Harro W. Wong Fong Sang

Synthesis and degradation of phytochelatins in cadmium-sensitive and cadmium-tolerant Silene vulgaris

In response to external Cd supply Cd-tolerant Silene vulgaris plants produce three times less phytochelatins than Cd-sensitive ones. Phytochelatin synthase activity in roots of both ecotypes was measured in order to determine whether a lower enzyme activity could be responsible for the lower phytochelatin production. The phytochelatin synthase activity in tolerant plants, grown in the absence or in the presence of Cd, tended to be slightly lower than that in sensitive ones. However, the difference was too small to account for the differential phytochelatin contents observed. Observations on phytochelatin synthesis in excised roots and on the recovery of the level of glutathione, the precursor of phytochelatins, after arresting the Cd exposure, suggested that tolerant plants did not exhibit a decreased capacity to synthesize glutathione. Furthermore, the relative decrease in the phytochelatin concentration after arresting the Cd-exposure was identical for both populations, indicating that the difference in phytochelatin concentrations was not the result of a differential rate of phytochelatin degradation.

Studies on well coupled Photosystem I-enriched subchloroplast vesicles. Content and redox properties of electron-transfer components

Abstract Stable and well coupled Photosystem (PS) I-enriched vesicles, mainly derived from the chloroplast stroma lamellae, have been obtained by mild digitonin treatment of spinach chloroplasts. Optimal conditions for chloroplast solubilization are established at a digitonin/chlorophyll ratio of 1 ( w w ) and a chlorophyll concentration of 0.2 mM, resulting in little loss of native components. In particular, plastocyanin is easily released at higher digitonin/chlorophyll ratios. On the basis of chlorophyll content, the vesicles show a 2-fold enrichment in ATPase, chlorophyll-protein Complex I, P-700, plastocyanin and ribulose-1,5-bisphosphate carboxylase as compared to chloroplasts, in line with the increased activities of cyclic photophosphorylation and PS I-associated electron transfer as shown previously (Peters, A.L.J., Dokter, P., Kooij, T. and Kraayenhof, R. (1981) in Photosynthesis I (Akoyunoglou, G., ed.), pp. 691–700, Balaban International Science Services, Philadelphia). The vesicles have a low content of the light-harvesting chlorophyll-protein complex and show no PS II-associated electron transfer. Characterization of cytochromes in PS I-enriched vesicles and chloroplasts at 25°C and 77 K is performed using an analytical method combining potentiometric analysis and spectrum deconvolution. In PS I-enriched vesicles three cytochromes are distinguished: c -554 ( E ′ 0 = 335 mV), b -559 LP ( E ′ 0 = 32 mV) and b -563 ( E ′ 0 = − 123 mV); no b -559 HP is present (LP, low-potential; HP, high-potential). Comparative data from PS I vesicles and chloroplasts are consistent with an even distribution of the cytochrome b -563- cytochrome c -554 redox complex in the lateral plane of exposed and appressed thylakoid membranes, an exclusive location of plastocyanin in the exposed membranes and a dominant location of plastoquinone in the appressed membranes. The results are discussed in view of the lateral heterogeneity of redox components in chloroplast membranes.

Interactions of histatin 5 and histatin 5-derived peptides with liposome membranes: surface effects, translocation and permeabilization.

A number of cationic antimicrobial peptides, among which are histatin 5 and the derived peptides dhvar4 and dhvar5, enter their target cells and interact with internal organelles. There still are questions about the mechanisms by which antimicrobial peptides translocate across the membrane. We used a liposome model to study membrane binding, translocation and membrane-perturbing capacities of histatin 5, dhvar4 and dhvar5. Despite the differences in amphipathic characters of these peptides, they bound equally well to liposomes, whereas their membrane activities differed remarkably: dhvar4 translocated at the fastest rate, followed by dhvar5, whereas the histatin 5 translocation rate was much lower. The same pattern was seen for the extent of calcein release: highest with dhvar4, less with dhvar5 and almost none with histatin 5. The translocation and disruptive actions of dhvar5 did not seem to be coupled, because translocation occurred on a much longer timescale than calcein release, which ended within a few minutes. We conclude that peptide translocation can occur through peptide-phospholipid interactions, and that this is a possible mechanism by which antimicrobial peptides enter cells. However, the translocation rate was much lower in this model membrane system than that seen in yeast cells. Thus it is likely that, at least for some peptides, additional features promoting the translocation across biological membranes are involved as well.

Monovalent cations differentially affect membrane surface properties and membrane curvature, as revealed by fluorescent probes and dynamic light scattering

The effects of monovalent cations on the interfacial electrostatic potential (psi d), hydrodynamic shear boundary distance (ds), and membrane curvature were studied in large unilamellar phospholipid and galacto/sulfolipid liposomes containing different fractions of negatively charged lipids. The differential effects of alkali metal ions on psi d could be accurately determined at physiological surface charge densities with a surface-anchored fluorescent probe. Li+ and Na+ more effectively decrease psi d and exhibit higher association constants (Kas) than K+ and Cs+. These two groups of cations display qualitatively different perturbations of the interfacial structure. Combining Kas values with the electrokinetic (zeta) potentials yielded the respective ds values. At low ionic strength ds more substantially increases with Li+ or Na+ than with K+ or Cs+. Increasing surface charge density causes increased membrane curvature in the presence of K+ or Cs+, but this is largely prevented by Li+ or Na+. Membrane binding of the amphiphilic cation acridine orange decreases surface charge and membrane curvature more extensively than H3O+, Li+, and Na+. The differential interface-perturbing behavior of monovalent cations is discussed with regard to their different hydration tendencies that will modulate the extent and stability of the hydrogen-bond network along the charged membrane surface.

