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Keywords:

  • apoplastic barriers;
  • Oryza sativa;
  • outer part of root;
  • oxygen permeability;
  • waterlogging

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • Despite the importance of the barrier to oxygen losses of the roots of hygrophytes growing in wet environments devoid of oxygen, there are few data available on permeability coefficients for O2 across outer root cell layers (POPR) and how they may change in response to low O2.
  • A gas perfusion technique was used to measure the POPR of rice (Oryza sativa) plants grown in either aerated or deoxygenated solution. The contributions of the apoplast and of living cells to the overall POPR were characterized either by blocking apoplastic pores with precipitates of brown Cu2[Fe(CN)6] or by killing cells with 0.1 N HCl.
  • Compared with that of plants from aerated hydroponics, the POPR of plants grown in deoxygenated medium was smaller by an order of magnitude. Precipitates resulting from CuSO4/K4[Fe(CN)6] treatment only formed in plants grown in aerated solution, where they reduced the POPR by 5–20%. Killing of root segments with HCl increased POPR in plants grown in both conditions by 20–55%.
  • The results indicated that apoplastic barriers effectively restricted radial O2 loss. The relative role of the respiratory O2 consumption of root peripheral layers increased as POPR decreased.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

When faced with oxygen depletion in flooded soil, rice (Oryza sativa L.), like other wetland species, develops numerous morphological and biochemical responses to reduce the impact of the stress. The aerenchyma is of particular importance. It facilitates the internal transport of gasses from well-aerated aerial shoots to underground organs exposed to anaerobic surroundings. The effectiveness of the aerenchyma can be increased by the formation of barriers in the outer cell layers of roots. These barriers inhibit radial O2 loss (ROL) to the rhizosphere and enhance longitudinal diffusion of O2 towards the root apex (Armstrong, 1979; Colmer, 2003b).

Roots of some wetland species constitutively express barriers to ROL (e.g. Juncus effusus and Eleocharis acuta; Visser et al., 2000; McDonald et al., 2002). In others, barriers are induced during growth in stagnant, deoxygenated media (e.g. Oryza sativa, wild Hordeum species and Lolium multiflorum; Colmer et al., 1998; McDonald et al., 2002; Colmer, 2003a; Garthwaite et al., 2003; Kotula et al., 2009). The barrier to ROL in roots has usually been linked to suberization and lignification of the walls of the peripheral cell layers (De Simone et al., 2003; Soukup et al., 2007; Garthwaite et al., 2008). Kotula et al. (2009) combined measurements of ROL with histochemical and biochemical analyses of the outer part of roots (OPR) of rice. They showed that, when grown in deoxygenated solution, the amounts of suberin and lignin in OPR sleeves were several folds greater than those of plants grown in aerated solution. This correlated with the pattern of ROLs. It was concluded that the suberized exodermis and lignified sclerenchyma of rice roots formed a strong barrier to ROL.

Despite the importance of the barrier to ROL for roots of wetland plants, there are few data available on the permeability coefficient of O2 across cell layers exterior to the aerenchyma. In Phragmites australis, this permeability coefficient was assessed using O2 concentration profiles across the hypodermis/epidermis, in combination with rates of O2 consumption in these layers (Armstrong et al., 2000). Using roots of Hordeum marinum, Garthwaite et al. (2008) developed another method to determine the diffusivity of O2 across outer cell layers. Diffusivities were derived from measurements of ROL obtained while either varying shoot O2 partial pressure or cooling the rooting medium to cancel respiratory effects. In the present study, the O2 permeability coefficient of the OPR (POPR) of rice has been measured directly using the method recently developed by Kotula & Steudle (2009). The technique was based on perfusing the aerenchyma of root segments with gas mixtures (O2:N2) of known O2 concentration while, at the same time, measuring radial losses of oxygen using a root-sleeving platinum electrode (Armstrong, 1994). The O2 permeability of the OPR could be calculated, as both the O2 flow across the OPR and the O2 concentration gradient between the aerenchyma and the surrounding medium were known.

