Cold storage of isolated class C chloroplasts: optimal conditions for stabilization of photosynthetic activities

Preservation of photosynthetic activities (photophosphorylation, electron transport, fluorescence induction, 0.3-second delayed light emission) of isolated broken (class C) chloroplasts by low temperature storage was investigated under a wide range of conditions in order to optimize long time activity retention.The more labile functions (photophosphorylation and electron transport) required very low temperatures (below -79 C) and relatively high (above 20%, v/v) concentrations of cryoprotectives for satisfactory stabilization. Fluorescence induction and delayed light emission were less sensitive, especially during the 1st month of storage.Taking into account the effect of cryoprotectives on absolute activities prior to freezing, optimum activity retention was observed with a medium containing ethylene glycol (30%, v/v) and a storage temperature of -100 C or below. In this case, given fast thawing and high chloroplast concentration, practically 100% preservation of all of the photosynthetic activities investigated was obtained for at least 10 months, even with very simple freezing and storage procedures.The same optimal medium at somewhat higher temperatures (-79 C and to a lesser extent at -41 C) caused a dramatic uncoupling effect: photophosphorylation was inhibited in a few hours, while electron transport increased 3- to 5-fold. The enhanced electron transport was stable for almost a month and then declined sharply. This uncoupling effect was specific only to ethylene glycol.     

Strategies for the cryopreservation of microencapsulated cells

The major challenge in developing cryopreservation protocols for microencapsulated cells is that the relatively large size (300-400 microm) and the fragile semipermeable membrane of microcapsules makes them particularly prone to cryodamage. Rapid-cooling cryopreservation protocols with high DMSO concentrations (3.5M, 25% v/v) resulted in low post-thaw cell viability (<10%), which did not improve with higher concentrations (4.5M, 32% v/v) and longer exposure to DMSO, even though the majority of microcapsules (60-80%) remained intact. Subsequent investigations of slow cooling with a range of DMSO and EG concentrations resulted in a much higher post-thaw cell viability (80-85%), with the majority of the microcapsules remaining intact ( approximately 60%) when DMSO was used at a concentration of 2.8M (20% v/v) and EG at a concentration of 2.7M (15% v/v). The presence of 0.25M sucrose significantly improved post-thaw cell viability upon slow cooling with 2.8M (20% v/v) DMSO, although it had no effect on microcapsule integrity. Multistep exposure and removal of sucrose did not significantly improve either post-thaw cell viability or microcapsule integrity, compared to a single-step protocol. Ficoll 20% (w/v) also did not significantly improve post-thaw cell viability and microcapsule integrity. Hence, the optimal condition for microcapsule cryopreservation developed in this study is slow cooling with 2.8M (20% v/v) DMSO and 0.25M sucrose.     

Inhibition of photophosphorylation and electron transport by N,N-dimethylformamide

The basal electron transport of pea chloroplasts was inhibited by 78% by 7% (v/v) N,N-dimethylformamide; the inhibition was partially reversed by NH4Cl. N,N-Dimethylformamide also inhibited the Pi-ATP exchange, ATP synthesis and to a smaller extent Mg2+-ATPase activity. Light induced proton uptake was not affected by up to 30% (v/v) N,N-dimethylformamide. Uncoupled electron transport in photosystem II was inhibited to a larger extent by N,N-dimethylformamide than in photosystem I. These results indicate that N,N-dimethylformamide acts as an inhibitor of energy transfer and electron transport.     

Effects of heterocyclic and tertiary permeant amines on the electron transfer in thylakoid membranes

The effect of low concentrations (up to 50 μM) of lipophilic permeant amines on the electron transfer in thylakoid membranes of pea chloroplasts has been investigated. In the presence of heterocyclic amines (9-aminoacridine and neutral red), the electron transfer, initiated from H₂O to PS I acceptors, has been shown to be inhibited to a level amounting to less than 50% of control, this taking place for both the basal (at alkaline pH) and the gramicidin-uncoupled transport (at pH 6.5–8.5). Under the same conditions, tertiary amines (dibucaine, tetracaine) cause only a 10–15% inhibition of transport. With all the amines, the rate of electron transport from H₂O to DCBQ, PS II acceptor is decreased to 80–90% of control at pH above 8.0, but this effect is completely removed when pH is lowered to 7.7–6.5. In the region of PS I, all the amines accelerate the basal transport, but do not influence the uncoupled electron transfer. A conclusion has been drawn that, parallel with uncoupling, heterocyclic and tertiary amines also cause an inhibition of PS II, appearing at alkaline pH values. Additionally, heterocyclic amines seem to brake electron flow at the level of plastoquinone reduction. This revised version was published online in June 2006 with corrections to the Cover Date.     

