Assessment of O2 diffusivity across the barrier to radial O2 loss in adventitious roots of Hordeum marinum

Authors

  • Alaina J. Garthwaite,

    1. School of Plant Biology and
    2. Future Farm Industries CRC, Faculty of Natural and Agricultural Sciences, The University of Western Australia, Crawley, 6009 Western Australia;
    Search for more papers by this author
  • William Armstrong,

    1. Department of Biological Sciences, University of Hull, Hull HU6 7RX, UK
    Search for more papers by this author
  • Timothy D. Colmer

    1. School of Plant Biology and
    2. Future Farm Industries CRC, Faculty of Natural and Agricultural Sciences, The University of Western Australia, Crawley, 6009 Western Australia;
    Search for more papers by this author

Author for correspondence:
Timothy D. Colmer
Tel:+61 8 6488 1993
Fax:+61 8 6488 1108
Email: tdcolmer@cyllene.uwa.edu.au

Summary

  • • The subapical regions of wetland plant roots can develop a barrier to radial O2 loss (ROL), but barrier resistance has rarely been quantified in terms of its O2 diffusivity.
  • • Barrier resistance in adventitious roots of the waterlogging-tolerant Hordeum marinum was assessed from measurements of ROL using cylindrical platinum electrodes while either varying shoot O2 partial pressures or cooling the rooting medium. Anatomical features were examined using fluorescence microscopy.
  • • When grown in stagnant agar nutrient solution, a barrier to ROL was induced over the basal half of 100–120-mm-long roots. Autofluorescence in hypodermal cell walls indicated that putative suberin deposition was coincident with barrier expression. Root cooling revealed a significant respiratory component in barrier resistance. Eliminating the respiration effect by manipulating shoot O2 partial pressures revealed an O2 diffusivity for the barrier of 5.96 × 10−7 cm2 s−1, 96% less than that at the apex, which was ≥ 1.59 × 10−5 cm2 s−1.
  • • It is concluded that the ROL barrier is a manifestation of two components acting synergistically: a physical resistance caused principally by secondary cell-wall deposits in the outer hypodermal layer; and respiratory activity in the hypodermal/epidermal layers; with physical resistance being the dominant component.

Introduction

In numerous wetland species, the external cell layers in the subapical regions of adventitious roots develop a very low radial permeability to O2, a barrier to radial O2 loss (ROL) (Armstrong, 1979; Armstrong et al., 2000; Colmer, 2003a). In combination with large volumes of aerenchyma, a barrier to ROL enhances longitudinal O2 diffusion towards the root tip and thus root penetration into waterlogged soils (Armstrong, 1979; Armstrong & Beckett, 1987) that are anoxic and chemically reduced (Ponnamperuma, 1984). ROL near the tip of the main root axis, as well as along any lateral roots, oxidizes the rhizosphere and may protect against phytotoxins (Armstrong, 1979; Jackson & Armstrong, 1999) as well as supporting nitrification (Kirk & Kronzucker, 2005).

In some species, the barrier to ROL is constitutively present in adventitious roots (e.g. Juncus effusus, Echinochloa crus-galli, Schoenoplectus validus) (Visser et al., 2000; McDonald et al., 2001a, 2002) while in others, e.g. Oryza sativa, the barrier to ROL is inducible, forming in adventitious roots subjected to stagnant, deoxygenated conditions, but not (or less so) in roots in aerated conditions (Colmer, 2003b; Colmer et al., 2006). The barrier to ROL is also inducible in a number of wild Hordeum species inhabiting waterlogging-prone areas (Garthwaite et al., 2003). In contrast to these wild Hordeum species, their cultivated relatives (barley, Hordeum vulgare and wheat, Triticum aestivum) are much less tolerant of waterlogging and do not form a barrier to ROL (McDonald et al., 2001a).

Despite the apparent importance of a barrier to ROL for roots of wetland species, little is known about its O2 permeability (Colmer, 2003a), yet there appears to be significant variation among species in the ‘strength’ of the barrier. Our current understanding of variation in radial O2 permeability comes largely from measures of O2 flux obtained with root-sleeving O2 electrodes (summarized by Colmer, 2003a). For example, rates of O2 flux from the basal regions of adventitious roots with a barrier to ROL vary from < 0.2 ng cm−2 root surface min−1 in O. sativa (Colmer, 2003b) to 58 ng cm−2 root surface min−1 in Phalaris aquatica (McDonald et al., 2002). The decline in radial O2 permeability across the exterior of roots with a barrier to ROL has been linked to secondary wall deposits in the outer cell layers: hypodermis, exodermis, and epidermis (Clark & Harris, 1981; De Simone et al., 2003; Armstrong & Armstrong, 2005; Soukup et al., 2007) and coupled to the O2 consumption in the external cell layers, which diminishes O2 loss (Armstrong et al., 2000). In maize roots, this synergism between physical resistance and respiratory activity in the hypodermal/epidermal cell layers is also evident, but more so in roots grown in solid agar than in aerated culture solution (Darwent et al., 2003). The only data available so far on the O2 diffusivity of a ROL barrier are for Phragmites australis (Armstrong et al., 2000), and were deduced from microelectrode-derived O2 profiles across hypodermal/epidermal layers and estimates of ‘root wall’ respiratory demand. These data indicated that respiratory activity in the external cell layers might sometimes play an important part in determining rates of ROL from roots with a barrier.

In the present study, we chose to investigate the O2 permeability of the barrier in adventitious roots of the waterlogging-tolerant grass Hordeum marinum, using cylindrical platinum electrodes for measuring ROL while manipulating internal O2 partial pressures by either cooling roots to 4°C (to reduce respiration), or increasing O2 partial pressure around the shoot to cancel out the effects of any respiratory component. By means of these various manipulations, and in conjunction with internal resistance values calculated from root porosity data, it is possible to reveal whether there is a significant respiratory component, and the value of the nonrespiratory resistance, expressed as a resistance or as an overall O2 diffusion coefficient for the cell-wall layers exterior to the cortical gas spaces (epidermal/hypodermal cell cylinders) that form the barrier. These results are discussed in the context of the anatomy of the external cell layers in adventitious roots of H. marinum when grown in aerated or in stagnant (0.1% w/v agar), deoxygenated nutrient solution. The study demonstrated that putative suberin deposition in the outer hypodermal layer was coincident with reduced O2 diffusivity in basal root zones of plants in stagnant agar nutrient solution, and presented a method using root-sleeving O2 electrodes and manipulative treatments to quantify apparent O2 diffusivity across the external cell layers of roots.

