New evidence for large negative xylem pressures and their measurement by the pressure chamber method

Pressure probe measurements have been interpreted as showing that xylem pressures below c. –0.4 MPa do not exist and that pressure chamber measurements of lower negative pressures are invalid. We present new evidence supporting the pressure chamber technique and the existence of xylem pressures well below –0.4 MPa. We deduced xylem pressures in water-stressed stem xylem from the following experiment: (1) loss of hydraulic conductivity in hydrated stem xylem (xylem pressure = atmospheric pressure) was induced by forcing compressed air into intact xylem conduits; (2) loss of hydraulic conductivity from cavitation and embolism in dehydrating stems was measured, and (3) the xylem pressure in dehydrated stems was deduced as being equal and opposite to the air pressure causing the same loss of hydraulic conductivity in hydrated stems. Pressures determined in this way are only valid if cavitation was caused by air entering the xylem conduits (air-seeding). Deduced xylem pressure showed a one-to-one correspondence with pressure chamber measurements for 12 species (woody angiosperms and gymnosperms); data extended to c. –10 MPa. The same correspondence was obtained under field conditions in Betula occidentalis Hook., where pressure differences between air- and water-filled conduits were induced by a combination of in situ xylem water pressure and applied positive air pressure. It is difficult to explain these results if xylem pressures were above –0.4 MPa, if the pressure chamber was inaccurate, and if cavitation occurred by some mechanism other than air-seeding. A probable reason why the pressure probe does not register large negative pressures is that, just as cavitation within the probe limits its calibration to pressures above c. –0.5 MPa, cavitation limits its measurement range in situ.     

Evidence for discontinuous water columns in the xylem conduit of tall birch trees

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) ¹H nuclear magnetic resonance imaging of slice pieces; (iii) infusion experiments (dye, ⁸⁶Rb⁺, D₂O) 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.     

植物体内水分长距离运输的生理生态学机制

植物体内长距离水分运输是植物生理生态学研究中的一个重要问题,长期为植物生理学家和生理生态学家所关注。木质部探针技术的问世,掀起了近年来植物生理学界最为激烈的一场争论。提出了已经有100多年,风行40年的内聚力-张力(Cohesion-Tension, C-T)学说受到质疑。随后维护派和质疑派围绕木质部探针技术、压力室技术(C-T理论的主要支撑实验技术)的可靠性展开辩论。进一步从物理学原理和各种实验上就C-T理论的3个支柱(木质部导管或管胞中巨大的张力、沿树高的压力梯度、连续水柱)进行争论。这场争论似暂告一段落,C-T理论没有被推翻,但仍留有问题期待以后的研究。     

Comparative measurements of the xylem pressure ofNicotiana plants by means of the pressure bomb and pressure probe

Determination of the pressure in the water-conducting vessels of intactNicotiana rustica L. plants showed that the pressure probe technique gave less-negative values than the Scholander-bomb method. Even though absolute values of the order of −0.1 MPa could be directly recorded in the xylem by means of the pressure probe, pressures between zero and atmospheric were also frequently found. The data obtained by the pressure probe for excised leaves showed that the Scholander bomb apparently did not read the actual tension in the xylem vessles ofNicotiana plants. The possibility that the pressure probe gave false readings was excluded by several experimental controls. In addition, cavitation and leaks either during the insertion of the microcapillary of the pressure probe, or else during the measurements were easily recognized when they occurred because of the sudden increase of the absolute xylem tension to that of water vapour or to atmospheric, respectively. Tension values of the same order could also be measured by means of the pressure probe in the xylem vessels of pieces of stem cut from leaves and roots under water and clamped at both ends. The magnitude of the absolute tension depended on the osmolarity of the bathing solution which was adjusted by addition of appropriate concentrations of polyethylene glycol. Partial and uniform pressurisation of plant tissues or organs, or of entire plants (by means of the Scholander bomb or of a hyperbaric chamber, respectively) and simultaneous recording of the xylem tension using the pressure probe showed that a 1∶1 response in xylem pressure only occurred under a few circumstances. A 1∶1 response required that the xylem vessels were in direct contact with an external water reservoir and/or that the tissue was (pre-)infiltrated with water. Corresponding pressure-probe measurements in isolated vascular bundles ofPlantago major L. orP. lanceolata L. plants attached to a Hepp-type osmometer indicated that the magnitude of the tension in the xylem vessels was determined by the external osmotic pressure of the reservoir. These and other experiments, as well as analysis of the data using classical thermodynamics, indicated that the turgor and the internal osmotic pressure of the accessory cells along the xylem vessels play an important role in the maintenance of a constant xylem tension. This conclusion is consistent with the cohesion theory. In agreement with the literature (P.E. Weatherley, 1976, Philos. Trans. R. Soc. London Ser. B23, 435–444; 1982, Encyclopedia of plant physiology, vol. 12B, 79-109), it was found that the tension in the xylem of intact plants under normal and elevated ambient pressure (as measured with the pressure probe) under quasi-stationary conditions was independent of the transpiration rate over a large range, indicating that the conductance of the flow path must be flow-dependent.     