Inhibition of chloroplast electron transport reactions by trifluralin and diallate

The herbicides trifluralin (alpha,alpha,alpha-trifluoro-2,6-dinitro-N, N-dipropyl-p-toluidine) and diallate (S-[2,3-dichloroallyl] diisopropylthiocarbamate) inhibit electron transport, ATP synthesis, and cytochrome f reduction by isolated spinach (Spinacia oleracea L.) chloroplasts. Both compounds inhibit noncyclic electron transport from H(2)O to ferricyanide more than 90% in coupled chloroplasts at concentrations less than 50 mum. Neither herbicide inhibits electron transport in assays utilizing only photosystem I activity, and the photosystem II reaction elicited by addition of oxidized p-phenylenediamine or 2,5-dimethylquinone is only partially inhibited by herbicide concentrations which block electron flow from H(2)O to ferricyanide. Inhibition of ATP synthesis parallels inhibition of electron flow in all noncyclic assay systems, and cyclic ATP synthesis catalyzed by either diaminodurene or phenazine metho-sulfate is susceptible to inhibition by both herbicides. These results indicate that trifluralin and diallate both inhibit electron flow in isolated chloroplasts at a point in the electron transport chain between the two photosystems.     

Mechanism of dimethylsulfoxide protection against the teratogenicity of secalonic acid D in mice

Dimethylsulfoxide (DMSO) is known to antagonize the teratogenic effects of secalonic acid D (SAD) in mice. To establish the optimum protective dose of DMSO, pregnant CD-1 mice were treated, i.p., with 30 mg/kg of SAD in 5% (w/v) NaHCO3, containing 0, 10, 20, or 30% (v/v) DMSO on day 11 of gestation. Data indicate that at 10% and 20% levels, DMSO affords an apparent dose-related protection against SAD-induced cleft palate, whereas 30% DMSO enhanced fetal resorption with no reduction in the incidence of cleft palate. Ultraviolet spectra and TLC mobility indicated that DMSO at 20% did not directly interact with SAD. Distribution and elimination of 14C-SAD was studied in fetal and maternal tissues from pregnant mice at 24 and 48 hr after exposure to 30 mg/kg of 14C-SAD, i.p., in NaHCO3 (control) or in 20% DMSO. Compared with those not receiving DMSO, maternal exposure to DMSO: 1) significantly reduced (42-75%) radioactivity in fetal heads and bodies, placenta, and maternal tissues other than liver; 2) significantly increased (up to 222%) the radioactivity in maternal liver; and 3) significantly reduced (44-58%) fecal and urinary elimination of SAD-derived radioactivity. These results suggest that the antiteratogenic effect of DMSO against SAD may be at least partly mediated by increased SAD (or its metabolites) retention by maternal liver leading to reduced SAD uptake by the fetus.     

Calmodulin antagonists inhibit electron transport in photosystem II of spinach chloroplasts

Chlorpromazine, phenothiazine and trifluoperazine, known as calmodulin antagonists, inhibit electron transport in Photosystem II of spinach chloroplasts in concentrations from 20–500 μM. The inhibition site is located on the diphenyl carbazide to indophenol pathway in Tris-treated chloroplasts, indicating that water oxidation is not affected by these drugs. Ca²⁺ ions, bound to chloroplast membranes before the addition of calmodulin antagonists, can protect against inhibition up to 25% of the electron transport rate. In presence of A23187, the Ca²⁺-specific ionophore, Ca²⁺ ions provide less protection against inhibition by the 3 calmodulin antagonists used. A possible role of a calmodulin-like protein in spinach chloroplasts is postulated.     