Materials and Methods

Plant materials and culture

Hordeum marinum ssp. gussoneanum (accession H 819, Nordic Gene Bank, Alnarp, Sweden) was used in all experiments.

Seeds (caryopses) were placed on plastic mesh floating on 0.1 strength aerated nutrient solution in darkness in a phytotron at 20 : 15°C (12 h day : night). The composition of the nutrient solution at full strength was (mol m−3): K+ 5.60, Ca2+ 1.50, Mg2+ 0.40, inline image 0.625, inline image 4.375, inline image 1.90, inline image 0.20, Na+ 0.20, inline image 0.10; and the micronutrients (mmol m−3): Cl 50, B 25, Mn 2, Zn 2, Ni 1, Cu 0.5, Mo 0.5, Fe-Na-EDTA 50. The solution also contained 2.5 mol m−3 2-[N-morpholino]ethanesulfonic acid (MES) and the pH was adjusted to 6.5 with KOH (the final K+ concentration as above). All chemicals used were analytical grade.

The seedlings were germinated in darkness as described above for 4 d before being exposed to natural sunlight within the phytotron (located in Perth, Australia, with experiments conducted August–March). Seedlings were given 0.25 strength nutrient solution for an additional 4 d, before being transferred to pots of aerated, full-strength nutrient solution. Each 4.5-dm3 plastic pot initially held five seedlings, with one removed at the time of the initial harvest when treatments were imposed (see below). Plants were held in the lids using flexible polystyrene foam. The pots and lids were covered with aluminium foil to ensure the roots were in darkness.

Root-zone O2 treatments

Experiments were conducted using a common procedure. Fifteen days after germination, an initial harvest of one plant was taken from each replicate pot (leaving four plants per pot); shoot and root dry mass were recorded. Root-zone O2 treatments were initiated immediately after the plants were sampled for the initial harvest. In the pots designated for the aerated treatment, the nutrient solution continued to be bubbled with air. The pots designated for the stagnant treatment were initially given a ‘hypoxic pre-treatment’ whereby pots were flushed with N2 to reduce the O2 concentration in solution to approx. 0.03 mol m-3, then left unstirred overnight. The following day, the nutrient solution in the stagnant pots was replaced with deoxygenated nutrient solution containing 0.1% (w/v) agar; the agar prevented convective movements in the solution (Wiengweera et al., 1997). Solutions in the aerated pots were also renewed at this time (solution without agar). All solutions were renewed every 7 d. Treatments were maintained for 21–28 d, with measurements/harvests during the final 7 d (exact times depending on the experiment; see figures and tables).

Growth parameters

Plants were harvested after 24–25 d treatment. The numbers of stems and adventitious roots, and the longest adventitious root per plant, were recorded. The dry mass of shoots and adventitious and seminal roots were measured. The whole-plant relative growth rate (RGR) was calculated from the dry mass data taken at initial and final harvests, using the formula given by Venus & Causton (1979).

Adventitious root porosity

Porosity (percentage gas volume per root volume) was measured for adventitious roots of plants grown in aerated or stagnant nutrient solution, using the method of Raskin (1983) with equations as modified by Thomson et al. (1990). Adventitious roots 90–140 mm long were cut into 40–50-mm segments and a subsample of approx. 0.8 g was used.

Anatomy of the external cell layers

Fresh sections were taken using a handheld razor to compare the external cell layers (exterior to the aerenchyma) in adventitious roots from H. marinum grown in aerated or in stagnant nutrient solution. The transverse sections were taken 70 mm behind the tip, where the barrier to ROL was induced in stagnantly treated roots. Sections were left unstained and mounted in tapwater, and viewed under blue fluorescence (450–490 nm) using a microscope (Zeiss Axiophot Photomicroscope, Germany). Filter combinations used for fluorescence were UV exciter and UV barrier. Epifluorescent optics show up lignified and suberized walls: smaller molecular-weight phenolic compounds or lipid complexes (O’Brien & McCully, 1981). Photomicrographs were recorded in colour using Agfachrome RSX 100 slide film. Tranverse sections were also taken at other positions behind the root tip and viewed with white light under the microscope, to assess the dimensions of various tissues.

Rates of radial O2 loss along intact adventitious roots when in an O2-free medium

Plants were sealed into rubber lids fitted in 100 × 100 × 170-mm Perspex chambers filled with an O2-free solution containing 0.1% (w/v) agar, 0.50 mol m−3 CaSO4 and 5.0 mol m−3 KCl. The intact root system was immersed in the O2-free solution while the shoot remained in air. A root-sleeving O2 electrode (height 5 mm; internal diameter 2.25 mm) (Armstrong & Wright, 1975; Armstrong, 1979, 1994), fitted with root-centralizing guides, was placed around a selected adventitious root. Root lengths ranged from 100 to 120 mm. The equilibrium flux of O2 from root to electrode was measured with the centre of the electrode positioned at 5, 10, 20, 30, 40, 60 and 80 mm behind the tip. The first measurements were taken at least 2 h after plants were transferred into the system. All measurements were taken at 20°C, with photon-flux density at shoot height of 100 µmol m−2 s−1.

When in an O2-free root medium, ROL at any position along a root depends on: the adjacent concentration of O2 within the root; the permeability of the outer tissues of the root to O2 diffusion; and the respiratory demand in the outer layers. For a root dependent on internal diffusion of O2 from the shoots as the only source, the concentration of O2 at a given position within the root is determined by the amount of gas space along the diffusion path, O2 consumption rates along the path, and the distance from the most basal O2 entry point in the shoots. Oxygen concentrations within the cortical gas spaces will decline in a curvilinear fashion from the root base to the apex (Armstrong, 1979). For roots without a barrier to ROL, the ROL also decreases with distance from the root–shoot junction as the concentration of O2 inside the root declines towards the apex (Armstrong, 1979). For roots with a barrier to ROL, ROL is typically very low in the basal regions of the main axis towards the root–shoot junction and relatively high near the apex, even though internal O2 declines towards the apex (as shown for rice; Armstrong, 1971).

