Received for publication October 29, 1991
Accepted January 4, 1992
Plant Physiol. (1992) 99, 886-894
0032-0889/92/99/0886/09/$01 .00/0
Effects of NaCI and CaCI2 on Water Transport across Root
Cells of Maize (Zea mays L.) Seedlings'
Hassan Azaizeh, Benito Gunse, and Ernst Steudle*
Lehrstuhl fur Pflanzenokologie, Universitat Bayreuth, Universitatsstrasse 30, D-8580 Bayreuth, Federal Republic of
Germany (H.A., E.S.); and Laboratorio de Fisiologia Vegetal, Facultad de Ciencias, Universidad Autonoma de
Barcelona, E-08193 Bellaterra, Spain (B.G.)
ABSTRACT
Salt stress may reduce plant growth by causing water
deficits, ion toxicity, ion imbalance, or a combination of any
of these adverse factors (6). Investigations of the effects of
salt stress on different plant crops have been increasing
rapidly during the past few years (4). However, the exact
mechanisms by which high salinity inhibits the growth of
different crops are still poorly understood (3). The effect of
salt stress on nutrition is particularly interesting because Ca
is an important factor involved in the resistance of plants to
salt stress (6, 9). Numerous studies have shown that the
growth of plant crops is seriously inhibited by high ratios of
Na+/Ca2" characteristic of sodic conditions (2, 9, 10). For
example, maize plants grown in nutrient solutions salinized
with NaCl exhibited severe symptoms of Ca deficiency at the
four-leaf stage (12). The symptoms disappeared when an
extra 10 mM CaCl2 was added to the medium. Symptoms of
Ca deficiency were correlated with low concentrations in the
maize leaf tissue. Recent studies showed that 100 mm NaCl
in the growth medium caused reduction of the growth of
primary maize roots that was accompanied by reductions in
the length of the root tip elongation zone, in the length of
the epidermal cells, and in the apparent rate of cell production
(27). Each of these reductions was partially reversed when
extra Ca was added to the medium. Possibly because of its
role in maintaining membrane integrity, contributes to the
ability of different plants to resist salt stress. In maize, the
increase of cation concentrations imposed by salt stress
caused a displacement of Ca from root cell membranes (11).
Increasing the external concentrations counteracted this displacement, which may account for the protective effects of
Ca in salt-stressed plants as proposed earlier (10).
The salt concentrations of root media are known to affect
plant water status and thus might also affect Lpr2. In a recent
The effect of salinity and calcium levels on water flows and on
hydraulic parameters of individual cortical cells of excised roots of
young maize (Zea mays L. cv Halamish) plants have been measured
using the cell pressure probe. Maize seedlings were grown in onethird strength Hoagland solution modified by additions of NaCI
and/or extra calcium so that the seedlings received one of four
treatments: control; +100 millimolar NaCI; +10 millimolar CaC12;
+100 millimolar NaCI + 10 millimolar CaC12. From the hydrostatic
and osmotic relaxations of turgor, the hydraulic conductivity (Lp)
and the reflection coefficient (a.) of cortical cells of different root
layers were determined. Mean Lp values in the different layers
(first to third, fourth to sixth, seventh to ninth) of the four different
treatments ranged from 11.8 to 14.5 (Control), 2.5 to 3.8 (+NaCI),
6.9 to 8.7 (+CaCI2), and 6.6 to 7.2. 10-7 meter per second per
megapascal (+NaCI + CaC12). These results indicate that salinization of the growth media at regular calcium levels (0.5 millimolar)
decreased Lp significantly (three to six times). The addition of extra
calcium (10 millimolar) to the salinized media produced compensating effects. Mean cell as values of NaCI ranged from 1.08 to
1.16, 1.15 to 1.22, 0.94 to 1.00, and 1.32 to 1.46 in different root
cell layers of the four different treatments, respectively. Some of
these a, values were probably overestimated due to an underestimation of the elastic modulus of cells. os values of close to unity
were in line with the fact that root cell membranes were practically
not permeable to NaCI. However, the root cylinder exhibited some
permeability to NaCI as was demonstrated by the root pressure
probe measurements that resulted in as,r of less than unity. Compared with the controls, salinity and calcium increased the root
cell diameter. Salinized seedlings grown at regular calcium levels
resulted in shorter cell length compared with control (by a factor
of 2). The results demonstrate that NaCI has adverse effects on
water transport parameters of root cells. Extra calcium could, in
part, compensate for these effects. The data suggest a considerable
apoplasmic water flow in the root cortex. However, the cell-to-cell
path also contributed to the overall water transport in maize roots
and appeared to be responsible for the decrease in root hydraulic
conductivity reported earlier (Azaizeh H, Steudle E [1991] Plant
Physiol 97: 1136-1145). Accordingly, the effect of high salinity on
the cell Lp was much larger than that on root Lpr.
2Abbreviations and symbols: Lpr, root hydraulic conductivity; P,
cell turgor pressure; P., stationary cell turgor pressure before hydrostatic or osmotic experiments; Pr, root pressure; d, cell diameter; 4
cell length; A, cell surface area; V, cell volume; Lp, cell hydraulic
conductivity; T1/2, half-time of water exchange of cells; T1/2, halftime of water exchange of roots; k8, rate constant of solute exchange;
E, elastic modulus of cells; 1r', external osmotic pressure; 7r', osmotic
pressure of cell; Pmin, minimum cell turgor pressure reached after
equilibrium in osmotic experiments; tm,, time required to reach Pmi,
in osmotic experiments; a,, reflection coefficient of cell; a,,, reflection
coefficient of root. Superscripts 'en' and 'ex' denote flows from the
medium into the cell or from the cell into the medium, respectively.
