Plant Physiol. (1999) 119: 775-784
Separating Growth from Elastic Deformation during
Cell
Enlargement1
Timothy E. Proseus,
Joseph K.E. Ortega, and
John S. Boyer*
College of Marine Studies, University of Delaware, Lewes, Delaware
19958 (T.E.P., J.S.B.); and Department of Mechanical Engineering,
University of Colorado, Denver, Colorado 80224 (J.K.E.O.)
 |
ABSTRACT |
Plants change size by deforming
reversibly (elastically) whenever turgor pressure changes, and by
growing. The elastic deformation is independent of growth because it
occurs in nongrowing cells. Its occurrence with growth has prevented
growth from being observed alone. We investigated whether the two
processes could be separated in internode cells of Chara
corallina Klien ex Willd., em R.D.W. by injecting or removing
cell solution with a pressure probe to change turgor while the cell
length was continuously measured. Cell size changed immediately when
turgor changed, and growth rates appeared to be altered. Low
temperature eliminated growth but did not alter the elastic effects.
This allowed elastic deformation measured at low temperature to be
subtracted from elongation at warm temperature in the same cell. After
the subtraction, growth alone could be observed for the first time.
Alterations in turgor caused growth to change rapidly to a new, steady
rate with no evidence of rapid adjustments in wall properties. This
turgor response, together with the marked sensitivity of growth to
temperature, suggested that the growth rate was not controlled by inert
polymer extension but rather by biochemical reactions that include a
turgor-sensitive step.
| |
INTRODUCTION |
This study was undertaken to determine whether growth can be
distinguished from elastic deformation when plants enlarge. Both processes are present in plants, but they occur together and are superimposed on each other when a plant becomes larger. Nevertheless, they are fundamentally different because growth results from
irreversible enlargement, whereas elastic enlargement is not permanent
and reverses when the deforming force is removed. At the cell level growth extends the wall permanently, whereas elastic wall deformation is reversible. Both involve water uptake because growth is associated mostly with increased cell water content, whereas elastic deformation is caused mostly by changes in P that result from changes in
water content. These similar origins make the two phenomena hard to separate but, without separation, it is not possible to accurately study the growth process.
Some efforts to separate growth from elastic deformation involved
plasmolyzing or freezing and thawing excised tissues to remove elastic
effects of P (Ursprung and Blum, 1924
; Thimann and
Schneider, 1938; Ordin et al., 1956; Cleland, 1958, 1959; Brouwer,
1963; Ray and Ruesink, 1963; Burström et al., 1967; Hohl and
Schopfer, 1992). Other efforts involved stretching isolated cell walls
or dead or live tissues in an external apparatus (Probine and Preston,
1962; Cleland, 1967; Lockhart, 1967; Haughton and Sellen, 1969;
Yamamoto et al., 1970; Fujihara et al., 1978; Kutschera and Briggs,
1988; Nonami and Boyer, 1990a). Typically, the residual enlargement
after subtracting the elastic component was considered to be the growth
of the plant material. As it became increasingly possible to monitor
rapid changes in the dimensions of cells (Green et al., 1971; Ortega et
al., 1989; Zhu and Boyer, 1992) and tissues (Hsiao et al., 1970;
Vanderhoef and Stahl, 1975; Boyer and Wu, 1978, Kuzmanoff and Evans,
1981), it became necessary to rapidly distinguish growth from elastic
effects. However, the usual plasmolytic and stretching techniques were
either too slow or could not be adapted to intact plants, and rapid
changes in enlargement increasingly were interpreted solely as growth.
Most plant enlargement results from cell enlargement in localized
growing regions. Because of the complexities of these regions, Lockhart
(1965a, 1965b) modeled the growth of single cells surrounded by free
water. He assumed that the cell walls behaved as inert polymers
stretched by P, and that wall biosynthesis was independent of growth. He considered elastic effects to be rapid and ignored them
by applying the model several minutes after a new rate was achieved.
Ortega (1985, 1990) extended the Lockhart treatment to account
explicitly for the elastic properties of the wall. This allowed rapid
effects on enlargement to be modeled, but the model remains untested
because methods were unavailable to rapidly separate growth from
elastic effects in experiments. The present work provides a rapid
method to make this separation in live cells.
