TY - JOUR
AU1 - Wei, C.
AU2 - Tyree, M.T.
AU3 - Bennink, J.P.
AB - Abstract In earlier work tobacco leaves were placed in a Scholander–Hammel pressure bomb and the end of the petiole sealed with a pressure transducer in order to measure pressure transmission from the compressed gas (Pg) in the bomb to the xylem fluid (Px). Pressure bomb theory would predict a 1 : 1 relationship for Pg:Px when tobacco leaves start at a balance pressure of zero. Failure to observe the expected 1 : 1 relationship has cast doubt on the pressure‐bomb technique in the measurement of the xylem pressure of plants. The experimental and theoretical relationship between Px and Pg was investigated in Tsuga canadensis (L) branches and Nicotiana rustica (L) leaves in this paper. It is concluded that the non 1 : 1 outcome was due to the compression of air bubbles in embolized xylem vessels, evaporation of water from the tissue, and the expansion of the sealed stem segment (or petiole) protruding beyond the seal of the pressure bomb. The expected 1 : 1 relationship could be obtained when xylem embolism was eliminated and stem expansion prevented. It is argued that the non 1 : 1 relationship in the positive pressure range does not invalidate the Scholander pressure bomb method of measuring xylem pressure in plants because Px never reaches positive values during the determination of the balance pressure. Pressure bomb, embolism, Tsuga canadensis, Nicotiana rustica Introduction Many efforts have been made to demonstrate or to measure the negative pressure in xylem vessels. The most important and widely accepted method is the pressure bomb technique (Scholander et al., 1965). The pressure bomb is capable of measuring only equilibrium values of negative pressure in harvested plant parts, for example, leaves or shoots. If a transpiring leaf is harvested for pressure bomb measurement, time must be allowed for equilibrium of water potential gradients before a measurement can be made. When a previously transpiring leaf or shoot is placed inside a pressure bomb (=a dark, humid chamber) water flow ceases and hence any gradients of Px and gradients of water potential in living cells in the shoot will dissipate given sufficient time. The value of Px ultimately measured in the pressure bomb is determined by the equilibrium water potential achieved by the shoot after the gradients equalize; at this point Px is equal to average water potential minus the average osmotic pressure of the xylem fluid. This difference between the dynamic state of shoots with gradients and the equilibrium state without gradients in non‐transpiring leaves has been known for decades (Begg and Turner, 1970). In the pressure bomb technique, the shoot is placed in a metal vessel with the cut end protruding through a pressure seal to the outside air. The basic assumption of the pressure bomb techniques is that Px in vessels can be increased by applying a gas pressure, Pg, to the shoot within the bomb. For every unit increase in Pg, Px will become less negative by one unit until the balance pressure (Pb) is reached when water is ‘balanced’ on the cut end (at this moment the Px=0). The rise of Px to a value of zero can be expressed as Px=Pg–Pb. Thus the gas pressure Pg needed to force water just to appear at the cut end is equal to the negative pressure previously existing in the xylem. The invention of this technique provided a simple tool to measure the water tension in plants. This technique has been successfully compared with other methods such as temperature‐corrected psychrometers (Dixon and Tyree, 1984), centrifugal tension measurement (Holbrook et al., 1995) and other methods. It has been suggested that the Scholander pressure bomb did not actually measure xylem pressure (Balling and Zimmermann, 1990). Using pressure probes or pressure transducers they could measured Px directly in xylem vessels or at the cut surface of petioles of leaves enclosed in a pressure bomb, respectively. Many different experiments were reported in this work (Balling and Zimmermann, 1990) and this paper deals with only a subset of these, where leaves were fully hydrated so that Px should be zero prior to admitting gas into the pressure bomb as shown in Fig. 1A. The apparatus shown in Fig. 1A can be used to test the hypothesis that gas pressure in the bomb should be directly transmitted to the xylem in the positive pressure range, i.e. Px from 0 to 700 kPa. The response of this pressure transducer (PT2 in Fig. 1A) to increasing chamber pressure was not 1 : 1 in Balling and Zimmermann (Balling and Zimmermann, 1990); under most situations the value of Px measured by the pressure transducer was lower than Pg (measured with PT1; Fig. 1A). At first this outcome appeared to be in total contradiction to the underlying hypothesis of pressure bomb technique, and the bomb seemed to overestimate xylem pressure. The purpose of this paper is to examine the reasons for the disagreement between Pg and Px in the positive pressure range and to determine if this disagreement negates the use of the pressure bomb for the determination of Px under normal circumstances. The experimental set‐up used by Balling and Zimmermann, Fig. 1A, should change the dynamics