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Roots of higher plants provide the shoot with nutrients and water. In exchange, they receive photosynthates, which serve both as energy source and building blocks for maintenance and growth. While studies in plant bioenergetics used to focus on photosynthesis, several more recent findings also aroused or renewed interest in energy conversion and allocation in roots. Root building costs were identified as a long-undervalued trait, which turned out to be highly relevant for stress tolerance and nutrient use efficiency. Reduced building costs per root length (e.g., by aerenchyma formation or by increasing the cell size) are beneficial for exploring the soil for nutrient-rich patches, especially in low-input agrosystems. Also, an apparent mismatch was frequently found between the root energy budget in the form of the ATP pool on the one side and the apparent costs on the other side, particularly the costs of membrane transport under stress conditions, e.g., the Na + detoxification costs resulting from Na + sequestration at the plasma membrane. Ion transport across the plasma membrane (and also endomembranes) is coupled to the proton motive force usually believed to be exclusively generated by H + ATPases. Recently, an alternative mechanism, the biochemical pH clamp, was identified which relies on H + formation and binding in the apoplast and the cytosol, respectively, driven by metabolism (so-called active buffering). On this background, several aspects of root bioenergetics are discussed. These are (1) root respiration in soil, with a critical view on calorimetric vs. gas exchange measurements; (2) processes of energy conversion in mitochondria with a special focus on the role of the alternative oxidases, which allow adjusting carbon flow through metabolic pathways to membrane transport processes; and (3) energy allocation, in particular to transport across the plasma membrane forming the interface to soil solution. A concluding remark is dedicated to modeling root bioenergetics for optimizing further breeding strategies. Apparent "energy spoilers" may bestow the plant with a yet unidentified advantage only unfolding their beneficial effect under certain environmental conditions., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wegner.)

Complex, multigenic biological traits are shaped by the emergent interaction of proteins being the main functional units at the molecular scale. Based on a phenomenological approach, algorithms for quantifying two different aspects of emergence were introduced (Wegner and Hao in Progr Biophys Mol Biol 161:54-61, 2021) describing: (i) pairwise reciprocal interactions of proteins mutually modifying their contribution to a complex trait (denoted as weak emergence), and (ii) formation of a new, complex trait by a set of n 'constitutive' proteins at concentrations exceeding individual threshold values (strong emergence). The latter algorithm is modified here to take account of protein redundancy with respect to a complex trait ('full redundancy'). Irreducibility is considered a necessary and sufficient criterion for strong biological emergence; if one constitutive protein is missing, or its concentration drops below the threshold the trait is lost. A definition based on 'unpredictability' is dismissed, because this criterion is irrelevant for the evolution of a complex trait, and apparent unpredictability may rather reflect our basic deficits in understanding unless we can provide an unequivocal proof for it. The phenomenological approach advocated here allows to identify hidden rules according to which strongly emergent traits may be organized. This is of high value for understanding the evolution of complex traits which seems to require the saltational advent of all constitutive proteins 'in one turn' to arrive at a functional trait providing for an improved fitness of the organism. Rather than being a purely random process, it may be guided by fundamental structural principles., (© 2021. Springer Nature B.V.)

Linking complex, multigenic traits to protein activity is an important challenge in current biology, including applications in the medical sciences, agriculture, or forestry. Two simple algorithms are presented here to establish that link. The first one describes synergistic interactions of n proteins in shaping a complex trait ('weak emergence') as opposed to a simply additive 'modular' contribution of these proteins. A coefficient κ is defined that allows to quantify the degree of emergent interaction. For cases of strong emergence a separate formalism is introduced, implying that a number of n proteins at concentrations exceeding individual threshold values are required to spontaneously form a new, complex trait. Threshold concentrations are allowed to vary, depending on the concentrations of the other constitutive proteins. The experimental effort is estimated to provide a corresponding database for applying both formalisms, including high-throughput phenomics, and manipulation of protein concentrations using the molecular toolbox. Future efforts will be directed to overcome current limitations of the models that ignore the dynamics of protein-trait relationships with time, and the importance of the spatial arrangement of proteins for emergent interaction., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

P-type H + ATPases mediate active H + efflux from plant cells. They generate a proton motive force across the plasma membrane, providing the free energy to drive the transport of other solutes, partly by coupling to H + influx. Wegner & Shabala (2020) recently suggested that passive H + influx can exceed pump-driven efflux due to 'active buffering', that is, cytosolic H + scavenging and apoplastic H + generation by metabolism ('biochemical pH clamp'). Charge balance is provided by K + efflux or anion influx. Here, this hypothesis is extended to net H + efflux: even though H + pumping is faster than backflow via symporters and antiporters, a progressive increase in the transmembrane pH gradient is avoided. Cytosolic H + release is associated with bicarbonate formation from CO 2 . Bicarbonate serves as substrate for the PEPCase, catalyzing the reaction from phosphoenolpyruvate to oxaloacetate, which is subsequently reduced to malate. Organic anions such as malate and citrate are released across the plasma membrane and are (partly) protonated in the apoplast, thus limiting pump-induced acidification. Moreover, a 'biophysical pH clamp' is introduced, that is, adjustment of apoplastic/cytosolic pH involving net H + fluxes across the plasma membrane, while the gradient between compartments is maintained. The clamps are not mutually exclusive but are likely to coexist., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)