Fluorescent probes of membrane surface properties.

We have studied the properties of two new fluorescent probes, 7-dimethylaminocoumarin derivatives, 4-[N, N-dimethyl-N-(n-tetradecyl)ammoniummethyl]-7-(N,N-dimethylamino)co umarin chloride (TAMAC) and 4-(n-dodecylthiomethyl)-7-(N,N-dimethylamino)coumarin (DTMAC) in model membrane systems. Both probes are sensitive to solvent polarity. The TAMAC probe has a quaternary ammonium function to position it at a fixed location with respect to the membrane interface. In membranes of dipalmitoleoylphosphatidylethanolamine (DiPoPE), both probes detect marked increases in surface hydrophobicity as the bilayer to hexagonal phase transition temperature is approached. This does not occur when the probes are embedded in dipalmitoleoylphosphatidylcholine (DiPoPC) in which case the fluorescence emission is found to be largely independent of temperature. A nitroxide quencher covalently linked to the 5 position of the sn-2 acyl chain of phosphatidylcholine quenches the fluorescence of DTMAC in DiPoPC more than in DiPoPE, indicating the deeper insertion of this probe in DiPoPC. As the temperature is increased the DTMAC fluorophore moves even further out of the membrane. These findings indicate that DTMAC, which does not contain a group to fix its location along the bilayer normal, adjusts its position to small changes in environment polarity, so as to maintain an environment of a fixed dielectric constant. However, with greater changes in membrane interfacial polarity the environment of the probe will be altered. Thus, in addition to the sensitivity of these probes to solvent polarity, the ability of a fixed nitroxide to quench DTMAC becomes another parameter with which to characterize membrane properties with these probes.

Electric surface charge dynamics of chloroplast thylakoid membranes. Temperature dependence of electrokinetic potential and aminoacridine interaction

Abstract Electric surface charge dynamics of unstacked broken chloroplasts at low-ionic strength were studied by free-flow electrophoresis and aminoacridine fluorescence and binding changes over the temperature range 4–36°C. Both illumination and ATP hydrolysis in the dark cause a significant increase of net negative surface charge. The light and dark electrokinetic (ζ) potentials have a broad temperature optimum between 20 and 36°C. The decline at lower temperature shows a transition at about 18°C. The ATP-induced increase of the ζ potential requires preactivation of the ATPase and is dicyclohexylcarbodiimide sensitive. Aminoacridine binding shows a quite different temperature dependence. At lower temperatures there is an increased number of binding sites with a decreased affinity and the binding becomes positively cooperative. It is demonstrated that aminoacridines aggregate to dimers upon binding to the membranes. This phenomenon is stimulated by light and favoured at lower temperatures. The light-dependent extra binding increases sigmoidally with increasing temperature, similar to the increase of ζ potential, but with a less abrupt transition. The different effects of temperature on the electrokinetic and binding data are explained in terms of surface charge screening in the electric double-layer of the thylakoid membrane.

Use of azolla as a test organism in a growth chamber of simple design

SummaryThe construction and application of a new type of growth chamber, in which different growth conditionsi.e.: temperature, humidity, pH, light intensity, light colour, change in nutrient composition and gas exchange can easily be controlled, are presented.The method has previously been applied to twoAzolla speciesviz. Azolla filiculoides, which is cold tolerant andAzolla pinnata (distinguished in Vietnam as the form Xanh), which is heat tolerant.In the growth chamber natural growth conditions of the Azolla —Anabaena azollae symbiotic association were imitated as much as possible.For testing the system, methods discussed earlier8,14 and some previously presented data, concerning photosynthetic activities, such as oxygen evolution and nitrogen fixation (acetylene reduction) of twoAzolla species39, were partially used. Biomass ofA. filiculoides was measured and reactions to its environment at conditions when grown in the field and in the growth chamber, were studied.Growth and photosynthesis measurements were performed under special light conditions and with whole plants grown under laboratory conditions.Anthocyanin synthesis was studied in relation with humidity.Anthocyanin spectra were analyzed by means of a spectrum-deconvolution method.

Temperature Dependence of Electric Events in Thylakoid Membranes: Comparison of Membrane Potential, Surface Potential and Aminoacridine Binding

Various methods are available for monitoring electric potential generation in illuminated photosynthetic membranes. These include the use of electrodes, free-flow electrophoresis, distribution or binding of amphiphilic dyes and optical changes of intrinsic or added field-sensitive probes. Some of these events seem to be correlated kinetically but without certainty about their precise mechanism and origin they remain difficult to accomodate in a single physical model. As part of such correlation studies we have determined the temperature dependence of some of these electric events that are thought to reflect thylakoid membrane potentials or interfacial electric potentials.