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The continuity of the xylem water columns was studied on 17- to 23-m tall birch trees (trunk diameter about 23 cm; first branching above 10 m) all year round. Fifty-one trees were felled, and 5-cm thick slices or 2-m long boles were taken at regular, relatively short intervals over the entire height of the trees. The filling status of the vessels was determined by (i) xylem sap extraction from trunk and branch pieces (using the gas bubble-based jet-discharge method and centrifugation) and from trunk boles (using gravity discharge); (ii) (1)H nuclear magnetic resonance imaging of slice pieces; (iii) infusion experiments (dye, (86)Rb(+), D(2)O) on intact trees and cut branches; and (iv) xylem pressure measurements. This broad array of techniques disclosed no evidence for continuous water-filled columns, as postulated by the Cohesion-Tension theory, for root to apex directed mass transport. Except in early spring (during the xylem refilling phase) and after extremely heavy rainfall during the vegetation period, cohesive/mobile water was found predominantly at intermediate heights of the trunks but not at the base or towards the top of the tree. Similar results were obtained for branches. Furthermore, upper branches generally contained more cohesive/mobile water than lower branches. The results suggest that water lifting occurs by short-distance (capillary, osmotic and/or transpiration-bound) tension gradients as well as by mobilisation of water in the parenchymatic tissues and the heartwood, and by moisture uptake through lenticels.

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Investigation of 67 gymnosperm and angiosperm species belonging to 25 orders shows that epistomatal mucilage plugs are a widespread phenomenon. Measurements of the leaf water status by using the leaf patch clamp pressure technique suggest that the mucilage plugs are involved in moisture uptake and buffering leaf cells against complete turgor pressure loss at low humidity.

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Seasonal variations in osmolality and components of xylem sap in tall birch trees were determined using several techniques. Xylem sap was extracted from branch and trunk sections of 58 trees using the very rapid gas bubble-based jet-discharge method. The 5-cm long wood pieces were taken at short intervals over the entire tree height. The data show that large biphasic osmolality gradients temporarily exist within the conducting xylem conduits during leaf emergence (up to 272 mosmol x kg(-1) at the apex). These gradients (arising mainly from glucose and fructose) were clearly held within the xylem conduit as demonstrated by (1)H NMR imaging of intact twigs. Refilling experiments with benzene, sucrose infusion, electron and light microscopy, as well as (1)H NMR chemical shift microimaging provided evidence that the xylem of birch represents a compartment confined by solute-reflecting barriers (radial: lipid linings/lipid bodies; axial: presumably air-filled spaces). These features allow transformation of osmolality gradients into osmotic pressure gradients. Refilling of the xylem occurs by a dual mechanism: from the base (by root pressure) and from the top (by hydrostatic pressure generated by xylem-bound osmotic pressure). The generation of osmotic pressure gradients was accompanied by bleeding. Bleeding could be observed at a height of up to 21 m. Bleeding rates measured at a given height decreased exponentially with time. Evidence is presented that the driving force for bleeding is the weight of the static water columns above the bleeding point. The pressure exerted by the water columns and the bleeding volume depend on the water-filling status of (communicating) vessels.

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A high-precision pressure probe is described which allows non-invasive online-monitoring of the water relations of intact leaves. Real-time recording of the leaf water status occurred by data transfer to an Internet server. The leaf patch clamp pressure probe measures the attenuated pressure, P(p), of a leaf patch in response to a constant clamp pressure, P(clamp). P(p) is sensed by a miniaturized silicone pressure sensor integrated into the device. The magnitude of P(p) is dictated by the transfer function of the leaf, T(f), which is a function of leaf patch volume and ultimately of cell turgor pressure, P(c), as shown theoretically. The power function T(f)=f(P(c)) theoretically derived was experimentally confirmed by concomitant P(p) and P(c) measurements on intact leaflets of the liana Tetrastigma voinierianum under greenhouse conditions. Simultaneous P(p) recordings on leaflets up to 10 m height above ground demonstrated that changes in T(f) induced by P(c) changes due to changes of microclimate and/or of the irrigation regime were sensitively reflected in corresponding changes of P(p). Analysis of the data show that transpirational water loss during the morning hours was associated with a transient rise in turgor pressure gradients within the leaflets. Subsequent recovery of turgescence during the afternoon was much faster than the preceding transpiration-induced water loss if the plants were well irrigated. Our data show the enormous potential of the leaf patch clamp pressure probe for leaf water studies including unravelling of the hydraulic communication between neighbouring leaves and over long distances within tall plants (trees).

