The electrochemical proton gradient and its influence on citrate uptake in tonoplast vesicles of Hevea brasiliensis

The relationship between the electrochemical proton gradient, _H+ ^– , and citrate transport has been studied in tonoplast vesicles from Hevea brasiliensis (the rubber tree). Vesicles were generated from lyophilized samples of fresh vacuoles obtained from the latex sap. Methylamine was used to measure intravesicular pH and lipophilic ions to determine the electrical potential difference () across the tonoplast. When incubated at pH 7.5 in the absence of ATP, the tonoplast vesicles showed a pH of 0.6 units (interior acid) and a of about-100 mV (interior negative). This potential is thought to be made up of contributions from an H⁺ diffusion potential, diffusion potentials from other cations and a Donnan potential arising from the presence of internal citrate. In the presence of 5 mol m^-3 MgATP the pH was increased to about 1.0 unit and the to about-10 mV. Under these conditions the proton-motive force ( _{p H+} ^– /F) became positive and reached +50 mV. These effects were specific to MgATP (ADP and Mg²⁺ having no significant effect) and were prevented by the protonophore p-trifluoromethoxycarbonylcyanidephenylhydrazone (FCCP). Citrate uptake by the vesicles was markedly stimulated by MgATP; ADP and Mg²⁺ again had no effect. Nigericin greatly increased pH and this was associated with a large increase in citrate accumulation. The results indicate that the vesicle membrane possesses a functional H⁺-translocating ATPase. The _H+ ^– generated by this ATPase can be used to drive citrate uptake into the vesicles. The properties of the tonoplast vesicles are compared with those of the fresh latex vacuoles.Abbreviations _H+ ^– electrochemical proton gradient - electrical potential difference across membrane - p proton-motive force ( _H+ ^– /F) - FCCP p-trifluoromethoxycarbonylcyanidephenylhydrazone - TPMP⁺ triphenylmethylphosphonium ion     

Kinetics of the flash-induced electrochromic absorbance change in the presence of background illumination. Turnover rate of the electron transport. I. Isolated intact chloroplasts

We investigated the flash-induced electrochromic absorbance change, A ₅₁₅, of isolated intact chloroplasts in continuous monochromatic background light of different intensities and wavelengths. From the variation of the amplitude of A ₅₁₅ in background illumination the steady-state turnover time of electron transport was found to be around 100 msec and the slowest process could be assigned to a photosystem 1 driven cycle. The change of pH induced by nigericin, ATP, or ADP did not modify substantially the turnover time.In contrast to earlier observations the slow rise (10 msec) of A ₅₁₅ in untreated chloroplasts persists also at high-intensity background illumination exciting both photosystems. The proportion of the slow rise of A ₅₁₅ in nigericin-treated chloroplasts increases in the presence of background light. This result on the slow rise is discussed in terms of two different models existing in the literature.     

Identification of Ferredoxin-Dependent Cyclic Electron Transport around Photosystem I Using the Kinetics of Dark P700<Superscript>+</Superscript> Reduction

Kinetic curves of absorbance changes induced by far-red light (FR, 830 nm) (A ₈₃₀), which reflect redox transformations of PSI primary electron donor, P700, were examined in intact barley (Hordeum vulgare L.) leaves. In intact leaves, FR induced the biphasic increase in absorbance related to P700 photooxidation. Leaf treatment with methyl viologen or antimycin A suppressed the slow phase of P700 photooxidation, which was attained in such leaves within the first second of light exposure. With FR turned off, the previously increased absorbance at 830 nm dropped down to its initial level, thus reflecting P700⁺ reduction. In the control leaves, the kinetics of P700⁺ reduction consisted of three exponentially decaying components, with the corresponding half-times of 8.8 s (the slow component, with its magnitude comprising 24% of the total A ₈₃₀ signal), 0.73 s (the middle component, 49% of A ₈₃₀), and 0.092 s (the fast component, 26% of A ₈₃₀). The rate of the fast component of P700⁺ reduction, following FR irradiation of leaves, was about ten times lower than that of the noncyclic electron transfer from PSII to PSI computed from A ₈₃₀ relaxation after the abrupt offset of white light. The treatment of leaves with methyl viologen or antimycin A completely abolished the fast component of A ₈₃₀ relaxation after FR exposure. It was concluded that the fast component is determined by the operation of ferredoxin-dependent cyclic electron transport around PSI. This study represents the first report on the identification of this pathway of electron transport in vivo and the estimation of its rate.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 3, 2005, pp. 325–330.Original Russian Text Copyright © 2005 by Bukhov, Egorova.     