The model of Ortega (1985, 1990) was based on the superposition
principle from polymer physics and showed that, for a single cell whose
water uptake was not limiting, growth and elastic effects could be
added according to:
|
(1)
|
where (dV/dt)/V is the relative
volumetric rate of enlargement (m3
s
1 m3, or
s1), Pc is the
critical turgor pressure below which growth does not occur (MPa),
is the relative irreversible extensibility of the cell
wall (s1 MPa1), and
is the volumetric elastic modulus (MPa). On the right side of the equation, the first term represents growth, which is the
irreversible enlargement at a steady "effective" turgor (P 
Pc). The second term
is the reversible elastic enlargement, which is important when
P changes. Note that when P is constant, the
second term becomes zero and the equation takes on the form of the
Lockhart equation (dV/dt)/V = (P Pc).
Conversely, when a cell matures, becomes zero and the
irreversible enlargement disappears so that only elastic effects are
seen. In this equation it is important to point out that is a coefficient representing all of the biological and physical
factors contributing to growth. It is not restricted to inert polymer
effects, as originally proposed by Lockhart (1965a, 1965b).
Many studies of cell enlargement use external osmotica to vary the
P. Osmotica change both the P and the solute
environment in the wall, rendering it difficult to determine which
factor controls enlargement. A better approach would be to alter only P without changing the environment of the wall. Ortega et
al. (1988, 1989) were the first to do this kind of experiment, and they
varied P by injecting silicone oil into the vacuole of cells of Phycomyces. Zhu and Boyer (1992) used a pressure probe to
inject or remove cell solution to raise or lower P in the
internode cells of Chara corallina. The wall environment was
unaltered. Enlargement was continuously monitored. The cells were
surrounded by water, causing the growth-induced water potentials
associated with water uptake to be negligible (Zhu and Boyer, 1992).
This latter system is the one used in the present work because it
allowed P effects to be studied without considering water
uptake, thus simplifying the analysis.
C. corallina grows primarily in length, at a rate
essentially independent of the total length of the cell, and Equation 1 can be revised to:
|
(2)
|
where dL/dt is the elongation rate (m
s1), m is the irreversible
longitudinal extensibility of the cell wall (m
s1 MPa1),
Lo is the original cell length (m), and
L is the longitudinal component of the
elastic modulus (MPa). Using this system to vary and control
P, we were able to separate growth and elastic effects, and
explore the mechanism of wall elongation with the model of Ortega
(1985, 1990).
| |
MATERIALS AND METHODS |
Plant Materials
Several cultures of Chara corallina Klien ex Willd., em
R.D.W. were grown in liquid medium as described in Zhu and Boyer
(1992). Fluorescent lights and ambient sunlight above the cultures
provided continuous PAR of 10 to 15 µmol photons
m2 s1 at the surface of
the water. The culture temperature was 22°C to 23°C and pH was 8.0 to 8.5. We conducted the experiments in an environmentally controlled
chamber in which the temperature and light intensity were the same as
those used for the cultures. We used a single internode cell, dissected
by hand from a healthy thallus, for each experiment. Young, growing
internode cells were from near the thallus apices, and older,
nongrowing (mature) cells were from lower portions of the thallus. All
of the experiments were conducted in the culture medium taken directly
from the cultures.
Experimental Apparatus
The experimental apparatus was similar to that described in Zhu
and Boyer (1992), with some modifications. A trough to hold a single
internode cell was made from clear acrylic. A vertical, scissors-like
acrylic gate was mounted at one end of the trough (Fig.
1). A hole (800 µm in diameter) was
drilled at the interface of the top and bottom jaws of the gate.
Clamping the basal end in the gate hole immobilized the internode cell.
In this position the node of the cell protruded from the hole in the
gate and the remaining internode was suspended horizontally within the
trough (Fig. 1). The hole was sealed with petroleum jelly to prevent the leakage of medium around the cell. We attached a thin steel wire to
the free, apical end of the cell and passed it out the end of the
trough opposite the gate. This wire was attached to a Kevlar thread
that was wrapped once around a vertical plastic wheel on a position
transducer (see "P and L Measurement" for details). A small weight (2.3 g) on the end of the thread ensured good
contact with the transducer wheel. The trough was supported at the gate
end on a vertical piece of acrylic that we attached to the top of an
adjustable jack, allowing the height of the apparatus to be altered.
