1. The xylem pressure probe: direct measurement of xylem pressure in intact plants
The xylem pressure probe is a variant of the cell turgor pressure probe developed by Räde and Zimmermann and applied initially to the determination of turgor pressure and other water relations parameters of giant algal cells from turgor pressure relaxation and clamp experiments (Zimmermann et al., 1969; Steudle & Zimmermann, 1971; Zimmermann & Steudle, 1974; Zimmermann, 1978, 1989; Wendler & Zimmermann, 1982, 1985; Ortega et al., 1992; Murphy & Smith, 1998; Tomos & Leigh, 1999). The electrical properties of the tonoplast and the plasmalemma can also be determined separately if the probe is combined with vacuolar microelectrodes and perfusion assemblies (Wang et al., 1997; Ryser et al., 1999; Heidecker et al., 2003a,b; Mimietz et al., 2003). Measurements of the water relations parameters of tissue cells of higher plants were first reported by Hüsken et al. (1978) using a sophisticated modification of the probe. However, this micro-pressure probe failed the practical tests and the present version (used by many laboratories for turgor pressure measurements as well as for sap sampling from single cells) is identical to the original version (see Tomos & Leigh 1999). The ‘new’ device of Steudle (see Fig. 2 in his review article of 2002a) is a re-invention of the pressure probe of Räde and Zimmermann and deserves, therefore, no further discussion. This also applies to the so-called root pressure probe (Steudle & Jeschke, 1983; Steudle, 1993) that was adopted from Zimmermann & Mehlan (1976) and is used for measuring bulk water relations parameters of excised roots. This approach does not yield information at the single-cell level and, due to the multi-phase root compartment, interpretation of data obtained by the root pressure probe is based on many unproved assumptions.
Figure 2. Schematic diagrams of the cell turgor pressure probe (a), xylem pressure probe (b), xylem pressure-potential probe (c) and ion-selective xylem pressure probe (d). Abbreviations: c = cell, = cell turgor (= Pc − Pam), Mc = microcapillary, Pt = pressure transducer, Mr = metal rod, Ms = micrometer screw, x = xylem vessel, Px = xylem pressure, TRP = trans-root potential, dbMc = double-barrelled microcapillary, ISM = ion selective matrix, aK+ = K+ activity in the xylem sap, El1 = Ag/AgCl electrode for TRP recording and El2 = electrode for aK+ or pH recordings. For more details, see text.
Download figure to PowerPoint
The cell turgor pressure probe senses the turgor pressure by a pressure transducer mounted in a 50-µl perspex chamber and sealed to a glass microcapillary that is inserted into the cell (Fig. 2a). The entire probe system is filled with low-viscosity oil. Upon probing, the turgor pressure pushes cell sap into the very tip of the microcapillary. When the position of the oil/cell sap boundary is clamped close to the cell surface (by displacement of a metal rod introduced into the probe chamber) the pressure within the probe equals the turgor pressure of the cell. In pressure relaxation experiments turgor pressure changes are induced by osmotic challenges or transpirational changes while keeping the position of the oil/cell sap boundary constant. Changes in cell volume and turgor pressure, respectively, are brought about by deliberately changing the position of the oil/cell sap boundary (for details, see Zimmermann, 1989).
In the case of the xylem pressure probe (Fig. 2b), it must be emphasised that the components of the probe need to be carefully manufactured and thoroughly cleaned before use to promote complete wetting. Similarly, special care must be taken to avoid gas nucleation at the interfaces between the different components of the probe. For the same reason the oil must be replaced by de-ionised and degassed water. If these crucial factors are not considered, cavitation can easily be induced at the metal rod and at the perspex surface (see e.g. Wei et al., 1999b). For measuring xylem pressure a water-wettable pressure transducer is required. Generation of negative pressures down to −1.4 MPa showed that the water-wettable transducers operate linearly (Balling & Zimmermann, 1990; Zimmermann et al., 1994a). The ability of the xylem pressure probe to measure −1 MPa has been demonstrated by using a Hepp-type osmometer (see section II.1) and other model systems (Thürmer et al., 1999). Further effort to test whether the xylem pressure probe can measure even more negative pressures as demanded by Steudle (2003) is obviously wasting time because stable negative xylem pressures being significantly below −0.6 MPa have not been found for higher plants. Wei et al. (2001) claimed to have measured stable xylem pressures down to −1 MPa, but only an absolute pressure value of −0.62 MPa was documented (Wei et al., 1999a,b).
