Tree Physiology 20, 701–707
© 2000 Heron Publishing—Victoria, Canada
Reduction in turgid water volume in jack pine, white spruce and black
spruce in response to drought and paclobutrazol
J. G. MARSHALL,1,3 R. G. RUTLEDGE,2 E. BLUMWALD1 and E. B. DUMBROFF3,4
1
Department of Botany, University of Toronto, Toronto, Ontario M5S 3B2, Canada
2
Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Center, 1055 du P.E.P.S., Sainte-Foy, Quebec G1V 4C7, Canada
3
Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada
4
Present address: Kennedy-Leigh Center for Horticultural Research, Faculty of Agriculture, Hebrew University of Jerusalem, P.O. Box 12, Rehovot
76100, Israel
Received May 18, 1999
Summary Significant reductions in needle water content
were observed in white spruce (Picea glauca (Moench) Voss),
black spruce (Picea mariana (Mill) B.S.P.), and jack pine
(Pinus banksiana Lamb.) seedlings in response to a 10-day
drought, although turgor was apparently maintained. When the
seedlings were re-watered after the drought, jack pine needles
regained their original saturated volume, whereas white spruce
and black spruce needles did not. Significant drought-induced
reductions in turgor-loss volume (i.e., tissue volume at the
point of turgor loss) were observed in shoots of all three species, especially jack pine. Repeated exposure to 7 days of
drought or treatment with the cytochrome P450 inhibitor,
paclobutrazol ((2RS,3RS)-1-(4-chlorophenyl)-4,4-dimethyl2-(1,2,4-triazol-1-yl)-pentan-3-ol), reduced seedling height
relative to that of untreated controls in all three species. The reductions in saturated and turgor-loss needle volumes in the
paclobutrazol-treated seedlings were comparable with those of
seedlings subjected to a 10-day drought. The treatment-induced reductions in shoot and needle water contents enabled
seedlings to maintain turgor with tissue volumes close to, or
below, the turgor-loss volume of untreated seedlings. Paclobutrazol-treated seedlings subsequently survived drought treatments that were lethal to untreated seedlings.
Keywords: conifers, dehydration, needles, Picea glauca,
Picea mariana, Pinus banksiana, saturated volume, shoots,
turgor-loss volume.
Lamb.) and detected a decrease in relative water content in
jack pine seedlings in response to drought. Furthermore, compared with untreated controls, treatment of jack pine seedlings
with ABA (abscisic acid) or paclobutrazol resulted in less negative water potentials despite lower water contents (Marshall
et al. 1991). Changes in cell volume measured by means of the
pressure chamber have been shown to correspond with
changes in turgor measured directly by pressure probe analysis (Murphy and Smith 1995).
Paclobutrazol belongs to a class of cytochrome P450 inhibitors, termed triazoles, that inhibit gibberellin biosynthesis
(Coolbaugh and Hamilton 1976). More recently, it has been
shown that triazoles stimulate accumulation of ABA (AsareBoamah et al. 1986, Rademacher 1992) by inhibiting the
hydroxylation step (Zeevaart et al. 1990) required for metabolic deactivation of ABA (Gillard and Walton 1976). Thus,
triazoles seem to cause a transient activation of mechanisms
that increase resistance to heat and drought stresses (Marshall
et al. 1991).
We have compared the effects of paclobutrazol with that of
drought to assess whether they evoke a common mechanism
of stress resistance. We found that treatment with paclobutrazol induced volumetric decreases similar to those observed in response to a 10-day drought.
Materials and methods
Plant materials
Introduction
Plant tissues have been observed to shrink during desiccation
by amounts that apparently exceed the volume of water that
could be transferred from the free spaces or cell walls (Huck et
al. 1970, Kozlowski 1972, Buxton et al. 1985, Eze et al. 1986,
Levitt 1986, Kozlowski 1991). It is not clear if this phenomenon reflects a physiological adaption to drought. Buxton et al.