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| Figure 1.
Apparatus for measuring P and
L in C. corallina. A, Perspective view.
B, Top view. The cell is inserted in the trough as shown by the thick
arrows. The gate is closed to immobilize the end to be probed with the
capillary for measuring P. The other end is attached to
a wire and thread leading to a weight. The thread is placed on a
transducer wheel that turns and measures changes in L.
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|
Temperature Control
A peristaltic pump delivered the medium to the trough (20 mL
min1) through a closed circuit of insulated
tubing. Before entering the trough, the medium flowed through a
temperature control unit constructed of stainless steel tubing. The
tubing was bent into a flat spiral (1.5-mm i.d. of tubing bent into
6-cm flat spiral) and was clamped to the cold side of an electronic
Peltier chiller (Thermoelectrics Unlimited, Wilmington, DE). The
chiller could be set to give any desired medium temperature in the
range of 4°C to 22°C. We used cool tap water as the heat sink for
the hot side of the Peltier block. Temperatures above 23°C could be
obtained by replacing this tap water with temperature-controlled warm
water and switching off the Peltier block. The temperature of the
medium around the cell was continuously monitored with a
copper/constantan thermocouple (0.1 mm in diameter) mounted in the side
of the trough. We designed (and tested) the system to minimize thermal
disturbance that could be mistaken for cell elongation. Culture medium
lost by evaporation from the trough was replaced by gravity-fed medium from a reserve container.
P and L Measurement
P was measured with a large pressure probe (Steudle and
Zimmermann, 1974) as described in Zhu and Boyer (1992). The pressure transducer in the probe was calibrated with compressed
N2 and the output was linear from 0 to 0.9 MPa.
The tip of the glass microcapillary was ground at a
25o angle (relative to the long axis) until the
opening had a diameter of 75 µm. This minimized plugging during
P changes but had no effect on the viability of the cells.
The probe was mounted on a micromanipulator to allow fine control while
moving and inserting the microcapillary tip. Before beginning each
experiment, the microcapillary was filled with silicone oil, and cell
solution was sucked in from a growing cell (this cell was then
discarded). We placed another growing cell in the trough and connected
it to the position transducer (Fig. 1). We filled the trough with culture medium, inserted the microcapillary tip into the basal end of
the cell immobilized in the scissors-like gate, repositioned the
plunger in the probe to return the oil/cell-solution meniscus to its
preinsertion position, and obtained a reading of the original cell
P.
The L was continuously recorded with a position transducer
(radial voltage induction transducer, RVIT, Lucas Control Systems, Hampton, VA), which was calibrated to give a linear output throughout the expected range of L.
The P Clamp
The effect of a step change in P was investigated with
the P clamp method developed by Zhu and Boyer (1992). An
upward P clamp involved a step increase in P
followed by small further injections of cell solution to keep the new
P from decreasing. After 8 to 10 min, sufficient water had
moved out of the cell to concentrate the cell solution. The
P was now balanced by the new osmotic potential of the cell,
remaining steady at the new value, and needed no further injections. A
downward P clamp involved the removal of cell solution
followed by small further removals. When enough solution had been
removed, the P remained at the new lower level and needed no
further removals. Before each experiment, we measured the
Lo and monitored L during the
experiment to determine the elongation rate
(dL/dt).
dL/dt at Various Temperatures
We selected growing internode cells from cultures at 23°C and
exposed them to temperatures above and below the culture temperature. We measured the steady elongation rates at temperatures below 23°C by
decreasing the setting in 3oC to
4oC increments with the Peltier chiller until we
found a low temperature that completely inhibited elongation. The
temperature was returned to 23°C and then raised incrementally until
a high temperature was reached that again inhibited elongation.
Elastic Behavior of Cell Walls at Various Temperatures
We examined the elastic behavior of the cell walls with pulses of
P short enough to keep growth negligible, as described by Ortega (1994) and shown in Figure 2.
Using the P clamp before each set of pulses, we adjusted the
P to approximately the same value. While the cell was
growing at a constant rate and constant P, cell solution was
injected to increase P by 0.04 MPa for 10 s.
