Skip to main content
NCBI home page
Plant Physiology logoLink to Plant Physiology
. 1998 Apr;116(4):1403–1412. doi: 10.1104/pp.116.4.1403

Root Growth and Oxygen Relations at Low Water Potentials. Impact of Oxygen Availability in Polyethylene Glycol Solutions1

Paul E Verslues 1, Eric S Ober 1, Robert E Sharp 1,*
PMCID: PMC35048  PMID: 9536058

Abstract

Polyethylene glycol (PEG), which is often used to impose low water potentials (ψw) 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 ψw, 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 ψw. 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 ψw, compounding the effect of PEG on O2 supply. Under O2-sufficient conditions root elongation was substantially less sensitive to the low ψw imposed by PEG than that imposed by dry vermiculite.

In studies of plant responses to water deficit, low ψw 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 ψw 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 ψw 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 ψw, 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 ψw 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 ψw 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 ψw ≤ −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 ψw 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 ψw. 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 ψw (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 ψw. 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 ψw, but substantially increased root elongation in PEG (Mr 8000; Sigma) solution at a ψw 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.

Figure 1.

Figure 1

Open in a new tab

Effect of root guides on increase of primary root length in vigorously aerated solutions at ψw 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 (ψw = −0.02 MPa). Imposition of low ψw 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 ψw of −0.3, −0.8, or −1.6 MPa were used. The rate of ψw decline for each treatment was adjusted so that the final ψw was reached 8 h after imposition of low ψw was begun (unless otherwise noted). To convert PEG concentrations to ψw, the ψw of a series of PEG solutions were measured using isopiestic thermocouple psychrometry (Boyer and Knipling, 1965). A time course of ψw decline was then calculated for each ψw treatment; measurements of samples of the growth media that were withdrawn periodically from the box confirmed the accuracy of the predicted ψw.

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 min−1. 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 ψw 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 ψw and is shortened at low ψw 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 α-aminobutyric acid or α-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 (ψw = −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 ψw 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 min−1. (During low-ψw 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 ψw 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 ψw, either in PEG solution with supplemental O2 (but not at ambient pO2) or in vermiculite, were thinner than roots at high ψw in either medium (for a detailed analysis of the thinning response to low ψw 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 × ψw) 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 ψw (−0.02 MPa, no PEG used) as well as in PEG solutions at ψw of −0.3, −0.8, and −1.6 MPa (Fig. 2). In all treatments, root elongation rate was approximately 2 mm h−1 during the first 2 h after transfer to solution culture (before low-ψw 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-ψw 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-ψw 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 ψw, 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 ψw 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 ψw, 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 ψw itself became the dominant limiting factor for root elongation.

Figure 2.

Figure 2

Open in a new tab

Time courses of primary root elongation rate in solutions of various ψw (imposed by PEG) and pO2. A, ψw = −0.02 MPa (no PEG); B, ψw = −0.3 MPa; C, ψw = −0.8 MPa; D, ψw = −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 ψw. Solid lines in the insets represent the calculated change in solution ψw; data points are ψw measurements of growth media sampled during the experiments. Solution ψw was constant at −0.02 MPa throughout the experiments shown in A.

Figure 3.

Figure 3

Open in a new tab

Response of steady-state root elongation rate to solution pO2 and low ψw 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 ψw 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 ψw 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 ψw 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 ψw (data not shown). Thus, the stimulation of growth at elevated pO2 in PEG was not a general feature of roots at low ψw, 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 ψw; 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 ψw 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 ψw (Fig. 4A). At higher ψw, 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 ψw. 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 h−1 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).

Figure 4.

Figure 4

Open in a new tab

Response of root-tip Ala (A) and Pro (B) concentrations to solution pO2 and low ψw 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 ψw, 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 ψw. 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 ψw 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 ψw 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 ψw 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 ψw (−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.)

Figure 5.

