Directory UMM :Data Elmu:jurnal:T:Tree Physiology:Vol16.1996:

Tree Physiology 16, 713--718
© 1996 Heron Publishing----Victoria, Canada

Growth, water relations and solute accumulation in osmotically
stressed seedlings of the tropical tree Colophospermum mopane
J. M. JOHNSON,1,3,5 J. PRITCHARD,2,4 J. GORHAM1 and A. D. TOMOS2
1

Centre for Arid Zone Studies, Prifysgol Cymru, Bangor, Gwynedd LL57 2UW, U.K.

2

Ysgol Gwyddorau Bioleg, Prifysgol Cymru, Bangor, Gwynedd LL57 2UW, U.K.

3

Present address: School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, U.K.

4

Present address: School of Biological Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, U.K.

5

Formerly J. M. Tuohy

Received October 18, 1995

Summary Root and hypocotyl elongation, water status and
solute accumulation were studied in osmotically stressed seedlings of the tropical tree, Colophospermum mopane (Kirk ex
Benth.) Kirk ex J. Léonard, which grows in hot arid areas of
southern and central Africa. Seeds were imbibed for 24 h and
then subjected to a polyethylene-glycol-generated osmotic
stress of −0.03 (control), −0.2, −0.8, − 1.6 or −2.0 MPa for 60 h.
Seedlings subjected to moderate water stress (−0.2 MPa) had
higher root growth rates (2.41 ± 0.24 mm h−1), greater final root
lengths (111 ± 3.8 mm) and longer cells immediately behind
the root elongation zone than control seedlings (1.70 ± 0.15
mm h −1 and 93 ± 3.9 mm, respectively). Root lengths of
seedlings in the −0.8 and −1.6 MPa treatments were similar to
those of control seedlings, whereas the −2.0 Mpa seedlings had

significantly shorter roots. Both root and hypocotyl tissues
exhibited considerable osmotic adjustment to the external
water potential treatments. Seedlings in the − 0.03, −0.2, and
−0.8 MPa treatments had similar cell turgor pressures (0.69 ±
0.10, 0.68 ± 0.07 and 0.57 ± 0.04 MPa, respectively), whereas
the −2.0 MPa treatment lowered cell turgor pressure to 0.17 ±
0.04 MPa. Root vacuolar osmotic pressures were generally
similar to sap osmotic pressures, indicating that the increased
root elongation observed in moderately water-stressed seedlings was not caused by increased turgor pressure difference.
Neutral-fraction solute concentrations, including the osmoticum pinitol, increased approximately two-fold in root sap in
response to a low external water potential. In hypocotyl sap of
seedlings in the −2.0 MPa treatment, pinitol more than doubled, sucrose increased from about 2 to 75 mol m −3 but glucose
and fructose remained unchanged and, as a result, total sugars
increased only slightly. The benefits of rapid early root elongation and osmoticum accumulation under conditions of water
stress are discussed in relation to seedling establishment.
Keywords: osmotic stress, pinitol, root elongation, turgor pressure, solute accumulation, Zimbabwe.

Introduction
Colophospermum mopane ((Kirk ex Benth.) Kirk ex J. Léonard) is widespread in the low-altitude, high-temperature regions of southern Africa (Coates Palgrave 1983) and is able to
establish and flourish in habitats characterized by arid or sodic

soils (Bennett 1985). It is a preferred fuelwood species (Reh et
al. 1989) and is also used as fodder (Coates Palgrave 1983).
Colophospermum mopane is capable of germinating and establishing root growth at water potentials lower than those
tolerated by species from similar regions, e.g., 80% germination at − 0.51 MPa compared to less than 30% in Acacia tortilis
(Forsk.) Hayne. (Choinski and Tuohy 1991). Under moderate
water stress (−0.2 MPa) radicle elongation was greater after 3
days than that of control seeds grown at −0.03 MPa (Choinski
and Tuohy 1991).
Generally, root extension growth is reduced at the onset of
osmotic stress (Hsiao 1973, Pritchard et al. 1987, Pritchard et
al. 1991, Spollen and Sharp 1991); however, in some studies it
has been observed that root elongation growth is enhanced
under conditions of moderate water stress (Sharp and Davies
1979, Meyer and Boyer 1981, N’guyen and Lamant 1989,
Triboulot et al. 1995). It is not clear how this drought-induced
increase in root elongation is achieved. Although cell and
organ expansion rate may be controlled by several factors,
including water transport (Boyer 1985) and cell wall properties
(Hsiao and Jing 1987, Passioura and Fry 1992, Pritchard et al.
1993), turgor pressure is the main driving force (Lockhart