For probing a vessel in an intact plant the microcapillary is inserted slowly into the tissue, usually at an angle of about 20° to the transverse plane. If the vessels are filled with water (see e.g. the cross-sectional 1H NMR image of the water distribution in the shoot of tobacco in Fig. 3a) impalement is always detected by a rapid change of the pressure readings from above-atmospheric values to sub-atmospheric, positive or negative values (Fig. 3b). The advance of the microcapillary is then immediately stopped. Pressure equilibrium between the vessel and the probe is established within a few seconds. It is evident from Fig. 3b that the vessels of this tobacco plant were under a moderate negative pressure of −0.1 MPa (light irradiance 6 µmol m−2 s−1). Subsequent pressure recordings are usually stable for several hours (part of a pressure trace is shown in Fig. 3b), even though weak oscillations can occur at low light irradiance as exemplified for tomato in Fig. 4a. Volume (pressure) pulses injected by displacement of the metal rod in the probe dissipate very quickly indicating that the punctured vessels are connected to other conducting vessels (Balling & Zimmermann, 1990; Benkert et al., 1991). In herbaceous plants, such as tomato (Fig. 4a), an increase in the light irradiation to more than about 50–100 µmol m−2 s−1 commonly results in a marked decrease in xylem pressure to more negative values (around −0.3 to −0.4 MPa) which is frequently followed by a back-regulation to a slightly less negative value (see Fig. 4a). This typical ‘overshoot’ reaction is apparently linked with changes in stomatal conductance (Farquhar & Cowan, 1974; Raschke, 1975; Schneider et al., 2004). At very high light irradiations strong oscillations in transpiration can occur that are reflected somewhat delayed in xylem pressure (Wegner & Zimmermann, 1998; Schneider et al., 2000a; Zimmermann et al., 2002a; see also Fig. 14 further below). Xylem pressure changes can also be induced by changes in the ambient relative humidity. Decrease in relative humidity results in a drop of xylem pressure towards more negative values which can be reversed by increasing the relative humidity again (Zimmermann et al., 2002a; Schneider et al., 2004).
Figure 3. Typical T1-weighted 1H NMR spin echo images of the water distribution and xylem pressure recordings (given in absolute values) in the shoot of well-watered (a, b) and drought-stressed (c, d) about 2-month-old Nicotiana tabacum plants. The 1H NMR images (repetition time 0.2 s, echo time 9.8 ms, slice thickness 1 mm, spatial resolution = 20 × 20 µm2) demonstrate that most xylem vessels (‘x’) of the well-watered plant were filled with water (a; high signal intensity), while more than 80% of the vessels were embolised after 1-wk drought (c; no signal intensity). Correspondingly, repeated xylem probing showed that most vessels of the well-watered plant were under negative absolute pressure (b; c. −0.1 MPa), whereas in the drought-subjected plant only slightly sub-atmospheric pressure values were recorded indicating that most vessels were embolised. Only occasionally, stable xylem pressures down to −0.62 MPa could be recorded for some time before cavitation ended the experiment (horizontal arrow in d). Note that drought resulted in shrinkage of the shoot cross-sectional area and in the formation of extended intercellular cavities in the pith (‘pi’) evidencing considerable water loss from the tissue cells (c). Dashed arrows in (b) and (d) indicate vessel impalement. (d) reproduced from Zimmermann et al. (1994a), with kind permission of Blackwell Science, Oxford, UK.
Download figure to PowerPoint
Figure 4. Typical xylem pressure recordings on shoots of a 2-month-old Lycopersicon esculentum plant (a) and on roots of 2-wk-old hydroculture Zea mays seedlings (b, c). Vessel impalements are indicated by downward-directed dashed arrows. (a) Response of the xylem pressure when the plant was subjected to repeated short-term light regimes (6 and 150 µmol m−2 s−1 light irradiation; arrows). (b, c) Xylem pressure recordings by two pressure probes (PA and PB) inserted consecutively into the same vessel (b) and in two adjacent vessels (c). Measurements were performed at a light irradiation of 700 µmol m−2 s−1. In (c) the xylem pressure was further lowered before insertion of probe PB by addition of 25 mm NaCl to the nutrient medium (asterisk). As indicated in the insets, insertion of the second probe PB resulted in a pressure spike in probe PA when the same vessel was pierced (b). In the case of two adjacent punctured vessels (c) a pressure spike could only be recorded by PA upon removal of probe PB (upward-directed dashed arrow), but not upon insertion or reinsertion several micrometers away from the first impalement site. Note that both probes read always the same xylem pressure value independent of the various manipulations. For further details, see Schneider et al. (1997a). (b) and (c) redrawn after Schneider et al. (1997a), Oxford University Press, Oxford, UK.
Download figure to PowerPoint
Figure 14. Oscillations in the transpiration rates of Triticum aestivum hydroculture plants (Tr; dashed trace in a) occurring at a light irradiation > 300 µmol m−2 s−1 resulted in corresponding, somewhat delayed oscillations of root xylem pressure (Px; solid traces in a and c), of the turgor pressure of root cortical cells (; b) and of the trans-root potential (TRP; dash-dotted trace in c). Measurements were performed by using a conventional steady-state porometer, the xylem pressure probe, cell turgor pressure probe and the xylem pressure-potential probe, respectively. For more details, see text and Schneider et al. (1997b), Wegner & Zimmermann (1998), Zimmermann et al. (2002a) and Zimmermann (2003). Taken from Zimmermann et al. (2002a), with kind permission of Kluwer Academic Publishers, Dordrecht, The Netherlands.