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The water supply to leaves of 25 to 60 m tall trees (including high-salinity-tolerant ones) was studied. The filling status of the xylem vessels was determined by xylem sap extraction (using jet-discharge, gravity-discharge, and centrifugation) and by (1)H nuclear magnetic resonance imaging of wood pieces. Simultaneously, pressure bomb experiments were performed along the entire trunk of the trees up to a height of 57 m. Clear-cut evidence was found that the balancing pressure (P(b)) values of leafy twigs were dictated by the ambient relative humidity rather than by height. Refilling of xylem vessels of apical leaves (branches) obviously mainly occurred via moisture uptake from the atmosphere. These findings could be traced back to the hydration and rehydration of mucilage layers on the leaf surfaces and/or of epistomatal mucilage plugs. Xylem vessels also contained mucilage. Mucilage formation was apparently enforced by water stress. The observed mucilage-based foliar water uptake and humidity dependency of the P(b) values are at variance with the cohesion-tension theory and with the hypothesis that P(b) measurements yield information about the relationships between xylem pressure gradients and height.

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Video rasterstereography has been developed for optical back shape measurement and for biomechanical analysis of spinal and pelvic geometry. Analysis of one single measurement permits 3-dimensional reconstruction of the back surface and calculation of shape parameters including pelvis tilt and torsion. In addition, estimates of the lateral deviation of the spinal midline and of vertebral rotation are provided. Its extended analytic potential makes rasterstereography a very appropriate tool for functional examinations. The term "functional examinations" refers in this context to biomechanical analysis of functional movements of the spine and pelvis caused by quasi-continuous changes of posture, if these can be observed as changes in back shape. Two examples are given to illustrate the aim and performance of functional examinations. Shoe elevation is used to correct leg-length discrepancy and is therefore prescribed for prevention and correction of scoliosis produced by pelvic obliquity. In a previous study it was shown that simulating leg-length discrepancy by raising a foot causes the pelvis to perform a torsional movement about the transverse axis. In effect, this movement reduces to some extent the effect of shoe elevation; thus a larger elevation might give better results. 42 scoliotic patients underwent functional examination. Leg-length discrepancies were simulated in 7 steps, and the resulting back shape was analysed by rasterstereography. The measurements were corrected for pelvic torsion. This method provided satisfactory correspondence with radiographically recorded leg-length discrepancies, i.e. 0.7 mm +/- 11.2 mm. One specific advantage of this procedure is that it covers aspects relating to spinal lateral deviation and vertebral rotation. It is concluded, however, that the 7 measurements used are hardly sufficient for this application and that better results are therefore to be expected from extended series. Kyphosis and lordosis clearly depend on posture. This is confirmed in a functional examination where these angles are measured under voluntary changes of posture. In forward bending, the trunk straightens and kyphosis and lordosis angles decrease. The reverse case applies to backward bending. If this effect is taken into quantitative consideration, an improved accuracy of measurements is obtained by reference to a standardised, mathematically defined posture. The resulting rms-error of kyphosis/lordosis measurement is then reduced from 3.10 degrees/2.95 degrees to 1.65 degrees/1.40 degrees. These figures open up new applications in the follow-up of kyphotic and lordotic deformities.

