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Keywords:

  • aerenchyma;
  • Melilotus siculus;
  • micro-CT;
  • multiscale model;
  • oxygen;
  • phellem;
  • waterlogging

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
  • Internal root aeration enables waterlogging-tolerant species to grow in anoxic soil. Secondary aerenchyma, in the form of aerenchymatous phellem, is of importance to root aeration in some dicotyledonous species. Little is known about this type of aerenchyma in comparison with primary aerenchyma.
  • Micro-computed tomography was employed to visualize, in three dimensions, the microstructure of the aerenchymatous phellem in roots of Melilotus siculus. Tissue porosity and respiration were also measured for phellem and stelar tissues. A multiscale, three-dimensional, diffusion–respiration model compared the predicted O2 profiles in roots with those measured using O2 microelectrodes.
  • Micro-computed tomography confirmed the measured high porosity of aerenchymatous phellem (44–54%) and the low porosity of stele (2–5%) A network of connected gas spaces existed in the phellem, but not within the stele. O2 partial pressures were high in the phellem, but fell below the detection limit in the thicker upper part of the stele, consistent with the poorly connected low porosity and high respiratory demand.
  • The presented model integrates and validates micro-computed tomography with measured radial O2 profiles for roots with aerenchymatous phellem, confirming the existence of near-anoxic conditions at the centre of the stele in the basal parts of the root, coupled with only hypoxic conditions towards the apex.

Introduction

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

Plants in waterlogged soils need to cope with the lack of O2 available to roots from the soil. Waterlogging-tolerant species possess mechanisms that enhance internal root aeration and thus growth in anoxic soil; tissues can also tolerate short-term O2 deficiency via anaerobic catabolism of sugars in fermentation, as well as avoid or repair oxidative damage (Blokhina et al., 2003; Gibbs & Greenway, 2003; Bailey-Serres & Voesenek, 2008). Internal O2 transport to roots can avoid an energy crisis and maintain growth (Vartapetian & Jackson, 1997; Colmer & Voesenek, 2009). Aerenchyma development and the formation of a barrier to radial O2 loss enhance internal O2 supply to roots (Armstrong, 1979; Drew et al., 2000; Colmer, 2003; Shiono et al., 2008). Several studies have shown a relationship between waterlogging tolerance and aerenchyma formation in primary cortical tissues (e.g. Justin & Armstrong, 1987; Gibberd et al., 2001; McDonald et al., 2002; Garthwaite et al., 2003; Seago et al., 2005). Recent studies have also demonstrated the importance of secondary aerenchyma, in the form of aerenchymatous phellem, in enabling internal O2 movement in the roots of some dicotyledonous species (Shimamura et al., 2010; Teakle et al., 2011); although less is known about this form of aerenchyma in comparison with the extensively studied primary aerenchyma.

Aerenchymatous phellem forms externally from a phellogen and contains large volumes of intercellular gas-filled spaces (Jackson & Armstrong, 1999); it can be observed easily as ‘white spongy tissue’ on the outer parts of stems, hypocotyl, roots and nodules (Walker et al., 1983; Stevens et al., 1997; James & Sprent, 1999; Shiba & Daimon, 2003; Thomas et al., 2005; Teakle et al., 2011). The highly porous aerenchymatous phellem provides gas space continuity for the internal diffusion of O2 from shoots to roots. The removal of phellem from stem or hypocotyl prevents internal O2 diffusion to roots of Lythrum salicaria (Stevens et al., 2002) and Melilotus siculus (Teakle et al., 2011). Similarly, for soybean in waterlogged substrate, covering the phellem at the stem base with petroleum jelly reduces O2 transport to roots and decreases root dry mass (Shimamura et al., 2003). Despite the apparent importance of aerenchymatous phellem for waterlogging tolerance in some dicotyledonous species, the structure of gas spaces and the O2 profiles within phellem and adjacent stelar tissue have not been well documented to date.

Although light and electron microscopy have rendered very detailed images of root anatomy in wetland species (Webb & Jackson, 1986; Justin & Armstrong, 1987; Stevens et al., 1997; Schussler & Longstreth, 2000; Seago et al., 2005), the question of the shape, size and connectivity of gas-filled spaces would be better resolved by three-dimensional (3-D) rather than by two-dimensional (2-D) techniques (Verboven et al., 2008; Dhondt et al., 2010), particularly for complex tissue such as phellem. Furthermore, 3-D images can be used in mathematical models to accurately calculate the gas flows in tissues (Ho et al., 2011). In particular, a discussion of the relative importance of respiratory regulation and gas diffusion in submerged plants (Armstrong & Beckett, 2010) relies on mathematical models that today use estimates of gas diffusivity of different tissues. Gas diffusivity can be explicitly calculated using the 3-D images of the gas space network in the tissue (Ho et al., 2011). To this end, a 3-D imaging method is required that can distinguish clearly the gas-filled spaces from cells within tissues. Computed tomography (CT) uses X-ray radiation, which can penetrate plant tissues. The level of transmission of these rays depends mainly on the mass density and mass absorption coefficient of the material. Because absorption is considerably different in gas and water, gas-filled spaces can be visualized by CT on samples taken directly from the tissue without significant preparation (Verboven et al., 2008). Micro-CT has now been established as an accurate in vivo method to visualize the 3-D microstructure of plant organs and tissues with pixel sizes close to, and below, 1 μm (Stuppy et al., 2003; Cloetens et al., 2006; Mendoza et al., 2007; Verboven et al., 2008; Dhondt et al., 2010). In previous studies with micro-CT, a well-connected network of intercellular spaces has been observed in fruits (Mendoza et al., 2007; Verboven et al., 2008), with pores of various sizes and shapes interconnected in different arrangements. The understanding of gas exchange related to metabolic process has benefited to a large extent from the quantitative 3-D analysis using these high-resolution images (Ho et al., 2010b; Ho et al., 2011). X-Ray CT has recently been applied to visualize the complete root architecture in situ (Tracy et al., 2010), but these images did not resolve gas-filled spaces in the roots, and the study was not an analysis of gas exchange. Micro-CT images are used not only to visualize plant structures, but also to predict gas fluxes through tissues (Ho et al., 2011). Such models have been used in a multiscale framework to investigate the gas exchange of plant organs with their environment (Ho et al., 2006, 2009, 2010a). Structurally, phellem can be a very complex type of porous tissue, and a high-resolution 3-D imaging technique is desirable if its gas diffusion properties are to be compared quantitatively with those of more simple gas channels, such as the typical aerenchyma found in wetland plants.

