Cryoprotection of spinach chloroplast membranes by dextrans

The cryoprotective properties of dextrans have been investigated in freezing experiments with isolated spinach thylakoids (Spinacia oleracea L.). The activity of cyclic photophosphorylation was used as an assay for membrane integrity.Dextrans of average molecular weights between 10,000 and 70,000 daltons proved to be fairly nontoxic to chloroplast membranes. On a molar basis, cryoprotective action increased with increasing molecular weight; on a unit weight basis, the cryoprotective effectiveness of different dextrans was comparable. In the presence of low dextran concentrations which are not sufficient for complete membrane preservation, the effectiveness of the polymers could be considerably increased by the addition of electrolytes. This is in contrast to cryoprotection exerted by sugars. At a given dextran concentration, membrane activity is a function of the electrolyte concentration and follows an optimum curve. If membrane-toxic action of the electrolytes and salt crystallization during freezing which complicate the situation, are not taken into consideration, the increase in membrane protection during freezing by salts was dependent on the concentration of the salts and was not much influenced by the nature of the cations and anions. At 0 °C, dextrans delayed the inactivation of thylakoids suspended in NaCl solutions.From the results it is concluded that cryoprotection produced by dextrans is caused in part by specific membrane stabilization.     

Degradation of stachyose,raffinose, melibiose and sucrose by different tempe-producing Rhizopus fungi

Forty-six strains of tempe-forming Rhizopus species were screened for their ability to grow on raffinose as the sole carbon source. Six of the strains showed good growth and sporulation. These isolates were one Rhizopus oligosporus, one Rhizopus microsporus var. chinensis, three Rhizopus oryzae and one Rhizopus stolonifer. These six moulds and R. oligosporus strain NRRL 2710 were investigated for their metabolism of the raffinose family of -galactoside carbohydrates. Degradation experiments were performed in submerged culture in a medium containing soybean -protein, sodium phytate and either stachyose, raffinose or melibiose. R. oryzae and R. stolonifer completely consumed the tested carbohydrates as carbon source. R. microsporus var. chinensis failed to hydrolyse the -galactosidic bonds of raffinose, stachyose or melibiose, whereas it was able to use sucrose and the fructose moiety of raffinose or stachyose for growth. R. oligosporus NRRL 2710 was unable to hydrolyse any of the tested carbohydrates. The results of the oligosaccharide degradation experiments could be verified during tempe production from soybeans with the selected fungal species.     

Light-, pH- and uncoupler-dependent association of chloride with chloroplast thylakoids

Studies of the association of Cl^? with Photosystem (PS) II in CF₁-containing thylakoid membranes revealed that photosynthetically active Cl^? is retained in a Cl^?-free medium unless it is sufficiently alkaline, uncoupling conditions are established and light is excluded. After treatment under such conditions, electron transport from water became dependent on added Cl^? under all conditions. Quantitative measurements of ³⁶Cl^? retention in the light revealed that there were about five Cl^? anions present in Cl^?-sufficient chloroplasts per PS II reaction center, and one-fourth of that in Cl^?-deficient samples. Uncouplers representing three different types of uncoupling mechanism were found to be effective mediators of Cl^? release from thylakoids. Since the ability to collapse a proton gradient probably is the only property shared by all the tested uncouplers, a proton gradient may be involved in the retention of Cl^?. As uncoupler-mediated Cl^? release did not depend on preillumination of our samples, a long-lived proton gradient must exist in dark-adapted chloroplasts which may not span the whole thickness of the thylakoid membrane. It is postulated that the Cl^? active in PS II reactions resides in a special membrane domain from which protons slowly equilibrate with those in the bulk solutions. Cl^? is thought to be released to the bulk phases only when the pH of the membrane domain is raised above a certain threshold by the action of uncouplers. This domain may be identical to the intramembranous compartment which has been postulated to be associated with PS II (Prochaska, L.J. and Dilley, R.A., (1978) Front. Biol. Res. Energ. 1, 265–274).     