(1985) compared mechanisms of water stress resistance in
white spruce (Picea glauca (Moench) Voss), black spruce
(Picea mariana (Mill) B.S.P.) and jack pine (Pinus banksiana
Seedlings of white spruce, black spruce, and jack pine were
raised in a growth chamber in plastic forms (Rigi-Pot Model
76-50, IPL Products, Brampton, Ontario, Canada) for
12 weeks before being transferred to 1-liter pots (10-cm top diameter) containing 550 ml of 3:2:2:1 (v/v) peat:perlite:vermiculite:water. The pots, each containing three seedlings,
were placed in trays of water for 12 h. Thereafter, the seedlings were watered every 3 days with a nutrient solution containing 150 ppm each of N, P, and K. Seedlings were
maintained at 80% relative humidity with day/night tempera-
702
MARSHALL, RUTLEDGE, BLUMWALD AND DUMBROFF
tures of 23/18 °C and a 16-h photoperiod. The seedlings received 212 µmol m –2 s –1 of PAR from a mixture of very high
output (VHO) fluorescent (F96T12-CW-1500, General Electric, Cleveland, OH), standard incandescent (I-line 130, General Electric) and red incandescent (75R30 P1, General
Electric) light during the day. At least 12 pots were randomly
assigned to each treatment, of which six pots were marked for
sampling at Day 0 of the 10-day drought treatment and the remaining pots were marked for sampling at Day 10 of the
10-day drought treatment.
Pretreatments
Sixteen-week-old seedlings were subjected to drought and
paclobutrazol pretreatments. Control seedlings were watered
three times a week and fertilized weekly. Seedlings in the
7-day drought cycle pretreatment (7DD) were watered to capacity and fertilized once per week for 4 weeks (i.e., Weeks 16
to 19 inclusive). Paclobutrazol-pretreated seedlings received a
total of 5 mg of active ingredient ((2RS,3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4,-triazol-1-yl)-pentan-3-ol) (ICIChipmann, Stoney Creek, ON) in two applications with the
first two waterings of Week 16. Each application comprised
2.5 mg suspended in 100 ml water per pot. No attempt was
made to prevent leaching of excess paclobutrazol during subsequent watering and fertilization.
Ten-day drought treatment
At Week 20, half of the well-watered controls and half of the
paclobutrazol-treated seedlings were subjected to a 10-day
drought period (well-watered + 10DD and paclobutrazol +
10DD, respectively). The other well-watered controls and
paclobutrazol-treated seedlings were kept well watered (wellwatered control and paclobutrazol + well-watered treatments,
respectively). At the same time, all of the seedlings subjected
to the 7-day drought cycle pretreatment were exposed to a
10-day drought period before sampling (7DD + 10DD) and
one half of these seedlings were re-watered 12 h before sampling (7DD + 10DD + RW).
Needle water content
The fresh mass (FW) of 25 needles from each seedling was
measured before the needles were placed on the bottom of a
20-ml scintillation vial, covered with water and capped for
24 h. The needles were then blotted dry on kraft paper and reweighed to obtain saturated weight. Subsequently, the needles
were dried in a forced draft oven at 70 °C for 48 h and reweighed to determine dry mass (DW). The water contents
(ml gDW–1) of fresh and saturated needles (FWC and SWC,
respectively) were then calculated.
Survival
At each sampling day, percent survival was measured by
re-watering 3–5 pots per treatment (i.e., 9 to 15 seedlings), for
a minimum of 3 weeks after the cessation of drought. Seedlings that remained wilted, turned brown or became brittle
were judged to be dead, whereas seedlings that remained green
and supple were judged to be alive.
Transpiration
Transpiration was measured with an LI-1600 steady state
porometer (Li-Cor Inc., Lincoln, NE) fitted with a conifer
cuvette. Just before the measurements, the desiccant chambers
were filled with silica gel that had been dried in a forced draft
oven at 70 °C for 24 h. Measurements were made on the leading shoot. At the start of the experiment, some needles were
removed to allow a close seal of the cuvette. Measurements
were made inside the controlled environment chamber at constant temperature and humidity 2 to 4 h after the onset of the
light cycle. Other details were as decribed previously (Marshall et al. 1991).
Pressure–volume curves
Pressure–volumes curves were measured with the precautions
of Ritchie and Hinckley (1975) as decribed by Buxton et al.
(1985). In addition, to reduce transpirational water flow and
avoid disequilibrium in water potentials across the shoot and
between the apoplast and symplast, we removed the seedlings
from the growth chamber 4 h after the onset of the light cycle
on the sampling date and incubated them in the dark for ≥ 4 h.
