Journal of Experimental Botany, Vol. 51, No. 343, pp. 309–316, February 2000
The transmission of gas pressure to xylem fluid pressure
when plants are inside a pressure bomb
C. Wei1, M.T. Tyree1,3 and J.P. Bennink2
1 Department of Botany, University of Vermont, Burlington, VT 05405, USA
2 USDA Forest Service, Aiken Forestry Sciences Laboratory, PO Box 968, S. Burlington, VT 05402, USA
Received 13 May 1999; Accepted 7 October 1999
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 (P ) in the bomb to the xylem fluid (P ). Pressure
g
x
bomb theory would predict a 151 relationship for P :P
g x
when tobacco leaves start at a balance pressure of
zero. Failure to observe the expected 151 relationship
has cast doubt on the pressure-bomb technique in the
measurement of the xylem pressure of plants.
The experimental and theoretical relationship
between P and P was investigated in Tsuga canadx
g
ensis (L) branches and Nicotiana rustica (L) leaves in
this paper. It is concluded that the non 151 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 151 relationship could
be obtained when xylem embolism was eliminated and
stem expansion prevented. It is argued that the non
151 relationship in the positive pressure range does
not invalidate the Scholander pressure bomb method
of measuring xylem pressure in plants because P
x
never reaches positive values during the determination
of the balance pressure.
Key words: 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
P and gradients of water potential in living cells in the
x
shoot will dissipate given sufficient time. The value of P
x
ultimately measured in the pressure bomb is determined
by the equilibrium water potential achieved by the shoot
after the gradients equalize; at this point P is equal to
x
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 P in vessels can be
x
increased by applying a gas pressure, P , to the shoot
g
within the bomb. For every unit increase in P , P will
g x
become less negative by one unit until the balance pressure
(P ) is reached when water is ‘balanced’ on the cut end
b
(at this moment the P =0). The rise of P to a value of
x
x
zero can be expressed as P =P –P . Thus the gas pressure
x
g b
P needed to force water just to appear at the cut end is
g
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
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: [email protected]
© Published by Oxford University Press
310 Wei et al.
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 P directly in xylem
x
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
P should be zero prior to admitting gas into the pressure
x
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. P from 0 to
x
700 kPa. The response of this pressure transducer (PT2
in Fig. 1A) to increasing chamber pressure was not 151
in Balling and Zimmermann (Balling and Zimmermann,
Fig. 1. 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 P <−100 kPa the bubble occupies
x
the entire volume of the tracheid, but when P >0 the bubble is
x
compressed by displacement of fluid from the water-filled tracheids to
the embolized tracheid.
1990); under most situations the value of P measured by
x
the pressure transducer was lower than P (measured with
g
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 P and P in the
g
x
positive pressure range and to determine if this disagreement negates the use of the pressure bomb for the
determination of P under normal circumstances.
x
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 P will
g
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 P equal to P because if the water
g
b
were removed from the tube then the P required to bring
g
water to the surface of the stem or petiole would be P
b
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 P increases then the symplast will not
g
dehydrate because there would be nowhere for the water
to flow. Under these special conditions, the pressure
bomb hypothesis would make us predict that P =P .
x
g
But, if the volume of water in the apoplast should increase
then the symplast would be dehydrated and P would be
x
less then P and equal to P −P . Xylem tissue is freg
g
b
quently 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 P increases >0 and hence the
x
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 P is increased causing a
g
corresponding dehydration (reduction in volume) of the
symplast because of (1) pressure-induced expansion of
the tissues outside the pressure bomb and (2) pressureinduced compression of air bubbles in the xylem tissue
anywhere in the leaf or shoot. As P increases the xylem
x
Xylem pressure measurement using a pressure bomb 311
vessels outside the bomb would increase in volume
because the pressure difference between the vessels and
outside air would increase. As P increases the water
x
potential of living cells would become positive causing an
increase in turgor pressure and cell volume. As P
x
increases the volume of any air bubbles trapped in
embolized vessels would decrease until the pressure of the
gas bubbles $P , the relationship between volume change
x
and gas pressure would be given approximately by the
ideal gas law. Hence, as the value of P increases water
x
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 P and make
b
P less then P .
x
g
This study will provide the mathematical analysis to
predict the magnitude of the deviation of P from P and
x
g
will show that P =P under experimental conditions
x
g
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 25251 (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.
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
(P ) of <20 kPa when measured in a Scholander–Hammel
b
pressure bomb. Nicotiana leaves were at P <10 kPa 8 h after
b
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 P . The water was expressed
b
from the stem by over-pressurizing the stems beyond P and
b
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 P was measured. Cumulative water loss was
b
plotted versus P .
b
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, P , and xylem fluid pressure, P )
g
x
were measured with model P 136GV100 pressure transducers
X
(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, P , was increased linearly with
g
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 P and P was recorded every 2 or 3 s. A
g
x
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 P increased
x
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 P >700 kPa and were placed entirely within the pressure
b
bomb as in Fig. 1C and pressurized to P =700 kPa. This was
g
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
312 Wei et al.
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 P
b
below 20 kPa.