1965). Thus, we compared effects of osmotic stress on root and
hypocotyl growth of C. mopane seedlings with effects of
osmotic stress on cell turgor to determine the role of turgor
pressure in root elongation of seedlings under conditions of
water stress.
Maintenance of turgor pressure under conditions of external
water stress requires an accumulation of solutes within cells,
and hence an increase in internal osmotic pressures. An increase in vacuolar osmotic pressure can be achieved by accumulating a wide variety of solutes, whereas osmotic

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JOHNSON, PRITCHARD, GORHAM AND TOMOS

adjustment in the cytoplasm is generally achieved by an accumulation of organic compounds, often sugars and amino acids.
Plants also accumulate osmotically active low molecular
weight compounds such as sugar alcohols (including pinitol),
proline and glycine betaine (Hellebust 1976, Gorham et al.
1981, Wyn Jones and Gorham 1983) in response to abiotic
stress. There have been few studies on pinitol in roots and none
on tropical tree roots, but the compound has been found to

increase in Pinus pinaster L. roots in response to drought
(N’guyen and Lamant 1988) and in soybean roots in response
to high temperature stress (Guo and Oosterhuis 1995). To
obtain a more detailed understanding of the role of osmotic
accumulation during seedling establishment under conditions
of water stress, we measured the concentrations of pinitol and
other neutral-fraction sugars in roots and hypocotyls of unstressed and osmotically stressed C. mopane seedlings and
calculated their contribution to osmotic pressure.

Materials and methods
Plant material and collection site
Colophospermum mopane seeds were collected from the Sapi
Game Reserve in the Zambezi Valley, Zimbabwe (altitude
550 m, mean annual rainfall 750 mm, and mean annual maximum and minimum temperatures of 34.1 and 19.6 °C, respectively), and dispatched to Wales.
Germination and growth of seedlings
Fruit walls were removed and the seeds surface-sterilized for
3 min in 0.35% sodium hypochlorite containing a drop of
detergent and then rinsed five times with distilled water. Surface-sterilized seeds were imbibed for 24 h in vermiculite
mixed with 0.1 mol m −3 CaCl2 solution to give a water potential of − 0.03 MPa (i.e., 3.72 ml CaCl2 solution per g dry
vermiculite) (Sharp et al. 1988) at 30 °C. The imbibed seeds

were then subjected to various external water potential treatments for 60 h. Polyethylene glycol (PEG) was made up in 0.1
mol m −3 CaCl2 solution to give water potentials of −0.03,
−0.21, −0.8 and −2.1 MPa at 30 °C and mixed with vermiculite
in the same ratio as used for seed imbibition. The PEG--vermiculite system placed the seeds in contact with PEG at the
designated water potential, while allowing good aeration.
Water potentials of the PEG-vermiculite mixes were measured
with a thermocouple psychrometer (Model 85, J.R.D. Merrill,
Inc., Logan, UT). For the pressure probe and cell length measurements, germinated seeds were placed in the PEG--vermiculite mix in tall beakers (sealed with clingfilm to minimize
evaporation), covered with aluminium foil and incubated at
30 °C for 60 h. For the growth studies, germinated seeds were
grown in PEG--vermiculite mix in angled Plexiglas (Perspex)
chambers (Sharp et al. 1988) so that root elongation could be
determined in situ. Root elongation data are from a minimum
of three roots per treatment from each of three separate experiments.