Figure 5

Open in a new tab

Effect of solution pO2 on the pO2 profile across the boundary layer and into roots at high ψw (−0.02 MPa) (A) and low ψw (−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 ψw 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-ψw 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 ψw 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 ψw 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 ψw, mucilage was usually barely detectable at all positions at low ψw, 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 ψw 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 ψw (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 ψw and solution pO2 of 45.9 kPa than at high ψw at any solution pO2. This resulted in a comparable decrease in pO2 from the bulk solution to the root surface at high and low ψw despite the much greater thicknesses of the mucilage and boundary layers at high ψw. A possible explanation of these results is that the influx of O2 and O2 consumption were higher at low ψw (see Discussion).

In summary, at both high and low ψw, 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 ψw 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 ψw (Fig. 6). The response of root elongation rate to decreasing ψw was strikingly different in vermiculite compared with PEG. At all ψw 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 ψw 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 ψw (compare Fig. 4 with figure 1 of Voetberg and Sharp, 1991). Pro accumulation at −1.6 MPa, however, was similar in the two systems.

Figure 6.

Figure 6

Open in a new tab

Response of root elongation rate to low ψw imposed by PEG or vermiculite. Steady-state elongation rates of roots grown in PEG at a pO2 of 43 kPa (○, after low ψw 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-ψw imposition can affect Pro accumulation (Naidu et al., 1990) and could possibly affect steady-state root elongation, the time over which ψw 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 ψw 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 ψw was imposed with PEG over a period as long or longer than that required for roots to adapt to low ψw 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 ψw 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-ψw 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 ψw in vermiculite and PEG were not explained by the rate at which low ψw was imposed.

We also investigated whether mannitol or melibiose, which are sometimes used to impose low ψw 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 ψw using PEG solutions.

Assessment of Root O2 Status

At a ψw 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 ψw had slightly greater pO2 at all measured depths across the cortex than roots at high ψw (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 ψw; this could have determined the root growth and metabolic responses regardless of cortical pO2. Even at high ψw 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 ψw acclimated to this condition, the roots at low ψw 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 ψw.

At high ψw 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 ψw (Sealey et al., 1995), although, consistent with our PEG-grown roots, not at lower ψw (McCully and Boyer, 1997). Second, root hairs were observed in the same region in roots growing in vermiculite at high ψw as in roots at high solution pO2. Arguably, therefore, even at high ψw 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 ψw 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 ψw 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 ψw. 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 ψw 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 ψw 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 ψw, 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 ψw, 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 ψw. 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 ψw most likely reflects increased O2 consumption. The pO2 gradient from the root surface to the interior was also steeper at low than at high ψw, 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 ψw, although possible effects of low ψw 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 ψw 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 ψw. Under O2-sufficient conditions, root elongation was less sensitive to low ψw imposed by PEG than by vermiculite at all ψw tested (Fig. 6). This finding emphasizes that demonstration of similar growth rates in the two media at a given ψw 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 ψw gradient between the expanding tissue and its water source (Nonami and Boyer, 1993), and any increase in resistance to water flow will increase the ψw 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 ψw of −0.3 MPa had a ψw 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 ψw was nearly the same as solution ψw, indicating that only a very small ψw gradient was needed to drive water uptake (P.E. Verslues and R.E. Sharp, unpublished data). Root-tip ψw 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 ψw are more “stressed” than those in PEG solution of the same ψw. The extent to which the different responses of root-tip ψw explain the difference observed between PEG and vermiculite in the response of root elongation to the ψw 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 ψw 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 ψw.

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.

Abbreviations:

pO2

O2 partial pressure(s)

ψw

water potential(s)

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.