Agriculture is expanding into regions that are affected by salinity. This review considers the energetic costs of salinity tolerance in crop plants and provides a framework for a quantitative assessment of costs. Different sources of energy, and modifications of root system architecture that would maximize water vs ion uptake are addressed. Energy requirements for transport of salt (NaCl) to leaf vacuoles for osmotic adjustment could be small if there are no substantial leaks back across plasma membrane and tonoplast in root and leaf. The coupling ratio of the H + -ATPase also is a critical component. One proposed leak, that of Na + influx across the plasma membrane through certain aquaporin channels, might be coupled to water flow, thus conserving energy. For the tonoplast, control of two types of cation channels is required for energy efficiency. Transporters controlling the Na + and Cl - concentrations in mitochondria and chloroplasts are largely unknown and could be a major energy cost. The complexity of the system will require a sophisticated modelling approach to identify critical transporters, apoplastic barriers and root structures. This modelling approach will inform experimentation and allow a quantitative assessment of the energy costs of NaCl tolerance to guide breeding and engineering of molecular components., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Solute uptake and release by plant cells are frequently energized by coupling to H + influx supported by the proton motive force (pmf). The pmf results from a stable pH difference between the apoplast and the cytosol, with bulk values ranging from 4.9 to 5.8 and from 7.1 to 7.5, respectively, in combination with a negative electrical membrane potential. The P-type H + ATPases pumping H + from the cytosol into the apoplast at the expense of ATP hydrolysis are generally viewed as the only pmf source, exclusively linking membrane transport to energy metabolism. However, recent evidence suggests that pump activity may be insufficient to energize transport, particularly under stress conditions. Indeed, cytosolic H + scavenging and apoplastic H + generation by metabolism (denoted as 'active' buffering in contrast to the readily exhausted 'passive' matrix buffering) also stabilize the pH gradient. In the cytosol, H + scavenging is mainly associated with malate decarboxylation catalyzed by malic enzyme, and via the GABA shunt of the tricarboxylic acid (TCA) cycle involving glutamate decarboxylation. In the apoplast, formation of bicarbonate from CO 2 , the end-product of respiration, generates H + at pH ≥ 6. Membrane potential is stabilized by K + release and/or by anion uptake via ion channels. Finally, thermodynamic aspects of active buffering are discussed., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Current concepts of growth hydraulics in higher plants are critically revisited, and it is concluded that they partly fail to interpret the experimental data adequately, particularly in the case of hydroponics-grown roots. Theoretical considerations indicate that the growth rate in roots is controlled by the extensibility of the cell wall, excluding water availability (i.e. hydraulic conductance) as a major constraint. This is supported by the findings that the growth rate does not scale with turgor, and that no radial nor axial water potential gradients have been observed in the root elongation zone. Nevertheless, a water potential deficit ranging from -0.2 to -0.6MPa has repeatedly been reported for growing cells that by far exceeds the shallow trans-membrane water potential difference required for the uptake of growth water. Unexpectedly, growth was also shown to depend on the hydraulic conductance (LP) of the plasma membrane of root cells, even though LP should generally be too large to have an impact on growth. For leaves, similar observations have been reported, but the interpretation of the data is less straightforward. Inconsistencies associated with the current model of growth hydraulics prompt the author to suggest a revised model that comprises, in addition to a passive mechanism of water transport across the plasma membrane of growing cells mediated by aquaporins ('leak') a secondary active water transport ('pump'), in analogy to a mechanism previously demonstrated for mammalian epithelia and postulated for xylem parenchyma cells in roots. Water is hypothesised to be secreted against a trans-membrane water potential difference by cotransport with solutes (salts, sugars, and/or amino acids), taking advantage of the free energy released by this transport step. The solute concentration gradient is supposed to be maintained by a subsequent retrieval of the solutes from the apoplast and back-transport at the expense of metabolic energy. Water secretion tends to reduce the turgor pressure and retards growth, but turgor and, in turn, growth can be upregulated very rapidly independent from any adjustment in the osmolyte deposition rate by increasing LP and/or reducing secondary active water transport, e.g. when the root is exposed to mild osmotic stress, as confirmed by experimental studies.

A series of recent publications has launched a debate on trans-membrane water secretion into root xylem vessels against a water potential gradient, energised by a cotransport with salts (e.g. KCl) that follow their chemical potential gradient. Cation-chloride-cotransporter -type transporters that function in this way in mammalian epithelia were detected in root stelar cells bordering on xylem vessels. Using literature data on barley (Hordeum vulgare L.) seedlings, one study confirmed that K+ and Cl- gradients across stelar cell membranes favour salt efflux. Moreover, the energetic costs of putative water secretion into the xylem (required for maintaining ionic gradients) would amount to just 0.12% of the energy captured by photosynthetic C assimilation if transpirational water flow relied exclusively on this mechanism. Here, a detailed thermodynamic analysis of water secretion into xylem vessels is undertaken, including an approach that exploits its analogy to a desalinisation process. Water backflow due to the passive hydraulic conductivity of stelar cell membranes is also considered. By comparing free energy consumption by putative water secretion with (i) the free energy pool provided by root respiration and (ii) stelar ATPase activity, the feasibility of this mechanism is confirmed but is shown to depend critically on the plant's energy status.

Membrane permeabilization by pulsed electric fields (electroporation), that is supposed to be caused by the formation of aqueous pores, is widely used in biomedicine and biotechnology. It is detected most precisely by measuring membrane conductance. When whole-cell patch-clamp experiments are used to screen a wide voltage range, poration becomes manifest by large currents elicited at extreme hyper-/depolarization. The slope conductance, G(slope), can be obtained from non-linear current-voltage relations by differentiation of the current-voltage curve. Alternatively, the chord conductance, G(chord), is defined as the slope of straight lines connecting each point on the current-voltage curve with the zero-current (reversal) potential on the voltage axis. Here, Boltzmann functions were fitted to plots of G(chord) versus voltage recorded on protoplasts from bright-yellow-2 tobacco cells. These plots are supposed to reflect transition from a non-porated to a porated membrane state. Consistently, G(chord) saturated at extremely negative and positive voltages at values well below those expected for a complete demolition of the membrane (half-maximum voltages: ~-332 mV and +294 mV, respectively). The slope factor allowed inferring the change in dipole moment associated with water intrusion into the bilayer. It was -6.19 10(-4) and 3.35 10(-4)C ∗ m ∗ mol(-1), respectively. Outside-out patches rendered similar results, but half-maximum voltages were shifted to more extreme voltages with respect to whole-cell experiments., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Treatment of biological material by pulsed electric fields is a versatile technique in biotechnology and biomedicine used, for example, in delivering DNA into cells (transfection), ablation of tumors, and food processing. Field exposure is associated with a membrane permeability increase usually ascribed to electroporation, i.e., formation of aqueous membrane pores. Knowledge of the underlying processes at the membrane level is predominantly built on theoretical considerations and molecular dynamics (MD) simulations. However, experimental data needed to monitor these processes with sufficient temporal resolution are scarce. The whole-cell patch-clamp technique was employed to investigate the effect of millisecond pulsed electric fields on DC-3F cells. Cellular membrane permeabilization was monitored by a conductance increase. For the first time, to our knowledge, it could be established experimentally that electroporation consists of two clearly separate processes: a rapid membrane poration (transient electroporation) that occurs while the membrane is depolarized or hyperpolarized to voltages beyond so-called threshold potentials (here, +201 mV and -231 mV, respectively) and is reversible within ∼100 ms after the pulse, and a long-term, or persistent, permeabilization covering the whole voltage range. The latter prevailed after the pulse for at least 40 min, the postpulse time span tested experimentally. With mildly depolarizing or hyperpolarizing pulses just above threshold potentials, the two processes could be separated, since persistent (but not transient) permeabilization required repetitive pulse exposure. Conductance increased stepwise and gradually with depolarizing and hyperpolarizing pulses, respectively. Persistent permeabilization could also be elicited by single depolarizing/hyperpolarizing pulses of very high field strength. Experimental measurements of propidium iodide uptake provided evidence of a real membrane phenomenon, rather than a mere patch-clamp artifact. In short, the response of DC-3F cells to strong pulsed electric fields was separated into a transient electroporation and a persistent permeabilization. The latter dominates postpulse membrane properties but to date has not been addressed by electroporation theory or MD simulations., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The thermodynamics of root pressure remains an enigma up to the present day. Water is transported radially into xylem vessels, under some conditions even when the xylem sap is more dilute than the ambient medium (soil solution). It is suggested here that water secretion across the plasma membrane of xylem parenchyma cells is driven by a co-transport of water and solutes as previously shown for mammalian epithelia (Zeuthen T. 2010. Water-transporting proteins. Journal of Membrane Biology 234, 57-73.). This process could drive volume flow 'energetically uphill', against the free energy gradient of water. According to the model, solutes released by xylem parenchyma cells are subsequently retrieved from the sap at the expense of metabolic energy to maintain the concentration gradient that drives the water secretion. Transporters of the CCC type known to mediate water secretion in mammalian cells have also been found in Arabidopsis and in rice. The mechanism proposed here for root pressure could also explain refilling of embolized vessels. Moreover, it could contribute to long-distance water transport in trees when the cohesion-tension mechanism of water ascent fails. This is discussed with respect to the old and the more recent literature on these subjects.

