J.PlantPhysiol. Vol. 132.pp. 653-657{1988}
A New Method of Measurement of Changes in Turgor Pressure
in the Mesophyll Cells of Leaves
WITOLD PIWOWARCZYK
Laboratory of Physiology and Biochemistry of Plants, Jan Zurzycki, Institute of Molecular Biology, Jagiellonian
University, Krakow
Received May 18, 1987 . Accepted November 18, 1987
Summary
The aim of this paper is to report a convenient indicator of turgor changes in the mesophyll cells of the
leaves of Vicia /aba L. It has been demonstrated that Young's modulus of the leaf blade being a measure if
its stiffness may be such an indicator. A new method of a fast measurement of the changes in Young's
modulus has been proposed. The new method made it possible to carry out measurements which could
not be realized by other techniques available. In the discussion the presented method is compared with
the commonly applied «pressure probe» of Hiisken et a1. (1978).
As an example of the application of the method the results of measurements of light-induced turgorpressure changes are presented.
Key words: Vicia /aba L., method 0/ measurement, turgor pressure.
Introduction
The turgor-pressure changes of cells may be caused by a
number of factors. These may be external factors such as
water relations in the environment, temperature or light intensity, and internal factors such as ion transport across the
plasmalemma changing the osmotic potential of the protoplast (Piwowarczyk 1986). The rate of the ion exchange may
be modified by such factors as light intensity (Rahat and
Reinhold 1983) and light quality (Blakeley et al. 1983) or the
osmotic potential of the medium (Reinhold et al 1984). In
the case of a mesophyll cell of a leaf it will be the influence of
the osmotic potential of water bound in the cell wall of the
protoplast. The known methods of indirect estimation of
turgor pressure by means of osmotic measurements - i.e.
turgor pressure was determined either from measurements
of the internal and external osmolarity (freezing-point depression measurements) (Kesseler 1959) or calculated from
the osmotic pressure of the external medium - required to
induce «incipient» plasmolysis (Stadelmann 1966). Similarly,
measurements based on the pressure bomb technique (Hellkvist et al. 1974, Cheung et al. 1975), resonance frequency
technique (Virgin 1955, Falk et al. 1958) and the vapor pressure equilibration technique (Boyer and Knipling 1965,
© 1988 by Gustav Fischer Verlag, Stuttgart
Boyer 1966) are not applicable for measuring rapid changes
in turgor caused by the action of external factors.
From among the known methods the best results of the
measurements of turgor changes of mesophyll cells of leaves
seem to be obtained by applying the suitable adjusted technique of the pressure probe (Hiisken et al. 1978, Zimmermann et al. 1980). This very elegant method permits measurement of the value of turgor pressure in a single cell with a
simultaneous recording of the kinetics of the turgor changes.
It consists of the introduction of a microcapillary filled with
oil into the cell and measurement of hydrostatic pressure existing inside the capillary. This pressure corresponds to the
cell turgor pressure. However, when applying this method
to some kinds of investigations of the changes in turgor pressure in the mesophyll cells of the leaves some problems may
appear. For example, the removal of epidermis and the measurement of the changes in turgor pressure of mesophyll cells
must take place under water. This essentially changes the
water relations in the tissue. Moreover, even a brief exposure
of the naked mesophyll cells to the action of air may result in
their damage. The problem becomes still more acute upon
the examination of the effect of high intensities of light, because 99 % of the absorbed light energy changes into heat
and accelerates the drying process.
654
WITOLD PIWOWARCZYK
In the present paper a method of measuring the changes in
the stiffness (Young's modulus) of the leaf blade has been presented. Changes in the stiffness of the leaf blade are a sensitive indicator of turgor changes in the cells. Thus it is possible to regard the changes in stiffness as a function, in
certain areas even a linear one, of the turgor changes in the
cells. An application of the presented indirect method of
measuring the turgor changes is justified when the application of the pressure probe method is not possible.