'This work was supported by a grant to H.A. from the Minerva
Foundation, MPI fur Kemphysik, FRG, and by a grant to B.G. from
Programa Nacional de Formaci6n de Personal Investigador en el
Extranjero, Subprograma de Perfeccionamiento de Doctores y Tecn6logos, Ref. PF91 40970420, Spanish Ministry of Education.
886
EFFECTS OF NaCI AND CaCI2 ON WATER RELATIONS OF MAIZE ROOT CELLS
study, the root pressure probe technique was used to determine the effects of salinity at regular (0.5 mM) and at high
(10 mM) levels on the Lpr of maize roots (1). Salinization of
the growth medium by 100 mm NaCl caused reductions in
Lpr of as large as 30 to 60%. Changes of Lpr occurred using
both hydrostatic and osmotic pressure gradients across the
maize root, which in turn have been shown to result in
different values of Lpr (1, 14, 18). However, from the study
of Azaizeh and Steudle (1), it was not clear whether the
changes of Lpr were due to changes in cell Lp, changes in
root structure, or some other effects.
The aim of this research was to determine the effects of
salinity at regular (0.5 mM) and at high (10 mM) calcium levels
on the Lp of maize root cells and on other water relation
parameters. The cell pressure probe was used to measure the
P in different layers of the cortex and at different distances
behind the root tip. The water relation parameters of root
cells both in hydrostatic and osmotic experiments were determined as well as a, for NaCl. The effects of salt stress on
cell dimensions were also investigated.
MATERIALS AND METHODS
Plant Material and Treatments
Maize kernels (Zea mays L. cv Halamish) were germinated
for 2 to 3 d in the dark at 270C on wetted filter paper as
described earlier (1). Seedlings were transferred to plastic
tanks (6 L) containing aerated one-third strength Hoagland
nutrient solution, and were maintained in a growth chamber
at 26 ± 20C, at a 12-h photoperiod for 4 to 17 d (two-to-six
leaf stage). Nutrient solutions were modified by adding NaCl
and/or extra CaCl2 so that the growing seedlings received
one of four different treatments: control; +100 mm NaCl (in
increments of 25 mm NaCl every 24 h); + 10 mm CaCl2; and
+100 mM NaCl + 10 mm CaCl2. The 7r' (Table II) of the
media were kept constant during the growing period by
adding fresh solutions. The seminal roots used for the cell
pressure probe experiments were varied in their ages for the
four different treatments and ranged between 4 and 17
(control), 2 and 12 (+NaCl), 2 and 16 (+CaCl2), and 2 and 9
d (+NaCl + CaCl2).
Freehand cross- and longitudinal sections at distances of
30 to 90 mm from the main root tips were made to determine
the mean dimensions of cortical cells. The roots of salinized
plants that were grown at regular Ca level were shorter.
Therefore, the sections were made 25 to 55 mm behind the
tip in these roots. The hand-cut root sections were transferred
into mesh-bottomed holders, and then into staining plates
containing 0.5% Toluidine blue 0 for 2 to 3 min (13). The
sections were rinsed several times in distilled water before
mounting. They were mounted in water and photographs
were taken to determine the cell dimensions using a computerized digitizing tablet program. The means of A and V of
cells of the different cortical root layers were determined
from the appropriate d and /
Cell Pressure Probe Measurements
The cell pressure probe (Fig. 1) was used to determine P,
T1/2, and water relation parameters (Lp, E, and a,) of individ-
A
~
887
microscope
A
motor
metal clamps-
oulet
Figure 1. Cell pressure probe used for measuring cell turgor, and
water and solute parameters of individual maize root cells. The cell
turgor was determined by inserting a microcapillary into a root cell
under the microscope. A meniscus, which served as a reference
point during the measurements, formed in the tip of the capillary
(see inset, A). Water and solute parameters were measured by
either changing the P with the aid of a metal rod (hydrostatic
experiments) or by changing the osmotic pressure of the medium
(osmotic experiments) and following the subsequent relaxations of
cell turgor (for further explanations, see text).
ual cortical cells (7, 16, 19, 26). The cell pressure probe (Fig.
1) was filled with silicone oil. The glass capillaries used in
the cell pressure probe had external diameters of 1 mm. They
were pulled on a puller for obtaining thinner tips. The capillaries were filled with silicone oil and attached to the probe.
Their tips were broken to yield final tip diameters of 4 to 8
Am. A root segment was mounted inside a glass tube (diameter 3 mm). Solutions were circulated along the segment in
the tube as shown in Figure 1. Root segments employed for
determination of cell P, Lp, E, as, and T1/2 were taken from
the mature region of the main roots at distances 225 mm
behind the root tip. Roots varied in length depending on the
treatment they received and were (±SD): 70 ± 19 (n = 72,
control), 28 ± 5 (n = 39, + NaCl), 54 ± 20 (n = 44, + CaCl2),
and 68 ± 16 mm (n = 64, + NaCl + CaCl2). The microcapillary of the cell pressure probe was pushed into a cortical
cell of the mounted root and the cell sap formed a meniscus
with the silicone oil inside the capillary. The P0 was recorded
after a few minutes. When P became stationary, hydraulic
parameters of the cell were determined (PO, E, and T1/2). The
position of the measured cortical cell was estimated from the
depth of the insertion of the microcapillary tip inside the
root.