Pressure–volume curves (Scholander et al. 1964) from the upper 12 cm of the shoot were obtained with a PMS pressure
chamber (Corvalis, OR). Shoots were excised with a scalpel
and needles removed from around the base of the stem with
scissors. The tissue was rapidly weighed, inserted through a
drilled rubber stopper and sealed in the chamber, which was
lined with moist paper. The pressure was increased until water
appeared at the cut surface of the stem, slowly released until
the moisture disappeared, and then subsequently reapplied to
determine the balance pressure. Extruded sap was collected on
dry tissue paper contained in disposable sample tubes, capped
with vapor-tight lids and weighed. Extruded sap was collected
every 0.2 MPa from the balance pressure up to 4.0 MPa of applied pressure. The sampled shoots were sealed in envelopes
and dried in a forced draft oven at 70 °C for 48 h and weighed
(DW).
The relationship between the reciprocal of the applied pressure and the weight of extruded water (EW) was subjected to
regression analysis. Turgor was calculated at each point, and
the relationship between turgor and volume developed for
each curve. The volume at zero turgor (EWr = 0) was calculated by regression. The computer-fitted values, where the
maximum possible regression error in the calculated turgor
loss point was typically about 0.02 ml g –1, were used in the
statistical analysis. Volumetric elastic coefficient, ε, was calculated as (dP/dv)V (Dainty 1976), where P is pressure, dv is
change in volume, and V is water volume. We report water potentials, osmotic potentials, turgor, TLV and ε values of the
unsaturated tissue at the actual water content, i.e., fresh water
content (FWC) of the equilibrated tissue for Days 0 and 10 of
the 10-day drought treatment. Water and osmotic potentials
were calculated by extrapolating the P–V curve to zero extruded volume as described by Dainty (1976). Turgor pressure
was calculated as the difference between water and osmotic
potentials (Dainty 1976). The fresh water content (FWC;
TREE PHYSIOLOGY VOLUME 20, 2000
CELL WALL ADJUSTMENT IN SPRUCE AND PINE
ml gDW –1) of the shoots was calculated as (FWC = (FW –
DW)/DW) and the turgor-loss volume (TLV; ml gDW –1) was
defined as TLV = (FWC – EWΨρ=0 – DW)/DW.
Statistical analysis
Results were analyzed by ANOVA followed by t-tests for the
comparison of two means, the Tukey-Kramer Highly Significant Difference (HSD) test, where three or four means were
compared, and by Hsu’s Multiple Comparison with Best
(MCB) test, where multiple comparisons were made. All statistics were performed with the SAS software package (SAS
Institute, Cary, NC).
Results
Both the paclobutrazol pretreatment and the 7-day drought cycle pretreatment (7DD) caused similar reductions in height
growth in all three species (Table 1).
The 10-day drought treatment (10DD) caused large reductions in water volume of previously untreated (well-watered +
10DD) plants of all three species. In response to the 10DD
treatment, the fresh water content (FWC) of white spruce
needles declined to less than half the volume of the control
(well-watered) needles and there was a significant decline in
FWC in black spruce and jack pine needles (Table 2). The
10DD treatment also caused significant reductions in needle
saturated water content (SWC) in black spruce and white
spruce needles, but not in jack pine needles. Pretreatment of
Table 1. Effects of 7-day cyclic drought (7DD) and paclobutrazol
pretreatments on the height growth (cm) of 24-week-old seedlings of
black spruce, white spruce and jack pine. Values are means (n ≥ 6).
Pretreatment
Jack pine
White spruce
Black spruce
Control
Drought
Paclobutrazol
15.3 a1
11.7 b
11.6 b
24.8 a
20.0 b
18.3 b
28.2 a
25.0 ab
24.1 b
1
Different letters within columns indicate means that differ by the
Tukey Kramer HSD test, α = 0.05.
703
well-watered plants with paclobutrazol resulted in reductions
in needle fresh water content and needle saturated water content in all three species and the subsequent 10DD treatment
had little further effect (Table 2).