Cation selectivity of the cellular membrane of tobacco culture cells (cell line 'bright yellow-2') exposed to pulsed electric fields in the millisecond range was investigated. The whole cell configuration of the patch clamp technique was established on protoplasts prepared from these cells. Ion selectivity of the electroporated membrane was investigated by measuring the reversal potential of currents passing through field-induced pores. To this end the membrane was hyper- or depolarized for 10ms (prepulse); subsequently the voltage was driven to opposite polarity at a constant rate (+40 or -40mV/ms, respectively). The experiment was started by polarizing the membrane to moderately negative or positive voltages (prepulse potential ±150mV) that would not induce pore formation. Subsequently, an extended voltage range was scanned in the porated state of the membrane (prepulse potential ±600mV). IV curves in the porated and the non-porated state (obtained at the same prepulse polarity) were superimposed to determine the voltage at which both curves intersected ('Intersection potential'). Using a modified version of the Goldmann-Hodgkin-Katz equation relative permeabilities to Ca(2+) and various monovalent alkali and organic cations were calculated. Pores were found to be fairly cation selective, with a selectivity sequence determined to be Ca(2+)>Li(+)>Rb(+)≈K(+)≈Na(+)>TEA(+)≈TBA(+)>Cl(-). Relative permeability to monovalent cations was inversely related to the ionic diameter. By fitting a formalism suggested by Dwyer at al. (J. Gen. Physiol. 75 (1980), 469-492) the effective average diameter of field induced pores was estimated to be about 1.8nm. Implications of these results for biotechnology and electroporation theory are discussed., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The patch clamp technique in the whole cell configuration is potentially a powerful tool to investigate electroporation (electric-field-induced membrane permeabilization). Membrane polarization beyond certain threshold voltages leads to a steep conductance increase either indicating field-induced pore formation or being due to patch clamp artifacts (seal resistance breakdown). Protoplasts derived from tobacco culture cell lines (Bright Yellow-2, BY-2; Virginia bright Italian-0, VBI-0) were stained with the voltage-sensitive dye ANNINE-6. After establishing the whole cell patch clamp configuration 50-ms command voltage (Ucomm) steps ranging from -500 mV to +500 mV were applied while simultaneously exposing protoplasts to light at 475 nm wavelength. Pulse-induced currents and fluorescence intensity (known to be linearly related to the trans-membrane voltage, Um) were recorded. Plotting fluorescence intensity against Ucomm revealed saturation of the curve at values<-300 mV and >+300 mV and close correlation with theoretical Um values calculated on the basis of membrane pore formation. For BY-2 and VBI-0 protoplasts ANNINE-6 voltage sensitivity was calculated to be -0.0014 mV(-1) and -0.0012 mV(-1), respectively. Voltage ramp experiments revealed cation-selectivity of field-induced pores. Anions are conducted poorly independent of their size. In conclusion, the patch clamp technique is validated as a useful tool in electroporation research., (Copyright © 2013 Elsevier B.V. All rights reserved.)

By insertion into an individual xylem vessel at the root base, the multifunctional xylem probe allows the monitoring of the xylem pressure, the radial electrical gradients in the root (the so-called trans-root potential, TRP), as well as the activity of a particular ion such as K(+) in the xylem sap of intact, transpiring plants. The biophysical and physiological significance of these parameters with respect to salt stress is briefly explained, and the assembly of the probe, the setup used for these measurements, and the experimental procedure are outlined in detail.

Permeabilization of biological membranes by pulsed electric fields ("electroporation") is frequently used as a tool in biotechnology. However, the electrical properties of cellular membranes at supra-physiological voltages are still a topic of intensive research efforts. Here, the patch clamp technique in the whole cell and the outside out configuration was employed to monitor current-voltage relations of protoplasts derived from the tobacco culture cell line "Bright yellow-2". Cells were exposed to a sequence of voltage pulses including supra-physiological voltages. A transition from a low-conductance (~0.1 nS/pF) to a high-conductance state (~5 nS/pF) was observed when the membrane was either hyperpolarized or depolarized beyond threshold values of around -250 to -300 mV and +200 to +250 mV, respectively. Current-voltage curves obtained with ramp protocols revealed that the electro-permeabilized membrane was 5-10 times more permeable to K+ than to gluconate. The K+ channel blocker tetraethylammonium (25 mM) did not affect currents elicited by 10 ms-pulses, suggesting that the electro-permeabilization was not caused by a non-physiological activation of K+ channels. Supra-physiological voltage pulses even reduced "regular" K+ channel activity, probably due to an increase of cytosolic Ca2+ that is known to inhibit outward-rectifying K+ channels in Bright yellow-2 cells. Our data are consistent with a reversible formation of aqueous membrane pores at supra-physiological voltages., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Early events in NaCl-induced root ion and water transport were investigated in maize (Zea mays L) roots using a range of microelectrode and imaging techniques. Addition of 100 mm NaCl to the bath resulted in an exponential drop in root xylem pressure, rapid depolarization of trans-root potential and a transient drop in xylem K(+) activity (A(K+) ) within ∼1 min after stress onset. At this time, no detectable amounts of Na(+) were released into the xylem vessels. The observed drop in A(K+) was unexpected, given the fact that application of the physiologically relevant concentrations of Na(+) to isolated stele has caused rapid plasma membrane depolarization and a subsequent K(+) efflux from the stelar tissues. This controversy was explained by the difference in kinetics of NaCl-induced depolarization between cortical and stelar cells. As root cortical cells are first to be depolarized and lose K(+) to the environment, this is associated with some K(+) shift from the stelar symplast to the cortex, resulting in K(+) being transiently removed from the xylem. Once Na(+) is loaded into the xylem (between 1 and 5 min of root exposure to NaCl), stelar cells become more depolarized, and a gradual recovery in A(K+) occurs., (© 2011 Blackwell Publishing Ltd.)