Long-term xylem pressure measurements in the lianaTetrastigma voinierianum by means of the xylem pressure probe

Diurnal changes of xylem pressure in the lianaTetrastigma voinierianum have been measured under greenhouse conditions by means of the recently developed xylem pressure probe. During the early morning hours, tensions in the vessels developed more or less rapidly with time, depending on light intensity. On sunny days, absolute negative pressures down to about -0.4 MPa (atmospheric = 0.1 MPa) were recorded around noon in petiolar or stem xylem vessels, whereas on rainy or cloudy days the xylem pressure remained in the positive sub-atmospheric or slightly negative pressure range. Towards the evening the tension in the vessels always decreased, i.e. the xylem pressure shifted to about atmospheric, or even above-atmospheric, values during the night. Simultaneous xylem pressure recordings at heights of 1 and 5 m frequently yielded either no gradient in tension at all, or far less than expected from the Cohesion Theory. Occasionally, tension gradients were even opposite to those predicted by this theory. Stem-toleaves pressure gradients in accord with the Cohesion Theory were recorded only when tension had been developed during sunny days in the upper branches of the liana, because increases in tension were not immediately propagated to the xylem of the leaves at ground level, as would be expected from a strictly coupled hydraulic system. Parallel recordings of the xylem tension using the pressure chamber yielded rather variable values ranging from 0.1 to 1 MPa; diurnal pressure changes could not be detected at all. The data are discussed on the basis of the equation for the chemical activity of water. They strongly suggest that the xylem tension induced by transpiration is not the sole force for water ascent. Other forces, such as osmotic pressure or convectional and interfacial forces, which to a remarkable extent have already been postulated for decades, seem to be equally important.Abbreviation R.H. relative humidity The authors are very grateful to Professor D. Fürnkranz, Institut für Botanik der Universität Salzburg, for his interest and help with the greenhouse facility, to Walter Gigerl for expert technical assistance, to Heike Schneider and Notburga Gierlinger for the petiolestaining experiments. This work was supported by a grant of the Deutsche Forschungsgemeinschaft to U.Z. (NMR-Graduiertenkolleg Ha 1232/8-1).     

Diurnal changes in xylem pressure and mesophyll cell turgor pressure of the lianaTetrastigma voinierianum: The role of cell turgor in long-distance water transport

Summary Long-term xylem pressure measurements were performed on the lianaTetrastigma voinierianum (grown in a tropical greenhouse) between heights of 1 m and 9.5 m during the summer and autumn seasons with the xylem pressure probe. Simultaneously, the light intensity, the temperature, and the relative humidity were recorded at the measuring points. Parallel to the xylem pressure measurements, the diurnal changes in the cell turgor and the osmotic pressure of leaf cells at heights of 1 m and 5 m (partly also at a height of 9.5 m) were recorded. The results showed that tensions (and height-varying tension gradients) developed during the day time in the vessels mainly due to an increase in the local light intensity (at a maximum 0.4 MPa). The decrease of the local xylem pressure from positive, subatmospheric or slightly above-atmospheric values (established during the night) to negative values after daybreak was associated with an almost 1 1 decrease in the cell turgor pressure of the mesophyll cells (on average from about 0.4 to 0.5 MPa down to 0.08 MPa). Similarly, in the afternoon the increase of the xylem pressure towards more positive values correlated with an increase in the cell turgor pressure (ratio of about 1 1). The cell osmotic pressure remained nearly constant during the day and was about 0.75–0.85 MPa between 1 m and 9.5 m (within the limits of accuracy). These findings indicate that the turgor pressure primarily determines the corresponding pressure in the vessels (and vice versa) due to the tight hydraulic connection and thus due to the water equilibrium between both compartments. An increase in the transpiration rate (due to an increase in light intensity) results in very rapid establishment of a new equilibrium state by an equivalent decrease in the xylem and cell turgor pressure. From the xylem, cell turgor, and cell osmotic pressure data the osmotic pressure (or more accurately the water activity) of the xylem sap was calculated to be about 0.35–0.45 MPa; this value was apparently not subject to diurnal changes. Considering that the xylem pressure is determined by the turgor pressure (and vice versa), the xylem pressure of the liana could not drop to — in agreement with the experimental results — less than -0.4 MPa, because this pressure corresponds to zero turgor pressure.     