In contrast to the previous study of Kotula & Steudle (2009), where plants were grown in an aerated solution, in the present study we examined the effects of different growth conditions on POPR by growing rice in either aerated or stagnant, deoxygenated medium. In order to estimate the contribution of the apoplast and living cells to the overall movement of O2 across the OPR, the POPR was varied either by partially blocking apoplastic pores in the OPR with salt precipitates or by killing living tissue with 0.1 N HCl. As far as we are aware, this is the first direct quantitative comparison of O2 permeability coefficients of peripheral layers in roots subjected to aerated or deoxygenated treatments. Our study provides evidence of the effectiveness of apoplastic barriers in reducing O2 and ion permeability across the root outer cell layers, when plants are grown in deoxygenated medium.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Plant material

Seeds of an upland rice cultivar (Oryza sativa L. cv. Azucena; International Rice Research Institute, Manila, Philippines) were germinated and cultivated in either aerated hydroponics (control) or deoxygenated 0.1% agar nutrient solution (treatment) as previously described by Kotula et al. (2009). After 30–40 d of growth, roots were excised and used in experiments.

Measurements of oxygen permeability coefficients (PdO2) in rice root segments

The O2 permeability coefficients of the outer part of rice root segments grown in either aerated or deoxygenated solution were measured as described previously by Kotula & Steudle (2009). Root segments of a length of 50 mm were excised from adventitious roots at distances of 20–70 mm from the apex, where the aerenchyma was sufficiently developed for axial gas perfusion, and inserted through a cylindrical platinum (Pt) electrode. Both cut ends of segments were fixed to glass capillaries and firmly secured on the rack in a Perspex chamber. The chamber was filled with deoxygenated 0.1% (w/v) agar solution containing 5 mol m−3 KCl and 0.5 mol m−3 CaSO4 and closed with a lid. The basal end of the root segment was connected to tanks with mixtures of compressed O2 : N2 of oxygen concentrations (v/v) of 10, 21 and 40%. The other end was open to the atmosphere. Perfusion of moistened gas mixtures through the aerenchyma of the root segments was conducted at an overpressure of 20 kPa. The corresponding rates of radial O2 flows (JO2 in nmol m−2 s−1) were measured along the segments at distances of 30, 40, 50 and 60 mm from the apex. Permeability coefficients were calculated from the slopes of JO2/Ci curves, where Ci (mol m−3) is the concentration of O2 in the liquid phase of the aerenchyma at the inner surface of the OPR, derived from the partial oxygen pressure (Henry’s law). Measurements were taken at 25°C in a temperature-controlled room.

Calculation of the diffusivity of oxygen across the OPR (POPR)

Measured values of permeability coefficients obtained from the perfusion experiment included diffusion of O2 across the shell of 0.1% agar (Pagar) between the root and the electrode surface, which acted as an unstirred layer (USL). These effects may be substantial, and were found to account for 16 to 33% of the measured PdO2 by Kotula & Steudle (2009). As the diffusion coefficient of O2 in the 0.1% agar solution was known (2.3 × 10−9 m2 s−1 at 25°C; Kotula & Steudle, 2009), and the diffusional resistances of the OPR and of the agar solution were in series, POPR was calculated using the formula:

  • image( Eqn 1)

where Pagar is the O2 permeability coefficient in the shell of agar, and POPR is the O2 permeability coefficient of the OPR.

Blocking the apoplast of the OPR with inorganic salt precipitates

In order to investigate the contribution of the apoplast to O2 movement across the OPR, apoplastic pores were blocked with insoluble salt precipitates using the technique of Ranathunge et al. (2005). Root segments (20–70 mm from the apex), previously used to measure POPR, were fixed to glass capillaries (inner diameter 1.3 mm) using polyacrylamide glue (UHU, Bühl, Germany) and connected to the perfusion apparatus. One of the glass capillaries (inlet side) was connected to a syringe while the outlet remained open. The syringe, filled with 1 mM K4[Fe(CN)6], was mounted on a 12-step Braun–Melsungen pump that produced defined pumping rates between 1.7 × 10−9 and 1.1 × 10−11 mm3 s−1. Solutions of K4[Fe(CN)6] were perfused through the aerenchyma of the root segments at rates of c. 1 × 10−10 mm3 s−1, displacing air, while the root segment was bathed in 0.5 mM CuSO4 solution. Perfusion was conducted for 3 h. The reaction between CuSO4 and K4[Fe(CN)6] resulted in rusty brown insoluble precipitates of Cu2[Fe(CN)6] or Cu[CuFe(CN)6]. Following the treatment, root segments were fixed to the gas perfusion set-up, all solution in the aerenchyma was exchanged by gas perfusion and POPR re-measured.