Turgor pressure provides a sensitive indicator for irrigation scheduling. Leaf turgor pressure of Musa acuminate was measured by using the so-called leaf patch clamp pressure probe, i.e. by application of an external, magnetically generated and constantly retained clamp pressure to a leaf patch and determination of the attenuated output pressure P(p) that is highly correlated with the turgor pressure. Real-time recording of P(p) values was made using wireless telemetric transmitters, which send the data to a receiver base station where data are logged and transferred to a GPRS modem linked to an Internet server. Probes functioned over several months under field and laboratory conditions without damage to the leaf patch. Measurements showed that the magnetic-based probe could monitor very sensitively changes in turgor pressure induced by changes in microclimate (temperature, relative humidity, irradiation and wind) and irrigation. Irrigation effects could clearly be distinguished from environmental effects. Interestingly, oscillations in stomatal aperture, which occurred frequently below turgor pressures of 100 kPa towards noon at high transpiration or at high wind speed, were reflected in the P(p) values. The period of pressure oscillations was comparable with the period of oscillations in transpiration and photosynthesis. Multiple probe readings on individual leaves and/or on several leaves over the entire height of the plants further emphasised the great impact of this non-invasive turgor pressure sensor system for elucidating the dynamics of short- and long-distance water transport in higher plants.

Control of ion loading into the xylem has been repeatedly named as a crucial factor determining plant salt tolerance. In this study we further investigate this issue by applying a range of biophysical [the microelectrode ion flux measurement (MIFE) technique for non-invasive ion flux measurements, the patch clamp technique, membrane potential measurements] and physiological (xylem sap and tissue nutrient analysis, photosynthetic characteristics, stomatal conductance) techniques to barley varieties contrasting in their salt tolerance. We report that restricting Na(+) loading into the xylem is not essential for conferring salinity tolerance in barley, with tolerant varieties showing xylem Na(+) concentrations at least as high as those of sensitive ones. At the same time, tolerant genotypes are capable of maintaining higher xylem K(+)/Na(+) ratios and efficiently sequester the accumulated Na(+) in leaves. The former is achieved by more efficient loading of K(+) into the xylem. We argue that the observed increases in xylem K(+) and Na(+) concentrations in tolerant genotypes are required for efficient osmotic adjustment, needed to support leaf expansion growth. We also provide evidence that K(+)-permeable voltage-sensitive channels are involved in xylem loading and operate in a feedback manner to maintain a constant K(+)/Na(+) ratio in the xylem sap.

We have analyzed the cellular effects of nanosecond pulsed electrical fields on plant cells using fluorescently tagged marker lines in the tobacco cell line BY-2 and confocal laser scanning microscopy. We observe a disintegration of the cytoskeleton in the cell cortex, followed by contraction of actin filaments towards the nucleus, and disintegration of the nuclear envelope. These responses are accompanied by irreversible permeabilization of the plasma membrane manifest as uptake of Trypan Blue. By pretreatment with the actin-stabilizing drug phalloidin, the detachment of transvacuolar actin from the cell periphery can be suppressed, and this treatment can also suppress the irreversible perforation of the plasma membrane. We discuss these findings in terms of a model, where nanosecond pulsed electric fields trigger actin responses that are key events in the plant-specific form of programmed cell death.

An advanced non-invasive, field-suitable and inexpensive leaf patch clamp pressure probe for online-monitoring of the water relations of intact leaves is described. The probe measures the attenuated output patch clamp pressure, P(p), of a clamped leaf in response to an externally applied input pressure, P(clamp). P(clamp) is generated magnetically. P(p) is sensed by a pressure sensor integrated into the magnetic clamp. The magnitude of P(p) depends on the transfer function, T(f), of the leaf cells. T(f) consists of a turgor pressure-independent (related to the compression of the cuticle, cell walls and other structural elements) and a turgor pressure-dependent term. T(f) is dimensionless and assumes values between 0 and 1. Theory shows that T(f) is a power function of cell turgor pressure P(c). Concomitant P(p) and P(c) measurements on grapevines confirmed the relationship between T(f) and P(c). P(p) peaked if P(c) approached zero and assumed low values if P(c) reached maximum values. The novel probe was successfully tested on leaves of irrigated and non-irrigated grapevines under field conditions. Data show that slight changes in the microclimate and/or water supply (by irrigation or rain) are reflected very sensitively in P(p).

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.

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.

Nutrient acquisition in the mature root zone is under systemic control by the shoot and the root tip. In maize, exposure of the shoot to light induces short-term (within 1-2 min) effects on net K+ and H+ transport at the root surface. H+ efflux decreased (from -18 to -12 nmol m(-2) s(-1)) and K+ uptake (approximately 2 nmol m(-2) s(-1)) reverted to efflux (approximately -3 nmol m(-2) s(-1)). Xylem probing revealed that the trans-root (electrical) potential drop between xylem vessels and an external electrode responded within seconds to a stepwise increase in light intensity; xylem pressure started to decrease after a approximately 3 min delay, favouring electrical as opposed to hydraulic signalling. Cutting of maize and barley roots at the base reduced H+ efflux and stopped K+ influx in low-salt medium; xylem pressure rapidly increased to atmospheric levels. With 100 mm NaCl added to the bath, the pressure jump upon cutting was more dramatic, but fluxes remained unaffected, providing further evidence against hydraulic regulation of ion uptake. Following excision of the apical part of barley roots, influx changed to large efflux (-50 nmol m(-2) s(-1)). Kinetin (2-4 microM), a synthetic cytokinin, reversed this effect. Regulation of ion transport by root-tip-synthesized cytokinins is discussed.

The investigation of transport processes in roots has so far been hampered by a lack of adequate methods to study water and solute transport simultaneously in intact, transpiring plants. The role of xylem tension in regulating volume flow and nutrient transport could not be addressed properly. In order to overcome limitations of conventional, massive-invasive methods, a gravimetric technique was used to measure water uptake by maize roots while simultaneously recording xylem pressure and xylem K(+) activity in individual xylem vessels by means of a K(+)-selective xylem probe. This minimal-invasive approach allowed the calculation of the radial K(+)flux into the root xylem and the radial root hydraulic conductance on transpiring seedlings. By changing the light regime or the osmotic pressure of the external solution, radial water and K(+) flux could be varied in order to study the interaction between water and solute transport. A major finding was that both radial K(+) transport and hydraulic conductance strongly depended on radial volume flow, whereas xylem pressure had little (if any) effect on these parameters. Results are discussed with respect to relevant membrane transport processes and their regulation by volume flow.