Hydrostatic Experiments
In the hydrostatic experiments, the Lp(TI/2) of the endosmotic (Lpen) and exosmotic (Lpex) water flows were determined by moving the meniscus backward or forward, respectively. The meniscus was kept stable at a certain position
during the hydrostatic relaxation with the aid of the motor.
The Lp values in the hydrostatic experiments were determined from the relationship:
888
AZAIZEH ET AL.
Plant Physiol. Vol. 99, 1992
CELLS
ROOTS
0.20-
0.10
0~
cL
:2
T,,/2=8S
Tr,
-
01
+52 mOsmol
/2=
7s
0.05 4 Osr= 0.7
0.10
I
0.08
I
-52 mOsmol
I
I
I
Ti12=13s
+43 mOsmol
10-
0.06-
0.8-
T 1/2= 92s
tn
0.04
a-
0.02T,1/2= 16s
Tasr=I0.7
:3
2nd
-
,
0.45 us= 0. 90
-43 mOsmol
1I/2= 1 0.5S
°1 ,
;
L-
0.25
0.20 A
0.15
0.10-
T,r/2=7s i+44
(fi3
+50mOsmol
NaCI
0.6-
-
CL0
0)
rI
layer
T,/2=8.6s
I
-50 mOsmol
i
I
,
NaCI
,
mOsmol
'/"V\T,112= 85s
Tr,i/2= 14s
I
-44 mOsmol
Us =0O . 7
<
(r
(
0.16
1
1
1/" I
T,/2=7s
I
I
I
I
I
+37 mOsmol
1
0.14
0.12
0.10
rI,2= 86s
Trl/=
I.0
0
0
Tr=0.
13s37
1
1 2 3 4 5 15
30
45
60
mOsmol
75
Time,t (min)
90
105
Timet (s)
Time,t (min)
Figure 2. Typical examples of hydrostatic and osmotic relaxation experiments performed with either the root pressure probe or the cell
pressure probe on excised maize roots grown in control (A); control + 100 mm NaCI (B); control + 10 mm CaCI2 (C); and control + 100 mm
NaCI + 10 mm CaCI2 (D) growth solutions. The T1/2 and Tr 1/2 in the hydrostatic experiments (left side of traces) for the four different treatments
were used to calculate the Lp and Lpr, respectively. Half-times of water phases of cells and of whole roots in the osmotic experiments (right
side of traces) are given. Osmotic experiments using the cell pressure probe indicated that NaCI caused only
monophasic responses (water phases) in turgor. On the other hand, typical biphasic response curves (water and solute phases) were obtained
in the whole root relaxations (right side of traces) for all four treatments by using the root pressure probe. The as and asr were calculated
from maximum changes of turgor and root pressure, respectively.
In(2)
Lp = AT()+
1
(1)
where V =r .d2/4, volume of a cylindrical cell; A = 7r d .4
cell surface area; T1/2, half-time of water exchange of the cell;
e = V dP/dV, elastic modulus of cell, and dP/dV
AP/AV
is the change in pressure measured in the system per change
in volume that was induced by pushing the meniscus forward
or backward;
osmotic pressure of the cell. The 7' was
measured using the Nanolitre Osmometer (Clifton Technical
Physics, Hartford, NY) where small amounts of solutions are
used to determine their osmotic concentration. To determine
7ri, cell sap was sucked out from root cortical cells with the
aid of the cell pressure probe. The measured sample was
ejected into holes made in a golden holder that were already
loaded with immersion oil. The sap was frozen and afterwards thawed slowly. The osmotic concentration of the sample was determined by the osmometer (which already was
calibrated with NaCl standards) when the last small ice
crystal disappeared in the solution droplet. 7ir was also estimated from the relationship = P. + 7r', where P0 is the
stationary cell turgor prior to the experiment and 7r' is the
external osmotic pressure of the growth solution (Table II).
Both techniques used to determine
resulted in similar
values.
Osmotic Experiments
-,
In osmotic experiments, the original nutrient solutions were
rapidly exchanged for media containing extra NaCl at known
concentrations. Solutions were circulated along the root segments as shown in Figure 1 to minimize unstirred layers
outside the root. During the osmotic experiments, the meniscus was kept stable to avoid changes in cell volume. The
apparent cell Lp in the osmotic experiments could be, in
principle, determined using Equation 1. However, these
measurements would include considerable unstirred layer
effects and other effects caused by the distance that the solute
889
EFFECTS OF NaCI AND CaCI2 ON WATER RELATIONS OF MAIZE ROOT CELLS
would have to pass across the tissue, depending on the
position of the measured cell. The as was calculated at zero
water flow from the relationship (22):
AP
iv E + ir1
E
as= .i'~.
Air0
exp(ks tmin)
Table I. Dimensions of Cortical Cells of Maize Roots
Values are the means ± SD of measurements taken at different
distances from the root tip. Numbers in parentheses denote the
numbers of cells measured.
(2)
where AP = P. - Pmin is the maximum change in P measured
in response to the change of the osmotic pressure of the
external medium (Air0). The term (E + ir1)/e corrects for
concentration changes in the cell induced by volume changes
and the exponential term for the passive solute flow. However, when measuring cells in osmotic experiments we obtained monophasic responses (Fig. 2) after changing the
original growth solutions for solutions containing extra NaCl
around the root segments. Thus, the permeability of the cells
for NaCl was virtually zero and hence k5 also was zero and
the exponential term in Equation 2 was unity. The a, would
be that of a cell surrounded by a tissue that would also
incorporate unstirred layer effects. Accordingly, 20 to 30 min
were allowed for equilibration of diffusional gradients of
NaCl across the cortical apoplast.