LITERATURE  CITED

  1. Armstrong W, Strange ME, Cringle S, Beckett PM. Microelectrode and modelling study of oxygen distribution in roots. Ann Bot. 1994;74:287–299. [Google Scholar]
  2. Atwell BJ, Thomson CJ, Greenway H, Ward G, Waters I. A study of the impaired growth of roots of Zea mays seedlings at low oxygen concentrations. Plant Cell Environ. 1985;8:179–188. [Google Scholar]
  3. Bates LS, Waldren RP, Teare ID. Rapid determination of free proline for water stress studies. Plant Soil. 1973;39:205–207. [Google Scholar]
  4. Baumgärtl H, Lubbers DW. Microaxial needle sensor for polarographic measurement of local O2 pressure in the cellular range of living tissue: its construction and properties. In: Gnaiger E, Forstner H, editors. Polarographic Oxygen Sensors: Aquatic and Physiological Applications. New York: Springer-Verlag; 1983. pp. 37–65. [Google Scholar]
  5. Boyer JS, Knipling EB. Isopiestic technique for measuring leaf water potentials with a thermocouple psychrometer. Proc Natl Acad Sci USA. 1965;54:1044–1051. [PMC free article] [PubMed] [Google Scholar]
  6. Carpita N, Sabularse D, Montezinos D, Delmer DP. Determination of the pore size of cell walls of living plant cells. Science. 1979;205:1144–1147. doi: 10.1126/science.205.4411.1144. [DOI] [PubMed] [Google Scholar]
  7. 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
  8. Drew MC. Sensing soil oxygen. Plant Cell Environ. 1990;13:681–693. [Google Scholar]
  9. Ebert RF. Amino acid analysis by HPLC: optimized conditions for chromatography of phenylthiocarbamoyl derivatives. Anal Biochem. 1986;154:431–435. doi: 10.1016/0003-2697(86)90010-2. [DOI] [PubMed] [Google Scholar]
  10. Greenway H. Effects of slowly permeating osmotica on metabolism of vacuolated and nonvacuolated tissue. Plant Physiol. 1970;46:254–258. doi: 10.1104/pp.46.2.254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Henriksen GH, Raj Raman D, Walker LP, Spanswick RM. 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. 1992;99:734–747. doi: 10.1104/pp.99.2.734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hohl M, Schopfer P. Water relations of growing maize coleoptiles. Comparison between mannitol and polyethylene glycol 6000 as external osmotica for adjusting turgor pressure. Plant Physiol. 1991;95:716–722. doi: 10.1104/pp.95.3.716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lawlor DW. Absorption of polyethylene glycols by plants and their effects on plant growth. New Phytol. 1970;69:501–513. [Google Scholar]
  14. Liang BM, Sharp RE, Baskin TI. Regulation of growth anisotropy in well-watered and water-stressed maize roots. I. Spatial distribution of longitudinal, radial and tangential expansion rates. Plant Physiol. 1997;115:101–111. doi: 10.1104/pp.115.1.101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. McCully ME, Boyer JS. The expansion of maize root-cap mucilage during hydration. 3. Changes in water potential and water content. Physiol Plant. 1997;99:169–177. [Google Scholar]
  16. Mexal J, Fisher JT, Osteryoung J, Reid CPP. Oxygen availability in polyethylene glycol solutions and its implications in plant-water relations. Plant Physiol. 1975;55:20–24. doi: 10.1104/pp.55.1.20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Naidu BP, Palig LG, Aspinall D, Jennings AC, Jones GP. Rate of imposition of water stress alters the accumulation of nitrogen-containing solutes by wheat seedlings. Aust J Plant Physiol. 1990;17:653–664. [Google Scholar]
  18. Nonami H, Boyer JS. Direct demonstration of a growth-induced water potential gradient. Plant Physiol. 1993;102:13–19. doi: 10.1104/pp.102.1.13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Nye PH, Tinker PB. Solute movement in the soil-root system. In: Anderson DJ, Greig-Smith P, Pitelda FA, editors. Studies in Ecology, Vol 4. Berkeley, CA: University of California Press; 1977. pp. 8–13. [Google Scholar]
  20. Ober ES, Sharp RE. A microsensor for direct measurement of O2 partial pressure within plant tissues. J Exp Bot. 1996;47:447–454. [Google Scholar]
