by relatively low content of Chl a-protein of PS I (CP I) and beta-subunit of ATP-synthase complex, the high content of proteins in the 33-30.5 kDa region and LHC polypeptides (28-24.5 kDa), the intensive fluorescence at 740 nm and more high photochemical activity of PS II under normal irrigation compared with Azamatli-95. However, the content of CP I (M(r) 115 kDa) and apoprotein of P700 with M(r) 63 kDa insignificantly increases in the drought-resistant genotype Azamatli-95 under extreme water supply condition while their content decreases in drought-sensitive cv Giymatli-2/17. Intensity of synthesis alpha- and beta-subunits of CF(1) (55 and 53.5 kDa) also decreases in Giymatli-2/17. The levels of the core antenna polypeptides of FS II with M(r) 46 and 44.5 kDa (CP47 and CP43) remains stable both in normal, and stressful conditions. At the same time the significant reduction is observed in the content of polypeptides in the 33-30.5 kDa region in the more sensitive genotype Giymatli-2/17. There is an increase in the LHC II polypeptides level in tolerant genotype Azamatli-95 in contrast to Giymatli-2/17 (where the content of these subunits is observed decreasing). The intensity of short wavelength peaks at 687 and 695 nm sharply increases in the fluorescence spectra (77 K) of chloroplasts from sensitive genotype Giymatli-2/17 under water deficiency and there is a stimulation of the ratio of fluorescence band intensity F687/F740. After exposure to drought, cv Giymatli-2/17 shows a larger reduction in the actual PS II photochemical efficiency of chloroplasts than cv Azamatli-95.     

Structural-functional state of thylakoid membranes of wheat genotypes under water stress

Plants were grown in field conditions in the wide area under normal water supply and severe water deficit. Two wheat (Triticum aestivum L.) genotypes contrasting by architectonics and differing in drought-resistance were used: Giymatli-2/17, short stature, with broad and drooping leaves, drought-sensitive, and Azamatli-95, short stature, with vertically oriented small leaves, drought-tolerant). It was found out that Giymatli-2/17 was characterized by relatively low content of Chl a-protein of PS I (CP I) and β-subunit of ATP-synthase complex, the high content of proteins in the 33-30.5 kDa region and LHC polypeptides (28-24.5 kDa), the intensive fluorescence at 740 nm and more high photochemical activity of PS II under normal irrigation compared with Azamatli-95. However, the content of CP I (M_r 115 kDa) and apoprotein of P700 with M_r 63 kDa insignificantly increases in the drought-resistant genotype Azamatli-95 under extreme water supply condition while their content decreases in drought-sensitive cv Giymatli-2/17. Intensity of synthesis α- and β-subunits of CF₁ (55 and 53.5 kDa) also decreases in Giymatli-2/17. The levels of the core antenna polypeptides of FS II with M_r 46 and 44.5 kDa (CP47 and CP43) remains stable both in normal, and stressful conditions. At the same time the significant reduction is observed in the content of polypeptides in the 33-30.5 kDa region in the more sensitive genotype Giymatli-2/17. There is an increase in the LHC II polypeptides level in tolerant genotype Azamatli-95 in contrast to Giymatli-2/17 (where the content of these subunits is observed decreasing). The intensity of short wavelength peaks at 687 and 695 nm sharply increases in the fluorescence spectra (77 K) of chloroplasts from sensitive genotype Giymatli-2/17 under water deficiency and there is a stimulation of the ratio of fluorescence band intensity F687/F740. After exposure to drought, cv Giymatli-2/17 shows a larger reduction in the actual PS II photochemical efficiency of chloroplasts than cv Azamatli-95.     

Lipid composition of thylakoid membranes of cadmium treated wheat seedlings.

Cadmium (200 ppm) applied through the rooting medium to 30-day-old wheat plants decreased chlorophyll content, net CO2 exchanges and PSII activity by 34, 54 and 43% respectively. Thylakoid total lipids, total glycolipids, total phospholipids and total neutral lipids decreased by 22, 23, 12 and 25%, respectively, under cadmium treatment. Thylakoid membrane glycolipids had three major constituents, viz. monogalactosyl diacylglycerol, digalactosyl diacylglycerol and sulphoquinovosyl diacylglycerol. Monogalactosyl diacylglycerol and digalactosyl diacylglycerol contents decreased by 32 and 27%, respectively, under cadmium. Cadmium application also decreased the concentration of phosphatidyl glycerol and phosphatidyl choline to the extent of about 57 and 31%, respectively. On the other hand, phosphatidic acid and free fatty acids content showed an increase. These compositional changes in thylakoid membranes might be responsible for reduced PSII activity and rate of photosynthesis as observed under cadmium treatment.     