In apparent contrast to the pronounced 10DD-induced reduction in water contents observed in the needles, pressure–
volume analysis indicated large positive turgor values in the
shoots of all species. The 10DD treatment lowered the turgorloss volume (TLV) from 1.95 to 1.65 ml g –1 in black spruce,
from 2.2 to 1.8 ml g –1 in white spruce, and from 3 to 2.1 ml g –1
in jack pine (Table 3). The 10DD-induced reduction in TLV
was similar to or exceeded that produced by paclobutrazol in
well-watered plants. The reduction in TLV produced by
paclobutrazol was not augmented by the subsequent 10DD
treatment. Jack pine seedlings in the 7DD pretreatment had
similar values of FWC and TLV to the well-watered control
seedlings and the subsequent 10DD treatment resulted in significant reductions in FWC and TLV in both groups of seedlings (Table 3). In contrast, compared with the corresponding
well-watered controls, black spruce and white spruce seedlings in the 7DD pretreatment lost little or no water in response
to the subsequent 10DD treatment. Because the water potentials required to produce declines in TLV and needle saturated
water content in all three species were above –1.7 MPa, and
the needle water content was similar to, or less than, that of
whole shoots, we conclude that release of water by cavitation
of xylem cells in the stem and migration to the symplast of the
needles was not an important process under the conditions of
our study (Tyree and Dixon 1986).
In both the 10DD and paclobutrazol treatments, seedlings of
all three species maintained turgor, and in some cases close to
full turgor, at sharply reduced needle water contents. Moreover, shoot fresh water contents (FWC) were near or below the
TLV of the well-watered controls. Well-watered black spruce
and white spruce seedlings showed moderate decreases in
TLV in response to the 10DD treatment, whereas much
sharper decreases in TLV were recorded in jack pine. Paclobutrazol treatment also resulted in significantly reduced TLV
in all species (Table 3).
The 10DD treatment reduced turgor of jack pine seedlings
by about 50%, despite causing water contents to decline far
Table 2. Effects of a 10-day drought on saturated water content (SWC; ml gDW–1) and fresh water content (FWC; ml gDW–1) of untreated (control)
and paclobutrazol-treated jack pine, white spruce and black spruce needles. Needles were measured on Day 0 of the 10-day drought treatment 12 h
after the pots had been watered to field capacity and on Day 10 of the 10-day drought treatment (i.e., 10 days after the pots had been saturated to
field capacity). Values are means (n ≥ 8).
Pretreatment
Drought treatment (day)
Jack pine
White spruce
Black spruce
SWC
FWC
SWC
FWC
SWC
FWC
Control
0
10
3.79 a1
3.99 a
3.23 a
2.07 c
3.03 a
2.26 b
2.60 a
1.08 b
2.88 a
2.34 b
2.58 a
1.79 b
Paclobutrazol
0
10
3.19 b
3.33 b
2.84 b
2.88 b
2.35 b
2.26 b
2.15 b
1.91 b
2.47 b
2.39 b
2.13 b
1.95 b
1
Different letters within columns indicate significant differences by the Tukey Kramer HSD test, α = 0.05.
TREE PHYSIOLOGY ON-LINE at http://www.heronpublishing.com
704
MARSHALL, RUTLEDGE, BLUMWALD AND DUMBROFF
Table 3. Effects of a 10-day drought on shoot water relations of black spruce, white spruce and jack pine seedlings pretreated with paclobutrazol,
subjected to four 7-day drought cycles, or kept well watered (control). Seedlings were measured on Day 0 of the 10-day drought treatment 12 h after the pots had been watered to field capacity and on Day 10 of the 10-day drought treatment (i.e., 10 days after the pots had been saturated to field
capacity). Values are means (n ≥ 6).
Pretreatment
Black spruce
Control
Paclobutrazol
Drought
White spruce
Control
Paclobutrazol
Drought
Jack pine
Control
Paclobutrazol
Drought
1
Drought treatment
(day)
FWC
(ml gDW–1)
TLV
(ml gDW–1)
Ψs
(MPa)
Ψw
(MPa)
Ψp
(MPa)
ε
(MPa)
0
10
0
10
0
10
2.1 a1
1.8 b
1.9 a
1.7 b
2.4 a
2.1 a
1.9 a
1.6 b
1.6 b
1.6 b
2.2 a
1.9 a
–2.4
–2.6
–2.3
–2.4
–2.5
–2.5
–0.9 a
–1.7 b
–0.6 a
–1.4 b
–1.3 a
–1.3 a
1.4 a
0.8 b
1.7 a
1.0 b
1.2 a
1.2 a
19 a
16 a
30 a
22 a
11 b
10 b
0
10
0
10
0
10
2.4 a
2.1 b
2.1 b
2.0 b
2.5 a
2.3 a
2.2 a
1.8 b
1.9 b
1.8 b
2.2 a
2.1 a
–2.3 a
–2.4 a
–2.5 a
–2.6 b
–2.6 a
–2.4 a
–0.7 a
–1.6 b
–0.7 a
–1.3 b
–0.7 a
–1.0 a
1.6 a
0.8 b
1.7 a
1.3 a
1.9 a
1.4 a
15 a
8b
20 a
12 a
14 a
13 a
0
10
0
10
0
10
3.3 a
2.3 b
3.0 a
2.7 b
3.4 a
2.7 b
3.0 a
2.1 b
2.4 b
2.4 b
3.1 a
2.5 b
–1.8 a
–2.3 b
–1.9 a
–2.1 b
–1.6 a
–1.9 a
–0.5 a
–1.6 b
–0.5 a
–0.9 a
–0.5 a
–1.3 b
1.3 a
0.7 b
1.4 a
1.2 a
1.1 a
0.6 b
5
4
5
5
5
7
Within a species, different letters within columns indicate a significant difference by Hsu’s MCB test, α = 0.05.