Basolateral membrane potential and conductance in frog skin exposed to high serosal potassium

Summary In studies of apical membrane current-voltage relationships, in order to avoid laborious intracellular microelectrode techniques, tight epithelia are commonly exposed to high serosal K concentrations. This approach depends on the assumptions that high serosal K reduces the basolateral membrane resistance and potential to insignificantly low levels, so that transepithelial values can be attributed to the apical membrane. We have here examined the validity of these assumptions in frog skins (Rana pipiens pipiens). The skins were equilibrated in NaCl Ringer's solutions, with transepithelial voltageV _t clamped (except for brief perturbations V _t) at zero. The skins were impaled from the outer surface with 1.5m KCl-filled microelectrodes (R _el>30 M). The transepithelial (short-circuit) currentl _i and conductanceg _t=–I _t/V _t, the outer membrane voltageV _o (apical reference) and voltage-divider ratio (F _o=V _o/V _t), and the microelectrode resistanceR _el were recorded continuously. Intermittent brief apical exposure to 20 m amiloride permitted estimation of cellular (c) and paracellular (p) currents and conductances. The basolateral (inner) membrane conductance was estimated by two independent means: either from values ofg _i andF _o before and after amiloride or as the ratio of changes (–I _c/V _i) induced by amiloride. On serosal substitution of Na by K, within about 10 min,I _c declined andg _t increased markedly, mainly as a consequence of increase ing _p. The basolateral membrane voltage (V _i(=–V _o) was depolarized from 75±4 to 2±1 mV [mean±sem (n=6)], and was partially repolarized following amiloride to 5±2 mV. The basolateral conductance increased in high serosal K, as estimated by both methods. Essentially complete depolarization of the basolateral membrane and increase in its conductance in response to high [K] were obtained also when the main serosal anion was SO₄ or NO₃ instead of Cl. On clampingV _t over the range 0 to +125 mV in K₂SO₄-depolarized skins, the quasi-steady-stateV _o V _t relationship was linear, with a mean slope of 0.88±0.03. The above results demonstrate that, in a variety of conditions, exposure to high serosal K results in essentially complete depolarization of the basolateral membrane and a large increase in its conductance.     

Cellular and shunt conductances of toad bladder epithelium

Summary Toad urinary bladders were mounted in Ussing-type chambers and voltage-clamped. At nonzero voltages only, small fluctuations in current, I, and therefore in tissue conductance, G _t, were detected. These fluctuations were caused by the smooth muscle of the underlying tissue which could be monitored continuously and simultaneously with the current,I. Inhibition of the smooth muscle contraction with verapamil (2×10^–5 m) abolished the fluctuations inI andG _t. Amiloride (10^–4 m) had no significant effect on the magnitude of G _t, oxytocin increasedG _t without affecting G _t, and mucosal hypertonicity produced by mannitol increased G _t. These results are consistent with the hypothesis that two parallel pathways exist for passive current flow across the toad urinary bladder: one, the cellular pathway, was not affected by smooth muscle activity; the other, the paracellular pathway, was the route whose conductance was altered by the action of the smooth muscle.Thus the relationship between the cellular and shunt conductances of the epithelium of the toad urinary bladder, under a variety of conditions, can be investigated by utilizing the effects of the movement of the smooth muscle.     

Kinetics of the flash-induced electrochromic absorbance change in the presence of background illumination. Turnover rate of the electron transport. II. Higher plant leaves

The flash-induced electrochromic absorbance change (A ₅₁₅) was measured in leaves of higher plants in the absence and presence of continuous monochromatic background illumination of different intensities and wavelengths. The variation of the amplitude of A ₅₁₅ in background light was used to estimate the steady-state turnover time of the electron transport. In red light we obtained about 5 msec which was accounted for by the turnover of the linear electron transport. With far red background illumination or in the presence of the photosystem 2 inhibitor, DCMU, the steady-state turnover time tentatively assigned to photosystem 1 cyclic electron transport was much larger (100 msec).Increasing the intensity of background illumination with far red light gradually diminished the slow rise of A ₅₁₅ in parallel with suppression of the initial rise generated by photosystem 1. At high intensities of the red light, however, while A ₅₁₅ was attenuated, the slow rise was not eliminated and its proportion relative to the initial rise did not vary appreciably.     