The present study aimed to enhance the understanding of O2 supply to phellem-containing roots in anoxic media. To this end, micro-CT was used to image, in three dimensions, the phellem gas space structure. These images were used to calculate O2 fluxes and model O2 profiles, which were compared with O2 microelectrode traces. Tissue porosity and respiration were also measured. Experiments used an annual legume, M. siculus, as this species produces extensive aerenchymatous phellem on hypocotyl and roots (Teakle et al., 2011), and is of interest as a pasture species for waterlogged, saline land (Rogers et al., 2008).

Materials and Methods

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

Plant growth and nutrient solution composition

Seeds of M. siculus (accession SA 36983) were scarified using fine sandpaper, washed in 0.04% NaHClO solution and rinsed thoroughly in deionized water. Seeds were imbibed in aerated 0.5 mM CaSO4 solution for 3 h before being transferred to mesh over aerated 10% nutrient solution (composition given below) in darkness for 3 d. The mesh with seedlings was then transferred to aerated 25% nutrient solution and exposed to light. One week after germination, seedlings were transplanted into pots containing 4.5 l of aerated 50% nutrient solution. Each pot contained eight plants held in place by polyethylene foam at the hypocotyl. Pot bases and pot lids were covered in aluminium foil so that roots were in darkness. One week after transplanting, solutions were changed to 100% aerated nutrient solution. The nutrient solution at full concentration consisted of the macronutrients (mM) KH2PO4 (0.5), KNO3 (3.0), Ca(NO3)2 (4.0) and MgSO4 (1.0), and the micronutrients (μM) FeNa3EDTA (37.5), H3BO3 (23.0), MnCl2 (4.5), ZnSO4 (4.0), CuSO4 (1.5) and MoO3 (0.05). The solution pH was buffered with 2.5 mM 2-(N-morpholino)ethanesulfonic acid (MES) adjusted with KOH to pH 6.3. Nutrient solutions were renewed weekly and topped up with deionized water as required. Twenty-one days after imbibition, a hypoxic pretreatment was imposed in the nutrient solution by bubbling with N2 until the dissolved O2 level was c. 10% of an air-saturated solution, so as to avoid subsequent ‘anoxic shock’ (Gibbs & Greenway, 2003). After 24 h, solutions were changed to a stagnant deoxygenated (i.e. anoxic) 0.1% (w/v) agar nutrient solution. This method simulates the decrease in dissolved O2 and accumulation of plant-produced ethylene, which occurs in waterlogged soils (Wiengweera et al., 1997). Pots were filled to the top with agar nutrient solution, so that the lower portion of foam and hypocotyl were partially submerged. Plants were grown in a temperature-controlled, naturally lit (average photosynthetically active radiation at midday, 1057 μmol m-2 s-1) phytotron (20 : 15°C day : night) for 42 d in Perth, Western Australia, during September to November 2010 for all experiments, except for the 3-D imaging (details given below).

Dry mass and light microscopy

Plants were harvested when 6 wk old (3 wk in stagnant agar solution) and photographed before being separated into shoot, tap root and lateral roots for biomass measurements. Transverse sections of hypocotyl and tap roots were taken with a hand-held razor blade. Unstained sections were viewed using an optical microscope (Zeiss Axioscope II) with a UV filter at 10× objective (total magnification, ×100), photographed with a digital camera (Zeiss AxioCam) and analysed using Zeiss AxioVision software (Carl Zeiss Pty Ltd., Thornwood, NY, USA). Samples were oven dried for 3 d at 70°C and the dry mass was recorded.

Porosity based on the buoyancy method

The porosity of phellem and stele from tap roots of 6-wk-old plants was measured using the buoyancy method of Raskin (1983), with calculations as described by Thomson et al. (1990) and modifications to the sample holder as described in Visser & Bögemann (2003). A tap root segment c. 0.5–1 cm in length was taken just below the hypocotyl–root junction and the porosity was measured (intact segment of root). The stele porosity was measured on a similar root segment after removing the phellem using the blunt side of a scalpel. The phellem porosity was calculated from the difference in porosity between intact root segments and stelar samples, based on the volume of phellem as a proportion of the total volume of these root segments.

Respiration of stele and phellem in air

Phellem and stele tissue were taken from hypocotyls of 6-wk-old plants and respiration was measured in a micro-respiration system (MicroResp, Unisense A/S, Aarhus, Denmark). Hypocotyls were used instead of tap roots as it was easier to remove phellem as strips of 1–2 cm from the hypocotyl; lateral roots impeded such removal from the tap root. Strips were cut, using a scalpel, from two plants per replicate (total of eight plants) to obtain sufficient tissue for accurate weighing. Fresh weights of phellem and stele were recorded and the tissues were added to preweighed 4-ml glass chambers that contained a small, glass-coated magnetic stir bar and a lid with a capillary hole in the glass stopper (MR Ch-4000, Unisense A/S). Samples were placed on a mesh platform. Twenty microlitres of deionized water were added to the bottom of each chamber to prevent the dehydration of tissues and this volume was subtracted from the air volume used for the calculations. An O2 microelectrode (OX-MR, Unisense A/S) was inserted through the capillary hole of the glass stopper so that the tip of the electrode was in air and not touching the hypocotyls. The gap between the electrode shaft and hole in the lid was sealed with one or two drops of glycerol. O2 was monitored continuously until O2 consumption rates were constant, or for a maximum of 4 h. The signals from the two, two-channel pA meters (PA2000, Unisense A/S) were logged every 10 s (ADC16, Pico Technology, St Neots, Cambridgeshire, UK). Four chambers were run simultaneously at 20°C in darkness. The calculations were based on both the volume and mass of tissues. For calculations on a volume basis, the volume of the phellem samples was corrected for compression of the tissue during excision by measurement of the tissue porosity using the buoyancy method (already described) and by using the difference in the gas-filled spaces of removed phellem and of intact phellem on root segments to estimate this compression (correction of 34%).