P was then rapidly returned to the original value, producing a P pulse of 10 s. This P pulse was repeated
two or three times with 2 min between each pulse. Following the set of
P pulses at 23°C, additional sets were performed at
various temperatures for each cell. We determined the total change in
length (
LT) when P was
increased during the P pulse. We established the reversible, elastic change in Lr when P
was decreased at the end of the P pulse. We considered the
total change in length to be the total of the reversible change and any
irreversible change (Li) as demonstrated
in Figure 2:
|
(3)
|
From the set of replicate P pulses, we calculated the
mean LT and
Lr for each temperature. When
comparisons were made among cells of different lengths,
Lr, Li,
and LT were expressed as relative length
changes
Lr/Lo,
Li/Lo, and
LT/Lo,
respectively. The longitudinal component of the elastic modulus was
calculated from L = Lo(P/Lr)
for each cell.
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| Figure 2.
Change in L of C. corallina internode cells when P was changed
rapidly with a P pulse. P pulses were
given for 10 s with a pressure probe (A) and L was
recorded (B). During each pulse, L increased rapidly at
first and then more slowly to give the total length increase
LT. When the P was
returned to the original level, L decreased rapidly to
give the reversible component Lr. The
Li was a small, irreversible component
seen whenever P increased rapidly. The variations in
P (after it was returned to the original level) were
generated intentionally to test for continuity between the probe
contents and the cell contents. Temperature was 23°C. This cell was
13 mm long and growing at 0.014 µm s1 for 20 min before
the experiment. L is shown as the length beyond
Lo at the beginning of the trace.
|
|
Data Processing
We recorded the voltage outputs from the position transducer,
pressure probe, and thermocouple with a datalogger (model CR7X, Campbell Scientific, Logan, UT) and displayed them continually on a
laptop computer. Voltages were recorded every 5 s. Following each
experiment, we downloaded the stored data to a desktop computer for
processing. The voltages were converted to L (µm),
dL/dt (µm s1),
P (MPa), and temperature using the calibration factors
determined during the construction of the equipment. A two-pen chart
recorder gave real-time monitoring of the P and L
during the experiments. Overall, the datalogger and computerized data
management provided simpler and more sensitive measurements than those
described by Zhu and Boyer (1992), who used only a recorder.
| |
RESULTS |
Immediately after placing the isolated internode cells into the
apparatus, elongation rates were larger than in the intact plant (Zhu
and Boyer, 1992). After 30 to 40 min, the elongation rate slowed to the
range for intact plants and was relatively stable for the next 10 to
20 h. All of our measurements were done after the first 30 to 40 min in the apparatus.
Components of Elongation in Single Cells
The cells changed in L when P was changed
with the P clamp (Fig. 3). In
a growing cell, a negative P clamp caused an instantaneous decrease in L followed by a steady elongation that was
slower than before the P clamp (Fig. 3B). After a positive
P clamp, an instantaneous increase in L was
followed by a transition period of several minutes to a new steady
elongation that was faster than before the P clamp. Similar
instantaneous and transitional changes were seen in the mature cell
(Fig. 3D), indicating that they were independent of the growth process.
The instantaneous responses were reversible and thus elastic
(Lr). The transitional response was not
reversible and appeared to be an extended expression of
Li in Figure 2, which is sometimes
termed a viscoelastic change. Note that the wall environment was
unaltered during these measurements because cell solution was injected
only into the interior of the cell. The P change was
permanent, supported by a slight change in the concentration of solute
normally in the cell.
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| Figure 3.
Changes in L during
P steps in a growing (A and B) and a mature (C and D)
C. corallina internode cell. The applicable equations
are shown in B and D from Equation 2. The steps were generated with a
pressure probe after which P was held constant by
removing or injecting cell solution (P clamp). The
temperature was 23°C. The cell in B was 12 mm long and growing at
0.0116 µm s1 before P was changed. The
cell in D was 21 mm long and not growing (mature). L is
shown as the length beyond Lo at the
beginning of the trace.
|
|
The growth rate changed with temperature but the elastic deformation
did not. Figure 4A shows that growth was
markedly affected between 8°C and 37°C, and was maximum in the
range of 30°C to 35°C. We did not observe growth at temperatures
below 5°C or above 37°C. After exposure to the low temperature,
growth resumed when the cells were re-warmed. However, after exposure
to the highest temperature, they were unable to grow again when cooled.