Cell length determination
Roots were fixed in 3% glutaraldehyde in 50 mol m −3 cacodylate buffer for 24 h under vacuum, dehydrated in 25% steps of
ethanol over 3 days, infiltrated with 50/50 (v/v) ethanol/Historesin (Reichert-Jung Ltd) overnight, left in pure resin for
2 days then embedded in fresh resin containing hardener. Longitudinal sections were made of three roots from each treatment and stained with toluidine blue. Cell lengths were
measured with the aid of a microscope using an eyepiece

graticule; 20 cells from each side of the central vascular strand
at a point where cells had ceased to elongate (approximately
9 mm behind the root tip) were measured from four sections of
root per treatment.
Bulk sap osmotic pressures
Root tip (first 10 mm) and hypocotyl (total) samples of seedlings that had been osmotically stressed for 60 h were placed
in Eppendorf tubes, frozen in liquid N2, and stored at −18 °C
until required. Sap was collected as described by Gorham et al.
(1984). A minimum of three samples of both root and hypocotyl tissue per treatment were prepared and osmotic pressures
of 10-µl sub-samples were measured with a vapor pressure
osmometer (Model 5100B, Wescor, Inc., Logan, UT).
Pressure probe measurements
Roots of intact seedlings were exposed to a solution of PEG
with a water potential identical to that of the PEG--vermiculite
mix in which the seedlings had been grown for 60 h, in a
Plexiglas holder as described previously (Pritchard et al.
1987). Turgor pressures were measured with a cell pressure
probe (Hüsken et al. 1978, Pritchard et al. 1987) along and
across the growing zone on 1--5 cells per root and 3--4 roots
per treatment. Vacuolar osmotic pressures were measured

cryoscopically on sap collected from vacuoles using the pressure probe (Malone et al. 1989). Three measurements per root
comprising 2--3 epidermal and cortical cell vacuoles mixed
were made.
Bulk solute concentrations
Seedlings were separated into root tip (first 10 mm) and hypocotyl and replicate samples of between 200 to 400 mg of tissue
from each treatment from two separate experiments were
placed in Eppendorf tubes. Each tube containing the tissue
sample was weighed, frozen in liquid N2, returned to room
temperature and sap expressed as described for the osmotic
pressure measurements. Expressed sap was immediately refrozen in liquid N2 to minimize invertase activity. Just before
analysis, sap was thawed, a 40-µl aliquot was diluted with
120 µl of deionized water, centrifuged through 0.45 µm mesh
micro centrifuge filters (Whatman, UK) and analyzed by
HPLC on a Sarasep sodium-form carbohydrate column at
80 °C with 25 mol m −3 Na2SO4 as eluent with a Marathon auto
sampler and Shodex refractive index detector.

ROOT ELONGATION IN DROUGHTED MOPANE SEEDLINGS

715

Results
Root length, growth rate and cell length
Roots of seedlings that had been moderately stressed
(−0.2 MPa) for 60 h were significantly (P < 0.05) longer than
those of control seedlings (−0.03 MPa) (111 ± 3.8 versus
93 ± 3.9 mm, respectively). Root growth rates of seedlings in
the −0.2 MPa treatment were also greater than those of control
seedlings (2.41 ± 0.24 versus 1.70 ± 0.15 mm h −1) (Table 1).
Root lengths of seedlings in the −0.8 and −1.6 MPa treatments
were similar to those of control seedlings (Table 1). Root cell
lengths in the zone of maximum elongation were greater in
moderately stressed roots than in control roots (Figure 1A).
There were no significant differences in root cell lengths between the other treatments.
In contrast to roots, hypocotyl extension was lower in seedlings in all of the water stress treatments than in control
seedlings. Even the moderate water stress (0.2 MPa) trea tment
caused an approximately 30% reduction in hypocotyl extension (Table 1).
Osmotic and turgor pressures
Tissue sap osmotic pressures increased in both roots and hypocotyls in response to decreasing external water potential (Figure 1B), indicating that osmotic adjustment occurred in both
tissues. A differential in osmotic pressure was maintained

between the root and hypocotyl tissues, with the sap osmotic
pressure of the root being lower than that of the hypocotyl in
all treatments (1.26 ± 0.07 MPa in root tissue versus 1.43 ±
0.08 MPa in hypocotyl tissue).
Turgor pressures of cells in the root growing zone were
similar in seedlings in the −0.03, −0.2 and −0.8 MPa treatments, ranging from 0.71 ± 0.13 MPa at −0.03 MPa to 0.57 ±
0.04 at −0.8; however, the 2.0 MPa treatment caused root
turgor pressure to drop to 0.17 ± 0.08 (Figure 1D). None of the
treatments had a significant effect on hypocotyl turgor pressure
(Figure 1D).
Root vacuolar osmotic pressure increased with increasing
stress (Figure 1C). Vacuolar osmotic pressures were similar to
sap pressures except in the −2.0 MPa treatment where the
vacuolar osmotic pressure was about 0.4 MPa higher than the
sap pressure (2.5 ± 0.36 versus 2.1 ± 0.04 MPa). Osmotic

Table 1. Effects of external water potential on elongation growth in
hypocotyls and roots, and root growth rates in mopane seedlings.
Lengths were measured to the nearest mm; values are given as mean
± SE. ND = not determined. Within each column, means without a

common letter are significantly different at P ≤ 0.05.
External water
potential
(MPa)

Root
length
(mm)

Hypocotyl
length
(mm)

Root
growth rate
(mm h −1)

−0.03
−0.2
−0.8
−1.6
−2.0

93 ± 3.9a
111 ± 3.8b
86 ± 6.4a
91 ± 6.1a
57 ± 3.5c

25 ± 2.4a
17 ± 1.8b
10 ± 0.8c
8 ± 0.87c
8 ± 1.0c

1.70 ± 0.15a
2.41 ± 0.24b
1.55 ± 0.12a
ND
0.38 ± 0.04c

Figure 1. Effects of external water potentials on root growing zone cell
lengths (A), tissue osmotic pressures measured on expressed root tip
and hypocotyl sap (B), vacuolar osmotic pressures measured in individual root cell vacuoles in the growing zone (C), and turgor pressures
in root and hypocotyl cells (D) of mopane seedlings. Roots = d;
hypocotyls = s. Data are means ± SE.

pressures were essentially constant along the apical 8 mm of
the root (data not shown), with values similar to those measured 4--6 mm from the tip (see Figure 1D).
Solute concentrations
Total sugar (sucrose, fructose and glucose) and pinitol concentrations (mol m −3) were consistently higher in hypocotyl sap
than in root sap (Figures 2A and 2B), except in seedlings in the
− 2.0 MPa treatment where total sugar concentrations were
similar in both organs (Figure 2A). In the root sap, concentrations of pinitol, total sugars, and the individual sugars, sucrose,
fructose and glucose increased with increasing external stress.
Both pinitol and total sugar concentrations were over three
times higher in roots in the −2.0 than in the −0.03 MPa
treatment (Figures 2A and 2B and Table 2).
The effects of the treatments on solute concentrations was
less clear in the hypocotyl sap than in the root sap. Although
the pinitol concentration of hypocotyl sap increased with increasing external stress, total sugar concentrations were similar in control and moderately stressed plants (about 230 mol
m −3) and only slightly higher (about 300 mol m −3) in the −0.8
and − 2.0 MPa treatments (Figure 2A). Hypocotyl glucose and
fructose concentrations remained similar in all treatments, but
sucrose concentrations were about 15 times higher in the −2.0
MPa treatment than in the other treatments (75 versus approximately 5 mol m −3) (Table 2). In contrast to the other solutes
measured, sucrose concentrations were lower in hypocotyl sap
than in root sap, except in the −2.0 MPa treatment (Table 2).

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JOHNSON, PRITCHARD, GORHAM AND TOMOS

Table 2. Concentrations (mol m − 3) of individual sugars in root and hypocotyl sap of mopane seedlings stressed for 60 h at various external water
potentials.
External water

Sucrose (mol m −3)

Glucose (mol m −3)

potential (MPa)

Root

Hypocotyl

Root

Hypocotyl

Root

Hypocotyl

−0.03
−0.2
−0.8
−2.0

13 ± 1.3
15 ± 0.2
29 ± 2.8
44 ± 3.4

1.2 ± 0.33
4.9 ± 3.3
2.0 ± 0.2
75 ± 6.3

21 ± 0.5
31 ± 0.5
65 ± 2.5
140 ± 12

103 ± 8.1
102 ± 5.8
131 ± 10
96 ± 13

43 ± 1.8
67 ± 2.6
106 ± 2.5
176 ± 8.4

110 ± 9.9
129 ± 12
157 ± 14
138 ± 18

Figure 2. Effects of external water potentials on total sugar (A) and
pinitol (B) concentrations (mol m − 3) in roots and hypocotyls of
mopane seedlings. Roots = shaded bars; hypocotyls = open bars. Data
are means ± SE.

The combined contribution of sugars and pinitol to osmotic
pressure, calculated according to the van’t Hoff equation, was
about 30% of the total root osmotic pressure at an external
stress of −0.03 MPa and 41% at −2.0 MPa. The corresponding
values for the hypocotyl were 70 and 45%, respectively. Although the concentration of pinitol increased with increasing
stress, the overall contribution of pinitol to the pinitol + sugars
component of root osmotic pressure did not increase significantly, being 30% in control roots and 33% in roots grown at
−2.0 MPa. In the hypocotyl, the osmotic contribution of pinitol
to the pinitol + sugars component of osmotic pressure doubled
from 22% in control plants to 44% at −2.0 MPa.

Discussion
As in other plant species, primary root elongation of mopane
seedlings was less sensitive to low external water potentials
than hypocotyl extension (cf. Westgate and Boyer 1985, Sharp
et al. 1988, Spollen et al. 1993). Water stress generally leads to
a reduction in root growth (Pritchard et al. 1991, Spollen et al.
1993); however, in mopane, there was a significant stimulation
of root elongation under conditions of moderate water stress
(−0.2 MPa) (Table 1). Increased root mass, lateral branching
and depth of rooting in response to long-term water deficits
have also been observed in other plant species (Sharp and

Fructose (mol m −3)

Davies 1979, Osonubi and Davies 1981, Reader et al. 1992)
but during early radicle development in mopane (Choinski and
Tuohy 1991) and Pinus pinaster (Triboulot et al. 1995). Enhanced radicle development in response to mild water stress is
not typical of all tropical African trees. In two species of
African Acacia, including A. tortilis a species common to more
arid regions, PEG treatment inhibited radicle development
(Choinski and Tuohy 1991). Although roots were longer than
controls in the moderately stressed (−0.2 MPa) mopane seedlings 60 h after stress was imposed, seedlings in the −0.8 MPa
treament had the longest roots 14 days later (data not shown).
Similarly, maize roots grown in vermiculite at − 2.0 MPa were
intially shorter than roots of control seedlings but after 44 days
there was no difference in length (Sharp et al. 1988). There
may also be differences in both the timing and the location of
root growth responses to water stress. For example, water
stress may cause an increase in elongation growth in younger
cells, while causing a decrease in older cells, leading to either
an overall increase or an overall decrease in root elongation.
In the root growing zone, turgor pressure and expansion
decrease immediately after drought stress is imposed (Hsiao
and Jing 1987, Frensch and Hsiao 1994). Thereafter, depending on the severity of the stress, partial or total turgor recovery
occurs. Following 24 h of exposure to 300 mol m −3 mannitol,
root turgor of wheat plants recovered to unstressed values in
the region of accelerating root growth, but growth rates were
reduced (Pritchard et al. 1991). In maize roots subjected to
long-term exposure to −1.6 MPa water stress, growth rates
were maintained in the region of accelerating growth, but
turgor pressures were about 0.3 MPa lower than in unstressed
controls (Spollen and Sharp 1991). Hence, in wheat, cell wall
tightening must account for the reduction in growth, whereas
in maize the cell walls must loosen in response to stress to
maintain growth at reduced turgor. In mopane roots, growth
rates increased under conditions of moderate stress but turgor
pressures were not significantly different to those of unstressed
controls. This suggests that increased root growth in response
to moderate water stress results from increased cell wall loosening. Increased root growth rates at a turgor pressure similar
to that of unstressed controls also occurs in moderately water
stressed (− 0.15 MPa) Pinus pinaster (Triboulot et al. 1995),
but not in maize (Sharp et al. 1988).
At an external stress of − 0.8 MPa, neither root growth rates
nor turgor pressures were significantly different from those of
unstressed controls. In other species, such severe water stress
can lead to sharp reductions in root elongation rates, e.g.,