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Activities of the tonoplast ATPase (V-ATPase EC 3.6.1.3) and PPase (V-PPase EC 3.6.1.1) provide the proton gradient driving the accumulation of various metabolites, organic and inorganic ions in the plant vacuole. We used anion exchange chromatography, liquid-phase isoelectric focusing (IEF), and continuous-elution native polyacrylamide gel electrophoresis (preparative PAGE) to enrich the V-PPase from solubilized tonoplast proteins from suspension cultured cells of Chenopodium rubrum L.The fractions were identified by their enzymatic activity, sensitivity towards the specific PPase inhibitor aminomethylenediphosphonate, apparent molecular weight, and immunological reactivity with an antibody raised against mung bean V-PPase. All these different methods used for the separation of solubilized tonoplast proteins revealed the existence of two physically separable V-PPase proteins exhibiting substrate specific enzymatic activity and 66 kDa apparent molecular weight after sodium dodecyl sulfate(SDS)-PAGE. The isoelectric points of the active V-PPase forms were 5.05 and 5.48 (V-ATPase 6.1). On the basis of the observation of high recoveries of enzymatic activity after different preparations we suggest that the V-PPase proteins separated may represent physiologically occurring forms of the enzyme which cannot be distinguished by SDS-PAGE and Western blot.

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The resurrection plant Myrothamnus flabellifolia has the ability to recover from repeated prolonged and extreme desiccation cycles. During the dry state the inner walls of the xylem vessels seemed to be covered, at least partly, by a lipid film as shown by Sudan III and Nile Red staining. The lipid film apparently functioned as an 'internal cuticle' which prevented the adjacent parenchyma ray cells from complete water loss. The hydrophobic nature of the inner xylem walls was supported by the finding that benzene ascended as rapidly as water in the xylem of dry Myrothamnus branches. On watering, numerous lipid bodies were found in the water-conducting vessels, presumably formed from the lipid film and/or from lipids excreted from the adjacent living cells into the vessels. The presence of lipid bodies within the vessels, as well as the hydrophobic properties of the inner xylem walls, could explain the finding that the xylem pressure of hydrated, well watered plants (measured both under laboratory and field conditions with the xylem pressure probe) never dropped below c. -0.3 MPa and that cavitation occurred frequently at low negative xylem pressure values (-0.05 to -0.15 MPa). The xylem pressure of M. flabellifolia responded rapidly and strongly to changes in relative humidity and temperature, but less obviously to changes in irradiance (which varied between 10 and c. 4000 µmol m m-2 s-1 ). The morphological position of the stomata in the leaves could explain the extremely weak and slow response of the xylem pressure of this resurrection plant to illumination changes. Stomata were most abundant in the furrows, and were thus protected from direct sunlight. Simultaneous measurements of the cell turgor pressure in the leaf epidermal cells (made by using the cell turgor pressure probe) revealed that the xylem and the cell turgor pressure dropped in a ratio of 1:0.7 on changes in the environmental parameters, indicating a quite close hydraulic connection and, thus, water equilibrium between the xylem and cellular compartments. An increase in irradiance of c. 700 µmol m-2 s-1 resulted in a turgor pressure decrease from 0.63 to 0.48 MPa. Correspondingly, the cell osmotic pressure increased from 1.03 to 1.22 MPa. From these values and by assuming water equilibrium, the osmotic pressure of the xylem sap was estimated to be 0.25-0.4 MPa. This value seems to be fairly high but may, however, be explained by the reduction of the water volume within the vessels due to the floating lipid bodies.

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The dielectric structure of mature pollen of the angiosperm Lilium longiflorum was studied by means of single-cell electrorotation. The use of a microstructured four-electrode chamber allowed the measurements to be performed over a wide range of medium conductivity from 3 to 500 mS m-1. The rotation spectra of hydrated pollen grains exhibited at least three well-resolved peaks in the kHz-MHz frequency range, which obviously arise due to the multilayered structure of pollen grains. The three-shell model can explain the complex rotational behavior of pollen grains in terms of conductivities, permittivities and thicknesses of the following compartments: the exine and intine of the pollen grain wall as well as the membrane and cytoplasm of the vegetative cell. However, the number of unknown parameters (more than 8) was too large to allow unambiguous values to be assigned to any of them. Therefore, to facilitate the evaluation of the pollen grain parameters, additional rotational measurements were made on isolated vegetative and generative cells. The rotation spectra of these cells could be fitted very accurately on the basis of the single-shell model by assuming a dispersion of the cytoplasm. The data on the membrane and cytoplasmic properties of isolated vegetative cells were then used for modeling the rotation spectra of pollen grains. This greatly facilitated the fitting of the theoretical model to the experimental data and allowed the dielectric properties of the major structural units to be determined. The dielectric characterization of pollen is of enormous interest for plant biotechnology, where pollen and isolated germ cells are successfully used for production of transgenic crop and drug plants of economic importance by means of electromanipulation techniques.