Protein phosphorylation and redox sensing in chloroplast thylakoids

Transduction of light dependent signals to redox sensitive kinases in photosynthetic membranes modulates energy transfer to the photochemical reaction centres and regulates biogenesis, stability and turnover of thylakoid protein complexes. The occupancy of the quinol-oxidation site of the cytochrome bf complex by plastoquinol and the redox state of protein thiol groups act as elements of the signal transducing chains.     

Lateral distribution and diffusion of plastocyanin in chloroplast thylakoids

The lateral distribution of plastocyanin in the thylakoid lumen of spinach and pea chloroplasts was studied by combining immunocytochemical localization and kinetic measurements of P700+ reduction at high time resolution. In dark-adapted chloroplasts, the concentration of plastocyanin in the photosystem I containing stroma membranes exceeds that in photosystem II containing grana membranes by a factor of about two. Under these conditions, the reduction of P700+ with a halftime of 12 microseconds after a laser flash of saturating intensity indicates that to greater than 95% of total photosystem I a plastocyanin molecule is bound. An analysis of the labeling densities, the length of the different lumenal regions, and the total amounts of plastocyanin and P700 shows that most of the remaining presumable mobile plastocyanin is found in the granal lumen. This distribution of plastocyanin is consistent with a more negative surface charge density in the stromal than in the granal lumen. During illumination the concentration of plastocyanin in grana increases at the expense of that in stroma lamellae, indicating a light-driven diffusion from stroma to grana regions. Our observations provide evidence that a high concentration of plastocyanin in grana in the light favors the lateral electron transport from cytochrome b6/f complexes in appressed grana across the long distance to photosystem I in nonappressed stroma membranes.     

Superoxide anion permeability of phospholipid membranes and chloroplast thylakoids

The permeability of phospholipid membranes to the superoxide anion (O₂^?) was determined using soybean phospholipid vesicles containing FMN in the internal space. The efflux of O₂^? generated by the illumination of FMN was so slow that more than 90% of the radicals were spontaneously disproportionated within the vesicles before they could react with cytochrome c at the membrane exterior. The amount of diffused O₂^? was proportional to the intravesicular concentration of O₂^? over a range from 1 to 10 μm which was deduced from its disproportionation rate. The permeability coefficient of the phospholipid bilayer for O₂^? was estimated to be 2.1 × 10^?6 cm s^?1 at pH 7.3 and 25 ° C. Superoxide dismutase trapped inside vesicles was not reactive with extravesicular O₂^? unless Triton X-100 was added. O₂^? generated outside spinach chloroplast thylakoids did not interact with superoxide dismutase or cytochrome c which had been enclosed in the thylakoids. Thus, chloroplast thylakoids also showed little permeability to O₂^?.     

Simultaneous measurement of deltapH and electron transport in chloroplast thylakoids by 9-aminoacridine fluorescence

Electron transport and the electrochemical proton gradient across the thylakoid membrane are two fundamental parameters of photosynthesis. A combination of the electron acceptor, ferricyanide and the DeltapH indicator, 9-aminoacridine, was used to measure simultaneously electron transport rates and DeltapH solely by changes in the fluorescence of 9-aminoacridine. This method yields values for the rate of electron transport that are comparable with those obtained by established methods. Using this method a relationship between the rate of electron transport and DeltapH at various uncoupler concentrations or light intensities was obtained. In addition, the method was used to study the effect of reducing the disulfide bridge in the gamma-subunit of the chloroplast ATP synthase on the relation of electron transport to DeltapH. When the ATP synthase is reduced and alkylated, the threshold DeltapH at which the ATP synthase becomes leaky to protons is lower compared with the oxidized enzyme. Proton flow through the enzyme at a lower DeltapH may be a key step in initiation of ATP synthesis in the reduced enzyme and may be the way by which reduction of the disulfide bridge in the gamma-subunit enables high rates of ATP synthesis at low DeltapH values.     