Material and Methods
Seeds of Vicia /aba L. were soaked in water for 48 h and subsequently placed in plastic pots filled with garden soil. The seedlings
were grown in light provided by fluorescent tubes of the Flora type
produced by Polam. The light intensity was about 20W· m -2. The
photoperiod applied was: 16 h in light and 8 h in darkness. A leaf on
a well-developed plant, about 30 days old, was selected for measurements.
In all experiments the relative humidity of the air was about
80%.
A part of the leaf with large vascular bundles was removed in
order to eliminate the leaf stiffness associated with these bundles.
The left fragment of the leaf blade without the large vascular
bundles was 5 . 10 - 3 m in width and 2· 10 - 2 m in length. This fragment was connected with the central vascular bundle through one
of its shorter sides. This end was immobilized while the other end
remained free. Since the described leaf fragment remained in contact
with the rest of the plant and the central vascular bundle supplied
the examined tissue with water, it remained alive for several weeks.
The free end of the leaf fragment was fastened to the arm of an analytical balance (Fig. 1). The distance between the support point of
the leaf and the point at which the leaf was fastened to the balance
arm was about 1 cm. The free end of the leaf fragment was initially
deformed with a force 1.96 .10- 3 N, and left again for 24 h in darkness for adaptation. During this time the evaporation from the cut
tissue became reduced due to the drying up of the wound edges. The
applied force brought about the bending of the leaf blade by about
3.10- 3 m (see Fig. 1). The bending of the leaf caused slight deformation of the shape of the mesophyll cells and the cells of the epidermis. It has been assumed that external factors modifying the cell
turgor will affect this turgor in a similar way as in the undeformed
cells.
The swings of the balance pointer were recorded automatically.
In the control experiment the fragment of the leaf blade was cut
in such a way that two large vascular bundles bronching off the central vascular bundle passed along its whole length. The leaf blade,
because of its stiffness associated with large bundles, became <<less
sensitive» in comparison with that deprived of large bundles data
not shown.
According to the results of Falk et al. (1958) and Nilsson et al.
(1958) for thin strips of tissue with the cross section less than
1.10- 5 m 2 Hook's law is valid. The examined fragment of the leaf
blade with the thickness S·1O- 2 m has a cross section 2.S·10- 6 m 2 •
Hence it should be assumed that the theory of the beams can be applied here (Landau and Lifszic 1953).
The value of the deflection(u) of the beam at a distance(z) from
the point at which the beam is fastened is given by the formula:
u
f
6E· I
= - - Z2
(31-z)
(1)
where:
I - total length of the beam
f - concentrated force acting on the beam at a distance I
from the point of attachment
E - Young's modulus of the beam material
I - moment of the cross section inertia.
If an experiment is carried out in which Young's modulus is influenced by any factor (x) and in this experiment care is being taken
that the deflection (u) of the beam is constant (by changing the force
acting on the beam), then this means that du = o. Then from formula(1) there follows the differential equation:
dE(x) _ ~
df(x) - f(x)
(2)
The general solution of this equation has the form:
E(x) = c·f(x)
(3)
where (c) is a certain constant.
A close relationship between Young's modulus and turgor can be
obtained by considering a model in which some definite assumptions are made concerning the anatomical structure of the leaf, including Young's and Poisson's moduli for the cell walls. Finding the
relation between the turgor and Young's modulus will require complicated and time-consuming numerical computations. Nevertheless, for small changes of pressure in comparison with the initial
Fig. 1: Mode of fastening the leaf to the arm
of an analytical balance.
Measurement of changes in turgor pressure
pressure, the relationship between turgor and Young's modulus is
linear (Falk et al. 1958, Nilsson et al. 1958). If these assumptions are
accepted, then from (2) it follows that:
P(x)
=
a·f(x) + b
655
flu
10
(4)
where:
P (x) - turgor as a function of the factor x
a, b - constants.