Step-Down Experiments with Roots Grown at High Salinity
In the experiments described in this paper, changes of the
osmotic concentration were relatively small (40-60 mOsmolkg-1, which have equivalent 0.1-0.15 MPa of osmotic pressure, respectively). In some cases, osmotic experiments were
also performed in which the roots were first grown in + 100
mM NaCl or + 100 mm NaCl + 10 mm CaCl2. Then salinity
was reduced by 180 to 190 mOsmol * kg-', which is equivalent
to an osmotic step-down of 0.45 to 0.48 MPa, respectively
(4.5-4.8 bars). Changes in P were followed. The experiments
were performed to determine if the system (cell) would be
stable during these large changes and also if it would react
as an ideal osmometer exhibiting one phase.
RESULTS
Cell Dimensions and P
Cell dimensions of roots were determined from cross and
longitudinal sections. d and / of cells of different layers of
the root cortex of seedlings (3-9 d old) are summarized in
Table I for the four different treatments. In the range between
25 and 90 mm behind the root apex, there was a slight
tendency for the cells to become shorter toward the tip, but
these differences were not significant. Therefore, the data
were pooled and averages are given. Also, the different root
ages did not significantly affect the cell dimensions. Table I
shows that mean cell diameters ranged from 29 to 41 ,um and
that cells of the fourth to sixth root layers were slightly, but
significantly, larger than those of both outer and inner cortex
layers. This was true for three of four treatments. Salinized
plants grown at a regular Ca level had the shortest cell lengths
(/= 98-115 Am) and the control plants had the longest cells
(/= 185-209 Am). Thus, they were different by a factor of
2. Mean V were calculated from the mean values of d and
/taken from the different cell layers of roots of the four
different treatments. They ranged from 126 to 171 (control),
Treatment
Control
Layer
d
Am
1-3 31.1 ± 3.7 efa 208.5 ± 15.7 aa
(32)
(60)
4-6 34.3 ± 4.6 d 184.6 ± 20.3 bc
(56)
Control + 100 mm NaCI
7-9 29.3 ± 3.2 f
(48)
1-3 34.9 ± 3.9 dc
(72)
4-6 39.5 ± 3.0 ab
(72)
7-9 33.5 ± 5.5 de
(72)
Control + 10 mm CaCI2
(32)
187.0 ± 16.4 b
(20)
114.7 ± 17.2 f
(48)
111.8 ± 33.1 f
(52)
98.1 ± 8.1 f
(52)
1-3 37.4 ± 2.0 bc 172.5 ± 25.7 bcd
(72)
4-6 40.8±2.5a
(72)
Control + 100 mm NaCI
+ 10 mM CaCI2
I
(48)
165.3±19.5cde
(44)
7-9 35.9 ± 3.3 cd 166.2 ± 25.5 cde
(40)
(68)
1-3 35.0 ± 4.0 cd 172.5 ± 14.5 bcd
(44)
(44)
4-6 35.3 ± 1.7 cd 157.8 ± 17.3 de
(44)
(48)
7-9 33.7 ± 2.9 de 151.4 ± 18.0 e
(44)
(36)
Values in same column followed by different letters differ
significantly (Duncan test, probability = 0.05).
a
86 to 135 (+NaCl), 168 to 216 (+CaCl2), and 135 to 166.
10-15 m3 (+NaCl + CaCl2). Seedlings grown at high salt and
regular Ca levels had the smallest mean V. Mean values of
V and A calculated from mean d and / were used to evaluate
the e and the Lp of the different treatments.
P of the main roots of the four different treatments were
measured using the cell pressure probe (Fig. 1). P values were
also calculated from the difference between the 7i and the
7r'. The measured P values appeared to show some gradient
(of about 0.1 MPa) across the entire cortex, but these differences were not significant due to high variability in turgor
between cells and roots. Therefore, the data were pooled and
presented as means ±SD for each treatment (Table II). Measured P values showed that control plants had the highest cell
P compared with the other three treatments. Salinized plants
grown at high Ca levels had the smallest cell P. The data
show that NaCl and Ca levels had significant effects on the
measured turgor pressure (Table II).
Water Relation Parameters and or, of Cortical Cells
Typical examples of hydrostatic and osmotic relaxation
curves used to determine the cell water relation parameters
for the four different treatments are shown in Figure 2 for
roots and cells. Hydrostatic experiments are shown on the
left side of each of the traces given. On the right side, osmotic
responses are given using NaCl as the osmotic solute. In the
Plant Physiol. Vol. 99, 1992
AZAIZEH ET AL.
890
hydrostatic experiments, the T1/2 were usually somewhat
smaller than those of Tr1/2, although both parameters were
of the same order of magnitude (2-16 s). Hydrostatic T1/2
were much shorter than those obtained in osmotic experiments, at least for cells sitting deeper in the roots.
Table Ill. Hydrostatic Experiments: Lp of Maize Root Cortical Cells
as Determined by Cell Pressure Probe
Mean values ± SD for TI,2 and for Lp of the different cell layers
are given. Numbers in parentheses denote the numbers of cells
measured.
