Plant Physiol. (1999) 120: 143-152
Water Stress Inhibits Hydraulic Conductance and
Leaf Growth
in Rice Seedlings but Not the
Transport of Water via
Mercury-Sensitive
Water Channels in the Root1
Zhongjin Lu and
Peter M. Neumann*
Plant Physiology Laboratory, Lowdermilk Faculty of Agricultural
Engineering, Technion Israel Institute of Technology, Haifa 32000, Israel
 |
ABSTRACT |
The mechanisms by which moderate
water stress (adding polyethylene glycol 6000 to the root medium)
induces a sustained inhibition of growth in emerging first leaves of
intact rice (Oryza sativa) seedlings was investigated
under growth-chamber conditions. Early (24 h) inhibition of leaf growth
was not related to changes in root size or in osmotic potential
gradients and cell wall-yielding characteristics in the leaf-expansion
zone of stressed seedlings. However, reductions in root-to-leaf
hydraulic conductance (L) were measured in two rice
cultivars after 4 or 24 h at various levels of water stress, and
these reductions correlated well with the inhibition of leaf growth. We
assayed L by a psychrometric method and, in intact
seedlings, by a novel osmotic-jump method. The addition of 0.5 mM HgCl2 to the root medium to inhibit water transport through Hg-sensitive water channels in the roots did not
inhibit leaf growth in unstressed seedlings. However, both leaf growth
and L were additionally reduced (by 49% and 43%,
respectively) within minutes of adding HgCl2 to roots of
water-stressed seedlings. Water stress therefore appeared to increase
the transport of water via Hg-sensitive water channels. Other
mechanisms were apparently involved in inhibiting overall
L and leaf growth.
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INTRODUCTION |
Moderate (nonlethal) water stress can be defined as a situation in
which reduced water availability leads to the inhibition of plant
growth. Intermittent episodes of leaf-growth inhibition, e.g. between
irrigation events, are clearly undesirable during irrigated crop
production (for review, see Neumann, 1995
, 1997). Although water-stress
episodes often adversely affect rice (Oryza sativa)
production, relatively little is known about the underlying mechanisms
of stress-induced growth inhibition in this important food crop
(O'Toole and Chang, 1979; Hanson et al., 1990; Lilley et al., 1996).
The early (24 h) inhibition by moderate water stress of leaf growth in
seedlings of rice, maize, and barley was found to be uniformly
associated with reductions in cell production and cell expansion (Lu
and Neumann, 1998). In the emerging first leaves of maize and barley
seedlings this growth inhibition was also associated with
stress-induced decreases in the extensibility of the expanding cell
walls (Neumann, 1993; Chazen and Neumann, 1994; Bogoslavsky and
Neumann, 1998). However, cell wall extensibility in the emerging first
leaves of rice seedlings did not decrease in response to water stress,
although leaf growth was inhibited (Lu and Neumann, 1998). Therefore,
different growth-inhibitory mechanisms appeared to regulate rice leaf
responses to water stress.
Water stress may increase the threshold pressure for wall yielding in
expanding cells (Lockhart, 1965; Cosgrove, 1993; Neumann et al., 1994;
Kramer and Boyer, 1995). Alternatively, water stress could induce
hydraulic limitations to water uptake. These could then limit water
availability for volume increases in expanding cells (Shultz and
Matthews, 1988; Evlagon et al., 1990; Cruz et al., 1992; North and
Nobel, 1996; Nonami et al., 1997). For example, Cutler et al. (1980)
showed that root pressurization, which should increase
w gradients in the leaf-expansion zone, caused
immediate increases in leaf growth of osmotically stressed rice plants. This suggested that water uptake might be a primary rate-limiting factor for leaf growth. Similarly, Yeo et al. (1991) suggested that
early inhibition of rice leaf growth by salt stress might result from
hydraulic limitations. There are apparently no reports, however, that
directly measure any early inhibitory effects of water stress on
L in rice plants.
The physiological mechanisms that might underlie any stress-induced
reductions in the L of rice seedlings are also unknown. One
possibility is that transmembrane water-channel proteins (aquaporins) are involved (for review, see Chrispeels and Agre, 1994; Steudle and
Henzler, 1995; Maurel, 1997; Schäffner, 1998). Thus, cDNA sequences from the aquaporin gene family have been identified in
numerous animal and plant species (including rice; Liu et al., 1994).
