Journal of Experimental Botany, Vol. 48, No. 306, pp. 45-58, January 1997
Journal of
Experimental
Botany
Cell turgor, osmotic pressure and water potential in the
upper epidermis of barley leaves in relation to cell
location and in response to NaCI and air humidity
Wieland Fricke1
The Swedish University of Agricultural Sciences, Department for Production Ecology, PO Box 7042,
S-75007 Uppsala, Sweden
Received 26 February 1996; Accepted 22 July 1996
Abstract
Previous single-cell studies on the upper epidermis of
barley leaves have shown that cells differ systematically in their solute concentrations depending on their
location relative to stomatal pores and veins and that
during NaCI stress, gradients in osmotic pressure (n)
develop (Fricke et al., 1995, 1996; Hinde, 1994). The
objective of the present study was to address the
question to which degree these intercellular differences in solute concentrations and n are associated
with intercellular differences in turgor or water potential [i//). Epidermal cells analysed were located at various positions within the ridge regions overlying large
lateral or intermediate veins, in the trough regions
between those veins or in between stomata (i.e. interstomatal cells). Turgor pressure of cells was measured
using a cell pressure probe, and n of extracted cell
sap was determined by picolitre osmometry. For both
large and intermediate lateral veins, there were no
systematic differences in turgor between cells located
at the base, mid or top of ridges, regardless of whether
plants were analysed at low or high PAR ( < 1 0 or
300-400 /imol photons m 2 s~ 1 ). However, turgor
within a ridge region was not necessarily uniform, but
could vary by up to 0.14 MPa (1.4 bar) between adjacent cells. In 60 out of 63 plants, turgor of ridge cells
was either slightly or significantly higher than turgor
of trough (lowest turgor) or interstomatal cells (intermediate turgor). The significance and magnitude of
turgor differences was higher in plants analysed under
high PAR or local air flow than in plants analysed under
low PAR. The largest (up to 0.41 MPa) and consistently
significant differences in turgor were found in plants
treated for 3-9 d prior to analysis with 100 mM NaCI.
For both NaCI-treated and non-treated (control) plants,
differences in turgor between cell types were mainly
due to differences in n since differences in <p were
1
Fax: +46 18 673037. E-mail: Wieland.Fncke©emc.s/u.se
© Oxford University Press 1997
negligible (0.01-0.04 MPa). Epidermal cell \fi in NaCItreated plants was about 0.38 MPa more negative than
in control plants due to higher n. Turgor pressures
were similar. Following a sudden change in rootingmedium \f/ or air humidity, turgor of both ridge and
trough cells responded within seconds and followed
the same time-course of relaxation. The half time (T1/2)
of turgor relaxation was not limited by the cell's T 1/2
for water exchange.
Key words: Barley leaf epidermis, cell turgor, heterogeneity, NaCI stress, osmotic pressure, water potential.
Introduction
Differences in turgor pressure between different leaf tissues
(e.g. mesophyll and epidermis in Tradescantia, Frensch
and Schulze, 1988; Nonami et al, 1990) or root tissues
(cortex and stele in maize roots, Pritchard et al., 1989)
might be explained by differences in the water relations
of these tissues or the existence of turgor-dependent
processes that are associated with differences in tissue
physiology. The question arises whether systematic
differences in turgor may also exist between cells that
belong to the same (non-growing) tissue and which are
apparently uniform in structure and function.
Some pressure-probe studies on roots have shown
radial turgor gradients between cortical cells (Rygol et al.,
1993; Zimmermann et al., 1992), whereas others have not
(Jones et al, 1983; Steudle and Jeschke, 1983; Pritchard
et al, 1989). Differences in results may have been due to
differences in the species analysed or differences in measurement conditions. For example, intercellular gradients
in turgor were only observed under considerable transpiration. Turgor (and osmotic) pressures increased from the
outermost layers of the cortex towards the xylem which
was explained by a transpiration stream-dependent
46
Fncke
solvent drag effect on solutes (Zimmermann et al., 1992).
An alternative explanation for the direction of gradients
has been provided by the results of Meshcheryakov et al.
(1992) which suggest that in the mature region of the
hypocotyl of Ricinus communis, gradients in turgor (and
77) are related to the proximity of cells to the phloem
representing the major source of organic osmotica. A
similar explanation has been given by Frensch and Hsiao
(1994) for the observation that cells located in the inner
cortex of osmotically-stressed maize roots recovered faster
in turgor than cells located in the outer cortex.
Pressure-probe studies on leaves (mature tissue of the
leaf epidermis of Tradescantia virginiana, Tomos et al.,
1981; Shackel and Brinckmann, 1985; mesophyll of
KalanchoS daigremontiana, Steudle et al., 1980), suggest
that there are no gradients in turgor or n between cells
belonging to the same tissue. However, recent single-cell
sampling and solute-analysing studies of the upper epidermis of barley leaves have shown that cells located at
various positions relative to the vascular bundles and
stomatal pores differ systematically in their vacuolar
solute concentrations (Hinde, 1994; Fricke et al., 1995)
and that during NaCl stress, intercellular differences in n
can develop (Fricke et al., 1996). This indicates that
intercellular differences in turgor or <>
/ also exist. If so,
this would add another level of complexity to our understanding of leaf water and solute relations (Leigh and
Tomos, 1993) and its response to changes in nutrient
supply, water supply or evaporative demand.
In the present study on the upper epidermis of barley
leaves, this possibility was investigated by analysing cells
located at various well-defined positions relative to (i)
stomatal pores and (ii) large lateral veins (LV) and
intermediate veins (IV) for turgor pressure, n and ifi.
Large and intermediate lateral veins were compared since
these veins can differ in their solute concentrations (particularly for NO3", Fricke et al., 1995) and may differ in
their capacities for water transport (Altus et al., 1985).
To cover a range of measurement environments, leaves
were analysed under low or high PAR, or under low or
high local illumination and air flow. In some experiments,
plants were treated for 3-9 d before analysis with 100 mM
NaCl to see to which degree the differential accumulation
of N a + and Cl~ in epidermal cell types (Fricke et al.,
1996) affects their turgor and if> relations. To characterize
the water relations under a fluctuating (/(-environment,
plants were subjected to short-term changes in external <>
/
and the response (relaxation) of epidermal cell turgor
followed with time. The TU2 of turgor relaxation was
compared with the cell's Ti/2 for water flux equilibrium.