Radial O2 profiles of stele and phellem

Six-week-old plants were transferred into a horizontal chamber (length, 0.3 m) made from an 80-mm-diameter Perspex pipe sliced lengthways in half, with caps at each end and a divider fitted 20 mm from one end (Colmer & Pedersen, 2008). The hypocotyl–root junction was positioned 10 mm above the divider, so that the roots were submerged in deoxygenated 0.1% agar nutrient solution (described above). Deoxygenated agar nutrient solution was continuously passed (600 ml h−1) into and along the root compartment to strip away external O2 resulting from radial oxygen loss (ROL), thereby keeping the root medium hypoxic (below 5 kPa). The hypocotyl was partly in the foam plug and partly protruding into air in the shoot compartment. The root compartment was covered with glass plates and a small opening enabled the insertion of an O2 microelectrode. The shoot base was held in place within the divider using a combination of BlueTac putty and polyethylene foam. Experiments were conducted at 20°C in darkness.

Clark-type O2 microelectrodes with a guard cathode and tip diameter of 10 μm (Revsbech, 1989; OX-10, Unisense A/S) were used. The electrode was fixed in a motorized micromanipulator stage and motor controller (Unisense A/S) and connected to a pA meter (Unisense Multimeter, Unisense A/S). The electrode was advanced in steps of 50 μm every 6 s and the signal was logged with SensortracePRO (Unisense A/S). Radial O2 profiles through phellem and stele were obtained from the hypocotyl–root junction and in the main root axis at 0.5, 1.0, 2.0, 3.0 and 9.0 cm below the hypocotyl–root junction.

Micro-computed tomography of stele and phellem

Plants were grown as above, except for a temperature of 18–25°C and photosynthetically active radiation of 350 μmol m−2 s−1. Seven-week-old plants were wrapped in damp towels and in bubble plastic, packed in cardboard boxes (three plants in each box to avoid crushing) and priority shipped from Denmark to Belgium. In Belgium, root samples were taken for micro-CT imaging (Skyscan 1172, Kontich, Belgium). Disc-shaped cross-sectional samples with a minimum thickness of 5 mm were cut from the hypocotyl and main root axis (tap root) with a scalpel and further manipulated with tweezers. Samples were taken from three random plants at different distances from the hypocotyl–root junction. If the disc-shaped samples were too large to scan, pie-shaped subsamples from the discs were taken to reduce the sample size and increase the imaging resolution. The samples were wrapped in parafilm to prevent dehydration. To mount samples on the imaging stage, they were placed inside a hollow Styrofoam cylinder (diameter, 1 cm) which was sealed onto the stage with double-sided tape. The Styrofoam is almost invisible on the micro-CT images. Micro-CT scans were taken at 55 keV and 180 μA; the exposure time was 560 ms and the rotation step was 0.3°. The total scan time was 35 min, resulting in 2000 cross-sectional image slices of 2000 × 2000 pixels each. The image pixel size was dependent on the sample size: from 2.5 μm for a sample of 5 mm3 to 7.5 μm for a sample of 15 mm³. Volume renderings and quantitative calculation of porosity on the sample were performed by 3-D image segmentation and isosurface representations with Avizo Fire software (Visualization Group Sciences, Merignac, France). The microscale gas exchange model was solved over the 3-D microstructural geometry using the finite volume method (Versteeg & Malalasekera, 1995).

Multiscale calculation of tissue O2 diffusivity

The oxygen diffusivities of the phellem and stele were calculated using a microscale model. 3-D-rendered tomographic images of root tissues with edge dimensions of 660 μm were discretized into 100 × 100 × 100 cubical control volumes with an edge of 6.6 μm. Diffusion model equations (see Ho et al., 2011) were discretized over the finite volume grid to yield a linear system of algebraic equations on the unknown concentrations at the nodes. The linear equation system was solved by the conjugate gradient method available in Matlab (The Mathworks, Natick, MA, USA). The program was run on a 16-GB RAM node (Opteron 250; Xenon 5420 and Xenon 5560) of the high-performance computer at K.U. Leuven (Leuven, Belgium).

O2 partial pressure differences of 5 kPa were applied over the microscale geometry and the corresponding fluxes were calculated by means of the microscale model. From these fluxes and the length of the sample, the apparent diffusion coefficients of the tissues in the microscale sample were calculated. The diffusivity values of the phellem, stele and phellem–stele interface region were calculated according to the positions outlined in Fig. 5(a).

Simulation of 3-D pO2 profiles in the root

A macroscale diffusion–reaction model, developed previously (Ho et al., 2008, 2009), was used to predict O2 profiles in the tap root as a result of diffusion and respiration in the roots. The aim of the model was to demonstrate the 3-D nature of the observed radial pO2 profiles along the tap root. Root dimensions were approximated from the measured radial pO2 profiles, as detailed in Supporting Information Notes S1 and Fig. S1. The corresponding model takes these average dimensions into account by means of two conical cylinders for the two distinct regions (0–4 and 4–9 cm). The root model had a total length of 13 cm (4 cm below the measurement position at 9 cm) (see Fig. 7 for the final geometry used in the model).