In some cases, putative aquaporin genes have expressed their protein
products in the plasma membrane of Xenopus oocytes. The
expression of aquaporins then led to greatly increased rates of
osmotically induced water movement across the oocyte membrane, which
may be inhibited by Hg ions.
A physiological role for Hg-sensitive water channels in facilitating
transmembrane water transport in plants has been directly demonstrated
at the whole-organism level with giant algal cells (Wayne and Tazawa,
1990; Steudle and Henzler 1995; Tazawa et al., 1996). Similar roles for
water channels have been demonstrated only in parts of higher plants,
e.g. excised roots, coleoptile segments, epidermal cells, and root
membrane vesicles (Maggio and Joly, 1995; Steudle and Henzler, 1995;
Carvajal et al., 1996; Hejnowicz and Sievers, 1996; Niemietz and
Tyerman, 1997; Tazawa et al., 1997). Experiments with transgenic
Arabidopsis expressing an antisense construct targeted to the
water-channel gene PIP1b, however, have recently suggested
that there is long-term physiological importance to aquaporins in
intact higher plants. The transgenic plants developed much larger roots
than control plants, apparently to compensate for the inhibited water
permeability of the plasma membranes in the root cells (Kaldenhoff et
al., 1998).
Recent evidence indicates that the level of aquaporin gene transcripts
and the water-transport capacity of aquaporins themselves can be
affected by relatively short periods (hours to days) of water stress
(Yamaguchi-Shinozaki et al., 1992; Liu et al., 1994; Yamada et al.,
1995, 1997; Johansson et al, 1996, 1998). We therefore became
interested in the hypothesis that induced reductions in the activity of
root water channels in intact rice seedlings might help to regulate
early reductions in L and thereby facilitate sustained
inhibition of leaf growth by moderate water stress.
Here we report the possible relationships between the early inhibitory
effects of water stress on leaf growth in intact rice seedlings and (a)
yield-threshold pressure in the leaf-elongation zone, (b) L
of the water pathway from the root to the leaf-elongation zone, and (c)
water transport through Hg-sensitive water channels in the roots.
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MATERIALS AND METHODS |
Plant Growth
We investigated the leaf growth characteristics of rice
(Oryza sativa L.) genotypes that show differing resistance
to water stress under field conditions. Rice cv IR20, a lowland paddy
rice, is grown under semiaquatic conditions and is considered to be relatively sensitive to water stress. Rice cv Salumpikit, a more water-stress-resistant rice, has become adapted to upland conditions. Both of these crop cultivars were supplied by the International Rice
Research Institute (Manila, Philippines).
Seeds were germinated in the dark on filter paper discs moistened with
0.4 mM CaCl2 at 27°C ± 2°C
for 3 d. Germinated seedlings then grew hydroponically on a 0.1×
dilution of aerated nutrient solution with additional Ca (2.5 mM CaCl2), in an environmentally regulated growth chamber with a 12-h photoperiod, light intensity of
150 µmol s
1 m2 PAR at
plant height, temperature of 27°C ± 2°C, and RH of 35%/60%, day/night, respectively.
Water Stress
Rice seedlings were taken for water-stress or control treatments
on the 4th d after transfer to hydroponic culture when the emerging
first leaf protruded 12 ± 1 mm above the surrounding coleoptile
sheath (means ± SE, n = 20). Uniform
reductions in water availability to the seedling roots were generated
by adding PEG, a nonpenetrating osmolyte, to the nutrient solution
(Chazen et al., 1995). To minimize osmotic shock, PEG was raised to the desired level by 0.1 MPa steps at 1-h intervals.
Leaf Elongation
We assayed the treatment effects on long-term elongation growth of
the first true leaves by measuring distances from a mark at the base of
the coleoptile to the leaf tip at 12-h intervals.
We assayed the short-term rates of leaf elongation using a computerized
whole-plant extensiometer, as previously described (Neumann, 1993;
Chazen and Neumann, 1994; Lu and Neumann, 1998). Each seedling was
firmly fitted into a suitable plastic holder so that the root alone was
bathed in aerated nutrient solution. The seed was positioned so that
the shoot (coleoptile and protruding first leaf) extended vertically
above it. The leaf tip was stuck to an aluminum foil tab connected to a
small alligator clip, which was in turn connected to a thread looped
over a low-resistance pulley wheel and joined to the core of a linearly
variable displacement transducer (Instruments and Control, Haifa,
Israel). Changes in leaf position were stored at 1-s intervals and
displayed graphically using data acquisition and graphical display
software (Viewdac 2.1, Keithley Metrabyte, Taunton, MA). The accuracy
of leaf-position measurements was within ±2 µm.