Hydrostatic Experiments
Table III summarizes T1/2 and Lp obtained in the hydrostatic experiments. The longest T1/2 were obtained for salinized plants grown at low and at high Ca levels. Mean T1/2
values of all treatments ranged between 2.4 and 5.0 s. In
some treatments, significant differences were observed for
T1/2 of the different root layers (Table III). However, the
hydraulic conductivities of cells (Lpen and Lpex) of different
cortical root layers were similar for each treatment. Therefore,
the data were pooled and were given as hydrostatic Lp values
(Table III). Also, ratios of Lpen/Lpex (data not shown) indicated
no polarity in water movement across the cell membranes.
Mean values of Lp of control seedlings were considerably
larger (by a factor of 3-6) than those obtained in the other
three treatments. Mean Lp values of the salinized plants
grown at regular Ca levels were significantly smaller compared with the other three treatments. The mean Lp values
of the salinized seedlings grown at high Ca levels were similar
to those values of the control seedlings grown at high Ca
levels. Table III shows that, in general, salinization of the
growth media caused reductions of hydrostatic Lp. The addition of extra Ca to the salinized growth solutions had a
partially compensating effect on the Lp values. The volumetric e of the root cortical cells of the different treatments were
measured after the P was stable. The e values of the four
treatments used to calculate cell Lp according to Equation 1
ranged between 0.8 and 2.0 (control), 0.8 and 3.6 (+NaCl),
1.2 and 8.9 (+CaCl2), and 0.8 and 2.1 MPa (+NaCl + CaCl2).
The propagated error in e values (1, 26) of the different
treatments ranged between 20 and 40%.
The Lp values in Table III are given as means ± SD and
were calculated according to Equation 1 without considering
the propagated errors of the five different independent vari-
Treatment
Layer
T%
s
1-3
(29)
7-9
3.8 ± 1.3 bc
(24)
Control + 100 mm NaCI
1-3 4.5 ± 1.6 ab
(20)
4-6 3.9 ± 1.2 bc
(11)
7-9 3.1 ± 1.0 d
(9)
Control + 10 mm CaCI2
3.2 ± 1.3 cd
(20)
4-6 2.5 ± 0.7 e
1-3
(11)
7-9 2.4±0.9e
(12)
Control + 100 mm NaCI +
10 mM CaCI2
5.0 ± 1.5 a
(19)
4-6 4.9±1.4a
1-3
(30)
7-9
4.8 ± 1.5 a
(17)
(29)
12.0 ± 4.1 b
(24)
3.4 ± 1.3 e
(1 1)
2.5 ± 1.0 e
(8)
3.8 ±1.1 e
(8)
6.9 ± 2.0 cd
(12)
7.6 ± 2.5 cd
(8)
8.7±2.6c
(9)
6.6 ± 1.8 d
(19)
6.8±1.8cd
(30)
7.2 ± 1.9 cd
(17)
a Values in same column followed by different letters differ
significantly (Duncan test, probability = 0.05).
cells measured.
r0
1'r
Measured P
0.02
0.57 ± 0.05
0.63 ± 0.10 aa
(16)
(73)
(16)
0.52 ± 0.10 b
0.58 ± 0.08 a
Calculated P
MPa
Control
MPa-1
3.3 ± 0.7 cda 11.8 ± 4.3 ba
(17)
(17)
4-6 3.3 ± 1.0 cd 14.5 ± 4.7 a
Control
Table II. Osmotic Pressures and Turgor Pressures of Root Cells
Effects of salinity at different levels on P of maize root cortical cells, measured using the cell
pressure probe or as calculated from the difference between 7r and 7r using the Nanolitre Osmometer
technique. The values are given as means ± SD. Numbers in parentheses denote the numbers of
Treatment
Lp. 107
m-s1
0.55 ± 0.05 aa
Control + 100 mM NaCI
0.51
1.09 ± 0.06
(26)
(48)
(26)
Control + 10 mm CaCI2
0.08
0.62 ± 0.08
0.48 ± 0.10 b
0.54 ± 0.08 a
(9)
(45)
(9)
0.48 ± 0.08 a
0.41 ± 0.09 c
1.06 ± 0.08
0.58
Control + 100 mm NaCI + 10
(16)
(64)
mM CaCI2
(16)
a
Values in same column followed by different letters differ significantly (Duncan test, probability
= 0.05).
EFFECTS OF NaCI AND CaCI2 ON WATER RELATIONS OF MAIZE ROOT CELLS
Table IV. Osmotic Experiments: Effects of Salinity on the Tv, and on
a, for NaCI of Maize Roots as Determined by Cell Pressure Probe
Values are mean ± SD for T½, and for as of maize root cortical
cells. Numbers in parentheses denote the numbers of cells measured.
Treatment
Layer
aS
Ta
S
Control
1-3 148 ± 101 abca 1.16 ± 0.12 cda
(7)
(7)
4-6 126±34bcd 1.14±0.22cd
(7)
(8)
Control + 100 mm NaCI
7-9 170 ± 142 ab
(11)
1-3 92 ± 40 cd
(8)
4-6 71 ±44d
Control + 10 mm CaCI2
7-9 146 ± 45 abcd 1.15 ± 0.22 cd
(1 0)
(7)
1-3 87 ± 29 cd
1.00 ± 0.08 de
(12)
(15)
4-6 93 ± 48 bcd 0.94 ± 0.09 e
Control + 100 mm NaCI
+ 10 mM CaCI2
7-9 155±101abc
(12)
1-3 142 ± 45 abcd
(15)
4-6 155±67abc
(18)
7-9 207 ± 51 a
(9)
(9)
(9)
1.08 ± 0.31 cde
(11)
1.22 ± 0.05 bc
(10)
1.17±0.14bc
(8)
(10)
0.94±0.13e
(12)
1.32 ± 0.14 ab
(15)
1.46±0.19a
(17)
1.38 ± 0.15 a
(11)
aValues in same column followed by different letters differ
significantly (Duncan test, probability = 0.05).
ables (d, 4 T1/2, dP/dV, and ir). The propagated relative errors
in Lp values were 26 to 39% for all treatments. The errors
for determination of the d and / for the different treatments
were 15 to 25% and 14 to 23%, respectively, whereas the
errors in the determination of T1/2, dP/dV, and w' were
calculated to be 10 to 17%, 6 to 15%, and 2 to 3%, respectively. It is obvious that the errors in d and / that were used
to calculate A and V contributed most to the total error,
whereas the errors due to the measurements made with the
cell pressure probe (T1/2 and dP/dV) were relatively small.