Response to low temperature: effects of cooling to 4°C on respiratory rate and ROL

Plants were raised at 20 : 15°C day : night in aerated solution for 15 d, then in aerated or in stagnant agar nutrient solution for an additional 21–24 d. The effect on root O2 consumption of decreasing the temperature from 20 to 4°C was evaluated on segments of excised roots, and the consequences of reduced respiration for ROL and barrier expression were then assessed by taking ROL measurements along intact adventitious roots in an O2-free medium at 20 and at 4°C.

Rates of O2 consumption by adventitious roots were measured polarographically using the technique and apparatus described by Lambers & Steingröver (1978). Adventitious root segments, 20–40 mm from the root tip and of known diameter, were excised from H. marinum grown in aerated or stagnant nutrient solution. This tissue was chosen as the cells here would be fully expanded, and the barrier against ROL is not yet formed in this region even in stagnantly grown H. marinum. This eliminated the possibility that differences in impedance to O2 uptake might influence the measurements. Measurements were commenced in an air-saturated nutrient solution, having the same composition as the culture solution given above, at 20°C ± 0.5 or 4°C ± 1.0°C.

The effect of excision and subsequent ‘ageing’ (or ‘healing’; Gronewald et al., 1979) on O2 consumption was tested in excised tissues. O2 consumption rates were measured in segments immediately after excision, and 3 h after excision following healing in aerated nutrient solution with 20 mol m−3 glucose also added during this period. Healing did not affect O2 consumption rates. O2 consumption measurements were therefore completed within 30 min of excision. From the rate of O2 depletion of the medium and the volume of the tissues, a respiratory rate per unit volume of root was determined.

Radial O2 losses along intact adventitious roots were initially measured at 20°C at 5, 10, 20, 30, 40, 60 and 80 mm behind the tip to determine the ROL profiles for the roots. The Perspex chambers containing the plants were then submerged in an ice–water slurry to reduce the temperature of the solution in the chambers to 4°C. Measurements of ROL were then taken on the same adventitious roots at the lower temperature, at 5 and 80 mm behind the tip. The temperature was monitored throughout the measurements and maintained at 4 ± 1°C by adding or removing ice from the water bath around chambers.

Analyses of the effects of cooling on radial O2 loss

Based on certain assumptions, the O2 partial pressure inside the cortical gas space of roots aerated by diffusion from the shoot can be predicted for any point along the root from the following equation (Greenwood, 1967; equation 30 of Armstrong, 1979):

Cx = Co – [Mx (2l – x)]/2D0ɛ(Eqn 1)

where Cx = internal O2 concentration (g cm−3) at distance x from root base (cm), Co = external (shoot) O2 concentration (g cm−3) (273 × 10−6 g cm−3 in moist air at 20°C; 293 × 10−6 g cm−3 at 4°C), M = O2 consumption rate (g O2 cm−3 s−1), l = total root length (cm), D0 = diffusion coefficient of O2 in air (cm2 s−1) (0.201 cm2 s−1 at 20°C; 0.184 cm2 s−1 at 4°C), and ɛ = fractional porosity of the root (proportion of total volume). O2 consumption rates and fractional porosity values were obtained as described earlier. The assumptions are that respiratory demand and porosity are distributed uniformly along the root and that respiration is unaffected by O2 concentration until the point of anoxia. As the roots did not reach anoxia and were aerenchymatous for much of their length, it was assumed that by using average values for porosity and respiratory demand, the data for H. marinum would approximately satisfy these assumptions. Support for the applicability of the model for predicting internal O2 partial pressures comes from the close agreement of the model when predicting lengths of roots dependent on internal O2 when in an O2-free rooting medium (McDonald et al., 2001b). The exact profile of internal O2 within roots, however, might deviate from the situation described in equation 1 (Armstrong, 1979; Armstrong et al., 2000; Darwent et al., 2003).

For the stagnantly grown plants, equation 1 was used to predict the O2 partial pressures in the adventitious roots at 5 and 80 mm behind the tip when at the two temperatures; the results were used for comparison with the corresponding effects of temperature on ROL.

Estimating the diffusivity of O2 across the external cell layers

To evaluate the physical resistance of any barrier to ROL (to obtain an overall measure of O2 diffusivity across the hypodermal/epidermal layers), it is necessary to eliminate any effect of respiratory O2 consumption across the layers. To this end, we adopted a procedure that was a modification of the two-point gas-mixture method suggested by Armstrong & Wright (1975) for estimating root pore-space resistance in intact plants. The method makes no attempt to curtail respiration, but eliminates its influence by combining two equations. These describe ROL in terms of root pore-space resistance, hypodermal/epidermal (external cell layers) resistance, liquid shell resistance between root and electrode surface, and respiration, at each of two shoot source O2 concentrations, both chosen to be sufficiently high to saturate root respiration. It has been shown previously that the critical O2 pressure for respiration at the cellular level in intact roots can be very low (≤ 1 kPa: Greenwood, 1967; Armstrong & Gaynard, 1976), hence shoot O2 concentrations were chosen to maintain O2 levels at all times substantially higher than this.

The two equations are:

ROL1 = [Ci1/(RPS + RECL + RLS)] – Resp  (Eqn 2)
ROL2 = [Ci2/(RPS + RECL + RLS)] – Resp  (Eqn 3)

where Ci1 = shoot O2 concentration for treatment 1 (e.g. shoots in 31.5% O2) (4.09 × 10−4 g cm−3) and Ci2 = shoot O2 concentration for treatment 2 (e.g. shoots in 100% O2) (1.30 × 10−3 g cm−3), ROL1 and ROL2 are the radial O2 diffusion rates (ng s−1) corresponding with these treatments (equation 6), RECL = resistance to O2 diffusion across the external cell layers (s cm−3), RLS = resistance to O2 diffusion in the liquid agar between the root surface and the electrode surface (equation 8), RPS is the pore-space resistance (to O2 diffusion) of the root between the shoot and the position at which ROL1 and ROL2 were measured (for RPS see equation 7) and Resp. is respiration.