of pressure transmission in the petiole or stem, because the pressure transducer prevents the free flow of water from the cut surface. Imagine a situation in which the fluid in the xylem is continuous with fluid in a tube attached to the cut end of the petiole or stem (Fig. 1B). When the cut surface is open to the air, the application of Pg will dehydrate the living cells (symplast) in the leaf and the water will flow out of the cells at atmospheric pressure. The volume of the symplast will decrease and the volume of water in the apoplast (water in cell walls, vessel lumens and tube) will increase by an equal amount. When equilibrium has been achieved, the dehydration of the symplast will make Pg equal to Pb because if the water were removed from the tube then the Pg required to bring water to the surface of the stem or petiole would be Pb by definition. Now consider what happens if the tube at the end is filled with water and closed off with a pressure transducer as in the experiment by Balling and Zimmermann. If the volume of water in the apoplast does not change as Pg increases then the symplast will not dehydrate because there would be nowhere for the water to flow. Under these special conditions, the pressure bomb hypothesis would make us predict that Px=Pg. But, if the volume of water in the apoplast should increase then the symplast would be dehydrated and Px would be less then Pg and equal to Pg−Pb. Xylem tissue is frequently filled with air bubbles in vessels or tracheids, and sometimes wood fibre cells are embolized (Tyree and Sperry, 1989). Figure 1D illustrates the status of air in an embolized trachied of Tsuga wood under two conditions, i.e. when xylem water is under tension and when xylem water is pressurized. Any bubbles present in xylem will be compressed as Px increases >0 and hence the volume of water in the apoplast will be increased at the expense of an equal loss of water from the symplast. It was hypothesized that the volume of water in the apoplast should increase as Pg is increased causing a corresponding dehydration (reduction in volume) of the symplast because of (1) pressure‐induced expansion of the tissues outside the pressure bomb and (2) pressure‐induced compression of air bubbles in the xylem tissue anywhere in the leaf or shoot. As Px increases the xylem vessels outside the bomb would increase in volume because the pressure difference between the vessels and outside air would increase. As Px increases the water potential of living cells would become positive causing an increase in turgor pressure and cell volume. As Px increases the volume of any air bubbles trapped in embolized vessels would decrease until the pressure of the gas bubbles ≅Px, the relationship between volume change and gas pressure would be given approximately by the ideal gas law. Hence, as the value of Px increases water would have to flow from the symplast inside the pressure bomb to increase the volume of water in the apoplast inside the bomb by bubble compression. Also water would flow from the symplast inside the pressure bomb to increase the volume of the apoplast and symplast outside the pressure bomb by tissue swelling and bubble compression. This water flow would increase Pb and make Px less then Pg. This study will provide the mathematical analysis to predict the magnitude of the deviation of Px from Pg and will show that Px=Pg under experimental conditions where the volume of water in the apoplast does not change. Materials and methods Plant species Y‐shaped shoots each having two similar branches 40–50 cm in length, 4 mm in diameter and about 22 g in fresh weight were collected from Tsuga canadensis trees growing at the Proctor Maple Research Center, Underhill Center, Vermont, USA, and immediately placed in water and transported to the laboratory. Leaves of 13–15‐week‐old Nicotiana (about 250–350 mm tall) were also used for this study. The plants were grown in the greenhouse of the United States Forest Service, Northeastern Forest Experiment Station, South Burlington, Vermont, in 25 cm pots in soil consisting of a 2 : 2 : 1 (by vol.) mixture of vermiculite, peat moss and processed bark ash, respectively, and watered daily. About 8 h before the experiments, the plants were brought to the University of Vermont, watered well and covered with a plastic bag. Fig. 1. View largeDownload slide Experimental set‐up resembling the Scholander‐Hammel bomb method. A pressure transducer (PT2) was sealed to the cut end of the stem of a Tsuga branch (or the petiole of a Nicotiana leaf). Another pressure transducer (PTI) was used to measure the chamber pressure, which increased linearly from 100 kPa to 700 kPa (absolute) within 24 min. (B) Similar to (A), but the pressure transducer is replaced by an open tube and apoplast volume is allowed to expand into the tube. (C) Experiment set‐up designed to avoid the expansion of the stem segment (or petiole) protruding through the bomb chamber of set‐up (A). The whole plant material and the PT2 were now placed in the gas chamber. (D) An illustration showing the fate of embolisms during pressurization of xylem fluid. While Px <−100 kPa the bubble occupies the entire volume of the tracheid, but when Px>0 the