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The activity and the voltage dependence of the inorganic pyrophosphatase (PPase) was measured on intact vacuoles of Chenopodium rubrum cells using the patch-clamp technique. With K+ at the cytoplasmic side a negative current representing the forward mode of the pump was measured after addition of pyrophosphate (PPi). The pump was reversed and a positive current was detected after addition of orthophosphate (Pi) in the presence of K+ at the vacuolar side when a pH gradient across the tonoplast was applied. The PPase operates as a constant current source, because no voltage dependence was observed (-60 to 60 mV). The K+ dependence of the PPi-induced current was investigated by substitution of cytoplasmic K+ by other cations. The selectivity sequence was: K+ > or = Rb+ > NH4+ = Cs+ > Na+ > Li+ = choline+, and was independent of the membrane voltage and pHcyt. With Cs+ or Li+ in the bath and K+ inside the vacuole the PPi-induced current became voltage-dependent, and positive currents were observed even if the pump was geared to operate in the forward mode. We suggest a "tunneling' effect through a channel-like domain in the PPase molecule which, under defined electrochemical gradient conditions and in the presence of PPi, allows K+ ions to cross the energy barrier usually separating the cytoplasmic from the vacuolar face of the pump.

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Voltage- and Ca(2+)-dependence of the slow-activating SV-K+ channel in the vacuolar membrane of Chenopodium rubrum suspension cells has been analyzed using the patch clamp technique in the vacuole-attached, outside-out and whole-vacuolar configuration. Patch-pipette perfusion was applied to measure Ca2+ dependence of single channels in the attached-configuration. Using the PCLAMP-software (Axon Instruments), an algorithm was developed to extract reliable individual channel data from multi-channel activity records, including open probability, mean open and closed times, as well as time constants for open and closed distributions. The channel conductance of the major open state was about 83 pS (seal resistance > 8 G omega) at 30 mV (transmembrane voltage Vm, vacuole negative), and symmetrical 100 mM KCl. the channel exhibited a strong voltage- and a weak Ca(2+)-activation: increasing Vm from 40 to 100 mV is equivalent to a Ca2+ concentration change from 10(-7) to 10(-4) M. Mean open probabilities at Vm = 30 mV were 0.03 with 1 microM and 0.09 with 100 microM Ca2+. Mean open times were approx. 7 ms, and almost independent of both, voltage and Ca2+. Mean closed times, however, varied in a strongly voltage- and Ca(2+)-dependent manner, e.g., at Vm = 30 mV dropped from 205 to 67 ms, if Ca2+ was raised from 10(-6) to 10(-4) M. Open and closed distributions of events within bursts could be fitted by the sum of two exponentials with time constants between 0.3 and 11 ms.

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Single channel performance and deactivation currents have been analyzed in the presence of cation channel blockers to reveal pharmacological properties of the slow-activating (SV) cation-selective ion channel in the vacuolar membrane (tonoplast) isolated from suspension cells of Chenopodium rubrum L. At a holding potential of -100 mV, the SV channel showed half-maximal inhibition with 20 mM tetraethylammonium (TEA), 7 microM 9-amino-acridine, 6 microM (+)-tubocurarine, 300 nM quinacrine, and 35 microM quinine, respectively. The SV channel is also blocked by charybdotoxin (20 nM at -80 mV) but not by apamine. 9-Amino-acridine, (+)-tubocurarine and quinacrine act in a voltage-dependent fashion, binding to the open channel and to different sites along the transmembrane voltage profile according to Woodhull (J. Gen. Physiol. 61:687-708, 1973). No binding site could be specified for charybdotoxin, which binds to the closed channel, and for quinine. Except for quinine, all tested blockers were effective only if added to the cytoplasmic side of the tonoplast. A structural relationship between the SV channel and Maxi-K channels in animal systems is inferred.