Excitation energy transfer in native and unstacked thylakoid membranes studied by low temperature and ultrafast fluorescence spectroscopy

In this work, the transfer of excitation energy was studied in native and cation-depletion induced, unstacked thylakoid membranes of spinach by steady-state and time-resolved fluorescence spectroscopy. Fluorescence emission spectra at 5 K show an increase in photosystem I (PSI) emission upon unstacking, which suggests an increase of its antenna size. Fluorescence excitation measurements at 77 K indicate that the increase of PSI emission upon unstacking is caused both by a direct spillover from the photosystem II (PSII) core antenna and by a functional association of light-harvesting complex II (LHCII) to PSI, which is most likely caused by the formation of LHCII-LHCI-PSI supercomplexes. Time-resolved fluorescence measurements, both at room temperature and at 77 K, reveal differences in the fluorescence decay kinetics of stacked and unstacked membranes. Energy transfer between LHCII and PSI is observed to take place within 25 ps at room temperature and within 38 ps at 77 K, consistent with the formation of LHCII-LHCI-PSI supercomplexes. At the 150–160 ps timescale, both energy transfer from LHCII to PSI as well as spillover from the core antenna of PSII to PSI is shown to occur at 77 K. At room temperature the spillover and energy transfer to PSI is less clear at the 150 ps timescale, because these processes compete with charge separation in the PSII reaction center, which also takes place at a timescale of about 150 ps.     

Exogenously-supplied trehalose protects thylakoid membranes of winter wheat from heat-induced damage

The effects of trehalose pretreatment on thylakoid membranes of winter wheat were investigated under heat stress. Under normal growth conditions, the winter wheat synthesized 502 μg g⁻¹(f.m.) trehalose, which increased to 1250 μg g⁻¹(f.m.) under heat stress and to 1658 μg g⁻¹(f.m.) in trehalose-pretreated seedlings. Under heat stress, proteins in the thylakoid membranes and the photosynthetic capacity were protected by trehalose pretreatment. Moreover, the electrolyte leakage, contents of malondialdehyde, superoxide anion and hydrogen peroxide, and lipoxygenase activity in trehalose-pretreated seedlings were lower than in the non-pretreated plants.     

Structural consequences of genetically engineered saturation of the fatty acids of phosphatidylglycerol in tobacco thylakoid membranes. An FTIR study

The role of phosphatidylglycerol (PG) in protein-lipid interactions and membrane dynamics has been studied in the thylakoids of wild type and manipulated tobacco plants transformed with complementary DNAs for glycerol-3-phosphate acyltransferases (GPATs) from squash and Arabidopsis. The expression of the foreign enzymes resulted in the level of saturation of the PG molecules being higher in the squash and lower in the Arabidopsis transformants, as compared with the level in wild-type tobacco. For the analysis of fatty acyl chain dynamics in the thylakoid membranes, the nu(sym)CH(2) vibration bands of the infrared specta were decomposed into two components, corresponding to ordered and disordered fatty acyl chain segments. With this approach, it was shown that in squash GPAT-transformed tobacco thylakoids a rigid lipid domain exists below 25 degrees C. Above 25 degrees C, the dynamics of all thylakoid membranes were very similar, regardless of the manipulations. PG seems to tune the dynamics at the protein-lipid interface rather than to affect the structure of the proteins directly. Above 50 degrees C, the frequencies of the disordered nu(sym)CH(2) component bands were decreased. This lipid-related phenomenon correlated with protein denaturing. It is demonstrated that the protein aggregation appearing upon heat denaturing changes the conformational distribution of the disordered lipid population. The data also reveal that the protein stability does not depend on the fatty acid composition of the PG molecules; other lipids should provide the environment governing the protein stability in the thylakoid membrane. This is the first such detailed analysis of the infrared spectra of biological membranes that permits a differentiation between structurally different lipid populations within a membrane.     