bubble is compressed by displacement of fluid from the water‐filled tracheids to the embolized tracheid. Fig. 1. View largeDownload slide Experimental set‐up resembling the Scholander‐Hammel bomb method. A pressure transducer (PT2) was sealed to the cut end of the stem of a Tsuga branch (or the petiole of a Nicotiana leaf). Another pressure transducer (PTI) was used to measure the chamber pressure, which increased linearly from 100 kPa to 700 kPa (absolute) within 24 min. (B) Similar to (A), but the pressure transducer is replaced by an open tube and apoplast volume is allowed to expand into the tube. (C) Experiment set‐up designed to avoid the expansion of the stem segment (or petiole) protruding through the bomb chamber of set‐up (A). The whole plant material and the PT2 were now placed in the gas chamber. (D) An illustration showing the fate of embolisms during pressurization of xylem fluid. While Px <−100 kPa the bubble occupies the entire volume of the tracheid, but when Px>0 the bubble is compressed by displacement of fluid from the water‐filled tracheids to the embolized tracheid. Rehydration of material Branches or leaves were covered with a plastic bag and either rehydrated with the cut stem or petiole immersed in a beaker of water for 2 h or perfused from the cut end with degassed water pressurized at 200 kPa for 2 h. All pressures mentioned in this paper are absolute pressure except balance pressures measured with a Scholander‐Hammel pressure bomb. After rehydration by either method, branches had a balance pressure (Pb) of <20 kPa when measured in a Scholander–Hammel pressure bomb. Nicotiana leaves were at Pb<10 kPa 8 h after irrigation and covering of shoots with plastic bags. Water potential isotherms Water potential isotherms were measured on T. canadensis branches to get the relationship between weight of water lost from the symplast of shoots versus Pb. The water was expressed from the stem by over‐pressurizing the stems beyond Pb and collecting the water expressed into preweighed vials stuffed with absorbent paper. Weight of the vials before and after collection of water was measured to the nearest 0.1 mg with a model AE200 Mettler balance (Mettler Instrument Corporation, Highstown, NJ). After each period of application of overpressure, the resulting Pb was measured. Cumulative water loss was plotted versus Pb. Pressure transmission from gas to xylem fluid After rehydration or perfusion, the two branches cut from one Y‐shaped shoot were used in the experiments with one having the stem segment protruding through the pressure bomb (Fig. 1A) and another one having a whole branch in the bomb (Fig. 1C). The bark was removed from the entire length (50 mm) of stem segment protruding from the pressure bomb. All pressures (gas pressure, Pg, and xylem fluid pressure, Px) were measured with model PX136GV100 pressure transducers (Omega Engineering, Stamford, CT) as shown in Fig. 1. Similar experiments were done on Nicotiana leaves with petioles either outside or inside the bomb as shown in Fig. 1. Petioles or woody stems were connected to the pressure transducers using Omnifit connectors (Rannin Inst, Lexington, Ma) which sealed the stem using an O‐ring leaving the apical 10 mm of stem or petiole above the seal. In the typical experiment, Pg, was increased linearly with time from 100–700 kPa over a period of 24 min. This rate of pressurization was achieved by use of a pressure regulator set at 4 MPa admitting air to the pressure bomb via a needle valve. During this time Pg and Px was recorded every 2 or 3 s. A linear pressurization was used rather than a timed, stepwise pressure increase because of the difficulty of maintaining constant pressure after a rapid step increase. As Px increased air bubbles would compress (approximately according to the idea gas law) and simultaneously air bubbles would dissolve. The time‐course for bubble dissolution would take several hours (Yang and Tyree, 1992). So a compression period of 24 min was selected so that bubble compression would be the dominant process. Some Tsuga branches and Nicotiana leaves were dehydrated to Pb>700 kPa and were placed entirely within the pressure bomb as in Fig. 1C and pressurized to Pg=700 kPa. This was to verify that by doing so the chamber pressure did not ‘directly’ transmit pressure to the xylem pressure by gas diffusion and embolism formation in the Omnifit connector or pressure transducer. Wood density measurements Wood densities were measured on stem segments 30 mm long with bark removed by the Archimedean principle, i.e. the stem segments were weighed in air and weighed while immersed and suspended in water. The weight of the segment while immersed and suspended in water divided by the density of water gave the volume of the segment. The stem density was computed from the weight of the segment in air divided by the volume. Densities were measured on excised segments from portions of stem protruding from the pressure bomb and from segments inside the bomb. Attempts were made to measure the maximum density of stem segments, i.e. in segments presumed to have no air in the wood. For this purpose, shoots were perfused with degassed water at pressures up to 600 kPa for periods