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Using the patch-clamp technique, we studied the action of charybdotoxin which blocks Ca(2+)-activated large-conductance K+ channels in animal tissue on the slow-activating (SV), Ca(2+)-activated cation channel in the vacuolar membrane of suspension-cells of Chenopodium rubrum L. The toxin reversibly reduced the vacuolar current with EC50 approximately 20 nM suggesting structural similarities between ion channels in animal and plant membranes.

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The patch-clamp technique was applied to vacuoles isolated from a photoautotrophic suspension cell culture of Chenopodium rubrum L. and vacuolar clamp currents, which are predominantly carried by the previously identified Ca(2+)-dependent slow vacuolar (SV) ion channels, were recorded. These currents, which were activated by 1-s voltage pulses of -100 mV (vacuolar interior negative) in the presence of 100 µM Ca(2+) (cytosolic side), could be blocked completely and reversibly by the calmodulin antagonist W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide] and its chlorine-deficient analogue W-5; half-maximum inhibition was found at approx. 6 µM for W-7 and 70 µM for W-5. Inhibition was reversed by addition of 1 µg · ml(-1) calmodulin purified from Chenopodium cell suspensions; reversal by bovine brain calmodulin was scarcely appreciable. We conclude that cytosolic calmodulin mediates the Ca(2+) dependence of the SV-channel in the Chenopodium tonoplast.

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The effect of the acetylcholine antagonist and channel blocker (+)-tubocurarine on the calcium-dependent slow vacuolar (SV) cation channels in the tonoplast of suspension-cultured cells of Chenopodium rubrum L. was examined using the patch-clamp technique. In whole-vacuolar recordings the drug strongly suppressed the potassium conductance (EC50: 6 microM) and altered the kinetics of channel inactivation. In excised membrane patches (+)-tubocurarine evokes channel-'flickering' without affecting the single-channel conductance (approx. 80 pS).

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The potassium ion is an indispensible cytosolic component of living cells and a key osmolyte of plant cells, crossing the plasmalemma to drive physiological processes like cell growth and motor cell activity. K(+) transport across the plasmalemma may be passive through channels, driven by the electrochemical gradient, K(+) equilibrium potential (E(K) ) - membrane potential (V(m) ), or secondary active by coupling through a carrier to the inward driving force of H(+) or Na(+) . Known K(+) channels are permeable to monovalent cations, a permeability order being K(+) > Rb(+) > NH(4) (+) > Na(+) ≥ Li(+) > Cs(+) . The macroscopic K(+) currents across a cell or protoplast surface commonly show rectification, i.e. a V(m) -dependent conductance which in turn, may be controlled by the cytosolic activity of Ca(2+) , of K(+) , of H(+) , or by the K(+) driving force. Analysis by the patch clamp technique reveals that plant K(+) channels are similar to animal channels in their single channel conductance (4 to 100 pS), but different in that a given channel population slowly activates and may not inactivate at all. Single-channel kinetics reveal a broad range of open times (ms to s) and closed times (up to 100 s). Further progress in elucidating plant K(+) channels will critically depend on molecular cloning, and the availability of channel-specific (phyto)toxins.

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Substrate specifity of the proton-driven hexose cotransport carrier in the plasmalemma of photoautotrophic suspension cells of Chenopodium rubrum L. has been studies through the short-term perturbation of (14)C-labelled efflux of 3-O-methyl-D-glucose. Efflux, occurring exclusively via carrier-mediated exchange diffusion, is trans-stimulated by the substrate and trans-inhibited by the glucose-transport inhibitors phlorizin (K 1/2=7.9 mM) and its aglucon phloretin (K 1/2=84 µM); with both inhibitors, 3-O-methyl-D-glucose efflux may be blocked completely. Trans-stimulation of efflux (up to fourfold) by a variety of the D-enantiomers of neutral hexoses, including glucose (K 1/2=48 µM), 3-O-methyl-D-glucose (K 1/2=139 µM), and fructose (K 1/2=730 µM), but not by, for instance, D-allose, and L-sorbose, shows that carrier-substrate interaction critically involves the axial position at C-1 and C-3, respectively. We suggest that substrate binding by the Chenopodium hexose carrier involves both hydrophobic interaction with the pyran-ring and hydrogen-ion bonding at C-1 and C-3 of the D-glucose conformation.