(3) The pressure transducer PT2 was mounted on the base of
the stem (Fig. 1A or C ).
(4) P versus P was measured while ramping P from 0 to
x
g
g
800 kPa over 1500 s.
(5) P was returned to zero; the balance pressure was measured
g
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.
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
Rehydrated in beaker for 2 h
Perfused 200 kPa for 2 h
Perfused 500 kPa for 5 h
Vacuum for 3 d
1.139±0.011
1.150±0.001
1.161±0.008
1.207±0.011
1.234±0.039
(20)
(14)
(14)
(7)
(15)
The P value between density-fresh and density-rehydrated was 0.043.
The P values of any other pairing were <0.01.
Representative balance pressure isotherms are shown
in Fig. 2. The relationship between volume expressed
versus balance pressure was linear for P <500 kPa. The
b
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, P , and the xylem fluid, P , in Tsuga stems
g
x
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 P to P had much to do with the P of the
x
g
b
Results
Although the two different rehydation procedures used
on Tsuga both succeeded in making P @0, the procedures
b
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.
Fig. 2. 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
P <500 kPa where water expressed was a linear function of P .
b
b
Differences in slope probably reflect differences in mass of fresh leaf
material attached to each branch (not measured ).
Xylem pressure measurement using a pressure bomb 313
Fig. 3. 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, P , versus time. Main graph: (curve A) xylem
g
pressure, P , of Tsuga branch perfused with degassed water (200 kPa,
x
2 h) versus P during linear pressurization; (curve B) P of a Tsuga
g
x
branch rehydrated in a beaker for 2 h; (curve C ) Measurement done
on Tsuga branch with P >700 kPa. Each curve consists of 300–360
b
points and all pressures are absolute.
shoot as well as how the plant material was rehydrated.
When P reached 700 kPa, P was 422±65 kPa in
g
x
branches rehydrated in a beaker for 2 h, and P was
x
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 P from 0 to 700 kPa, the
x
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 150.967 for Nicotiana leaves
and 150.984 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 waterstressed 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 P versus P . Extrapolation of the curve
x
g
Table 2. End values of P at P =700 kPa under various conditions
x
g
for Tsuga and Nicotiana and Tsuga wood volume
All errors are standard deviations. All pressures in kPa and volume in ml.
Experiment as in Fig. 1A
P @P =700
x
g
Volume of Tsuga wood in
typical branch 22 g FW
Experiment as in Fig. 1C
P @P =700
x
g
Tsuga
(perfused)
Tsuga
(rehydrated)
Nicotiana
502±44
(n=10)
4.48±0.22
(n=7)
422±65
(n=11)
512±44
(n=10)
689±12
(n=10)
392±53
(n=9)
677±6
(n=12)
Fig. 4. 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, P versus time. Main
x
graph: (curve A) xylem pressure, P , versus P of branch perfused with
x
g
degassed water (200 kPa, 2 h). The offset from the 151 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 151 relationship. Each curve consists of
300–360 points and all pressures are absolute.
314 Wei et al.
tissues (Tsuga stems), as would be expected. The portion
of the stem inside the bomb presumably contracted whenever P <P , although there was no way of measuring
x
g
the contraction with the instruments available.
With the whole plant material placed in the chamber,
it was possible to obtain a 151 relationship between xylem
pressure and chamber pressure ( Fig. 5, curve A) when
there was no air in the wood. In this case P $P so there
x
g
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 P increase so P =P −P .
x
x
g
b
Thus either the air content in the plant material or the
expansion of the stem segment, or both, could cause the
non 151 relationship. The effects of these two factors can
be quantified separately and in combination through the
following theoretical considerations. Let P =balance
b
pressure=P −P , V =volume of water expressed from
g
x e
the symplast ( living cells), V =the volume increase of the
s
stem segment due to swelling, V =the volume of air in
a
the wood (inside plus outside the bomb), and P =the
a
pressure of the air in the wood. Three cases will be
considered.
Case 1: No air in the wood but the stem segment swelled
Fig. 5. (A) Similar to the experiment in Fig. 3 (curve B). The thin
diagonal line is the 151 relationship. The solid line gives P versus P
x
g
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.
back to atmospheric pressure suggested a net weight loss
of 0.2 g. Tissue dehyration was confirmed by weighing
shoots before and after measuring P versus P .
x
g
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
P exceeded atmospheric pressure. This would cause a
x
mechanical expansion of dead tissues and an increase of
turgor pressure and expansion of living tissues because
an increase of P above atmospheric would cause positive
x
water potentials in living cells. The swelling was much
more for soft tissues (Nicotiana petioles) than lignified
This case is similar to Fig. 1A with perfused samples.