For the V, errors in determining the d contributed by a factor
of 2 to the total propagated error.
Osmotic Experiments
Table IV summarizes the effects of salinity and calcium
levels on the T1/2 and on the a. obtained in osmotic experiments as demonstrated in Figure 2. The right side of the
figure shows that individual cells behaved differently than
whole roots during osmotic experiments when gradients were
imposed by changing the osmotic pressure of the external
solution for solutions containing extra NaCl. The addition of
NaCl as an osmoticum in the medium caused monophasic
relaxations of cell turgor, ie. only the water phase was
present. However, in whole root relaxations, NaCl caused
typical biphasic curves (water and solute phases present) in
891
all four treatments (see left side of Fig. 2). This means that
the cell's permeability coefficient for NaCl was much smaller
than that of the root. The mean T1/2 in response to osmotic
changes of the media markedly increased with increasing
distance from the root surface (Table IV). The mean T1/2
values of the inner layers (seventh to ninth) were the largest
in all four treatments. The mean T1/2 in the osmotic experiments of the different treatments ranged between 71 and 207
s, depending on the cell layers that were measured.
The a, was determined from the maximum changes in
turgor in response to changes of the osmotic pressure of the
medium (Fig. 2). The a. values were calculated using Equation
2 and are summarized in Table IV. The mean a. values of the
different treatments ranged between 0.94 and 1.46. These
high values of a. were probably overestimated because of
uncertainties in the correction factor [(e + ri)/e] that ranged
between 1.07 and 1.75. An underestimation of E could have
yielded a. values that were even larger than unity.
Step-Down Experiments with Roots Grown at High Salinity
Step-down experiments were conducted to determine the
effects of extreme reduction of the osmotic pressures on cell
membrane integrity, stability of the system, and the behavior
of single cells as an osmometer in short-term experiments.
These experiments were performed on roots grown in salinized media at regular and high Ca levels (Fig. 3). The original
growth solutions in both treatments were exchanged for
solutions containing only 1 mi NaCl (i.e. + 100 mm NaCl
growth solution [195 mOsmol-kg-1] was replaced by a + 1
mM NaCl solution [8 mOsmol.kg-1], Fig. 3A; and the +100
mM NaCl + 10 mm CaC12 [229 mOsmol.kg-1] solution was
replaced by + 1 mm NaCl + 10 mm CaC12 [45 mOsmol-kg-'],
Fig. 3B). The time course of the changes in P for individual
1.2 ,
1.0-
vA)
a
0.8-
T,-2577sIf56
cL
0.6-
_m
+ 187 mOsmol
m- 0.40.2 -,t
musmoi
I-Io /
00
I
I
20
40
60
80
0
20
Time, t (min)
40
60
80
Figure 3. Typical step-down (hypotonic change) and step-up (hypertonic change) experiments showing P as a function of time for
cortical root cells (fourth to fifth layer) of maize seedlings grown in
control + 100 mm NaCI (A) and seedlings grown in control + 100
mM NaCI + 10 mm CaCI2 (B) growth solutions. The changes of the
7r0 and of the T,12 are also given. Step-up experiments were performed after the P was stable by exchanging the modified external
solutions for the original growth solutions (right side of traces). The
reductions in P to lower values indicated that some damage probably has been caused to the root cell membrane by both treatments
because of the extreme osmotic stresses applied.
892
AZAIZEH ET AL.
root cells in both treatments was followed up to 60 min after
stepping down the osmotic pressure of the external solutions.
Figure 3 shows that P increased in the cells and reached a
maximum value after a few minutes (the rate of increase
depended on the measured cell layer) and then remained
stable without any noticeable decline in P, at which time the
external solution was exchanged again. The P was considered
stable because it reached a maximum value after changing
the 7r0, and no decline in P was noticed after 60 min (Fig.
3A). The result demonstrated that there was no biphasic
response. This criterion was used to distinguish a stable P
from a situation in which a decline in P could be interpreted
as a biphasic response for root cells. The increase in P values
was similar to those values of the changes in the external
osmotic pressure (Ar°) in both treatments, i.e. it was valid
that AP/Air' = 1 as one would expect for a nearly perfect
osmometer. After the step-down, external solutions were
exchanged again for the original solution after reaching a
stationary P (Fig. 3, A and B, right side of traces). This osmotic
step-up caused P to decline again and to reach a minimum
lower than the original P.. The difference may indicate some
damage to the cell membranes due to the extreme osmotic
changes (Airo +4.5 bars). The T1/2 of the step-down and
step-up experiments shown in Figure 3 were estimated to be
577 and 356 s for the salt plants grown at regular Ca levels,
and 140 and 133 s for the plants grown at high Ca levels,
respectively. There is no doubt that Ca has some effects on
the change of P as a result of the step-down and step-up
experiments. However, this needs more study. These kinds
of experiments were repeated more than three times with
similar results.