The model distinguished the phellem and stele regions. The calculated O2 diffusivity values were applied to the different parts. The spatial variations and anisotropy of the O2 diffusivity within each part (phellem and stele) were neglected. The distinction of different tissue layers with different properties (Armstrong & Beckett, 2010) was not taken into account. Radial oxygen loss and oxygen diffusion to lateral roots were taken into account by a mass transfer coefficient at the external surface of the phellem layer of the tap root. The value of this mass transfer coefficient was estimated to fit the computed pO2 profiles to the measured profiles. The sensitivity of the model with respect to this coefficient was computed (see Notes S1). The pO2 value around the submerged root was approximately 5 kPa. This condition was also applied at the lower end of the root model (at 13 cm from the hypocotyl). At the top of the root (at the hypocotyl–root junction), the average measured pO2 values in the phellem (15.5 kPa) and stele (0.06 kPa) were applied.

O2 consumption was modelled by means of a Michaelis–Menten equation with maximum consumption rates in each tissue equal to the measured rates (Table S1). The mitochondrial Km value was assumed to be equal to 0.148 μM (0.0108 kPa) (Armstrong & Beckett, 2010). The sensitivity of the computed pO2 profiles to Km is reported in Notes S1.

The 3-D model of the tap root was developed and solved using finite elements in COMSOL 3.5a (Comsol B.V., Stockholm, Sweden). The mesh sensitivity of the model results is reported in Notes S1 (Fig. S2).

Results

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

Plant growth and anatomy of phellem

After 6 wk of growth (3 wk aerated and 3 wk stagnant deoxygenated agar nutrient solution), M. siculus had mean (± SE, = 4) shoot and root dry masses of 1.96 g ± 0.16 g and 1.07 g ± 0.07 g per plant, respectively (data not shown). Lateral roots comprised 30% of the total plant dry mass. The maximum root length was 20 ± 0.81 cm. The hypocotyl, tap root and older laterals from the upper tap root were covered by a thick aerenchymatous phellem (Fig. 1a). Transverse sections of a phellem-covered lateral root showed extensive gas-filled spaces in the phellem and some spaces also in the phellogen (Fig. 1b).

image

Figure 1. Examples of a 6-wk-old Melilotus siculus plant grown in stagnant agar nutrient solution for the final 3 wk, and a transverse section of phellem and stele from a lateral root. (a) Photograph of the shoot base and hypocotyl–root junction, showing the first 10 cm of the root system (total root length average, 20 ± 0.81 cm). Arrow indicates the approximate location of the hypocotyl–root junction. (b) Unstained partial transverse section of a phellem-covered lateral root showing the aerenchymatous phellem (ap), phellogen (pg) and secondary xylem (sx).

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Respiration of stele and phellem in air

Phellem and stelar tissues were excised from hypocotyls and the O2 consumption rates were measured in moist air (Fig. 2). The difference in O2 uptake rates between stele and phellem tissues was greater when calculated on a volume basis because of the highly porous phellem being extremely light compared with the dense stele. After correcting for the reduction in phellem volume as a result of removal with a scalpel (see Materials and Methods), the phellem tissue had an O2 consumption rate of 0.83 nmol cm−3 s−1. The stele had a five-fold higher O2 consumption rate of 4.03 nmol cm−3 s−1.

image

Figure 2. Rates of O2 consumption by phellem and stele tissues from Melilotus siculus hypocotyl. Hypocotyls were excised from 6-wk-old plants and O2 consumption was measured in humid air in darkness. O2 uptake was calculated based on both the volume (light grey bars) and mass (white bars) of tissue, and phellem samples were corrected for reduced volume (dark grey bars) caused by compression during excision of strips of phellem using a scalpel (see the Materials and Methods section). Values are means ± SE (= 4).

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Micro-CT scans of stele and phellem

Fig. 3 plots a CT cross-section and the rendered 3-D volume obtained from one of the root samples. Video S1 displays a 3-D rendering of the root volume obtained from the CT scan. The denser stele region can be clearly distinguished from the porous phellem tissue on both tap and lateral roots (Fig. 3a).

image

Figure 3. Computed tomography (CT) cross-section and reconstructed three-dimensional view of a quarter section of the tap root with phellem at a position 1.5 cm below the hypocotyl of Melilotus siculus: (a) CT scan; (b) stele; (c) complete phellem; (d) subsample of the phellem layer (pixel size, 7.3 μm). The figures show the tap root (tr) and lateral roots (lr) with the central stele (s) covered by aerenchymatous phellem (ap); on the lateral roots, some remains of agar (a) are present and the whole sample is wrapped in parafilm (pa). Colours are green for stele and yellow for phellem tissue.

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During the work, the root images were segmented into two layers: ‘phellem’ and ‘stele’. ‘Phellem’ was defined as the outer region in which gas spaces were abundant. The inner ‘stele’ was defined from the radial position at which the gas spaces disappeared abruptly from the CT images. Inside the stele, only smaller pores were present.

The 3-D reconstructions (Fig. 3b–d) were made by thresholding the CT images to separate cell regions in both the stele and phellem from air-filled spaces. Based on the resulting images (e.g. Fig. 4a), the porosities of the stele and phellem were calculated as the proportion of the gas volume to the total volume of the respective tissue (Table 1). The calculated porosity depends on the imaging resolution; gas spaces that are smaller than the imaging resolution are not visible in the images. Mendoza et al. (2007) showed that the porosity will converge to a steady value as the imaging resolution is reduced to the micrometre scale. Here, the porosity was calculated on images with a pixel resolution of 2.4–3.3 μm for both the phellem and stelar regions.

image

Figure 4. Three-dimensional reconstructions of the roots of Melilotus siculus grown in stagnant solution. (a) At 1.5 cm below the hypocotyl–root junction (pixel size, 2.4 μm).The figure shows the central stele (s) covered by aerenchymatous phellem (ap). Colours are green for stele, yellow for phellem tissue and blue for air. (b) Pore network in the phellem of the sample in (a). The skeleton shows the connections of the pores; the colours indicate the radii of the pores (1.65 μm, blue; 18 μm, red). (c) Pore network in the stele of the sample in (a). The skeleton shows the connections of the pores; the colours indicate the radii of the pores (1.22 μm, blue; 10 μm, red). (d) Pore network across the phellem–stele interface of the sample in (a). The arrow indicates small connections between the pores in the phellem and in the stele (1.22 μm, blue; 23.96 μm, red).