In vivo magnetic resonance imaging of xylem vessel contents in woody lianas

Previous reports suggest that in some plant species the refilling of embolized xylem vessels can occur while negative pressure exists in the xylem. The aim of this experiment was to use non‐destructive nuclear magnetic resonance imaging (MRI) to study the dynamics of xylem cavitation and embolism repair in‐vivo. Serial ¹H‐MRI was used to monitor the contents of xylem vessels in stems of two dicotyledonous (Actinidia deliciosa and Actinidia chinensis, kiwifruit) and one monocotyledonous (Ripogonum scandens, supplejack) species of woody liana. The configuration of the horizontal wide bore magnet and probe allowed the imaging of woody stems up to 20 mm in diameter. Tests using excised stems confirmed that the image resolution of 78 µm and digital image subtraction could be used to detect the emptying and refilling of individual vessels. Imaging was conducted on both intact plants and excised shoots connected to a water supply. In the case of Ripogonum the excised shoots were long enough to allow the distal end of the shoot, including all leaves, to be exposed to ambient conditions outside the building while the proximal end was inside the MRI magnet. In total, six stems were monitored for 240 h while the shoots were subjected to treatments that included light and dark periods, water stress followed by re‐watering, and the covering of all leaves to prevent transpiration. The sudden emptying of water‐filled vessels occurred frequently while xylem water potential was low (below ?0.5 MPa for Actinidia, ?1.0 MPa for Ripogonum), and less frequently after xylem water potential approached zero at the end of water‐stress treatments. No refilling of empty vessels was observed at any time in any of the species examined. It is concluded that embolism repair under negative pressure does not occur in the species examined here. Embolism repair may be more likely in species with narrower xylem vessels, but further experiments are required with other species before it can be concluded that repair during transpiration is a widespread phenomenon.     

The plant cell pressure probe

The pressure probe is a micro manometer for the simultaneous direct recording and manipulation of plant cell hydrostatic pressure. It is used to map in space and time the turgor pressures of individual cells within tissues and organs of intact plants. This is used to study the hydraulic architecture of tissues, tissue movement and the responses of tissues to water stress. The approach can be augmented by simultaneous measurement of individual cell osmotic pressure. This permits the hydraulic driving forces across selectively permeable membranes and walls to be assessed fully. By manipulating manually the pressure, cell wall elasticity and its properties can also be mapped. Under some conditions this can be extended to plastic behaviour.     

Canny's compensating pressure theory fails a test

Canny's compensating pressure theory for water transport (American Journal of Botany 85: 897–909) has evolved from the premise that cavitation pressures are only a few tenths of a megapascal negative (approximately −0.3 MPa). In contradiction, “vulnerability curves” indicate that xylem pressures can drop below −3 MPa in some species without causing a loss of hydraulic conductivity. Canny claims these curves do not measure the limits to negative pressure by cavitation, but rather the limits to the compensating tissue pressure that otherwise quickly refills cavitated conduits. Compensating pressure is derived from the turgor pressure of the living cells in the tissue. To test this claim, we compared vulnerability curves of Betula nigra stems given three treatments: (1) living control, (2) killed in a microwave oven, and (3) perfused with a −1.5 MPa (10% w/w) mannitol solution. According to Canny's theory, the microwaved and mannitol curves should show cavitation and loss of conductance beginning at approximately −0.3 MPa because in both cases, the turgor pressure would be eliminated or substantially reduced compared to controls. We also tested the refilling capability of nonstressed stems where compensating pressure would be in full operation and compared this with dead stems with no compensating pressure. According to Canny's interpretation of vulnerability curves, the living stems should refill within 5 min. Results failed to support the compensating tissue theory because (a) all vulnerability curves were identical, reaching a −1.5 MPa threshold before substantial loss of conductance occurred, and (b) killed or living stems had equally slow refilling rates showing no significant increase in conductivity after 30 min. In consequence, the cohesion theory remains the most parsimonious explanation of xylem sap ascent in plants.     