Length of Cell-Elongation Zone
We determined the lengths of the cell-elongation zones at the base
of the leaves by pricking intact leaves (n = 10) at
regular intervals with fine wires (77 µm in diameter), which
penetrated through the surrounding hypocotyl. After 24 h of PEG or
control treatment the pricked leaves were excised at the base and
separated from the coleoptile. The distances between the holes were
then compared using a binocular microscope and eyepiece scale to
determine the approximate point above the leaf base at which no further differences in spacing could be observed, i.e. where cell elongation ended (Neumann, 1993; Lu and Neumann, 1999). A final determination of
the end of the elongation zone (to within 1 mm) was obtained in a
repeat assay in which only five pricks per leaf were made at 1-mm
intervals, from 2 mm above to 2 mm below the initially estimated end
point. Thus, a relatively small number of pinpricks were confined to
the upper end of the elongation zone and inhibitory effects on leaf
elongation were thus minimized (
8% reduction).
w, 
, and Yield Threshold
The technique used to measure bulk w in
live tissues from the elongation zone at the base of the first leaf and
bulk of the frozen-thawed tissues was
slightly modified from that previously described by Chazen et al.
(1995). Excised rice leaf segments were assayed by thermocouple
psychrometry using four 6-mm segments per leaf-cutter chamber (volume
0.5 mL, series 76, J.D. Merrill, Logan, UT), and sampling was carried
out in a humidity chamber. The first leaves were carefully separated
from other leaves, after excision at the base of the elongation zone.
Segments were then cut and rapidly sealed into the psychrometer
chambers. We connected the chambers to a microvoltometer (Wescor,
Logan, UT) and took readings after a 3-h equilibration period at 27°C
in a water bath. At this time the electrical output was constant (data
not shown). No corrections were made for possible effects of apoplastic
solution. Each reported value is the mean for n 
5 replicate probes.
Wall-loosening processes (i.e. cell wall relaxation) with consequent
changes in w and decreases in turgor pressure
can occur after excision of growing tissues and consequent separation
from their external sources of water. This wall relaxation is reported to stop after turgor decreases to the threshold pressure required to
initiate wall yielding (Lockhart, 1965; Cosgrove, 1993; Neumann et al.,
1994; Kramer and Boyer, 1995). We calculated the relaxed turgor
pressure by subtracting from relaxed
w and used it as a measure of the yield
threshold pressure in the expanding rice leaf.
Psychrometric Assay of L
Water flux into expanding tissues (J) can be described
by the relationship J = L
(e 
i), where
e i represents the
difference between the external water source
(e) and the internal cell
(i) w (Lockhart,
1965). L in rice seedlings was assayed when the first
leaves were beginning to emerge from the surrounding coleoptile and
still tightly rolled. Thus, the leaf surface area available for
transpiration was small. Water flux into the cell-elongation zone was
considered to be primarily used for increasing leaf volume, i.e.
J = dV/dt = L
(e i). Moreover,
the relative rate of leaf volume increase (1/V) × (dV/dt) should be approximately equivalent to the
RGR; therefore, the flux equation can be expressed in the form:
|
(1)
|
The RGR (min1) is calculated by dividing
the leaf-elongation rate by the length of the cell-elongation zone at
the base of the leaf. A value for L
(MPa1 min1) can then be
obtained when RGR is divided by the w
difference (e i).
Because we determined plant w
psychrometrically with excised segments, wall relaxation may have led
to more negative values of i than those in the
intact plant (Shultz and Matthews, 1988; Cosgrove, 1993; Neumann et
al., 1994; Kramer and Boyer, 1995). Conductance values determined in
this way may thus be underestimated.
Osmotic-Jump Assay of L
To avoid excision artifacts, we used a novel, alternative approach
to determine L in intact rice seedlings. Short-term
differences (minutes) in leaf elongation were assayed in intact plants,
which had equilibrated for up to 24 h with external solutions of
0.2 or 0.4 MPa PEG. Leaf elongation was assayed over 3-min periods before and 30 s after replacing the root medium with fresh solution at
a less negative w, i.e. after an "osmotic
jump." A fine stream of air bubbles continuously stirred the medium
around the root. Thus, external, unstirred layer effects were
minimized. Rapid leaf growth responses to an osmotic jump of only 0.1 MPa were clearly detectable using sensitive position transducers
(linearly variable displacement transducers), as shown in Figure
1.
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| Figure 1.
