Leaf water relations and anatomy of a tropical rainforest tree species vary with crown position

Summary Leaf water potentials, osmotic properties and structural characteristics were examined in the Australian tropical rainforest tree species, Castanospermum australe. These features were compared for individuals growing in the understorey and canopy of the undisturbed forest and in an open pasture from which the forest had been cleared. Leaf water potentials during the day declined to significantly lower values in the open-grown and canopy trees than in the understorey trees. During most of the day the opengrown tree experienced the lowest water potentials. These differences were paralleled by significant differences in tissue osmotic properties. The tissue osmotic potential at full hydration was lowest in the open-grown tree (-1.80 MPa), intermediate in the canopy trees (-1.38 MPa), and highest in the understorey trees (-0.80 MPa). As a result, the degree to which high and positive turgor pressures were maintained as water potentials declined was highest in the open-grown tree, intermediate in the canopy trees, and lowest in the understorey trees. The differences in tissue osmotic properties between individuals in the three crown positions were paralleled, in turn, by differences in leaf structual characteristics. Relative to leaves of the canopy and open-grown trees, leaves of the understorey trees had significantly larger epidermal cells with thinner cell walls, larger specific leaf areas and turgid weight: dry weight ratios, and a higher proportion of intercellular air space.Abbreviations ₁ Leaf tissue water potential - _min Lowest value of ₁ during the day ( noon) - _{P=0 1} zero turgor - R Relative water content - P Tissue turgor pressure - Tissue osmotic potential - ₀ at full hydration     

The mitochondrial membrane potential (deltapsi(m)) in apoptosis; an update

Mitochondrial dysfunction has been shown to participate in the induction of apoptosis and has even been suggested to be central to the apoptotic pathway. Indeed, opening of the mitochondrial permeability transition pore has been demonstrated to induce depolarization of the transmembrane potential (_m), release of apoptogenic factors and loss of oxidative phosphorylation. In some apoptotic systems, loss of _m may be an early event in the apoptotic process. However, there are emerging data suggesting that, depending on the model of apoptosis, the loss of _m may not be an early requirement for apoptosis, but on the contrary may be a consequence of the apoptotic-signaling pathway. Furthermore, to add to these conflicting data, loss of _m has been demonstrated to not be required for cytochrome c release, whereas release of apoptosis inducing factor AIF is dependent upon disruption of _m early in the apoptotic pathway. Together, the existing literature suggests that depending on the cell system under investigation and the apoptotic stimuli used, dissipation of _m may or may not be an early event in the apoptotic pathway. Discrepancies in this area of apoptosis research may be attributed to the fluorochromes used to detect _m. Differential degrees of sensitivity of these fluorochromes exist, and there are also important factors that contribute to their ability to accurately discriminate changes in _m.     

Growth at reduced turgor: irreversible and reversible cell-wall extension of maize coleoptiles and its implications for the theory of cell growth

The relationship between steady-state elongation rate (G) and turgor pressure (P; G/P curve) was investigated using isolated segments of maize (Zea mays L.) coleoptiles incubated in osmotic solutions of a water potential range of 0 to -10 bar (polyethylene glycol 6000 as osmoticum). Short-term elongation measurements revealed curvilinear G/P curves with a steep slope at high turgor and a shallow slope at low turgor. Owing to a decrease of osmotic pressure and turgor, there was a tendency for straightening of the G/P curves during long-term elongation. An elongation rate of zero was adjusted by lowering the turgor by 4.5 bar at a constant osmotic pressure of 6.7 bar. Auxin increased — whereas abscisic acid decreased — the slope of the G/P curve but these hormones had no effect on the threshold turgor of growth (Y = 2.2 bar). It is concluded that extensibility of the growing cell walls represented by the yielding coefficient of Lockhart's growth equation is turgor-dependent and therefore decreases to a very low value as the turgor approaches Y. When the turgor was kept at Y, a constant segment length was maintained over at least 6 h. However, separation of reversible (l_rev) and irreversible (l_irr) components of total (in vivo) length (l_tot = l_rev + l_irr) W measuring segment length before and after freezing/thawing revealed that l_irr increased continuously and l_rev decreased continuously at constant l_tot. After a step-down in turgor the segments grew in l_irr although they shrank in l_tot over the whole turgor range of 0irr irreversible length - l_rev reversible length - l_tot total length (= l_irr + l_rev) - _i osmotic pressure of cell sap - _i water potential of tissue - _o water potential of incubation medium - ABA abscisic acid - G growth rate - m yielding coefficient - P turgor pressure - PEG polyethylene glycol 6000 - Y yield threshold Supported by Deutsche Forschungsgemeinschaft (SFB 206). We thank R. Hertel for helpful comments.     