Download figure to PowerPoint
When a xylem vessel is targeted in an inappropriate way, cavitation can occur upon piercing. Frequently, there is a subsequent leak. Probe-related cavitation can also occur during xylem pressure measurements. Leaks can easily be detected and identified by a pressure increase to atmospheric while cavitation leads to an instantaneous shift of the pressure to +2 kPa (Fig. 3d and also Figs 12a,c further below). Cavitations within the xylem can clearly be distinguished from cavitations within the probe. In the latter case no pressure is built up in the probe when a volume pulse is applied and gas bubbles can be seen in the probe.
Doubts about probe function have also been allayed by numerous other experiments. When the probe was placed into a vessel of an excised shoot segment of tobacco that was sealed at both ends and bathed in PEG 6000 solutions of increasing concentrations, xylem pressures down to about −0.4 MPa could be established (Balling & Zimmermann, 1990). Insertion of the probe into the xylem of a cut leaf sealed to the lower end of the glass capillary of a Hepp-type osmometer (Balling et al., 1988; Balling & Zimmermann, 1990) yielded negative pressure values that corresponded to the osmotic pressure in the PEG 6000 solution reservoir (up to 0.3 MPa; Balling & Zimmermann, 1990). Consecutive insertion of two probes into the root xylem of intact maize hydroculture plants also excluded artefacts arising from probing (Schneider et al., 1997a). Fig. 4b shows an experiment performed in nutrient solution at a light irradiation of about 700 µmol m−2 s−1. Placement of two probes in the same vessel was proved by filling the capillary tip of one probe with fluorescein solution, the other one with alcian blue. The inset in Fig. 4b shows that the introduction of the second probe (PB) induced a small pressure spike in the vessel that was detected by the first probe (PA). After equilibration both probes read the same value. In Fig. 4c a negative pressure of about −0.3 MPa was established by NaCl addition to the nutrient solution before insertion of probe PB. This time probe PB punctured an adjacent vessel. In this case the reading of probe PA remained unaffected, even when PB was gently removed after about 4 min and then reinserted several micrometers away from the first insertion site. However, intermediate removal of PB led to a small pressure spike registered by PA (see inset in Fig. 4c). Upon replacement of the saline solution by nutrient medium both probes responded identically (data not shown).
Probing of water-conducting vessels is further supported by insertion of probes filled partly with degassed (low- and high- molecular weight) dye solutions. Owing to the tension in the xylem, the dye solution is sucked from the microcapillary into the vessel upon impalement (Balling & Zimmermann, 1990; Benkert et al., 1991; Zimmermann et al., 1993a,b, 1994a,b, 2002a). Inspection of the cross-sections of roots, shoots and leaf petioles of various herbaceous and woody plants under the light microscope always revealed that the dye was confined to a single vessel at the insertion point (Fig. 5). In the case of fluorescein (Fig. 5a), the dye was transported with the transpiration stream as revealed by cross-sections made above the insertion point at regular intervals (Benkert et al., 1991). The flow rates were in the same order of magnitude as determined by the heat pulse technique (Huber, 1932) and by NMR flow imaging (Kuchenbrod et al., 1996, 1998).
Figure 5. Light microscopy cross-sections of probed xylem vessels of a shoot of Solanum tuberosum (a), a seedling stem of Rhizophora mangle (b), a twig of Fagus sylvatica (c), a leaf petiole of Anacardium excelsum (d) and a shoot of Nicotiana tabacum (e). Before insertion the tip of the probe microcapillary was loaded with Na-fluorescein (a), alcian blue (b) or India ink (c–e) solution. Note that the dyes were sucked into only one of the vessels. Bars: 50 µm. (c) and (d) were taken from Zimmermann et al. (1993a,b), with kind permission of BIOS Scientific Publishers, Oxford, UK, and The Royal Society, London, UK, respectively.
Download figure to PowerPoint
Zimmermann and co-workers (1991) have also shown that pneumatic pressure applied to the roots of tobacco immersed in water was transmitted to the xylem. At low bomb pressures the xylem pressure probe (inserted into a vessel outside the bomb) responded immediately and almost linearly, whereas at higher overpressures the response curve was more sigmoid. Additionally, when the roots were cut, 1 : 1 responses of the xylem pressure probe over a large pressure range were recorded upon pressurisation (Balling & Zimmermann, 1990).