below the TLV of well-watered seedlings (3.0 ml g –1) (Table
3). Jack pine seedlings in the paclobutrazol treatment maintained full turgor at a TLV of 2.7 ml g –1, well below that of untreated seedlings. None of the treatments had a significant
effect on ε values in jack pine (Table 3). Paclobutrazol-treated
black spruce and white spruce maintained turgor at or below
the TLV of the untreated controls. Turgor maintenance was
accompanied by a significant reduction in TLV (Table 3). No
role for osmotic adjustment in the maintenance of turgor was
observed in any of the species. The small declines in osmotic
potentials could be accounted for by loss of water volume
alone.
All untreated black spruce seedlings and most white spruce
seedlings died after 12 days of drought, whereas most untreated jack pine seedlings, which displayed a greater 10DDinduced decrease in TLV than the other species, survived
15 days of drought (Figure 1). Moreover, paclobutrazoltreated seedlings, which exhibited a significant treatment-induced decline in TLV, survived drought conditions much longer than untreated seedlings. Among the species, paclobutrazol-treated white spruce showed the greatest capacity to
survive soil drying. However, these results were confounded
by differences in size between the pine and spruce seedlings,
which caused different rates of water loss from the pots. Similarly, the paclobutrazol effect may have been confounded by
differences in size between species and differences in transpi-
ration rates between treatments (Tables 1 and 4), resulting in
slower soil drying in pots containing jack pine or paclobutrazol-treated seedlings (Figure 1b).
Figure 1. Effects of drought on survival (A) and soil water content
(B). Percent mortality values are means for ≥ 9 seedlings up to 10 days
and 12 or 15 seedlings per sampling date thereafter. Soil water content
values are means ± SE (n ≥ 3). Symbols: 䊊, untreated control pots; 䊉,
paclobutrazol–treated pots.
TREE PHYSIOLOGY VOLUME 20, 2000
CELL WALL ADJUSTMENT IN SPRUCE AND PINE
Discussion
In response to soil drying, conifer needles and shoots maintained positive turgor despite decreases in fresh water content
(Tables 2 and 3) that equaled or exceeded the amount of water
that is generally thought to be available in the apoplast, estimated to be about 15% of fresh weight (Kozlowski 1972,
Levitt and Ben Zaken 1975, Levitt 1986, Kozlowski 1991). It
is possible that the apparent volumetric contraction of the cell
walls in response to drought merely reflects transfer of water
from the apoplast to the symplast (Weisz et al. 1989). However, this explanation cannot account for the decrease observed in well-watered plants treated with paclobutrazol.
Furthermore, it is not consistent with the often moderate water
potentials observed in drought-treated seedlings. We postulate
that this phenomenon is not merely a symptom of drought but
indicates a rearrangement of cell walls to prevent loss of turgor
during drought (Buxton et al. 1985, Eze et al. 1986, Marshall
et al. 1991).
In response to a 10-day drought, the fresh water content of
needles decreased by up to 36% in jack pine, by more than
50% in white spruce and by 30% in black spruce (Table 2);
however, the seedlings remained visibly turgid. To determine
if the reduction in turgid volume resulted from adjustment in
cell wall elasticity or TLV, we measured pressure–volume relationships of drought-treated seedlings that had not been
re-saturated before analysis. We found that turgor-loss volume
decreased in the control seedlings by up to 16% in black
spruce, 18% in white spruce and 30% in jack pine in response
to a 10-day drought and by comparable amounts in well-watered seedlings treated with paclobutrazol. The drought-induced decreases in TLV, especially in jack pine, occurred at
shoot water potentials that were not sufficiently negative
(around –1.6 MPa) to induce extensive xylem cavitation and
consequent water transfer from the vascular tissues to the liv-
Table 4. Effects of a 10-day drought on transpiration (mg min–1) of
untreated (control) and paclobutrazol-treated jack pine, white spruce
and black spruce seedlings. Seedlings were measured on Day 0 of the
10-day drought treatment 12 h after the pots had been watered to field
capacity and on Day 10 of the 10-day drought treatment (i.e., 10 days
after the pots had been saturated to field capacity).