Characterization of the non-native trifluoroethanol-induced intermediate conformational state of the Shiga toxin B-subunit

The effect of increasing concentrations of 2,2,2-trifluoroethanol (TFE) on the conformational stability of the Shiga toxin B-subunit (STxB), a bacterial homopentameric protein involved in cell-surface binding and intracellular transport, has been studied by far-, near-UV circular dichroism (CD), intrinsic fluorescence, analytical ultracentrifugation, and differential scanning calorimetry (DSC) under equilibrium conditions. Our data show that the native structure of STxB is highly perturbed by the presence of TFE. In fact, at concentrations of TFE above 20% (v/v), the native pentameric conformation of the protein is cooperatively transformed into a helix-rich monomeric and partially folded conformational state with no significant tertiary structure. Additionally, no cooperative transition was detected upon a further increase in the TFE concentration (above 40% (v/v)). The thermal stability of STxB was investigated at several different TFE concentrations using DSC and CD spectroscopy. Thermal transitions at TFE concentrations of up to 20% (v/v) were successfully fitted to the two-state folding/unfolding coupled to oligomerization model consistent with the transition between a pentameric folded conformation to a monomeric state of the protein, which the presence of TFE stabilizes as a partially folded conformation.     

Development of photosynthetic electron transport in Pinus silvestris

Photosynthetic electron transport activity has been measured in chloroplasts isolated from dark-grown seedlings of Pinus silvestris L. and in chloroplasts isolated from seedlings subjected to illumination for periods of up to 48 h. Activities of photosystem 2, photosystem 1 and photosystem 2 plus 1 have been measured. Chloroplasts isolated from dark-grown seedlings showed significant electron transport activity through both photosystems and through the entire electron transport chain from water to NADP. Illumination of the seedlings for only 5 min markedly promoted photosystem 2 activity. The artificial electron donor, diphenylcarbazide. promoted activity in chloroplasts from dark-grown seedlings and in chloroplasts from seedlings illuminated for up to 30 min. In comparison to photosystem 2 and overall electron transport from water to NADP, photosystem 1 activity increased only slightly during illumination. Measurements of electron transport and fluorescence kinetics have confirmed that photosynthetic electron transport capacity is limited on the water splitting side of photosystem 2 in dark-grown seedlings, whereas the primary and secondary electron acceptors of photosystem 2 are fully synthesized and functioning in darkness. Polyethylene glycol must be used as a protective agent when isolating photoactive chloroplasts from secondary needles of conifers. However, the presence of polyethylene glycol, when isolating chloroplasts from dark-grown pine cotyledons, caused a total inhibition of the activity of photosystem 2. The failure of others to show a substantial electron transport activity in chloroplasts from dark-grown Pinus silvestris might depend on their use of polyethylene glycol in the preparation medium and/or on their use of suboptimal reaction conditions for the electron transport measurements.     

Delayed chloroplast luminescence. Activation and suppression

Effect of diethyl ether, detergent triton X-100, glycerine, sucrose and preliminary heating on delayed luminescence (DL) of chloroplasts has been studied. Ether and triton X-100 in concentrations activating electron transport inhibit DL acting similarly to photophosphorylation uncouplers. Preliminary heating to 42-42C, glycerine and sucrose activate both the electron transport and DL of chloroplasts. Activation of electron transport under these agents is suggested to result not from photophosphorylation uncoupling, but from the changes in the conformation of chloroplast memebranes.     

Inhibition by triphenyltin chloride of a tightly-bound membrane component involved in photophosphorylation.