The stacking of chloroplast thylakoids: Effects of cation screening and binding,studied by the digitonin method

The differential action of digitonin on stacked and unstacked chloroplast thylakoids was used to investigate the molecular interactions between thylakoid membranes. The yield of the heavy fraction which is obtained from chloroplasts after digitonin incubation and differential centrifugation was taken as a measure of the degree or tightness of membrane appression. The effects of various mono-, di-, and trivalent cations on the yield of the heavy fraction were studied, and the results interpreted in terms either of electrostatic screening or ion binding to the thylakoid membrane surface: Although there was some degree of cation specificity in the degree of thylakoid appression indicative of cation binding, the nonspecific screening effect was much more important in determining the overall balance of forces. It is postulated that stacking occurs in regions of low net surface charge density, with a possible segregation of excess negative charges into nonstacked regions.     

Microbial transformation of sucrose and glucose to erythritol

Summary Two strains of osmophilic yeast which were isolated from honey-comb, produced good yields of erythritol as a main product. These strains were identified as Trichosporonoides sp., 150-5 and 331-1.From the fermentation studies with these strains using glucose and sucrose as substrate, strain 331-1 produced more erythritol as the sole polyhydric product,with trace quantities of glycerol, than strain 150-5.     

A protein oxidase catalysing disulfide bond formation is localized to the chloroplast thylakoids

In chloroplasts, thiol/disulfide-redox-regulated proteins have been linked to numerous metabolic pathways. However, the biochemical system for disulfide bond formation in chloroplasts remains undetermined. In the present study, we characterized an oxidoreductase, AtVKOR-DsbA, encoded by the gene At4g35760 as a potential disulfide bond oxidant in Arabidopsis. The gene product contains two distinct domains: an integral membrane domain homologous to the catalytic subunit of mammalian vitamin K epoxide reductase (VKOR) and a soluble DsbA-like domain. Transient expression of green fluorescent protein fusion in Arabidopsis protoplasts indicated that AtVKOR-DsbA is located in the chloroplast. The first 45 amino acids from the N-terminus were found to act as a transit peptide targeting the protein to the chloroplast. An immunoblot assay of chloroplast fractions revealed that AtVKOR-DsbA was localized in the thylakoid. A motility complementation assay showed that the full-length of AtVKOR-DsbA, if lacking its transit peptide, could catalyze the formation of disulfide bonds. Among the 10 cysteine residues present in the mature protein, eight cysteines (four in the AtVKOR domain and four in the AtDsbA domain) were found to be essential for promoting disulfide bond formation. The topological arrangement of AtVKOR-DsbA was assayed using alkaline phosphatase sandwich fusions. From these results, we developed a possible topology model of AtVKOR-DsbA in chloroplasts. We propose that the integral membrane domain of AtVKOR-DsbA contains four transmembrane helices, and that both termini and the cysteines involved in catalyzing the formation of disulfide bonds face the oxidative thylakoid lumen. These studies may help to resolve some of the issues surrounding the structure and function of AtVKOR-DsbA in Arabidopsis chloroplasts.     

Protein tyrosine phosphorylation in the transition to light state 2 of chloroplast thylakoids

Redox dependent protein phosphorylation in chloroplast thylakoids regulates distribution of excitation energy between the two photosystems of photosynthesis, PS I and PS II. Several thylakoid phosphoproteins are known to be phosphorylated on N-terminal threonine residues exposed to the chloroplast stroma. Phosphorylation of light harvesting complex II (LHC II) on Thr-6 is thought to account for redistribution of light energy from PS II to PS I during the transition to light state 2. Here, we present evidence that a protein tyrosine kinase activity is required for the transition to light state 2. With an immunological approach using antibodies directed specifically towards either phospho-tyrosine or phospho-threonine, we observed that LHC II became phosphorylated on both tyrosine and threonine residues. The specific protein tyrosine kinase inhibitor genistein, at concentrations causing no direct effect on threonine kinase activity, was found to prevent tyrosine phosphorylation of LHC II, the transition to light state 2, and associated threonine phosphorylation of LHC II. Possible reasons for an involvement of tyrosine phosphorylation in light state transitions are proposed and discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Probing the events of photoinhibition by altering electron-transport activity and light-harvesting capacity in chloroplast thylakoids

Abstract. The effect of photoinhibition on the activity of photosystem II (PSII) in spinach chloroplasts was investigated. Direct light-induced absorbance change measurements at 320 nm (Δ A ₃₂₀) provided a measure of the PSII charge separation reaction and revealed that photoinhibition prevented the stable photoreduction of the primary quinone acceptor Q_A. Sensitivity to photoinhibition was substantially enhanced by treatment of thylakoids with NH₂OH which extracts manganese from the H₂O-splitting enzyme and prevents electron donation to the reaction centre. Incubation with 3-(3,4,-dichlorophenyl)-1,1-dimethylurea (DCMU) during light exposure did not affect the extent of photoinhibitory damage. The chlorophyll (Chl) b -less chlorina (2 mutant of barley displayed a significantly smaller light-harvesting antenna size of PSII (about 20% of that in wild type chloroplasts) and, simultaneously, a lower sensitivity to photoinhibition. These observations suggest that photoinhibition depends on the amount of light absorbed by PSII and that the process of photoinhibition is accelerated when electron donation to the reaction centre is prevented. It is postulated that the probability of photoinhibition is greater when excitation energy is trapped by P680⁺, the oxidized form of the PSII reaction centre. The results are discussed in terms of the D1/D2 heterodimer which contains the functional PSII components P680, pheophytin, Q_A and Q_B.     

Correlation between membrane-localized protons and flash-driven ATP formation in chloroplast thylakoids

Flash-driven ATP formation by spinach chloroplast thylakoids, using the luciferin luminescence assay to detect ATP formed in single turnover flashes, was studied under conditions where a membrane protein amine buffering pool was either protonated or deprotonated before the beginning of the flash trains. The flash number for the onset of ATP formation was delayed by about 10 flashes (from 15 to about 25) when the amine pool was deprotonated as compared to the protonated state. The delay was substantially reversed again by reprotonating the pool upon application of 20–30 single-turnover flashes and 8 min of dark before addition of ADP, P_i, and the luciferin system. In the case of deprotonation by desaspidin, the uncoupler was removed by binding to BSA before the reprotonating flashes were given. Reprotonation was carried out before addition of ADP and P_i, to avoid a possible interference by the ATP-ase, which can energize the system by pumping protons. The reprotonated state, as indicated by an onset lag of about 15 flashes rather than 25 for the deprotonated state, was stable in the dark over extended dark times. The number of protons released by 10 flashes is approximately 30 nmol H⁺ (mg chl)^–1, an amount similar to the size of the reversibly protonated amine group buffering pool. The data are consistent with the hypothesis that the amine buffering groups must be in the protonated state before any protons proceed to the coupling complex and energize ATP formation. Other work has suggested that the amine buffering pool is sequestered within membrane proteins rather than being exposed directly to the inner aqueous bulk phase. Therefore, it is possible that the sequested amine group array may provide localized association-dissociation sites for proton movement to the coupling complex.     

Transport of glucose,fructose and sucrose by Streptanthus tortuosus suspension cells

Streptanthus tortuosus Kell. suspension cells will grow in a medium with sucrose as carbohydrate source. It was investigated whether the cells are able to take up sucrose or whether sucrose has to be hydrolyzed to glucose and fructose which eventually are taken up. The detailed quantitative analysis of sugar-uptake rates in the low concentration range up to 1 mM showed the following features: (i) There is definitely no sucrose-uptake system working in the low concentration range; any uptake of radioactivity from labelled sucrose proceeds via hydrolysis of sucrose by cell-wallbound invertase. (ii) Hexoses are taken up by two systems, a glucose-specific system with a K _m of 45 M and a high V _max for glucose and a K _m of 6 mM and a low V _max for fructose, and a fructosespecific system with a K _m of 500 M and high a V _max for fructose and a K _m of 650 M and a low V _max for glucose. (iii) There is a more than tenfold preference for uptake of the fructose derived from sucrose versus uptake of free fructose, with the result that the kinetic disadvantage of the fructoseuptake system compared to the glucose-uptake system is diminished if sucrose is supplied as the carbon source. It is speculated that invertase might work as an enzyme aiding in fructose transport.Abbreviations FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - FW fresh weight     

An analysis of proton fluxes coupled to electron transport and ATP synthesis in chloroplast thylakoids

Electron transport, phosphorylation and internal proton concentration were measured in illuminated spinach chloroplast thylakoid membranes under a number of conditions. Regardless of the procedure used to vary these parameters, the data fit a simple chemiosmotic model. Protons from Photosystem II did not appear to be utilized differently from those derived from Photosystem I. The maximal phosphorylation efficiency ($\text{P}\text{e}{}_2$) for photophosphorylation in washed thylakoids under oxidizing conditions is likely to be $\text{4}\text{3}$. This value is consistent with a proton-to-electron-pair ratio of 4 for electron flow through both photosystems and a proton-to-ATP ratio of 3 for the chloroplast proton-ATPase.     

Differential scanning calorimetric investigation of pea chloroplast thylakoids and thylakoid fractions.

High sensitivity differential scanning calorimetry (DSC) was employed to study the thermal denaturation of components of pea chloroplast thylakoid membranes. In contrast to previous reports utilizing spinach thylakoids, several transitions are reversible, and deconvolution of the calorimetric curves indicates nine transitions in both first and second heating scans, but overlapping transitions obscure at least three transitions in the first heating scans of control thylakoids. Glutaraldehyde fixation increases the denaturation temperature of several transitions which is consistent with a reported increase in thermal stability of thylakoid function due to fixation. Acidic pH treatment has little effect on the DSC curves, although it has been reported to have a significant effect on membrane structure. Separation of grana from stroma thylakoids indicates that components responsible for transitions centered at approximately 56, 73, 77, and 91 degrees C are predominantly or exclusively associated with grana thylakoids, whereas components responsible for transitions centered at approximately 63 and 81 degrees C are predominantly associated with stroma thylakoids. A broad transition centered at 66 degrees C is associated with grana thylakoids, whereas a sharp transition at the same temperature is due to a component associated with stroma thylakoids. Evidence obtained by washing treatments suggests the latter transition originates from the denaturation of the thylakoid ATPase (CF1). Analysis of the calorimetric enthalpy values indicates most components of the grana thylakoids denature irreversibly at high temperature, whereas components associated with the stroma thylakoids have a considerable degree of thermal reversibility.     

Selective modification of coupling factor 1 in spinach chloroplast thylakoids by a fluorescent maleimide

N-(1-Anilinonaphthyl-4)maleimide (ANM) has been used to modify coupling factor 1 (CF1), the terminal coupling factor of photophosphorylation in chloroplasts. As with other monofunctional maleimides, incubation of thylakoids with ANM in the light, but not in the dark, causes energy transfer inhibition of photophosphorylation. In the dark, sites on both the gamma and epsilon subunits of CF1 are modified. The light-accessible site is also on the gamma subunit. Trypsin digestion of the enzyme after dithiothreitol activation reveals that the dark-and light-accessible sites on the gamma subunit are different amino acid residues. Fluorescence of ANM bound at the dark-and light-accessible sites has been measured after isolation of CF1 from thylakoids. The fluorescence emission maximum of ANM at the light-accessible site is blue-shifted and the quantum yield is increased 2-fold relative to ANM bound at dark-accessible sites. On the soluble enzyme, fluorescence polarization is high and equivalent for ANM bound at both dark-and light-accessible sites. Fluorescence energy transfer from a tryptophan in a hydrophilic region of the epsilon subunit to ANM bound to the epsilon subunit but not to the gamma subunit has been observed. The significance of these observations is discussed with respect to the structure of the gamma subunit and its role in conformational transitions within CF1 that occur during energization of the membrane.     

Octanol-water partition coefficient of glucose, sucrose, and trehalose

The octanol-water partition coefficients (P) of glucose, sucrose, and trehalose were measured at temperatures between 5 and 20 degrees C using an enzymatic method. The measured log P is compared with calculated and experimental data previously reported. In the case of trehalose, the measured log P differs considerably from the theoretically estimated values and agrees with the expected value for a disaccharide. Some methods of assessing the partition coefficients are also analyzed and it is concluded that the atom/fragment contribution method overestimates the hydrophilicity of disaccharides and, probably in a larger extension, that of trisaccharides. The knowledge of P for these sugars is valuable both for basic or applied purposes, including food and biomolecules stabilization.