In contrast, when measurements were made with P pulses as in
Figure 2, the relative elastic deformation was constant at temperatures
between 7oC and 30°C. This behavior was the
same for growing and mature cells (Fig. 4B). The relative elastic
deformation was larger for growing cells than for mature cells.
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| Figure 4.
Growth rate (A) and relative elastic cell wall
deformation (B) at various temperatures in C. corallina
internode cells. Growth rates (A) were obtained from nine cells growing
at 0.0095 to 0.024 µm s1 at the reference temperature
of 23°C (rate = 100%). Data for one individual cell are shown
by  . Relative elastic deformation in B was obtained with
P pulses as in Figure 2 from cells growing at 0.010 and
0.014 µm s1 (closed symbols) and from mature cells that
were not growing at 23°C (open symbols). Data in A are individual
measurements and data in B are means ± SD of four
repetitions in individual cells, as shown in Figure 2.
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|
Figure 5 shows that the relative elastic
deformation was linearly related to the original steady elongation
rate. Cells growing rapidly at 23°C had a
LT/Lo of
0.00083, but others with lower growth rates had progressively smaller
LT/Lo (Fig.
5A). Mature cells had a
LT/Lo of
only 0.0002 or 0.0003. Most of the variation came from differences in
the elastic component
Lr/Lo (Fig.
5B). The irreversible component also varied
(Li/Lo, Fig.
5C) and was always smaller in the mature cells. Figure
6 summarizes the elastic responses of the
cells in terms of the longitudinal elastic modulus and shows that
mature cells had a larger modulus than growing cells, i.e. mature walls
were less deformable than growing walls.
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| Figure 5.
Relationship between growth rates at 23°C and
relative length changes caused by P pulses in C. corallina internode cells. A, total; B, reversible (elastic);
and C, irreversible change in length caused by P pulses
as in Figure 2. Each cell was originally growing at the rate shown.
Corresponding points in A, B, and C were measured in the same cell.
Data are means ± SD of four repetitions in individual
cells.  , Mature cells; , growing cells.
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| Figure 6.
Longitudinal elastic modulus
(L) at 23°C for the C. corallina internode cells in Figure 5B, calculated from the
right-hand term of Equation 2. Data are means ± SD of
four repetitions in individual cells, as shown in Figure 2.
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Comparing Elastic Effects and Growth
When elongation was compared at high and low temperature in the
same cell, only the rapid elastic and viscoelastic responses were
detected at 7.3°C, and they were nearly the same as the rapid response at 23.7°C (Fig. 7). Whether
P was stepped down (Fig. 7, A and B) or up (Fig. 7, C and
D), these responses at cold temperatures always accounted for most of
the transient response at warm temperatures. Only a slight amount of
the viscoelastic response remained after the rapid response at cold
temperature; this could be seen as the difference in cell length at the
two temperatures between min 3.5 and 6.0 in Figure 7D. However, growth
occurred at 23.7°C but was completely eliminated at 7.3°C (Fig.
7B). The time interval was kept short (20 min) between temperatures to
minimize any changes in wall composition.
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| Figure 7.
Comparison of elongation at 23.7°C and 7.3°C
when a C. corallina internode cell was subjected to a
P step of 0.04 MPa downward (A and B) or upward (C and
D). At 23.7°C (heavy trace), the cell was growing. At 7.3°C (light
trace), the same cell was not growing. A and C show the superimposed
P step for the two temperatures in the same cell. B and
D show the rapid elongation responses to the P step
superimposed in the same cell. L has been adjusted for
the superposition in B and D.