Treatment of root segments with 0.1 N HCl to kill living cells: effects of respiration

In order to quantify the contribution of respiratory O2 consumption to diffusion of O2 across the OPR, 0.1 N of HCl (pH = 1) was used to kill living cells. Root segments previously used to measure POPR were attached to a syringe containing the acid (via a glass capillary and a Teflon tube). Hydrochloric acid was gently injected into the aerenchyma, displacing air. Then the entire root segments were immersed in 0.1 N HCl for 20–30 min. Subsequently, root segments were carefully washed and perfused with distilled water to remove HCl from the aerenchyma and root surface. After fixing HCl-treated segments to the perfusion set-up, O2 permeability was re-measured.

Vitality test

As the O2 permeability experiments lasted for several hours (6–7 h) and even low Cu2+ concentrations during precipitation experiments may be toxic to living cells (Murphy et al., 1999), it was essential to check the viability of cells constituting the OPR at the end of the experiments. The segments were cut into 5-mm-long pieces and soaked in 0.5% Evan’s blue (w/w; Sigma-Aldrich, St Louis, MO, USA) for 30 min (Taylor & West, 1980), allowing the dye to diffuse through the aerenchyma. After thoroughly rinsing the stained segments with water several times, freehand cross-sections and longitudinal sections were made and examined using an Axioplan light microscope (Zeiss, Oberkochen, Germany). Photographs were taken with a digital camera system (D60; Nikon, Göttingen, Germany). Evan’s blue stains nuclei and cytoplasm dark blue in dead cells but it does not penetrate the healthy plasmalemma of living cells. Living cells remained unstained while dead cells took on a blue coloration (Fischer et al., 1985).

Statistical analysis

Data on O2 permeability coefficients were analysed using repeated measures analysis of variance (ANOVA) to examine the effects of growth conditions or treatments (between-subject factor) and position (within-subject factor). Data are presented as means with standard errors. Means were compared at the  0.05 level using a paired t-test (effect of distance within growth condition or treatment and effect of treatment within distance) and a two sample t-test (effect of growth condition within distance).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Oxygen permeability coefficient of the OPR of plants grown in aerated and stagnant solutions

Measurements were made at 30, 40, 50 and 60 mm from the apex of rice plants grown in either aerated or deoxygenated solutions. Permeability coefficients of the outer part of the root (POPR) were highest at 30 mm from the apex and decreased along the root axis, reaching the lowest values at 60 mm (F3,141 = 80.4, < 0.001; Fig. 1). For plants grown in the aerated solution, values of Popr were always significantly different along the root (paired t-test at  0.05). The mean value of POPR at 60 mm was 3.8-fold smaller than that at 30 mm. During stagnant treatment, the POPR decreased by a factor of 5.5 from 30 to 60 mm; however, a significant decrease was not observed between distances of 50 and 60 mm (paired t-test at  0.05). Apparently, a ‘tight barrier’ to ROL was already present at these positions. At all distances, when compared with roots grown in the aerated solution, the stagnant treatment resulted in a several-fold lower O2 permeability coefficient across the OPR (F1,47 = 204.1, < 0.001). At 30 mm, the mean POPR values were 5.2 ± 0.2 and 0.6 ± 0.1 × 10−6 m s−1 for plants grown in the aerated and stagnant solutions, respectively. At 60 mm, these values decreased to 1.4 ± 0.2 and 0.1 ± 0.01 × 10−6 m s−1 (SE; = 19–30 roots). It should be noted that the POPR of plants grown in both solutions should be highest at positions closer to the root apex. However, using the perfusion technique it was not possible to measure POPR at distances below 30 mm from the apex (Colmer, 2003a; Kotula et al., 2009).