Materials and methods
CaSO4. Eight seedlings were then transferred to 5 1 of aerated,
modified Hoagland solution containing 1 mM NaCl (Fricke
et al., 1994) and grown at a PAR of 300-400 jumol photons m~ 2
s~' and a 16 h day/8 h dark period. Plants were grown in the
same room where measurements were carried out. Plants were
analysed when they were 12-22-d-old. Second, third or fourth
leaves were analysed at the time they represented the youngest,
fully-, or nearly fully-expanded leaf. Leaves were analysed halfway along the expanded blade at the adaxial (upper) epidermis,
within a small region covering the first large lateral vein (LV),
counted from the midrib, the adjacent (outward) intermediate
vein (IV) and the second large lateral vein. For details of the
NaCl treatments see Figure legends and Tables.
Location of epidermal cells
The location of epidermal cells analysed is illustrated and
specified in Fig. 1 which gives a cross-sectional view of a barley
leaf (A) and a view of the surface of the upper epidermis (B).
The ridge regions consisted typically of a total of six cells to be
analysed; those cells that were located at the very top of a
LV-ridge were not analysed since these cells were considerably
smaller (shorter and narrower) than the other ridge cells and
capilllaries became quite often clogged during turgor measurements (IV-ridges generally did not show those small cells at the
very top); those cells that were located at the very base of a
IV- or LV-ridge, at the transition from the ridge to the stomatal
cell files, were not included in the analyses either. These cells
are likely to be exposed at their leaf-inner surface to the
substomatal cavity and might therefore be more comparable
with interstomatal cells (see also Fricke et al., 1995).
Measurement environment
Plants were transferred from their growth site to the measurement site. After 15-20 min analyses were started. Throughout
the analyses, the leaf area to be analysed was illuminated with
a cold light. Cells were viewed through a zoom stereomicroscope
(type Wild MZ8, Leica AG, Heerburg, Switzerland), at
magnifications ranging from x 128 to x 200. Leaves were
viewed either horizontally or vertically and four different
measurement set-ups were used. (lst + 2nd set-up) When cells
were analysed only for turgor, leaves were viewed horizontally.
The illumination of plants was reduced to dim room light (<; 10
fimol photons m " 2 s" 1 at plant level: 'low PAR' set-up) or
comparable to that at the growth site (illumination of 300-400
fimol photons m~ 2 s" 1 at plant level: 'high PAR' set-up). (3rd
set-up) When cells were analysed only for 77, the leaf (vertical
viewing) was kept near to the stage on which the picolitre
osmometer was mounted. The overall illumination of the plant
was dim and the illumination with the cold light source was
kept at a minimum. This set-up was chosen to compare the -rr
data with the turgor data obtained under low PAR. (4th setup) When cells were analysed for turgor and -n, to derive </i free
of errors introduced by calculating tf> from turgor and TT data
obtained from different plants and cells, the leaf area to be
analysed was exposed to a continuously by-passing stream of
dry air and illuminated brightly with the cold light source
(vertical viewing and dim overall illumination; the stream of air
was pointing towards the part of the picolitre osmometer where
samples were placed under liquid paraffin).
Details of the experiments where plants were subjected to
sudden changes in external (root medium or atmospheric) </i are
given in the Results section and Figure legends.
Plant material
Turgor and osmolality measurements
Seeds of barley (Hordeum vulgare L. cv. Golf) were imbibed
overnight in water and germinated for 4-5 d on aerated 0.5 mM
Turgor measurements (Husken et al., 1978) were performed
with a pressure probe obtained from Bangor University
Turgor in leaf epidermal cells
47
RIDGE
LE
B
IV-RIDGE
VI
IS
TRoU6H
IS LV—RIDGE
*•*
\
Fig. 1. Cross-section (A) and view of the upper epidermis (B) of a barley leaf. The cross-section was cut fresh by hand from the third leaf of
barley, 1 d following full expansion. The same leaf was used to obtain a view of the upper leaf surface using a double-reprint technique (Fncke
el al., 1995). Ridges that cover large lateral veins (LV), with a bundle sheath extension stretching from the bundle to the centre top of the ridgeepidermal surface are referred to as LV-ridges; ridges that cover intermediate veins (IV), which lack such a far-stretching bundle sheath extension,
are referred to as IV-ridges. At the transition from a ndge (R-cells) to trough region (TR-cells) is typically one file of stomata, and interstomatal
cells (IS) are located in between stomata. RT, RM and RB refers to ridge cells located at the top-, mid- or base-position analysed within ndges; UE,
upper epidermis; LE, lower epidermis; MS, mesophyll; SC, substomatal cavity. The bar represents 50 ^m.
(Bangor, North Wales, UK). To minimize vibration during
turgor measurements, all pieces of equipment were tightened
to a metal plate that rested on inflated rubber (bicycle)
tubes. Probe capillaries were pulled from borosilicate glass
capillaries (outer diameter, 1.0 mm; inner diameter, 0.58 mm;
Clark Electromedical Instruments, Pangbourne, UK) using a
capillary puller (type 50-2013, Harvard Apparatus Ltd,
Edenbridge, UK) and broken with a microforge (type MF-83,
Narashige Scientific Laboratories, Tokyo, Japan). Non-silanized
capillaries were used and filled, as was the rest of the probe,
48
Fricke
with silicone oil (type AS4, Wacker Chemie, MOnchen,
Germany). The underestimation of cell turgor caused by the
volume of cell solution outside the cell (in the capillary) during
turgor measurements should be highest for the smallest cells,
i.e. here interstomatal cells (Fig. IB). The volume of interstomatal cells was generally between 31-50 pi (calculated from
leaf cross-sections and reprints of the epidermal surface; troughcell volume generally between 400-520 pi and ridge-cell volume
between 750-980 pi). The volume of cell solution outside the
cell was less than 0.078 pi, i.e. less than 0.16-0.25% of the
interstomatal cell volume. Assuming a volumetric elastic
modulus of 5 MPa (see Malone and Tomos [1990] for leaf
epidermal cells of wheat) this would result in an underestimation
of interstomatal cell turgor of 0.008-0.013 MPa. This is about
an order of magnitude smaller than the differences in turgor
found here between cell types and, therefore, does not interfere
with the interpretation of results.