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A microsomal vesicle fraction (GV) markedly enriched by the Golgi marker enzyme latent inosine diphosphatase (IDPase) has been isolated from photoautotrophic suspension-cell protoplasts ofChenopodium rubrum L. Addition of ATP creates a substantial pH gradient across the GV membrane as measured by accumulation of acridine orange. The GV showed a density of 1.14 g·cm(-3) by equilibrium density centrifugation on sucrose gradients. Coincidence of acridine-orange accumulation and IDPase activity was confirmed on Percoll gradients. Formation of the pH gradient half-saturates at 0.3 mM MgATP, peaks at pH 7, and is competitively inhibited by ADP (k i≤0.1 mM), but not by Pi; it is hardly inhibited by orthovanadate, quickly dissipated by monensink 2=18 nM), nigericin (k 1/2=25 nM), and sluggishly by N-ethylmaleimide (k 1/2≈35 µM). Inhibition by KNO3 (k 1/2≈6.7 mM) is incomplete (60%). Uridine 5'-diphosphate (UDP)-glucose, UDP-galactose, but not UDP-mannose and the pertinent sugars, dissipate the ATP-generated pH gradient (k 1/2≈10-20 mM UDP-glucose; optimum pH at 7.8). This UDP-glucose activity is accompanied by release of Pi, but not of glucose or sucrose. UDP-glucoseinduced Pi release from the GV saturates (k 1/2=1 mM UDP-glucose; optimum pH at 7) and is completely inhibited by the anion-channel blocker 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS;k 1/2=140 µM). The GV incorporates UDP-[U-(14)C]glucose into an acid-labile, alkaline-stable macromolecular compound; this process is like-wise inhibited by DIDS. We propose a model including, inter alia, a UDP-glucose/uridine-5'-monophosphate translocator and a phosphate-permeable anion channel to operate in Golgi vesicles ofChenopodium rubrum.

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The tonoplast of isolated vacuoles from photoautotrophic suspension cells of Chenopodium rubrum L. was studied by means of the patch-clamp technique. In a symmetrical K(+) concentration of 46 mM, similar to in vivo conditions, the tonoplast displayed a membrane potential near zero and a linear current-voltage relationship with a mean slope of 1.0 S/m(2). ATP at 2 mM hyperpolarized the tonoplast (vacuole positive) by 15-20 mV and, in a parallel experiment, acidified the vacuole (outside pH 7.0) to pH 5.0, as monitored by accumulation of acridine orange. Analysis of the voltage-clamp current indicates a 2-fold, ATP-dependent increase of the membrane capacitance, from 4 to 8 mF/m(2), and an ATP-independent, unidentified ion channel having a mean opening time of about 5 msec and a conductivity of 0.5-1.0 pS.

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From photoautotrophic cell suspension cultures of Chenopodium rubrum L. microsomal fractions were prepared by differential and isopycnic sucrose gradient centrifugation. ATP-generated proton-accumulation was studied on a microsomal fraction (MF) of proton-translocating vesicles with a density of 1.11 to 1.12 gem(-3). This activity did not overlap with mitochondrial or chloroplast markers. MF is presumably of tonoplast origin. The assay was based upon pH-dependent acridine orange (AO) absorption shifts at 490 nm, Δ(490), due to AO-accumulation and polymerization within the vesicles, and was calibrated using pK and K(D) for AO polymers given by Zanker (Z. physikal. Chemie 199, 225, 1952). Addition of 1 mM ATP to MF yields a transmembrane ΔpH of about 2 units within 10 min (kinetics with a single time constant, τ=400s) and collapses after addition of the protonophor CCCP (τ=22s). The proton pumping activity is insensitive to vanadate and highly specific for ATP compared with GTP, UTP, CTP, and ITP, and half-saturates at 0.38 to 0.46 mM ATP, indicated by both initial rate (1 min) and steady state value of Δ(490) (10 min). Proton pumping into and proton leakage from the vesicles was cast into a feedback-scheme showing that the pump is working at a constant rate, that is, independently of the size of the created ΔpH.