  21. Plaut Z, Federman E. A simple procedure to overcome polyethylene glycol toxicity on whole plants. Plant Physiol. 1985;79:559–561. doi: 10.1104/pp.79.2.559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Radin JW, Loomis RS. Ethylene and carbon dioxide in the growth and development of cultured radish roots. Plant Physiol. 1969;44:1584–1589. doi: 10.1104/pp.44.11.1584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Reinhardt DH, Rost TL. Primary and lateral root development of dark- and light-grown cotton seedlings under salinity stress. Bot Acta. 1995;108:457–465. [Google Scholar]
  24. Ricard B, Couee I, Raymond P, Saglio PH, Saint-Ges V, Pradet A. Plant metabolism under hypoxia and anoxia. Plant Physiol Biochem. 1994;32:1–10. [Google Scholar]
  25. Saab IN, Sharp RE, Pritchard J, Voetberg GS. Increased endogenous abscisic acid maintains primary root growth and inhibits shoot growth of maize seedlings at low water potentials. Plant Physiol. 1990;93:1329–1336. doi: 10.1104/pp.93.4.1329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Saglio PH, Rancillac M, Bruzan F, Pradet A. Critical oxygen pressure for growth and respiration of excised and intact roots. Plant Physiol. 1984;76:151–154. doi: 10.1104/pp.76.1.151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sealey LJ, McCully ME, Canny MJ. The expansion of maize root-cap mucilage during hydration. I. Kinetics. Physiol Plant. 1995;93:38–46. [Google Scholar]
  28. 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
  29. Sharp RE, Davies WJ. Solute regulation and growth by roots and shoots of water-stressed maize plants. Planta. 1979;147:43–49. doi: 10.1007/BF00384589. [DOI] [PubMed] [Google Scholar]
  30. Sharp RE, Hsiao TC, Silk WK. 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. 1990;93:1337–1346. doi: 10.1104/pp.93.4.1337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sharp RE, Silk WK, Hsiao TC. Growth of the maize primary root at low water potentials. I. Spatial distribution of expansive growth. Plant Physiol. 1988;87:50–57. doi: 10.1104/pp.87.1.50. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Spollen WG, Sharp RE. Spatial distribution of turgor and root growth at low water potentials. Plant Physiol. 1991;96:438–443. doi: 10.1104/pp.96.2.438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Stewart CR, Hanson AD. Proline accumulation as a metabolic response to water stress. In: Turner NC, Kramer PJ, editors. Adaptation of Plants to Water and High Temperature Stress. New York: John Wiley & Sons; 1980. pp. 173–190. [Google Scholar]
  34. Termaat A, Munns R. Use of concentrated macronutrient solutions to separate osmotic from NaCl-specific effects on plant growth. Aust J Plant Physiol. 1986;13:509–522. [Google Scholar]
  35. Thompson JF, Stewart CR, Morris CJ. Changes in amino acid content of excised leaves during incubation. I. The effect of water content of leaves and atmospheric oxygen level. Plant Physiol. 1966;41:1578–1584. doi: 10.1104/pp.41.10.1578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Voetberg GS, Sharp RE. Growth of the maize primary root at low water potentials. III. Role of increased proline deposition in osmotic adjustment. Plant Physiol. 1991;96:1125–1130. doi: 10.1104/pp.96.4.1125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Westgate ME, Boyer JS. Osmotic adjustment and the inhibition of leaf, root, stem and silk growth at low water potentials in maize. Planta. 1985;164:540–549. doi: 10.1007/BF00395973. [DOI] [PubMed] [Google Scholar]
  38. Xia J-H, Roberts JKM. 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. 1994;105:651–657. doi: 10.1104/pp.105.2.651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Yang C-Y, Sepulveda FI. Separation of phenylthiocarbamoyl amino acids by high-performance liquid chromatography on Spherisorb octadecylsilane columns. J Chromatogr. 1985;346:413–416. doi: 10.1016/s0021-9673(00)90532-6. [DOI] [PubMed] [Google Scholar]

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES