AOBPreview originally published online on August 10, 2005
Annals of Botany 2005 96(4):591-612; doi:10.1093/aob/mci213
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Stem Photosynthesis not Pressurized Ventilation is Responsible for Light-enhanced Oxygen Supply to Submerged Roots of Alder (Alnus glutinosa)
Biological Sciences, University of Hull, Kingston upon Hull HU6 7RX, UK
* For correspondence. E-mail w.armstrong{at}hull.ac.uk
Received: 28 January 2005 Returned for revision: 5 April 2005 Accepted: 2 May 2005 Published electronically: 10 August 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONBACKGROUNDMATERIALS AND METHODSRESULTS AND DISCUSSIONGENERAL DISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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• Background and Aims Claims that submerged roots of alder and other wetland trees are aerated by pressurized gas flow generated in the stem by a light-induced thermo-osmosis have seemed inconsistent with root anatomy. Our aim was to seek a verification using physical root–stem models, stem segments with or without artificial roots, and rooted saplings.
• Methods Radial O2 loss (ROL) from roots was monitored polarographically as the gas space system of the models, and stems were pressurized artificially. ROL and internal pressurization were also measured when stems were irradiated and the xylem stream was either CO2 enriched or not. Stem photosynthesis and respiration were measured polarographically. Stem and root anatomy were examined by light and fluorescence microscopy.
• Key Results Pressurizing the models and stems to 
10 kPa, values much higher than those reportedly generated by thermo-osmosis, created only a negligible density-induced increase in ROL, but ROL increased rapidly when ambient O2 concentrations were raised. Internal pressures rose by several kPa when shoots were exposed to high light flux and ROL increased substantially, but both were due to O2 accumulation from stem photosynthesis using internally sourced CO2. Increased stem pressures had little effect on O2 transport, which remained largely diffusive. Oxygen flux from stems in high light periods indicated a net C gain by stem photosynthesis. Chloroplasts were abundant in the secondary cortex and secondary phloem, and occurred throughout the secondary xylem rays and medulla of 3-year-old stems. Diurnal patterns of ROL, most marked when light reached submerged portions of the stem, were modified by minor variations in light flux and water level. Low root temperatures also helped improve root aeration.
• Conclusions Pressurized gas flow to submerged roots does not occur to any significant degree in alder, but stem photosynthesis, using internally sourced CO2 from respiration and the transpiration stream, may play an important role in root aeration in young trees and measurably affect the overall carbon balance of this and other species.
Key words: Alnus glutinosa, root aeration, diffusion, pressure flow, stem photosynthesis, radial oxygen loss, thermal transpiration, thermo-osmosis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONBACKGROUNDMATERIALS AND METHODSRESULTS AND DISCUSSIONGENERAL DISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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The aeration of the belowground parts of some wetland plant species is known to be much improved by various forms of pressurized gas flow from the aboveground parts. The flows known to be most effective involve a through-flow of gases, sometimes between emergent stems via the rhizome system (Armstrong and Armstrong, 1990; Armstrong et al., 1992
; Brix et al., 1992; Sorrell and Boon, 1994), or between emergent leaves via the underground stem to other emergent leaves (Dacey, 1980, 1981; Grosse and Bauch, 1991; Bendix et al., 1994), or even from one part of an emergent leaf via the underground system and back through other channels in the same petiole and leaf (Mevi-Schultz and Grosse, 1988). The major pressurizing mechanisms driving these flows have been identified as thermal transpiration (=thermo-osmosis), humidity-induced diffusion and Venturi-induced pressure differences (Dacey, 1987; Armstrong et al., 1992, 1996a, b; Grosse, 1996). The roots are not considered to be a part of the through-flow system, but they can derive obvious benefit from it, since the gas stream will usually raise the O2 concentration at the root–rhizome junction, thus increasing O2 diffusion into the roots. The removal of CO2, C2H4, CH4 and other products will also be enhanced (Brix et al., 1996).