Xylem sap under high tension is in a metastable state and tends to cavitate, frequently leading to an interruption of the continuous water columns. Mechanisms of cavitation repair are controversially discussed. Magnetic resonance (MR) imaging provides a noninvasive, high spatial and temporal resolution approach to monitor xylem cavitation, refilling, and functionality. Spin density maps of drought-stressed maize taproots were recorded to localize cavitation events and to visualize the refilling processes; c. 2 h after release of the nutrient solution from the homemade MR imaging cuvette that received the root, late metaxylem vessels started to cavitate randomly as identified by a loss of signal intensity. After c. 6 h plants were rewatered, leading to a repair of water columns in five out of eight roots. Sap ascent during refilling, monitored with multislice MR imaging sequences, varied between 0.5 mm min(-1) and 3.3 mm min(-1). Flow imaging of apparently refilled vessels was performed to test for functional repair. Occasionally, a collapse of xylem vessels under tension was observed; this collapse was reversible upon rewatering. Refilling was an all-or-none process only observed under low-light conditions. Absence of flow in some of the apparently refilled vessels indicates that functionality was not restored in these particular vessels, despite a recovery of the spin density signal.

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.

Potassium is an inevitable component of plant life, and potassium channels play a pivotal role in plant growth and development. The role of potassium and of K(+) channels in plant cell division and cell-cycle progression, however, has not been determined so far. K(+) channel blocker studies with synchronized tobacco BY-2 cells revealed that K(+) uptake is required for proper cell-cycle progression during the transition from G(1) to S phase. Electrophysiological studies (patch-clamp and voltage-clamp techniques) showed a cell-cycle dependency of K(+) channel activities and reduced driving force for K(+) uptake in dividing cells. Among the four Shaker-like K(+) channel genes expressed in BY-2 cells, NKT1 represents an inwardly rectifying K(+) channel that mediates K(+) uptake. NKT1 is transcriptionally induced during G(1) phase, while transcripts of the outward-rectifier NTORK1 dominate S phase. Elongating BY-2 cells appeared hyperpolarized (-101 +/- 11 mV), and had elevated osmotic pressure and approximately twice the turgor pressure when compared with depolarized (-64 +/- 8 mV) dividing cells. This indicates that cells have to gain a threshold K(+) level to re-enter the cell cycle. Based on these findings, turgor regulation through modulation of K(+) channel density in plant cell division and cell-cycle progression is discussed.

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.

Exposure of the giant marine alga Valonia utricularis to acute hypo-osmotic shocks induces a transient increase in turgor pressure and subsequent back-regulation. Separate recording of the electrical properties of tonoplast and plasmalemma together with turgor pressure was performed by using a vacuolar perfusion assembly. Hypo-osmotic turgor pressure regulation was inhibited by external addition of 300 microM of the membrane-permeable ion channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). In the presence of 100 microM NPPB, regulation could only be inhibited by simultaneous external addition of 200 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a membrane-impermeable inhibitor of Cl(-) transport. At concentrations of about 100 microM, NPPB seems to selectively inhibit Cl(-) transporters in the tonoplast and K(+) transporters in the plasmalemma, whereas 300 microM NPPB inhibits K(+) and Cl(-) transporters in both membranes. Evidence was achieved by measuring the tonoplast and plasmalemma conductances (G(t) and G(p)) in low-Cl(-) and K(+)-free artificial seawater. Inhibition of turgor pressure regulation by 300 microM NPPB was accompanied by about 85% reduction of G(t) and G(p). Vacuolar addition of sulfate, an inhibitor of tonoplast Cl(-) transporters, together with external addition of DIDS and Ba(2+) (an inhibitor of K(+) transporters) also strongly reduced G(p) and G(t) but did not affect hypo-osmotic turgor pressure regulation. These and many other findings suggest that KCl efflux partly occurs via electrically silent transport systems. Candidates are vacuolar entities that are disconnected from the huge and many-folded central vacuole or that become disconnected upon disproportionate swelling of originally interconnected vacuolar entities upon acute hypo-osmotic challenge.

The concept of encapsulated-cell therapy is very appealing, but in practice a great deal of technology and know-how is needed for the production of long-term functional transplants. Alginate is one of the most promising biomaterials for immunoisolation of allogeneic and xenogeneic cells and tissues (such as Langerhans islets). Although great advances in alginate-based cell encapsulation have been reported, several improvements need to be made before routine clinical applications can be considered. Among these is the production of purified alginates with consistently high transplantation-grade quality. This depends to a great extent on the purity of the input algal source as well as on the development of alginate extraction and purification processes that can be validated. A key engineering challenge in designing immunoisolating alginate-based microcapsules is that of maintaining unimpeded exchange of nutrients, oxygen and therapeutic factors (released by the encapsulated cells), while simultaneously avoiding swelling and subsequent rupture of the microcapsules. This requires the development of efficient, validated and well-documented technology for cross-linking alginates with divalent cations. Clinical applications also require validated technology for long-term cryopreservation of encapsulated cells to maintaining a product inventory in order to meet end-user demands. As shown here these demands could be met by the development of novel, validated technologies for production of transplantation-grade alginate and microcapsule engineering and storage. The advances in alginate-based therapy are demonstrated by transplantation of encapsulated rat and human islet grafts that functioned properly for about 1 year in diabetic mice.

In higher plants the pH of the xylem sap plays an important role in drought signaling, growth regulation, and plant nutrition. However, the interpretation of the data is very controversial. The main reason for this is that the xylem pH in intact plants was not directly accessible hitherto. We present here a novel, minimally-invasive probe based on the xylem pressure-potential probe (used for measuring directly xylem pressure and the electrical potential between root xylem sap and medium). Single-tipped, double-barreled capillaries were used, one barrel served as H(+)-selective electrode, whereas pressure and electrical potential were recorded by the other one. Upon insertion of the probe into the root xylem of maize (Zea mays) seedlings, pH values ranging between about 4.2 and 4.9 were monitored when the roots were immersed in standard nutrient solution. The pH did not respond to changes in light irradiation (up to 300 micromol m(-2) s(-1)), but increased upon exposure of the root to 5 or 20 mm bicarbonate in the bath solution. Changes in pH could also be recorded in transpiring plants when the root was cut below the insertion point of the probe and placed in media with different pH. The data support the hypothesis of Mengel ([1994] Plant Soil 165: 275-283) that upon external supply with bicarbonate Fe is immobilized in the leaf apoplast via changes in xylem pH.

The Cohesion Theory considers plant xylem as a 'vulnerable pipeline' isolated from the osmotically connected tissue cells, phloem and mycorrhizas living in symbiosis with plant roots. It is believed that water is pulled exclusively by transpiration-induced negative pressure gradients of several megapascals through continuous water columns from the roots to the foliage. Water under such negative pressures is extremely unstable, particularly given the hydrophobicity of the inner xylem walls and sap composition (lipids, proteins, mucopolysaccharides, etc.) that prevents the development of stable negative pressures larger than about -1 MPa. However, many plant physiologists still view the Cohesion Theory as the absolute and universal truth because clever wording from the proponents of this theory has concealed the recent breakdown of the Scholander pressure bomb (and other indirect methods) as qualified tools for measuring negative pressures in transpiring plants. Here we show that the arguments of the proponents of the Cohesion Theory are completely misleading. We further present an enormous bulk of evidence supporting the view that - depending on the species and ecophysiological context - many other forces, additional to low tensions, can be involved in water ascent and that water can be lifted by a series of watergates (like ships in staircase locks). Contents I. Introduction 576 II. Can water sustain negative pressures? 577 III. Negative xylem pressures of several megapascals: fact or mystery? 579 IV. The continuity of the xylem water columns: fact or hypothesis? 588 V. The 'Multi-Force' or 'Watergate' Theory 590 VI. Conclusions 604 Acknowledgements 605 References 605 Appendix 1 612 Appendix 2 613.