Phloretin and phloretin analogs: Mode of action in planar lipid bilayers and monolayers

Summary Phloretin and other neutral phloretin-like molecules are able to decrease the electrostatic potential within neutral lipid bilayers and monolayers. The relationship between the change in the dipole potential and the aqueous concentration of the molecule is well described by a Langmuir isotherm. From the Langmuir isotherm, the apparent dissociation constants (K _D ^A ) and the maximum dipole potential change ( _max) are obtained for the different phloretin-like molecules tested. Considering the phloretin analogs as derivatives of acetophenone containing two kinds of substituents, one on the benzene ring and another on the carbon chain, it is found that (a)K _D ^A is related to the hydrophobicity of the compound and is also a function of the position of the hydroxyl substituent in the ring; (b) from the dependence ofK _D ^A on the length of the acyl chain, it is estimated that the free-energy change is 650 cal/mole CH₂; (c) _max is not a simple function of the dipole moment of the molecule but depends on the substituent on the carbon chain and on the position and number of hydroxyl groups on the benzene ring; (d) phloretin adsorption parameters are a function of membrane lipid composition. The results are discussed in terms of the effect of these compounds on chloride transport in red blood cells.     

Subcellular heterogeneity of mitochondrial membrane potential in anther tapetum

Studies on animal material have revealed that changes in the mitochondrial permeability transition pore (PTP), which cause a reduction in the mitochondrial transmembrane potential (_m) followed by release of cytochrome c, belong to the earliest manifestations of some types of apoptosis. We have attempted to monitor the _m of mitochondria during programmed cell death (PCD) of the secretory tapetum using JC-1, a fluorochrome dye that detects mitochondrial membrane potential and to relate changes in this potential to mitochondrial ultrastructure. Analysis of tapetal cells isolated from Ornithogalum virens anthers revealed that the _m of mitochondria in the tapetal cells alters during development; the change, however, is not uniform in the mitochondrial population within a single tapetal cell. In young tapetal cells, at the tetrad stage, we detected only the red fluorescence of JC-1 aggregates in all tapetal mitochondria, which indicates highly negative _m. In an advanced stage of PCD at the late microspore stage, in each tapetal cell we detected both mitochondria with red (as formerly) and mitochondria with green fluorescence. The green fluorescence of JC-1 monomers indicates mitochondria with depolarised membranes. These changes in _m are related to observed changes in mitochondria ultrastructure. This is the first documentation of intracellular heterogeneity of _m during anther tapetum development. Alteration in _m suggests a relationship between mitochondrial function and PCD processes in tapetal cells.     

Transport phenomena in a model membrane accompanying a conformational change: Membrane potential and ion permeability

Summary Membrane potential _m ion permeabilityP and d-c conductanceG _p ⁰ of a model membrane composed of a filter paper and a synthetic lipid analogue, i.e. dioleylphosphate (DOPH), were observed in various concentrations of 11 type electrolyte solution. With successive increases of salt concentration in the external solution, _m ,P andG _p ⁰ changed their values rather discontinuously at a certain critical value of the concentrationc _t . When the concentration was decreased successively, the values of _m ,P andG _p ⁰ were returned to the original levels at a concentration much lower thanc _t . Thus, the transport phenomena observed across the membrane showed an appreciable hysteresis loop.The discontinuous variations of _m ,P andG _p ⁰ were interpreted in terms of a transition of DOPH adsorbed in the filter paper from an oil membrane to a charged membrane. This transformation of DOPH at the critical salt concentration is consistent with the previous argument that the DOPH impregnated in the filter paper changes its conformation from oil droplets to a number of bilayer films, which deduced from a theoretical analysis of frequency dependency of the electrical capacitance of the membrane.     