Xylem pressure changes induced by mechanical destruction of the tissue cells due to the impalement process were excluded by using a probe incorporating an Ag/AgCl microelectrode and filled with degassed electrolyte solution (Fig. 2c; Wegner & Zimmermann, 1998; Wegner et al., 1999; Schneider et al., 2000a; Zimmermann et al., 2002a). Insertion of this xylem pressure-potential probe into the root xylem of hydroculture plants yielded resistance values that were on average even higher than those published in the literature (e.g. Anderson & Higinbotham, 1976). Location of the probe in a xylem vessel was indicated by changes in xylem pressure and in the trans-root potential upon illumination (Fig. 6a). Except for probe sites at the root base and along the root of very young wheat plants the trans-root potential responded usually about 1–3 min faster than the xylem pressure. Very recently, Wegner & Zimmermann (2002) additionally integrated a K+-selective electrode into the xylem pressure-potential probe (Fig. 2d). On-line measurements of the K+ activity in individual vessels of hydroculture plants probed at the root base showed (Fig. 6a) that an increase in light irradiation resulted in a concomitant drop of the xylem pressure and xylem sap K+ activity accompanied by a depolarisation of the trans-root potential. The effects of light on all three parameters were reversible. The K+ activity values at low irradiation agreed well with those deduced from xylem exudates of cut leaf veins and of xylem sap collected with the conventional xylem pressure probe (Lohaus et al., 2000). Correct reading and placement of the probe in a vessel without injury was also demonstrated by immersing cut roots in media containing elevated K+ activities. Furthermore, use of xylem pressure-potential probes, in which a pH-sensitive electrode had been integrated (see Fig. 2d; Wegner et al., 2004), also yielded pH-values (Fig. 6b) which were in the order of magnitude expected for the xylem (Davis & Higinbotham, 1969; Miller, 1985). In contrast to K+, the pH remained constant upon short-term illumination. However, it increased when the root was exposed to bicarbonate (data not shown), providing evidence for the hypothesis of Mengel et al. (1994) that the reduced availability of Fe in plants growing on calcareous soil (‘lime chlorosis’) is due to an alkalinisation of the xylem sap when bicarbonate is taken up.
Figure 6. Typical on-line recordings of xylem pressure (Px), trans-root potential (TRP), K+ activity (aK+) and pH, respectively, in the root xylem of Zea mays plants by using the ion-selective xylem pressure probe. Vessel impalements were performed at a light irradiation of 10 µmol m−2 s−1 and are indicated by downward-directed dashed arrows. (a) Changes in Px, TRP and aK+ in the root xylem of a 21-d-old plant when subjected to a repeated short-term light regime (downward-directed arrows: increase of light irradiance from 10 to 300 µmol m−2 s−1; upward-directed arrows: return to low light irradiation). (b) Analogous pH measurements in the root xylem of a 9-d-old seedling. The responses of Px and TRP upon an increase in light irradiation were similar to those in (a) and are therefore not given. Note that changes in Px are reflected in corresponding changes of aK+, but not of pH. For more details, see Wegner & Zimmermann (2002) and Wegner et al. (2004).
Download figure to PowerPoint
In the light of the bulk of evidence given above doubts about the proper location and function of the xylem pressure probe are obviously unfounded. Despite this, the leading proponents of the Cohesion Theory persistently argue that the probe is not placed in a conducting vessel (see Appendix 1) because of the xylem pressures around 0 and −0.1 MPa measured in mangroves and at large heights of tall forest trees. So far, stable xylem pressures down to c.−0.6 MPa were only found in laboratory-sized herbaceous plants when exposed to extremely strenuous conditions or severed from roots before probing (see Fig. 3d and Zimmermann et al., 1994a, 1995a; Schneider et al., 1997a, 2004). It is important to note that at these negative pressures the leaves were nearly turgorless, independent of the plant species investigated. Furthermore, these pressure values could only be recorded in very few vessels. Most of the vessels that were impaled appeared to be gas-filled since their pressure was close to atmospheric. T1-weighted 1H NMR images of various heavily stressed plants confirmed the results of the xylem pressure probe experiments. As exemplified for an initially well-watered tobacco plant (Fig. 3a), more than 80% of the xylem vessels were filled with air after 1-week drought (Fig. 3c). Consistent with the assumption that the majority of the vessels was cavitated, flow-weighted 1H NMR imaging of severely drought-stressed, wilted plants revealed that the xylem volume flow had dropped dramatically. Interestingly, upon watering flow signals could be recorded within several hours accompanied with the reoccurrence of moderate negative pressures (data not shown). If such flow-weighted NMR imaging measurements are not made (Holbrook et al., 2001; Clearwater & Clarke, 2003), erroneous conclusions may be drawn about the functional state of the vessels.
As discussed in section V.3, it is extremely unlikely that stable negative xylem pressures can exist when the turgor pressure dropped to zero. Nature has obviously limited the magnitude of negative pressures that can develop in the xylem sap. This conclusion is also supported by the average xylem pressure values measured with the leafy twig/vacuum line technique (Renner, 1925; Scholander et al., 1955, 1962).