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Table 1.   Porosity (% gas volume per unit tissue volume) of the hypocotyl and roots of Melilotus siculus grown in stagnant solution, obtained from computed tomography (CT) images and by the buoyancy method (Raskin, 1983)
 PhellemSteleEntire root
CTBuoyancyCTBuoyancyCTBuoyancy
  1. 1Root samples at 0.5 cm contained lateral roots.

  2. Representative subsamples are depicted in Fig. 2. The porosity based on the buoyancy method was measured on tap root samples from 0.5 to 1 cm below the hypocotyl–root junction. Values are means ± SE, with = 3 subsamples of the same plant (CT method) or = 4 replicate plants (buoyancy method).

Hypocotyl48.4 ± 0.8 1.8 ± 0.3 43.5 ± 1.0 
Root at 0.5 cm154.4 ± 2.151.8 ± 2.42.1 ± 0.42.2 ± 0.636.8 ± 1.224.1 ± 1.2
Root at 1.5 cm44.9 ± 1.2 5.5 ± 0.5 34.2 ± 0.1 

Videos S2 and S3 display the 3-D structure of the air spaces of small samples of phellem and of the stelar tissue close to the phellem. The size and 3-D connections of the gas spaces in the phellem are displayed in Fig. 4(b), supported by Video S2. Pore sizes up to c. 20 μm in diameter are found.

The stele contains only a very small amount of gas spaces. The gas spaces are mostly concentrated in a ring zone beneath the phellem–stele interface (under the phellogen); additional gas spaces appear at the central axis of the root (Fig. 4a). Fig. 4(c) displays the size and 3-D connections of the pores in the stele under the phellogen, supported by Video S3. The pores are generally smaller than in the porous phellem with fewer visible connections. The pores in the stele have a few tiny connections to the pores in the phellem; these connections are marked in Fig. 4(d) by the arrow (see also Video S4). The present study cannot confirm the existence of capillaries that are smaller than the imaging resolution of 2.4 μm.

The calculated porosity of the phellem (Table 1) ranged from 45% to 54%, depending on the position along the tap root. The calculated stele porosity was much smaller, from 1.8% to 5.5%, the larger value further from the hypocotyl–root junction. These values were similar to those measured using the buoyancy method, with the phellem porosity calculated to be 52% and the stele porosity only 2% at 0.5–1 cm below the hypocotyl–root junction (Table 1). The total porosity varied significantly from the measured value. This was a result of the fact that the diameters of the stele and phellem of the micro-CT plants differed considerably from those in the buoyancy method.

O2 diffusivity of stele and phellem

By application of the microscale gas diffusion model to the micro-CT images (Fig. 5b), the radial O2 diffusivity of the tissues was calculated for the different samples. The effective tissue values are listed in Table 2. The apparent values (c. 8 × 10−11 m2 s−1) are very low in the stele, and approach the low diffusivity of O2 in water (2.01 × 10−9 m2 s−1, or 6.7 × 10−11 m2 s−1 on a gas equilibrium basis, using Dgas = RTHDliquid, where = 8.314 J mol−1 K−1 is the universal gas constant, T is the temperature in kelvin and = 0.0137 mol m−3 Pa−1 is the Henry constant for water; this 30-fold change in diffusivity equals the 30-fold drop in oxygen concentration at an air–water interface). As a result of the abundant presence of gas spaces in the phellem, the sample across the phellem–stele interface has a much higher O2 diffusivity (c. 1 × 10−6 m2 s−1) and the highest value (c. 5 × 10−6 m2 s−1) is found in the phellem, closer to that of O2 in air (2.02 × 10−5 m2 s−1). The Supplementary Information further explains the relative importance of the porosity and tortuosity of the gas spaces on tissue diffusivity (Ho et al., 2010b; Pham et al., 2008).

image

Figure 5. pO2 profile calculation for roots of Melilotus siculus in O2-free solution (i.e. reliant on internal O2 diffusion from the stem base into the roots): (a) position of the samples (0.66 mm side) on the computed tomography images; from left to right: stele, stele–phellem interface, phellem; (b) O2 profile (mol m−3) in the gas spaces of the phellem for a pO2 gradient of 2 kPa (from 7 to 5 kPa) across a sample 0.66 mm thick.

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Table 2.   Apparent radial gaseous diffusivity of O2 in phellem and stele of Melilotus siculus, estimated using the microscale model
PositionNumber of samplesDirectioninline image (m2 s−1)1
PhellemPhellem–stele interfaceStele
  1. 1Average ± SE.

  2. 2Root samples at 0.5 cm contained lateral roots.

Hypocotyl3Radial(50.4 ± 2.9) × 10−7(9.0 ± 2.8) × 10−7(73.3 ± 1.8) × 10−12
Longitudinal(51.7 ± 2.2) × 10−7(20.4 ± 2.0) × 10−7(95.6 ± 17.0) × 10−12
0.5 cm22Radial(49.7 ± 24.8) × 10−7(80.7 ± 1.4) × 10−12
1.5 cm6Radial(18.0 ± 4.7) × 10−7(12.6 ± 5.1) × 10−7(81.2 ± 3.8) × 10−12

The diffusivity in the phellem decreases with distance down the root: at 1.5 cm, it is less than one-half of the value at the hypocotyl–root junction (Table 2). In the stele, it is equal at all measured positions. Diffusion in the radial direction was compared with diffusion in the longitudinal direction, that is, the direction along the axis of the root. At the stele–phellem interface, the longitudinal diffusivity has twice the value of that in the radial direction. At the other positions, there is no anisotropy.

Radial pO2 profiles of stele and phellem

The capacity of the phellem to facilitate radial or longitudinal O2 diffusion when immersed in deoxygenated agar was studied by taking sequences of radial pO2 profiles along the roots. At the hypocotyl–root junction, the radial O2 loss was significant, as indicated by the semi-linear profile starting at 5 kPa and increasing to c. 17 kPa at the phellem surface when the electrode was moved from the bulk solution towards the root (Fig. 6). Inside the phellem, pO2 ranged between 15 and 19 kPa, but dropped steeply across the peripheral layers of the stele, and the bulk of the stele was generally severely hypoxic (< 0.2 kPa) or anoxic. When the electrode again entered the phellem, after penetrating across the stele (i.e. right side of the stele in Fig. 6), pO2 again increased, but not to the same level as on the side of first entry (i.e. the left side; Fig. 6). In addition, the electrode apparently penetrated a lateral root emerging from the pericycle and this caused a steep decline in pO2 with severe hypoxia/anoxia in the stele of the lateral root. The pattern with high pO2 in the phellem and much lower pO2 in the stele continued further down the tap root at 0.5, 1.0, 2.0 and 3.0 cm below the hypocotyl–root junction. However, the stele of the tap root contained significant levels of O2 at 4.0 and 9.0 cm below the hypocotyl–root junction, presumably owing to the narrower diameter of the stele further from the root–hypocotyl junction, as evidenced in Fig. S1. Given the often irregular shape of the phellem (i.e. not always a perfect cylinder), it is possible that some of the electrode traces (e.g. Fig. 6c at 1.0 cm) are actually tangential, which could explain the higher O2 levels measured in the stele for these samples.

image

Figure 6. Radial and longitudinal pO2 profiles in the phellem and stele of a tap root of Melilotus siculus in O2-free solution (i.e. reliant on internal O2 diffusion from the stem base into the roots). Radial pO2 profiles were obtained at the hypocotyl–root junction (a) and at 0.5 cm (b), 1.0 cm (c), 2.0 cm (d), 3.0 cm (e), 4.0 cm (f) and 9.0 cm (g) below the hypocotyl–root junction using a microelectrode with a tip diameter of 10 μm at steps of 50 μm from the surrounding hypoxic agar solution, into the phellem, through the stele, and into the phellem of the other side of the tap root. pO2 traces were obtained for three individual plants and an example trace is presented.

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Radial O2 profiles at the root–hypocotyl junction and 0.5 cm below show sharp peak O2 concentrations between 6000 and 8000 μm, and 4000 and 6000 μm, respectively (Fig. 6a,b). These are believed to correspond to measurements inside pores that have connections to the phellem. Some minor peaks in the radial profiles could also be measurements inside smaller pores in the stele. These pores and the connections to the air spaces in the phellem are visualized in Fig. 4. At 2 and 9 cm, the high O2 plateau on the right-hand side appears wider than on the left-hand side, but they both include a sharp dip in O2 pressure. Here, the oxygen probe most probably penetrated a lateral root (Fig. 6d,g).

In addition to the radial O2 profiles described above, there was also a longitudinal gradient (declining pO2) with distance from the hypocotyl–root junction. The longitudinal decline in phellem pO2 was not obvious before 3.0 cm below the hypocotyl–root junction, but, at 9.0 cm, pO2 was less than one-half of that in the upper 2–3 cm of the phellem in the tap root. The longitudinal O2 gradient in the stele differed from that in the phellem. O2 was below detection in the inner tissues of the upper area of the (thicker) stele close to the hypocotyl–root junction, whereas further down the root (i.e. at 4.0 and 9.0 cm below the hypocotyl–root junction), where the stele is narrower, it contained 3.6-8.2 kPa O2 (Fig. 6).

The diffusion–respiration model was solved for the 3-D root model plotted in Fig. 7(a) to obtain radial pO2 profiles through the stele and phellem at different positions along the root. According to the results above, in the phellem, a constant diffusivity value of 5.0 × 10−6 m² s−1 was applied; in the stele, a value of 8 × 10−11 m² s−1 was taken. Maximum O2 consumption rates were equal to 4 and 0.8 mmol m−3 s−1 in the stele and phellem, respectively. In Fig. 7, the resulting distribution of O2 in the root model is shown in a contour graph plotted in an axial plane through the root. The model predicts high pO2 in the phellem which is radially uniform. pO2 decreases in the phellem from the hypocotyl–root junction down the root. Inside the stele, there are large radial gradients in the upper thicker part of the root and smaller gradients in the lower part. The O2 level in the stele has a maximum at the 4-cm position.

image

Figure 7. pO2 profiles from a three-dimensional root model that solves diffusion and respiration by the finite element method to elucidate the O2 status in the roots of Melilotus siculus when in O2-free solution (i.e. reliant on internal O2 diffusion from the stem base into the roots). The simplified root consists of a tap root section, 13 cm in length, with diameters of 9 and 2.5 mm at the root–hypocotyl junction and at 9 cm from the hypocotyl, respectively (corresponding to the dimensions in Supporting Information Fig. S1). The section represents the upper (i.e. older) portion of the tap root with secondary aerenchyma. The steady O2 distribution within the roots in anoxic medium is plotted.

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The computed radial and axial profiles are compared with the measurements in Fig. 8 (only the measurement data inside the phellem and stele of the tap root were plotted). The uniform radial pO2 in the phellem is predicted well; the phellem pO2 decreases with distance from the hypocotyl–root junction and the gradients in the stele are comparable. Finally, the predicted and measured profiles show a maximum of the axial stelar pO2 at the 4-cm position. The peaks in stelar pO2 at 0.5 cm cannot be reproduced by the model because the stele is treated as a homogeneous material with a constant diffusivity, whereas the peaks are a result of discrete pores in the microstructure.

image

Figure 8. (a) Predicted vs measured radial profiles of pO2 from the three-dimensional results in Fig. 7 plotted at 0.5, 3.0, 4.0 and 9.0 cm below the root–hypocotyl junction. The longitudinal profiles (b) are plotted on the axis of the stele and in the phellem. Measurements in (b) are averages with standard error bars from all data points in the phellem (= ± 50) and at a few data points in the centre of the stele (= 5). The solid lines are the result of the model. In (a), the symbols are the measured values at 0.5 cm (open squares), 3 cm (closed squares), 4 cm (open circles) and 9 cm (closed circles). In (b), the symbols are the average measured values in the stele centre (diamonds) and in the phellem (closed squares).

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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
  9. Supporting Information

This study used micro-CT images to examine aerenchymatous phellem gas space structure and to model O2 fluxes to enhance the understanding of the O2 supply to roots with this type of secondary aerenchyma using M. siculus as the case study. Although phellem is visible to the naked eye (Fig. 1a), the 3-D micro-CT images enabled the resolution of the complex structure of the gas spaces in three dimensions within this tissue (Figs 3, 4), which forms in hypocotyl, upper tap root and basal portions of older lateral roots (Teakle et al., 2011). 3-D reconstruction of the roots revealed that the aerenchymatous phellem is a very spongy tissue, particularly in comparison with the dense stele. Furthermore, the images in Fig. 4 highlight the well-connected gas spaces in the phellem that are aligned with the root axis to provide a direct pathway for longitudinal O2 transport. Such low-resistance pathways for O2 diffusion via aerenchyma increase the internal aeration capacity of the roots, a critical trait that determines the waterlogging tolerance in plants (Armstrong, 1979; Sorrell et al., 2000; Colmer, 2003). Therefore, the aerenchymatous phellem probably contributes substantially to the waterlogging tolerance of M. siculus (Rogers et al., 2008, 2011; Teakle et al., 2011).

The micro-CT images were also successfully used to calculate the gas-filled porosity for the phellem and stele tissues, and these results were compared with measurements obtained using the more common ‘buoyancy method’. The micro-CT image-derived porosity values matched those measured using the buoyancy method (Raskin, 1983), validating the use of micro-CT for the assessment of the porosity of roots. As expected, based on the large amounts of gas spaces in the 3-D images, the porosity of the phellem was high (45–54%) and the stele had only 1.8–5.5% porosity (Table 1). The porosities of organs that have extensive aerenchymatous phellem tissues have been measured for soybean roots (21%; Thomas et al., 2005), soybean hypocotyl (24–32%; Shimamura et al., 2003) and L. salicaria stems (49%; Stevens et al., 1997). Given that the root and hypocotyl tissues above have high ratios of phellem to stele, it is probable that the porosity of phellem in these species would be similar to the c. 50% measured in the present study for M. siculus phellem. Overall, the porosity of these phellem-containing roots is close to the uppermost values reported for the porosity of whole adventitious roots of wetland species (Justin & Armstrong, 1987).

The large differences in porosity between the stele and phellem are likely to produce differences in the respiratory demand of the tissues when expressed on a volume basis (cf. Armstrong, 1979). The O2 consumption rates of the phellem (0.83 nmol cm−3 s−1) were five-fold lower than those of the stele (Fig. 2). The rates measured for the roots of M. siculus with tissues in air are in the same range as those of mature root segments in air-saturated solutions for Trifolium (Gibberd et al., 2001) and Lotus (Teakle et al., 2010) species. To our knowledge, these are the first data on the respiration of separated phellem and stele tissues; however, the values can be compared with the respiration rates of separated primary cortex and stele measured for two monocotyledonous species. The respiration rate of the cortex of banana roots was six-fold lower than that of the stele on a volume basis (Aguilar et al., 2003). Similar results were modelled for maize roots, with respiratory values of the cortex four-fold lower than those of the stele (Armstrong et al., 1994). In these studies, the lower respiratory demand of the cortex on a volume basis was also attributed to the presence of aerenchyma in the cortex. Even on a mass basis, the O2 consumption by the stele of M. siculus was c. two-fold that of the phellem tissue (Fig. 2), reflecting the known higher respiratory demand of the more active tissues within the stele compared with the cortex (e.g. Armstrong et al., 1991).

Diffusion model equations were applied to 3-D-rendered tomographic images of root tissues to calculate the O2 diffusivity through the phellem and stele. The diffusivity values in Table 2 clearly reflect the observations made on the micro-CT images in Fig. 4. In the stele, the number and fraction of gas spaces appear to be small, and the O2 diffusivity is low. The gas spaces in the stele are also not well connected at the scan resolution, which also contributes to the decrease in the O2 diffusivity. The micro-CT images indicate that there is no continuous gaseous path to the centre of the root through the stele. The diffusivity of the stele is very low, from 7 × 10−11 to 10 × 10−11 m² s−1. Armstrong & Beckett (2010) also used a value for the stele equal to or somewhat lower than that of O2 in water (6.7 × 10−11 m2 s−1 on a gas equilibrium basis). The combination of low rates of O2 supply because of low diffusivity and high O2 consumption rates results in very low O2 concentrations in the stele (Fig. 2). This is confirmed by the radial pO2 profiles in Fig. 6. In contrast with the stele, phellem tissue contains a large proportion of gas spaces that are well connected (Fig. 3). Here, the diffusivity value approaches that of O2 in air (Table 2). The gas space connectivity exists in both the radial and longitudinal directions. Close to the stele, the connectivity of spaces is strongest in the longitudinal direction, which is reflected in somewhat higher O2 diffusivity values. The O2 diffusivity of the phellem is between 9 × 10−7 and 52 × 10−7 m² s−1, and these high values result in O2 being rapidly redistributed over the entire phellem, as also evidenced by the relatively flat radial profiles of high O2 in the phellem. As a result of low diffusivity values of the surrounding external liquid medium, as well as in the stele, there are high resistances for radial O2 movement from the phellem, resulting in sharp gradients across both the internal and exterior phellem interfaces. pO2 in the phellem decreases slowly with distance along the length of the tap root as a result of the O2 consumption of the phellem, radial O2 losses to the medium, O2 diffusion into the stele and O2 diffusion into lateral roots. It appears that the phellem acts as the main O2 delivery route and steadily supplies the stele with O2 at low levels in the upper part of the root. This is the first experimental finding of near-anoxic conditions at the centre of the stele in the basal parts of a root, coupled with only hypoxic conditions towards the apex. The possibility of this occurring was first predicted by Armstrong & Beckett (1987).