A non-invasive probe for online-monitoring of turgor pressure changes under field conditions

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.     

Simultaneous recording of xylem pressure and trans-root potential in roots of intact glycophytes using a novel xylem pressure probe technique

The radial electrical potential difference between the root xylem and the bathing solution, i.e. the so-called trans-root potential, was measured in intact maize and wheat plants using a xylem pressure probe into which an Ag/AgCl electrode was incorporated. Besides other advantages (e.g. detection and removal of tip clogging; determination of the radial root resistance), the novel probe allowed placement of the electrode precisely in a single xylem vessel as indicated by the reading of sub-atmospheric or negative pressure values upon penetration. The trans-root potentials were of the order of 0 to – 70 mV and + 40 to – 20 mV for 2- to 3-week-old maize and wheat plants, respectively. Osmotic experiments performed on maize demonstrated that addition of 100 mM mannitol to the solution resulted in a decrease of xylem pressure associated with a slow, but continuous depolarization. The depolarization was reversible upon removal of the mannitol. For wheat plants it could be shown that the oscillations of the xylem pressure described recently by Schneider et al. (1997, Plant, Cell and Environment 20, 221–229) were accompanied by (rectangular, saw-tooth and/or U-shaped) oscillations in the trans-root potential (but not by corresponding changes of the membrane potential of the cortical cells measured simultaneously with conventional microelectrodes). Increase of the light intensity (up to 550 μmol m^–2 s^–1) resulted in a drop of the xylem pressure in wheat, whereas the trans-root potential showed a biphasic response: first hyperpolarization (by about 10 mV) was observed, followed by depolarization (by up to about + 40 mV). Similar light-induced biphasic (but often less pronounced) changes in the trans-root potential were also recorded for maize plants. Most interestingly, the response of the trans-root potential was always faster (by about 1–3 min) than the response of the xylem pressure upon illumination, suggesting that changes in the transpiration rate are reflected very quickly in the electrical properties of the root tissue. The impact of this and other findings on long-distance transport of solutes and water as well as on long-distance signalling is discussed.     

An artificial osmotic cell: a model system for simulating osmotic processes and for studying phenomena of negative pressure in plants

Abstract An artificial osmotic cell has been constructed using reverse osmosis membranes. The cell consisted of a thin film of an osmotic solution (thickness: 100 to 200 μm) containing a non-permeating solute and was bounded between the membrane and the front plate of a pressure transducer which continuously recorded cell turgor. The membrane was supported by metal grids to withstand positive and negative pressures (P). At maximum, negative pressures of up to –0.7 MPa (absolute) could be created within the film on short-term and pressures of up to –0.3 MPa could be maintained without cavitation for several hours. As with living plant cells, the application of osmotic solutions of a non-permeating solute resulted in monophasic relaxations of turgor pressure from which the hydraulic conductivity of the membrane (Lp) and the elastic modulus of the cell (?) could be estimated. The application of solutions with permeating solutes resulted in biphasic pressure relaxation curves (as for living cells) from which the permeability (P_s) and reflection (σ_s) coefficients could be evaluated for the given membrane. Lp, P_s, and σ_s were independent of P and did not change upon transition from the positive to the negative range of pressure. It is concluded that the artificial cell could be used to simulate certain transport properties of living cells and to study phenomena of negative pressure as they occur in the xylem and, perhaps, also in living cells of higher plants.     

Xylem pressure response in maize roots subjected to osmotic stress: determination of radial reflection coefficients by use of the xylem pressure probe

The absolute pressure in conducting xylem vessels of roots of 2-week-old, slowly transpiring intact maize plants (bathed in nutrition medium) was determined to be +0·024 ± 0·044 MPa using the xylem pressure probe. When the roots were subjected to osmotic stress (NaCI, KCI or sucrose), the xylem pressure decreased immediately and became more negative. However, the response of xylem pressure to osmotic stress was considerably attenuated, indicating that the radial reflection coefficients, σ₁₃ of the maize root for these solutes were rather low (between 0·2 and 0·4 depending on the concentration of the osmoticum). The low values of a, may be caused (partly) by unstirred layer effects. In repeated osmoticum/nutrition regimes a complex pattern of changes in xylem pressure was observed which was apparently linked to the interplay between transpiration and (passive and/or active) solute loading of the xylem. These processes were not observed when the roots were subjected to osmotic stress after excision. In this case, a biphasic response was observed comparable to that found for excised roots using the root pressure probe.     