Water relations and growth of three grasses during wet and drought years in a tallgrass prairie

Summary The water relations and growth of three tallgrass prairie species Panicum virgatum, Andropogon gerardii and A. scoparius were examined in irrigated and unwatered prairie in eastern Kansas (USA). Measurements of the osmotic potential at full turgor, ¹⁰⁰ , at zero turgor, ⁰, and growth of vegetative and reproductive tillers were made in a year with above-normal precipitation and a drought year to evaluate: 1) the ability of these grasses to osmotically adjust in response to water stress and 2) the effect of drought or supplemental water on growth of these species. Although these grasses adjusted osmotically even in the wet year, the degree of adjustment of ¹⁰⁰ and ⁰ in the drought year was relatively large (0.60–0.78 MPa and 0.88–1.34 MPa, respectively) compared to reports for other species. Seasonal minimum values of ¹⁰⁰ and ⁰ for these grasses in the drought year were lower than in most mesic species and seasonal fluctuations in ¹⁰⁰ and ⁰ were greater than reported for most mesic or xeric species. The relatively frequent occurrence of drought in sub-humid tallgrass prairies may partially explain the greater than expected magnitude of osmotic adjustment in these grasses.Irrigation in the wet year increased reproductive biomass in the mesic grass P. virgatum, but had no effect on A. gerardii or the more xeric grass A. scoparius. However, irrigation in the drought year increased maximum shoot biomass in all three grasses significantly with the largest increase in P. virgatum. Reproduction in P. virgatum was also increased more by irrigation in the drought year compared to the other grasses. Irrigation did not increase season's end production of A. gerardii in the wet year, but in the drought year production was 28% greater in irrigated than unwatered prairie. The combination of these water relations and growth responses of the three grasses to wetter than normal and drought years supports their reported distribution along a moisture gradient in tallgrass prairies.     

Some characteristics of anion transport at the tonoplast of oat roots,determined from the effects of anions on pyrophosphatedependent proton transport

The effects of anions on inorganicpyrophosphate-dependent H⁺-transport in isolated tonoplast vesicles from oat (Avena sativa L.) roots were determined. Both fluorescent and radioactive probes were used to measure formation of pH gradients and membrane potential in the vesicles. Pyrophosphate hydrolysis by the H⁺-translocating pyrophosphatase was unaffected by anions. Nonetheless, some anions (Cl^-, Br^- and NO₃-) stimulated H⁺-transport while others (malate, and iminodiacetate) did not. These differential effects were abolished when the membrane potential was clamped at zero mV using potassium and valinomycin. Stimulation of H⁺-transport by Cl^- showed saturation kinetics whereas that by NO₃- consisted of both a saturable component and a linear phase. For Cl^- and NO₃-, the saturable phase had a K _m of about 2 mol·m^-3. The anions that stimulated H⁺-transport also dissipated the membrane potential (.) generated by the pyrophosphatase. It is suggested that the stimulatory anions cross the tonoplast in response to the positive generated by the pyrophosphatase, causing dissipation of and stimulation of pH, as expected by the chemiosmotic hypothesis. The work is discussed in relation to recent studies of the effects of anions on ATP-dependent H⁺-transport at the tonoplast, and its relevance to anion accumulation in the vacuole in vivo is considered.Abbreviations and symools BTP 1,3-bis[tris(hydroxymethyl)-methylamino]-propane - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid - IDA iminodiacetate - membrane potential - pH pH gradient - PPase inorganic pyrophosphatase - PPi morganic pyrophosphate     

Correlation between loss of turgor and accumulation of abscisic acid in detached leaves