DISCUSSION
In an earlier study (1), salinization of the growth media of
maize roots grown hydroponically at low Ca levels revealed
that the Lpr was reduced by 30 to 60% as compared with
control seedlings. Addition of Ca (10 mM) to the salinized
media was ameliorative and resulted in higher Lpr. The
present study was conducted to determine whether the reduction in Lpr was due to changes of Lp. The cell pressure
probe was used to determine P and water relation parameters
of individual cortical maize root cells. The results were compared for seedlings grown in salinized media containing low
or high Ca levels with values obtained from control plants.
As one may expect, salinization of the growth media of
maize roots at different levels of Ca significantly reduced the
mean measured P compared with the values obtained from
control seedlings (Table II). Our data show that osmoregulation obviously took place, but seedlings grown in control
solutions had the greatest P values, although the differences
were small. Thus, osmoregulation was not complete. Also in
Nicotiana tabacum, salinization of the growth medium caused
reduction in the mean P values of the root cortical cells (23).
Measured values of turgor were slightly different from those
calculated from the difference ir - ino (Table II). Gradients in
P and also in the 7i of the different treatments cannot be
completely excluded, as already pointed out (19). However,
if existing, they should have been <0.1 MPa across the entire
cortex (thickness 270-360 ,um), and were not significantly
Plant Physiol. Vol. 99, 1992
different. In wheat roots, Pritchard et al. (14) demonstrated
that P was constant along the radius of the cortical cells
within the elongation zone, irrespective of the nature of the
bathing solution. However, in the mature regions of the roots,
gradients were found. In the mature zone, the P of the stelar
cells was greater than that of the cortex.
Mean values of Lp obtained in the hydrostatic experiments
of the control seedlings were three to six times greater than
those obtained from plants grown in salinized media with
regular Ca levels (Table III). Seedlings grown either in salinized media at high Ca levels or in control solutions at high
Ca levels revealed similar Lp values. However, these values
were also significantly smaller compared with the control
seedlings grown at regular Ca levels. Cells from all layers
measured in the different treatments showed differences in
mean Lp values in the same direction.
This is the first time that changes of Lp of membranes of
a higher plant are reported in response to changes of the
osmotic concentration. Our data suggest that osmotic concentrations have some effects on Lp. In addition, the ionic species
used may have some toxic effects that could determine the
magnitude of the changes. For the bladder cells of the halophytic species Mesembryanthemum crystallinum (20), it was
found that Lp values were not affected by concentration
changes of as large as 400 mm NaCl in the medium.
The reductions in Lp caused by salinity in maize roots were
in agreement with reductions found earlier for Lpr (1), although the latter were much less pronounced. High Ca levels
had a compensating effect on Lp as well as on Lpr. However,
the Lp values were four to five times greater than those of
Lpr in all treatments. Thus, our data suggest a significant
bypass of water around cells at least when hydrostatic gradients are imposed. Otherwise, the root Lpr should be a factor
of 20 (for 10 layers of cortical, epidermal, and endodermal
cells to be crossed) smaller than Lp.
However, the data indicate that the cell-to-cell (transcellular plus symplasmic) path cannot be neglected, and there
was a cell-to-cell component also in the hydrostatic experiments (for calculations, see ref. 17). The mean Lp values of
another maize cultivar were calculated to be more than five
times greater than those of Lpr measured in hydrostatic
experiments (26) where the authors found that Lp can vary
among cortical cell layers, which is also indicated by our data
(Table III).
Using cell and root pressure probe techniques, values of
cell Lp and root Lpr of different cultivars of maize have been
determined for both hydrostatic and osmotic experiments (5,
18, 21, 26). Differences between osmotic and hydrostatic Lpr
have been interpreted in terms of a composite membrane
model for the root, i.e. by assuming different parallel and
serial membrane-like barriers in the root (e.g. the apoplasmic
and cell-to-cell paths of the different cell layers). In hydrostatic experiments, water flow appeared to be mostly apoplasmic, i.e. bypassing root protoplasts. However, in the
presence of osmotic gradients, there was a substantial radial
cell-to-cell transport of water (18, 24).
In the roots of rice seedlings grown under salinized conditions, apoplastic flow contributed substantially to the total
quantity of Na+ that reached the xylem (25). These authors
suggested that apoplastic transport may increase under salt
EFFECTS OF NaCI AND CaCI2 ON WATER RELATIONS OF MAIZE ROOT CELLS
stress conditions. Jones et al. (8) measured the Lpr of maize
and wheat roots using an osmotically induced back-flow
technique, and the cell Lp of the roots were determined using
a pressure probe. Their results showed that near the root tips,
water apparently flowed through the apoplasmic pathway,
although earlier data showed a predominant cell-to-cell component. However, further from the tips, the measured Lpr
were consistent with flow either through the cell-to-cell or
the apoplasmic pathways.
Radin and Matthews (15) compared the hydrostatic cell Lp
and root Lpr of cotton. They concluded that there was a
considerable apoplasmic bypass during water flow similar to
the situation found in maize roots (see above, 26). However,
in other systems, such as in bean and barley roots, both the
hydrostatic and osmotic flows were from cell to cell, as
indicated by a large cell Lp compared with root Lpr (17, 19).
Thus, depending on the species and on the nature of the
driving force (hydrostatic or osmotic), there can be large
differences in the pattern of radial water transport across
roots.
Salinization of the growth media had no significant effects
on the water relation parameters of tobacco seedlings (23).