As the respiration is the same in both equations, then:

[Ci1/(RPS + RECL + RLS)] – ROL1 = [Ci2/(RPS + RECL + RLS)]                – ROL2
C2 − C1/(ROL2 – ROL1) = RPS + RECL + RLS (Eqn 4)
RECL = [C2 − C1/ROL2 – ROL1] – (RPS + RLS) (Eqn 5)

Data for the right-hand side of equation 5 are found as follows:

  • (i) ROL1 and ROL2, the data from the root-sleeving O2 electrodes, are expressed as diffusion rate (ng s−1) using equation 6 (Armstrong, 1979):

ROL = (4.974 × (Idiff – Iresid))/60 (Eqn 6)

where Idiff = equilibrium current (µA) generated by electrolytic O2 reduction at the electrode surface, and Iresid = residual current (close to zero) where the root is replaced by a glass rod of similar dimensions to the root.

RPS = l/D0ɛτAr(Eqn 7)

where l = apparent length of the diffusion path between the shoot base and the position along the root at which ROL1 and ROL2 are measured (cm); D0 = the diffusion coefficient of O2 in air (0.201 cm2 s−1 at 20°C); ɛ = the fractional porosity of the root between the shoot and the position at which ROL1 and ROL2 are measured; Ar = the mean cross-sectional area of the root between the shoot and the position at which ROL1 and ROL2 are measured (cm2); τ = tortuosity factor (~1). RPS ignores resistance to longitudinal O2 diffusion resulting from the stem or the root–shoot junction (RRSJ). However, it should be noted that where the barrier is greatest, RECL will be so large as to be many times greater than RPS and/or RRSJ, and any error from only estimating these parameters can be all but disregarded.

  • (iii) RLS, the resistance to O2 diffusion in the liquid agar between the root surface and the electrode surface (s cm−3), adjusted for relativity to RPS by incorporating a correction for gas:liquid phase concentration differences, is calculated using equation 8 (Armstrong & Wright, 1975):

RLS = ((rr × ln(re/rr))/(Dw × Ars))       × (Cgasphase/Cwaterphase) (Eqn 8)

where rr = radius of the root (cm), re = internal radius of the root-sleeving electrode (cm), Dw = diffusion coefficient for O2 in water at measurement temperature = 2.10 × 10−5 cm2 s−1 at 20°C, Ars = surface area of the root within the electrode (cm2), Cgasphase = atmospheric gas-phase O2 concentration at 20°C (272.8 × 10−6 g cm−3) and Cwaterphase = concentration of O2 in air-saturated water at 20°C (9.08 × 10−6 g cm−3).

The diffusive resistance of the external cell layers (RECL) can now be determined by substituting appropriate values for C1 and C2 into equation 5, together with the data obtained from equations 6–8. The diffusive resistance RECL can also be defined using the following equation:

RECL = (roec × ln(rr/roec)/DECL × Aoec)       × (Cgasphase/Cwaterphase) (Eqn 9)

where roec = radius to the outer edge of the porous cortex in the root (cm), rr = radius of the root (cm), DECL = diffusivity of O2 across the external cell layers (cm2 s−1), and Aoec = inner surface area of external cell layers on radius roec (cm2). On rearrangement, the overall O2 diffusion coefficient, DECL, for the outer cell layers (inclusive of the barrier) is given by:

DECL = ((roec × ln(rr/roec))/(RECL × Aoec))         × (Cgasphase/Cwaterphase) (Eqn 10)

The experimental procedure for determining DECL was as follows. Measurements were on adventitious roots of H. marinum previously grown in stagnant solution (with barrier to ROL induced). The O2 partial pressure within the root gas space was increased in steps by raising O2 partial pressure around the shoots. The quasi-steady-state ROL was recorded after each step. Initially, ROL was measured for intact adventitious roots with the shoots in air at 5, 10, 20, 30, 40, 60 and 80 mm behind the root tip, as described earlier. A Perspex hood with an inlet and an outlet tube was then placed over the shoot of each plant and sealed at the base using a plug of wet cotton wool. Oxygen and air were mixed using flow meters and the partial pressure of O2 in the gas around the shoot was increased from that in air (approx. 21%) to 26.3% (= 1.25 × 21%); 31.5% (= 1.5 × 21%); 42% (= 2 × 21%); and finally to 100%. At each new quasi-steady-state, ROL was measured at 5 and 80 mm behind the root tip.

With the shoot O2 concentration and pore-space resistance of the root known, and the flux rate for ROL measured for two known shoot O2 partial pressures (as above), the resistance (RECL) to O2 movement across the cell layers exterior to the aerenchyma at the position of ROL measurements is calculated from equation 5. Once RECL resistance is known, a value for O2 conductance, or the diffusivity of O2 across the outer cell layers (DECL) can be determined from equation 10. An alternative procedure for deriving RECL and DECL from the data for the gas-mixture method depends on the cessation of O2 consumption in the roots when shoot O2 concentration is zero. If, for the 5- and 80-mm positions on the root (but only for ROL values > 0), the ROL vs shoot O2 concentration data are plotted and the regression lines for the two plots extended to the y-axis (i.e. to zero shoot O2 concentration, e.g. Fig. 3b), the negative ROL values at the intercepts represent the amounts by which ROL (with a change in sign from minus to plus) would have been raised if there had been no root O2 consumption. For each of the two sampling positions, 80 and 5 mm, the total effective physical diffusive resistance from shoot (via the diffusion path within the root) to the electrode surface RT can be expressed as follows. At 80 mm, for example:

Figure 3.

(a) Profile of radial O2 loss (ROL) along intact adventitious roots of Hordeum marinum in an O2-free medium with shoots in air, and the effect of stepwise increases in shoot O2 partial pressure on ROL from 5 or 80 mm behind the root tip. (b) Regression of actual ROL against calculated O2 partial pressures inside root cortical gas spaces for stagnantly treated H. marinum exposed to stepwise increases in shoot O2 partial pressure. Dashed lines in (b) show the predicted relationships, in the absence of respiration, between ROL (at 5 and 80 mm behind the root tip) and shoot O2 partial pressure. Before ROL measurements, plants were raised to the approx. 2.5-leaf stage in aerated solution, then grown in stagnant, deoxygenated nutrient solution for a further 25 d. The percentage of O2 in the gas phase surrounding the shoot was 21% (control), 26.3, 31.5, 42 or 100%. Measurements taken at 20°C for intact roots 100–120 mm long. Values are means ± SE (n = 4). For ROL data, P(shoot O2 partial pressure) < 0.001; LSD = 71. O2 partial pressures inside root cortical gas spaces were calculated as described in Materials and Methods, using equation 1 (Armstrong, 1979).