Pretreatment
Drought
treatment
(day)
Jack
pine
Black
spruce
White
spruce
Control
0
10
4.199 a1
0.0150 A
1.429 a
0.0378 A
1.917 a
0.0263 A
Paclobutrazol
0
10
3.024 a
0.0002 B
1.118 a
0.0004 B
1.721 a
0.0001 B
1
2
Different lower-case letters within the same column indicate significant differences between watered seedlings by paired t-test. Different upper-case letters within the same column indicate significant
differences between 10-day drought-treated seedlings by paired
t-test.
Values are means (n ≥ 4).
705
ing cells. Hence it is possible that a contraction of the cell
walls may have occurred. Similarly, jack pine seedlings
pretreated with four 7-day cycles of drought still lost considerable water during a subsequent 10-day drought and a large decrease in TLV was observed. However the TLV decrease in
jack pine, which was observed at a water potential of about
–1.3 MPa (Table 3), seems too small to be associated with extensive cavitation (Tyree and Dixon 1986). In contrast, compared with well-watered controls, black spruce and white
spruce seedlings pretreated with four 7-day drought cycles lost
little or no water during a subsequent 10-day drought and no
significant changes in TLV were observed.
Additional evidence that changes in tissue volume represent
a physiological mechanism of dehydration tolerance included
the finding that TLV declined in well-watered seedlings
treated with paclobutrazol (Table 3). A further line of evidence is the failure of dried spruce needles to regain their
previous volume on re-saturation (Table 2). Also, paclobutrazol-induced reductions in needle-saturated water content were
observed in well-watered seedlings of all three species (Table 2). Taken together, we interpret these data to indicate that
some volumetric adjustment may have occurred at the level of
the cell walls. Moreover, because these volumetric adjustments occurred in response to either a 10-day drought, or to
paclobutrazol treatment in the presence of an adequate water
supply, we conclude that volumetric adjustment may represent an active mechanism of drought resistance. Volumetric
contractions were not observed in black spruce and white
spruce seedlings subjected to a mild drought regime, followed
by rehydration, although inhibition of growth was comparable
to that observed in paclobutrazol-treated seedlings, indicating
that the volumetric contractions were not linked solely to
growth inhibition.
A major difficulty in interpreting the decline in TLV as a
mechanism of drought resistance is the confounding effect of
paclobutrazol on seedling morphology (Table 1). Paclobutrazol treatment caused substantial reductions in shoot extension and needle length, although much smaller effects were
observed in seedlings that were actively growing before treatment. In nearly all cases, paclobutrazol markedly reduced
TLV even in seedlings that had stopped growing before the
treatment was applied and thus were not susceptible to paclobutrazol-induced effects on morphology. Moreover, paclobutrazol-treated seedlings that showed little inhibition of
height growth compared with untreated seedlings survived a
prolonged drought that killed control seedlings. It is possible
that paclobutrazol induced a form of dehydration tolerance
that altered turgid water content independently of its effects on
shoot extension. We conclude that the paclobutrazol-induced
reduction in shoot TLV and needle SWC of well-watered
seedlings, which closely matched the physiological response
to drought of untreated seedlings, cannot be explained by
paclobutrazol-induced changes in morphology.
Seedlings pretreated with paclobutrazol appear to have been
more resistant to drought than untreated seedlings because of
their lower transpiration rate (Table 4), despite more positive
water potentials (Table 3) at higher soil water contents (Fig-
TREE PHYSIOLOGY ON-LINE at http://www.heronpublishing.com
706
MARSHALL, RUTLEDGE, BLUMWALD AND DUMBROFF
ure 1). Similar effects have been reported in white spruce seedlings treated with triadimefon (a triazole related to
paclobutrazol) (Sailerova and Zwiazeck 1993). Paclobutrazol
belongs to a class of cytochrome P450 inhibitors that have been
shown to induce ABA accumulation (Asare-Boamah et al.