Mature leaves of Phaseolus vulgaris L. (red kidney bean), Xanthium strumarium L. (cocklebur), and Gossypium hirsutum L. (cotton) were used to study accumulation of abscisic acid (ABA) during water stress. The water status of individual, detached leaves was monitored while the leaves slowly wilted, and samples were cut from the leaves as they lost water. The leaf sections were incubated at their respecitive water contents to allow ABA to build up or not. At least 8 h were required for a new steady-state level of ABA to be established. The samples from any one leaf covered a range of known water potentials (), osmotic pressures (), and turgor pressures (p). The and p values were calculated from pressure-volume curves, using a pressure bomb to measure the water potentials. Decreasing water potential had little effect on ABA levels in leaves at high turgor. Sensitivity of the production of ABA to changes in progressively increased as turgor approached zero. At p=1 bar, ABA content averaged 4 times the level found in fully turgid samples. Below p=1 bar, ABA content increased sharply to as much as 40 times the level found in unstressed samples. ABA levels rose steeply at different water potentials for different leaves, according to the at which turgor became zero. These differences were caused by the different osmotic pressures of the leaves that were used; must cqual - for turgor to be zero. Leaves vary in , not only among species, but also between plants of one and the same species depending on the growing conditions. A difference of 6 bars (calculated at =0) was found between the osmotic pressures of leaves from two groups of G. hirsutum plants; one group had previously experienced periodic water stress, and the other group had never been stressed. When individual leaves were subsequently wilted, the leaves from stress-conditioned plants required a lower water potential in order to accumulate ABA than did leaves from previously unstressed plants. On the basis of these results we suggest that turgor is the critical parameter of plant water relations which controls ABA production in water-stressed leaves.Abbreviations ABA abscisic acid - me-ABA abscisic-acid methyl ester - leaf water potential - osmotic pressure - p volumeaveraged turgor - volumetric modulus of elasticity     

Assessment of membrane potential changes using the carbocyanine dye,diS-C3-(5): synchronous excitation spectroscopy studies

The fluorescence of the voltage sensitive dye, diS-C₃-(5), has been analyzed by means of synchronous excitation spectroscopy. Using this rather rare fluorescence technique we have been able to distinguish between the slightly shifted spectra of diS-C₃-(5) fluorescence from cells and from the supernatant. It has been found that diS-C₃-(5) fluorescence in the supernatant can be selectively monitored at _exc = 630 nm and _em= 650 nm, while the cell associated fluorescence can be observed at _exc= 690 nm and _em = 710 nm. A modified theory for the diSC₃-(5) fluorescence response to the membrane potential is presented, according to which a linear relationship exists between the logarithmic increment of the dye fluorescence intensity in the supernatant, In I/I°, and the underlying change in the plasma membrane potential, _p=_p-°_p. The theory has been tested on human myeloid leukemia cells (line ML-1) in which membrane potential changes were induced by valinomycin clamping in various K⁺ gradients. It has been demonstrated that the membrane potential change, _p,can be measured on an absolute scale. Offprint requests to: J. Plasek     

Ecophysiological analysis of woody species in contrasting temperate communities during wet and dry years

This study employed an intensive sampling regime in which leaf gas exchange and tissue-water relations were measured simultaneously on the same leaf at midday on 19 tree species from three distinct forest communities during wet (1990) and dry (1991) growing seasons. The study sites were located on a xeric barrens, a misic valley floor, and a wet-mesic floodplain in central Pennsylvania, United States. The xeric, mesic, and wetmesic sties had drought-related decreases in gravimetric soil moisture of 53, 34 and 27%, respectively. During the wet year, xeric and mesic communities had high seasonal mean photosynthetic rates (A) and stomatal conductance of water vapor (g _wv) and low midday leaf water potential (), whereas the wet-mesic community had low A and g _wv and high midday . The mesic and wet-mesic communities had dry year decreases in predawn , g _wv and A with the greatest drought effect occurring in the mesic community. Regression analysis indicated that species from each site that exhibited high wet-year A and g _wv tended to have low midday . This trend was reversed only in the mesic community in the drought year. Despite differences in midday , all three communities had similar midday leaf turgor pressure (_p) in the wet year attributable to lower osmotic potential at zero turgor ( ⁰ ) with increasing site droughtiness. Lower wet year ⁰ in the xeric community was due to low symplast volume rather than high solute content. Species with the lowest ⁰ in the wet year often did not have the lowest ¹⁰⁰ possibly related to differences in tissue elasticity. Moreover, increased elasticity during drought may have masked osmotic adjustment in ¹⁰⁰ but not in ⁰ , via dilution of solutes at full hydration in some species. Despite the sampling regime used, there were no relationships between gas exchange and osmotic and elastic parameters that were consistently significant among communities or years. This result questions the universal, direct effect of osmotic and elastic adjustments in the maintenance of photosynthesis during drought. By including a large number of species, this study provided new insight to the ecophysiology of contrasting forest communities, and the community-wide impact of drought on contrasting sites.     

Kinetics of Δψ-Dependent Sucrose Transport in Plasma Membrane Vesicles from Higher Plant Cells

The inside-out vesicles of plasma membranes were isolated from pumpkin stem cells, and the kinetics of sucrose efflux induced by the K⁺-diffusion potential (_D) was studied by measuring light transmission. Two phases differing in their rates and duration were identified in _D-dependent changes of light transmission. The increase in _Delevated the rate and magnitude of the fast phase in light transmission changes but did not markedly affect the rate of the slow phase. These two phases also differed in their sensitivity to inhibitors and to changes in sucrose concentration in the external medium. Measurements of _Dduring sucrose transport by means of the fluorescence probe dis-C₃-(5) revealed differences in the magnitude of _Dand its stability in vesicles loaded with sucrose and mannitol, as well as under the action of inhibitors. The two-phase dependence of sucrose efflux from vesicles on the applied diffusion potential is discussed in the context of modern concepts on the functioning of sucrose carriers in the membranes.     

Preparations of Ψ-peptide bond and peptide-aldehyde inhibitors of atrial granule serine proteinase, a candidate processing enzyme of pro-atrial natriuretic factor

Pseudo-peptide bond inhibitors (-bond inhibitors) and peptide-aldehyde inhibitors of atrial granule serine proteinase, the candidate processing enzyme of pro-atrial natrieuretic factor, are prepared in high yield and purity by novel synthetic routes. The -bond compounds retain essential residues for enzyme binding, but place the enzyme inhibition site in the midst of the peptide sequence. Thus, Bz-APR--LR and Bz-APR--SLRR can be considered readthrough inhibitors of atrial granule serine proteinase. The most potent -peptide, Bz-APR--SLRR (IC₅₀=250 M), is about fivefold less potent than the best peptide-aldehyde inhibitor (EACA-APR-CHO), and both the -bond and peptide-aldehyde compounds are competitive, reversible inhibitors of the enzyme. The -bond peptides containing two C-terminal Arg residues are three-to tenfold more potent than the analogous compounds containing only one C-terminal Arg residue, confirming the importance of both Arg residues in the enzyme processing recognition site. As expected, because of their moderate potencies, the -peptides are not useful affinity ligands for purification of atrial granule serine proteinase, but both peptide aldehydes are effective affinity ligands [Damodaran and Harris (1995),J. Protein Chem., this issue].Abbreviations AGSP atrial granule serine proteinase - ANF atrial natriuretic factor - Bz benzoyl - DIEA diisopropylethylamine - DIPCDI diisopropylcarbodiimide - DMF dimethylformamide - DMSO dimethylsulfoxide - EACA 6(e)-aminocaproic acid - EtOAc ethyl acetate - HEPES N-2-hydroxyethylpiperazine-N-propanesulfonic acid - HOBt N-hydroxybenzotriazole - HPLC high-performance liquid chrornatography - NMR nuclear magnetic resonance - PEG polyethylene glycol-3350 - PyBOP benzotriazole-1-yl-oxy-trispyrrolidino-phosphonium-hexafluorophospate - TEA triethylamine - TFA trifluoroacetic acid - THF tetrahydrofuran - TLC thin-layer chromatography - UV ultraviolet - pseudo-peptide bond -CH₂-NH-. Single-letter abbreviations are used to denote amino acids     

Chloroplast volume: cell water potential relationships and acclimation of photosynthesis to leaf water deficits

Studies were undertaken to determine if there is an association between nonstomatally-mediated acclimation of photosynthesis to low water potential (w) and the maintenance of chloroplast volume during water stress. Spinach plants either kept well watered throughout their growth (non-acclimated), or subjected to water stress such that leaf w dropped to -1.5 megapascals (MPa) and then were rewatered (acclimated) were subjected to drought episodes. During these stress periods, photosynthesis was maintained to a greater extent in acclimated plants as compared to non-acclimated plants at w below -1 MPa.Estimates of internal leaf [CO₂] suggested that photosynthetic acclimation to low w was not primarily due to altered stomatal response. As w dropped from initial values, a decline in steady state levels of ribulose 1,5-bisphosphate (RuBP) occurred in both non-acclimated and acclimated plants. RuBP decline was less severe in acclimated plants.Low w effects on chloroplast volume in non-acclimated and acclimated plants were estimated by measuring the volume of intact chloroplasts isolated from plants in solutions which were made isotonic to declining leaf osmotic potential during the drought episodes. Chloroplast volume was maintained to a greater extent at low w in acclimated, as compared with non-acclimated plants. Although substantial osmotic adjustment occurred in both non-acclimated and acclimated plants, the extent of osmotic adjustment was the same. These data were interpreted as supporting the hypothesis that cellular-level acclimation to low w is associated with chloroplast volume maintenance, and this physiological acclimation is correlated with enhanced photosynthetic capacity of the leaf at low w.Abbreviations [CO₂]_i internal leaf CO₂ concentration - s osmotic potential - RWC relative water content - RuBP ribulose 1,5-bisphosphate - w water potential     

Estimation of Membrane Potential Δψ in Reconstituted Plasma Membrane Vesicles Using a Numerical Model of Oxonol VI Distribution

A model of membrane potential-dependent distribution of oxonol VI to estimate the electrical potential difference across Schizosaccharomyces pombe plasma membrane vesicles (PMV) has been developed. was generated by the H⁺-ATPase reconstituted in the PMV. The model treatment was necessary since the usual calibration of the dye fluorescence changes by diffusion potentials (K⁺ + valinomycin) failed. The model allows for fitting of fluorescence changes at different vesicle and dye concentrations, yielding in ATP-energized PMV of 80 mV. The described model treatment to estimate may be applicable for other reconstituted membrane systems.     

Chloroplast acclimation to low osmotic potential

Photosynthetic potential of isolated chloroplasts was investigated during in situ water deficits. An eight day stress cycle imposed on spinach plants reduced leaf _w by 0.57MPa, and leaf by 0.50MPa, resulting in partial turgor maintenance during the stress cycle. Pressure/volume curves confirmed the occurrence of osmotic adjustment. Leaf depression was associated with an altered response of chloroplasts to low in vitro. Optimum reaction medium for photosynthesis shifted from –1.04 to –1.57MPa, and low was not as inhibitory to photosynthesis of plastids pre-exposed to stress in situ. These data indicate that chloroplasts acclimate to low external in response to leaf water deficits. This response was still evident four days after a stress cycle ended, but was nearly reversed eight days after stress. Repeated stress cycles in situ did not increase the degree of chloroplast acclimation to low in vitro. Fast dehydration of leaves did not induce this apparent chloroplast acclimation.Abbreviations osmotic potential - _w water potential - PEG polyethylene glycol 8000 - MPa megapascals     

Effects of shoot and root application of thiamin on salt-stressed sunflower plants

Plants of sunflower (Helianthus annuus L. cv Giza2) were salt-stressed with a combination of NaCl and CaCl₂ inconcentrations having different osmotic potentials (s from 0 to –1.0MPa) and were treated with 5 and 10mg L^–1 of thiamin either sprayed on the shoot orapplied to the root. The membranes of leaf discs from salt-stressed plantsappeared to be less stable (more injured) under heat(51°C) and drought (40% polyethylene glycol6000) stresses than control plants. Salinity slowed the rate of growth (lengthand dry mass production), lowered leaf relative water content (RWC) and leafandroot water potential (w), decreased the contents of chlorophyll (Chl),soluble sugars (SS) and the K⁺/Na⁺ ratio butenhanced total free amino acids (TAA), Na⁺,Ca²⁺and Cl^– accumulation in the shoot and root system. Root orshoot application of thiamin reduced membrane injury by either heat ordehydration stress, lowered leaf w, improved uptake of K⁺,and increased leaf RWC, Chl, SS, TAA contents and dry mass production. Theeffects of salinity (s), thiamin (Thi.) and their interaction(s×Thi) on the parameters tested were significant.Salinity was dominant (as indicated by ² values) in affectingthe contents of Ca²⁺, Cl^–, TAA and membranestability to heat and leaf w. The role of thiamin was dominant forNa⁺, K⁺ and SS contents and the contribution ofinteraction was dominant for growth parameters, Chl. and root w.     

Leaf water potential,component potentials and relative water content in a xeric grass,Agropyron dasystachyum (Hook.) Scribn.

Summary Leaf water potential ( _l ), osmotic potential ( _s ), pressure potential ( _p , turgor pressure), relative water content (R) and their interrelationships were determined for a xeric grass (Agropyron dasystachyum) found in the grasslands of Canada. Thermocouple psychrometers were used to measure _l and _s ; _p was obtained by subtraction. _l dropped from near 0 bars to about-28 bars as R went from 90% to 75%. R greater than 90% was not observed, perhaps because of a systematic error in determination of turgid water content. R remained relatively high in A. dasystachyum, even at low _l . The slope of the _l -R relationship was similar to other species which are generally considered to be drought tolerant. _p as high as 14 bars was observed. Most of the decrease in _l was accounted for by a decline in _p . The ability of A. dasystachyum to adjust to fluctuating water stress over the growing season is probably as much related to changes in tissue structure and turgor relationships as to simple changes in osmotic potential.     

Measurements of electrical potential differences across yeast plasma membranes with microelectrodes are consistent with values from steady-state distribution of tetraphenylphosphonium inPichia humboldtii

Summary Electrical potential differences across the plasma membrane () of the yeastPichia humboldtii were measured with microelectrodes (filled with 0.1m KCl) inserted into cells immobilized in microfunnels. The registered signals were reproducible and stable for several minutes. On attainment of stable reading for the specific membrane resistanceR _sp was determined by applying square-current pulses to the preparation. Both andR _sp were pH dependent and displayed equal but opposite deflection, reaching its maximal value of –88±9 mV (n=13) andR _sp its minimal value of 10 k·cm² (maximal conductance) at pH 6.5. Uncouplers and the polyene antibiotic nystatin depolarized the cells, decreasing to –21±15 mV (n=10) with concomitant decrease ofR _sp. Comparison of values from microelectrode measurements with those calculated from the steady-state distribution of tetraphenylphosphonium ions agreed within 10 mV under all physiological conditions tested, except at pH values above 7.0. During microelectrode insertion transient voltage signals (a few msec long) were detected by means of an oscilloscope. These voltage signals were superimposed on the stable recordings described above. These short voltage signals disappeared in uncoupled cells. The closely related values obtained by two independent methods (direct measurements with microelectrodes and calculation from steady-state distribution of a lipophilic cation) provide evidence that these values reffect the true membrane potential of intact cells.     

Effects of water stress on the gas exchange,the activities of some enzymes of carbon and nitrogen metabolism,and on the pool sizes of some organic acids in maize leaves

Water-stressed maize (Zea mays L.) leaves showed a large decrease in leaf conductance during photosynthesis. Net CO₂ uptake and evaporation declined fast at mild stress (=–0.6 to –1.0 MPa) and slower at more severe stress (=–1.0 to -1.2 MPa), whereas the CO₂ concentration in the intercellular spaces (C_i) did not drop to the CO₂ compensation point. The activities of the enzymes of photosynthetic carbon metabolism tested in this study dropped by approx. 30% at =-1.2 MPa. Glutamine synthetase activity was unaffected by water stress, whereas the activity of nitrate reductase was almost completely inhibited. The decline of enzyme activities in relation to was correlated with a concomitant decrease in the content of total soluble protein of the stressed leaves. The total leaf pools of malate, pyruvate and oxaloacetate decreased almost linearly in relation to , thus obviously contradicting the almost constant C_i. In comparison to the controls (=0.6 MPa) the content of citrate and isocitrate increaed markedly at =-0.9 MPa and decreased again at =-1.2 MPa.Abbreviations PCR photosynthetic carbon reduction cycle - PCO photosynthetic carbon oxidation cycle - PEP phosphoenolypyruvate - RuBP ribulose-1,5-bisphosphate     

Auxin causes oscillations of cytosolic free calcium and pH inZea mays coleoptiles

In epidermal cells of maize (Zea mays L.) coleoptiles, cytosolic pH (pH_c), cytosolic free calcium, membrane potential and changes thereof were monitored continuously and simultaneously (pH_c/, _m, Ca²⁺/ _m) using double-barrelled ion-sensitive microelectrodes. In the resting cells the cytosolic pH was 7.3–7.5 and the concentration of free calcium was 119±24 nM. One-micromolar indole-3-acetic acid (IAA), added to the external medium at pH 6.0 triggered oscillations in _m, pH_c and free calcium with a period of 20 to 30 min. Acidification of the cytosolic pH increased the cytosolic free calcium. The _m oscillations are attributed to changes in activity of the H⁺-extrusion pump at the plasmalemma, triggered off by pH and controlled by pH regulation (pH oscillation). The origin of the pH_c and Ca²⁺ changes remains unclear, but is possibly caused by auxin-receptor-induced lipid breakdown and subsequent second-messenger formation. It is suggested that the observed cytosolic pH and Ca²⁺ changes are intrinsically interrelated, and it is concluded that this onset of regulatory processes through the phytohormone IAA is indicative of calcium and protons mediating early auxin action in maize coleoptiles. It is further concluded that the double-barrelled ion-sensitive microelectrode is an invaluable tool for investigating in-vivo hormone action in plant tissues.Abbreviations and symbols FC fusicoccin - IAA indole-3-acetic acid - Mes 2-(N-morpholino)ethanesulfonic acid - pH_c cytosolic pH - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol - _m membrane potential difference (mV)     

The mechanistic nature of the membrane potential dependence of sodium-sugar cotransport in small intestine

Summary Methods are described which demonstrate the use of unidirectional influx of¹⁴C-tetraphenylphosphonium (¹⁴C-TPP⁺) into isolated intestinal epithelial cells as a quantitative sensor of the magnitude of membrane potentials created by experimentally imposed ion gradients. Using this technique the quantitative relationship between membrane potential () and Na⁺-dependent sugar influx was determined for these cells at various Na⁺ and -methylglucoside (-MG) concentrations. The results show a high degree of dependence for the transport Michaelis constant but a maximum velocity for transport which is independent of . No transinhibition by intracellular sugar (40mm) can be detected. Sugar influx in the absence of Na⁺ is insensitive to 1.3mm phlorizin and independent of . The mechanistic implications of these results were evaluated using the quality of fit between calculated and experimentally observed kinetic constants for rate equations derived from several transport models. The analysis shows that for models in which translocation is the potential-dependent step the free carrier cannot be neutral. If it is anionic, the transporter must be functionally asymmetric. A model in which Na⁺ binding is the potential-dependent step (Na⁺ well concept) also provides an appropriate kinetic fit to the experimental data, and must be considered as a possible mechanistic basis for function of the system.     

Potential-sensitive response mechanism of diS-C3-(5) in biological membranes

Summary The potential-sensitive response mechanism of 3,3-dipropylthiodicarbocyanine iodide (diS-C₃-(5)) was examined based on our previous model of diS-C₃-(5) interaction with brush border membrane vesicles (BBMV) in the absence of a membrane potential. The model contained binding (6 msec), reorientation (30 msec), dimerization (<10 nsec), and translocation (1 sec) reaction steps (Cabrini & Verkman, 1986.J. Membrane Biol. 90:163–175). Transmembrane potentials () were induced in BBMV by K⁺ gradients and valinomycin. Steady-state diS-C₃-(5) fluorescence (excitation 622 nm, emission 670 nm) increased linearly with . The reorientation and translocation reaction steps were resolved by the stopped-flow technique as a biexponential decrease in fluorescence following mixture of diS-C₃-(5) with BBMV at varying . The fractional amplitude of the faster exponential increased from 0.36 to 0.73 with increasing (–17 to 87 mV); the time constant for the faster exponential (30–35 msec) was independent of . There were single exponential kinetics (0.5–1.5 sec) for diS-C₃-(5) fluorescence response to a rapid (<2 msec) change in in the absence of a diS-C₃-(5) concentration gradient. These results, and similar findings in placental brush border vesicles, red cell vesicles, and phosphatidylcholine vesicles, support a translocation mechanism for diS-C₃-(5) response, where induced membrane potentials drive diS-C₃-(5) redistribution between sites at the inner and outer membrane leaflets, with secondary effects on diS-C₃-(5) dimerization and solution/membrane partitioning. Fluorescence lifetime and dynamic depolarization measurements showed no significant change in diS-C₃-(5) rotational characteristics or in the polarity of the diS-C₃-(5) environment with changes in . Based on the experimental results, a mathematical model is developed to explain the quantitative changes in diS-C₃-(5) fluorescence which accompany changes in at arbitrary dye/lipid ratios.