Guard cell pressure/aperture characteristics measured with the pressure probe

Pressure within guard cells in strips of intact epidermis of Tradescantia virginiana was controlled with a pressure probe apparatus after the guard cells had been filled with silicone oil. Pressure was increased and decreased incrementally between 0.0 and 4.1 MPa to cause inflation and deflation of the guard cells. At steady-state guard cell pressures, the width of the stomatal pore was recorded and plotted against pressure. The pressure required for near-maximum aperture was 4.1 MPa. Aperture as a function of pressure was sigmoidal.     

Relax and refill: xylem rehydration prior to hydraulic measurements favours embolism repair in stems and generates artificially low PLC values

Diurnal changes in percentage loss of hydraulic conductivity (PLC), with recorded values being higher at midday than on the following morning, have been interpreted as evidence for the occurrence of cycles of xylem conduits' embolism and repair. Recent reports have suggested that diurnal PLC changes might arise as a consequence of an experimental artefact, that is, air entry into xylem conduits upon cutting stems, even if under water, while under substantial tension generated by transpiration. Rehydration procedures prior to hydraulic measurements have been recommended to avoid this artefact. In the present study, we show that xylem rehydration prior to hydraulic measurements might favour xylem refilling and embolism repair, thus leading to PLC values erroneously lower than those actually experienced by transpiring plants. When xylem tension relaxation procedures were performed on stems where refilling mechanisms had been previously inhibited by mechanical (girdling) or chemical (orthovanadate) treatment, PLC values measured in stems cut under native tension were the same as those measured after sample rehydration/relaxation. Our data call for renewed attention to the procedures of sample collection in the field and transport to the laboratory, and suggest that girdling might be a recommendable treatment prior to sample collection for PLC measurements.     

Hydraulic propagation of pressure along immature and mature xylem vessels of roots of Zea mays measured by pressure-probe techniques

Turgor pressure was measured in cortical cells and in xylem elements of excised roots and roots of intact plants of Zea mays L. by means of a cell pressure probe. Turgor of living and hence not fully differentiated late metaxylem (range 0.6–0.8 MPa) was consistently higher than turgor of cortical cells (range 0.4–0.6 MPa) at positions between 40 and 180 mm behind the root tip. Closer to the tip, no turgor difference between the cortex and the stele was measured. The turgor difference indicated that late-metaxylem elements may function as nutrient-storage compartments within the stele. Excised roots were attached to the root pressure probe to precisely manipulate the xylem water potential. Root excision did not affect turgor of cortical cells for at least 8 h. Using the cell pressure probe, the propagation of a hydrostatic pressure change effected by the root pressure probe was recorded in mature and immature xylem elements at various positions along the root. Within seconds, the pressure change propagated along both early and late metaxylems. The half-times of the kinetics, however, were about five times smaller for the early metaxylem, indicating they are likely the major pathway of longitudinal water flow. The hydraulic signal dissipated from the source of the pressure application (cut end of the root) to the tip of the root, presumably because of radial water movement along the root axis. The results demonstrate that the water status of the growth zone and other positions apical to 20 mm is mainly uncoupled from changes of the xylem water potential in the rest of the plant.Abbreviations and Symbols CPP cell pressure probe - EMX early metaxylem - LMX Late metaxylem - P_c cell turgor - P_r root pressure - RPP root pressure probe - t_1/2,c half-time of water exchange across a single cell - t_1/2 half-time of water exchange across multiple cells We thank Antony Matista for his expert assistance in the construction and modification of instruments. The work was supported by grant DCB8802033 from the National Science Foundation and grant 91-37100-6671 from USDA, and by the award of a Feodor Lynen-Fellowship from the Alexander von Humboldt-Foundation (Germany) to J.F.     

Comparison of plant cell turgor pressure measurement by pressure probe and micromanipulation

The conventional method of measuring plant cell turgor pressure is the pressure probe but applying this method to single cells in suspension culture is technically difficult and requires puncture of the cell wall. Conversely, compression testing by micromanipulation is particularly suited to studies on single cells, and can be used to characterise cell wall mechanical properties, but has not been used to measure turgor pressure. In order to demonstrate that the micromanipulation method can do this, pressure measurements by both methods were compared on single suspension-cultured tomato (Lycopersicon esculentum vf36) cells and generally were in good agreement. This validates further the micromanipulation method and demonstrates its capability to measure turgor pressure during water loss. It also suggests that it might eventually be used to estimate plant cell hydraulic conductivity.