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Analyzing Growth with the Elongation Growth Equation (Eq. 2)
From the behavior described above it can be seen that a simple
subtraction of the rapid response at 7.3°C from the total response at
23.7°C would produce a curve lacking elastic and rapid viscoelastic effects, showing growth alone. Accordingly, we subtracted them (heavy
lines in Fig. 8C) from the total
elongation (Fig. 8B) to obtain the growth in Figure 8D. The rate of
rapid change was similar when P was stepped down (0.13 and
0.14 µm s1) or up (0.15 and 0.20 µm
s1) at the two temperatures. After the
subtraction there was no evidence of rapid transients in the response
to P. Growth changed immediately and smoothly. The immediate
change indicated that P altered a step involved in growth
that was not part of the elastic or rapid viscoelastic responses. The
graphs indicate the applicable form of the growth equation (Eq. 2) and
show the growth component, i.e. m(P Pc), that was changed by P after
the subtraction in Figure 8D.
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| Figure 8.
Growth behavior in response to P
steps for the cell in Figure 7. A, P steps down and up;
B, total elongation at 23.7°C; C, total elongation at 7.3°C in the
same cell; and D, growth obtained by subtracting heavy lines in C from
corresponding locations in B. The relevant form of Equation 2 is shown
in each graph. Data for thin lines are running averages of 20 s
for measurements every 5 s, and data for heavy lines are
regressions for the data in the thin lines underneath. Numbers beside
heavy lines are rates obtained from slopes of the regressions.
L is shown as the length beyond
Lo at the beginning of the trace.
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DISCUSSION |
Separating Elastic Effects from Growth
The cell walls of C. corallina internodes
displayed elastic deformation whenever P changed during
growth. The deformation was an inevitable consequence of the change in
force on the walls. It combined with growth to create a complex
response mixing early rapid elongation, viscoelastic deformation, and,
later, steady elongation that blurred interpretation. Because elastic
and viscoelastic deformation occurred when the cells were not growing,
they could be separated from the growth process. It is important that
the deformation of mature cells could not be used to correct for
elastic and viscoelastic behavior in growing cells. Mature cells always displayed elastic responses that were smaller than in growing cells,
probably because of differences in wall composition. Instead, we
measured the elastic responses alone when growth did not occur at cold
temperatures, and subtracted it from the total elongation in the same
cell when growth did occur at warm temperatures. The residue gave the
actual elongation due to growth. With this method, the growth that
emerged responded to changes in P. If P
decreased, the growth rate immediately decreased, and if P
increased, the growth rate immediately increased. There was no evidence
for rapid transients of growth rate or rapid changes in m or
Pc as P changed, and growth
smoothly came to a new steady rate.
The key to the method was the invariable nature of the elastic
component as temperature changed. Inert polymers often display stable
elastic behavior over a considerable range of temperatures above the
glass-transition temperature (Sperling, 1992). Temperatures in the
range around room temperature that we used with C. corallina are above the glass-transition temperature. Cross-linking and semicrystalline components likely to be present in cell walls tend
to increase the thermal stability of elastic behavior (Sperling, 1992).
Tomos et al. (1981) similarly observed stable elastic effects at
temperatures around room temperature in Tradescantia
virginiana cells. The thermal stability suggests that elastic
behavior is purely physical and can be expected to be present in all
plant cells.
The viscoelastic behavior also appeared to be largely physical because
it was present in mature cells and thus was independent of growth. In
contrast to the reversible elastic responses, it was largely
irreversible and probably can be attributed to a displacement of wall
polymers that was not reversed when P returned to its original level. It was particularly apparent during a step-up in
P, but with our method the rapid portion of this
viscoelastic component was subtracted from elongation, removing it from
observation. There remained only a small residual portion expressed
slowly, and we could observe little if any effect of it on the steady growth of the cells.
Green et al. (1971) did not report elastic responses in single cells of
growing Nitella, although Kamiya et al. (1963), Probine and
Preston (1962), and Metraux et al. (1980) observed such responses in
nongrowing cell walls of Nitella. Probine and Preston (1962) found that the magnitude of the elastic response of the isolated cell
walls was correlated with the previous growth rate of the intact cells,
as we also observed. Green et al. (1971) interpreted cell elongation
entirely as growth, and rapid changes in growth rate were attributed to
alterations in Pc. However, P
was changed with osmotica during the experiments, and the need to
change bathing solutions around the cells may have obscured elastic
effects in the first seconds after the change (Cleland, 1971). Ortega
et al. (1989, 1991) attempted to quantify the elastic response in single cells of Phycomyces to step-up and pulse-up in
P produced with a pressure probe and by using the equation
reported in Ortega (1985). However, they encountered technical
difficulties that complicated the interpretation of the results (Ortega
et al., 1991). Zhu and Boyer (1992) reported elastic changes in growing C. corallina cells but lacked a method for
quantitatively separating them from growth.