Mechanisms of voltage transients during current clamp inNecturus gallbladder

Summary Microelectrode techniques were employed to study the mechanisms of the transepithelial voltage transients (V _ms ) observed during transmural current clamps in the isolatedNecturus gallbladder. The results indicate that: a) part of V _ms is due to a transepithelial resistance change (R _t ), and part to a tissue emf change. b) R _t is entirely caused by changes of the resistance of the paracellular pathway. At all current densities employed, the measured changes are probably due to changes in both fluid conductivity and width of the lateral intercellular spaces. At high currents, in addition to the effects on the lateral spaces, the resistance of other elements of the pathway (probably the limiting junction) drops, regardless of the direction of the current. c) The magnitude and polarity of the R _t -independent transepithelial and cell membrane potential transients indicate that the largest emf change takes place at the basolateral membrane (E _b ), with smaller changes at the luminal membrane (E _a ) and the paracellular (shunt) pathway (E _s ). It is shown that two-thirds of the transient are caused by E _s , and one-third by (E _b –E _a ). E _s can be explained by a diffusion potential generated by a current-dependent NaCl concentration gradient across the tissue. E _a and E _b are caused by [K] changes, mainly at the unstirred layer in contact with the basolateral membrane.     

Modelling based method for pharmacokinetic hypotheses test

The aim of this work is to propose methods to test mechanism of synergy of toxic agents in bees. A synergy between prochloraz, an imidazole fungicide, and deltamethrin, a pyrethroid insecticide, was demonstrated experimentally. The hypothesis is that prochloraz modifies the penetration or the metabolism of deltamethrin. This hypothesis is tested using a pharmacokinetic box model. A previous experimental work showed that bee instantaneous mortalities were higher, from the time t ₁ to the time t ₂ after spraying, in groups sprayed with deltamethrin at dose D ₀ in the presence of prochloraz (+P) than in those sprayed with deltamethrin alone at a dose time as high (). We postulate that accrued mortality is proportional to the cumulated internal deltamethrin (ID ₂). ID ₂ of treatment (+P) had to be greater than ID ₂ of treatment () during the period from t ₁ to t ₂ so that the hypothesis would be consistent with the experimental data. The limit, for which the hypothesis is conceivable, is the ID ₂₍₎ = ID _2(+P ) curve. We study, in particular, the asymptotic behaviour of the limit curve when different parameters of the kinetic model tend to 0 or . These limits allow to verify quickly and easily whether a mechanism is conceivable or not As the limits are calculated with algebraic values, the test can be used for other synergies.     

Estimation of microbial cell concentration in suspension culture by the osmotic pressure measurement of culture broth

Summary A method for estimating microbial cell concentration in suspension cultures even under heterogeneous conditions was developed on the basis of changes in osmotic pressure of the medium. During batch cultivation of Saccharomyces cerevisiae and Candida brassicae, there was a linear relationship between increase in cell concentrations (X) and the difference between osmotic pressure change in the broth (P_c) and the osmotic pressure increase due to product accumulation (P_p) regardless of the product (ethanol) concentration in the broth. A linear relationship between (X) and (P_c — P_p) was also observed when medium containing solid substrate (wheat germ) was used. An enzymatic method for separating cells from the solids was developed and cell concentrations in broths containing solid substrates could be measured accurately. During the batch production of bialaphos (a herbicide) by Streptomyces hygroscopicus using a medium containing solid substrates, the cell concentration could also be estimated by the developed methods.     

Dependence of energization of thylakoids on frequency of exciting flashes in intact chloroplasts

We investigated the frequency-dependence of the flash-induced electrochromic absorbance change, A₅₁₅, and of the pH-indicating absorbance change of neutral red in isolated intact chloroplasts. The energization pattern of thylakoids depended strongly on the frequency (f) of the exciting flashes, tested between 0.05 and 2 s^–1. When the frequency was increased from 0.1 to 1 s^–1 the total initial change and the slow rise of A₅₁₅ decreased by about 30% and 70%, respectively, and both the slow rise and decay were considerably accelerated. These changes were fully reversible, even after prolonged excitation at 1 s^–1, if the frequency was decreased again to 0.1 s^–1. Accumulation of an appreciable transmembrane electric field strength could not be detected in any of our experiments, at high frequency, since the decay of A₅₁₅ was considerably accelerated when the frequency was increased. In contrast, pH significantly increased at higher frequencies of the exciting flashes. In the steady-state (after about 100 flashes) pH was about 0.5–0.8 pH unit higher than in the dark or at low frequencies. In the presence of nigericin or dithionite, both of which prevented accumulation of protons in the lumen, the total initial change in A₅₁₅ at f=1 s^–1 relative to that at f=0.1 s^–1 decreased to a similar extent as in the control. The proportion of the slow rise relative to the initial amplitude, however, did not decrease. Our data support the suggestion that pH controls the amplitude of the slow rise of A₅₁₅. However, contrary to a previous statement (B. Bouges-Bouquet (1981) Biochim. Biophys. Acta 535, 327–340), we show that the pH effect cannot be accounted for by variation of the rate of this kinetic component of A₅₁₅.Abbreviations DCMU 3(3,4-dichlorophenyl)-1,1-dimethylurea - f frequency of the exciting flashes - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PS photosystem     