In 1985, Grosse and Schroder reported that ‘... experiments show evidence of [in addition to diffusion] an additional manifold higher O2 supply due to a gas transport in leaved as well as leafless trees of Alnus glutinosa (L.). This gas transport is directed from the stems to the roots and is driven by a thermo-osmotic pressurization within the air space system of the stems resulting from temperature gradients up to 3·6 °C between the stem and the ambient air following absorption of light energy by the brownish pigments of the bark’. In subsequent papers, it was claimed that the O2 flow to the roots of several other wetland tree species was improved by a thermo-osmotically driven pressure flow, i.e. Taxodium distichum, Betula pubescens and Populus tremulus (Grosse et al., 1992), Alnus japonica and Alnus hirsuta (Grosse et al., 1993) and Salix alba, S. cinerea and S. viminalis (Grosse et al., 1996). However, no evidence of improved root ventilation was found in Alnus incana, Fraxinus excelsior or Acer pseudoplatanus (Schröder, 1989a), or in Acer pseudopatanus and Ilex aquifolium (Grosse et al., 1992).
For several reasons, we doubted that there could be a significant pressure flow into submerged tree roots. For example, to create a through-flow, high surface tension pressures would have to be overcome to force gas from submerged lenticels, and the internal gas pressures measured by Grosse and Schröder (1985) in the alder stem did not exceed 17 Pa. In these circumstances, we would anticipate only transitory non-through-flow convection to the roots as they are pressurized. Similarly, it is difficult to envisage how a significant pressure flow could enter submerged non-woody roots since, in ventilation terms, they are effectively blind-ended tubes (Beckett et al., 1988).
If pressure flow was not responsible for the reported improvements in the O2 supply to the roots of alder and the other species, an obvious alternative explanation could be that photosynthetically generated O2 might have been the cause. Although the brown stems of alder do not immediately prompt such an explanation, abundant chloroplasts can be found in the secondary cortex, the secondary phloem, secondary rays and the medulla of stems up to 4 years old (see below). However, if the only CO2 source for photosynthesis is that diffusing through lenticels from the atmosphere, the photosynthetically generated O2 would simply counter-diffuse away; any increase in O2 concentration in the secondary cortex could be expected to be tiny and not sufficient to have a significant influence on O2 transport to the roots (Armstrong, 1979). A marked increase in O2 concentration in the stem would require either that there should be an internal CO2 source and/or that the resistance to O2 escape should be greater than the resistance to CO2 entry. The latter occurs where leaves and stems are submerged (Waters et al., 1989). However, Stringer and Kimmera (1993) have reported significant petiolar photosynthesis in Populus deltoides dependent upon CO2 carried in the xylem stream; concentrations up to 9 mM were recorded in the xylem stream in plants from submerged soils. Hibbard and Quick (2002) have reported C4-type photosynthesis in the petioles of tobacco, with the carbon source being supplied from the vascular system and not from the stomata. Preliminary studies on willow (S. viminalis) and alder in our laboratory (Moir, 1993; Dockerty, 1994; Hughes, 1996) found that ROL from roots increased significantly only if (a) partially submerged stems were illuminated; (b) gas pressures applied to a cut emergent end of the stem were sufficient to cause bubbling below the water line (pressures >2000 Pa); or (c) the emergent stems were exposed to above ambient O2 concentrations. We also found that alder twigs fresh from the field would produce a regular bubbling from submerged lenticels and the cut end when they were partially submerged and illuminated (W. Armstrong and J. Armstrong, unpubl. res.).
We have now extended these observations and report here on (a) the pressurization generated by stem photosynthesis in the black alder, Alnus glutinosa (L.) Gaertn.; (b) the effect that stem photosynthesis has on O2 diffusion from the stems to the roots; and (c) the insignificant effect pressurization per se has on transport to submerged roots. The latter was tested using (a) a purely physical artificial root model; (b) a combined model in which alder stems were equipped with artificial roots; and (c) whole plants with submerged root systems.
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BACKGROUND |
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TOPABSTRACTINTRODUCTIONBACKGROUNDMATERIALS AND METHODSRESULTS AND DISCUSSIONGENERAL DISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Thermal transpiration (thermo-osmosis)
Thermal transpiration (thermo-osmosis) and humidity-induced diffusion and pressurization in plants require the presence of a microporous partition between the aerial atmosphere and the internal atmosphere of the plant with pores sufficiently small so as to offer more resistance to a pressure flow than to gas phase diffusion. For thermal transpiration to generate pressures within these plants, it is necessary for the internal atmosphere under the micropores to be constantly warmer than the external atmosphere (Grosse, 1996
). Solar warming will tend to cause gas expansion and pressurization of the internal atmosphere. Pressure is not readily dissipated through the micropores because of their resistance (although they may be leaky to some extent) and, if the gases can escape more readily along a more distal venting path with lower resistance, the internal gas density will become less than the external atmosphere, so inducing a diffusive inflow of atmospheric gases. Provided that the solar warming is continuous, the inwardly diffusing gases will be sufficient to rise and sustain above ambient internal pressures and continuously drive a pressurized gas flow along the venting path.
In alder, Buchel and Grosse (1990) identified the phellogen under the lenticels as a microporous partition that should support thermo-osmosis: pores through the vascular cambium were not smaller than 100 nm, but the mean diameters of pores through the sub-lenticellular phellogen were as low as 14 nm. Pores as large as 3 µm diameter can be sufficient to support some pressurization (Takaishi and Sensui, 1963) and flow (Armstrong et al., 1996b), but they could be expected to be very leaky.
Original evidence underlying the supposition of a significant pressure flow to the roots of alder
The suggestion that thermal transpiration causes a thermo-osmotically driven pressure flow into the roots of alder and other wetland tree species was based largely on the results of two types of data. Both were obtained from experimental assemblies similar to that shown in Fig. 1. Six-month- to 1-year-old seedlings were used. For plants in leaf, the lower half of the woody shoot above the root collar was enclosed in an upper chamber as shown; for leafless plants, the whole of the woody shoot above the root collar was enclosed (Grosse and Schröder, 1984, 1985; Schröder, 1989a). The shoot base and the whole of the root system were in a lower chamber, the stem base and several root bases being enclosed in a gas pocket above either saturated soil or a culture solution; the remainder of the root system was submerged. The top chamber was fitted with an injection port and the lower was fitted with a sample port. Pressure gradients between the chambers and the external atmosphere were minimized using long narrow ‘breather’ tubes. Ethane was injected into the upper chamber and sampled from the head space in the lower chamber, and the experiments were carried out in the dark and with a radiant light source (a 150 W incandescent lamp) as illustrated. Grosse and Schröder (1985) and Grosse et al. (1992) measured differences in temperature (
T °C) between the inside of the stems and the surrounding atmosphere. Grosse and Schröder (1984) also recorded differences in pressure (P) between the inside of the stem and the atmosphere. Grosse et al. (1992) did not measure pressure differences but did record changes in O2 concentration in the soil solution around the roots using a Clark-type electrode sealed into the bottom of the root chamber.
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The results showed that ethane was transported from the upper to the lower chamber through the plant and that the rates of ethane release into the lower chamber increased rapidly when the stems were irradiated by incandescent light, reaching a new equilibrium after approx. 20 min. For those species that showed a marked response, the increased rate as a percentage of the dark condition was 189–261 for Alnus glutinosa, 177 for Taxodium distichum and 144 for Populus tremula. In A. glutinosa,
Ts of up to 3·6 °C were accompanied by Ps of up to 17 Pa, and the latter was considered to be the driving force increasing the transport to the lower chamber. O2 uptake by roots was less when the stems were irradiated, and this also was taken as indicative of an increased O2 transport from stem to root due to a pressurized flow. Translated into an increased O2 supply through the stem, examples of the readings obtained under irradiance as a percentage of the dark condition were T. distichum, 453 and for Betula pubescens, 252. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONBACKGROUNDMATERIALS AND METHODSRESULTS AND DISCUSSIONGENERAL DISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Plant material
Stem material
One metre leafless lengths of stems were harvested from mature partially coppiced black alder at North Cliffe Woods, East Yorkshire between February and May 2004, cut into 20–25 cm long segments and the lower ends placed in tap water for transference to the laboratory. The water level was maintained at a depth of 1–2 cm as necessary until a cutting was required for an experiment. The cutting would then be trimmed back at both ends by approx. 1 cm, and a 1 cm length of the ‘bark’ was stripped back from what had been originally the lower end. This end was now placed uppermost and a small water reservoir was attached to it by means of rubber tubing sleeving on to the stripped surface (e.g. see Fig. 2B). This reservoir was topped up with tap water or CO2-enriched de-ionized water as required. The water usually began to percolate immediately through the xylem. If percolation failed, the ends of the cutting were re-cut and the procedure was repeated. These cuttings were employed for various types of experiment as described later; sometimes they were fitted with artificial roots and with pressure take-off and application tubes.
