Heavy Metal Resistance of the Extreme Acidotolerant Filamentous Fungus Bispora sp.

To obtain information on the importance of membrane and zeta potentials as repelling or facilitating forces during the uptake of cationic trace elements, the heavy metal content and the growth resistance of the acidotolerant fungus Bispora. sp. to heavy metals were compared at pH 1.0 and pH 7.0. Cu, Co, Ni, Cd, Cr, and La contents of the fungus were significantly lower at pH 1.0 than at pH 7.0. A similar pH effect occurred with cationic macro elements such as Na, Mg, Ca, Fe, and Mn. Only K and Zn exhibited higher levels at pH 1.0 in the fungus than at pH 7.0. Macro and micro elements present in the medium in anionic form (sulfate, chloride) showed the opposite pattern to cations: Contents were higher at pH 1.0 than at pH 7.0. Minerals present at pH 1.0 predominantly in the electrical neutral, protonated form (phosphate, borate) exhibited a similar cell content at both acid and neutral pH (P) or a higher content at neutral pH than at acid pH (B). The resistance of fungal growth to the cations Cu, Zn, Ni, Co, Cr, and Cd was significantly higher at pH 1.0 than at pH 7.0. Such a difference was not observed with Hg, present in the medium at both pH values as electrically silent HgCl2. The anionic tungstate exhibited the opposite pattern to cationic heavy metals: The resistance of growth was higher at pH 7.0 than at pH 1.0. A greater growth resistance to heavy metals was correlated with a lower uptake of these elements, and vice versa; Uptake of heavy metals correlated with a lower resistance of fungal growth to these elements. The results are in agreement with the hypothesis that membrane and zeta potentials of the fungus are important factors controlling the uptake of heavy metals and thereby the resistance of growth to these elements: At pH 1.0 positive potentials of fungal hyphae impede the uptake of cationic heavy metals, but facilitate the uptake of anionic species. At neutral pH values the negative potentials facilitate the uptake of cations, but impede the uptake of anions.

Primary sodium plasma membrane ATPases in salt-tolerant algae: facts and fictions.

For thermodynamic reasons algae growing in media of both high salinity and high alkalinity require active export of sodium. However, experimental evidence for an active Na+-dependent cycle was scarce until recently, in contrast to the situation in marine bacteria (including cyanobacteria), fungi and animals. However, a review of literature reveals that some progress has been made in this respect, recently: data demonstrate that at least in two marine algae, Tetraselmis (Platymonas) viridis and Heterosigma akashiwo (syn. Olisthodiscus luteus), active Na+-export is carried out by means of a plasma membrane localized Na+-pump (apparent molecular mass 100-140 kDa). Biochemical characteristics of this vanadate-sensitive, but ouabain-resistant primary P-type Na+-ATPase are described and compared with the corresponding properties of Na+-ATPase from prokaryotes and animals. Alternative mechanisms for Na+-pumping are discussed.

Cell wall adaptations to multiple environmental stresses in maize roots.

A municipal solid-waste bottom slag was used to grow maize plants under various abiotic stresses (high pH, high salt and high heavy metal content) and to analyse the structural and chemical adaptations of the cell walls of various root tissues. When compared with roots of control plants, more intensive wall thickenings were detected in the inner tangential wall of the endodermis. In addition, phi thickenings in the rhizodermis in the oldest part of the seminal root were induced when plants were grown in the slag. The role of the phi thickenings may not be a barrier for solutes as an apoplastic dye could freely diffuse through them. The chemical composition of cell walls from endodermis and hypodermis was analysed. Slag-grown plants had higher amounts of lignin in endodermal cell walls when compared to control plants and a higher proportion of H-type lignin in the cell walls of the hypodermis. Finally, the amount of aliphatic suberin in both endo- and hypodermal cell walls was not affected by growing the plants on slag. The role of these changes in relation to the increase in mechanical strengthening of the root is discussed.

Na+-ATPase from the plasma membrane of the marine alga Tetraselmis (Platymonas) viridis forms a phosphorylated intermediate.

Plasma membranes isolated from the marine unicellular alga Tetraselmis (Platymonas) viridis were phosphorylated by [gamma-32P]ATP, and membrane proteins were then analyzed by PAGE in SDS, under acidic conditions. Three radioactive components with apparent molecular masses of 100 kDa, 76 kDa, and 26 kDa were detected. The phosphorylation of one of them, the 100 kDa polypeptide, was specifically stimulated by Na+. Vanadate almost completely inhibited the Na+-mediated phosphorylation of the peptide. The phosphate bound to this peptide underwent rapid turnover and was discharged by hydroxylamine. The 100 kDa phosphopeptide was sensitive to ADP. The conclusion is drawn that the 100 kDa phosphopeptide is a phosphorylated intermediate of the Na+-transporting ATPase in the T. viridis plasma membrane.