2. The leafy twig/vacuum line technique
In Renner's technique an excised leafy twig is attached to a potometer (A in Fig. 7a). When water uptake has stabilised, the flow resistance is increased by squeezing or by making saw-cuts into the twig from opposite sides. This leads to a temporary drop in water flow (associated with a transient xylem tension increase as shown by the pressure probe; Benkert et al., 1991). When the temporary drop has passed, the leaves are removed and a vacuum line is attached to the decapitated end of the twig (B in Fig. 7a). This is again accompanied by a change in water consumption. The unknown tension in the xylem is calculated from the ratio of water volume flow through the leafy twig to that through the twig/vacuum line system, by assuming that the vacuum line creates an absolute sub-atmospheric pressure value of +0.01 MPa. The average xylem pressure data initially published by Renner (1911, 1912) ranged between −1 and −2 MPa. During the following years, however, he received severe criticism by his colleagues (e.g. Ursprung & Blum, 1916; Nordhausen, 1916, 1919a,b, 1921). As a consequence, Renner improved his technique and published in 1925 an overview of tension values measured on twigs of various higher plants and under various environmental conditions. The majority of the xylem pressure values ranged between −0.2 and −0.4 MPa. The most negative xylem pressure of −0.8 MPa was measured on a cut twig of Forsythia suspensa (after introduction of a double clamp). Renner (1925) also measured positive, sub-atmospheric values under some circumstances. However, modern literature only cites the very negative pressure values of 1911 and 1912, presumably because these pressure values are more consistent with those measured by the pressure bomb technique.
Figure 7. Schematic diagrams of the leafy twig/vacuum line technique (a) and the pressure bomb (b). For details, see text.
Download figure to PowerPoint
An important artefact that can falsify the tension data is the entry of air into the xylem upon cutting (see Pfeffer, 1881, p. 133). This problem was eliminated by Renner by cutting the twig under water. A further problem involved in the interpretation of the leafy twig/vacuum line data is the assumption that the effective cross-sectional area for water flow remained unchanged during the entire experiment. This is not necessarily the case because the leaves were not replaced by a corresponding number of vacuum pumps (see set-up B in Fig. 7a). According to the Hagen-Poiseuille law, the volume flow, but not the flow velocity is directly proportional to the square of the water-conducting area. Therefore, despite an increase in the flow velocity the total flow in the twig/vacuum line system could be lower than that in the intact twig, if the number of conducting xylem vessels decreases due to the experimental manipulations (see Zimmermann et al., 1993a).
Nevertheless, the method of Renner allows at least the magnitude of the average xylem tension to be estimated that can develop in a cut transpiring leafy twig that is continuously watered by an attached potometer.
3. The pressure bomb technique: a conceptual artefact
The pressure bomb technique (Scholander et al., 1965) was never validated in appropriate, well-defined air-water model systems in which negative pressures of known magnitude could be established. The method has only been compared with the root pressurisation (Passioura & Munns, 1984) and the psychrometric method (see e.g. Kaufmann, 1968a,b; Klepper & Ceccato, 1969; Barrs et al., 1970; West & Gaff, 1971; Turner et al., 1984), but these techniques have also never been calibrated and tested in model experiments. Agreement between the data obtained by the various approaches only suggests that ‘sometimes these [...] methods are estimating the same quantity, but this is not proof that the quantity is the tension that prevailed in the xylem water columns before cutting’ (Canny, 1995b, p. 344). It is also surprising retrospectively that publication of many conflicting data (Tobiessen et al., 1971; Hellkvist et al., 1974; Connor et al., 1977; Koch et al., 1994) and some critical evaluations of the pressure bomb (Begg & Turner, 1970; Ritchie & Hinckley, 1971; Janes & Gee, 1973; Baughn & Tanner, 1976; Turner & Long, 1980) have not evoked a broadly based critical reassessment of the pressure bomb technique and the other methods.
With the pressure bomb, publication of stable negative xylem pressure values of −1 MPa down to −17 MPa became commonplace (e.g. Kappen et al., 1972). The method is simple and thus very popular. A leafy twig is placed into a steel chamber with the cut end protruding through a pressure-tight seal to the ambient atmospheric pressure (Fig. 7b). Gas pressure is applied to the specimen with a rate of about 0.3 MPa/min. The overpressure at which water appears at the cut surface, the so-called balancing pressure, Pb (defined in relation to atmosphere), is postulated to be numerically equal to the tension (=−Px + 0.1 MPa) that existed in the xylem before cutting. This explanation implies (see e.g. Scholander et al., 1965; Boyer, 1967; Passioura, 1982; Jones, 1992; Holbrook et al., 1995; Tyree, 1997; Steudle, 2003) that the tension in the xylem is instantaneously relieved upon cutting of the leaf. Water is osmotically forced from the xylem into the adjacent tissue cells and the xylem sap is withdrawn from the cut end. The balancing overpressure is needed for forcing the water from cells back into the xylem elements and to re-establish the original tension.
