Plant Physiol. (1998) 116: 1403-1412
Root Growth and Oxygen Relations at Low Water Potentials. Impact
of Oxygen Availability in Polyethylene Glycol
Solutions1
Paul E. Verslues,
Eric S. Ober, and
Robert E. Sharp*
Department of Agronomy, Plant Science Unit, 1-87 Agriculture
Building, University of Missouri, Columbia, Missouri 65211
 |
ABSTRACT |
Polyethylene glycol (PEG), which is
often used to impose low water potentials (
w) in
solution culture, decreases O2 movement by increasing
solution viscosity. We investigated whether this property causes
O2 deficiency that affects the elongation or metabolism of
maize (Zea mays L.) primary roots. Seedlings grown in
vigorously aerated PEG solutions at ambient solution O2
partial pressure (pO2) had decreased
steady-state root elongation rates, increased root-tip alanine
concentrations, and decreased root-tip proline concentrations relative
to seedlings grown in PEG solutions of above-ambient
pO2 (alanine and proline accumulation are
responses to hypoxia and low w, respectively).
Measurements of root pO2 were made using an
O2 microsensor to ensure that increased solution pO2 did not increase root
pO2 above physiological levels. In
oxygenated PEG solutions that gave maximal root elongation rates, root
pO2 was similar to or less than (depending
on depth in the tissue) pO2 of roots growing
in vermiculite at the same w. Even without PEG, high
solution pO2 was necessary to raise root
pO2 to the levels found in vermiculite-grown
roots. Vermiculite was used for comparison because it has large air
spaces that allow free movement of O2 to the root surface.
The results show that supplemental oxygenation is required to avoid
hypoxia in PEG solutions. Also, the data suggest that the
O2 demand of the root elongation zone may be greater at low
relative to high w, compounding the effect of PEG on
O2 supply. Under O2-sufficient conditions root
elongation was substantially less sensitive to the low w
imposed by PEG than that imposed by dry vermiculite.
| |
INTRODUCTION |
In studies of plant responses to water deficit, low
w is often imposed by decreasing the supply of
water in the soil or other solid media in which the plants are grown.
Our previous studies of maize (Zea mays L.) primary root
growth at low w were conducted by
transplanting seedlings to vermiculite containing limited amounts of
water (e.g. Sharp et al., 1988
). However, in certain types of
experiments there are advantages to imposing low
w using osmotica in solution culture, e.g.
when radiolabeled compounds must be supplied to the roots in a
controlled manner. Despite its convenience, a liquid medium could
potentially complicate the results because terrestrial plants such as
maize do not normally grow in an environment in which the roots are
surrounded by water. Solution culture has been used extensively at both
high and low w, but there have been few
attempts to verify that plants grown under such conditions are
physiologically similar to those grown in solid media.
When studying the behavior of roots at low w
in solution culture, two factors are centrally important: the osmoticum
used and aeration of the solution. It is desirable to use a compound that does not interact with plants in any way other than lowering the
w of the medium. Thus, slowly penetrating
osmotica such as mannitol or sorbitol (Hohl and Schopfer, 1991) or
inorganic salts (Termaat and Munns, 1986) are not ideal, especially for
experiments extending beyond a few hours. Polymers of PEG have been
used for many years, principally because PEG molecules with a
Mr 
6000 cannot penetrate the cell wall
pores (Carpita et al., 1979). Because PEG does not enter the apoplast,
water is withdrawn not only from the cell but also from the cell wall.
Therefore, PEG solutions mimic dry soil more closely than solutions of
low-Mr osmotica, which infiltrate the cell wall
with solute. Although some studies indicate that PEG could contain
toxic contaminants that inhibit plant growth (e.g. Plaut and Federman,
1985), other studies have found that deleterious effects occurred only
if PEG entered the tissue; for instance, if roots were damaged (Lawlor,
1970).
A potential disadvantage is that the high viscosity of PEG solutions
limits the movement of O2, thereby increasing the
likelihood of root O2 deficiency. Even in pure
water, O2 transport to the root surface is
limited by its low mobility (104 times less than
that in air [Nye and Tinker, 1977]) and by the presence of an
unstirred boundary layer at the root surface (Drew, 1990). Outside of
the boundary layer O2 is carried largely by bulk
movement of the solution, but within the layer molecular diffusion is
the dominant transport component. The thickness of the boundary layer
is determined by several factors, including the degree of stirring and
solution viscosity. Therefore, viscous solutions of PEG tend to
diminish the contribution of mass transport and increase the importance
of diffusion to overall O2 transport. Based on
these principles and on measurements of O2
transport in PEG solutions, Mexal et al. (1975) warned that roots
growing in stirred, air-saturated solutions of PEG
(Mr 4000 and w 
0.7 MPa) could be severely O2 limited.
However, despite large numbers of studies using PEG, to our knowledge
the effects of PEG on root O2 status have never
been quantified.
Our objectives were to determine whether O2
deficiency limits growth or alters the metabolism of maize primary
roots growing at low w in PEG solutions, and,
if O2 is limiting, to determine the conditions
necessary to ensure adequate oxygenation. To address these questions we
used three approaches. First, the effects of elevated solution
pO2 on root elongation were measured
at various w. Second, in the same experiments
we measured the root-tip concentrations of two metabolites: Ala, which
accumulates under O2 deficiency (Ricard et al.,
1994), and Pro, which accumulates at low w
(Stewart and Hanson, 1980). Third, we directly measured tissue
pO2 in the tips of intact, growing
roots using an O2 microsensor (Ober and Sharp,
1996) to quantify the effects of PEG and supplemental
O2 on root O2 status. The
results establish that above-ambient solution pO2 is required to avoid alterations
in root growth and metabolism caused by low O2
availability in PEG solutions.
| |
MATERIALS AND METHODS |
Plant Culture Conditions and Root Elongation
Measurements
Seedlings were grown in the dark in a chamber maintained at 29°C
and near-saturation humidity; when necessary, illumination was provided
by a dim-green safelight (Saab et al., 1990). Kernels of maize
(Zea mays L. cv FR27 × FRMo17) were germinated for
40 h in moist vermiculite. Seedlings with primary roots 20 to 25 mm long were then transferred to solution (5 mm Mes, 0.5 mm CaSO4, 6 µm
H3BO4, adjusted to pH 6.0 with NaOH); root elongation rates were nearly identical to those in a
complete nutrient solution. The solution was contained in Plexiglas
boxes that were 20 cm long, 1.2 cm wide, and 18 or 25 cm tall. The
taller boxes were used to accommodate greater root elongation at higher
w. Twenty seedlings were arranged on a
Plexiglas holder at the top of the box so that the caryopses were
suspended above the solution. A Plexiglas cover enclosed the shoots.
The primary roots grew downward through transparent root guides
fashioned from plastic drinking straws (i.d. 6 mm), which facilitated
measurements of root elongation rate. The solution was vigorously
aerated through a perforated plastic tube extending along the bottom of
the box. O2 and air were mixed in various
proportions before entering the box to give a solution
pO2 of 20.4 to 67 kPa. In most
experiments one of three solution pO2
treatments was used: 20.4 kPa (ambient), 28 ± 2.4 kPa, or 43 ± 4.3 kPa (means ± sd). The total flow rate was
always 1100 mL min
1, and solution
pO2, measured with an
O2 probe (ISO2, World Precision Instruments,
Sarasota, FL), was constant throughout each experiment.
The root guides were perforated with holes (diameter approximately 0.5 mm) large enough to allow exchange of solution, yet small enough to
prevent most roots from growing through them. Preliminary experiments
showed that the guides minimally affected root elongation at high
w, but substantially increased root elongation in PEG (Mr 8000; Sigma) solution at a
w of 0.8 MPa (Fig.
1). This beneficial effect may be
explained by prevention of damage to the roots from the vigorous
aeration. Lawlor (1970) reported that root damage caused PEG uptake and
growth inhibition. Likewise, in our experiments PEG could have entered
roots that were grown without guides. To check that the guides did not
excessively hinder solution mixing, food-coloring dye was injected into
the PEG solution (1.6 MPa) in one of the guides. The dye dispersed
evenly throughout the box and within the guides in less than 1 min.
Root elongation rates were quantified by marking the positions of the
root apices on the side of the box at various times. In the preliminary
experiments without guides, seedlings were periodically removed
(without replacement) from the box to measure root length.
View larger version (27K):
[in this window]
[in a new window]
| Figure 1.
Effect of root guides on increase of primary root
length in vigorously aerated solutions at w
of 0.02 (no PEG) and 0.8 MPa (imposed by PEG). All treatments were
at ambient (20.4 kPa) solution pO2. Without
the guides, marking the positions of the root apex was only possible at
the first four time points. Data for the last two time points were
obtained by destructively harvesting a portion of the roots in the box.
Data points are means ± se (n = 20-40) combined from two experiments. Error bars are not shown where
they are smaller than the symbols.
|
|
In all treatments, seedlings were grown without PEG for the first
2 h after transfer to solution culture (w = 0.02 MPa). Imposition of low w was then
begun by pumping a solution of PEG (dissolved in growth solution) into
the bottom of the box. Aeration mixed the contents and the excess
solution drained from a tube near the top of the box. Solutions having
w of 0.3, 0.8, or 1.6 MPa were used. The
rate of w decline for each treatment was
adjusted so that the final w was reached
8 h after imposition of low w was begun
(unless otherwise noted). To convert PEG concentrations to
w, the w of a series
of PEG solutions were measured using isopiestic thermocouple
psychrometry (Boyer and Knipling, 1965). A time course of
w decline was then calculated for each
w treatment; measurements of samples of the
growth media that were withdrawn periodically from the box confirmed
the accuracy of the predicted w.
Experiments to test the effect of supplemental O2
on roots growing in vermiculite were conducted in Plexiglas boxes as
described previously (Sharp et al., 1988). Humidified mixtures of air
and O2 passed through a perforated tube along the
bottom of the box and into the vermiculite at a rate of 500 mL
min1. The pO2
within the vermiculite was monitored by inserting the O2 probe so that its tip was at the same depth as
the root apices. Various vermiculite w were
obtained by thorough mixing with different amounts of water (Sharp et
al., 1988), and were measured by isopiestic psychrometry.
HPLC Analysis of Amino Acids
At the end of some experiments, the apical 10 mm of two to five
roots growing at approximately the mean elongation rate for that
particular treatment were collected in preweighed microcentrifuge tubes. The sampled region encompassed most or all of the root elongation zone, which extends 10 to 12 mm from the apex at high w and is shortened at low
w in both solution culture and vermiculite (Sharp et al., 1988; E.S. Ober and R.E. Sharp, unpublished data). Samples were immediately frozen in liquid N2 and stored at
20°C. At the time of analysis, samples were weighed, freeze-dried,
and reweighed to obtain the mass of water. Samples were then ground and
-aminobutyric acid or -aminoadipic acid was added as an internal
standard. The samples were extracted overnight at 4°C in
methanol:chloroform:water (12:5:3, v/v) and free amino acids were
recovered by phase separation. The aqueous phase was then applied to a
Sep-Pak Light C18 cartridge (Waters) equilibrated with 50% methanol, and amino acids were eluted with 50% methanol. Samples were then derivatized with phenylisothiocyanate, and the phenylthiocarbamoyl amino acids were separated by HPLC (ISCO, Lincoln,
NE) on a Spherisorb ODS-2 column (3 mm, 4.6 × 150 mm, Alltech
Associates, Deerfield, IL) and detected by
A254 (procedures were modified from Yang
and Sepulveda, 1985; Ebert, 1986).
In some experiments root tips were analyzed for Pro content by the
ninhydrin assay (Bates et al., 1973). Preliminary tests showed that
results from the ninhydrin assay and HPLC analysis were similar.
O2 Microsensor Measurements
Commercially available O2 sensors are too
large for direct measurement of pO2
within root tissue, and previous studies using various types of bare
O2 microelectrodes have been problematic (for
review, see Baumgärtl and Lubbers, 1983). Therefore, root pO2 was measured with a newly
developed Clark-type O2 microsensor with a tip
diameter of 1 to 5 µm (Ober and Sharp, 1996). The microsensor was
calibrated before and immediately after use using a two-point calibration method: first in N2, then in either
air-saturated water or air-saturated PEG (w = 1.6 MPa). If pre- and postmeasurement calibrations differed by more
than 10% the data were discarded. The calibrations were linear from 0 to 100 kPa. Data were corrected for a small offset (approximately 3%
of signal) that occurred in PEG solutions relative to water, perhaps as
a result of an effect of osmotic pressure on the microsensor membrane.
This correction did not affect interpretations or conclusions drawn
from the data. Measurements of pO2 in
solution-cultured roots were made at various solution
pO2 and w of
0.02 and 1.6 MPa in a small (30 mL) Plexiglas chamber. Conditions
were identical to those described above for the root elongation
measurements except that solutions were aerated in an adjacent chamber
and pumped through the chamber housing the root at a rate of 3 mL
min1. (During low-w
imposition the root chamber was aerated directly at the same ratio of
gas-flow rate to chamber volume used in the growth experiments.) These
conditions resulted in steady-state elongation rates identical to those
obtained in the larger-volume root boxes and permitted vibration-free
measurements to be made. Measurements of vermiculite-grown roots were
made at ambient pO2 and
w of 0.02 and 1.6 MPa in Plexiglas
cylinders described by Spollen and Sharp (1991). Root lengths at the
time of measurement were 80 to 100 mm.
The microsensor was attached to a micromanipulator (MO-203, Narishige
Ltd., Tokyo, Japan) and impaled perpendicularly between 4 and 10 mm
from the apex of vertically oriented primary roots elongating at
approximately the mean rate for each treatment as observed in the
growth experiments. No longitudinal gradients in
pO2 were observed along the root tip,
so at each depth of impalement, measurements from all positions were
averaged. The root and microsensor were viewed through a microscope
mounted horizontally in front of the apparatus, and the depth of
impalement was measured with the micromanipulator, subtracting any
movement of the root itself as measured with an eyepiece reticle.
Measurements were made across the outer 150 µm of the roots; the
maximum depth of impalement remained within the cortex in all
treatments (based on micrographs of fresh cross-sections taken at 7 mm
from the apex). Measurements were not made at greater depths to
minimize tissue damage caused by the taper of the microsensor. Root
pO2 values at particular depths cannot
be strictly compared between treatments because of differences in root
diameter. Roots at low w, either in PEG solution with supplemental O2 (but not at ambient
pO2) or in vermiculite, were thinner
than roots at high w in either medium (for a
detailed analysis of the thinning response to low
w in vermiculite-grown roots, see Liang et al.
[1997]).
Statistical Analysis
Steady-state root elongation rates and Ala and Pro concentrations
were analyzed by analysis of variance using a 3 × 4 (pO2 × w)
factorial design. All differences in mean values reported in the text
as significant have P 0.05.
| |
RESULTS |
Effect of Elevated pO2 on Root
Elongation
Time-course measurements of root elongation rate after transfer to
solution culture at ambient (20.4 kPa) and above-ambient (28 and 43 kPa) pO2 showed that elevated
solution pO2 stimulated root
elongation at high w (0.02 MPa, no PEG used)
as well as in PEG solutions at w of 0.3,
0.8, and 1.6 MPa (Fig. 2). In all
treatments, root elongation rate was approximately 2 mm
h1 during the first 2 h after transfer to
solution culture (before low-w imposition);
steady-state rates were reached by 40 h after transfer and, except
at 1.6 MPa, were greater than the initial rate. In the
high-w treatment, roots reached maximum
elongation rates sooner at elevated
pO2, indicating that during the first 30 h, O2 supply limited root growth (Fig.
2A). The steady-state elongation rate was not significantly affected by
pO2, however (Fig.
3). After the addition of PEG in the
three low-w treatments at ambient
pO2, root elongation rate first
decreased and then recovered to varying extents (Fig. 2, B-D). When
solution pO2 was elevated, elongation
increased more rapidly (Fig. 2, B-D) and steady-state elongation rates
were significantly greater (Fig. 3). At all w,
steady-state elongation rates were not significantly different at
solution pO2 levels between 28 and 43 kPa (Fig. 3). To confirm that this range of solution
pO2 was optimal for root growth,
elongation was examined when solution
pO2 was further elevated to 67 kPa. At
w of 0.02, 0.3, and 0.8 MPa, root
elongation at 67 kPa was inhibited relative to that at 43 kPa, both in
terms of the steady-state elongation rate and the time required to
reach steady state, and at 1.6 MPa there was no difference in
elongation rate between 43 and 67 kPa (data not shown). Because the
viscosity of the PEG solution increases with decreasing
w, one might expect that the greatest relative
stimulation by O2 (ratio of steady-state growth
at 43 kPa to that at ambient pO2)
would occur at 1.6 MPa. This was not the case; in fact, a slightly
greater stimulation occurred at 0.8 MPa (Fig. 3, inset). This result
indicates that at 1.6 MPa, the low w itself
became the dominant limiting factor for root elongation.
View larger version (28K):
[in this window]
[in a new window]
| Figure 2.
Time courses of primary root elongation rate in
solutions of various w (imposed by PEG) and
pO2. A, w = 0.02 MPa
(no PEG); B, w = 0.3 MPa; C, w = 0.8
MPa; D, w = 1.6 MPa. Data points are means ± se (n = 13-40) from two experiments.
Error bars are not shown where they are smaller than the symbols. The
insets in B, C, and D show the time courses of solution
w. Solid lines in the insets represent the calculated
change in solution w; data points are w
measurements of growth media sampled during the experiments. Solution
w was constant at 0.02 MPa throughout the experiments
shown in A.
|
|
View larger version (24K):
[in this window]
[in a new window]
| Figure 3.
Response of steady-state root elongation rate to
solution pO2 and low w
imposed by PEG. Data were obtained by calculating the average
elongation rate for the last 17 to 23 h of the experiments shown
in Figure 2. Data are means ± se
(n = 13-37). Statistical differences in the data
were analyzed by analysis of variance (see ``Results''). Inset,
Relative stimulation of root elongation rate at solution
pO2 of 43 kPa compared with 20.4 kPa.
|
|
Steady-state root elongation rates at w of
0.02 and 0.3 MPa were not significantly different at solution
pO2 of either 28 or 43 kPa (Fig. 3).
This shows that elongation of solution-cultured roots can fully adapt
to a w of 0.3 MPa if supplemental
O2 is supplied, and indicates that there were no
toxic effects of PEG on root growth, at least at that concentration.
The growth data show that root elongation was O2
limited in PEG solutions at ambient
pO2. To test whether supplemental
O2 could also stimulate root growth at low
w in a solid medium, roots growing in
vermiculite were supplied with an elevated
pO2 of 28 kPa. This treatment had a
negligible effect on root growth at several w
(data not shown). Thus, the stimulation of growth at elevated
pO2 in PEG was not a general feature
of roots at low w, and was probably
attributable to alleviation of hypoxia.
Amino Acid Measurements
As an additional test for root hypoxia in PEG solutions without
supplemental O2, we measured the effects of
solution pO2 on root-tip Ala and Pro
levels. Ala was measured because it often accumulates under hypoxic
conditions (Thompson et al., 1966; Ricard et al., 1994; Xia and
Roberts, 1994). Pro was measured because it generally increases in
concentration in tissues at low w; in the
primary root tip of maize Pro accounts for as much as 50% of the
osmotic adjustment (Voetberg and Sharp, 1991).
In PEG solution at a w of 1.6 MPa, Ala
concentration was significantly higher at ambient solution
pO2 than at solution
pO2 of 28 or 43 kPa and was also
significantly higher than at any other w (Fig.
4A). At higher
w, Ala concentration did not vary significantly as solution pO2
decreased, suggesting that roots at 1.6 MPa were affected by
O2 limitation to a greater extent than roots at
the other w. To confirm that the trend in Ala
accumulation observed at 1.6 MPa continued under more severe
O2 limitation, Ala was also measured at
subambient solution pO2 (12 kPa,
achieved by mixing N2 with the air flowing into
the solution). The steady-state root elongation rate decreased to 0.22 mm h1 and the Ala concentration increased
further to 31.3 millimolal; which is similar to the values reported in
maize roots exposed to pO2 of 3 kPa
for 4 h followed by 2.5 h of anoxia (Xia and Roberts, 1994).
View larger version (18K):
[in this window]
[in a new window]
| Figure 4.
Response of root-tip Ala (A) and Pro (B)
concentrations to solution pO2 and low
w imposed by PEG. The apical 10 mm of roots with
elongation rates approximately equal to the mean were collected at the
end of the experiments shown in Figure 2 and analyzed by HPLC. Data are
means ± se (n = 3-8) and were
analyzed by analysis of variance (see ``Results'').
|
|
To discern whether low O2 availability also had
an effect on metabolic changes that normally occur in response to low
w, changes in the level of Pro in the root tip
were analyzed. In 1.6 MPa PEG, Pro concentration was significantly
decreased at ambient solution pO2
compared with solution pO2 of 28 or 43 kPa (Fig. 4B). Levels of Gln, which may provide substrate for Pro synthesis (through conversion to Glu), were also decreased (data not
shown). As with Ala, no significant differences in Pro concentration between different solution pO2 levels
were observed at higher w. When the
pO2 of the 1.6-MPa solution was
reduced to 12 kPa, Pro accumulation was further inhibited to 19.1 millimolal. The inhibition of Pro accumulation at lower
pO2 is consistent with the results of
a previous study that showed that Pro accumulation in wilted turnip
leaves was inhibited by O2-limited conditions (Thompson et al., 1966).
The amino acid data show that roots in PEG at a
w of 1.6 MPa and ambient solution
pO2 exhibited metabolic signs of
O2 deficiency in addition to inhibition of
elongation. However, root elongation was more sensitive than Ala or Pro
accumulation to low O2 supply because only
elongation was affected by ambient solution
pO2 at w of
0.3 and 0.8 MPa.
Root pO2
To directly assess the effects of PEG and solution
pO2 on the
O2 status of the root elongation zone,
pO2 was measured across the outer 150 µm of the cortex with an O2 microsensor in
roots grown at 0.02 and 1.6 MPa. Measurement of root
O2 status was essential to ensure that
supplemental O2 did not increase tissue pO2 above normal physiological levels.
To aid in interpreting the data for solution-cultured roots, the
pO2 of roots growing in vermiculite at
ambient pO2 and the same
w were measured for comparison. Vermiculite
was used because the large air spaces allow much more rapid movement of
O2 to the root surface than is possible in liquid
media. Given the lack of root-growth stimulation with supplemental
O2, as noted above, the
pO2 of vermiculite-grown roots appears
to be optimal for growth and metabolism.
At high w (0.02 MPa), root
pO2 was much lower in solution culture
at ambient pO2 (20.4 kPa) than in
vermiculite (Fig. 5A). This difference
was attributable to a lower root-surface
pO2, which was associated with a
pO2 gradient extending from the bulk solution through the boundary layer next to the root. When the solution
pO2 was increased by 6.4 kPa to 26.8 kPa, root-surface and internal pO2
increased by approximately the same extent. Unexpectedly, when the
solution pO2 was further increased by
20.8 kPa to 47.6 kPa, root-surface and internal
pO2 increased by only 5 kPa, which gave values similar to those in vermiculite-grown roots. This result
was associated with an increase in boundary-layer thickness from
approximately 500 µm at solution pO2
of 20.4 and 26.8 kPa to 1000 µm at solution
pO2 of 47.6 kPa (Fig. 5A).
(Boundary-layer thickness was measured as the distance from the root
surface at which pO2 began to decrease
from the bulk solution pO2.)
View larger version (27K):
[in this window]
[in a new window]
| Figure 5.
Effect of solution pO2
on the pO2 profile across the boundary layer
and into roots at high w (0.02 MPa) (A) and low
w (1.6 MPa, imposed by PEG) (B). The indicated bulk
solution pO2 are means from these specific
experiments. Profiles obtained with roots grown in vermiculite at
ambient pO2 and the same w
are also shown. Data were collected with a microsensor on a
perpendicular approach to the root surface between 4 and 10 mm from the
root apex. Values are means ± se
(n = 3-10). The inset in B shows a comparison of
the pO2 profiles across the cortex of roots
growing at ambient pO2 in high- or
low-w solution. The dashed line in A shows the effect of
removing most of the mucilage (by gently sliding the root tip past a
portion of the root guide) from the measured side of a root at a
solution pO2 of 26.8 kPa. These measurements were made at 4 mm from the apex, and the experiment was repeated with
similar results.
|
|
The increase in boundary-layer thickness at high solution
pO2 was associated with an increased
thickness of the mucilage layer. The mucilage was distinctly visible as
a layer coating the surface of the root when viewed through the
microscope, and the thickness of the layer could be measured with the
eyepiece reticle. The thickness varied greatly with distance from the
root apex and among different roots within each treatment, but was
generally less than 400 µm at solution
pO2 of 20.4 and 26.8 kPa, and in the
range of 400 to 800 µm at solution
pO2 of 47.6 kPa. The importance of the
mucilage layer for determining the thickness of the boundary layer is
illustrated for the 26.8-kPa treatment by the dashed line in Figure 5A,
which shows the effect of removing most of the mucilage from the
measured side of a root. This resulted in a substantial decrease in
boundary-layer thickness and, accordingly, an increase of approximately
3.5 kPa in root-surface pO2.
An additional factor that may have contributed to the thicker boundary
layer at solution pO2 of 47.6 kPa was
the presence of root hairs, which in this treatment were generally
observed starting at 10 mm from the root apex. Root hairs were not
observed in this region at lower solution
pO2. Root hairs increase the thickness
of the boundary layer by decreasing fluid velocity near the root
surface.
Within the roots at high w the
pO2 decreased steeply from the surface
to a depth of 100 µm, with a similar slope at all solution pO2 and in vermiculite, and then
exhibited little additional decrease from 100 to 150 µm. A similar
plateau of pO2 within the cortex of
maize primary roots was reported by Armstrong et al. (1994).
At a w of 1.6 MPa, root-surface and
internal pO2 were again much lower in
solution culture at ambient pO2 than
in vermiculite (Fig. 5B). However, root
pO2 at all measured depths were higher in the PEG solution than in the solution without PEG (Fig. 5B, inset).
This result was unexpected because the roots in PEG exhibited signs of
O2 deficiency (Figs. 3 and 4). Also, when the PEG
solution pO2 was increased by 10.0 to
30.4 kPa, root-surface pO2 increased only slightly and internal pO2 were
unaffected, even though this treatment increased root elongation rate
and alleviated the metabolic signs of hypoxia. Thus, the root
pO2, at least across the cortex, was
not a good indicator of O2 limitations to growth
and metabolism. The lack of effect of increasing solution
pO2 on root
pO2 was associated with a steeper
decrease in pO2 across the boundary layer and the outer region of the root, and probably reflected increasing O2 influx and consumption as the
O2 supply increased. The gradient in
pO2 was even steeper when the solution
pO2 was further increased to 45.9 kPa,
resulting in root-surface and internal pO2 that were similar to, or still
less than (depending on depth), those of roots in vermiculite. In
contrast to the roots at high w, mucilage was
usually barely detectable at all positions at low
w, and was not observed in any of the roots
studied at solution pO2 of 45.9 kPa.
Accordingly, there was no effect of increased O2
supply on the thickness of the boundary layer, which was 300 to 400 µm. As expected, the viscosity of the PEG solution increased the
thickness of the boundary layer, since at high
w the boundary layer was only around 200 µm
thick after most of the mucilage was removed (Fig. 5A, dashed line).
Comparison of the results at high and low
w (Fig. 5, A and B) shows that the
pO2 gradient across the boundary layer
and outer region of the root was considerably steeper at low
w and solution pO2 of 45.9 kPa than at high
w at any solution
pO2. This resulted in a comparable
decrease in pO2 from the bulk solution
to the root surface at high and low w despite
the much greater thicknesses of the mucilage and boundary layers at
high w. A possible explanation of these
results is that the influx of O2 and
O2 consumption were higher at low
w (see ``Discussion'').
In summary, at both high and low w, raising
the solution pO2 to around 47 kPa
increased the pO2 in the
root-elongation zone to values equal to or less than those of
vermiculite-grown roots, and therefore did not oxygenate the tissues
above normal physiological levels.
Comparison of PEG with Vermiculite
Because the vermiculite system for growing seedlings at low
w has been used extensively in our
laboratory, we compared the responses of vermiculite-grown roots with
those obtained using PEG. To do this, steady-state elongation rates of
O2-sufficient roots (43 kPa) in PEG were plotted
together with data from vermiculite-grown roots as a function of medium
w (Fig. 6). The
response of root elongation rate to decreasing
w was strikingly different in vermiculite compared with PEG. At all w tested, elongation
rate was less inhibited in PEG than in vermiculite. At 0.3 MPa,
elongation rate decreased by roughly one-third in vermiculite but was
not significantly affected in PEG. At 1.6 MPa, elongation rate was inhibited by 63% in PEG compared with 75% in vermiculite. Likewise, shoot growth was much less sensitive to low w
in PEG than in vermiculite (data not shown). Consistent with the growth
data, Pro concentration in the apical 1 cm of the primary root at 0.3 and 0.8 MPa PEG was considerably lower than that in vermiculite-grown roots at the same w (compare Fig. 4 with
figure 1 of Voetberg and Sharp, 1991). Pro accumulation at 1.6 MPa,
however, was similar in the two systems.
View larger version (15K):
[in this window]
[in a new window]
| Figure 6.
Response of root elongation rate to low
w imposed by PEG or vermiculite. Steady-state elongation
rates of roots grown in PEG at a pO2 of 43 kPa ( , after low w was imposed over 8 h) are
from Figure 3. The vermiculite response (dashed line) is from Sharp
(1990), and has been consistently reproduced. Also shown is the
steady-state elongation rate obtained after 1.6 MPa PEG was imposed
over 50 h at a solution pO2 of 43 kPa
( ) (mean ± se, n = 22 combined
from two experiments). Error bars are smaller than the symbols for all
data points.
|
|
Because the rate of low-w imposition can
affect Pro accumulation (Naidu et al., 1990) and could possibly affect
steady-state root elongation, the time over which
w was decreased in the PEG system was extended
from 8 to 50 h to determine if this could account for the
different responses of elongation rate to low w in PEG and vermiculite. A time of 50 h
was chosen to exceed the time required by maize roots transplanted into
dry vermiculite to reach steady-state root-tip osmotic potential
(approximately 35 h; Sharp et al., 1990). Thus, low
w was imposed with PEG over a period as long
or longer than that required for roots to adapt to low
w in the vermiculite system. The gradual
addition of PEG to attain 1.6 MPa over 50 h yielded a
steady-state root elongation rate that was slightly higher than that
obtained when the same w was reached after
8 h (Fig. 6). The concentration of Pro in the apical 1 cm of the
root was the same after the two rates of low-w
imposition (83.1 ± 4.8 millimolal [n = 3] after
the slow rate versus 82.5 ± 3.5 millimolal [n = 5] after the rapid rate; means ± se). Thus, the
different responses of root elongation rate to low
w in vermiculite and PEG were not explained by
the rate at which low w was imposed.
We also investigated whether mannitol or melibiose, which are sometimes
used to impose low w in solution culture,
could reproduce the results obtained with PEG solutions. However, both
mannitol (tested at 0.8 and 1.6 MPa) and melibiose (tested at 1.6
MPa) caused almost complete inhibition of root elongation by 50 h
after transfer to solution culture (data not shown), and thus
apparently had toxic effects.
| |
DISCUSSION |
Our results show that maize seedlings growing in PEG solutions at
ambient pO2 are
O2 deficient despite vigorous aeration. Because
it proved necessary to grow roots in guides (presumably to prevent root
damage and PEG uptake), it is not feasible to supply sufficient
O2 by increased solution mixing. Therefore, supplemental O2 must be supplied to have
confidence in experiments on responses to low
w using PEG solutions.
Assessment of Root O2 Status
At a w of 1.6 MPa and ambient solution
pO2, the roots exhibited signs of
O2 deficiency (decreased elongation rate, Ala
accumulation, and decreased Pro accumulation) relative to roots grown
with supplemental O2. In view of this finding, it
is paradoxical that at ambient solution
pO2, roots at low
w had slightly greater
pO2 at all measured depths across the
cortex than roots at high w (Fig. 5B, inset),
which, in the longer term, did not exhibit signs of hypoxia. It could
be that stelar pO2, which was not
measured, was in fact lower in the roots at low compared with high
w; this could have determined the root growth
and metabolic responses regardless of cortical
pO2. Even at high
w the roots appeared to be
O2 deficient early in the experiments, since
their elongation rate was inhibited relative to that of roots at higher
solution pO2 during the first 30 h after transfer to solution culture. However, whereas root growth at
high w acclimated to this condition, the roots
at low w exhibited maximal elongation rates
only at the higher tissue pO2 levels
that resulted from supplemental oxygenation. This suggests that the
ability to acclimate to low tissue pO2 may have been impaired at low w.
At high w it was unexpected that a solution
pO2 as high as 47 kPa was required to
raise cortical pO2 levels to the
levels of vermiculite-grown roots. It is important to note that because of the thick boundary layer at a solution
pO2 of 47 kPa, the root-surface pO2 was similar to that in
vermiculite. Thus, the root tips were exposed to the same local
O2 environment in the two conditions. Furthermore, the factors that contributed to the thickness of the
boundary layer at high solution pO2
(increased mucilage thickness and root hairs closer to the apex than at
lower pO2) are not unique to this
condition. First, extensive expansion of maize root mucilage can occur
in soils at high w (Sealey et al., 1995),
although, consistent with our PEG-grown roots, not at lower
w (McCully and Boyer, 1997). Second, root
hairs were observed in the same region in roots growing in vermiculite
at high w as in roots at high solution
pO2. Arguably, therefore, even at high
w the roots grown at a solution
pO2 of 47 kPa were physiologically
more similar to roots grown in solid media than those grown at lower solution pO2. Thus, supplemental
oxygenation may be an important consideration in any solution-culture
study in which normal oxygenation of root tissues is desired.
Consistent with this suggestion, previous studies of maize roots at
high w have reported that above-ambient solution pO2 was needed for maximal
O2 consumption (Saglio et al., 1984; Atwell et
al., 1985).
Taken together, our results confirm the view that bulk solution
pO2 is not a good indicator of root
O2 status (Drew, 1990), and, furthermore, a
static measure of root pO2 cannot
provide unambiguous evaluation of sufficient oxygenation. Data on
growth and metabolism are also required to assess
O2 status accurately.
Low w May Increase Root O2 Demand
In addition to the expected effect of PEG viscosity on
boundary-layer thickness, the results suggest that with adequate
oxygenation the root elongation zone may have an increased demand for
O2 at low compared with high
w. This would necessitate an even higher solution pO2 to provide adequate
oxygenation than what could be predicted from consideration of
O2-transport properties alone. Evidence for an
increased influx of O2 per unit surface area of the root tip at low relative to high w comes
from the finding that the decrease in
pO2 from the bulk solution to the root
surface at approximately 47 kPa was of similar magnitude at
w of 0.02 and 1.6 MPa, despite the fact
that the boundary layer was more than twice as thick at 0.02 MPa
(Fig. 5, A and B). The greater thickness of the boundary layer at high
w, which was associated with a thick mucilage
layer, presumably presented a greater overall resistance to
O2 transport from the bulk solution to the root surface than at low w, under which mucilage
was not observed. It is important to note that the diffusive resistance
to O2 movement within the boundary layer would
have been minimally altered by the addition of PEG, since the
diffusivity coefficient of O2 is similar in water
and PEG solutions (Mexal et al., 1975). Thus, to maintain the same
decrease in pO2 across the boundary
layer, but with a lower resistance to O2
movement, it seems likely that the O2 flux was
considerably greater at low compared with high w. This could not be quantified from the
pO2 gradients, however, because exact
knowledge of the extent of the unstirred boundary layer is required
(Henriksen et al., 1992).
An increased O2 flux into the roots at low
w most likely reflects increased
O2 consumption. The
pO2 gradient from the root surface to
the interior was also steeper at low than at high
w, both in O2-sufficient
roots in solution culture and in vermiculite-grown roots. This is
consistent with a higher rate of O2 flux and
consumption at low w, although possible
effects of low w on root permeability to
O2 could also be involved. There are reports of
increased O2 consumption by water-stressed
relative to well-watered roots of maize (root tips; Greenway, 1970) and
Arnica alpina (whole root systems; Collier and Cummins,
1992). Increased respiration at low w may
provide energy for adaptive processes such as osmolyte synthesis for
osmotic adjustment. Direct measurement of root respiration rates is
required to confirm that this occurs in our system because we do not
know what contribution shoot-supplied O2 may have
made to root O2 consumption (Saglio et al., 1984;
Armstrong et al., 1994). Treatment differences in
O2 flux from the solution into the root could
have been in response to differences in O2 supply from the shoot.
Comparison of PEG and Vermiculite
By investigating the O2 requirements of
roots growing in PEG solutions, our results allow a straightforward
comparison between PEG solutions and other methods of imposing low
w. Under O2-sufficient conditions, root elongation was less sensitive to low
w imposed by PEG than by vermiculite at all
w tested (Fig. 6). This finding emphasizes
that demonstration of similar growth rates in the two media at a given
w cannot be interpreted as evidence against hypoxia in PEG, but would in fact suggest the opposite.
It is not surprising that such different environments have different
effects on root growth. In another example, Reinhardt and Rost (1995)
found that primary roots of cotton seedlings responded differently to
salinity stress depending on whether they were grown in solution
culture or vermiculite. One major difference between PEG solutions and
vermiculite is the hydraulic contact between the root and the medium.
In solution culture, the entire surface of the root is in contact with
the medium, whereas in vermiculite only a portion of the root surface
contacts the vermiculite particles. All water uptake must then occur
through these limited areas of contact, which increases the resistance
to water flow into the root. Growth itself generates a
w gradient between the expanding tissue and
its water source (Nonami and Boyer, 1993), and any increase in
resistance to water flow will increase the w
gradient between the root and the medium. Preliminary measurements indicate that under the nontranspiring conditions used in this study,
root tips in vermiculite at a w of 0.3 MPa
had a w that was 0.27 MPa lower than that of
the vermiculite, whereas the mature region had equilibrated with the
vermiculite. In 0.3 MPa PEG, in contrast, root-tip
w was nearly the same as solution
w, indicating that only a very small
w gradient was needed to drive water uptake (P.E. Verslues and R.E. Sharp, unpublished data). Root-tip
w substantially lower than that of the
surrounding media have also been observed in soil-grown plants (Sharp
and Davies, 1979; Westgate and Boyer, 1985). In our system, these data
indicate that the root tips of seedlings in vermiculite at a given
w are more "stressed" than those in PEG
solution of the same w. The extent to which the different responses of root-tip w explain
the difference observed between PEG and vermiculite in the response of
root elongation to the w of the medium is not
known. Other factors may also be involved; for instance, the diffusion
of gases out of the root differs between the media. The concentrations
of ethylene and CO2, in particular, can affect
root elongation (Radin and Loomis, 1969).
In conclusion, our results show that PEG solutions with supplemental
oxygenation can be used to conduct experiments at low w without the confounding effects of root
O2 deficiency. However, caution must be used in
comparing results obtained using PEG with those obtained using other
methods of imposing low w.
| |
FOOTNOTES |
1
Supported by National Science Foundation grant
no. IBN-9306935 to R.E.S. and E.S.O. P.E.V. was supported by a
fellowship from the University of Missouri Maize Biology Training
Program, a unit of the Department of Energy/National Science
Foundation/U.S. Department of Agriculture Collaborative Research in
Plant Biology Program (grant no. BIR-9420688). This is journal series
no. 12,710 from the Missouri Agricultural Experiment Station.
*
Corresponding author; e-mail
robert_e._sharp{at}muccmail.missouri.edu; fax
1-573-882-1469.
Received September 5, 1997;
accepted December 18, 1997.
| |
ABBREVIATIONS |
Abbreviations:
pO2, O2
partial pressure(s).
w, water potential(s).
| |
ACKNOWLEDGMENTS |
We thank Dr. Gary Krause for assistance with the statistical
analysis, Dr. David Rhodes (Purdue University, West Lafayette, IN) for
advice concerning the amino acid analysis, Steven Wells for technical
assistance in constructing the root boxes, and Drs. Tobias Baskin and
Stephen Pallardy for constructive comments on the manuscript.
| |
LITERATURE CITED |
Armstrong W,
Strange ME,
Cringle S,
Beckett PM
(1994)
Microelectrode and modelling study of oxygen distribution in roots.
Ann Bot
74:
287-299
[Abstract/Free Full Text]
Atwell BJ,
Thomson CJ,
Greenway H,
Ward G,
Waters I
(1985)
A study of the impaired growth of roots of Zea mays seedlings at low oxygen concentrations.
Plant Cell Environ
8:
179-188
Bates LS,
Waldren RP,
Teare ID
(1973)
Rapid determination of free proline for water stress studies.
Plant Soil
39:
205-207
[CrossRef]
Baumgärtl H,
Lubbers DW
(1983)
Microaxial needle sensor for polarographic measurement of local O2 pressure in the cellular range of living tissue: its construction and properties.
In
E Gnaiger,
H Forstner,
eds, Polarographic Oxygen Sensors: Aquatic and Physiological Applications.
Springer-Verlag, New York, pp 37-65
Boyer JS,
Knipling EB
(1965)
Isopiestic technique for measuring leaf water potentials with a thermocouple psychrometer.
Proc Natl Acad Sci USA
54:
1044-1051
[Free Full Text]
Carpita N,
Sabularse D,
Montezinos D,
Delmer DP
(1979)
Determination of the pore size of cell walls of living plant cells.
Science
205:
1144-1147
[Abstract/Free Full Text]
Collier DE, Cummins WR (1992) Stimulation of respiration during
plant water deficits in sand-grown Arnica alpina. In H
Lambers, LHW van der Plas, eds, Molecular, Biochemical and Physiological Aspects of Plant Respiration. SPB Academic Publishers, The Hague, The Netherlands, pp 541-546
Drew MC
(1990)
Sensing soil oxygen.
Plant Cell Environ
13:
681-693
[CrossRef]
Ebert RF
(1986)
Amino acid analysis by HPLC: optimized conditions for chromatography of phenylthiocarbamoyl derivatives.
Anal Biochem
154:
431-435
[CrossRef][Web of Science][Medline]
Greenway H
(1970)
Effects of slowly permeating osmotica on metabolism of vacuolated and nonvacuolated tissue.
Plant Physiol
46:
254-258
[Abstract/Free Full Text]
Henriksen GH,
Raj Raman D,
Walker LP,
Spanswick RM
(1992)
Measurement of net fluxes of ammonium and nitrate at the surface of barley roots using ion-selective microelectrodes. II. Patterns of uptake along the root axis and evaluation of the microelectrode flux estimation technique.
Plant Physiol
99:
734-747
[Abstract/Free Full Text]
Hohl M,
Schopfer P
(1991)
Water relations of growing maize coleoptiles. Comparison between mannitol and polyethylene glycol 6000 as external osmotica for adjusting turgor pressure.
Plant Physiol
95:
716-722
[Abstract/Free Full Text]
Lawlor DW
(1970)
Absorption of polyethylene glycols by plants and their effects on plant growth.
New Phytol
69:
501-513
Liang BM,
Sharp RE,
Baskin TI
(1997)
Regulation of growth anisotropy in well-watered and water-stressed maize roots. I. Spatial distribution of longitudinal, radial and tangential expansion rates.
Plant Physiol
115:
101-111
[Abstract]
McCully ME,
Boyer JS
(1997)
The expansion of maize root-cap mucilage during hydration. 3. Changes in water potential and water content.
Physiol Plant
99:
169-177
Mexal J,
Fisher JT,
Osteryoung J,
Reid CPP
(1975)
Oxygen availability in polyethylene glycol solutions and its implications in plant-water relations.
Plant Physiol
55:
20-24
[Abstract/Free Full Text]
Naidu BP,
Palig LG,
Aspinall D,
Jennings AC,
Jones GP
(1990)
Rate of imposition of water stress alters the accumulation of nitrogen-containing solutes by wheat seedlings.
Aust J Plant Physiol
17:
653-664
Nonami H,
Boyer JS
(1993)
Direct demonstration of a growth-induced water potential gradient.
Plant Physiol
102:
13-19
[Abstract]
Nye PH,
Tinker PB
(1977)
Solute movement in the soil-root system.
In
DJ Anderson,
P Greig-Smith,
FA Pitelda,
eds, Studies in Ecology, Vol 4.
University of California Press, Berkeley, CA, pp 8-13
Ober ES,
Sharp RE
(1996)
A microsensor for direct measurement of O2 partial pressure within plant tissues.
J Exp Bot
47:
447-454
Plaut Z,
Federman E
(1985)
A simple procedure to overcome polyethylene glycol toxicity on whole plants.
Plant Physiol
79:
559-561
[Abstract/Free Full Text]
Radin JW,
Loomis RS
(1969)
Ethylene and carbon dioxide in the growth and development of cultured radish roots.
Plant Physiol
44:
1584-1589
[Abstract/Free Full Text]
Reinhardt DH,
Rost TL
(1995)
Primary and lateral root development of dark- and light-grown cotton seedlings under salinity stress.
Bot Acta
108:
457-465
Ricard B,
Couee I,
Raymond P,
Saglio PH,
Saint-Ges V,
Pradet A
(1994)
Plant metabolism under hypoxia and anoxia.
Plant Physiol Biochem
32:
1-10
Saab IN,
Sharp RE,
Pritchard J,
Voetberg GS
(1990)
Increased endogenous abscisic acid maintains primary root growth and inhibits shoot growth of maize seedlings at low water potentials.
Plant Physiol
93:
1329-1336
[Abstract/Free Full Text]
Saglio PH,
Rancillac M,
Bruzan F,
Pradet A
(1984)
Critical oxygen pressure for growth and respiration of excised and intact roots.
Plant Physiol
76:
151-154
[Abstract/Free Full Text]
Sealey LJ,
McCully ME,
Canny MJ
(1995)
The expansion of maize root-cap mucilage during hydration. I. Kinetics.
Physiol Plant
93:
38-46
[CrossRef]
Sharp RE (1990) Comparative sensitivity of root and shoot growth
and physiology to low water potentials. In WJ Davies, B
Jeffcoat, eds, Importance of Root to Shoot Communication in the
Responses to Environmental Stress. British Society for Plant Growth
Regulation, Bristol, UK, pp 29-44
Sharp RE,
Davies WJ
(1979)
Solute regulation and growth by roots and shoots of water-stressed maize plants.
Planta
147:
43-49
[CrossRef]
Sharp RE,
Hsiao TC,
Silk WK
(1990)
Growth of the maize primary root at low water potentials. II. Role of growth and deposition of hexose and potassium in osmotic adjustment.
Plant Physiol
93:
1337-1346
[Abstract/Free Full Text]
Sharp RE,
Silk WK,
Hsiao TC
(1988)
Growth of the maize primary root at low water potentials. I. Spatial distribution of expansive growth.
Plant Physiol
87:
50-57
[Abstract/Free Full Text]
Spollen WG,
Sharp RE
(1991)
Spatial distribution of turgor and root growth at low water potentials.
Plant Physiol
96:
438-443
[Abstract/Free Full Text]
Stewart CR,
Hanson AD
(1980)
Proline accumulation as a metabolic response to water stress.
In
NC Turner,
PJ Kramer,
eds, Adaptation of Plants to Water and High Temperature Stress.
John Wiley & Sons, New York, pp 173-190
Termaat A,
Munns R
(1986)
Use of concentrated macronutrient solutions to separate osmotic from NaCl-specific effects on plant growth.
Aust J Plant Physiol
13:
509-522
Thompson JF,
Stewart CR,
Morris CJ
(1966)
Changes in amino acid content of excised leaves during incubation. I. The effect of water content of leaves and atmospheric oxygen level.
Plant Physiol
41:
1578-1584
[Abstract/Free Full Text]
Voetberg GS,
Sharp RE
(1991)
Growth of the maize primary root at low water potentials. III. Role of increased proline deposition in osmotic adjustment.
Plant Physiol
96:
1125-1130
[Abstract/Free Full Text]
Westgate ME,
Boyer JS
(1985)
Osmotic adjustment and the inhibition of leaf, root, stem and silk growth at low water potentials in maize.
Planta
164:
540-549
[CrossRef]
Xia J-H,
Roberts JKM
(1994)
Improved cytoplasmic pH regulation, increased lactate efflux, and reduced cytoplasmic lactate levels are biochemical traits expressed in root tips of whole maize seedlings acclimated to a low-oxygen environment.
Plant Physiol
105:
651-657
[Abstract]
Yang C-Y,
Sepulveda FI
(1985)
Separation of phenylthiocarbamoyl amino acids by high-performance liquid chromatography on Spherisorb octadecylsilane columns.
J Chromatogr
346:
413-416
[Medline]
Top
Abstract
Introduction