RT(80) = ΔC/(measured ROL(80) + numerical ROL     value at intercept) (Eqn 11)

where ΔC is the O2 concentration difference between the O2 concentration around the shoot and that at the electrode surface. As the electrode imposes zero O2 at its surface, the value of ΔC is numerically that of the shoot O2 concentration. Provided the internal O2 concentrations within the root were above the respiratory critical O2 pressure, and that the plateau potentials for electrolytic O2 consumption had been set correctly, the total effective resistances derived at each imposed shoot O2 concentration should be the same for any particular sampling position. RECL can now be derived as follows. At 80 mm, for example:

RECL(80) = RT(80) – (RPS(80)+RLS(80)) (Eqn 12)

and DECL is obtained using equation 10 as before.

Statistics

anova was performed to evaluate treatment effects. For the ROL data, roots were treated as plots and the distance from the root tip (where measurements were taken) as subplots in the anova. Means are presented in tables and figures accompanied by standard errors, and where appropriate compared using the LSD (P = 0.05) (genstat, 6th edn, http://www.vsn-intl.com).

Results and Discussion

Growth in aerated or stagnant nutrient solution

Growth in the stagnant solution reduced the shoot dry mass by 42%, seminal root dry mass by 73%, and adventitious root dry mass by 33% (Table 1). Whole-plant RGR was reduced by 14% for plants in stagnant solution (Table 1). By contrast, stem number was not significantly affected by the stagnant solution treatment, but the number of adventitious roots produced increased by 26% in the stagnant solution (Table 1). Adventitious root porosity was higher when grown in stagnant, compared with aerated, nutrient solution (Table 1). Nevertheless, the maximum length of the main axis of adventitious roots in stagnant solution was 275 mm, being about half that in aerated solution (Table 1). This maximum root length in deoxygenated stagnant agar (O2-deficient medium) would have been determined largely by the capacity for internal O2 diffusion (cf. Armstrong, 1979). Interestingly, the length of 275 mm exceeds that expected for roots solely reliant on internal O2 diffusion (Armstrong, 1979) with the porosity (Table 1) and respiratory consumption (see below) as measured for 100–120-mm roots, indicating that in these older roots, porosity must have increased (e.g. to 40%) and/or respiration must have declined (e.g. by 50%) (examples of percentage change are based on calculations, data not shown).

Table 1.  Growth and root porosity of Hordeum marinum in aerated or stagnant deoxygenated nutrient solution
ParameterTreatment
AeratedStagnantPercentage change
  1. Plants were raised to the approx. 2.5-leaf stage in aerated nutrient solution before being transferred to aerated or stagnant deoxygenated nutrient solution for a further 24–25 d. Values are means ± SE (n = 4), where each replicate is a single plant. Significant differences between treatment means are indicated by: *, P < 0.05; **, P < 0.01; ns, not significant.

Shoot dry mass (g)3.13 ± 0.161.81 ± 0.09*−42
Adventitious root dry mass (g)1.03 ± 0.150.69 ± 0.02*−33
Seminal root dry mass (g)0.18 ± 0.050.05 ± 0.02*−73
Whole-plant relative growth rate (mg g−1 d−1)185 ± 3159 ± 2**−14
Stem number36 ± 3.531 ± 2.6ns
Adventitious root number68 ± 5.286 ± 2.3*+26
Longest adventitious root (mm)595 ± 13275 ± 7**−54
Adventitious root porosity (%)14.0 ± 3.619.8 ± 4.3*+41

Anatomy of external cell layers in adventitious roots

In the stagnantly treated roots, the cell layers external to the cortical gas spaces comprised an epidermis, an outer hypodermal layer, and an inner hypodermal layer. The one remaining outer layer of the cortical cells is not included in what we refer to as the external cell layers, as the intercellular gas spaces between these cells were relatively large. The radius to the outer edge of the porous cortex in the root (roec) was measured on three transverse sections taken 80 mm behind the root tip. On average, this distance was 85% of the root radius, with the external cell layers making up 15% of the root radius (15% of 0.4095 mm; distance across external cell layers = 61 µm). Cell packing in the external cell layers appeared to be tighter than in the mid-cortex. When grown in the stagnant solution, the stele occupied 26% of the total root cross-sectional area at 80 mm behind the root tip.

Under epifluorescence conditions, the hypodermal cell layers of adventitious roots of stagnantly treated plants fluoresced white (Fig. 1b), a feature largely absent in the same cell layers in roots of plants grown in aerated solution (Fig. 1a). Examination at higher magnification (stagnantly treated roots; Fig. 1c) showed the outermost layer of the hypodermis fluorescing white on the outer tangential walls and partially on the anticlinal walls; and the second-outermost layer of the hypodermis also fluorescing. The fluorescence suggests that these parts of the cell walls were lignified and/or contained suberin or other phenolic compounds. By comparison, the hypodermal cell layers in roots of aerated plants (Fig. 1a) did not show fluorescence, indicating that the lignin or suberin deposits present in the stagnantly treated roots were absent in the aerated roots.

Figure 1.

Fresh transverse sections of adventitious roots of Hordeum marinum grown in aerated or stagnant deoxygenated nutrient solution. Cross-sections were taken 70 mm behind the root tip, left unstained and photographed using epifluorescence. Plants used were raised to approx. 2.5-leaf stage in aerated nutrient solution before being transferred to aerated or stagnant deoxygenated nutrient solution for a further 28 d.

Rates of radial O2 loss along intact adventitious roots when in an O2-free medium

Subapically, a substantial barrier to ROL was induced in adventitious roots of stagnantly treated H. marinum (Fig. 2). Near to the base of these roots, ROL was low but increased sharply towards the root tip. By contrast, for roots previously in aerated solution, ROL was highest at the base of the root and, although ROL was relatively constant along these basal zones (indicative of some degree of barrier formation), it then declined towards the root tip. If the aerated roots had had no barrier in their basal parts, the ROL profile would have shown an uninterrupted increase from apex to base.

Figure 2.

Profiles of radial O2 loss (ROL) along intact adventitious roots in an O2-free medium with shoots in air for Hordeum marinum grown in aerated or stagnant deoxygenated nutrient solution. Before measurements, plants were raised to the approx. 2.5-leaf stage in aerated solution and then grown in either aerated or stagnant, deoxygenated nutrient solution for a further 21–24 d. Measurements were taken at 20°C using intact roots 100–120 mm long. Values are means ± SE (n = 3).

Rates of ROL at 80 mm behind the tip of H. marinum roots with the barrier to ROL were quite variable; replicates ranged from 0.38 to 4.8 ng cm−2 root surface min−1. Similar variability in basal ROL rates has previously been found for roots of H. marinum with the barrier to ROL induced (4.6 ± 4.2 ng cm−2 root surface min−1, McDonald et al., 2001a; 4.8 ± 1.5 ng cm−2 root surface min−1, Garthwaite et al., 2003). In addition, localized regions of relatively high ROL have been recorded in the basal zones of P. australis and O. sativa roots where the barrier to ROL was present (Armstrong, 1971; Armstrong et al., 2000). In O. sativa, these O2-permeable ‘windows’ in the barrier to ROL were associated with the presence of developing but nonemergent laterals in the pericycle and cortex, and thin-walled sections of exodermal cells (Armstrong, 1971). In P. australis, these ‘window-like’ regions were also related to passage cells in the exodermal Casparian bands (Armstrong et al., 2000). It is not known whether the observed variability in ROL from basal zones of roots of H. marinum coincides with the development of lateral roots or passage regions in the secondary thickening of the external cell layers.

Effect of low temperature on adventitious root O2-consumption rates and radial O2 loss

Oxygen consumption rates by root segments in an air-saturated medium were not influenced by previous growing conditions, regardless of the basis of expression of respiration (total soluble protein, fresh mass or volume; data not shown). Root O2-consumption rates were reduced from 101.5 ± 19 at 20°C to 30 ± 13 ng cm−3 s−1 at 4°C. That is, 4°C was not sufficient to suppress respiration completely, and the response to temperature demonstrated a Q10 of c. 2.

The effects of low temperature on ROL and the corresponding predicted internal O2 partial pressures at 5 and 80 mm from the apex of stagnantly grown roots are shown in Table 2. It should be noted that, hypothetically, if there had initially been no respiratory activity in the root at 20°C, the lowering of the temperature would have resulted in a lowering of ROL as rates of diffusion decrease (Armstrong & Drew, 2002) and, if a root were of sufficiently high porosity, some lowering of ROL could occur even if respiratory activity was reduced by cooling. At both positions on the H. marinum roots, however, the ROL was increased by the lowering of the temperature from 20 to 4°C. This is clear evidence of reduced respiratory demand in the root resulting in a significant raising of internal O2 partial pressures at the lower temperature. The predicted factorial rise in cortical gas-space O2 partial pressure at 5 mm behind the tip was 2.47 and the corresponding factorial increase in the ROL at this position was 2.72. In the barrier region 80 mm behind the tip, the predicted factorial rise in cortical O2 partial pressure was only 1.44, but the factorial rise in ROL was more than double this value (3.42).

Table 2.  Effect of low temperature on radial O2 loss (ROL) from intact adventitious roots of Hordeum marinum in an O2-free, stagnant solution, with shoots in air and predicted O2 partial pressures inside the cortical gas spaces
 5 mm80 mm
20°C4°CFactorial change20°C4°CFactorial change
  1. Before ROL measurements, plants were raised to the approx. 2.5-leaf stage in aerated solution, then grown in stagnant, deoxygenated nutrient solution for 21–24 d. Measurements were taken at 20 and 4°C, 5 and 80 mm behind the tip of roots 100–120 mm in length. ROL values are means ± SE (n = 3). Internal O2 partial pressures were calculated as described in the Materials and Methods section, using equation 1 (Armstrong, 1979).

ROL (ng cm−2 min−1)54.9 ± 4149.6 ± 132.721.54 ± 1.155.0 ± 0.963.25
Internal O2 partial pressure (kPa)6.716.62.4712.818.51.45

If ROL through the external cell layers had been dependent only on the diffusive resistance of these layers to O2, ROL and cortical O2 partial pressure should have shown similar factorial increases on cooling. However, in both positions the factorial increase in ROL exceeded that of the corresponding cortical O2 partial pressure. This indicates some depletion in potential ROL at 20°C due to O2 consumption within the external cell layers. At 80 mm, the results indicate that cooling to 4°C had removed a significant respiratory component to apparent resistance of the barrier that had been operating at 20°C. The overall effect of epidermal/hypodermal cell respiration on ROL at 5 mm appears to have been significantly less than at 80 mm, but this does not mean that respiratory activity in the external cell layers was lower at 5 than at 80 mm. Rather, this result suggests that the physical diffusive resistance through the external cell layers at 5 mm was less than at 80 mm.

Estimating diffusivity of O2 across the external cell layers

The gas-mixture method was applied to the stagnantly grown H. marinum plants with ROL from intact adventitious roots being sampled at the 5 and 80 mm positions behind the tip. Increasing the O2 partial pressure around the shoots was accompanied by concurrent increases in ROL at both positions (Fig. 3a,b). The presence of the barrier ensured that ROL from near the base of the root remained significantly lower than that from near the root tip, but the ROL values near the apex indicated clearly that O2 partial pressures throughout the roots were above the critical O2 pressure for respiration at the various shoot O2 concentrations. The linear increases in ROL with shoot O2 partial pressure 5 mm behind the tip (Fig. 3b) are also indicative of this (Armstrong & Gaynard, 1976).

Extrapolating the regressions for both positions to intercept the y-axis (zero shoot O2 partial pressure and hence zero respiration) should provide ROL values that represent the amounts by which respiration in the root (entire respiratory consumption, not just that in the external cell layers) had been reducing ROL at the two positions over the range of applied shoot O2 partial pressures: by 168 ng cm−2 min−1 (0.32 ng s−1) at the 5 mm position, and 45 ng cm−2 min−1 (0.0965 ng s−1) at the 80 mm position (Fig. 3b). If these values are added to the observed values of ROL, the new plots (dashed lines, Fig. 3b) would lie above and parallel to the original plots, but would intercept at the origin and represent the ROL vs shoot O2 partial pressure in the absence of any respiration. Applying equation 10 then reveals RT (physical resistance) values of approx. 6.92 × 105 s cm−3 for the 5 mm position and 30.2 × 105 s cm−3 at 80 mm behind the tip. If the liquid shell resistance (RLS) and pore-space resistance (RPS) through the cortical gas spaces are subtracted from these RT values (equation 12), we obtain values for the apparent resistance of the external wall layers (RECL) at 5 and 80 mm of 1.08 × 105 and 25.42 × 105 s cm−3, respectively. Ignoring any possible root–shoot junction resistance, and applying equation 10 (with an external cell layer thickness of 60 µm), yields apparent overall O2 diffusivity values for the external cell layers (DECL) at 5 and 80 mm behind the tip of 1.59 × 10−5 and 5.96 × 10−7 cm2 s−1, respectively. Thus barrier formation produced an approx. 96% (27-fold) reduction in effective O2 diffusivity across the external cell layers of the roots.

The results for the 80 mm position (region with an ROL barrier) are likely to be the more accurate as, at this position, the value of root pore-space resistance (RPS) is very much smaller relative to RECL than it is at 5 mm. Similarly, any reasonable estimate of root–shoot junction resistance will have much less influence on RECL and DECL at 80 mm than at 5 mm behind the root tip. Although the porosity of the space connection across root–shoot junctions is usually very much less than that of any aerenchymatous root base, the path length is very short, so this component of the overall resistance would be comparatively small. It is unlikely that the junction resistance would have been > 0.26 × 105 s cm−3, as the predicted DECL at 5 mm would then have equalled the diffusivity for O2 in water (2.1 × 10−5 cm2 s−1, a 32% increase). A resistance of 0.26 × 105 s cm−3 would require a porosity of < 2% for the junction, a path length of 5 mm, and junction diameter equal to that of the root base. However, this root–shoot junction resistance would only change our predicted value of DECL at the 80 mm position by 1.04%, to 6.02 × 10−7 cm2 s−1.

The study has also confirmed that respiratory O2 consumption in conjunction with the reduced permeability plays a significant part in determining the levels of ROL in these barrier zones, and we have been able to reveal the extent to which respiratory demand in the root and external cell layers had influenced ROL. With the shoot in 100% O2, the extrapolated plot for the 80 mm position in Fig. 2(b) shows that, if at 20°C all respiration in the root had ceased, the ROL would have risen from 156 to 201 ng cm−2 min−1. If we assume the derived O2 diffusivity value of 5.96 × 10−7 cm2 s−1, we can calculate that the latter figure equates to an internal O2 partial pressure of approx. 100% (the same as round the shoot – to be expected if there is no respiration and very little leakage). In the absence of a barrier (diffusivity ≥ 1.59 × 10−5 cm2 s−1, as near the apex), with no respiratory activity in the external cell layers and at this internal O2 partial pressure, we can predict that ROL would be approx. 1090 ng cm−2 min−1. We can tentatively deduce, therefore, that at the 80 mm position along the H. marinum roots, the physical nature of the barrier is accounting for approx. 95% of the ROL reduction and the respiratory activity for approx. 5%, thus the total fall in ROL due to respiration and barrier is 1090 – 156 = 934 ng cm−2 min−1, and the fall due to the barrier alone is 1090 – 201 = 889 ng cm−2 min−1. Therefore, as proportions, the barrier alone contributes 889/934 or approx. 95% of the fall in ROL, whereas respiration contributes (201 – 156)/934 or approx. 5%. This perception of relative proportions will change, however, depending on what O2 partial pressure there is around the shoot. With 42% O2 inside the root at the 80 mm position, the respective ROLs are 40 (barrier + respiration), 85 (barrier, no respiration) and 458 ng cm−2 min−1 (no barrier, no respiration). The physical nature of the barrier then appears to account for approx. 89% of the ROL reduction, and the respiratory activity approx. 11%. With 21% O2, the barrier per se appears to account for 84% of the reduction, and respiratory activity approx. 16%. It should be noted, however, that in the absence of a physical barrier, any reduction in ROL due to wall respiration would be very much less. For example, with 42% internal O2 at the 80 mm position, a pre-barrier figure of DECL 1.59 × 10−5 cm2 s−1 and a respiratory demand of 100 ng cm−3 s−1 in the root wall, we calculate (equation 1 in Armstrong & Webb, 1985) that the fall in ROL due to the respiration in the wall would amount to only 6 ng cm−2 s−1 (a fall in ROL of only 1.3%).

Functionally, in terms of ROL, barrier formation will conserve the internal O2 supply from root base to apex, and this might be particularly valuable to enhance O2 diffusion to the apex in roots with moderate gas-filled porosity (McDonald et al., 2001a; Colmer, 2003a). Moreover, O2 loss from apical regions into permanently flooded soils helps alleviate the reducing conditions: ROL precipitates potential phytotoxins such as FeII external to the root, and can reduce sulphide intake similarly by direct oxidation external to the root or by precipitation as FeS (Armstrong, 1979).

Perspectives and conclusions

This study has quantified the influence of barrier formation on ROL from roots of H. marinum, and in particular has provided quantitative measures of apparent O2 permeability across the external cell layers (exterior to cortical gas spaces). In plants grown in stagnant agar nutrient solution, the apparent permeability for O2 diffusion across the external cell layers of adventitious roots, expressed as diffusion coefficients, was reduced on average at least 27-fold; from ≥ 1.59 × 10−5 cm2 s−1 near the root tip, where ROL rates were relatively high, to 5.96 × 10−7 cm2 s−1 in the basal regions, which had very low rates of ROL. The respective permeability coefficients are 29 × 10−4 and 1.08 × 10−4 cm s−1. A diffusivity ≥ 1.59 × 10−5 cm2 s−1 in the apical region is relatively high (the diffusion coefficient of O2 in water at 20°C is 2.1 × 10−5 cm2 s−1). It might indicate some loss of permeability, relative to that in water, but as there are the tangential walls of at least three cell layers in the diffusion path, and not all cells will be fully vacuolated, any permeability loss must be slight at this stage.

The strong reduction in permeability in the barrier zone appears to coincide with secondary wall deposits, particularly in the outer tangential walls of the hypodermis (Fig. 1). These appear to contain small molecular-weight phenolic compounds or lipid complexes (O’Brien & McCully, 1981), such as are found in suberin or lignin. Suberin, in particular, is known to offer high resistance to O2 diffusion (De Simone et al., 2003; Soukup et al., 2007). As the deposits of suberin are obvious in some walls and not in others, resistance to O2 diffusion across the external cell layers of roots of H. marinum is unlikely to be uniform: thus the O2 diffusivity across the outer hypodermal cell layer could be significantly lower than the values we have calculated, which embrace the entire outer layers. If the bulk of the physical resistance resides in the inner and outer tangential walls of the hypodermis, and these have a notional thickness of 4 µm, then at the 80 mm position, if we subtract the resistance of water path through cell lumina of the external cell layers and that of the nonsuberized walls from the total resistance of the external cell layer path, we obtain a notional value of O2 diffusion coefficient for the suberized walls per se of approx. 1.36 × 10−9 cm2 s−1 and a permeability coefficient of approx. 3.4 × 10−6 cm s−1.

The results presented here for roots of H. marinum support the suggestion of Armstrong et al. (2000) that low ROL from basal regions of roots of P. australis results from both a physical impedance to O2 diffusion and respiratory consumption of O2 across the epidermal–hypodermal cell layers. The present study of H. marinum indicates that physical resistance is the dominant factor impeding O2 loss from the roots, but that respiration in the external cell layers contributes to low rates of ROL by mopping up leakages. The physical resistance resulting in low O2 permeability across the root exterior was associated with putative lignin and/or suberin deposits in the walls of the hypodermis, found only in the roots of stagnantly treated H. marinum. The cell-wall deposits of lignin or suberin in the basal zones of roots of H. marinum grown in stagnant solution were not present near the tips that were highly permeable to O2, nor were these observed in basal zones of roots grown in aerated solution (roots without a barrier to ROL). The extent to which suberin and other deposits may eventually totally restrict the release of O2 from roots without some synergistic effect of respiratory O2 demand in the external cell layers remains to be discovered. If the outer tangential wall of the exodermis was a complete barrier, one might expect to detect a significant O2 concentration on its inner surface and a concentration discontinuity to zero at some point across the barrier. Such barriers might develop as a response to soilborne phytotoxins: phytotoxins certainly make the expression of barrier resistance more extreme, both to O2 loss and to ion uptake (Armstrong & Armstrong, 2001, 2005), but it has not yet been possible to achieve microelectrode penetration at these points to observe the profiles.

These O2 diffusivity data for roots of H. marinum and those obtained earlier for Phragmites (Armstrong et al., 2000) might help to explain the apparent contradiction between ROL and nutrient- and water-uptake profiles along roots, that is, why the barrier can appear to be more effective in terms of reducing ROL than in terms of nutrient uptake. Reductions in water and/or nutrient uptake along roots with a barrier may be large (Armstrong & Armstrong, 2005; Soukup et al., 2007), or may be much less extreme than reductions in ROL (Rubinigg et al., 2002; Garthwaite et al., 2006). Similarly, absorption of water and nutrients can proceed across roots with a well developed hypodermis or exodermis (Ferguson & Clarkson, 1976; Clarkson et al., 1987; Gierth et al., 1998; Zimmermann & Steudle, 1998; Hose et al., 2001). In contrast to O2 movement across the external cell layers, however, nutrient and water transport across these layers will not be subject to the same degree of consumption (usage) along the transport path. Hence there will not be the same degree of effective (apparent) resistance caused by synergism between physical resistance and consumption as occurs with O2. It is interesting, however, that where more extreme barrier formation was induced in roots of rice by exposure to phytotoxins, rather than just to stagnant anaerobic/low-O2 media, both ROL and iron uptake were halted (Armstrong & Armstrong, 2005). Iron salts accumulated in the epidermis, apparently being halted by the outer exodermal wall.

Formation of the barrier to ROL in roots of H. marinum was induced by growth in stagnant conditions (Fig. 2), a finding consistent with earlier studies (McDonald et al., 2001a; Garthwaite et al., 2003). Recent studies on barrier induction in rice have concluded that neither low O2 per se, ethylene or CO2 accumulation stimulates barrier formation (Colmer et al., 2006); the effects of ethylene were studied as barrier formation and aerenchyma development often coincide, and ethylene is known to stimulate aerenchyma formation (Jackson & Armstrong, 1999). However, as aerenchyma formation is often induced or enhanced by growing roots in stagnant anaerobic media, and as phytotoxins (Armstrong & Armstrong, 2001, 2005) and the ethylene inhibitor silver can induce secondary wall deposits in hypodermal layers (Justin, 1990), it is tempting to suggest that perhaps it is the release (and because of the stagnant conditions) the accumulation of cell decomposition products from aerenchyma development or root exudates, such as citric acid, that help trigger barrier formation. Epidermal senescence products might also be involved. The involvement of exudates or senescence products would accord with the observation that apoplastic barrier formation can also occur when roots are grown in moist air (Clarkson et al., 1987).

Permeability coefficients for O2 diffusion across the external cell layers of aerenchymatous roots, in addition to data on ROL patterns along roots, would enable direct, quantitative comparisons of barriers to ROL in roots of different species or genotypes, and/or changes in response to environmental conditions (Colmer, 2003a). The method presented here to quantify apparent O2 diffusivity across the external cell layers of the roots, using data from manipulative experiments using root-sleeving O2 electrodes, will enable quantitative comparisons of the strength of the barrier to ROL along individual roots or among species.

Acknowledgements

We thank Mike Shane for help with root anatomy studies, Roland von Bothmer for providing seeds of H. marinum, the Grains Research and Development Corporation for providing a PhD scholarship to A.J.G., and UWA and the CRC for Plant-based Management of Dryland Salinity (now Future Farm Industries CRC) for funds that enabled T.D.C. to visit the University of Hull.

Ancillary