Relationships among violaxanthin deepoxidation,thylakoid membrane conformation,and nonphotochemical chlorophyll fluorescence quenching in leaves of cotton (Gossypium hirsutum L.)

The kinetics and temperature dependencies of development and relaxation of light-induced absorbance changes caused by deepoxidation of violaxanthin to antheraxanthin and zeaxanthin (Z; peak at 506 nm) and by light scattering (S; peak around 540 nm) as well as of nonphotochemical quenching of chlorophyll fluorescence (NPQ) were followed in cotton leaves. Measurements were made in the absence and the presence of dithiothreitol (DTT), an inhibitor of violaxanthin deepoxidase. The amount of NPQ was calculated from the Stern-Volmer equation. A procedure was developed to correct gross AS (S_g) for absorbance changes around 540 nm that are due to a spectral overlap with Z. This protocol isolated the component which is caused by light-scattering changes alone (S_n). In control leaves, the kinetics and temperature dependence of the initial rate of rise in S_n that takes place upon illumination, closely matched that of Z. Application of DTT to leaves, containing little zeaxanthin or antheraxanthin, strongly inhibited both S_n and NPQ, but DTT had no inhibitory effect in leaves in which these xanthophylls had already been preformed, showing that the effect of DTT on A_n and NPQ results solely from the inhibition of violaxanthin deepoxidation. The rates and maximum extents of S_n and NPQ therefore depend on the amount of zeaxanthin (and/or antheraxanthin) present in the leaf. In contrast to the situation during induction, relaxation of Z upon darkening was much slower than the relaxation of S_n and NPQ. The relaxation of S_n and NPQ showed quantitatively similar kinetics and temperature dependencies (Q₁₀=2.4). These results are consistent with the following hypotheses: The increase in lumen-proton concentration resulting from thylakoid membrane energization causes deepoxidation of violaxanthin to antheraxanthin and zeaxanthin. The presence of these xanthophylls is not sufficient to cause S_n or NPQ but, together with an increased lumen-proton concentration, these xanthophylls cause a conformational change, reflected by S_n. The conformational change facilititates nonradiative energy dissipation, thereby causing NPQ. Membrane energization is prerequisite to conformational changes in the thylakoid membrane and resultant nonradiative energy dissipation but the capacity for such changes in intact leaves is quite limited unless zeaxanthin (and/or antheraxanthin) is present in the membrane. The sustained S_n and NPQ levels that remain after darkening may be attributable to a sustained high lumen-proton concentration.Abbreviations A antheraxanthin - DTT dithiothreitol - F, F_m chlorophyll fluorescence yield at actual, full closure of the PSII centers - NPQ nonphotochemical chlorophyll fluorescence quenching - PFD photon flux density - PSII photosystem II - V violaxanthin - Z zeaxanthin - S_n, Z spectral absorbance change caused by light-scattering, violaxanthin deepoxidation We thank Connie Shih for skillful assistance in growing the plants, and for conducting HPLC analyses. A Carnegie Institution Fellowship and a Feodor-Lynen-Fellowship by the Alexander von Humboldt-Foundation to W. B. is gratefully acknowledged. This work was supported in part by Grant No. 89-37-280-4902 of the Competitive Grants Program of the U.S. Department of Agriculture to O.B. This is C. I. W. — D. P. B. Publication No. 1094.     

Carbon isotope discrimination in three semi-arid woodland species along a monsoon gradient

Leaf carbonisotope discrimination () was measured for three dominant, semi-arid woodland species along a summer monsoon gradient inthe southwestern United States over a 2-year period. We tested the hypothesis that decreased humidity levels during the growing season along this gradient resulted in lower leaf values. Sites of similar elevation along the transect were selected and the range in monsoon contribution to overall annual precipitation varied from 18 to 58%, while total annual precipitation differed by a maximum of only 25% across this gradient. Leaf values in Quercus gambelii were negatively correlated with , a seasonally-weighted estimate of the evaporative humidity gradient, suggesting that stomatal conductance declined as transpiration potential increased. For two other trees that co-occurred along this gradient, Pinus edulis and Juniperus osteosperma, remained relatively constant despite large variation in . These woodland species represent the full spectrum of responses of carbon isotope discrimination to increases in evaporative potential; that of decline where c _i /c _a (ratio of internal to ambient CO₂ concentration) and presumably stomatal conductance decrease, and that of constancy where whole plant internal adjustments allow c _i /c _a to remain stable.     

DNA/DNA hybridization studies of the carnivorous marsupials. I: The intergeneric relationships of bandicoots (Marsupialia: Perameloidea)

Summary A complete suite of comparisons among six bandicoot species and one outgroup marsupial was generated using the hydroxyapatite chromatography method of DNA/DNA hybridization; heterologous comparisons were also made with three other bandicoot taxa. Matrices of T_m's, modes, and T₅₀Hs were generated and corrected for nonreciprocity, homoplasy, and, in the case of T_m's, normalized percent hybridization; these matrices were analyzed using the FITCH algorithm in Felsenstein's PHYLIP (version 3.1). Uncorrected and nonreciprocity-corrected matrices were also jackknifed and analyzed with FITCH to test for consistency. Finally, sample scores for T_m, mode, and T₅₀H matrices were bootstrapped and then subjected to phylogenetic analysis. These manipulations were carried out, in part, to address criticisms of the statistics used to summarize DNA/DNA hybridization (especially T₅₀H) and the method itself. However, with the exception of an unresolved trichotomy among the twoEchymipera species andPeroryctes longicauda, all trees showed the same branchpoints. Except in the case of the tree generated from reciprocal-corrected T_m data, nodes were stable under jackknifing; and, again excepting the above-mentioned trichotomy, all nodes were supported by 95% or more of the bootstrapped trees. These results suggest that, despite arguments to the contrary, all three summary statistics can be valid for DNA/DNA hybridization data. Of taxonomic interest is the placement ofEchymipera spp. andPeroryctes longicauda together and separate from the more distantPeroryctes raffrayanus; the genusPeroryctes is thus at least paraphyletic. The trees further groupedEchymipera-plus-Peroryctes as the sister group ofIsoodon-plus-Perameles. Limited hybridizations withMacrotis lagotis suggest that its current position as representative of an entirely distinct family of perameloids is correct.This article was presented at the C.S.E.O.L. Conference on DNA-DNA Hybridization and Evolution, Lake Arrowhead, California, May 11–14, 1989     

Response of effective quantum yield of photosystem 2 to in situ temperature in three alpine plants

The response of effective quantum yield of photosystem 2 (F/F_m) to temperature was investigated under field conditions (1 950 m a.s.l.) in three alpine plant species with contrasting leaf temperature climates. The in situ temperature response did not follow an optimum curve but under saturating irradiances [PPFD >800 µìmol(photon) m^–2s^–1] highest F/F_m occurred at leaf temperatures below 10°C. This was comparable to the temperature response of antarctic vascular plants. Leaf temperatures between 0 and 15°C were the most frequently (41 to 56%) experienced by the investigated species. At these temperatures, F/F_m was highest in all species (data from all irradiation classes included) but the species differed in the temperature at which F/F_m dropped below 50% (Soldanella pusilla >20°C, Loiseleuria procumbens >25°C, and Saxifraga paniculata >40°C). The in situ response of F/F_m showed significantly higher F/F_m values at saturating PPFD for the species growing in full sunlight (S. paniculata and L. procumbens) than for S. pusilla growing under more moderate PPFD. The effect of increasing PPFD on F/F_m, for a given leaf temperature, was most pronounced in S. pusilla. Despite the broad diurnal leaf temperature amplitude of alpine environments, only in S. paniculata did saturating PPFD occur over a broad range of leaf temperatures (43 K). In the other two species it was half of that (around 20 K). This indicates that the setting of environmental scenarios (leaf temperature×PPFD) in laboratory experiments often likely exceeds the actual environmental demand in the field.This revised version was published online in March 2005 with corrections to the page numbers.     