Osmotically induced increase in the elongation of
an emerging first leaf in an intact cv IR20 rice seedling. To generate
an osmotic jump, the root nutrient solution containing PEG 6000 at
0.2 MPa w was drained and replaced at the time
indicated by the arrow with fresh nutrient solution containing PEG 6000 at 0.1 MPa. The solution exchange took less than 30 s. Leaf-tip
position was followed with linear variable displacement transducers,
and mean elongation rate was determined for 3 min before and after the
solution change, as shown by the straight line. Rice seedlings
were grown for either 4 or 24 h in PEG 6000 solutions at either
0.2 or 0.4 MPa prior to assay.
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Equation 1 was used to define the RGRs of the rice leaves before
(RGR1 = L
[e1 i]) and
after (RGR2 = L [e2 i]) the osmotic jump from the original external
w (e1) to a less
negative value (e2). We assumed that
i and L did not change greatly
during the brief 3-min position measurement, which follows the 0.1 MPa change in external .
By subtraction:
The above equation can then be reduced to:
A value for L can then be determined using:
The L in unstressed rice seedlings could not be
determined by this method, because their short-term growth could not be
further accelerated. Thus, the osmotic-jump method could be used only to compare L in rice seedlings previously exposed to
different levels of water stress.
The L determined by both the osmotic-jump and
psychrometric methods apply to water transport along the entire pathway
from the root surface into the expanding leaf cells. Both methods
assume that leaf growth (RGR) is proportional to volume increases via water influx and give results in comparative units of megapascals per
minute (Cosgrove, 1985). Therefore, the values represent
water-transport coefficients and are relative L. However,
they facilitate quantitative comparisons of the effects of water-stress
episodes on plant water-transport capacity.
Leaf Extensibility
We then investigated the possibility that the rapid increases in
rice leaf growth induced by the osmotic-jump technique were also
associated with rapid changes in extensibility parameters. Plants were
assayed using the same set-up as that described above for leaf
elongation. Detailed descriptions and characterization of the
computerized extensiometer used for measuring comparative wall-extensibility values in expanding leaf tissues and their relation
to leaf growth rates have been reported previously for rice (Lu and
Neumann, 1998) and other cereal species (Neumann, 1993, 1995; Chazen
and Neumann, 1994; Bogoslavsky and Neumann, 1998). A small additional
force was applied in the direction of leaf growth and then removed
after 3 min to determine comparative values of the reversible (elastic)
and apparently irreversible (plastic) extensibilities in growing
leaves.
A Student's t test was used to determine the significance
of differences between treatments. All of the experiments reported here
were repeated one or more times with similar results.
| |
RESULTS |
Leaf Growth
Figure 2 shows that the growth rates
of emerging first leaves of rice seedlings (cv IR20) declined with
increased water stress (as induced by adding different amounts of PEG
6000 to the root medium for 24 h). The inset in Figure 2 shows
that elongation kinetics for the emerging first leaves of control and
water-stressed rice seedlings were fairly linear for about 36 h.
We observed similar quasi-steady-state kinetics at other stress levels
and in the upland rice cultivar (data not shown).
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| Figure 2.
Growth rates of emerging first leaves of rice cv
IR20 seedlings exposed to indicated levels of water stress for 24 h. Water stress was induced by PEG 6000 additions to the root medium.
The inset shows typical long-term kinetics of leaf elongation at 0 MPa
( ) and 0.2 MPa ( ) w. Results are means ± SE, n = 20.
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The length of the cell-elongation zone at the base of rice leaves was
not significantly affected after 4 h of water stress. However,
values in paddy rice were slightly reduced after 24 h of water
stress by PEG at either 0.2 or 0.4 MPa. For example, the mean
length of the elongation zone decreased from 5.8 ± 0.3 to
5.0 ± 0.3 mm (means ± SE, n = 10) after 24 h of exposure to PEG at 0.4 MPa. Data for the more
stress-resistant upland rice were similar, except that the
leaf-elongation zone was affected only by 24 h at 0.4 MPa and
was not affected by PEG at 0.2 MPa. However, exposure of rice
seedlings to PEG at 0.6 MPa for 24 h caused large decreases in
the length of the elongation zone (from 6 to 2 mm in paddy rice or to 3 mm in upland rice). Water-stress effects on rice seedlings were
subsequently assayed during the early and quasi-linear period of leaf
growth, which occurred during seedling exposure to moderate
w of 0, 0.2, and 0.4 MPa PEG for up
to 24 h. This minimized potential complications associated with
the onset of large, morphological differences between seedlings in
water-stress and control treatments.
Differences and Wall Yield-Threshold Pressure
Figure 3 shows that bulk
and the relaxed
w, in tissues excised from the leaf-elongation
zone, adjusted to more negative values as cv IR20 plants were exposed
to increasingly more negative external w for
24 h. The stress-induced changes in internal
did not appear to result from water
efflux, because the leaves continued to elongate at each stress level
and we observed no signs of shrinkage. Because of these apparent
adjustments, the 
did not change
significantly at different levels of stress (Fig. 3, inset). Similar
results were obtained with upland rice (data not shown). The
progressive inhibition of leaf growth did not appear to be associated
with decreases in the maximum level of bulk turgor pressure (indicated
by ), which could be generated in the
expanding leaf tissues.
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| Figure 3.
Effects of increasing levels of PEG 6000-induced
water stress on ( ) and relaxed w
( ) in expanding rice cv IR20 leaf tissues. The inset shows the
relatively unchanging gradient between leaf
tissues and external medium. Results are means ± SE
(n 6).
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The yield-threshold pressure of the expanding cell walls was considered
equivalent to the relaxed turgor pressure measured in segments excised
from the leaf-elongation zone. The relaxed turgor pressure was
calculated by subtracting from relaxed w. The values were not significantly affected
by water stress. For example, in paddy rice the wall yield threshold
was 0.41 ± 0.07 MPa for control seedlings and 0.40 ± 0.07 MPa for seedlings stressed for 24 h in PEG at 0.4 MPa
w (means ± SE,
n = 6). Similarly, the equivalent values in upland rice
were 0.43 ± 0.04 MPa for the controls and 0.37 ± 0.06 MPa
for water-stressed seedlings. Thus, the stress-induced inhibition of
leaf growth occurred without associated reductions in
gradients or increases in wall yield
thresholds.
L
Exposure of paddy rice seedlings to increasing levels of
root-applied water stress (0, 0.2, or 0.4 MPa PEG) for 24 h
induced progressively greater reductions in L, as determined
by psychrometric assay (Fig. 4).
Reductions in L were also induced after 4 h. For example, the leaf-elongation rates in seedlings before and after water
stress for 4 h by PEG at 0.4 MPa were 6.3 ± 0.5 and
1.9 ± 0.3 µm min1, respectively. The
equivalent L values were reduced from 2.2 ± 0.2 min1 MPa1 × 103 in control seedlings to 1.4 ± 0.2 min1 MPa1 × 103 in water-stressed seedlings (means ± SE, n = 5). Water stress also
reduced leaf growth and L in the more water-stress-resistant upland rice cultivar. For example, leaf growth rates were reduced from
10 ± 1 mm d1 in the controls to 7 ± 1 mm d1 after a 24-h treatment with PEG at
0.4 MPa. The equivalent L values were reduced from
2.5 ± 0.2 to 1.9 ± 0.2 min1
MPa1 × 103 (means ± SE, n = 8).
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| Figure 4.
Effects of water stress on L in
paddy rice seedlings. L was estimated by psychrometric
assay, as described in ``Materials and Methods''. Results are means ± SE, n = 6. The inset shows the
effects of increasing levels of water stress on root growth rate. Root
growth rate was determined from root length increases over 4 d at
each level of water stress. Results are means ± SE,
n = 20.
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The inset in Figure 4 indicates that water stress in the range 0 to
0.4 MPa PEG did not have major inhibitory effects on root growth.
Thus, the reductions in root-to-shoot L and leaf growth,
which were caused by PEG at 0.2 and 0.4 MPa, did not seem to be
consequences of stress-induced reductions in the root area available
for water uptake.
In addition to the psychrometric assay, which was based on assaying
excised leaf segments, an osmotic-jump method assayed comparative
effects of water stress on L in intact plants.
The mean L assayed by the osmotic-jump method in rice
seedlings treated with 0.4 MPa PEG for 24 h was reduced in
comparison with plants treated with 0.2 MPa PEG (Table
I). Similar effects of water stress on
L (47% reduction in L at 0.4 MPa as compared with 0.2 MPa) were observed when isoosmotic mannitol solutions were
used in place of PEG from 30 min before and during the osmotic-jump assays. Thus, the stress-induced decreases in L were not
dependent on the type of osmoticum used.
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Table I.
L of rice seedlings (cv IR20) after 24 h
at indicated levels of water stress, as determined by the osmotic-jump
assay
GR1 and GR2 indicate leaf growth rates before and after the osmotic
jump, respectively. Results are means ± SE,
n = 8. Numbers in parentheses indicate equivalent
values determined by psychometric assay (means ± SE,
n = 6).
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Treatment-induced changes in leaf tissue w or
L were assumed to be negligible, because the osmotic jumps
were relatively small (0.1 MPa) and produced calculated volume changes
of only approximately 0.4% of the leaf-elongation zone during the
relatively short (3 min) assays. The volume changes were calculated by
multiplying the increase in leaf length by the leaf cross-sectional
area one-half way along the elongation zone.
Neither the elastic nor the apparently irreversible (plastic) component
of tissue extensibility changed significantly during the osmotic jumps.
For example, comparative in vivo elasticity for paddy rice seedlings
stressed by 0.4 MPa PEG for 24 h was 13 ± 3 µm (0.5 g/3
min) before the jump and 11 ± 2 µm (0.5 g/3 min) 5 min after
the jump (means ± SE, n = 8). The
equivalent plasticity values were 21 ± 3 µm (0.5 g/3 min) and
23 ± 3 µm (0.5 g/3 min), respectively. Thus, the rapid,
osmotically induced increases in rates of leaf expansion occurred
without significant changes in cell wall-extensibility parameters.
Most important, the stress-induced decreases in L values
measured by the osmotic-jump method were comparable to the decreases measured by the psychrometric method (Table I). Similarly, the L measured by both the psychrometric and osmotic-jump assays
showed good linear correlations with the leaf growth rates of paddy and upland rice cultivars (Fig. 5 and inset).
The correlation coefficients were 0.99 (n = 6, mean
results) using the psychrometric method and 0.89 (n = 32, individual seedlings) using the osmotic-jump method; both
correlations were significant at the 1% level.
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| Figure 5.
Correlations between changes in L
and growth rate. Average leaf growth rates of both paddy rice () and
upland rice ( ) after 24 h at 0, 0.2, and 0.4 MPa PEG are
plotted against average L, as determined by
psychrometric assay. Results are means ± SE
(n = 6, averaged values). The regression equation
Y = 0.81 + 4.24X and the
correlation coefficient is 0.99. The inset shows a similar plot using
L values obtained with the osmotic-jump method
for individual plants stressed for 24 h with PEG at either 0.2 or
0.4 MPa (means ± SE, n = 32). The regression equation is Y = 1.04 + 0.43X and the correlation coefficient is 0.88.
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Role of Hg-Sensitive Water Channels
It seemed possible that early stress-induced reductions in rice
seedling L and associated inhibition of leaf growth might be
the result of regulated reductions in the activity of water channels in
the roots. However, Figure 6 shows that
addition of a known water-channel inhibitor (0.5 mM HgCl2) to the root
medium for up to 60 min did not have any consistent inhibitory effect on leaf growth rates in unstressed control seedlings. Thus, water transport through Hg-sensitive water channels in the roots did not
appear to be essential for the maintenance of leaf growth in these
control seedlings.
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| Figure 6.
Kinetics of differential leaf growth responses to
adding Hg ions to root medium in control or water-stressed cv IR20 rice
seedlings. Hg (0.5 mM HgCl2) was added to root
medium at time 0. , Non-water-stressed treatment; , seedlings
water stressed by the addition of PEG 6000 (0.2 MPa) to root medium
24 h before the start of the experiment. Results are means ± SE (n = 6).
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In contrast, similar additions of 0.5 mM
HgCl2 rapidly induced a 49% reduction in leaf
growth rates of water-stressed rice seedlings. Moreover, the Hg ions
also induced rapid reductions (43%) in the L of the
root-to-leaf pathway, as measured by the osmotic-jump method 20 min
after Hg addition (Table II). A clear temporal association between reductions in L and early
reductions in leaf growth rate was therefore demonstrated.
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Table II.
Rapid effects of root addition of Hg ions on growth
rate and L in water-stressed rice
Seedlings of paddy rice (cv IR20) were stressed for 24 h by
exposure to PEG 6000 (0.2 MPa water potential) prior to also adding
Hg (0.5 mM HgCl2) to the aerated root medium.
Growth rates and L were assayed using the osmotic-jump
method after 20 min of Hg treatment (means ± SE,
n = 6). Numbers in parentheses are percentage reductions
caused by Hg ions.
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Added Hg ions had no significant effects on wall extensibility in the
leaves of water-stressed seedlings after 20 min (as compared with
negative Hg controls [data not shown]). Moreover, the fact that leaf
growth of non-water-stressed plants was not inhibited for at least 60 min after Hg addition to the roots (Fig. 6) indicates that any leaf
accumulation of Hg ions within this period was insufficient to induce
growth-inhibitory effects on the membranes or walls of the expanding
leaf cells. These findings suggest that the roots were the primary
locus of the early inhibitory effects of Hg ions on leaf growth of
water-stressed rice seedlings.
| |
DISCUSSION |
We have previously shown that both the cell size and the growth of
emerging first leaves of rice, maize, and barley seedlings were
inhibited under water-stress conditions equivalent to those reported
here (Lu and Neumann, 1998). Moreover, associated reductions in
cell wall extensibility in the leaves of maize and barley seedlings correlated well with water-stressed inhibition of leaf growth. In
contrast, growth-inhibitory levels of water stress did not inhibit wall
extensibility in rice leaves (Chazen and Neumann, 1994; Lu and Neumann,
1998).
According to the Lockhart growth model (Eq. 2), water stress might
inhibit cell expansion (RGR) and hence leaf growth by (a) affecting wall-yielding characteristics (i.e. decreasing the wall extensibility coefficient (m) and/or increasing the yield
threshold pressure for onset of wall yielding (Y); (b)
reducing the ; and (c) reducing the
L of the water-uptake pathway.
|
(2)
|
However, wall extensibility (related to, but not equivalent to,
m) in rice seedling leaves was not decreased by water
stress. Similarly, the present report shows that the rapid acceleration of rice leaf elongation induced by osmotic jumps was not accompanied by
the rapid increases in wall extensibility observed in the leaves of
other species (Neumann, 1993; Chazen and Neumann, 1994; Bogoslavsky and
Neumann, 1998, and refs. therein). Thus, the inhibition of rice leaf
growth did not appear to be caused by any stress-induced reduction in
wall extensibility. The present report also shows that water stress did
not increase the apparent yield threshold (Y) of rice
leaves. Moreover, the growing leaf tissues appeared to adjust their
during the 24-h water-stress episodes so that the potential was restored to
prestress values. A similar capacity for osmotic adjustment has often
been measured in rice leaves (Lilley et al., 1996).
The default possibility, that cell and leaf expansion in
water-stressed rice seedlings were at least partially inhibited by stress-induced reductions in root-to-leaf L, was supported
by the results obtained with both the psychrometric and osmotic-jump assays. Thus, the reductions in L assayed by both methods
correlated well with the inhibition of leaf growth rates over a range
of water-stress levels and in two different rice cultivars.
We considered the hypothesis that water stress may initiate early
reductions in root-to-leaf L by rapidly inducing reductions in the activity or number of Hg-sensitive water channels in the roots.
Hg ions have previously been shown to rapidly inhibit water transport
through excised (and unstressed) roots of other cereal plants such as
maize and wheat (Maggio and Joly, 1995; Carvajal et al., 1996).
However, root addition of a water-channel inhibitor (0.5 mM HgCl2) for up to 60 min
had no consistent inhibitory effect on leaf growth in unstressed rice
seedlings (without PEG addition). In this case, different water
pathways, including water channels that are not sensitive to inhibition
by Hg ions, may have supplied the water needed for ongoing leaf growth
(compare Daniels et al., 1994; Steudle and Peterson, 1998). Moreover,
these data are also consistent with a recent report that indicates that
growth of first leaves in non-water-stressed rice seedlings is
primarily limited by the low extensibility of the growing cell walls
(Lu and Neumann, 1999). None of our findings supported the idea
that imposition of water stress could rapidly induce the inhibition of
rice leaf growth by closing Hg-sensitive water channels in the roots.
In contrast, root treatment of water-stressed rice seedlings with Hg
ions caused further reductions in both leaf growth and L
after only 20 min. This experimental observation confirms that induced
reductions in L can limit leaf growth, at least in
previously water-stressed plants. Together with the finding that
L in rice is rapidly reduced by water stress, this report
provides strong support for previous suggestions that reduced
L might be a primary factor limiting the growth of leaves in
water-stressed rice (Cutler et al., 1980; Yeo et al., 1991).
The fact that leaf growth was inhibited within minutes of the addition
of Hg ions to the roots of water-stressed seedlings (Fig. 6)
suggests that rapid closure of root water channels was involved. It is
not clear whether this was a result of direct binding of Hg to
water-channel sulfhydryl groups in the roots or of indirect inhibition
by Hg of metabolic processes such as the ongoing phosphorylation, which
may be required to maintain channel openings (Johansson et al., 1996,
1998). However, the rapid inhibitory effect of Hg ions on L
indicated that the water channels in the roots of water-stressed
seedlings were open and functionally important prior to the addition of
Hg. The 24-h water-stress treatment therefore appeared to increase root
water-channel activity and/or number by comparison with unstressed
control seedlings. It did not appear to inhibit leaf growth by inducing
closure of these channels.
To our knowledge, this is the first report indicating that water stress
may increase the physiological functioning of water channels in roots
of intact plants. It is consistent with molecular evidence indicating
that water stress can also induce increases in levels of water-channel
transcripts in rice (Liu et al., 1994). Stress-induced increases in
water-channel activity might conceivably facilitate the transport of
water required for growth of expanding root tip cells and could be
associated with the fact that root growth was clearly maintained during
seedling exposure to levels of water stress, which strongly inhibited
leaf growth (compare Fig. 2 with inset of Fig. 4).
Given the apparent absence of stress-induced closure of root water
channels, the overall L of the root-to-leaf pathway in rice
seedlings was presumably inhibited via water-stress effects on other
parameters of water transport. Relatively small decreases in the length
of the leaf-elongation zone or root length after 24 h of water
stress were unlikely to have been a major cause of the measured
reductions in L. Moreover, significant reductions in
L and leaf growth were also measured after only 4 h of
water stress, when size differences between water-stressed and control plants were negligible.
Alternatively, the reductions in L could have been caused by
effects of water stress on the parameters of radial and axial water
transport through the root; for example (a) rapid stress-induced reductions in the water diffusivity of membrane lipid bilayers (Carvajal et al., 1996); (b) accelerated formation of Casparian bands
and suberin lamellae or delayed maturation of developing xylem cells
(Cruz et al., 1992; North and Nobel, 1996); and (c) stress-induced
formation of xylem embolisms (Shultz and Matthews, 1988; Neufeld et
al., 1992). The moderate levels of water stress used in our experiments
and the fact that stress was gradually applied when the emerging leaves
of young rice seedlings had very little potential for transpiration
make the last possibility seem unlikely.
An additional possibility is that water stress inhibited L
and leaf growth by specifically decreasing water-channel activity in
expanding leaf tissues. For example, plasmolysis by 0.4 M Suc rapidly decreased the in vivo
phosphorylation and hence the water-transport activity of a plasma
membrane water-channel protein (PM 28A) in spinach leaf discs
(Johannson et al., 1996, 1998). Similarly, exposure to levels of
salinity, which induced turgor loss, decreased levels of water-channel
transcripts in the leaves of ice plants (Yamada et al., 1995). Thus,
severe water stress has been associated with closure of water channels
in leaves. Direct measurements of water-stress effects on hydraulic
conductivity and water-channel activity in expanding rice leaf cells
will be required to substantiate their possible involvement in
regulating leaf growth responses.
In summary, several experimental approaches indicate that water stress
induces early reductions in the L of the root-to-leaf pathway of water transport in intact rice seedlings. Moreover, these
reductions appear to be a primary mechanism regulating the sustained
inhibition of leaf growth by moderate water stress. However, the
hypothesis that water stress acts at the cellular level to induce
decreases in the activity of Hg-sensitive water channels in the roots,
thereby inhibiting overall L, was rejected.
| |
FOOTNOTES |
1
This work was supported in part by the fund for
the promotion of research at Technion.
*
Corresponding author; e-mail agpetern{at}tx.technion.ac.il; fax
972-4-822-1529.
Received September 21, 1998;
accepted December 23, 1998.
| |
ABBREVIATIONS |
Abbreviations:
L, hydraulic conductance(s).
, difference between expanding
cells and external medium.
, osmotic
potential(s).
w, water potential(s).
RGR, leaf
growth-elongation rate.
| |
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Top
Abstract
Introduction
![<UP>RGR</UP><SUB>2</SUB>−<UP>RGR</UP><SUB>1</SUB>=L [(&PSgr;<SUB><UP>e<SUB>2</SUB></UP></SUB>−&PSgr;<SUB><UP>i</UP></SUB>)−(&PSgr;<SUB><UP>e<SUB>1</SUB></UP></SUB>−&PSgr;<SUB><UP>i</UP></SUB>)].](/content/vol120/issue1/fulltext/143/img002.gif)
