I. Dahse

Characterization of the activity of a plastid-targeted green fluorescent protein in Arabidopsis

In Arabidopsis thaliana the PALE CRESS (PAC) gene product is required for both chloroplast and cell differentiation. Transgenic Arabidopsis plants expressing a translational fusion of the N-terminal part of the PAC protein harboring the complete plastid-targeting sequence and the green fluorescent protein (GFP) exhibit high GFP fluorescence. Detailed analyses based on confocal imaging of various tissues and cell types revealed that the PAC-GFP fusion protein accumulates in chloroplasts of mature stomatal guard cells. The GFP fluorescence within the guard cell chloroplasts is not evenly distributed and appears to be concentrated in suborganellar regions. GFP localization studies demonstrate that thin tubular projections emanating from chloroplasts and etioplasts often connect the organelles with each other. Furthermore, imaging of non-green and etiolated tissue further revealed that GFP fluorescence is present in proplastids, etioplasts, chromoplasts, and amyloplasts. Even photobleaching of carotenoid-free plastids does not affect PAC-GFP accumulation in the organelles of the guard cells indicating that the protein translocation machinery is functional in all types of plastids. The specific accumulation of GFP in guard cell chloroplasts, their tubular connections, the translocation of the precursor polypeptide into the different types of organelles, as well as the use of a plastid-targeted GFP protein as a versatile marker is discussed in the context of previously described observations.

The Membrane Potential as Indicator for Transport and Energetic Processes of Leaf Cells of the Aquatic Plant Egeria densa I. Ion and Light Dependence and Reactions on Metabolic Inhibitors

Summary Leaf cells of Egeria densa were investigated electrophysiologically in detail in order to employ the electric cell potential as sensitive parameter which reveals essential living processes such as transport and metabolism. Data obtained by membrane potential measurements and flame-photometry under various conditions were used for a numerical approximation of the Goldman-Hodgkinkatz equation. In comparison to other plants, Egeria cells possess a high membrane potential with a substantial light-dependent component. Darkness and inhibitors of photosystem II depolarize the membrane potential to the same extent. The ATPase inhibitors DCCD, DES and orthovanadate also led to depolarization but with different kinetics and amplitudes, suggesting unequal effectiveness of inhibition. The uncouplers CCCP and DNP depolarize the cell potential far below its dark level. Their depolarizing effect was accompanied by a rapid electrolyte leakage from the leaves indicating irreversible processes. In contrast to auxin, the phytoeffec or fusicoccin, hyperpolarizing the membrane potential in the light, abolished the depolarizing action of darkness and inhibitors but not that of uncouplers. The results confirm the sensitivity of the cell potential to changes of the environment and metabolic processes. However, the interpretation of the electric cell response has to be undertaken with precaution.

The Interaction of Tentoxin with CF1 and CF1-e Isolated from Spinach Chloroplast

Summary Different HPLC techniques were applied to investigate the interaction of the cyclic tetrapeptide tentoxin with the chloroplast coupling factor 1 (CF 1 ) prepared from spinach. Tentoxin bound stoichiometrically 1:1 with the CF 1 , although dissociation of the e-subunit might correlate with an additional binding revealing a second, low-affinity binding site. Tentoxin binding did not depend on the presence of ADP and did not change the number of binding sites for ADP on the CF 1 , indicating a non-competitive binding. Binding of the natural derivative, dihydrotentoxin, was indirectly demonstrated. The calcium- and the magnesium-dependent ATPase activities of the CF 1 were strongly inhibited at low tentoxin concentration whereas at concentrations of 5 μM and above they were increasingly stimulated. The mechanism of action and the binding site of tentoxin on the β-subunit are discussed and a new hypothesis is presented.