Chlorophyll luminescence as an indicator of stress-induced damage to the photosynthetic apparatus. Effects of heat-stress in isolated chloroplasts

A brief review is given of investigations on stres-induced alterations of ms-to s-luminescence yield of chlorophyll in plants. Three different approaches are considered: phytoluminography, luminescence-temperature curves, and luminescence induction curves. The remainder of this article presents new results of the effect of heat stress on luminescence induction curves of isolated chloroplasts. Three parameters with widely different heat resistances were resolved from induction curves. A fast valinomycin sensitive transient, L'_i, with a 50% inhibition temperature of 33 to 34°C was correlated with the magnitude of the light-induced membrane potential after heat pretreatment. A slower nigericin sensitive transient, L'_m, with a 50% inhibition temperature of 39 to 40°C was mainly correlated with the light-induced proton gradient. An uncoupler resistant part of the induction curve, L₀, was enhanced by heat stress (half maximum after pretreatment at 46°C) and was correlated with the degree of inhibition of oxygen evolution. Since L₀ was also raised by other treatments impairing the oxygen evolving enzyme system, and since this rise was inhibited by DCMU and hydroxylamine, this type of luminescence was ascribed to the intrinsic backreaction. We conclude that luminescence induction curves can serve as an useful indicator of the intactness of the membrane potential, the proton gradient, and the oxygen evolving enzyme system in isolated chloroplasts after heat stress.Abbreviations 9-AA 9-aminoacridine - CCCP carbonylcyanide m-chlorophenylhydrazone - A₅₁₈ light-induced absorbance change at 518 nm - A_on, A_off rapid A₅₁₈ upon switching actinic light on or off, respectively - L_i, L_m, L₀ in this order, initial spike, main maximum, uncoupler insensitive transient of luminescence induction curve - L'_i = L_i – L₀ - L'_m = L_m – L₀ - P, P680 primary donor of PS II - PFD photon flux density (400–700 nm) - Q_A primary acceptor of PS II     

Lactic acid excretion via carrier-mediated facilitated diffusion in Lactobacillus helveticus

Summary During growth on a complex medium containing 2% (w/v) lactose, Lactobacillus helveticus produced about 180 mm lactate. Due to the acidification, the external pH decreased to 3.7. The pH remained constant at a level of 0.5–0.7 units (40 mV), and µ_Lac decreased gradually from –60 to 0 mV. The mechanism of lactate extrusion was studied with resting cells. Upon dilution of lactate-loaded cells in a buffer containing [¹⁴C]-lactate, a typical counterflow was observed, suggesting that a carrier system was employed in lactate excretion. Influx of lactate could not be driven by an artificial membrane potential, indicating that lactate was electroneutrally transported. By examining efflux under various lactate anion and lactic acid concentrations, the undissociated form of the acid was shown to influence the velocity of the transport process. A pH-dependent apparent K _m value of the carrier system was observed in efflux experiments with increasing internal lactate concentrations. It was concluded that the mode of end-product excretion can be defined as a carrier-mediated facilitated diffusion with the undissociated lactic acid or the lactate anion in symport with one proton, respectively, as the object of transport.Abbreviations L _tota total lactate - L _undissb free lactic acid - L _dissc lactate anion - pH_ed external pH - pH_ie internal pH - pH transmembrane H⁺ gradient - µ_Lacf transmembrane gradient of total lactate - µ_HLg transmembrane gradient of the free lactic acid - µ_Lh transmembrane gradient of the lactate anion - V _Effii efflux velocity Offprint requests to: G. Gottschalk     

Estimation of the canopy-gap size using two photographs taken at different heights

A new, convenient method to estimate canopy-gap size using a camera is proposed. Canopy-gap size was estimated from two images taken with a vertically mounted, digital camera at high and low points (P_h and P_l) beneath a canopy gap. The actual (A_OBS) and estimated (A_1.0) canopy-gap sizes were highly correlated (R² > 0.93) for 16 canopy gaps in the Cryptomeria japonica D. Don. plantations, and the relationship between A_1.0 and A_OBS was expressed as A_1.0 = 1.16A_OBS. Thus, the method overestimated actual canopy-gap size by an average of 16%.