Osmolalities of extracted cell sap were determined with
a picolitre osmometer (Bangor, North Wales, UK.) as
described by Malone et al. (1989) and Tomos et al. (1994).
Osmolalities were converted into -n assuming that 0.1 MPa
corresponds to c. 40.75 mOsmolkg"1 at 22°C (Nobel, 1991).
The accuracy of turgor and n determination was
0.002-0.003 MPa.
When cells were analysed for -n and turgor, the turgor of a
cell was measured immediately before cell sap was extracted
instantaneously using a rapid-sampling device on the probe
(Malone et al., 1989). The extracted sap was transferred within
seconds under liquid paraffin placed on to the osmometer stage
and analysed for osmolality within the following 70 min.
Results
Turgor profile within IV- and LV-ridges
The turgor relationship of the first and second LV
(counted from the midrib) was similar and results from
these two LVs were therefore pooled. Since IV- and
LV-ridges were analysed from the same leaves, their mean
cell turgor could be compared directly.
Both IV- and LV-ridges showed even distribution profiles of cell turgor regardless of whether plants were
analysed under low PAR (Fig. 2A, B) or high PAR
(Fig. 2C, D). There were no systematic differences in
turgor between cells. In most of the leaves analysed, cell
turgor within a ridge was within 0.1 MPa and cells located
in neighbouring positions were even closer in turgor and
sometimes identical. However, in some of the leaves
analysed, turgors of cells within one ridge differed by
more than 0.1 MPa (up to 0.16 MPa) and neighbouring
Low PAR
15
A
High PAR
1J
Intarmedlata vain
Intermediate vein
|
5.
1.2
o
0.9
iT
•
-
I
T
1.2
;
f
T
•
0.0
1.2
0.9
I
_i
1
T
r
0.0
B
D
Large lateral vein
1
r •—
1
1
Large latara vein
4T
1 ?
I
T ~~
-+—t
OJ
o.e
0.6
o
1
i
OJ
is
o.
'•
OJ
0J
15
T
OJ
0.6
o
C
OJ
0.0
0.0
Bate
Middle
Top
Top
Middle
Location of Ridge Cell
Bate
Bate
Middle
Top
Top
Middle
Bate
Location of Ridge Cell
Fig. 2. Turgor pressure profile within IV- and LV-ndges of the upper epidermis of barley leaves. Cells located at adjacent positions (R B , RM> ^ T )
within ridge regions overlying an IV (IV-ridge) or LV (LV-ridge) were analysed for turgor. The centre top of a ridge is indicated by a dashed line.
Analyses were carried out under low (A, B) or high PAR (C, D). Second, third and fourth leaves were analysed between 2 d preceding and
following full expansion. Cells were probed in such a way that first every other cell was analysed and thereafter the cells in between. Cells analysed
under low PAR conditions were located at direct neighbouring positions, whereas under high PAR conditions cells were displaced from each other
by one to two cell lengths up- or downstream along the vein. Results in (A) are expressed as means ± S D of 5-8 leaf analyses since not every leaf
analysed was composed of 6 ridge cells or a cell was lost during turgor measurement. Results in (B). (C) and (D) are expressed as means ± S D of
7, 5 and 7 complete leaf analyses, respectively.
Turgor in leaf epidermal cells
49
cells had turgors that were different by up to 0.15 MPa
(not shown). The latter was not an artefact caused by the
probing of neighbouring cells, i.e. there was no such
tendency that the turgor of a cell probed second was
lower than the turgor of its neighbour cell probed first.
The mean (±SD) ridge-cell turgor, calculated from
the values shown in Fig. 2, for IV and LV-ridges were:
low
PAR:
IV-ridge
1.03 ±0.02 MPa,
LV-ridge
1.04±0.03MPa; high PAR: IV-ridge 1.21 +0.03 MPa,
LV-ridge 1.19 ±0.03 MPa. Thus, under both measurement
conditions, IV- and LV-ridge cell turgor was almost
identical.
in four leaves significantly higher than trough-cell turgor
(indicated in Fig. 3A by '4/15'). The difference in turgor
was more pronounced and more often significant when
leaves were analysed under high PAR (Fig. 3B). On
average, ridge-cell turgor was 0.09 MPa higher than
trough- and 0.08 MPa higher than interstomatal-cell
turgor. The differences was in 6 of 8 plants significant
(mostly at 1% level). In some leaves, ridge-cell turgor
was up to 0.19 MPa higher than trough- or interstomatalcell turgor (not shown). The difference between
interstomatal- and trough-cell turgor was on average
minimal (0.01 MPa) and in none of the leaves statistically
significant. Plants were treated for 3-9 d before analysis
Turgor, n and i|/ in ridge, trough and interstomatal cells
with 100 mM NaCl and analysed under low PAR.
Treatment with NaCl increased turgor differences between
To give an idea of the cell-to-cell variation in turgor, n
cell
types. On average, ridge-cell turgor was 0.26 MPa
and <j> observed for each cell type during a leaf analysis,
higher
than trough- and 0.15 MPa higher than interone set of data is presented in its original form (Table 1).
stomatal-cell turgor (Fig. 3C). These turgor differences
The remaining data are presented as average of the means
were in all of the 13 plants analysed significant (mostly
of the individual leaf analyses together with the respective
at 0.1% level). In some leaves, differences reached
average of individual standard deviations (= error bars
0.41 MPa for trough and 0.33 MPa for interstomatal cells
in Figs 3-5). There was no obvious effect of leaf number
(not shown). Interstomatal-cell turgor was on average
or leaf-developmental stage on the results.
0.09 MPa higher than trough-cell turgor, though only in
Epidermal-cell turgor: In leaves analysed under condithree of 13 plants significantly (at 5% level).
tions of low PAR, ridge-cell turgor was on average only
0.04 MPa higher than trough-cell turgor, i.e. both cell
In (non-NaCl-treated) control plants analysed next to
types had rather similar turgor (Fig. 3A). However, ridgethe osmometer stage under conditions of high local illucell turgor was in almost all (14 of 15) leaves slightly and
mination and by-passing stream of dry air, ridge-cell
Table 1. Combined measurement of turgor and osmotic pressure, and inferred water potential (t/i) in cells of the upper epidermis of
barley leaves
The leaf region analysed was exposed during measurements to a by-passing stream of dry air and brightly illuminated with the
cold light source. The leaf number (L3, 4) is given with leaf developmental stage (d) expressed in days before (minus numbers) or
days following (plus numbers) full expansion. Turgor and osmotic pressure were measured from the same cell, and <p was calculated
as the difference between. Values are given as means ± S D of (n) cell analyses per cell type and plant. The absolute differences
between trough- and ridge-cell values are given in parentheses and their significance is shown at the 5% (*) and 1% (**) level.