White light treatment procedure
The leaf was illuminated with light provided from a halogen lamp
12 V, 75 W produced by Tungsram. Heat radiation was removed by
means of a CSOS filter (Carl Zeiss, Jena).
4
3
light
j
10
Results and Discussion
A detailed discussion of the relationship between turgor
and the operating external compressive force has been
carried out in their model study on a single cell of Chara by
Steudle et al. (1982). Ferrier and Dainty (1978) when investigating the measurement of the hydraulic conductivity
carried out a discussion of this relationship for a multilayer
tissue. Their investigations concerned a relatively simple
system such as represented by the fragments of the storage
tissue of red beet and artichoke. A theoretical analysis of similar relations in a complex tissue of a leaf blade would be
much more complicated, hence the present author has restricted himself to give an outline of the participation of the
particular tissues of a leaf, bent as in Fig. 1, in transferring external stresses, only at the first approximation.
It can be assumed that the cells of palisade parenchyma will
contribute more to Young's modulus than the spongy parenchyma on account of more compact packing. It appears that
the lower epidermis on account of its high stiffness, independently of the value of turgor, will transfer most of the
tensile stresses. On the other hand, in case of the upper epidermis, due to the presence of plane external walls, it may be
expected that it will not be very active in transferring the external compressive stresses. Turgor increase in cells of this
type will make the external walls convex and thereby reduce
their longitudinal dimensions which will manifest itself as
shrinking of the epidermis. This has been confirmed by observations (data not published) that fragments of the leaf
blade of Vicia faba with the lower epidermis removed, when
placed in a hypotonic solution bend their edges towards the
upper epidermis. The participation of spongy parenchyma
in transferring the forces will be small on account of the
small contact surface between the cells and the spatial position between the palisade parenchyma and the lower epidermis. Hence it appears that the observed changes in the
stiffness of the leaf blade are mainly the result of turgor
changes in the cells of the palisade mesophyll.
The applied method enables both the measurement of the
force needed to stop the turgor-induced change in the shape
of the leaf blade already bent, as well as the measurement of
the extent of the turgor-induced deflection of a fragment of
the leaf blade at constant force applied. It has been found,
20
30
40
50
60
time (s)
Fig. 2: Typical recordings of changes in the leaf turgor pressure ~ P
under the influence of white light. u-axis - relative values;
1O",,7.31O - 4 N. This axis illustrates the magnitude of the force
needed to stop the movement of the balance pointer, i.e. changes in
Young's modulus of the leaf blade caused by the turgor changes.
Light intensity = 1000W , m - 2 and SOW· m- 2 .
moreover, that the magnitude of the deflection is directly
proportional to the value of the operating force (data not
published), hence these values may be considered exchangeable.
Fig. 2 shows recordings which illustrate the effect of white
light on the stiffness of the leaf blade determined by the new
method.
The results obtained indicate that not later than 2 s after
exposure to light the changes in the turgor pressure in the
mesophyll cells of the leaves of Vicia faba are detectable
(Fig. 2).
After 20 s exposure to white light with the intensity of
50 - 1000 W . m - 2 of a fragment of leaf blade 5 . 10 - 3 m wide
and 5.10 - 4 m thick, when the distance between the supporting point and the point of attachment is about 1'10 - 2 m, the
force acting on the balance arm equals about 6.8 · 10 - sN for
50 W . m- 2 and 7.3' 10 - 4 N for 1000 W . m-2 (Fig. 3). It was
impossible to obtain such results by means of the former
methods.
Independently of the light intensity, the turgor pressure increase in the leaf cells did not take place in a uniform way
but stepwise (Fig. 2).
The half-time of relaxation after switching off the light
varied; it ranged from about 15 s after switching off white
light of the intensity 1000W· m - 2 up to about lOs after
switching off the light of 250W'm - 2 (data not shown) intensity. Evaluation of this time for lower light intensities
using the recordings would be within too great an error.
The force needed to stop the swing of the balance pointer
after the first 20 s of irradiation was arbitrarily adopted for
calculations. This proved more convenient then calculations
from the flattened phase while illustrating the kinetics of
turgor increment equally well.
Within a certain range of the light intensity (from about 50
up to about 500 W' m - 2) the magnitude of the swing ampli-
656
WlTOLD PrwOWARCZYK
5
500
1000 Wm-
2
Fig. 3: Effect of the intensity of white light on the turgor pressure
changes d P in the leaf tissue. u-axis - relative values;
10:::::7.310- 4 N . This axis illustrates magnitude of the force needed
to stop the movement of the balance pointer, i.e. changes in
Young's modulus of the leaf blade, caused by the changes in the leaf
turgor. Measurements were taken after 20 s continuous light. Standard deviation is marked. n = 5.
tude of the balance pointer was directly proportional to the
intensity of this light (Fig. 3). The deviation from linearity
did not appear until 500 W . m -2.
Whether the leaf was illuminated from above or from
below, the direction of leaf blade response remained the
same.
The method presented enables very accurate measurement
of the small turgor changes in the mesophyll cells of leaves
expressed in relative units, hence the application of this
method is justified when the application of the measurement
methods of absolute values is not possible or unnecessary.
To calculate absolute values it would require a more precise recognition of the physical properties of a leaf blade or
scaling by comparing at least two of the measured values (linear progress) over the range O-SOOW · m- 2 with corresponding values obtained using the pressure probe. In spite
of these disadvantages the method described has many advantages over the other methods available:
1. It permits measurement of turgor changes in the leaf
tissue in its physiological condition.
2. It eliminates the necessity of disturbing the continuity of
the plasmalemma caused by its perforation with the microcapillary. Although Husken et al. (1978) pointed out that the
pressure probe method has no effect on the cell physiology
as the damaged surface is small, however, in my opinion,
such an effect should be excluded separately with respect to
each examined factor causing a change of turgor pressure.
3. It permits measurement of the increment of the turgor
pressure in mesophyll cells without disturbing the continuity of the epidermis - this being of great importance because
of the drying up of tissue.
4. It permits measurement of the effect of very high intensities of light on the increase of turgor in mesophyll cells
without the risk of damaging them (see Introduction).
5. It eliminates the necessity to carry out measurements of
the changes in the turgor pressure of mesophyll cells in an
aquatic medium, which is necessary if the pressure probe
method is used.
6. It permits measurement of the turgor of small cells. For
the pressure probe the measurement in cells the volume of
which is small, presents problems if the volume is close to
the lower limit of detection of about 10- 2 nl (10- 14 m 3) because of the resistance of the capillary tip which has to be
kept small for the small cells (Zimmermann et al. 1980).
7. It permits carrying out of repeated, even hundreds of
measurements of turgor changes induce by different factors
on the same object for a number of days, which allows reduction of the number of repetitions necessary for statistic
purposes. The authors of the pressure probe make it clear
that their method permits measurement of the kinetics of
turgor changes in one object three times at the most (Zimmermann et al. 1980).
8. Finally it is an extremely simple method.
The effect of light on the increase of turgor in mesophyll
cells of the leaves of Vicia /aba is merely an illustration of the
proposed method which might be successfully applied for
the measurement of turgor changes caused by other factors
such as: transpiration, activity of hormones, disturbances in
water transport, or the influence of the soil solution, i.e. its
molarity and composition. For this reason a detailed discussion of the effect of light on turgor pressure changes including the results of additional investigations will be carried out
in a separate paper (Piwowarczyk unpublished).
Acknowledgements
The author wishes to express his thanks to Prof. Dr. hab. Stanislaw Wi,.ckowski for his encouraging interest in this work, and to
Doc. Dr. hab. Jozef Misiek for consultation of the physical part of
his work. He also wishes to express his thanks to both referees of
the paper for their valuable remarks.
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