From Fig. 2 it is justifiable to assume a linear relationship
between P and V :
b
e
V =m P =m (P −P )
(1)
e
1 b
1 g
x
Assuming a linear relationship between P and the
x
increase of stem volume (V ) outside the bomb:
s
V =m P
(2)
s
2 x
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. V =V . Thus m P =m (P −P )
s
e
2 x
1 g
x
and after solving for P the following is obtained.
x
m
1
(3)
P =P
x
g m +m
1
2
Because the ratio m /(m +m ) was always <1, the
1 1
2
xylem pressure would then be always lower than chamber pressure. The mean slope in Fig. 2, m , was
1
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 P =700 kPa corresponded to m =
x
2
5×10−5 cm3 kPa−1. Thus equation (3) predicts P =
x
672 kPa when P =700. Table 2 showed that the measured
g
P was 502±44 kPa. Hence more than stem expansion
x
must be assumed to account the non 151 relationship
between P and P . Since the density of these samples
x
g
was 1.16 gm cm−3 compared to a maximum density of
Xylem pressure measurement using a pressure bomb 315
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 P increases some air would
x
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 V =P*V*=nRT
(4)
a a
a a
Where P* and V* are the initial air pressure and air
a
a
volume in the wood, respectively. P* might be less than
a
atmospheric pressure if non-equilibrium conditions apply
in recently embolized xylem, but most likely P* will be
a
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 P would gradually compress the air
x
bubbles. Since it was assumed in case 2 that the stem
segment did not expand, the air volume decrease,
V *−V , must be totally occupied by the water expressed
a
a
from living cells. Hence
A
P*
V =V*−V $V* 1− a
e
a
a
a
P
a
B
Using Equation (1) to substitute for V and neglecting
e
surface tension effects we can substitute P $P giving:
a
x
P*
m (P −P )= V* 1− a
1 g
x
a
P
x
which can be written in the form of a quadratic equation:
A
B
m P2+(V*−m P )P −V*P*=0
1 x
a
1 g x
a a
(5)
Hence,
(m P −V*)+√(m P −V*)2+4m V*P*
1 g
a
1 g
a
1 a a (6)
2m
1
In the limiting case as P grows large, eventually
g
(m P −V*)2 would be much larger than 4m V*P*, and
1 g
a
1 a a
hence
P=
x
V*
(7)
P P − a
x
g m
1
This explains why air bubbles in the plant tissue could
cause the non 151 relationship, i.e. xylem pressure would
be lower than chamber pressure by V*/m . A Tsuga
a 1
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*=0.07×4.48=0.314 cm3 is estima
ated. From Fig. 2, a value of m =1.19×10−3 cm3 kPa−1
1
can be assigned. Putting these values into Equation (6)
with P*=100 and P =700 yields a value of P =364 kPa.
a
g
x
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 m were measured on similar, but not the
1
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=
x
(m P −V*)+√(m P −V*)2+4m V*P*
1 g
a
1 g
a
1 a a
2(m +m )
1
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 151 relationship of P 5P in experiments as in Fig. 1A. When the
g x
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 P versus P . The loss was
x
g
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 P and P .
x
g
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=
316 Wei et al.
rate of water loss (g s−1) and t=time is given in
Equation 9.
P=
x
(m P −V*+kt)+√(m P −V*+kt)2+4m V*P*
1 g
a
1 g
a
1 a a
2(m +m )
1
2
(9)
Conclusion
(1) Generally P <P in the positive pressure range even
x
g
when the initial P 0 in Tsuga shoots and Nicotiana leaves.
b
(2) The reasons for P being generally lower than P is
x
g
that air bubbles in the xylem tissue are compressed and
the portion of the stem or petiole outside the pressure
bomb swells as P increases. These two effects require the
x
displacement of water from the symplast to apoplast. The
displacement of water dehydrates the symplast raising the
balance pressure of the symplast such that P =P −P .
x
g
b
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 P cannot rise to positive values. Thus the
x
stem cannot expand and air present in xylem cannot be
compressed. Therefore the non 151 relationship in the
positive pressure range observed (Balling and
Zimmermann, 1990) does not invalidate the pressure
bomb technique for the determination of P in the negax
tive pressure region. With proper care evaporation can
also be reduced to negligible levels during pressure bomb
measurements.
(4) Deviations of P from what is expected by pressure
x
bomb theory when P is in the negative pressure range
x
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 import-
ant 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 151 relationship between P and P was not
x
g
found (Balling and Zimmermann, 1990). These experiments need to be reproduced; however, two independent
methods have been used to confirm the measurement of
negative P by the pressure bomb (Dixon and Tyree,
x
1984; Holbrook et al., 1995). For the moment the pressure
bomb can continue to be used to estimate P with
x
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).
References
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