Leaf/Dev
Cell type
MPa
Turgor pressure
L3/d-2
L3/d-l
L3/dl
L3/dl
L3/d2
L3/d2
L4/d-2
L4/d-l
L4/dl
Ridge (4)
Trough (5)
Ridge (5)
Trough (5)
Ridge (4)
Trough (5)
Ridge (5)
Trough (5)
Ridge (5)
Trough (7)
Ridge (6)
Trough (6)
Ridge (4)
Trough (4)
Ridge (8)
Trough (6)
Ridge (6)
Trough (6)
0.92±0.05
0 83 ±0.03* [-0.11]
.11+0.07
01 ±0.05* [-0.10]
.28 ±0.09
.18±0.05 [-0.10]
.37 ±0.08
.25 ±0.03* [-0.12]
.26±0.12
.09±0.06* [-0.17]
.27 ±0.05
.05±0.09" [-0.22]
.09±0.14
0.85±0.07* [-0.24]
0.72 ±0.08
0.58±0.05" [-0.14]
1.17±0.11
0.99±0.07* [-0.18]
[-O.15±O.O6f
Osmotic pressure
•A
1.24±0.05
1.18±O.O3 [-0.06]
1.31 ±0.04
1.30 ±0.03 [-0.01]
1.50 ±0.08
1.33±0.05* [-0.17]
1.53 ±0.04
1.50±0.04[-0.03]
1.57±0.15
1.39±0.07 [-0.18]
1.49 ±0.08
1.36±0.03" [-0.13]
1.41 ±0.10
1.21 ±0.03* [-0.20]
1 18±O.O3
1.09±0.01" [-0.09]
1.78 ±0.09
1.57 ±0.04** [-0.21]
[-0.12±0.08]
-0.33 ±0.08
-0.35±0.04 [-0.02]
-0.20 ±0.06
-0.28 ±0.05 [-0.08]
-0.22±0.04
-0.15±0.04* [+0.07]
-0.16 ±0.06
-0.25±0.04* [-0.09]
-0.31 ±0.14
-0.30±0.11 [+0.01]
-0.22 ±0.08
-0.31 ±0.10 [-0.09]
-0.32 ±0.09
-0.37 ±0.05 [-0.05]
-0.46 ±0.07
-0.50 ±0.05 [-0.04]
-0.58 ±0.15
-0.57±0.10 [+0.01]
-0.03 ±0.05]
'Means ±SD of the difference between the trough and ridge cell value (n = 9 leaf analyses).
50
Fricke
Horizontal viewing of plants
IV
O
Vertical viewing of plants
(high local illumination and air stream)
1.8
Control
1.5
1.2
'gor,
QL
0.9
a
0.6
03
0.0
TR
TR
IS
Cell Type
Fig. 3. Turgor pressure in leaf epidermal cells of barley. Plants were analysed while being viewed horizontally, either under low (A, C) or high
PAR (B), or while being viewed vertically, with the leaf area analysed exposed to high local illumination and stream of dry air (D, E). In control
plants (A, B, D), second, third and fourth leaves were analysed between 1 d preceding and 4 d following full expansion. NaCl-treated plants (C,
E) were treated for 3-9 d prior to analysis with 100 mM NaCl and third and fourth leaves were analysed between 2 d preceding and following full
expansion. NaCl-treatments were started at the time the third leaf emerged from the surrounding second-leaf sheath by two daily additions of
50 mM NaC1. Results are presented as means of 15 (A), 8 (B), 13 (C), 9 (D), and 5 (E) leaf analyses, with 4-11 cells analysed per cell type and
leaf. The error bars shown do nol represent the SD of the means of the n leaf analyses This would represent the leaf-to-leaf variation in cell turgor.
Instead, the error bars represent the mean cell-to-cell variation in turgor for a particular cell type ( = means of the SDs of the individual leaf
analyses). The number of leaves where turgor differences between ndge and trough, and between ridge and interstomatal cells were significant is
given in relation to the total number of leaves analysed fa/b').
turgor was on average 0.15 MPa higher than trough-cell
turgor (Fig. 3D). The difference was in 8 out of 9 plants
significant (mostly at 1% level). In NaCl-treated
plants analysed under the same conditions, ridge-cell
turgor was on average 0.28 MPa higher than trough- and
0.17 MPa higher than interstomatal-cell turgor (Fig. 3E).
Differences were in 4, and in 5 out of 5 plants significant
(1% level), respectively. Interstomatal-cell turgor was on
average 0.11 MPa higher than trough-cell turgor. This
difference was in 4 out of 5 leaves significant (1% level).
Epidermal-cell -n: Leaves were analysed under conditions of low local illumination and no by-passing air
stream, i.e. evaporative-demand conditions comparable
to the low PAR set-up. Ridge-cell n was on average only
0.04 MPa higher than trough-cell n and in none of the
17 leaves analysed significantly higher (Fig. 4A). In NaCltreated plants analysed under the same conditions, ridgecell 7T was on average 0.19 MPa higher than trough-, and
0.03 MPa higher than interstomatal-cell -n (Fig. 4B). The
difference in n between ridge and trough cells and between
ridge and interstomatal cells was in 7 out of 8, and in 2
out of 8 plants significant (mostly at 1%, and 5% level),
respectively.
Leaves were analysed under conditions of high local
Turgor in leaf epidermal cells
| Low local illumination and no ilr itraim|
Control
B
51
High local Illumination and air itream
c
100 mM NaCI
Control
D
100 mM NaCI
2/8
Q.