up to 8 h to dissolve all air present. In other cases stem segments were immersed in water in a vacuum flask while continually pulling a vacuum above the solution, trials indicated that 2–3 d were required to reach maximum density. The difference between maximum and ‘native’ stem densities gave information on the volume of air per unit volume of wood in native samples. Diameter changes of woody stems and petioles Diameter changes in stem segments and petiole segments protruding from the bomb were measured with digital calipers to the nearest 0.01 mm. All measurements were made 1.5 cm below the cut ends and were measured with bark removed in woody samples. Diameters were recorded versus chamber pressure. Diameter changes were used to estimate volume changes assuming cylindrical geometry and no length change; zero length change was confirmed experimentally. All measurements on one branch In order to provide more accurate data for a model (see Discussion) some experiments were done in which all parameters above were measured on the same Tsuga branch. The sequence of measurements were as follows: (1) A branch was harvested from the field and rehydrated in beaker of water for 2 h. (2) The branch was weighed then placed in a pressure bomb to determine the balance pressure to confirm full hydration. If the balance pressure was not zero a brief period (20 s) of perfusion of the shoot at 100 kPa was done to establish full hydration. Usually 2 or more 20 s perfusions were needed to reduce Pb below 20 kPa. (3) The pressure transducer PT2 was mounted on the base of the stem (Fig. 1A or C). (4) Px versus Pg was measured while ramping Pg from 0 to 800 kPa over 1500 s. (5) Pg was returned to zero; the balance pressure was measured and then the water potential isotherm was measured. (6) The branch was removed from the bomb and placed under water. Sample stem segments of 1, 2, 3, and 4 mm diameter were harvested and bark removed from the wood for determination of initial density and maximum wood density. The volume of all remaining wood was measured by the Archimedean principle. Results Although the two different rehydation procedures used on Tsuga both succeeded in making Pb@0, the procedures did not result in the same stem densities. The density of wood from shoots perfused with degassed water was significantly different from those rehydrated in a beaker (Table 1). Table 1 also showed the maximum densities recorded, most of them were found when the Tsuga branches had been perfused with 500 kPa degassed water for more than 8 h or when floated in water and a vacuum drawn for 3 d. The maximum density observed in this study (1.21–1.23 gm cm−3) agreed with a previous study in which debarked Tsuga stem segments 4 mm diameter were wetted for 10 d in Petri dishes while stored a 4 °C (MT Tyree and Alexander, unpublished results). Stems between 0.5 and 1 mm in diameter had maximum densities of 1.3–1.6 whereas stems 1.5–4 mm diameter were generally between 1.20 and 1.25. Representative balance pressure isotherms are shown in Fig. 2. The relationship between volume expressed versus balance pressure was linear for Pb<500 kPa. The slopes of three of the four curves shown differ from each other probably because of corresponding differences in leaf mass. Figure 3 shows the pressure transmission between the chamber gas, Pg, and the xylem fluid, Px, in Tsuga stems in an experiment much like that described previously (Balling and Zimmermann, 1990). Results for Nicotiana were similar (data not shown), hence this study's results confirm those of Balling and Zimmermann (1990). The closeness of Px to Pg had much to do with the Pb of the shoot as well as how the plant material was rehydrated. When Pg reached 700 kPa, Px was 422±65 kPa in branches rehydrated in a beaker for 2 h, and Px was 502±44 kPa for Tsuga branches perfused with degassed water for 2 h (Table 2). The volume increase of a Tsuga stem segment protruding from the pressure bomb could not be determined with accuracy because the change in diameter measured with a caliper was typically 0.05–0.15 out of 4 mm. Based on the mean diameter change for Px from 0 to 700 kPa, the volume of Nicotiana petioles had increased by 9±1% (n=10) compared to 6±2% (n=15) for Tsuga stems. The density of Tsuga stem (without bark) before and after the experiments like those in Fig. 3 and Fig. 1A were measured in some cases. The density (gm cm−3) before the experiments were 1.161±0.008 (n=14) and after the experiment the density of wood collected from inside the bomb did not change significantly (1.162±0.009, n=17) but did change significantly for samples collected outside the bomb (1.155±0.009, n=17, P=0.015). Since wood density did not change inside the bomb, the experiments (Figs 3–5) did not change the amount of air in most of the wood mass, hence it was possible to estimate gas volume from the ideal gas law assuming an approximately constant number of moles of gas (see Discussion). Figure 4 shows the typical result of experiments in which no part of the branch or petiole protruded from the chamber. When the chamber pressure reached 700 kPa, the xylem pressure of Tsuga branches which had been perfused was 689±12 kPa and the xylem pressure