ROOT ELONGATION IN DROUGHTED MOPANE SEEDLINGS

approximately 40% in soybean, 60% in squash (Spollen et al.
1993) and 50% in Pinus pinaster (Triboulot et al. 1995). In our
study, the most severe external water potential treatment,
−2.0 MPa, caused an approximately 60% reduction in root
turgor pressure and an approximately 75% reduction in root
growth rate. We do not have growth rates or turgor pressures
for roots grown at −1.6 MPa, but overall root length at −1.6
MPa was similar to that at −0.8 MPa. Hence, we conclude that
there was a threshold stress value between −1.6 and −2.0 MPa
at which the change from turgor and growth maintenance to
reduced turgor and growth occurred.
Although root growth was not reduced until the external
water stress was greater than −1.6 MPa, hypocotyl elongation
was reduced by about 33% even under conditions of moderate
stress (− 0.2 MPa); however, hypocotyl turgor pressures were
the same in all treatments. Hsiao and Jing (1987) suggested
that cell wall properties of shoots have the opposite response
to those of roots. There is evidence that in moderately waterstressed plants, despite the maintenance of turgor pressures at
control values, cell wall tightening in shoots is responsible for
reduced growth (Tomos and Pritchard 1994).
The changes in osmotic pressure required to maintain turgor
for extension growth are achieved by solute accumulation in
the cells of the growing region (see Figures 1B and 1C). The
compounds accumulated vary among species, but tend to be of
low molecular weight. For example, there is an accumulation
of proline in droughted wheat roots (Ober and Sharp 1994) and
of pinitol in legumes, including chickpea (Laurie and Stewart
1990), pigeon pea (Keller and Ludlow 1993) and soybean
(Guo and Oosterhuis 1995). Concentrations of pinitol vary
from 10 to 30 mol m −3 in stressed Mesembryanthemum crystallinum L. and Sesbania aculeata (L.) Merr. to 100 mol m −3
in leaves of unstressed Robinia pseudacacia L. (Thonke 1990,
cited in Popp and Smirnoff 1995). In mopane trees growing in
an arid area of Zimbabwe, leaf and twig pinitol concentrations
can be as high as 370 and 230 mol m −3, respectively (Popp,
unpublished data); however, pinitol concentrations in the seedlings we studied were lower, ranging from 50 (controls) to 125
mol m −3 (at −2.0 MPa) in hypocotyls and 25 to 85 mol m −3 in
roots (Figure 2C). On a dry weight basis we found that pinitol
concentrations of hypocotyls were similar in control seedlings
and seedlings in the −2.0 MPa treatment (550 ± 82 and 490 ±
60 mmol kg −1 respectively), whereas the corresponding values
for roots were 233 ± 5 and 550 ± 31 mmol kg −1. Hence the
higher root concentrations were brought about by an increase
in the amount of pinitol rather than a decrease in root water
content.
A concentration gradient in pinitol occurs in soybean plants
growing under ambient conditions and in response to high
temperature, with concentrations being highest in leaves,
lower in the stems and lowest in root tissue (Guo and Oosterhuis 1995). Guo and Oosterhuis suggested that the gradient
is a consequence of pinitol transport from leaves to stems and
roots. We found a concentration gradient of pinitol from the
hypocotyl to the root in mopane seedlings growing at external
water potentials of −0.03, 0.2 or 0.8 MPa, but the gradient was
not observed in seedlings in the − 2.0 MPa treatment.

717

Other soluble carbohydrates, particularly sucrose, accumulate in response to water deficit, possibly because of a reduced
shoot sink demand as a result of reduced growth (Popp and
Smirnoff 1995). In mopane, sucrose concentrations were low
in roots of seedlings in the − 0.03, − 0.2 and −0.8 MPa treatments but were higher in roots of seedlings growing at
− 2.0 MPa where growth was reduced.
We conclude that there is a relationship between osmotic
adjustment and pinitol accumulation in roots of mopane seedlings subjected to drought. However, we were unable to determine whether osmotic adjustment and pinitol accumulation
are related to root elongation or to increased drought resistance.
Acknowledgments
Thanks to the Forest Research Centre, Harare, Zimbabwe for seed
supply, Colin Ashcroft for excellent technical assistance (in particular
microscopy), and Prof. Dr. M. Popp, Dr. J.A.B. Prior and Prof. J.S.
Choinski Jr. for their interest in this work. J.P. was supported by a grant
from the BBSRC.
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