0/17
_I_
O
s/g
a.
o
o
R
TR
R
TR
IS
TR
TR
IS
Call Type
Fig. 4. Osmotic pressure (n) in leaf epidermal cells of barley. Plants were viewed vertically and the leaf area analysed was exposed to low local
illumination and no air stream (A, B) or high local illumination and dry-air stream (C, D) In control plants (A, C), second, third and fourth
leaves were analysed between 2 d preceding and 3 d following full expansion. NaCl-treated plants (B, D) were treated for 3-8 d prior to analysis
with 100 mM NaCI and third and fourth leaves were analysed between 1 d preceding and 4 d following fulJ expansion NaCl-treatments were
started at the time the third leaf emerged from the surrounding second-leaf sheath, by two daily additions of 50 mM NaCI. Results are presented
as means of 17 (A), 8 (B), 9 (C), and 5 (D) leaf analyses, with 4-8 cells analysed per cell type and leaf. The error bars represent the mean cell-tocell variation in -n for a particular cell type ( = means of the SDs of the individual leaf analyses). The number of leaves where TT differences between
ridge and trough, and between ridge and interstomatal cells were significant is given in relation to the total number of leaves analysed ('a/b').
illumination and by-passing stream of dry air. Ridge-cell
TT was on average 0.12 MPa higher than trough-cell n
(Fig. 4C) and in 5 out of 9 leaves significantly higher
(mostly at 1% level). In NaCl-treated plants analysed
under the same conditions, ridge-cell n was on average
0.24 MPa higher than trough- and 0.16 MPa higher than
interstomatal-cell n (Fig. 4D). The difference in TT between
ridge and troughs cells, and between ridge and interstomatal cells was in 5 out of 5, and in 4 out of 5 plants
significant (mostly at 1% level), respectively.
Epidermal-cell ip: Water potentials of cells were obtained
in two ways. Either by using turgor and n obtained from
different leaf analyses and under set-ups likely to be
comparable in evaporative demand (Fig. 5A) or by using
turgor and -n obtained from identical leaves and cells
(Fig. 5B). Calculation of </r from separate determinations
of turgor and TT gave identical mean tfi (-0.41 MPa) for
ridge and trough cells in control plants (Fig. 5A); in
NaCl-treated plants, mean ridge cell ifi (-0.76 MPa) was
0.07 MPa more positive than trough cell </. (-0.83 MPa)
and 0.12 MPa more positive than interstomatal cell </«
(-0.88 MPa; Fig. 5A). Calculation of 0 from combined
determinations of turgor and TT gave nearly identical mean
ip for ridge (-0.31 MPa) and trough cells (-0.34 MPa) in
control plants (Fig. 5B; see also Table 1 for data of
individual leaf analyses) and for ridge (-0.69 MPa),
trough (-0.73 MPa) and interstomatal cells in NaCl-
treated plants (-0.70 MPa; Fig. 5B). As for control plants,
differences in turgor between ridge cells (highest turgor)
and trough and interstomatal cells were almost entirely
due to differences in TT.
Short-term changes in turgor in ridge and trough cells
Plants were kept for 24 h in a plastic bag at saturated
humidity. Upon removal of the plastic bag, i.e. sudden
drop in atmospheric RH (room RH c. 25%), both ridge
and trough cell turgor dropped within minutes by about
0.7 MPa (Fig. 6A). Thereafter, turgor in ridge and trough
cells followed the same time-course of recovery and
reached a final value after 40-90 min, with a Tlj2 of 918 s
for the experiment shown (Fig. 6A). The T1/2 of the
immediate, exponential, decline in cell turgor following
the removal of the plastic bag was 78 s (Fig. 6B, separate
experiment). The following recovery in turgor was accompanied by a decrease in stomatal conductance (Fig. 6C;
TU2 of 702 s; separate experiments).
To lower the external </i of the root suddenly, 100 mM
NaCI was added to the medium while the plant was
analysed (room RH c. 34%). Turgor started to decrease
within 10 s, in both ridge (Fig. 6D) and trough cells
(Fig. 6E); 10 s were needed to add all the salt in the form
of a stock solution and to readjust the meniscus in the
probe to its initial position. Within the following 10-12
52
Fricke
Combined dtUrminttton of turgor and oimotk praitur*
Saparat* d*ttrmtnatk>n of turgor and otmotlc p r t u u r *
A
O«motie
Pr*»ura
2.1
Oimotic
• control
• 100 mM N*CI
1.8
Praiiur*
2.1
0 control
•
U
100 mM NaCt
Turgor
Turgor
1.5
1.8
I
1.2
\2
0.9
0.6
0J
0
1M
|1 |M|
1 II
R
TR
IS
0.9
0.6
0.3
R
R
TR
IS
-0J
TR
IS
I HI I I
1
III1
R
-0J
-0.9
-1.2
Wattr Potential
-1.2
IS
R
TR
TR
IS
IS
-0.6
-1.5
-13
-1.8
-1.8
-2.1
-2.1
Ctll Typ«
TR
-OJ
•II•
-0.6
R
0
Wattr PountlaJ
Cell Typ*
Fig. 5. Turgor pressure, osmotic pressure (IT) and inferred water potential (<j>) in leaf epidermal cells of barley. Turgor and n data were taken from
Figs 3 and 4 (in (A) from Fig. 3A, C and Fig. 4A, B; in (B) from Fig. 3D, E and Fig. 4C, D) Water potentials were inferred in two ways Either
(A) by comparing turgor data with n data obtained from different plant batches and cells (i.e separate determinations), or (B) by comparing turgor
data with n data obtained from the identical plants and cells (combined determinations). For details of NaCl-treatments, leaf number and leaf
developmental stage see legends to Figs 3 and 4. Water potentials (and turgor and n) in B represent the means of 9 (control) or 5 (NaCl-treated)
leaf analyses, and error bars give for each cell type the means of the SDs of the individual leaf analyses.
min, turgor in ridge and trough cells decreased by about
0.5 MPa and finally stabilized (Tm of 226 and 170 s for
the two experiments shown). Upon removal of NaCl,
turgor started to increase within 30 s in both ridge
(Fig. 6D) and trough cells (Fig. 6E); 30 s was needed to
exchange media and probe a new cell. Turgor continued
to increase and stabilized after about 10 min at a value
comparable to that previous to the experiment (T1/2 of
153 s and 215 s for the two experiments shown).
The RH was suddenly increased from 36% to >94%
RH by use of an ultrasonic humidifier. Epidermal (here:
ridge) cell turgor started to increase within 2 s and
attained within minutes a new stable value (Tl/2 of 62 s
for the experiment shown; Fig. 6F). Subsequent addition
of NaCl caused a similar response in cell turgor as under
34% RH (compare Fig. 6D, E). However, T{/2 was faster
(50 s) and the absolute decrease in turgor smaller than at
34% RH.
Fig. 6. Short-term response in epidermal-cell turgor and stomatal conductance to a sudden change in external ui. Plants were subjected under low
PAR to sudden changes in either the root-surrounding or atmospheric ip. This was achieved in three ways. Either by keeping the plant for 24 h
under a plastic bag at saturated humidity (bag with small holes for gas exchange) and then suddenly removing the bag (A, B, C; room RH c.
25%); or by adding 100 mM NaCl to, or removing it from the medium (D, E, F); or by suddenly increasing the leaf-surrounding RH from 36 to
^ 9 4 % RH through use of an ultrasonic humidifier (F). Each square represents the turgor of a separate cell, except the first 2-3 squares following
addition of NaCl in (D) and (E) and all the squares following increase in RH or addition of NaCl in (F), which were obtained from continuous
turgor measurement in the same cells. Stomatal conductance was measured with a portable photosynthesis system (LI-6200, Li-Cor, Lincoln, ME,
USA); results from four leaf analyses are plotted together, and each symbol represents the means of six determinations per leaf. Turgor relaxations
were fitted with curves (dotted lines) for a mono-exponential change plus residual to calculate 7", 2. Second or third leaves were analysed between
2 d preceding and following full expansion.
Turgor in leaf epidermal cells
Change in atmospheric
1.2
jrgor
a.
2
i-
o
Removal of bag
B
1.0
Removal of bag
0.8
0.6
0.4
T
t/i~
4
6
ri
T t / I - 918 .
Q.p
• Ridge e e l
• Trough ceB
0.2
0.0
•
«•
E
0
Removal of bag
o
omat al con<ductiince,
2
6
10 12 14
2.0
| o third l e a f |
Time, min
I
1.5
1.0
6\
0.5
Ti/i- 702 *
o.
b- • -Q. •o- ...<P
0.0
CO
-20
20
40
60
100
80
Time, min
| Change in root-medium 4>\
D
+ 100 mM
I
NaCI
1.0
-
0.8
o
at
3
^T
1 / t
"
226 i
153 t
0.6
0.4
•i
o
Naa
.
-
0J)
| Combined change in axtarnal <f>\
1.2
a.
2
1.6
\ , T 1 r t - 170 t
0.6
Cell
T 1 / I -82
r
1.2
ODD
o
\-
1.4
I
0.8
215 t
1.0
94 X
RH
0.8
0.4
t-
0.2
'
0.6
•I
•ifj Q
T,/ 2-50 t-
36 1
RH
0.4
0.0
-20
-10
10
•
NaCI
+ 100 mM
1.0
' F
20
Time, min
30
40
50
-
-20
-10
0
Time, min
10
53
54
Fricke
Discussion
Intercellular differences in n, turgor and \J* within the upper
epidermis of barley leaves
Epidermal-cell n: A previous study on the upper epidermis
of barley leaves showed that, as the leaf aged, ridge-cell
77 increased more than trough- and interstomatal-cell n.
This was generally accompanied by a particular high
accumulation of K + and Cl~ in ridge cells (Fricke et al.,
1995). Differences in n between cell types appeared to be
non-significant. This is consistent with the present results
obtained for (control) plants analysed under low local
illumination and no air flow (Fig. 4A). However, it
contrasts with the results obtained under conditions of
high local illumination and by-passing stream of dry air
(Fig. 4C) where 5 out of 9 plants showed a significantly
higher ridge- than trough-cell -n. This might indicate that
a change in the (local) evaporative demand or light
intensity affects solute ( K + and Cl~) export/import in
ridge and trough cells differentially and would imply that
osmotically-significant solute movement across epidermal
cell membranes is much faster than generally assumed
(here: within 20-90 min following transfer of plants to
the measurement site).
NaCl-treatment increased differences in n between ridge
(higher n) and trough cells. This is consistent with previous results on NaCl-treated barley which showed that, as
NaCl-treatment continued, ridge-cell n increased progressively more than trough-cell n, leading to n differences
of 0.2-0.45 MPa. Most likely, this resulted from higher
concentrations of Cl" and N a + in ridge compared to
trough cells (Fricke et al., 1996). The difference between
ridge- (higher n) and interstomatal-cell n was less pronounced and less often significant than the differences
between ridge- and trough-cell -n. This is in line with
previous results (Fricke et al., 1996). However, in the
previous study, ridge-cell -n was mostly slightly lower than
interstomatal-cell TT, whereas in the present study it was
mostly higher. This may indicate that, during NaCl
treatment, the barley cultivar 'Golf studied here has a
higher capacity for accumulation of Cl~ and N a + in
ridge cells or a lower capacity for accumulation of C\~
and Na + , or maintenance of NO 3 ~ in interstomatal cells
than the previously studied cultivar 'Klaxon'.
Epidermal-cell turgor: Nonami et al. (1990), studying
the water relations of Tradescantia leaves, found consistent, though non-significant differences in turgor between
subsidiary cells and other (unspecified) epidermal cells.
Shackei and Brinckmann (1985) reported for the same
species that there were no consistent differences in turgor
between ridge and trough cells and between cells located
at contrasting distances from stomata. This is in sharp
contrast to the present findings on barley and points to
a basic difference in the epidermal water and solute
relations between the two species.
The magnitude and significance of turgor differences
between cells was higher in (control) plants analysed
under high PAR than in plants analysed under low PAR,
and was highest in plants analysed under high local
illumination and by-passing stream of dry air (Fig. 3).
For each cell type, there were no statistically significant
differences in turgor between these three measurement
conditions (not shown). This indicates that turgor differences between cell types changed with measurement conditions since each cell type responded with (slightly)
different turgor. To what degree this was due to a direct
light effect on turgor or a changed transpiration rate, can
not be said. (Plants were not protected during analysis
from CO 2 from the investigators breath.)
The turgor difference between ridge and trough cells in
control plants analysed for turgor and -n was, on average,
attributable by 80% to a difference in n and attributable
by 20% to a difference in </. (Fig. 5B; Table 1). For NaCltreated plants, the values were 82% and 18%, respectively,
for the turgor difference between ridge and trough cells,
and 87% and 13%, respectively, for the turgor difference
between ridge and interstomatal cells (Fig. 5B). Similar
values for the contribution of n- and i/i-differences to
turgor differences between ridge and trough cells were
obtained using turgor and n measured separately in
different plants (Fig. 5A); however, only 20% of the
turgor difference between ridge and interstomatal cells
could be accounted for by their difference in n. The latter
underlines the potential 'weakness' of determining turgor
and n in separate plants and cells to derive i/i.
Together, the results show that the differences in turgor
between cell types are accompanied by only very small
differences (0.01-0.04 MPa) in </.. Instead, they reflect
mainly differences in n and, therefore, ultimately, differences in the cell's solute supply or ability to accumulate
and maintain Cl~ and K + (control plants) or Cl~, Na +
and NO 3 " (NaCl-treatment). Intercellular differences in
apoplastic transpiration tension or solute potential seem
to be far less important. (It is assumed that reflection
coefficients for osmolytes are close to one and comparable
between cell types.) The results are in line with the findings
of two other studies where intercellular gradients in turgor
and similar-sized gradients in n reflected the proximity of
cells to the source of solutes (Meshcheryakov et al., 1992)
or the site where water was ultrafiltrated into the xylem
(Zimmermann et al., 1992). In contrast, Nonami et al.
(1990) studying Tradescantia leaves observed that turgor
differences between subsidiary and other epidermal cells
were accompanied by differences in </i in the range of
0.1-0.2 MPa.
The type of turgor variation observed within ridge
regions where cells differed in turgor occasionally by
0.1-0.16 MPa, even in neighbouring positions, seems to
be random and comparable to the kind of turgor variation
noted for cells of other tissues (Steudle et al., 1980; Tomos
Turgor in leaf epidermal cells
1.0
55
1.0
NaCI
Q.
0.S
0-8
c(T,/i- 109 .
0.6
0.4
„
£ I
©-•
a
0.6
0.4
o, • rldga call
a, • trough call
-
"^
E
—
S
•.
M
0.2
0.0
S
o
•
0^
1
Q.
*
02
\TU1-
^^A
107 •
0.4
O
a.
0.6
A
rldga call
* trough call
0J
-20
-10
10
20
30
40
50
Time, min
Fig. 7. Short-term response in turgor, osmotic pressure and water potential in leaf epidermal cells of barley to the addition of 100 mM NaCI to the
medium. Each symbol represents the turgor (A), osmotic pressure (A) and inferred water potential (B) for a separate ridge or trough cell. During
each cell analysis, turgor was measured immediately before cell sap was extracted for determination of osmotic pressure and calculation of i/>. For
better visual comparsion, curves were fitted by first order (osmotic pressure) or third order (turgor, water potential) polynoms (dotted lines); 7*1/2
was calculated from curve fits for mono-exponential decline plus residual.
et al., 1981; Steudle and Jeschke, 1983; Malone and
Tomos, 1992; Frensch and Hsiao, 1994). The proximity
of ridge cells to stomata or bundle sheath extension did
not affect turgor. Similarity, trough-cell turgor showed
no obvious dependency on the proximity to stomata (not
shown). Thus, there may be at least three 'units' of turgor
relations within the upper epidermis of barley leaves:
the ridge-, the trough- and the interstomatal-cell unit
(other peristomatal cells and guard cells were not
considered).
Epidermal-cell i/i: Differences in if> between and within
non-growing leaf tissues have been reported for
Tradescantia and explained by the existence of transpiration-induced </r gradients (Frensch and Schulze, 1988;
Nonami et al., 1990). However, in the present study there
were no systematic differences in 0 between epidermal cell
types and regions. The difference in i/> between cells
obtained from calculation of I/I from combined analyses of
turgor and n ranged between 0.01 and 0.04 MPa (Fig. 5B)
and was statistically non-significant. A too low transpiration rate as a reason for missing epidermal <p gradients is
very unlikely. Even under low PAR where plants were
likely to transpire the least in the present study, the
'remaining' transpiration rate was still high enough to
lower epidermal cell turgor by 0.2 MPa (compared to
turgor at no transpiration; Fig. 6F). If the small differ-
ences in </i between cell types are transpiration-induced,
then the finding that >p is lowest in trough cells indicates
that interstomatal cells are lined at their inner walls with
a cuticle as observed in Tradescantia (Nonami et al., 1990)
and that the transpiration tension in the apoplast is highest
in trough cells. The difference between barley and
Tradescantia leaves concerning <<
/ gradients may partly
result from the generally slower Tl/2 of water exchange in
epidermal cells of Tradescantia (Tomos et al., 1981;
Tyerman and Steudle, 1982) compared to barley (Table 2).
Short-term regulation of epidermal-cell turgor, and T1/2 of
water flux equilibrium
The response of epidermal cell turgor to a sudden change
in external </r (Fig. 6) suggested that changes in the cell's
(vacuolar) solute content do not contribute to the turgor
response and that differences in I/I between ridge and
trough cells are at any time negligibly small. These
assumptions were proved correct by an experiment in
which turgor and -n (and inferred </«) were determined in
the same leaf and cells following addition of NaCI
(Fig. 7). While turgor and <>
/ decreased by about 0.4 MPa,
and with a similar TU2, -n remained almost constant
(<0.015 MPa increase). This also indicates that cell volumetric elastic modulus remained almost unchanged.
56
Fricke
The Tl/2 for local water flux equilibrium in the cells
studied was between 0.7-1.7 s (Table 2) and, thus, one
to two orders of magnitudes smaller (faster) than the T1/2
for epidermal turgor relaxations at plant level (Table 2).
This shows that water flux equilibration in the leaf
epidermis of barley is not time-limited by the water
exchange properties of epidermal cells. Falk (1966)
observed that following addition of 100 mM NaCl to the
root medium of wheat, water uptake dropped within
seconds and recovered within the following 15 min;
transpirational water loss also responded within seconds,
slowly increased during the following 5 min and decreased
thereafter such that, after 12-20 min, it balanced water
uptake almost perfectly. Thus, the T1/2 of 162 s (Table 2)
for thej turgor relaxation reported here following addition
of 100 mM NaCl reflects a mixture of root-water uptake
and stomatal responses. The latter seems to involve (i)
an early hydropassive opening stage which is due to the
removal of backpressure exerted from the surrounding
epidermis on guard cells (Franks et ai, 1995; Raschke,
1970; Dewar, 1995) and is represented by the initial
increase in transpirational water loss in Falk's experiments
(Falk, 1996; see also Malone, 1992); and (ii) a Mate'
(after 5-12 min) active closing stage which involves solute
movement across guard cell membranes (Raschke, 1970).