The pressure response of (plant) cells to osmotic challenges depends on the reflection coefficient, sigma, of osmotically active solutes; it is less than predicted by the van't Hoff equation if sigma < 1. In Valonia utricularis, sigma is significantly reduced by internal (and, to a lesser extent, by external) unstirred layers, protecting the cytoplasm against vacuolar osmotic fluctuations. As shown by scanning and transmission electron microscopy, diffusion-restricted spaces are formed by innumerable small vacuoles that are interconnected with each other and with the central vacuole. They are embedded in networks of cytoplasmic strands connecting and encircling the organelles. Unstirred layers are also created in the central vacuole by an extensive network of acid mucopolysaccharide filaments (visualized by alcian blue staining). Mucopolysaccharides apparently also affect steady-state turgor by reducing the water activity. When the effective vacuolar osmotic pressure was adjusted to that of the bath by perfusion with an artificial vacuolar sap (AVS), an "offset turgor pressure" of 17 +/- 5 kPa was recorded. Consistent with the ultrastructural data, sigma values less than unity were calculated from the pressure response upon vacuolar addition of KCl or sucrose by perfusion (sigma(iKCl) = 0.63 +/- 0.13; sigma(isuc) = 0.58 +/- 0.17). Dilution of AVS yielded slightly higher sigma(iKCl) values (0.73 +/- 0.35). External addition to the artificial sea water (ASW) indicated that sigma(e) > sigma(i) for these osmotica. However, even in this case, sigma(esuc) (0.86 +/- 0.09) and sigma(ePEG) (0.58 +/- 0.08) were significantly less than sigma(eNaCl) (0.94 +/- 0.05) and sigma(eKCl) (0.91 +/- 0.13), presumably due to unstirred layers within the 4 micro m thick cell wall. Consistent with the low sigma values, a partial replacement of NaCl by osmotically equivalent amounts of sucrose (ASW(suc)), PEG and dextran, respectively, as well as replacement of Cl(-) by the large anion MES(-) induced an 'anomalous' hyposmotic turgor pressure response followed by the usual backregulation of pressure. After a 2-day preincubation in ASW(suc), significantly lower sigma(e) values were obtained both hyperosmotically (sigma(eNaCl) = 0.78 +/- 0.14; sigma(esuc) = 0.72 +/- 0.15) and hyposmotically (sigma(eNaCl) = 0.70 +/- 0.17; sigma(esuc) = 0.63 +/- 0.09), probably due to long-term effects on membrane structure to be elucidated yet. The freshwater alga Chara corallina lacked these apparently closely related structural and biophysical features of Valonia.

The electrical properties of protoplasts of the turgor pressure-regulating giant marine alga Valonia utricularis were investigated by using the patch-clamp technique. In the whole-cell configuration, large inward currents were elicited by negative-going voltage pulses. The time-dependent component was predominantly carried by Cl-, as revealed by 'tail current' analysis. When experiments were performed on protoplasts directly after mechanical release from the 'mother cell', small outward currents were additionally observed at membrane voltages more positive than ECl-. These outward currents disappeared to a large extent after treatment of the protoplasts with a mixture of cell wall-degrading enzymes. Plots of the chord conductance versus the clamped membrane voltage revealed that enzymatic treatment affected the gating properties. By fitting Boltzmann distributions to the data, a midpoint potential of + 5 +/- 5 mV (n = 7) was obtained in symmetrical Cl- solutions for mechanically released protoplasts. In contrast, protoplasts treated additionally with enzymes exhibited a midpoint potential of -13 +/- 5 mV (n = 8). By varying the external and internal Cl- concentration, gating was also shown to depend on the Cl- gradient across the plasmalemma both in enzymatically treated and untreated protoplasts. Plotting of the midpoint potential against the Nernst potential of Cl- rendered a slope less than 1 (0.70 and 0.64, respectively) indicating that gating did not strictly depend on the electrochemical Cl- gradient. The voltage- and Cl--dependence as well as inhibition experiments with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) suggested that the Cl- conductance of the membrane is dominated by the Valonia Anion Channel 1 (VAC1) described by Heidecker, M., Wegner, L.H., Zimmermann, U. 1999: A patch-clamp study of ion channels in proto-plasts prepared from the marine alga Valonia utricularis. J. Membrane Biol. 172:235-247. The relevance of the findings for membrane potential control and turgor regulation in V. utricularis as well as the general implications of the data for electrophysiological work on protoplasts (that are usually obtained by enzymatic digestion of plant tissue) are discussed.

The ultrastructure of the several micrometers thick cytoplasmic layer of the giant marine alga Valonia utricularis displays characteristics which are apparently linked with the capability of this alga to regulate turgor pressure. Transmission and scanning electron microscopy of cells prefixed in different ways, including a protocol that allows prefixation of the alga in a turgescent state, revealed a highly dendritic network of cytoplasmic strands connecting and enveloping the chloroplasts and the nuclei. Innumerable vacuolar entities are embedded in the network, giving the cytoplasm a spongy appearance. Vacuolar perfusion of turgor-pressure-clamped cells with prefixation solution containing tannic acid presented evidence that these vacuolar entities together with the huge central vacuole form a large unstirred continuum. In contrast to the tonoplast, the plasmalemma followed smoothly the lining of the cell wall, even at the numerous cell wall ingrowths. Sucrose, but not polyethylene glycol 6000, induced chloroplast clustering. Acute hypoosmotic treatment (established by reduction of external NaCl or by replacement of part of the external NaCl by equivalent osmotic concentrations of sucrose or polyethylene glycol 6000) resulted in a local relocation of the chloroplasts and cytoplasm towards the central vacuole. This effect did not occur when the relatively low reflection coefficients of these two osmolytes were taken into account. The increase in spacing between the spongy cytoplasm and the plasmalemma by chloroplast relocation (viewed by confocal laser scanning microscopy) was associated with a speckled appearance of the affected surface area under the light microscope. As indicated by electron microscopy, hypoosmotically induced chloroplast relocation resulted from disproportionate swelling of the vacuolar entities located close to the plasmalemma. The cytoskeleton in the cytoplasm and the mucopolysaccharide network in the central vacuole apparently resisted swelling of these compartments. This finding has the important consequence that relevant hydrostatic pressure gradients can be built up throughout the entire multifolded vacuolar space. This gradient could represent the trigger for turgor pressure regulation which is manifested electrically first in the tonoplast.