However, from the beginning there have been many doubts about what the bomb in fact measures. The dilemma of interpreting pressure bomb data is that the contribution of the many processes to the Pb-value generated by cutting transpiring leaves and by subsequent application of gas pressure is not predictable and presumably highly species- and environment-dependent. Upon cutting capillary tension is created at the cut end which may considerably exceed the values that are predicted by the radii of the lumens of the conducting elements because of the varying diameters and/or the non-circular cross-sections of the vessels and tracheids (Finn, 1989; Zimmermann et al., 1994a; Schneider et al., 2000b). Tyree & Hammel (1972) assumed that Pb exclusively reflects the pressure at which the capillary force becomes zero, i.e. at which the cut surface becomes wetted (see their Eq. 6). Pressure probe work on air-cut leaves and roots has indeed shown (Zimmermann et al., 1995a; Benkert et al., 1995) that under well-hydrated conditions negative pressures in the xylem are not exclusively ‘transpiration-born’, but rather ‘capillarity-born’. Probing of excised roots of maize and barley yielded similar results (Zhu et al., 1995; Schneider et al., 1997b). Today Tyree (1997) and Wei et al. (2000b) postulate that the pressure bomb is measuring only equilibrium negative pressure values in leaves because of the dissipation of hydrostatic and osmotic pressure gradients in the multi-phase plant organs upon cutting and stop of transpiration when the leaf is placed into the bomb (see e.g. Rygol et al., 1993; Zimmermann et al., 1991, 1992, 1993a). This assumption implies that the balancing pressure is not numerically equal to the tension previously existing in the xylem of the intact plant at the site where the leaf is cut. The discrepancy between the two values may be quite large, in particular when leafy twigs are taken from heights above 10 m as done for example by Scholander et al. (1965; see also Tobiessen et al., 1971). In this case, the gravitational potential term vanishes upon cutting that should lead to large changes in the gradients of xylem pressure and in the turgor pressure of the hydraulically linked tissue cells provided that the fundamental assumption of the Cohesion Theory of the water columns being continuous over the total height of a tall tree is valid (see sections IV and V.1). Unfortunately, from the nomenclature used by Tyree and co-workers it is very often not clear whether the authors refer to the xylem tension in the intact plant or to the new artificial equilibrium value.
A further key problem is the transmission of external pressure to the xylem which is assumed by the users of the bomb technique to occur instantaneously. Scholander and his co-workers (1965) saw this quite clearly at the beginning of the ‘pressure bomb era’. Balling & Zimmermann (1990) studied the pressure transmission by sealing a pressure transducer onto the cut end of leaf petioles of tobacco protruding through the seal of the pressure bomb (Fig. 8a). Only in leaf petioles pre-infiltrated under vacuum with water, the pressure response upon increasing the bomb pressure was 1 : 1; otherwise, it was significantly less up to a bomb overpressure of about 0.25 MPa. In accordance with previous results of West & Gaff (1971, 1976), these findings evidence that air-filled spaces had to be compressed before pressure could be transmitted directly. Wei et al. (2000b) recently repeated the experiment of Balling & Zimmermann (1990) on excised, non-infiltrated leaf specimens. They traced the significant discrepancies between the applied bomb pressures and the pressure transducer readings back to the compression of air bubbles in embolised vessels. Surprisingly, they argued that this finding does not invalidate the pressure bomb as a measuring tool for negative xylem pressures when the cut surface is exposed to atmosphere. The finding of Balling & Zimmermann (1990) on detached leaves with their cut surface outside the bomb that the negative xylem pressure of non-infiltrated specimens − in contrast to infiltrated specimens − only responded to overpressures of 0.25 MPa (Fig. 8b) makes this statement obsolete.
Figure 8. External pressure transmission in leaves of Nicotiana tabacum plants. (a) Excised leaves were mounted into a pressure bomb, and a pressure transducer was directly sealed to the cut end of the petiole protruding through the seal of the bomb (inset). Pressure changes in the leaves upon external gas pressure application (ΔPb) are shown for a leaf preinfiltrated with water (dash-dotted line) and for an untreated leaf (dotted line). (b) Time course of changes in absolute xylem pressure (solid line) upon application of external gas pressure (dashed line; zero pressure = atmospheric pressure) measured by a xylem pressure probe inserted into the leaf petiole protruding through the seal of the bomb (inset). Vessel impalement is indicated by a dashed arrow. Double-headed arrow indicates pressurisation of the xylem sap associated with water release at the cut end. For discussion, see text and Zimmermann (2003). Taken from Balling & Zimmermann (1990), with kind permission of Springer-Verlag, Heidelberg, Germany.
Download figure to PowerPoint
1H NMR imaging of petioles of cut hydrated Epipremnum aureum leaves subjected to pressurisation also gave evidence that the overpressure cannot be set equal to the original (or equilibrium) xylem tension (Zimmermann et al., 2000; Schneider et al., manuscript in preparation). Leaves of this plant were selected because of their stiffness and low compressibility that prevented movement of the leaf within the bomb (mounted into the bore of a NMR magnet) during the measurements. Only data for uniform pressure application (0.1 MPa min−1) are shown in Fig. 9 because the Scholander bomb arrangement yielded the same results. It is obvious from Figs 9a–c that T1-weighted images showed a significant increase in signal intensity with increasing overpressure. By contrast, the spin density (i.e. the water content of the petiole) remained constant within the limits of accuracy (Fig. 9d). The effects of pressurisation on spin-lattice relaxation time, T1, were completely reversible. T1 depends on the compartment size confining the water (Mansfield & Morris, 1982) and on the oxygen concentration (Chen et al., 1998). Since pressure-induced changes in the oxygen concentration could be excluded by control experiments using N2-exposed leaves, the results can only be explained by a pressure-induced shift of water within the leaf petiole from large water-filled to air-filled compartments or to small cells (because of the volume dependence of the volumetric elastic modulus of the cell wall, Zimmermann, 1978). The criticism of Wei et al. (2000a, p. 147) that ‘a movement of water from the symplast into the tissue apoplast can be neither detected nor quantified because of a lack of resolution’ is irrelevant. What is relevant is that uniform pressure application leads to water movement within the leaf resulting in the generation of pressure gradients due to the anisotropic compressibility features of the multi-phase leaf system. Similar conclusions have been drawn from xylem pressure probe measurements in intact, weakly transpiring tobacco plants placed in a hyperbaric chamber (Balling & Zimmermann, 1990; Zimmermann et al., 1991). Changes in xylem pressure could be recorded over 2 h before the tissue equilibrated with ambient pressure (up to 0.5 MPa). The equilibrium xylem pressure corresponded to the original value as expected in the light of Eq. 4 stating that the absolute hydrostatic pressures in the water and solvent phase are independent of the ambient pressure (a ‘bizarre’ phenomenon according to Passioura, 1991). The observations of our group were recently confirmed by Wei et al. (2000b) by exposing Tsuga canadensis branches to uniform pressure in a pressure bomb. Astoundingly, even this did not provoke a shift in the persistent view of these authors that the pressure bomb is an appropriate tool for measuring xylem pressures.
Figure 9. Typical T1-weighted and spin-density-weighted 1H NMR experiments on well-hydrated leaves of the liana Epipremnum aureum subjected to uniform pressure regimes by using a pressure bomb placed in the bore of the NMR magnet. Note that T1-weighted images represent the compartment size confining the water whereas the spin density images reflect the water content of the tissue. In the case of T1-weighted experiments, application of overpressure (given in relative values) led to an increase of the signal intensity within the leaf petiole as indicated by the colour change from yellow to red (a = control; b = 0.5 MPa; signal intensity increase c. 20%). The pressure effects on the signal intensity ( •) were reversible as shown by intermediate pressure releases (▪ in c; mean values ± sd, n = 4). By contrast, in corresponding spin-density-weighted experiments the signal intensity remained unaffected by the pressure regime (d). Note that the values in (c) and (d) were normalised to the reference images which were taken before the first pressure application (□). Note further that the signal intensities of the water-filled reference capillary (rc) remained unaffected by the pressure regime both in T1-weighted and spin-density-weighted experiments. For discussion of the results, see text and Zimmermann (2003); for experimental details, see Zimmermann et al. (2000). (a,b) taken from Zimmermann et al. (2000), with kind permission of Elsevier, Amsterdam, The Netherlands.
Download figure to PowerPoint
Strong evidence for the interference of air-filled spaces with pressure transmission also arrived from the experiments of Melcher et al. (1998). In this study (Fig. 10a, left-hand side), one or two leaves of sugarcane or maize were covered with aluminium foil at predawn to prevent substantial transpiration during the day. In a nearby light-exposed, transpiring leaf of the same plant the xylem pressure probe was introduced into a vessel. As shown schematically in Fig. 10b (right-hand side), significant differences in xylem tensions between the neighbouring covered and light-exposed leaves can be excluded because of the hydraulic continuum of xylem sap (see also Passioura, 1982; Benkert et al., 1991; McCutchan & Shackel, 1992). For determination of Pb covered and uncovered leaves were excised simultaneously from the same plant. In order to prevent dehydration after excision, the leaves were covered with plastic bags just before excision. The excised leaves, still in plastic bags except for the cut end, were sealed into a pressure bomb. A nearly 1 : 1 relationship (up to a tension of 0.4 MPa; Px = −0.3 MPa) was found between xylem tension values measured with the xylem pressure probe and the bomb (Fig. 10b). By contrast, at noon when the transpiring leaves were exposed to light irradiations of 1500–2000 µmol m−2 s−1 and the proportion of the air-filled spaces within these leaves increased, the balancing overpressures of uncovered leaves were up to 0.6 MPa (200%!!) greater than those of the covered leaves and the tension values measured directly with the xylem pressure probe, respectively. This demonstrates that substantial overpressures were required to establish a hydraulic continuum within the periphery and the xylem of the uncovered leaves. Wei et al. (1999b, p. 1203) argued that ‘the presumption that adjacent leaves should have nearly identical pressures is clearly wrong’. These authors repeated the experiments of Melcher et al. (1998) under so-called ‘well-defined conditions’ by using maize plants being not irrigated for 1 d or more prior to the experiment. During probe measurements, the xylem pressure was artificially adjusted by root pressurisation in order to verify insertion of the probe tip into a xylem vessel. For determination of the balancing overpressure values leaf blade tissue was subsequently taken from the leaf in which the probe was inserted. In contrast to Melcher et al. (1998), Wei et al. (1999a,b) reported a 1 : 1 correlation between relative Pb-values and xylem tensions up to 0.7 MPa (Px =−0.6 MPa) also for the weakly transpiring leaf blade tissue (data for the non-transpiring leaves are not given for larger pressures, see Fig. 10c). Even though accurate details of the entire experimental procedure are not presented, it is clear that preceding root pressurisation led to a refilling of air-filled spaces and cells as illustrated in Fig. 10c on the right-hand side (see also the NMR results in Fig. 9). Under these highly artificial conditions excessive overpressure is, of course, not required. The question remains why Tyree, Steudle and Wei try to create the impression that the pressure bomb has been calibrated down to −1.0 MPa by using the pressure probe and can thus be employed for measuring xylem pressure even in highly transpiring and/or dehydrated plants.
Figure 10. Xylem pressure probe vs pressure bomb-based balancing overpressure measurements. The schematic diagrams in (a) show the experimental procedure used by Melcher et al. (1998). Before the experiment, when the leaves were well watered (upper drawing on the left-hand side), one leaf was covered with aluminium foil before onset of transpiration, while a pressure probe was inserted into the xylem of an adjacent, exposed leaf. Upon progressive increase in transpiration evaporational water loss leads to the development of large air-filled spaces in the uncovered leaves (illustrated by the large white areas in the lower drawing), but not in the covered leaf (upper drawing). Because of the hydraulic connection the xylem pressure in the covered leaf (Px,cov.) should be the same as in the adjacent uncovered leaves (Px,uncov.) independent of transpiration (right-hand side of (a); green leaf areas: full hydration; mottled leaf areas: incomplete hydration). (b) Plots of the balancing overpressure values, Pb, measured on the covered and an adjacent uncovered leaf vs the corresponding tension values yielded a nearly 1 : 1 relationship for covered leaves (▪; dotted lines indicate 95% confidence). Large discrepancies (that increased with transpiration) were found between the balancing overpressures and tension values of uncovered leaves (▿), since excessive overpressure is needed for compression of the air-filled spaces. (c) The approach of Wei et al. (1999b) differs essentially from that of Melcher et al. (1998) in that pneumatic pressure was applied to the roots before the Pb-values were determined. This leads to a refilling and compression of the air-filled spaces as shown in the drawings (right-hand side) and, because of the establishment of a hydraulic continuum in the leaves, also to a 1 : 1 relationship between the tension values and balancing overpressure values of uncovered leaves (left-hand side, redrawn from Wei et al., 1999b; • = covered leaves; ○ = uncovered leaves). Extrapolations of the straight lines to pressure values that were not measured were omitted. For a detailed discussion of the results, see text and Zimmermann (2003). (b) and (c) modified after Zimmermann et al. (2000) and Wei et al. (1999a), respectively, with kind permission of Elsevier, Amsterdam, The Netherlands.
Download figure to PowerPoint
Meinzer et al. (2001, p. 245) suggested recently that ‘pressure bomb measurements should routinely be made on both covered and exposed leaves, but even the covered leaf balance pressures can be ambiguous if substantial cavitation has occurred and if the anatomy of the stem to which the leaf is attached allows water to be forced into non-conducting tissue during pressurisation’. However, such bomb data are also ambiguous when the xylem sap does not consist of pure water. In this case, corrections of the Pb-values are required (Smith & Lüttge, 1985; Murphy & Smith, 1994) which can be quite dramatic if mucilage or proteins are present, as found in mangroves and other salt-tolerant trees (see sections IV and V.6). In Rhizophora mangle, excessive overpressures of about 3 MPa were required to compensate for the xylem mucilage, irrespective of the difference in overpressure between covered and uncovered leaves (0.6–0.8 MPa; Melcher et al., 2001). This clearly reflects the dilemma that applicants of the pressure bomb technique are faced with.
In the last years many desperate efforts were made by the proponents of the Cohesion Theory to ‘rescue’ the bomb as a measuring tool for xylem pressure. The inconsistencies of the various approaches are discussed in detail in Appendix 2. Tyree (1997), one of the outstanding proponents of the pressure bomb technique and Cohesion Theory, hit the mark by saying recently that the interpretation of pressure bomb data in terms of xylem pressure is based on hypothesis. This implies that stable negative pressures of the order of megapascals measured by this technique are not facts, but belong in the realm of science fiction.