Consequently, removal of NaCl may cause an early
closing and a late active opening stage.
NaCl was added to plants kept at >94% RH, or with
(all) leaves covered in stopcock grease. The Tl/2 in those
plants was only 46 s (Table 2). This shows that under the
conditions tested, control of stomatal aperture (water
loss) is the time-limiting process for water flux- and, thus,
turgor- and i/i-equilibration in the leaf epidermis of barley
(see also Fig. 6A, C). This may be a direct consequence
of the initial, non-controllable and 'counteracting', hydropassive movement of guard cells. The Tl/2 of 46 s is 2-17
times smaller than the Tl/2 obtained for excised barley
roots using the root pressure probe (Steudle and Jeschke,
1983). This points to a difference in hydraulic properties
between roots of intact plants and excised roots.
The turgor of a ridge cell next (axially or laterally) to
a previously punctured ridge cell was not obviously lower
than the turgor in that previously punctured cell: (i)
always, provided that at least 8-10 min were between the
two turgor measurements, but (ii) even sometimes when
the second cell was probed within the following 20 s (low
PAR conditions; not shown). This indicates that there is
very little symplasmic hydraulic contact between adjacent
ridge cells. If so, \\> of ridge (and trough and interstomatal ?) cells would equilibrate through the apoplast
(short Ti/2) or a symplasmic mesophyll connection.
Effect of 100 mM NaCl on epidermal cell turgor, n and v|/
During long-term treatment of plants with 100 mM NaCl,
the ifi change in the medium caused by addition of NaCl
(c. -0.45 MPa) resulted in a nearly equivalent change in
leaf-epidermal tp (c. -0.38 MPa). This was mainly due to
Table 2. Half times (T 1/2 ) of water flux equilibrium of (A) individual epidermal cells and (B) the hydraulic continuum between root,
xylem, leaf epidermis, and stomatal pore
Half-times of water flux equilibrium of individual cells were obtained by pressure relaxations using the pressure probe. For each
cell two Tl/2 determinations were carried out, one from endosmotic and one from exosmotic water flows. Results are presented as
means ± S D of 22-32 determinations per cell type. Third or fourth leaves were analysed, between 2 d preceding and 3 d following
full expansion, and NaCl plants were exposed for 3-9 d prior to analysis to 100 mM NaCl. Half-times of water flux equilibrium
of the hydraulic continuum between root, xylem, leaf epidermis, and stomatal pore were calculated from curves (mono-exponential
change plus residual) fitted to epidermal turgor relaxations obtained during short-term imposed changes in external 0; results are
presented as means ± S D of (n) experiments and the range of Tl/2 is given in '[ ]'.
Treatment/Experiment
TUi
(A)
TV2 of individual cells
R-cells
TR-cells
IS-cells
Control-plants
lOOmM NaCl-plants
0.74 ±0.12
0.89±0.22
1.31 ±0.30
1.70 ±0.42
0.78±0.18
not analysed
(B)
Tu2 of hydraulic continuum between root, xylem, leaf epidermis, and stomata
Addition of NaCl to medium (c. 34% RH)
(£94% RH)
(leaf greased)'
Removal of NaCl from medium (c. 34% RH)
Increase in RH from 34 to 2: 94%
Sudden drop in RH from 100% to 34%
Initial turgor drop
Subsequent turgor recovery
162 ±42 (6) [96; 228]
47±6(3) [45. 50]
46(1)
198±24 (3) [174; 216]
54±6(3) [48; 57]
78(1)
858±156(6) [618; 1008]
'Leaf covered in stopcock grease prior to addition of 100 mM NaCl
Turgor in leaf epidermal cells
an increase in epidermal -n since turgor was generally at
the control level (Fig. 5B; no significant difference in
turgor between control and NaCl treatment). The latter
might be indicative of turgor regulation in NaCl-treated
barley (osmotic adjustment). During short-term exposure
to 100 mM NaCl, changes in epidermal turgor seemed
also to be equivalent to changes in external </f (Fig. 6D,
E). However, this was rather coincidental since the recovery decreased with increasing RH to 48+17% (means
± S D of eight experiments at 94% RH; see also Fig. 6F,
and Thiel et al., 1988). Assuming that the pressure change
in root-xylem transmitted quantitatively into a change in
epidermal I/I, then the results give a radial root reflection
coefficient for NaCl of 0.48. This value is very similar to
values obtained with the root pressure probe on excised
roots (Steudle and Jeschke, 1983; Steudle 1993) and the
xylem pressure probe on intact plants (Zhu et al., 1995).
In conclusion, the present study on the upper epidermis
of barley leaves shows that the previously reported systematic heterogeneity in solute concentrations (Fricke
et al., 1995, 1996; Hinde, 1994) between ridge, trough
and interstomatal cells (and regions) is accompanied by
a systematic heterogeneity in turgor. In addition, n can
differ systematically between cells. However, </r of cells is
similar. Within ridge regions, where cells do not differ
systematically in solute concentrations (Fricke et al.,
1995), there is no systematic difference in cell turgor
either. Turgor differences between cell types increase with
NaCl-treatment. Following a sudden change in external
tp, water flux equilibration in epidermal cells is not limited
by their hydraulic properties or water supply via roots
but by the control of stomatal aperture. The elevated
turgor in ridge regions may well have a biomechanical
function since ridge regions are suited best as load-bearing
carriers. Some observations indicate that epidermal cells
are hydraulically isolated via their symplasts, though this
requires further experimental support.
Acknowledgements
This work was supported by a guest-researcher grant from the
Swedish Forest and Agricultural Reseach Council (SJFR).
Wieland Fricke would like to thank Jeremy Pritchard
(Birmingham, UK), Paul Richardson and Peter Hinde (Bangor,
UK) for teaching him the use of the pressure probe and
picolitre osmometer. Particular thanks go to Jurgen Frensch
(Bayreuth, Germany) and Deri Tomos (Bangor, UK) for their
very helpful comments on an earlier version of the manuscript.
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