In higher plants the xylem is the main pathway for anti-gravitational, long-distance transport of nutrients and water from the root through the shoot to the upper leaves. In the xylem conduit water is in a metastable state if tension larger than 0.1 MPa (i.e. negative pressure) is developed. While diurnal changes in negative pressure of individual xylem vessels can quite accurately be recorded by the minimal-invasive xylem pressure probe technique and water flow by non-invasive NMR techniques, the problem of continuous monitoring of solute flow remains a hitherto unresolved challenge. As shown here, integration of a K+ selective and a potential measuring microelectrode into the xylem pressure probe allowed on-line measurements of the K+ activity in individual xylem vessels of maize roots together with pressure and trans-root potential, the potential difference between the xylem and the external medium (i.e. the overall driving force of ions through the root tissue). When light irradiation was increased from 10 micro mol m(-2) s(-1) to 300 micro mol m(-2) s(-1) and negative pressure developed in the vessel, xylem K+ activity dropped from 3.6 +/- 2.6 mm to 0.9 +/- 0.7 mm (n = 16), whereas the trans-root potential depolarized from -2 +/- 11 mV to + 12 +/- 11 mV (n = 11), i.e. by + 14 +/- 7 mV. The effect of light on all three parameters was reversible. Exposure of the root to various K+ activities in the bath ranging from 0.1 to 43 mm revealed that the K+ activity of the xylem sap was shielded against short-term fluctuations in K+ supply to a large extent. In contrast, control experiments in which the root was cut 1 cm below the probe insertion point, allowing direct entry of external K+ into the xylem vessels, demonstrated that the xylem equilibrated rapidly with external K+. This was taken simultaneously as a proof for the correct reading of the probe.

We report here that NO(3)(-) in the xylem exerts positive feedback on its loading into the xylem through a change in the voltage dependence of the Quickly Activating Anion Conductance, X-QUAC. Properties of this conductance were investigated on xylem-parenchyma protoplasts prepared from roots of Hordeum vulgare by applying the patch-clamp technique. Chord conductances were minimal around -40 mV and increased with plasma membrane depolarisation as well as with hyperpolarisation. Two gates with opposite voltage dependences were postulated. When 30 mM Cl- in the bath was replaced by NO(3)(-), a shift in the midpoint potential of the depolarisation-activated gate by about -60 mV from 43 to -16 mV occurred (K(m) = 3.4 mM). No such effect was seen when chloride was replaced by malate. Addition of 10 mM NO(3)(-)to the pipette solution and reduction of [Cl-] from 124 to 4 mM (to simulate cytoplasmic concentrations) did not interfere with the voltage dependence of X-QUAC activation, nor was it affected by changes in external [K+]. If only the NO(3)(-) effect on gating was considered, an increase of the NO(3)(-) concentration in the xylem sap to 5 mM would result in an enhancement of NO(3)(-) efflux by about 30%. Although the driving force for NO(3)(-) efflux would be reduced simultaneously, NO(3)(-) efflux into the xylem through X-QUAC would be maintained with high NO(3)(-) concentrations in the xylem sap; a situation which occurs for instance during the night.

After Renner had shown convincingly in 1925 that the transpirational water loss generates tensions larger than 0.1 MPa (i.e. negative pressures) in the xylem of cut leafy twigs the Cohesion Theory proposed by Böhm, Askenasy, Dixon and Joly at the end of the 19th century was immediately accepted by plant physiologists. Introduction of the pressure chamber technique by Scholander et al. in 1965 enforced the general belief that tension is the only driving force for water lifting although substantial criticism regarding the technique and/or the Cohesion Theory was published by several authors. As typical for scientific disciplines, the advent of minimal- and non-invasive techniques in the last decade as well as the development of a new, reliable method for xylem sap sampling have challenged this view. Today, xylem pressure gradients, potentials, ion concentrations and volume flows as well as cell turgor pressure gradients can be monitored online in intact transpiring higher plants, and within a given physiological context by using the pressure probe technique and high-resolution NMR imaging techniques, respectively. Application of the pressure probe technique to transpiring plants has shown that negative absolute pressures (down to - 0.6 MPa) and pressure gradients can exist temporarily in the xylem conduit, but that the magnitude and (occasionally) direction of gradients contrasts frequently the belief that tension is the only driving force. This seems to be particularly the case for plants faced with problems of height, drought, freezing and salinity as well as with cavitation of the tensile water. Reviewing the current data base shows that other forces come into operation when exclusively tension fails to lift water against gravity due to environmental conditions. Possible candidates are longitudinal cellular and xylem osmotic pressure gradients, axial potential gradients in the vessels as well as gel- and gas bubble-supported interfacial gradients. The multiforce theory overcomes the problem of the Cohesion Theory that life on earth depends on water being in a highly metastable state.

The giant marine alga Valonia utricularis is a classical model system for studying the electrophysiology and water relations of plant cells by using microelectrode and pressure probe techniques. The recent finding that protoplasts can be prepared from the giant "mother cells" (Wang, J., Sukhorukov, V.L., Djuzenova, C.S., Zimmermann, U., Müller, T., Fuhr, G., 1997, Protoplasma 196:123-134) allowed the use of the patch-clamp technique to examine ion channel activity in the plasmalemma of this species. Outside-out and cell-attached experiments displayed three different types of voltage-gated Cl(-) channels (VAC1, VAC2, VAC3, Valonia Anion Channel 1,2,3), one voltage-gated K(+) channel (VKC1, Valonia K(+) Channel 1) as well as stretch-activated channels. In symmetrical 150 mm Cl(-) media, VAC1 was most frequently observed and had a single channel conductance of 36 +/- 7 pS (n = 4) in the outside-out and 33 +/- 5 pS (n = 10) in the cell-attached configuration. The reversal potential of the corresponding current-voltage curves was within 0 +/- 4 mV (n = 4, outside-out) and 9 +/- 7 mV (n = 10, cell-attached) close to the Nernst potential of Cl(-) and shifted towards more negative values when cell-attached experiments were performed in asymmetrical 50:150 mm Cl(-) media (bath/pipette; E(Cl(-)) -20 +/- 7 mV (n = 4); Nernst potential -28 mV). Consistent with a selectivity for Cl(-), VAC1 was inhibited by 100 micronM DIDS (4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid). VAC1 was activated by a hyperpolarization of the patch. Boltzmann fits of the channel activity under symmetrical 150 mm Cl(-) conditions yielded a midpoint potential of -12 +/- 5 mV (n = 4, outside-out) and -3 +/- 6 mV (n = 9, cell-attached) and corresponding apparent minimum gating charges of 15 +/- 3 (n = 4) and 18 +/- 5 (n = 9). The midpoint potential shifted to more negative values in the presence of a Cl(-) gradient. VAC2 was activated by voltages more negative than E(Cl(-)) and was always observed together with VAC1, but less frequently. It showed a "flickering" gating. The single channel conductance was 99 +/- 10 pS (n = 6). VAC3 was activated by membrane depolarization and frequently exhibited several subconductance states. The single channel conductance of the main conductance state was 36 +/- 5 pS (n = 5). VKC1 was also activated by positive clamped voltages. Up to three conductance states occurred whereby the main conductance state had a single channel conductance of 124 +/- 27 pS (n = 6). In the light of the above results it seems to be likely that VAC1 contributes mainly to the Cl(-) conductance of the plasmalemma of the turgescent "mother cells" and that this channel (as well as VAC2) can operate in the physiological membrane potential range. The physiological significance of VAC3 and VKC1 is unknown, but may be related (as the stretch-activated channels) to processes involved in turgor regulation.