Plants grown under salinity had longer T1/2. The Lp was
calculated for the salinized plants after the excised roots were
exposed to a step-down in NaCl concentrations. The authors
did not measure the Lp of the salinized plants before a stepdown experiment was performed, nor did they indicate which
particular root layers they measured. Their results showed
that the root cell volumes of the salinized plants were similar
to those values obtained in the control seedlings. However,
their results showed that 200 mOsmol -kg-' of NaCl caused
a reduction in root fresh weight of as large as 52% compared
with the controls, which should have had a significant effect
on cell dimensions and on the calculated Lp (23).
Our data indicated that salinized maize roots grown at low
Ca levels had significantly shorter cells compared with the
controls and with the salinized plants grown at high Ca levels
(Table I). Also, their growth rate was reduced by 75% compared with the control plants (27). This fact resulted in smaller
Lp when this parameter was calculated from similar T1/2
values obtained for the controls and for salinized seedlings
grown at regular Ca levels (Table III). The root cortical cells
of the salinized seedlings grown at low Ca levels were similar
in their lengths to those of the epidermal cells of the salinized
plants found earlier (27).
In the study of Radin and Matthews (15) on cotton roots,
growth-limiting deficiencies of nitrogen or phosphorus substantially decreased the cell Lp by a factor of 2 to 5 compared
with controls. The maize seedlings in our experiments were
grown in one-third Hoagland solution with sufficient nutrients; therefore, the reduction in Lp reported in this paper
was due to salinization, not to nutrient deficiency. The average age of maize plants used in the pressure probe experiments was 7 d and no deficiency symptoms were observed.
The e values obtained in our study ranged between 0.8 and
8.9 MPa in the different treatments, and these values were
relatively low when compared with earlier data obtained for
maize cortical cells (14, 21). However, low E values were also
obtained for another maize cultivar (26) and for tobacco root
cells of plants grown under salinity and in control solutions
893
(23). Ranges in e values were quite large, and some underestimations may have occurred due to the difficulties in the
estimation of V (see above) and to the fact that AV may be
underestimated because of some outflow of water while the
pressure inside the system was increasing.
Mean o, of the different treatments ranged between 0.94
and 1.46 (Table IV). These values may, in part, be overestimated due to underestimations of e values (Eq. 2). Using the
cell pressure probe without the root pressure probe could
have affected the mean cell a. values, which were even larger
than unity in some treatments (Table IV). The upper limit of
ar should be unity for a perfect osmometer. Preliminary
results showed that using the cell pressure probe when the
root pressure probe is still attached to the root segment to
measure AP/Awr in the osmotic experiments resulted in lower
values (10-25%) when compared with values obtained after
cutting the root from the root pressure probe. The a. values
(Table IV) were significantly greater compared with asr in all
four treatments (1). The lower a,,r values obtained when the
root was attached to the root pressure probe were explained
in terms of a parallel arrangement of different osmotic barriers in the maize root (for a composite membrane model of
the root, see above). This may also be true for cortical cells,
which would explain the differences in a. in the two types of
experiments and between a, and asr.
Step-down experiments in which the P of the root cortical
cells of salinized maize seedlings was followed over time
showed a monophasic response (Fig. 3). However, biphasic
responses in P have been obtained for the isolated epidermis
of Tradescantia virginiana leaves and for Chara corallina internodes that were subjected to changes of the external osmotic
pressure using permeating solutes (22, 24). Tobacco plants
(control and salinized seedlings) subjected to step-down osmotic experiments first showed an increase in root cell P, but
later P decreased to a new steady-state pressure (23). The
authors (23) concluded that tobacco root cells displayed some
biphasic responses to NaCl.
In our case, osmotic experiments with individual maize
root cortical cells have shown a monophasic response for all
four treatments. Nevertheless, NaCl permeated the root cylinder (Figs. 2 and 3). When entire roots from different treatments were subjected to NaCl as an osmoticum, typical
biphasic response curves (water and solute phases) were
obtained for the root pressure compared with cell pressure
(Fig. 2 and ref. 1). The reasons for these differences are not
known. The biphasic response in tobacco could be due to
either a rapid active export of sodium ions or a passive
leakage. The latter would be rather improbable because the
permeability of cell membranes for electrolytes is low.
Our results demonstrate that NaCl and CaC12 have similar
effects on Lpr and on Lp. NaCl caused more pronounced
reductions in Lp values compared with Lpr for all four
treatments, and CaCl2 had ameliorative effects when salt
stress was imposed. The data of Lpr and Lp suggest that there
was a significant apoplasmic water flow around the cells.
However, the cell-to-cell path in maize roots contributes to
the overall transport (see above). Thus, the effects of Ca on
Lpr could have been due mainly to effects on the hydraulic
conductivity of root cell membranes rather than on that of
the cell wall, although the latter cannot be completely ex-
AZAIZEH ET AL.
894
cluded. To date, the hydraulic conductivity of the cell wall
path is only indirectly accessible (26), and changes of this
parameter in response to salinity or Ca have not yet been
measured. The results may also indicate that Ca has small
effects on the apoplast Lp. Thus, the exact mechanisms by
which Ca affects Lpr and Lp still need more investigation;
possible effects on the apoplasmic path should be explored.
ACKNOWLEDGMENTS
The authors are indebted to Dr. Carol A. Peterson, Department of
Biology, University of Waterloo, Canada, for her help in the sectioning procedures and for reading the manuscript. We also thank Walter
Melchior for reading and discussing the paper.
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