image

Figure 1.  Oxygen permeability coefficients of the outer part of roots (POPR) of rice grown in either aerated (black bars) or deoxygenated (grey bars) nutrient solution for 30 to 40 d. Measurements of POPR were performed at different positions along the root segments. Values of POPR were corrected for the resistance of the shell of agar between the root and the electrode surface. In both growth conditions, POPR decreased along the root. At all distances, POPR was significantly lower for plants grown in the stagnant solution (significance level of  0.001 denoted by ***; two sample t-test). Different letters indicate a significant difference between distances within a growth condition (paired t-test). Data are means ± SE (= 19–30 roots).

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Blocking of the apoplastic path of the OPR with precipitated salt crystals

Irrespective of growth conditions, the surface of root segments untreated with CuSO4/K4[Fe(CN)6] appeared white along the entire length (Fig. 2a shows a root segment from an aerated plant). Because of the development of brown precipitates of Cu2[Fe(CN)6] in cell wall pores of the OPR, the surface of roots of plants grown in aerated solution turned brown after treatment with 1 mM K4[Fe(CN)6] and 0.5 mM CuSO4 (Fig. 2b). The brown colour was more intense on the surface of the immature (apical) part of the segments (30–50 mm from the apex). As apoplastic barriers developed in the basal part (50–70 mm), the brown colour gradually faded away. In contrast to roots grown in the aerated solution, no precipitates were observed on the surface of root segments of plants grown in the stagnant solution (Fig. 2c). Killing of root segments by exposing them to 0.1 N HCl resulted in intense brown precipitates along the entire surface of root segments in the aerated solution (Fig. 2d; compare with Fig. 2b). Precipitates in dead root segments were uniformly distributed in the immature part but irregular and patchy in more mature parts. In roots from the stagnant medium, no brown precipitates appeared even after exposing them for 30 min to 0.1 N HCl (Fig. 2e). After 24 h of treatment with CuSO4/K4[Fe(CN)6] only several dots of brown crystals were observed on the surface of the immature part of HCl-killed root segments, (Fig. 2f).

image

Figure 2.  Naked-eye views of the outer surface of rice root segments grown in either an aerated or a stagnant deoxygenated nutrient solution. Root segments of length 50 mm (20 to 70 mm from the apex) were placed in 0.5 mM CuSO4 and aerenchyma was perfused for 3 h with 1 mM K4[Fe(CN)6]. Brown copper ferrocyanide precipitates were formed in the cell walls of the inner cortical cell layers of the outer part of roots (OPR). (a) An untreated root segment. (b) Brown appearance of the surface of a root segment of a plant grown in the aerated solution. (c) No precipitates in a root segment of a plant grown in the stagnant solution. (d) Intense brown precipitates along a root segment of a plant grown in the aerated solution after exposure to 0.1 N HCl for 30 min. Precipitates were uniformly distributed in immature parts but irregular and patchy in mature parts. (e) No precipitates were formed in the root segments of plants grown in the stagnant solution, even after killing of the cells with 0.1 N HCl. (f) Several dots of brown crystals (white arrowheads) were observed in the immature parts of the HCl-killed root segments grown in the stagnant solution, even after 24 h of treatment. Bars, 10 mm.

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In cross-sections of aerated roots made after treatment with CuSO4/K4[Fe(CN)6], intense brown precipitates were observed in the walls of subsclerenchyma cortical cells at 30 mm from the apex (Fig. 3a). At 50 mm, less intense brown precipitates were observed in the tangential walls of the cortical layer beneath the sclerenchyma (Fig. 3b). By contrast, no visible brown precipitates were detected in the cell walls at either 30 or 50 mm in stagnantly grown plants (Fig. 3c,d). This indicated that apoplastic barriers, which developed in roots grown in deoxygenated solution, blocked ion movement across the OPR.

image

Figure 3.  Free-hand cross-sections of rice root segments of plants grown in the aerated (a, b) and stagnant (c, d) solutions, after they had been placed in 0.5 mM CuSO4 and the aerenchyma had been perfused with 1 mM K4[Fe(CN)6]. Cross-sections were made at 30 mm (a, c) and 50 mm (b, d) from the root apex. (a) Intense brown precipitates formed in the walls of subsclerenchyma cortical cells and (b) in the tangential walls of the cortical layer beneath the sclerenchyma of plants grown in the aerated solution. (c, d) No precipitates were formed in the root segments of plants grown in the stagnant deoxygenated solution. Bars = 50 μm.