The activation kinetics of outward currents in protoplasts from barley root xylem parenchyma was investigated using the patch-clamp technique. The K(+) outward rectifying conductance (KORC), providing the main pathway for K(+) transport to the xylem, could be described in terms of a Hodgkin-Huxley model with four independent gates. Gating of KORC depended on voltage and the external K(+) concentration. An increase in the external K(+) concentration resulted in a shift in the voltage dependence of gating. This could be explained by a K(+) dependence of the rate constant beta for channel closure, indicating binding of K(+) to a regulatory site exposed to the bath. Occasionally, KORC was observed to inactivate; this inactivation occurred and vanished spontaneously. In some of the whole cell and excised patch recordings, a stepwise increase in outward current was observed upon a depolarizing voltage pulse, indicating that several populations of 'sleepy' channels existed in the plasma membrane that activated with a certain lag time. It is discussed whether this observation can be explained by a putative subunit, which retards channel activation, or by a scheme of cooperative gating. A quantitative description of outward rectifying K(+) channels in xylem parenchyma cells is a major step forward towards a mathematical model of salt transport into the xylem.

Xylem parenchyma cells (XPCs) control the composition of the transpiration stream in plants and are thought to play a role in long-distance signaling as well. We addressed the regulation, selectivity, and dependence on the apoplastic ion concentrations of two types of outward rectifiers in the plasma membrane of XPCs, to assess the physiological role of these conductances. In whole-cell recordings, the membrane conductance at depolarization was under the control of cytosolic Ca2+: at physiological Ca2+ levels (150 nM) the K+ outward-rectifying conductance (KORC) predominated, whereas at elevated Ca2+ levels (5 [mu]M), only the nonselective outward-rectifying conductance (NORC) was active. No such regulatory effect of Ca2+ was observed in inside-out experiments. The voltage dependence of whole-cell KORC currents strongly depended on apoplastic K+ concentration: an increase in apoplastic K+ resulted in a positive shift of the current-voltage curve, roughly following the shift in Nernst potential of K+. KORC is impermeable to Na+, but does translocate Ca2+ in addition to K+. In contrast to KORC, NORC selected poorly among monovalent cations and anions, the relative permeability PC+/PA- being about 1.9. Gating of NORC was largely unaffected by the level of K+ in the bath. Under all ionic conditions tested, NORC tail currents or single-channel currents reversed close to 0 mV. Using an in vivo xylem-perfusion technique, tetraethylammonium (an inhibitor of KORC) was shown to block K+ transport to the shoot. These data support the hypothesis that release of K+ to the xylem sap is mediated by KORC. The molecular properties of these two conductances are discussed in the light of the distinct physiological role of XPCs.

Xylem parenchyma cells surround the xylem vessels and control the composition of the transpiration stream which flows through the vessels. In the plasma membrane of the xylem parenchyma cells, one inward rectifying channel (denoted KIRC) and two outward rectifying channels (denoted KORC and NORC) have been identified. In the present study it is shown that KIRC was activated by Gpp(NH)p, in contrast to the inward rectifier in guard cells. In the inside-out patch configuration, Gpp(NH)p elicited single channel KIRC activity as well and the conclusion is, therefore, that KIRC is G-protein regulated in a membrane-delimited fashion. NORC gating is affected by the calcium buffering capacity of the pipette solution as determined by the amount of EGTA. KORC conductance is shown to be strongly dependent upon the apoplastic K(+)-concentration. The role of the above-mentioned transporters and their regulation mechanisms are discussed in the light of root:shoot communication and long-distance signalling.

Xylem parenchyma cells are situated around the (apoplastic) xylem vessels and are involved in the control of the composition of the xylem sap by exporting and resorbing solutes. We investigated properties of the K+ inward rectifier in the plasma membrane of these cells by performing patch clamp experiments on protoplasts in the whole-cell configuration. Inward currents were sensitive to the K+ channel blocker TEA+ at a high concentration (20 mM). Barium, another "classical" K+ channel blocker, inhibited K+ currents with a Ki of about 1.3 mM. In contrast to guard cells, the cytosolic Ca2+ level proved to be ineffective in regulating the K+ conductance at hyperpolarization. External Ca2+ blocked currents weakly in a voltage-dependent manner. From instantaneous current-voltage curves, we identified a binding site in the channel pore with an electrical distance of about 0.2 to 0.5. Lanthanum ions reduced the inward current in a voltage-dependent manner and simultaneously displaced the voltage at which half of the channels are in the open state to more positive values. This finding was interpreted as resulting from a sum of two molecular effects, an interaction with the mouth of the channel that causes a reduction of current, and a binding to the voltage sensor, leading to a shielding of surface charges and, subsequently, a modulation of channel gating. A comparison between the K+ inward rectifier in xylem parenchyma cells, guard cells and KAT1 from Arabidopsis leads to the conclusion that these rectifiers form subtypes within one class of ion channels. The ineffectiveness of Ca2+ to control K+ influx in xylem parenchyma cells is interpreted in physiological terms.

To identify mechanisms for the simultaneous release of anions and cations into the xylem sap in roots, we investigated voltage-dependent ion conductances in the plasmalemma of xylem parenchyma cells. We applied the patch-clamp technique to protoplasts isolated from the xylem parenchyma by differential enzymic digestion of steles of barley roots (Hordeum vulgare L. cv Apex). In the whole-cell configuration, three types of cation-selective rectifiers could be identified: (a) one activated at membrane potentials above about -50 mV; (b) a second type of outward current appeared at membrane potentials above +20 to +40 mV; (c) below a membrane potential of approximately -110 mV, an inward rectifier could be distinguished. In addition, an anion-specific conductance manifested itself in single-channel activity in a voltage range extending from about -100 to +30 mV, with remarkably slow gating. In excised patches, K+ channels activated at hyperpolarization as well as at depolarization. We suggest that salt is released from the xylem parenchyma into the xylem apoplast by simultaneous flow of cations and anions through channels, following electrochemical gradients set up by the ion uptake processes in the cortex and, possibly, the release and reabsorption of ions on their way to the xylem.