At very low concentrations (less than 1 muM) triphenyltin chloride inhibits ATP formation and coupled electron transport in isolated spinach chloroplasts. Basal (-Pi) and uncoupled electron transport are not affected by triphenyltin. The membrane-bount ATP in equilibrium Pi exchange and Mg2+-dependent ATPase activities of chloroplasts are also completely sensitive to triphenyltin, although the Ca2+-dependent and Mg2+-dependent ATPase activities of the isolated coupling factor protein are insensitive to triphenyltin. The light-driven proton pump in chloroplasts is stimulated (up to 60%) by low levels of triphenyltin. Indeed, the amount of triphenyltin necessary to inhibit ATP formation or stimulate proton uptake is dependent upon the amount of chloroplasts present in the reaction mixture, with an apparent stoichiometry of 2-2.5 triphenyltin molecules/100 chlorophyll molecules at 50% inhibition of ATP formation and half-maximal stimulation of proton uptake. Chloroplasts partially stripped of coupling factor by an EDTA was are no longer able to accumulate protons in the light. However, low levels of triphenyltin can effectively restore this ability. The amount of triphenyltin required for the restoration of net proton uptake is also dependent upon the amount of chloroplasts, with a stoichiometry of 4-5 triphenyltin molecules/100 chlorophyll molecules at 50% reconstitution. On the basis of this and other evidence it is concluded that triphenyltin chloride inhibits phosphorylation.Atp in equilibrium Pi exchange and membrane-bound ATPase activities in chloroplasts by specifically blocking the transport of protons through a membrane-bound carrier or channel located in a hydrophobic region of the membrane at or near the functional binding site for the coupling factor.     

Effect of diethylsulphoxide on growth,survival and ion exchange of Escherichia coli

AIMS: To study the effect of diethylsulphoxide (DESO) on Escherichia coli growth, survival and ionic exchange in comparison with dimethylsulphoxide (DMSO). METHODS AND RESULTS: Bacterial survival was estimated by counting colony-forming units and by the most probable number (five-tube) technique; the K+ and H+ transport and H(2) formation were determined electrochemically. Diethylsulphoxide at concentrations between 0.01 and 0.5% (w/v) stimulated and above 5% decreased the anaerobic growth rate and survival. 2H+ : K+ exchange and H(2) formation were lost at 5% DESO. At 0.05% DESO the kinetic characteristics of H+ : K+ exchange and H(2) formation were typical for Delta micro (H(+)) -dependent TrkA uncoupled with F(0)F(1) under respiration. CONCLUSIONS: Diethylsulphoxide at low concentrations serves as an electron acceptor for an anaerobic respiratory chain stimulating bacterial growth and survival through the modulation of H+ : K+ exchange and H(2) formation activity. The effects of DESO were more pronounced than those of DMSO. SIGNIFICANCE AND IMPACT OF THE STUDY: Diethylsulphoxide determines essential biological and therapeutic properties that make its application preferable.     

Structural and functional stability of isolated intact chloroplasts

The effect of in vitro ageing on the ultrastructure, electron transport, thermoluminescence and flash-induced 515 nm absorbance change of isolated intact (type A) chloroplasts compared with non-intact (types B and C) chloroplasts was studied.When stored in the dark for 18 h at 5°C, the structural characteristics of intact and non-intact chloroplasts were only slightly altered. The most conspicuous difference between the two was in the coupling of the electron transport which was tighter and more stable in intact chloroplasts. Under dark-storage the activity of PS 2* decreased and the -20°C peak of thermoluminescence increased at the expense of the emission at +25°C. These changes were less pronounced in the intact chloroplasts. PS 1 activity and the flash-induced 515 nm absorbance change were not affected by dark-storage.When kept in the light (80 W m^-2 (400–700 nm) for 1 h at 5°C), the thylakoid system of chloroplasts rapidly became disorganized. Although the initial activity of electron transport was much higher in intact chloroplasts, after a short period of light-storage the linear electron transport and the electron transport around PS 2 decreased in both types of preparations to the same low level. These changes were accompanied by an overall decrease of the intensity of thermoluminescence. PS 1 was not inhibited by light-storage, while the flash-induced 515 nm absorbance change was virtually abolished both in preparations of intact and non-intact chloroplasts.The data show that in stored chloroplast preparations intactness cannot be estimated reliably either by the FeCy test or by inspection under the electron microscope. These tests should be cross-checked on the level and coupling of the electron transport.     

Permeabilization of Cinchona ledgeriana cells by dimethylsulphoxide. effects on alkaloid release and long-term membrane integrity

Dimethyl sulphoxide (DMSO) has been used to permeabilize cells of Cinchona ledgeriana in suspension culture and promote the release of intracellular alkaloids. 5–6% v/v is required before any release is seen, and greater than 20% DMSO is required for full release. Even at these high levels of DMSO release is slow, taking in excess of seven hours to reach completion. Conditions which produce significant release of alkaloids have a deleterious effect on cells. Many of the membranes permeabilized did not recover their ability to selectively exclude compounds such as mannitol when the DMSO was removed. It is concluded that DMSO is not a suitable material for inducing alkaloid release in any biotechnological exploitation of alkaloid production by C. ledgeriana.Abbreviations DMSO Dimethyl sulphoxide - 2,4D 2,4-Dichlorophenoxyacetic acid     

Electron paramagnetic resonance of electron transport in photosynthetic systems. XI. Effects of photosynthetic control: dependence of the rate of electron transport on the energization of bean chloroplast thylakoid membrane

The kinetics of light-induced P700 redox transients in bean chloroplast was studied. It has been shown that the rate of electron transport decreased during few seconds of illumination of coupled chloroplasts without addition of ADP and inorganic phosphate. The evidence were obtained that there is a feedback inhibition of electron transport governed by the internal pH of thylakoid. This results in the overshoot in the kinetics of P700 redox transients induced by continuous actinic light. Under the phosphorylation condition (addition of Mg-ADP and inorganic phosphate) the effect of decreasing of the rate of electron transport between two photosystems was not observed. Addition of uncouplers (FCCP or gramicidine) also increased the steady-state rate of noncyclic electron transport. After adding only Mg-ADP (without phosphate) or Mg-ATP to coupled chloroplasts the effect of the light-driven inhibition of electron transport was observed as in the case of chloroplasts without any additions. We showed that the regulation for the electron transport rate was realized at the step of the plastoquinol oxidation by photosystem 1. Light-driven energization of the thylakoid membrane also leads to the the slowing of the reduction of spin label TEMPO. Evidences were obtained that TEMPO interacts with the semiquinone localized in the acceptor side of photosystem 2. From the comparative study of P700+ and TEMPO reduction by photosystem 2 we have concluded that there are two points of inhibitory action of DCMU localized at the acceptor and donor sides of photosystem 2. The mechanisms of photosynthetic control and the role of transmembrane proton gradient for energy transmission in chloroplasts are discussed.     

TOLERANCE OF SIX MARINE MICROALGAE TO THE CRYOPROTECTANTS DIMETHYL SULFOXIDE AND METHANOL1

Knowledge of tolerance to cryoprotectants is important in determining viability after biological freezing of algae. Six taxonomically diverse marine microalgae were evaluated for their tolerance to the widely used cryoprotectants dimethyl sulfoxide (DMSO) and methanol. Tetraselmis chuii Butcher survived exposure to 30% (v/v) DMSO and 25% methanol for periods of up to 4 h. All other species were more sensitive to high concentrations of these cryoprotectants. DMSO was lethal at 25% after a 15-min exposure of Rhodomonas baltica Karsten, Isochrysis off. galbana (strain T-ISO) Parke, and Nannochloropsis gaditana Lubian. Nannochloris atomus Butcher could tolerate only a 1-min exposure at this concentration; Chaetoceros gracilis Schutt completely lost viability when exposed to 20% for 60 min. Safe concentrations for DMSO incubations were similar (about 5% lower) to lethal thresholds. Methanol incubations did not significantly decrease cell viability at concentrations of 5% (1 min) for R. baltica, 25% (up to 60 min) for T. chuii, 15% (up to 120 min) for I. galbana, 5% (up to 60 min) for N. gaditana, 15% (up to 240 min) for Ch. gracilis, and 15% (up to 120 min) for N. atomus. Nannochloris atomus has the potential to be cryopreserved without the need for any cryoprotectant. The other five species were clearly dependent on a 15% DMSO preincubation to achieve a growth response after thawing from ?196° C. Only N. atomus and N. gaditana could be grown after being cryopreserved in the presence of 5% methanol.     

Chloramphenicol as an energy transfer inhibitor in spinach chloroplasts

At concentrations of up to 300 mug/ml both d-threo- and l-threo-chloramphenicol act as energy transfer inhibitors in spinach chloroplasts, in that they inhibit both phosphorylation and phosphorylating electron transport, without affecting the nonphosphorylating electron transport which occurs either in the absence of a phosphate acceptor or in the presence of the uncoupler ammonium chloride. At higher concentrations, there appears to be an additional site of chloramphenicol inhibition of electron transport. If d-threo-chloramphenicol is to be used as a protein synthesis inhibitor in intact chloroplasts or tissues, control experiments with another chloramphenicol isomer seem to be necessary.     

Glucose transport into spinach chloroplasts

The uptake of radioactively labeled hexoses and pentoses into the sorbitol-impermeable (3)H(2)O space (the space surrounded by the inner envelope membrane) of spinach (Spinacia oleracea L.) chloroplasts has been studied using silicone layer filtering centrifugation. Of the compounds tested, d-xylose, d-mannose, l-arabinose, and d-glucose are transported most rapidly, followed by d-fructose and l-arabinose. The rate of l-glucose uptake is only about 5% of that of d-glucose.The transport of d-glucose and d-fructose shows saturation characteristics, the K(m) for d-glucose was found to be about 20 mm. All sugars transport and phloretin inhibit d-glucose transport. The temperature dependency of d-glucose transport appears to have an activation energy of 17 kcal/mol.With low external concentrations of d-glucose the transport into the chloroplasts proceeds until nearly the external concentration is reached inside the chloroplasts.d-glucose transport is inhibited by high d-glucose concentrations in the medium. It is concluded that d-glucose and other hexoses are transported by carrier-mediated diffusion across the inner envelope membrane. This transport is similar to the transport of d-glucose into erythrocytes.     

Transgenic tobacco plants expressing antisense ferredoxin-NADP(H) reductase transcripts display increased susceptibility to photo-oxidative damage

Ferredoxin-NADP(H) reductase (FNR) catalyses the final step of the photosynthetic electron transport in chloroplasts. Using an antisense RNA strategy to reduce expression of this flavoenzyme in transgenic tobacco plants, it has been demonstrated that FNR mediates a rate-limiting step of photosynthesis under both limiting and saturating light conditions. Here, we show that these FNR-deficient plants are abnormally prone to photo-oxidative injury. When grown under autotrophic conditions for 3 weeks, specimens with 20-40% extant reductase undergo leaf bleaching, lipid peroxidation and membrane damage. The magnitude of the effect was proportional to the light intensity and to the extent of FNR depletion, and was accompanied by morphological changes involving accumulation of aberrant plastids with defective thylakoid stacking. Damage was initially confined to chloroplast membranes, whereas Rubisco and other stromal proteins began to decline only after several weeks of autotrophic growth, paralleled by partial recovery of NADPH levels. Exposure of the transgenic plants to moderately high irradiation resulted in rapid loss of photosynthetic capacity and accumulation of singlet oxygen in leaves. The collected results suggest that the extensive photo-oxidative damage sustained by plants impaired in FNR expression was caused by singlet oxygen building up to toxic levels in these tissues, as a direct consequence of the over-reduction of the electron transport chain in FNR-deficient chloroplasts.     

Electron transport pathways in spinach chloroplasts. Reduction of the primary acceptor of photosystem II by reduced nicotinamide adenine dinucleotide phosphate in the dark.

Addition of NADPH to osmotically lysed spinach chloroplasts results in a reduction of the primary acceptor (Q) of photosystem II. This reduction of Q reaches a maximum of 50% in chloroplasts maintained under weak illumination and requires added ferredoxin and Mg2+. The reaction is inhibited by (I) an antibody to ferredoxin-NADP+ reductases (EC 1.6.7.1), (ii) treatment of chloroplasts with N-ethylmaleimide in the presence of NADPH, (iii) disulfodisalicylidenepropanediamine, (iv) antimycin, and (v) acceptors of non-cyclic electron transport. Uncouplers of phosphorylation do not affect NADPH-driven reduction of Q. It is proposed that electron flow from NADPH to Q may occur in the dark by a pathway utilising portions of the normal cyclic and non-cyclic electron carrier sequences. The possible in vivo role for such a pathway in redox poising of cyclic electron transport and hence in controlling the ATP/NADPH supply ratio is discussed.