Violaxanthin accessibility and temperature dependency for de-epoxidation in spinach thylakoid membranes

Using DTT and iodoacetamide as a novel irreversible method to inhibit endogenous violaxanthin de-epoxidase, we found that violaxanthin could be converted into zeaxanthin from both sides of the thylakoid membrane provided that purified violaxanthin de-epoxidase was added. The maximum conversion was the same from both sides of the membrane. Temperature was found to have a strong influence both on the rate and degree of maximal violaxanthin to zeaxanthin conversion. Thus only 50% conversion of violaxanthin was detected at 4 °C, whereas at 25 °C and 37 °C the degree of conversion was 70% and 80%, respectively. These results were obtained with isolated thylakoids from non-cold acclimated leafs. Pigment analysis of sub-thylakoid membrane domains showed that violaxanthin was evenly distributed between stroma lamellae and grana partitions. This was in contrast to chlorophyll a and -carotene which were enriched in stroma lamellae fractions while chlorophyll b, lutein and neoxanthin were enriched in the grana membranes. In combination with added violaxanthin de-epoxidase we found almost the same degree of conversion of violaxanthin to zeaxanthin (73–78%) for different domains of the thylakoid membrane. We conclude that violaxanthin de-epoxidase converts violaxanthin in the lipid matrix and not at the proteins, that violaxanthin does not prefer one particular membrane region or one particular chlorophyll protein complex, and that the xanthophyll cycle pigments are oriented in a vertical manner in order to be accessible from both sides of the membrane when located in the lipid matrix.     

The effect of freezing on thylakoid membranes in the presence of organic acids

The effect of salts of organic acids on washed and non-washed chloroplast membranes during freezing was investigated. Thylakoids were isolated from spinach leaves (Spinacia oleracea L.) and, prior to freezing, salts of various organic acids or inorganic salts or both were added. Freezing occurred for 3 to 4 hours at −25 C. After thawing membrane integrity was investigated by measuring the activity of cyclic photophosphorylation.     

Mechanism for binding of fatty acids to hepatocyte plasma membranes

The purpose of this study was to examine the interaction between fatty acids and plasma membranes from liver cells. We were unable to reproduce the reported effect of heating on the capacity of these membranes to bind [3H]oleate (Stremmel et al. 1985 Proc. Natl. Acad. Sci. USA. 82: 4-8). In fact, the distribution of [3H]oleate between plasma membranes and unilamellar vesicles of lipids extracted from these membranes was in favor of the lipids, indicating the absence of a detectable amount of binding to a putative fatty acid binding protein in plasma membranes. Radius of curvature of vesicles (125 A vs 475 A) had no effect on the partitioning of fatty acid. In addition, the distribution of [3H]oleate between plasma membranes and other phases had the properties of a partition coefficient over a 200-fold range of [3H]oleate. There was no evidence in this experiment for a binding isotherm, i.e., binding of [3H]oleate at a specific site, superimposed on the nonspecific partitioning of [3H]oleate into the lipids of the plasma membrane. There was no competition between [14C]oleate and [3H]palmitate for entry into plasma membranes. Finally, rates of uptake of [14C]oleate and [3H]palmitate by perfused rat liver were not affected by the presence of the other fatty acid in perfusates. These data indicate that the avidity of hepatocyte plasma membranes for [3H]oleate is a simple consequence of the physical chemical properties of oleate, lipids, and water. The data exclude the idea that the uptake of fatty acids into cells is the result of binding proteins and/or catalyzed reactions at the water-membrane interface of the cell or within the plane of the plasma membrane.     

Impact of salt adaptation on esterified fatty acids and cytochrome oxidase in plasma and thylakoid membranes from the cyanobacterium Anacystis nidulans

During adaptation of photoautotrophically growing fresh water cyanobacterium Anacystis nidulans to high salinity the cells showed a pronounced increase of proton-sodium antiporter activity, and of cytochrome c oxidase in isolated and purified plasma membrane. At the same time the concentrations of plasma membrane-bound EDTA-resistant copper and iron (determined by inductively coupled plasma atomic emission spectrometry) rose proportionately, accompanied by an increase in whole cell respiration. In plasma membranes from salt adapted cells lipid/protein ratios were markedly higher than in control cells, levels of esterified saturated and long-chain fatty acids being significantly higher than the respective levels of unsaturated and short-chain fatty acids which explains the higher lipid-phase transition temperatures derived from Arrhenius plots. Immunoblotting of the membrane proteins with antisera raised against the cytochrome c oxidases from Paracoccus denitrificans and A. nidulans gave two cross-reacting bands with apparent molecular weights around 50000 and 30000 (subunits I and II, respectively) which were more pronounced in plasma membranes from salt adapted cells when compared to control cells. The protein pattern of plasma membranes from salt adapted cells also showed the appearance of bands at apparent molecular weights of 44000–48000 and 54000–56000 which might stem from the proton/sodium-antiporter in this membrane.Abbreviations CM cytoplasmic or plasma membrane - ICM intracytoplasmic or thylakoid membrane - cyt cytochrome - DCCD N,N-dicyclohexylcarbodiimide - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonate - ICP-AES inductively coupled plasma atomic emission spectrometry - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - EPR electron paramagnetic resonance spectrometry