In addition to the measured radial O2 profiles, a 3-D model of O2 partial pressure in the stele and phellem was developed from the measured O2 consumption rates and calculated diffusivity values, based on typical dimensions obtained from the CT imaging and radial pO2 profiles for a tap root with several lateral roots. Measurements of radial pO2 profiles close to the hypocotyl–root junction show high levels of O2 in the phellem (15–19 kPa), which decrease rapidly to levels below detection (c. 0.05 kPa) in the stele (Fig. 6). The model predicts values as low as 0 kPa. This pattern is similar to, but more severely hypoxic than, the radial pO2 profiles measured in stems of soybean with secondary aerenchyma, in which the O2 partial pressure is constant at c. 18 kPa across the phellem and declines rapidly to c. 2 kPa in the stele (Shimamura et al., 2010). In the lower part of the M. siculus tap root, pO2 values were greater than 5 kPa in the stele, which is considerably higher than in the upper region of the stele. This pattern is a direct consequence of the root structure and the O2 consumption rates and gas diffusion properties of the phellem and stele, and such patterns of severe hypoxia within portions of the stele could impact on ion transport from the roots to the shoot (Colmer & Greenway, 2011). The modelled pO2 profiles of Figs 7 and 8 confirm the general observations above from the measured radial pO2 profiles (Fig. 6). The pO2 profile is flat in the phellem and decreases strongly when approaching the axis of the stele, as per the model. Despite the large decrease in the stele, the final pO2 levels in the stele are higher in the lower part of the root, in comparison with the very low values found close to the hypocotyl–root junction. The strong increase in stele pO2 in the lower part of the root is apparent from Fig. 8b and is caused by the decreasing diameter of the stele and the supply of O2 via the abundant gas spaces within the phellem over the entire length of the root. The model thus indicates that root aeration is a 3-D process. This is further demonstrated by calculating the axial and radial fluxes of O2 in the root, as detailed in the Supplementary Information. Other factors that affect the calculated profiles are the mass transfer coefficient for radial O2 loss and the Km value. The model sensitivity to these values is presented in Figs S3 and S4.

The model discussed above integrates and validates the data presented in this study, highlighting the value of micro-CT imaging for the understanding of O2 transport in roots. Previous studies have examined gas exchange properties of fruits using micro-CT (e.g. Verboven et al., 2008; Ho et al., 2011); however, this study is the first to apply the technology to O2 transport within roots. The unique structure and high O2 transport capacity of M. siculus phellem were demonstrated from the 3-D images and radial O2 profiles. The micro-CT imaging has improved our understanding of O2 fluxes through phellem, a key trait for waterlogging tolerance in M. siculus (Teakle et al., 2011) and certain other dicotyledonous species (e.g. soybean; Shimamura et al., 2010).

Acknowledgements

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

CT work was performed with financial support from the Research Council of K.U. Leuven (OT 08/023), the Flanders Fund for Scientific Research (project G.0603.08, post-doctoral fellowship for Q.T.H.) and the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT scholarship SB/0991469). Financial support of the Danish Science Council is acknowledged. Travel funds for O.P. from the A. W. Howard Memorial Trust are gratefully acknowledged.

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  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

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

Fig. S1 Dimensions of the measured tap root of Melilotus siculus.

Fig. S2 Effect of finite element size on the model result.

Fig. S3 Sensitivity of the radial oxygen profile in the root to the value of the mass transfer coefficient h.

Fig. S4 Sensitivity of the computed radial oxygen profiles to the value of Km used in the Michaelis–Menten model for respiration.

Table S1 Input parameters to the root diffusion–respiration model

Notes S1 Root dimensions, model parameters, tissue diffusivity values, sensitivity of the model, oxygen flux analysis.

Video S1 Three-dimensional rendering of a computed tomography scan of a quarter section of a tap root with lateral roots of Melilotus siculus, clearly showing the dense stele and porous phellem.

Video S2 Three-dimensional rendering of the pore network in the phellem. The skeleton shows connections of the pores; the skeleton colours indicate the radii of the pores (1.65 μm, blue; 18 μm, red).

Video S3 Three-dimensional rendering of the pore network in the stele. The skeleton shows connections of the pores; the skeleton colours indicate the radii of the pores (1.22 μm, blue; 10 μm, red).

Video S4 Three-dimensional rendering of the pore network across the phellem and stele. The skeleton shows connections of the pores; the skeleton colours indicate the radii of the pores (1.22 μm, blue; 23.96 μm, red).

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NPH_3934_sm_NotesS1-FigsS1-S4-TableS1.doc427KSupporting info item
NPH_3934_sm_VideoS1.gif36735KSupporting info item
NPH_3934_sm_VideoS2.mpg23758KSupporting info item
NPH_3934_sm_VideoS3.mpg15302KSupporting info item
NPH_3934_sm_VideoS4.mpg36546KSupporting info item