Light-stimulated Electrogenic Uptake of Neutral Amino Acids by Leaf Cells of Egeria densa and Vicia faba

Summary The kinetics and energetics of amino acid uptake into leaf tissues of Egeria and Vicia by means of 14C-amino acids (alanine, aminoisobutyric acid, leucine) and the accompanying electric response at the plasmalemma were compared. Amino acid uptake in the concentration range of 0.1 to 5 mmol/l at pH 6 exhibits biphasic isotherms (Egeria: saturable phase, [S]: 0.1-1.5 mmol/l, linear phase, [S]:, 1.5 mmol/l; Vicia: two Michaelis-Menten terms, without any linear component). Uptake of Ala, AIB and Leu is strongly pH-dependent and is enhanced in the light. Effects of light and various inhibitors on amino acid influx, medium-acidifying activity of the leaf tissues and on the membrane potential prove the existence of a light-stimulated H+ transport system at the plasmalemma, creating a protonmotive force capable to provide transport energy. The transient depolarization of the membrane potential during amino acid influx and the sensitivity of this influx to the membrane potential point to coupling of amino acid transport with electrochemical proton gradient. It is suggested that in Egeria as well as in Vicia neutral amino acids were taken up by proton symport in the range of pH 4 to 8.

On Possible Functions of Electron Transport in the Plasmalemma of Plant Cells

Summary Electron transport has now been established to occur not only in membranes of chloroplasts or mitochondria but also in the plasma membranes of prokaryotes and eukaryotes. Here the basis reactions and reaction participants of electron transport in the plasma membrane of plant cells - the plasmalemma - are reviewed and hypotheses on its functioning are presented and discussed. The question is addressed in what respect electron transport at the plasmalemma is involved in bioenergetics, growth, and other cellular processes of plants.

A Comparison of Tentoxin Action on the Delayed Fluorescence in Chloroplasts of Spinach, Chlorella and Anacystis

Summary The action of the energy transfer inhibitor tentoxin on the delayed fluorescence of chloroplast suspensions of the tentoxin-sensitive plant spinach as well as of suspensions of the green alga Chlorella and the blue-green alga Anacystis was investigated under different conditions of electron transport. Tentoxin increased the delayed fluorescence in all three species, indicating an unspecific effect, which cannot account for the observed sensitivity of some plants to this toxin. A clear distinction between delayed fluorescence of control and tentoxin-treated chloroplasts was achieved only under phosphorylating conditions (i.e., in the presence of ADP and inorganic phosphate). The curves for induction of delayed fluorescence suggest a decrease of the proton conductance of the thylakoid membrane after tentoxin treatment. A concomitant increase of the potassium conductance did not occur. The tentoxin effect on delayed fluorescence was qualitatively the same as that of the ATPase inhibitor DCCD. Consequently it was explained by the known inhibition of photo phosphorylation by the phytotoxin. The derivative dihydrotentoxin enhanced delayed fluorescence only slightly.

Lack of correlation between transplasmalemma electron transport rate and depolarisation in Egeria densa leaf cells

Summary Transplasmalemma electron transport induced by the impermeable electron acceptors, Na 3 [Fe(CN) 6 ], K 3 [Fe(CN) 6 ], K 2 [IrCl 6 ] and K 2 [IrBr 6 ], was investigated by means of microelectrode technique, pH-stat and colorimetric experiments using the waterweed, Egeria densa . No fix stoichiometry between protons and electrons exported was observed during acceptor reduction. The H + /e- ratio changed during the experiments with time and depended on a number of factors such as light conditions, preincubation and incubation medium. Application of ion channel blockers (BaCl 2 , tetraethylammonium, charybdotoxin), inhibitors (erythrosin B, NaCN+salicylhydroxamic acid) and stimulators of proton transport (ethanol, fusicoccin) showed that ions including protons are transported in order to compensate for the electron acceptor-induced charge imbalance. Electrically, acceptor reduction was accompanied by depolarisation of the plasmalemma at acid and neutral pH. Both, reduction and depolarisation displayed saturation kinetics under these conditions. Surprisingly, the depolarisation disappeared at alkaline pH, whereas the acceptor reduction continued. A different mechanism of charge compensation is proposed. The natural electron acceptor, nitrate, did not competitively inhibit ferricyanide reduction, by contrast, it enhanced the reduction rate. Furthermore, in the presence of nitrate no nitrite was produced in the medium. Nitrate did not depolarise the plasmalemma notwithstanding the required charge compensation. Nitrate uptake is accompanied by alkalinization of the medium and acidification of the cytosol. Hence, nitrate is not a substrate of the plasmalemma redox system and it is taken up by an apparent 1:1 H + /NO 3 - cotransport. The results demonstrate that the plasmalemma redox system of Egeria leaf cells is neither a proton pump nor a nitrate carrier or reductase.