Abscisic acid-lipid interactions: a phospholipid monolayer study.

Lipid monolayer studies were performed on a Langmuir trough in the absence and in the presence of the plant hormone abscisic acid (ABA). The ABA-induced effects on the lipid monolayers can be summarized as follows: (i) ABA as the free acid (pH below 5.3) increased the molecular area and slightly decreased the surface pressure in the collapse points of monolayers made of saturated, unsaturated and of mixed lipids; ABA as the anion showed only minor effects. (ii) The ABA-induced area increase of the lipid monolayers decreased when the surface pressure increased, but some ABA remained in the monolayers made of unsaturated phospholipids even at collapse pressure. (iii) The incorporation of ABA into the monolayers could be inhibited by adding the plant sterol beta-sitosterol to the monolayer forming phospholipids. (iv) There was no substantial difference of ABA action on plant phospholipids as compared with other phospholipids. (v) ABA had a much stronger influence on unsaturated phospholipids than on saturated ones. (vi) ABA decreased the phase-transition temperature of saturated phospholipids. These results, which agree with those obtained from phospholipid vesicle studies, indicate that the physical state of the lipid is important for the ability of ABA penetrating into the lipid monolayer. Finally, a possible relevance of these results is discussed in terms of the action of ABA on guard cell membranes of plants.

Inhibition of the plasma-membrane H(+)-ATPase from Dunaliella adidophila by omeprazole.

The acid-activated sulfhydryl reagent omeprazole inhibits light-induced H+ secretion at pH 1 in cells of the halotolerant alga Dunaliella acidophila. Plasma-membrane vesicles, prepared from omeprazole-treated cells, have impaired vanadate-sensitive ATPase and ATP-induced H+ uptake activities. Omeprazole inhibits ATPase activity also in isolated plasma-membrane vesicles. The inhibition is enhanced at acidic pH and can be prevented by protonophores indicating that it is promoted by internal acidification of the vesicles. Mercaptoethanol partially reverses omeprazole inhibition. ADP does not afford protection against omeprazole but it does protect against inhibition by N-ethylmaleimide, indicating that these reagents modify different sulfhydryl groups. It is suggested that omeprazole blocks SH groups of the D. acidophila plasma-membrane H(+)-ATPase, which face the outer side of the cell.

Properties of the Isolated Intact Chloroplast at Cytoplasmic K Concentrations : I. Light-Induced Cation Uptake into Intact Chloroplasts is Driven by an Electrical Potential Difference.

Photosynthesis, stroma-pH, and internal K(+) and Cl(-) concentrations of isolated intact chloroplasts from Spinacia oleracea, as well as ion (K(+), H(+), Cl(-)) movements across the envelope, were measured over a wide range of external KCl concentrations (1-100 millimolar).Isolated intact chloroplasts are a Donnan system which accumulates cations (K(+) or added Tetraphenylphosphonium(+)) and excludes anions (Cl(-)) at low ionic strength of the medium. The internally negative dark potential becomes still more negative in the light as estimated by Tetraphenylphosphonium(+) distribution. At 100 millimolar external KCl, potentials both in the light and in the dark and also the light-induced uptake of K(+) or Na(+) and the release of protons all become very small. Light-induced K(+) uptake is not abolished by valinomycin suggesting that the K(+) uptake is not primarily active. Intact chloroplasts contain higher K(+) concentrations (112-157 millimolar) than chloroplasts isolated in standard media. Photosynthetic activity of intact chloroplasts is higher at 100 millimolar external KCl than at 5 to 25 millimolar. The pH optimum of CO(2) fixation at high K(+) concentrations is broadened towards low pH values. This can be correlated with the observation that high external KCl concentrations at a constant pH of the suspending medium produce an increase of stroma-pH both in the light and in the dark. These results demonstrate a requirement of high external concentrations of monovalent cations for CO(2) fixation in intact chloroplasts.

The role of monovalent cations for photosynthesis of isolated intact chloroplasts.

The role of monovalent cations in the photosynthesis of isolated intact spinach chloroplasts was investigated. When intact chloroplasts were assayed in a medium containing only low concentrations of mono- and divalent cations (about 3 mval l(-1)), CO2-fixation was strongly inhibited although the intactness of chloroplasts remained unchanged. Addition of K(+), Rb(+), or Na(+) (50-100 mM) fully restored photosynthesis. Both the degree of inhibition and restoration varied with the plant material and the storage time of the chloroplasts in "low-salt" medium. In most experiments the various monovalent cations showed a different effectiveness in restoring photosynthesis of low-salt chloroplasts (K(+)>Rb(+)>Na(+)). Of the divalent cations tested, Mg(2+) also restored photosynthesis, but to a lesser extent than the monovalent cations.In contrast to CO2-fixation, reduction of 3-phosphoglycerate was not ihibited under low-salt conditions. In the dark, CO2-fixation of lysed chloroplasts supplied with ATP, NADPH, and 3-phosphoglycerate strictly required the presence of Mg(2+) but was independent of monovalent cations. This finding excludes a direct inactivation of Calvin cycle enzymes as a possible basis for the inhibition of photosynthesis under low-salt conditions.Light-induced alkalization of the stroma and an increase in the concentration of freely exchangeable Mg(2+) in the stroma, which can be observed in normal chloroplasts, did not occur under low-salt conditions but were strongly enhanced after addition of monovalent cations (50-100 mM) or Mg(2+) (20-50 mM).The relevance of a light-triggered K(+)/H(+) exchange at the chloroplast envelope is discussed with regard to the light-induced increase in the pH and the Mg(2+) concentration in the stroma, which are thought to be obligatory for light activation of Calvincycle enzymes.

Uptake and reduction of glycerate by isolated chloroplasts.

Intact chloroplasts of spinach (Spinacia oleracea L.) evolve O2 in the light in a glycerate-dependent reaction at rates usually close to 10 µmolxmg(-1) chlorophyllxh(-1). Glycerate isfirst phosphorylated and the resulting phosphoglycerate reduced to the sugar level. Products of the reaction are the intermediates of the Calvin cycle and glycolate. The ratio of triosephosphates to phosphoglycerate is higher under low light or at a low pH than under high light or at a high pH. Chloroplasts contain activities of glycerate kinase which approximately correspond to observed glycerate reduction rates at light saturation. The main part of the glycerate kinase of leaf cells is localized in the chloroplasts, but considerable activity also resides outside these organelles. Glycerate can enter intact chloroplasts of spinach as the anion and the undissociated acid. It can thus mediate indirect proton transfer across the chloroplast envelope. In the presence of slowly permeating cations it is taken up mainly in an anion exchange reaction. Chloride and acetate anions permeate faster than the glycerate anion. The relation between glycerate reduction and photorespiration is discussed.

Amino acid permeability of the chloroplast envelope as measured by light scattering, volumetry and amino acid uptake.

The amino acid permeability of the envelope of intact, functional spinach (Spinacia oleracea L.) chloroplasts was investigated by light scattering, volumetry and uptake of (14)C-labelled amino acids. The criterion for the functionally of the chloroplasts was their ability to reduce CO2, PGA and oxaloacetate in the light at high rates.Net uptake into the chloroplast interior of neutral amino acids such as alanine, glycine, serine, proline, threonine or valine occurred only at very low rates. The uptake was concentration dependent, indicating unspecific diffusion rather than carrier-mediated transport. The slowness of uptake is emphasized by the capability of neutral amino acids to provide osmotic support for intact chloroplasts during a considerable length of time. Back-exchange experiments also failed to indicate the existence of specific exchange carriers for the transport of neutral amino acids such as alanine or glycine through the envelope of intact chloroplasts. Dicarboxylic amino-acids are known to be taken up by the so-called dicarboxylate translocator. The same carrier was found to catalyze also the transfer of asparagine and glutamine.The data do not support current assumptions concerning fast carrier-mediated transport of neutral amino acids and their role in the transfer of carbon during photosynthesis.

Incorporation of P and C into Photosynthetic Products of Ankistrodesmus braunii as Affected by X-Rays.

The incorporation of (32)P and (14)C into organic compounds by Ankistrodesmus is strongly inhibited by X-rays. In the same phosphorylated compounds (32)P-incorporation apparently is more severely inhibited by X-rays than the (14)C-labelling. The (32)P-incorporation into organic compounds is more strongly inhibited than (32)P-labelling of inorganic phosphate in the cell. The inhibition of (32)P-incorporation into a number of compounds is strikingly uniform. It is concluded that the inhibition of (32)P-incorporation and of (14)C-incorporation into phosphorylated compounds in vivo is due to an uncoupling by X-rays of photophosphorylation as in vitro. The difference in X-ray sensitivity of (14)C- and (32)P-incorporation into one organic phosphorous compound is attributed to a dual action of X-rays on (32)P-incorporation in organic compounds (both via the uncoupling of photophosphorylation) and only a single effect on (14)C-incorporation and (32)P-labelling of inorganic phosphate. The effect of X-rays on (14)C-incorporation into organic compounds included inhibition in most cases but also stimulation as in the case of glycolic acid. These differences may be due to interference in the intercellular regulations following the application of X-rays. The inhibition of (14)C-incorporation in many cases exhibits different behaviour at low (<200 krad) and high doses. These changes are discussed on the assumption that at the lower doses X-rays cause uncoupling of photophosphorylation and at the higher doses an additional inhibition of electron transport.