1986, Rademacher 1992). Stomatal function, a major mechanism of stress resistance, is closely associated with the action
of ABA (Mittlehanser and Van Steveninck 1969), suggesting
that paclobutrazol is affecting stomatal function through the
accumulation of ABA and not the inhibition of GA synthesis.
Perhaps the paclobutrazol-induced decrease in TLV that was
observed in well-watered seedlings represents a mechanism of
stress resistance that is activated by the inhibition of ABA
turnover.
The volumetric changes in sclerophyllous conifer shoots in
response to drought (Kozlowski 1972) are much smaller than
those reported in roots (Huck et al. 1970) and cabbage leaves
(Levitt 1986). Although no general needle shrinkage was visible (Parker 1968), it is possible that volumetric changes were
localized in certain cells. It is unlikely that volumetric adjustment occurs in cells with well-developed secondary walls.
However, volumetric changes may have occurred in mesophyll cells that have only primary cell walls (Bacic et al.
1988), which allow cell shrinkage and expansion during freeze
dehydration and subsequent reversal (Pearce 1988). Microscopic studies of drying conifer needles have also shown alterations in response to drought in primary cell walls (Parker
1952, Levitt and Ben Zaken 1975). Hence primary cell walls
are the most likely locus of drought-induced changes in wall
structure affecting TLV, saturated volume and ε. The high ε
values observed in black and white spruce in response to
paclobutrazol were recorded at substantially reduced water
contents (Table 3), where mean ε values would tend to be lowered, not increased, and thus these changes likely reflected alterations in cell wall structure.
The reduction in TLV observed in well-watered seedlings
treated with paclobutrazol was also seen in untreated seedlings
exposed to a 10-day drought. These results suggest that reductions in TLV reflect a mechanism of drought tolerance and are
not merely a symptom of drought. If decreases in TLV represent adjustments in cell wall elasticity (Blake et al. 1991,
Zwiazek 1991, Edwards and Dixon 1995) that lead to decreased turgid volumes, then they must be accompanied by
changes in primary cell wall structure. Moreover, very large
contractions in jack pine root volume dehydrated with nonpenetrating osmotica are accompanied by microscopic
changes in the radius of the primary cells of the cortex (Marshall 1996). The oxidative insolubilization of de novo synthesized cell wall proteins could provide a mechanism for the cell
wall rearrangement (Marshall et al. 1999).
Acknowledgments
Support from the Natural Science and Engineering Research Council
of Canada is gratefully acknowledged.
References
Asare-Boamah, N.K., G. Hofstra, R.A. Fletcher and E.B. Dumbroff.
1986. Triadimefon protects bean plants from water stress through
its effects on abscisic acid. Plant Cell Physiol. 27:383–390.
Bacic, A., P.J. Harris and B.A. Stone. 1988. Structure and function of
plant cell walls. In The Biochemistry of Plants. A Comprehensive
Treatise. Vol. 14, Carbohydrates. Ed. J. Preiss, pp 315–361.
Blake, T.J., E. Bevilacqua and J.J. Zwiazek. 1991. Effects of repeated
stress on turgor pressure and cell elasticity changes in black spruce.
Can. J. For. Res. 21:1329–1333.
Buxton, G.F., D.R. Cyr, E.B. Dumbroff and D.P. Webb. 1985. Physiological responses of three northern conifers to rapid and slow induction of moisture stress. Can. J. Bot. 63:1171–1176.
Coolbaugh, R.C. and R. Hamilton. 1976. Inhibition of ent-kaurene
oxidation and growth by a cyclopropyl-α-(p-methoxyphenyl)-5pyrimidine methylalchohol. Plant Physiol. 57:245–248.
Dainty, J. 1976. Water relations of plant cells. In Encyclopedia of
Plant Physiology, Vol. 2A, New Series. Eds. A.P. Gottingen and
M.H. Zimmermann. Springer-Verlag, Berlin, pp 12–35.
Edwards, D.R. and M.A. Dixon. 1995. Mechanisms of drought response in Thuja occidentalis L. II Post-conditioning water stress
and stress relief. Tree Physiol. 15:129–133.
Eze, J.M.O., S. Mayak, J.E. Thompson and E.B. Dumbroff. 1986. Senescence in cut carnation flowers: temporal and physiological relationships among water status, ethylene, abscisic acid and
membrane permeability. Physiol. Plant. 68:323–328.