of Nicotiana leaves was 677±6 kPa (Table 2). These results showed that the relation between gas pressure and xylem pressure was about 1 : 0.967 for Nicotiana leaves and 1 : 0.984 for the perfused Tsuga branches (curve A). For Tsuga branches rehydrated in the beaker, the xylem pressure was only 392±53 kPa (curve B). For Tsuga branches or Nicotiana leaves freshly harvested from water‐stressed plants, the xylem pressures were much lower than chamber gas pressure (curve C). Figure 5 shows a repeat of the experiments above on samples collected in April 1999 when leaves were expanding on some angiosperms, but not advanced on Tsuga. The pressure–volume curve measured on the same branch suggested some tissue dehydration because the initial balance pressure had increased to 180 kPa after the determination of Px versus Pg. Extrapolation of the curve back to atmospheric pressure suggested a net weight loss of 0.2 g. Tissue dehyration was confirmed by weighing shoots before and after measuring Px versus Pg. Fig. 2. View largeDownload slide Balance pressure (relative to air pressure) versus water expressed from living cells of Tsuga branches. Fresh weights of the samples were all between 21.37 and 22.46 g. Regression lines shown were for Pb<500 kPa where water expressed was a linear function of Pb. Differences in slope probably reflect differences in mass of fresh leaf material attached to each branch (not measured). Fig. 2. View largeDownload slide Balance pressure (relative to air pressure) versus water expressed from living cells of Tsuga branches. Fresh weights of the samples were all between 21.37 and 22.46 g. Regression lines shown were for Pb<500 kPa where water expressed was a linear function of Pb. Differences in slope probably reflect differences in mass of fresh leaf material attached to each branch (not measured). Fig. 3. View largeDownload slide Typical time‐course of experiments on Tsuga stems when the cut end of the stem protruded from the pressure bomb. Relationship between chamber gas pressure and xylem pressure when the cut end of stem (or petiole) was protruding through the chamber. Inset graph: chamber gas pressure, Pg, versus time. Main graph: (curve A) xylem pressure, Px, of Tsuga branch perfused with degassed water (200 kPa, 2 h) versus Pg during linear pressurization; (curve B) Px of a Tsuga branch rehydrated in a beaker for 2 h; (curve C) Measurement done on Tsuga branch with Pb>700 kPa. Each curve consists of 300–360 points and all pressures are absolute. Fig. 3. View largeDownload slide Typical time‐course of experiments on Tsuga stems when the cut end of the stem protruded from the pressure bomb. Relationship between chamber gas pressure and xylem pressure when the cut end of stem (or petiole) was protruding through the chamber. Inset graph: chamber gas pressure, Pg, versus time. Main graph: (curve A) xylem pressure, Px, of Tsuga branch perfused with degassed water (200 kPa, 2 h) versus Pg during linear pressurization; (curve B) Px of a Tsuga branch rehydrated in a beaker for 2 h; (curve C) Measurement done on Tsuga branch with Pb>700 kPa. Each curve consists of 300–360 points and all pressures are absolute. Fig. 4. View largeDownload slide Typical relationships between chamber pressure and xylem pressure of Tsuga branches when the whole branch was placed inside the chamber. Inset graph: chamber gas pressure, Px versus time. Main graph: (curve A) xylem pressure, Px, versus Pg of branch perfused with degassed water (200 kPa, 2 h). The offset from the 1 : 1 relationship is caused by the non‐zero balance pressure of the sample. (curve B) Xylem pressure of plant material rehydrated in a beaker for 2 h. (curve C) Xylem pressure of plant material at lower water potential. The thin diagonal line is the reference 1 : 1 relationship. Each curve consists of 300–360 points and all pressures are absolute. Fig. 4. View largeDownload slide Typical relationships between chamber pressure and xylem pressure of Tsuga branches when the whole branch was placed inside the chamber. Inset graph: chamber gas pressure, Px versus time. Main graph: (curve A) xylem pressure, Px, versus Pg of branch perfused with degassed water (200 kPa, 2 h). The offset from the 1 : 1 relationship is caused by the non‐zero balance pressure of the sample. (curve B) Xylem pressure of plant material rehydrated in a beaker for 2 h. (curve C) Xylem pressure of plant material at lower water potential. The thin diagonal line is the reference 1 : 1 relationship. Each curve consists of 300–360 points and all pressures are absolute. Fig. 5. View largeDownload slide (A) Similar to the experiment in Fig. 3 (curve B). The thin diagonal line is the 1 : 1 relationship. The solid line gives Px versus Pg for a branch collected in the field and rehydrated for 2 h in a beaker; the line contains 360 points. The dotted line is the theoretical fit from Equation (8). The dashed line is the theoretical fit from Equation (9). (B) Pressure–volume curve measured on the same branch after the experiment shown in (A). Balance pressure points are relative to atmospheric. Note the non‐zero balance pressure, which indicates sample dehydration. Fig. 5. View largeDownload slide (A) Similar to the experiment in Fig. 3 (curve B). The thin diagonal line is the 1 : 1 relationship. The solid line gives Px versus Pg for a branch collected in the field and rehydrated for 2 h in a beaker; the line contains 360 points. The dotted line is the theoretical fit from Equation (8). The dashed line is the theoretical fit from Equation (9). (B) Pressure–volume curve measured on the same branch after the experiment shown in (A). Balance pressure points are relative to atmospheric. Note the non‐zero balance pressure, which indicates sample dehydration. Table 1. Densities (g cm−3) of Tsuga stems (without bark) from different conditions All errors are standard deviations. Stem densities were measured on wood samples of 3–4 mm diameter except the vacuum‐treated samples which is the mean of equal numbers of samples 1, 2, 3, and 4 mm diameter. Maximum stem density of wood with no air was assumed to be between 1.21 and 1.23 g cm−3. Sample condition   Density±SD (n)   Fresh  1.139±0.011 (20)  Rehydrated in beaker for 2 h  1.150±0.001 (14)  Perfused 200 kPa for 2 h  1.161±0.008 (14)  Perfused 500 kPa for 5 h  1.207±0.011 (7)  Vacuum for 3 d   1.234±0.039 (15)   Sample condition   Density±SD (n)   Fresh  1.139±0.011 (20)  Rehydrated in beaker for 2 h  1.150±0.001 (14)  Perfused 200 kPa for 2 h  1.161±0.008 (14)  Perfused 500 kPa for 5 h  1.207±0.011 (7)  Vacuum for 3 d   1.234±0.039 (15)   The P value between density‐fresh and density‐rehydrated was 0.043. The P values of any other pairing were <0.01. View Large Table 2. End values of Px at Pg=700 kPa under various conditions for Tsuga and Nicotiana and Tsuga wood volume All errors are standard deviations. All pressures in kPa and volume in ml.   Tsuga  Tsuga  Nicotiana    (perfused)   (rehydrated)     Experiment as in Fig. 1A  Px@Pg=700  502±44  422±65  512±44    (n=10)  (n=11)  (n=10)  Volume of Tsuga wood in  4.48±0.22  typical branch 22 g FW  (n=7)  Experiment as in Fig. 1C  Px@Pg=700  689±12  392±53  677±6    (n=10)   (n=9)   (n=12)     Tsuga  Tsuga  Nicotiana    (perfused)   (rehydrated)     Experiment as in Fig. 1A  Px@Pg=700  502±44  422±65  512±44    (n=10)  (n=11)  (n=10)  Volume of Tsuga wood in  4.48±0.22  typical branch 22 g FW  (n=7)  Experiment as in Fig. 1C  Px@Pg=700  689±12  392±53  677±6    (n=10)   (n=9)   (n=12)   View Large Discussion The difference between the two experimental set‐ups (Fig. 1A, C) was that in the first one the stem segment outside the bomb underwent expansion, while in the second one the stem may have contracted. The portion of the stem or petiole outside the bomb expanded because Px exceeded atmospheric pressure. This would cause a mechanical expansion of dead tissues and an increase of turgor pressure and expansion of living tissues because an increase of Px above atmospheric would cause positive water potentials in living cells. The swelling was much more for soft tissues (Nicotiana petioles) than lignified tissues (Tsuga stems), as would be expected. The portion of the stem inside the bomb presumably contracted whenever Px<Pg, although there was no way of measuring the contraction with the instruments available. With the whole plant material placed in the chamber, it was possible to obtain a 1 : 1 relationship between xylem pressure and chamber pressure (Fig. 5, curve A) when there was no air in the wood. In this case Px≅Pg so there was little contraction possible. The Tsuga stem (Fig. 4, curve B) must have had some air in it because the stem density was <1.21 gm cm−3, consequently water had to flow from the symplast to the apoplast to compress the air bubbles as Px increase so Px=Pg−Pb. Thus either the air content in the plant material or the expansion of the stem segment, or both, could cause the non 1 : 1 relationship. The effects of these two factors can be quantified separately and in combination through the following theoretical considerations. Let Pb=balance pressure=Pg−Px, Ve=volume of water expressed from the symplast (living cells), Vs=the volume increase of the stem segment due to swelling, Va=the volume of air in the wood (inside plus outside the bomb), and Pa=the pressure of the air in the wood. Three cases will be considered. Case 1: No air in the wood but the stem segment swelled This case is similar to Fig. 1A with perfused samples. From Fig. 2 it is justifiable to assume a linear relationship between Pb and Ve:   \[V_{e}{=}m_{1}P_{\mathrm{b}}{=}m_{1}{(}P_{g}{-}P_{\mathrm{x}}{)}\ {(}1{)}\] Assuming a linear relationship between Px and the increase of stem volume (Vs) outside the bomb:   \[V_{\mathrm{s}}{=}m_{2}P_{\mathrm{x}}{(}2{)}\] If there is no air in the wood, the volume increased due to swelling would be occupied by the water expressed from living cells, i.e. Vs=Ve. Thus m2Px=m1(Pg−Px) and after solving for Px the following is obtained.   \[P_{\mathrm{x}}{=}P_{\mathrm{g}}\frac{m_{1}}{m_{1}{+}m_{2}}\ {(}3{)}\] Because the ratio m1/(m1+m2) was always <1, the xylem pressure would then be always lower than chamber pressure. The mean slope in Fig. 2, m1, was 1.19×10−3 cm3 kPa−1. Typically 40 mm of a 4 mm diameter stem remained outside the bomb, so the volume change of 6% at Px=700 kPa corresponded to m2=5×10−5 cm3 kPa−1. Thus equation (3) predicts Px=672 kPa when Pg=700. Table 2 showed that the measured Px was 502±44 kPa. Hence more than stem expansion must be assumed to account the non 1 : 1 relationship between Px and Pg. Since the density of these samples was 1.16 gm cm−3 compared to a maximum density of 1.22, the present study had to account for the effect of air in the xylem. Case 2: Air in wood but no stem expansion This case is similar to conditions that might exist in experiments with the PT2 inside the bomb (Fig. 1C) with rehydrated branches. As Px increases some air would dissolve, but it is assumed over the time‐course of the experiment, that the loss of number of moles of air in air bubbles would be small because stem density changed little (see Results). So the volume of air would be given by the ideal gas law:   \[P_{\mathrm{a}}V_{\mathrm{a}}{=}{P_{a}^{{\ast}}}{V_{a}^{{\ast}}}{=}nRT\ {(}4{)}\] Where \(P_{a}^{{\ast}}\) and \(V_{a}^{{\ast}}\) are the initial air pressure and air volume in the wood, respectively. \(P_{a}^{{\ast}}\) might be less than atmospheric pressure if non‐equilibrium conditions apply in recently embolized xylem, but most likely \(P_{a}^{{\ast}}\) will be greater than atmospheric pressure because of capillary compression of the air bubbles and other possible effects that need not be considered here. In Equation 4, n=the number of moles of the air, R=the gas constant, and T=the Kelvin temperature. The increasing Px would gradually compress the air bubbles. Since it was assumed in case 2 that the stem segment did not expand, the air volume decrease, Va*−Va, must be totally occupied by the water expressed from living cells. Hence   \[V_{\mathrm{e}}{=}{V_{a}^{{\ast}}}{-}V_{a}{\cong}{V_{a}^{{\ast}}}{(}1{-}\frac{{P_{a}^{{\ast}}}}{\mathit{P}_{\mathrm{a}}}{)}\] Using Equation (1) to substitute for Ve and neglecting surface tension effects we can substitute Pa≅Px giving:   \[m_{1}{(}P_{g}{-}P_{x}{)}{=}{V_{a}^{{\ast}}}{(}1{-}\frac{{P_{a}^{{\ast}}}}{\mathit{P}_{a}}{)}\] which can be written in the form of a quadratic equation:   \[m_{1}{P_{x}^{2}}{+}{(}{V_{a}^{{\ast}}}{-}m_{1}P_{\mathrm{g}}{)}P_{x}{-}{V_{a}^{{\ast}}}{P_{a}^{{\ast}}}{=}0\ {(}5{)}\] Hence,   \[P_{\mathrm{x}}{=}\frac{{(}m_{1}P_{\mathrm{g}}{-}{V_{a}^{{\ast}}}{)}{+}\ \sqrt{{(}m_{1}P_{\mathrm{g}}{-}{V_{a}^{{\ast}}}{)}^{2}{+}4m_{1}{V_{a}^{{\ast}}}{P_{a}^{{\ast}}}}}{2m_{1}}\ {(}6{)}\] In the limiting case as Pg grows large, eventually (m1Pg− \(V_{a}^{{\ast}}\) )2 would be much larger than 4m1 \(V_{a}^{{\ast}}\) \(P_{a}^{{\ast}}\) , and hence   \[P_{\mathrm{x}}{\rightarrow}P_{g}{-}\frac{{V_{a}^{{\ast}}}}{m_{1}}\ {(}7{)}\] This explains why air bubbles in the plant tissue could cause the non 1 : 1 relationship, i.e. xylem pressure would be lower than chamber pressure by \(V_{a}^{{\ast}}\) /m1. A Tsuga branch rehydrated in a beaker for 2 h had a typical wood volume of 4.48 cm3 and a density of 1.15 gm cm−3 which was 0.07 less than the two estimates of maximum density in Table 1. From this \(V_{a}^{{\ast}}\) =0.07×4.48=0.314 cm3 is estimated. From Fig. 2, a value of m1=1.19×10−3 cm3 kPa−1 can be assigned. Putting these values into Equation (6) with \(P_{a}^{{\ast}}\) =100 and Pg=700 yields a value of Px=364 kPa. This should be compared to an observed value of about 400 for rehydrated samples (Table 2). Comparisons between theory and observed values are dependent on many uncertainties of measurements. The predictions of Equation (6) depend very strongly on small difference in density measurements (initial minus maximum). Initial and maximum densities were not measured on the same samples. Also water potential isotherms used to estimate m1 were measured on similar, but not the same, samples used to measure the curves in Figs 3 and 4. In order to address this problem experiments were repeated in which all parameters were measured on the same shoot (see below). Case 3: Both stem expansion and air in wood When both stem expansion and air in wood are taken into account the following quadratic solution results:   \[P_{\mathrm{x}}{=}\frac{{(}m_{1}P_{g}{-}{V_{a}^{{\ast}}}{+}\sqrt{{(}m_{1}P_{g}{-}{V_{a}^{{\ast}}}{)}^{2}{+}4m_{1}{V_{a}^{{\ast}}}{P_{a}^{{\ast}}}}}{2{(}m_{1}{+}m_{2}}{(}8{)}\] A sensitivity analysis of Equation (8) has revealed that bubble compression in the xylem is more important than stem swelling causing a deviation from the 1 : 1 relationship of Pg : Px in experiments as in Fig. 1A. When the stem density of Tsuga is 1.15, about 16 times as much water has to be displaced from the symplast to apoplast to cause bubble compression than tissue expansion outside the pressure bomb. In an attempt to get better quality data to input into Equation (8), the experiments were repeated in a way that permitted measurement of all parameters on the same shoot. When this was done, a previously unnoticed effect was observed, i.e. loss of water from the sample during the measurement of Px versus Pg. The loss was probably due to evaporation since liquid water was rarely noted around the portion of stem outside the bomb. The loss of water was confirmed by weight change of the sample. The dotted line in Fig. 5 is the solution of Equation 8 taking into account both stem expansion and air bubble compression. It can be seen that Equation 8 underestimated the deviation between Px and Pg. However, assuming that water evaporated at a uniform rate during the experiments, it was possible to get a reasonable agreement between theoretical and experimental results. The equation that applies to the case in which an additional water loss is given by kt, where k=rate of water loss (g s−1) and t=time is given in Equation 9.   \[p_{x}{=}\frac{{(}m_{1}P_{g}{-}{V_{a}^{{\ast}}}{+}kt{)}{+}\sqrt{{(}m_{1}P_{g}{-}{V_{a}^{{\ast}}}{)}^{2}{+}4m_{1}{V_{a}^{{\ast}}}{P_{a}^{{\ast}}}}}{2{(}m_{1}{+}m_{2}{)}}\ {(}9{)}\] Conclusion (1) Generally Px<Pg in the positive pressure range even when the initial Pb 0 in Tsuga shoots and Nicotiana leaves. (2) The reasons for Px being generally lower than Pg is that air bubbles in the xylem tissue are compressed and the portion of the stem or petiole outside the pressure bomb swells as Px increases. These two effects require the displacement of water from the symplast to apoplast. The displacement of water dehydrates the symplast raising the balance pressure of the symplast such that Px=Pg−Pb. In addition, in some cases there can be evaporation of water from the sample. (3) In the traditional pressure bomb technique, the cut end of stem or petiole is not sealed off with a pressure transducer so Px cannot rise to positive values. Thus the stem cannot expand and air present in xylem cannot be compressed. Therefore the non 1 : 1 relationship in the positive pressure range observed (Balling and Zimmermann, 1990) does not invalidate the pressure bomb technique for the determination of Px in the negative pressure region. With proper care evaporation can also be reduced to negligible levels during pressure bomb measurements. (4) Deviations of Px from what is expected by pressure bomb theory when Px is in the negative pressure range were also reported (Balling and Zimmermann, 1990). The reason such deviations have been addressed in recent publications and may be explained with the current understanding of pressure bomb theory (Melcher et al., 1998, Wei et al., 1999). Although most deviations between the pressure bomb and the pressure probe have been explained, some important deviations remain to be explained. For example, a pressure probe was inserted in the petiole xylem of a tobacco leaf while the leaf blade was inside a pressure bomb and a 1 : 1 relationship between Px and Pg was not found (Balling and Zimmermann, 1990). These experiments need to be reproduced; however, two independent methods have been used to confirm the measurement of negative Px by the pressure bomb (Dixon and Tyree, 1984; Holbrook et al., 1995). For the moment the pressure bomb can continue to be used to estimate Px with reasonable confidence. However, there must also be an element of caution in the interpretation of pressure bomb data and a reanalysis may be necessary if independent techniques, e.g. involving NMR spectroscopy, should ever reveal deviations from classical pressure bomb theory (Ulrich Zimmermann, personal communication). 3 Present address and to whom correspondence should be sent: USDA Forest Service, Aiken Forestry Sciences Lab, PO Box 968, S. Burlington, VT 05402, USA. Fax: +1 802 951 6368. E‐mail:MelTyree@aol.com References Balling A, Zimmermann U. 1990. Comparative measurements of the xylem pressure of Nicotiana plants by means of the pressure bomb and probe. Planta  182, 325–345. Google Scholar Begg JE, Turner NC. 1970. Water potential gradients in field tobacco. Plant Physiology  46, 343–346. Google Scholar Dixon MA, Tyree MT. 1984. A new temperature‐corrected stem hygrometer and its calibration against the pressure bomb. Plant, Cell and Environment  7, 693–697. Google Scholar Holbrook NM, Burns MJ, Field CB. 1995. Negative xylem pressures in plants: a test of the balancing pressure technique. Science  270, 1193–1194. Google Scholar Melcher PJ, Meinzer FC, Yount DE, Goldstein G, Zimmermann U. 1998. Comparative measurements of xylem pressure in transpiring and non‐transpiring leaves by means of the pressure chamber and the xylem pressure probe. Journal of Experimental Botany  49, 177–1760. Google Scholar Scholander PF, Hammel HT, Bradstreet ED, Hemmingsen EA. 1965. Sap pressure in vascular plants. Science  148, 339–346. Google Scholar Tyree MT, Sperry JS. 1989. Vulnerability of xylem to cavitation and embolism. Annual Review of Plant Physiology  40, 19–38. Google Scholar Yang S, Tyree MT. 1992. A theoretical model of hydraulic conductivity recovery from embolism with comparison to experimental data on Acer saccharum. Plant Cell and Environment  15, 633–643. Google Scholar Wei C, Tyree MT, Steudle E. 1999. Direct measurement of xylem pressure in leaves of intact maize plants: a test of the cohesion‐tension theory taking account of hydraulic architecture. Plant Physiology  (in press). Google Scholar © Oxford University Press
TI - The transmission of gas pressure to xylem fluid pressure when plants are inside a pressure bomb
JF - Journal of Experimental Botany
DO - 10.1093/jexbot/51.343.309
DA - 2000-02-01
UR - https://www.deepdyve.com/lp/oxford-university-press/the-transmission-of-gas-pressure-to-xylem-fluid-pressure-when-plants-yulVvLF02R
SP - 309
EP - 316
VL - 51
IS - 343
DP - DeepDyve
ER -