In our work elastic effects were clearly seen, but to evaluate them it
was essential to change P rapidly without other complicating factors. We used a single cell from an alga surrounded with water that
did not have large, growth-induced water potentials (Zhu and Boyer,
1992). These potentials are prevalent in growing multicellular tissues
and change when P changes, making interpretation difficult (Nonami and Boyer, 1990b; Boyer, 1993; Maruyama and Boyer, 1994; Nonami
et al., 1997). Our method of injecting solution from other C. corallina cells altered P alone in a natural
fashion, avoiding the complications of these potentials and changing
growth without altering the chemical environment of the wall. There
were no large solute concentrations from external osmotica that can
change wall behavior, as demonstrated by Zhu and Boyer (1992). The
cells were alive and displayed protoplasmic streaming during the
experiments (Zhu and Boyer, 1992). Thus, we observed normal growth and
elastic effects. By accurately and continuously measuring cell
dimensions, we could separate elastic effects experimentally and
analytically.
In multicellular plants it is more difficult to separate elastic
effects from growth. In some studies osmotica and freeze/thawing were
used to eliminate P after a period of growth, and the
remaining irreversible deformation was determined (Cleland, 1958, 1959; Hohl and Schopfer, 1992). This treatment prevented the tissue from
being used further and was not suitable for intact plants. As a
practical matter, it is worth noting that the method of estimating elastic deformation in C. corallina using P steps
has promise for estimating elastic deformation in multicellular
tissues.
Significance of the Temperature Response
There was a remarkable difference in the thermal response of
growth and elastic behavior. Growth rates varied from zero to maximum,
then returned to zero as the temperature rose from
5oC to 35oC to 37°C. The
elastic behavior was unaffected by the same range of temperatures.
This difference implies different mechanisms for the two processes.
Several models have been suggested to account for cell
enlargement (Passioura and Fry, 1992; Carpita and Gibeaut, 1993;
Passioura, 1994; Roberts, 1994; Carpita et al., 1996), and Figure
9 shows their central molecular features.
According to the models, during a P step-up, more tethers
come under tension in the matrix polysaccharides linking cellulose
microfibrils (Lr, Fig. 9, A and B) and
some tethers undergo displacement (Li,
Fig. 9, A and B). The increased tension decreases the range of kinetic
motion of the tethers. When the process is reversed, molecular motion
is regained (Lr, Fig. 9, B and C) but
the displacement is not reversed (Li,
Fig. 9C). These physical phenomena are present whether or not growth occurs. Their presence during growth does not imply that they control
or contribute to growth, but rather that they are expressed as separate
events while growth is ongoing.
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| Figure 9.
Diagrammatic representation of elastic changes
and growth in a molecular unit of the primary wall. A, Cellulose
microfibrils connected through hydrogen bonds
(>>>>) to tethering matrix polysaccharides,
two of which are load-bearing. B, More tethers become load-bearing
(Lr) and some tethers are displaced
(Li) when P increases. C,
Some tethers are released from load-bearing but displacements are not
fully reversed when P decreases. D, Wall at high
P (as in B) is loosened in E by breaking covalent bonds
( ) and hydrogen bonds () probably through enzymatic
action. F, Inserting new wall polymers and forming new hydrogen bonds
hardens the wall in E. Not shown are wall proteins or layering of
various polysaccharides in the wall.
|
|
By comparison, growth appears to involve many enzymatic events,
including the cutting of tethers (Wong and Maclachlan 1979; Huber and
Nevins, 1981; Yamamoto and Nevins, 1981; Hayashi and Maclachlan, 1984;
Fry, 1989; Fry et al., 1992; Wu et al., 1996) and possible enzymatic
breaking of hydrogen bonds (Cosgrove, 1993; McQueen-Mason and Cosgrove,
1995). The wall loosens as a result (Fig. 9E). The loosening is
probably countered by reconnecting cut tethers (Smith and Fry, 1991;
Nishitani and Tominaga, 1992; Hetherington and Fry, 1993) and
synthesizing and inserting new wall polymers (Roberts, 1994), hardening
the matrix (Fig. 9F). Hardening prevents rupture of the wall, which is
the disastrous end result of continued loosening. Temperature probably
has large effects on growth because the enzyme reactions involve
chemical bonds having high activation energies.