Gillard, D.F. and P.C. Walton. 1976. Abscisic acid metabolism by a
cell free preparation from Echinocystis lobata liquid endosperm.
Plant Physiol. 58:790–795.
Huck, M.G., B. Klepper and H.M. Taylor. 1970. Diurnal variations in
root diameter. Plant Physiol. 45:529.
Kozlowski, T.T. 1972. Shrinking and swelling of plant tissues. In
Water Deficits and Plant Growth, Vol. III. Ed. T.T. Kozlowski.
Academic Press, Toronto, pp 1–64.
Kozlowski, T.T. 1991. The physiological ecology of woody plants.
Academic Press, Toronto, 657 p.
Levitt, J. 1986. Recovery of turgor by wilted, excised cabbage leaves
in the absence of water uptake. Plant Physiol. 82:147–153.
Levitt, J. and R. Ben Zaken. 1975. Effects of small water stress on cell
turgor and intercellular space. Physiol. Plant. 34:273–279.
Marshall, J.G. 1996. Ph.D. Thesis, Univ. Waterloo, 198 p.
Marshall, J.G., J.B. Scarratt and E.B. Dumbroff. 1991. Induction of
drought resistance by abscisic acid and paclobutrazol in jack pine.
Tree Physiol. 8:415–422.
Marshall, J.G., E.B. Dumbroff, B.J. Thatcher, B. Martin, R.G. Rutledge and E. Blumwald. 1999. Synthesis and oxidative insolubilization of cell-wall proteins during osmotic stress. Planta
208:401–408.
Mittlehanser, C.J. and R.F.M. Van Steveninck. 1969. Stomatal closure and inhibition of transpiration induced by [RS]-abscisic acid.
Nature 221:281–282.
Murphy, R. and J.A.C. Smith. 1995. A critical comparison of the
pressure-probe and pressure-chamber technique for estimating
leaf-cell turgor pressure in Kalanchoe daigremontiana. Plant Cell
Environ. 17:15–29.
Parker, J. 1952. Desiccation in conifer leaves: anatomical changes
and determination of the lethal level. Bot. Gaz. 114:189.
Parker, J. 1968. Drought resistance mechanisms. In Water Deficits
and Plant Growth, Vol. I. Ed. T.T. Kozlowski. Academic Press,
Toronto, pp 195–234.
TREE PHYSIOLOGY VOLUME 20, 2000
CELL WALL ADJUSTMENT IN SPRUCE AND PINE
Pearce, R.S. 1988. Extracellular ice and cell shape in frost-stressed
cereal leaves: a low temperature scanning electron microscopy
study. Planta 175:313–324.
Rademacher, W. 1992. Biochemical effects of plant growth retardants. In Plant Biochemical Regulators. Ed. H.W. Gaussmann.
Marcel Dekker, Inc., New York, pp 169–199.
Ritchie, G.A. and T. M. Hinckley. 1975. The pressure chamber as an
instrument for ecological research. Adv. Ecol. Res. 9:165–254.
Sailerova, E. and J.J. Zwiazeck. 1993. Effect of triadimefon and osmotic stress on plasma membrane composition and ATPase activity in white spruce (Picea glauca) needles. Physiol. Plant. 87:
475–482.
Scholander, P.F., H.T. Hammel, E.A. Hemmingsen and E.D. Bradstreet. 1964. Hydrostatic pressure and osmotic potentials in the
707
leaves of mangroves and some other plants. Proc. Natl. Acad. Sci.
USA. 52:119–125.
Tyree, M.T. and M.A. Dixon. 1986. Water stress induced cavitation
and embolism in some woody plants. Physiol. Plant. 66:397–405.
Weisz, P.R., H.C. Randall and T.R. Sinclair. 1989. Water relations of
turgor recovery and restiffening of wilted cabbage leaves in the absence of water uptake. Plant Physiol. 91:433–439.
Zeevaart, J.A.D., D.A. Guage and R.A. Creelman. 1990. Recent studies in the metabolism of abscisic acid. In Plant Growth Substances.
Eds. R.P. Pharis and S.B. Rood. Springer-Verlag, Berlin, pp 233–
240.
Zwiazek, J.J. 1991. Cell wall changes in white spruce (Picea glauca)
needles subjected to repeated drought stress. Physiol. Plant.
82:513–518.
TREE PHYSIOLOGY ON-LINE at http://www.heronpublishing.com