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Oxygen permeability coefficient of the OPR after blocking the apoplast with inorganic salt precipitates

The blockage of apoplastic pores of the OPR caused a decrease in the O2 permeability coefficient (POPR) only in plants grown in the aerated solution (Fig. 4). For both control and treated root segments, POPR decreased along the root towards the base (F3,15 = 41.2; < 0.001). Compared with control, precipitates caused decreases of POPR of 20, 16, 11 and 5% at distances of 30, 40, 50 and 60 mm, respectively. At 30 mm, POPR decreased from 5.4 ± 0.5 to 4.3 ± 0.3 × 10−6 m s−1 and at 60 mm from 1.4 ± 0.3 to 1.3 ± 0.3 × 10−6 m s−1 (SE; = 6). Declines were relatively small, but were significant at all distances (paired t-test at  0.05). By contrast, there were no treatment effects on the POPR of stagnantly grown plants.

image

Figure 4.  Effect of treatment with insoluble salt precipitates on the oxygen permeability coefficient (POPR) of the outer part of rice roots (OPR). POPR was measured at different distances along root segments excised from plants grown for 30 to 40 d in either the aerated or the deoxygenated nutrient solution. Treatment with insoluble salt crystals resulted in the formation of brown precipitates only in the root segments of plants grown in the aerated solution. Hence, blockage of apoplastic pores of the OPR caused a decrease in the oxygen permeability coefficient only in these plants. Significance levels of  0.05 and  0.01 are denoted by * and **, respectively (paired t-test). Data are means ± SE (= 6 roots).

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Oxygen permeability coefficient of the OPR after killing the root segments with 0.1 N HCl

In both control and HCl-treated root segments, POPR decreased along the root from apical to basal zones for plants grown in either the aerated or the stagnant solution (F3,15 = 25.2, < 0.001 for aerated plants and F3,15 = 12.5, < 0.001 for stagnantly grown plants). Compared with the control, treatment with HCl caused a significant increase in POPR at all distances along the root segments of plants grown in either aerated or stagnant conditions (F1,5 = 66.9, < 0.001 for aerated plants and F1,5 = 46.5, = 0.001 for stagnantly grown plants; Fig. 5). For plants grown in the aerated solution, POPR increased by a factor of c.1.3. At 30 mm from the apex, the mean values were 4.8 ± 0.5 and 6.1 ± 0.6 × 10−6 m s−1 for control and HCl-killed root segments, respectively. At 60 mm POPR increased from 1.2 ± 0.5 to 1.7 ± 0.6 × 10−6 m s−1 (SE; = 6). When plants were grown in the stagnant solution, the POPR of HCl-treated root segments was 1.6- to 2.2-fold greater than that of nontreated roots. The mean POPR values for control and HCl-killed root segments at 30 mm were 0.6 ± 0.1 and 0.9 ± 0.2 × 10−6 m s−1, respectively. Values at 60 mm were 0.1 ± 0.03 and 0.3 ± 0.02 × 10−6 m s−1, respectively. The results indicate that effects of HCl treatments were significant but relatively small.

image

Figure 5.  Effects of 30 min of treatment with 0.1 N HCl on the oxygen permeability coefficient of the outer part of rice roots (POPR). POPR was measured at different distances along root segments excised from plants grown for 30 to 40 d in either the aerated or the deoxygenated nutrient solution. Regardless of growth conditions, there was an increase in the oxygen permeability coefficient at all distance from the apex after exposure to HCl. Significance levels of  0.01 and  0.001 are denoted by ** and ***, respectively (paired t-test). Data are means ± SE (= 6 roots).

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Tests for viability of cells in the OPR

After the experiments, the viability of cells in the OPR was examined by staining them with Evans blue. This stain cannot cross the intact plasma membrane and does not stain living cells. In killed cells lacking an intact plasma membrane, the stain could freely diffuse into the cell to stain the nucleus and cytoplasm dark blue. Unstained cells confirmed that the root segments used for permeability experiments were alive, although a few cells in the epidermis were found to be dead (Figs. 6a,b). Treatment with CuSO4/[K4Fe(CN)6] did not affect the viability of cells (Figs. 6c,d). By contrast, all cells of HCl-killed root segments stained dark blue, proving that they were dead (Figs. 6e,f).

image

Figure 6.  Free-hand longitudinal sections (a, c, e) and cross-sections (b, d, f) of rice root segments taken c. 60 mm from the apex, stained with Evans blue to check the viability of the cells at the end of the experiments. In dead cells, nuclei and cytoplasm were stained blue. (a, b) Following the gas perfusion experiment, only a few epidermal cells were found to be dead (arrowheads). (c, d) Treatment with insoluble crystals did not affect the viability of the cells. Only a few cells in the epidermis were found to be dead, as in controls. (e, f) Dead cells stained blue after 0.1 N HCl treatment (arrowhead). Bars, 50 μm.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In the present study, the gas perfusion technique developed by Kotula & Steudle (2009) was used to measure O2 permeability coefficients of the outer cell layers of roots (POPR) of rice plants grown in either aerated or stagnant, deoxygenated conditions. As far as we are aware, this is the first quantitative comparison of the permeabilities of the peripheral layers to O2 in rice roots and how these change when the OPR becomes modified. The results indicated that, when plants were grown in the stagnant deoxygenated solution, the POPR of all investigated zones was several folds smaller than that of plants grown in the aerated solution. When salt precipitates were formed in cell wall pores in roots of plants grown under aerated conditions, O2 permeability decreased. By contrast, POPR increased in response to the killing of root cells with 0.1 N HCl. The effect of the latter treatment was relatively small in roots in the aerated solution but larger in roots in the stagnant medium, suggesting a significant contribution of respiratory effects in the presence of low POPR. Overall, POPR was strongly affected by the apoplastic barriers in the roots of rice, which lowered oxygen diffusion across the peripheral cell layers.

The lower POPR in plants grown in the stagnant medium as well as the reduction of POPR along the roots of plants grown in both conditions strongly correlated with the development of apoplastic barriers in the OPR. In a quantitative examination, Kotula et al. (2009) showed that the amounts of suberin and lignin increased along the roots of rice plants grown in both aerated and stagnant solutions; however, absolute loads of these polymers were much greater in roots grown under deoxygenated conditions. In agreement with chemical analyses, detailed histochemical studies of the OPR of rice revealed early development of exodermal Casparian bands and suberin lamellae in plants grown in stagnant conditions. In addition to suberization, early lignification of walls of densely packed sclerenchyma was found closer to the root apex in these plants (Kotula et al., 2009). Apparently, apoplastic barriers impeded gas diffusion across the OPR. Suberin, in particular, is known to offer a high resistance to O2 diffusion (De Simone et al., 2003; Soukup et al., 2007). The present study supports these views.

Compared with the diffusion of water, O2 diffusion across the OPR of plants grown in the aerated solution was greater by an order of magnitude. This may result from the fact that, in contrast to the O2 molecule, water has a polar (dipole) structure, tending to reduce its diffusivity in suberized cell walls. Similar differences were found for cuticles (Lendzian, 1982; Lendzian & Kerstiens, 1991). The bulk (hydraulic) water permeability across the OPR of rice grown in an aerated solution was much greater than diffusional water flow (Ranathunge et al., 2004). Modification of the apoplast of the OPR by filling intermicrofibrillar spaces (cell wall pores) with precipitates reduced the diffusional permeability of both water and O2 by c. 20%. By contrast, this treatment caused a massive three- to four-fold reduction of hydraulic conductivity (LpOPR; Ranathunge et al., 2005). This indicated that precipitates affected bulk flows of water much more than diffusive flows of water and oxygen. In contrast to bulk water flow, the diffusion of O2 across outer cell layers ought to be appreciable over the whole inter-cell interface and was not restricted by cell membranes, which are thought to be highly permeable to O2 (Armstrong, 1979; Nobel, 2005). The diffusion of O2 across the OPR should be limited by the existence of apoplastic barriers such as suberin lamellae and Casparian bands. Hence, the OPR allows rather high water flow in the presence of a relatively high resistance to O2. This is achieved through differences in transport mechanisms: there is a bulk flow of water, but the flow of O2 is diffusional in nature (Kotula & Steudle, 2009). This suggested that rice has evolved an optimum balance between water uptake and O2 loss, with high rates of water uptake in the presence of low rates of O2 loss (Ranathunge et al., 2004; Kotula & Steudle, 2009). However, to date, the above conclusion had been reached only for rice grown in aerated conditions. When plants were grown in a stagnant deoxygenated solution, which mimics natural paddy-field conditions, roots showed several-fold greater amounts of suberin and lignin in the OPR and drastically reduced POPR. This means that pores were either completely absent in the apoplastic barriers under these conditions or the diameters of pores were rather small. This situation differs from that in the cuticles, for which the precipitation technique of Ranathunge et al. (2005) has been used to demonstrate the existence of pores (Schreiber et al., 2006). In future studies, the existence or absence of pores in apoplastic barriers of roots of plants grown under deoxygenated conditions needs to be determined by comparing the diffusional and bulk permeabilities of water.

In agreement with Ranathunge et al. (2005), precipitation treatment of roots from aerated hydroponics produced intense brown precipitates at the apical parts of the root segments which gradually faded along the root towards the base. At least in the immature parts of roots, ions could pass through the exodermis and sclerenchyma layer. Because of the development of apoplastic barriers, ion movement across the OPR was reduced in the more mature parts, but was still present. At distances of 50–60 mm, salt precipitates revealed a patchy structure, which may correlate with the maturation of the exodermis (Fig.2d). In cross-sections of salt-treated roots, brown precipitates were localized to the side where K4[Fe(CN)6] was applied, suggesting that Cu2+ rather than [Fe(CN)6]4− passed through the barrier of the OPR in plants grown in aerated conditions. Obviously, the ferrocyanide anion, with its four negative charges, moves across the barrier much more slowly than the positively charged copper cations, probably because of repulsion by the negative fixed charges of the cell wall matrix. In contrast to plants grown in aerated conditions, no precipitates were observed in the roots of plants grown in the deoxygenated medium. This is evidence that well-developed apoplastic barriers in the exodermis and lignified fibre cells impeded ion movement across the OPR. The concentration of Cu2+ and [Fe(CN)6]4 ions in the apoplast remained too low to cause precipitation. In addition, suberin lamellae may block ion transport through membranes. It was already suspected that the suberization/lignification of rice roots may form a barrier that reduces the uptake of ions, such as Fe2+ (Armstrong & Armstrong, 2005). Colmer & Bloom (1998) showed that NH4+ and NO3 uptake in basal regions of roots of O. sativa was c. 30% of that in Zea mays, even when plants were grown in aerated solution. The present results suggest that the barrier to ROL, which is induced during growth under deoxygenated conditions, reduces the transport of ions. Under stagnant conditions, the number and size of pores in the apoplast may also be reduced.

When cell wall pores were blocked with precipitates, the greatest reduction in POPR was observed close to the root apex, where the exodermis was not yet fully developed. The effect was reduced at the more basal parts of the root. The dense precipitates formed in the OPR hindered radial O2 diffusion through the transcellular pathway, which is also done by suberin lamellae in well-developed roots (Ranathunge et al., 2005). However, it should be noted that the precipitates may not have completely blocked the pores in the cell walls. Thus, the reduction in O2 permeability was relatively small. Overall, the effect of salt precipitates was twofold: firstly an additional barrier was created in series to the apoplastic barriers, and secondly pores within the barriers may have been blocked. No reduction in O2 permeability in plants grown in the deoxygenated solution resulted from the lack of salt precipitates in cell wall pores.

At all tested distances and growth conditions, the acid treatment caused a relatively small increase in POPR. The increase may have resulted from cancelling of respiratory O2 consumption by living cells in the OPR. It was possible to estimate the respiration rates of the OPR from the differences in radial O2 flows (in nmol m−2 s−1) between control and HCl-treated roots. Assuming the thickness of the OPR to be 85 μm and the root diameter to be 1 mm, the respiration rate of a 40-mm-long root segment at atmospheric O2 concentration would be 3.9 × 10−11 and 2.6 × 10−11 g O2 mm−3 s−1 for plants grown in aerated and deoxygenated solutions, respectively (for a ref. see Kotula and Steudle, 2009). Although there was a higher respiration rate in plants grown in the aerated solution, the relative increase in POPR after the cells had been killed was smaller than that found in plants grown in the stagnant medium. This suggested that, in plants grown in the aerated solution, the effect of respiration on POPRshould be relatively small in relation to high rates of JO2. This may differ from the situation in plants grown in stagnant conditions, where JO2 is relatively low. The data indicated that respiratory O2 consumption contributed much more in conjunction with a reduced O2 permeability. It may be argued that, in addition to the respiratory activity, the acid treatment may affect the integrity of the apoplastic barrier by causing disintegration of the suberin or lignin polymers, allowing higher O2 diffusion. However, as both suberin and lignin are known to be chemically resistant (Johansen, 1940; Ranathunge et al., 2008), 0.1N HCl probably only had a small effect on them. This conclusion agrees with the findings that, firstly, the POPR of HCl-killed roots grown in the deoxygenated solution was still significantly smaller than that of untreated roots grown in the aerated solution and, secondly, in root segments from plants grown in stagnant conditions, no precipitates were observed after killing with HCl. Overall, the ‘physical resistance’ played a dominant role in impeding O2 loss from rice roots, although respiration may also have an effect, when rates of radial O2 loss were low. The results support the findings of previous studies by Armstrong et al. (2000) and Garthwaite et al. (2008) on roots of Phragmites australis and Hordeum marinum, respectively. In H. marinum, the physical barrier appeared to account for 84% of the reduction and respiratory activity for 16% (Garthwaite et al., 2008).

In conclusion, the quantitative comparison of O2 permeability coefficients across the OPR (POPR) of rice grown in either an aerated or a deoxygenated solution indicated that the formation of apoplastic barriers strongly decreased POPR. Treatment with CuSO4/K4[Fe(CN)6] resulted in the formation of brown precipitates only in the roots of plants grown in the aerated solution, indicating that the apoplast of the OPR has a somewhat porous structure. By contrast, no precipitates were observed in roots of plants grown in the deoxygenated medium. It was concluded that well-developed apoplastic barriers in the exodermis, such as Casparian bands and suberin lamellae, as well as lignified fibre cells, blocked ion movement across the OPR. Even after killing of the root segments of plants grown in stagnant conditions, no brown precipitates were observed. This confirmed that apoplastic barriers had a substantial effect on ion movement. The formation of precipitates in the OPR of aerated plants reduced POPR by 5 to 20%, depending on the development of the apoplastic barriers along the root. Comparison with earlier findings concerning diffusional and hydraulic water permeabilities supports the view that apoplastic barriers provided substantial resistance to the diffusion of O2 across the OPR but allowed high bulk water flow. It appears that, in rice, the OPR allows rather high water flow in the presence of a high resistance to O2, which is an advantage for the plant. Killing of root cells by dilute hydrochloric acid increased POPR by up to 55%. This suggested a significant, although not dominant role of respiratory O2 consumption in living cells of the OPR. The fact that the POPR of HCl-treated roots of plants grown in the deoxygenated solution was still significantly lower than that of untreated aerated plants further supports the view that suberization and/or lignification provides a strong barrier to O2 flow across the OPR of rice.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors are indebted to Professor Lukas Schreiber (University of Bonn) for discussions of the manuscript. We thank Dr Hermann Heilmeier (Technical University Freiberg, Germany) for his valuable help with statistical analysis. KR is grateful to the Alexander-von-Humboldt-Foundation for a postdoctoral scholarship. The technical support of Burkhard Stumpf (University of Bayreuth) is gratefully acknowledged.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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