Elastic behavior has been observed in many plants (Kamiya et al., 1963;
Tomos et al., 1981; Steudle and Jeschke, 1983; Steudle et al., 1983;
Nonami and Boyer, 1990a), and its molecular nature suggests that
it will be present whenever the wall is placed under tension by
P. As a result, growth models incorporate it by adding elastic effects to other dimensional effects (Ortega, 1985, 1990; Nonami and Boyer, 1990a). When the elastic effects are
subtracted, the remaining ones are mostly the result of biochemical
loosening and hardening of the wall (Fig. 9, D-F). This suggests that
the term m(P Pc) of Equation 2 is mostly biochemically
controlled.
The Role of P
Lockhart (1965a, 1965b) noted the similarity between the extension
of inert polymers and cell growth when a force is applied. He proposed
that P is the force causing growth by acting on the wall as
an inert polymer. Accordingly, with an increase in P, growth
rate would increase; when P decreased, growth rate would decrease. Our work showed a similar P response during
growth. However, the deformation of inert polymers generally showed few thermal effects in the narrow range of temperatures that we used (Sperling, 1992). For example Lr clearly
is a property of inert materials and showed little thermal response in
the C. corallina wall. If the inert polymer model is
correct, growth similarly should have displayed little temperature
response. The large growth response actually observed
argues against the inert polymer model and suggests a
biochemical mechanism.
Increased P undoubtedly stretched the wall more, causing
inert elastic deformation as in Figure 9, A and B. We eliminated most
of it by subtraction, but a growth response remained and was apparent
within 1 min, suggesting that the magnitude of P may have
rapidly altered a growth factor involving biochemical events. The exact
way the magnitude of P might participate is unclear, but it
should be noted that Robinson and Cummins (1976) reported little
insertion of cellulose and matrix polymers into the wall of pea stem
cells when P was low. The delivery of matrix polymers
normally involved vesicles visible in the cytoplasm that fused with the
plasmalemma. Upon fusion, the vesicles opened to the wall and
immediately expelled their contents to the wall because of the force of
P. At low P the vesicle contents were inserted more slowly into the wall, and without P no wall insertion
occurred. Thus, the magnitude of P might play a role in wall
assembly (Fig. 9F) that could be highly temperature responsive.
From different evidence, others concluded that growth rates were
controlled more by biochemical factors than by the deformation of inert
wall polymers. Haughton and Sellen (1969) used temperature to vary the
deformation of isolated cell walls but found the effects to be too
small to account for the sensitivity of growth to temperature. Ray and
Ruesink (1962) suggested that there was a biochemical reaction close to
the terminal steps in wall enlargement because of the rapidity of the
growth response to temperature in living oat coleoptiles. Roberts
(1994) noted that nearly all wall polymers were newly synthesized in
primary walls of the outer, growth-limiting epidermal cells of
multicellular organisms, and concluded that this synthetic activity
must be central to wall growth. Zhu and Boyer (1992) used chemical
inhibitors to decrease energy metabolism in C. corallina,
and found growth to be inhibited despite high P. They
suggested that metabolism controlled growth.
Zhu and Boyer (1992) found that growth was eliminated below a threshold
P and only responded to P well above normal
levels. This behavior suggests that the growth process could be
entirely metabolic, with little involvement of P other than
as an initial triggering event. Although the present work confirms the
involvement of metabolism, the growth rate changed when
P changed, suggesting a pressure-sensitive step in
metabolism. This discrepancy needs further investigation.
| |
FOOTNOTES |
1
This study was supported by the National Science
Foundation (grant no. IBN-9603956 to J.K.E.O.) and the Department of
Energy (grant no. DE-FG02-87ER13776 to J.S.B.).
*
Corresponding author; e-mail boyer{at}udel.edu; fax
1-302-645-4007.
Received July 9, 1998;
accepted November 6, 1998.
| |
ABBREVIATIONS |
Abbreviations:
P, turgor pressure.
L, length.
| |
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