The Membrane Potential as Indicator for Transport and Energetic Processes of Leaf Cells of the Aquatic Plant Egeria densa: II. The Effect of the Specific Energy Transfer Inhibitor Tentoxin and its Derivative Dihydrotentoxin

Summary The effect of tentoxin, an energy transfer inhibitor produced by the phytopathogenic fungus Alternaria alternata , and of its natural derivative, dihydrotentoxin, on the membrane potential of leaf cells of the fresh-water plant Egeria densa was investigated. Tentoxin reversibly depolarizes the membrane potential in the light down to its dark level after a lag-phase of some 10 min. A second application of the phytotoxin shortens this lag-phase considerably. Dihydrotentoxin also depolarizes the membrane potential, but less effectively. In the dark, no influence on the PD of both substances could be observed. Fusicoccin repolarizes the membrane in the presence of tentoxin or dihydrotentoxin or, when first added, suppresses the toxin-induced response. Tentoxin as well as d1hydrotentoxin lower the ATP content of Egeria leaves significantly. The tentoxin action on the cell potential is discussed as a result of inhibition of photophosphorylation.

The Membrane Potential as Indicator for Transport and Energetic Processes of Leaf Cells of the Aquatic Plant Egeria densa

Summary The effect of 3 redox substances on the membrane potential, membrane integrity and proton extrusion of illuminated leaf cells of Egeria densa has been investigated. The herbicide, O,O-di-n-butyl-(1-n-butylamino-cyclohexyl)-phosphonate (PABT, 0.29 mol m-3), depolarizes the plasma membrane and inhibits the proton pumping in the initial phase followed by electrolyte leakage from the cells after about 90 min. For the formulation of PABT, nonylphenol-poly-oxyethylene adducts (EO) are used as nonionic surfactants. EO prove to be non-penetrating exogenous electron acceptors, causing membrane depolarization and a stimulation of H+-extrusion as is the case with ferricyanide. Concentrations up to two orders of magnitude above the cmc of the EO do not significantly disturb the membrane integrity in Egeria leaf cells. 1,4-Naphthoquinone (NQ), a general lipophilic electron acceptor, is used to modify plasmalemma electron transport. After accepting one electron, the resulting semiquinone radicals cause membrane disruption. NQ (0.1 mol m-3) depolarizes the membrane, inhibits the H+-extrusion, and leads to membrane disruption. Subtoxic concentrations (10-2 mol m-3) of NQ cancel the effects of PABT. Non-penetrating electron acceptors (EO, ferricyanide), however, do not impair PABT action. The effects of EO, PABT, and NQ are prevented by fusicoccin (FC, 10-3 mol m-3). A plasmalemma redox system in Egeria is considered to be the common site of action of EO, PABT, and NQ.

Light-induced Electric Potentials of Intact Anthoceros Chloroplasts and Their Modification in the Presence of the Energy Transfer Inhibitor tentoxin

Summary The photoelectric response of intact Anthoceros chloroplasts in situ was investigated using micro-electrode technique. The biphasic potential curve of dark-adapted chloroplasts could be modified by short preillumination. Tentoxin, a cyclic tetrapeptide known as energy transfer inhibitor, did not influence neither the biphasic character of the light-induced response nor the potential decay time constant τ in the dark. It prevented the transient undershoot which occurs during the potential decay after the end of illumination and led to some irregularities in the shape of the light-induced potential curve. The results are discussed in relation to possible tentoxin actions.