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Whole plants
Two- and three-year-old plants were purchased from a local nursery during December 2003, replanted in a sandy loam in 20 cm pots and placed in a cool greenhouse. In early spring before bud break, the pots were inserted into larger non-draining containers, the soil was flooded and maintained at approx. 4 cm above the surface, and the plants placed near the window of a south-facing laboratory until dormancy broke and beyond. Black plastic card was fitted around the stem base at the water level and over the rim of the pot to shade any adventitious roots that were produced. This also helped to prevent algal growth in the water. Whole plants were experimented upon in various ways as described later; before doing so they were removed from their pots and the soil was gently washed away from the roots.
Artificial root-stem model
To explore the effects on root radial O2 loss (ROL) of (a) pressurization per se; (b) a partial through-flow such as might arise if gas could be forced out through lenticels at some distant point, e.g. below the water table; and (c) an increased partial pressure at the O2 source, an artificial root and ‘stem’ system was constructed as shown in Fig. 2A. The gas space system of a stem/woody root was mimicked by two 1 µL microcap capillaries joined in series and connected at one end (the top) via a syringe tip and large bore connectors to an acrylic T-piece. At its bottom end, this capillary train was connected to one arm of a small glass T-piece. Two more 1 µL microcap capillaries tipped by a 0·5 cm length silicone rubber tubing (o.d. 1·0 mm; i.d. 0·5 mm) were connected to the other in-line arm of the glass T-piece to mimic the gas space system and O2-permeable apex of a short adventitious root. The silicone rubber tube was blocked at the tip by a piece of glass rod to prevent flooding. Provision for a return pressurized through-flow to the atmosphere from the junction between ‘stem’ and ‘adventitious root’ was made using a train of three 1 µL microcap capillaries connected to an elbow of the stem of the glass T-piece. This was to mimic the gas escape route through leaky unsubmerged lenticels or the situation where an internal pressure build-up might be sufficient to blow gas from submerged lenticels. To mimic the situation where gas pressure is insufficient to blow gas from submerged lenticels, the basal end of this capillary train was sealed with Terostat.
The ‘stem–root’ assembly was immersed in stagnant anaerobic 0·05 % agar : water (w/v) (+ supporting electrolyte: 7 mM KCl) with only the acrylic T-piece and the upper end of the by-pass capillary train remaining emergent. Compressed air, or air at atmospheric pressure, or raised partial pressures of O2 were supplied to one arm of the acrylic T-piece; any applied pressures were recorded using a pressure transducer (Digitron 2026P, Radiospares, UK) attached to the other arm. The 0·5 cm long O2-permeable tip of the artificial root was positioned exactly within a 0·5 cm long cylindrical Pt cathode (i.d. 0·225 cm) and ROL from the root tip was measured polarographically (Armstrong, 1979) using appropriate polarizing voltages chosen from current–voltage curves obtained using a polarograph with digital output (Barman Electronics, Skipsea, UK). A saturated Ag : AgCl half-cell was used as the anode.
Alder stems with artificial roots
In an attempt to simplify the logistics of this study, some alder stem cuttings were fitted with artificial roots (Figs. 2B, C). Alder cuttings do not readily form adventitious roots but the use of artificial roots meant that the complicating effects of root respiration could be avoided. To fit the root, a small disc of bark including a lenticel and subtending secondary cortex was first removed using a razor blade; the acrylic connector which formed the base of the artificial root was placed over the cavity and glued to the stem using quick-setting Araldite. To effect a good and long-lasting seal, it was necessary for the stem surface to be properly dry. On some occasions, the stems were also fitted with connectors and tubes for gas pressure sampling in, or application to, the secondary cortex; details of construction are provided later. Stem pressures were measured by pressure transducer (CPFM, FCO11 Mk. 2, Furness Controls, UK) with the analogue output directed to a suitably scaled chart recorder (BD 112, Kipp & Zonen, The Netherlands). Stems were pre-percolated with either 4 or 6·9 mM CO2-enriched water.
Measurements of ROL from the artificial roots could be used to calculate the O2 concentrations developed within the secondary cortex of the stem at the ‘root–shoot’ junction. At equilibrium, the ROL, Q/t (g s–1), from an artificial root can be expressed as:
 | (1) |
) and C = Cstem – Celectrode, where Cstem is the O2 concentration in the stem at the root base, and Celectrode is the O2 concentration at the electrode surface. Since Celectrode is always zero, C = Cstem.
R, the total diffusive resistance between the gas space in the root–shoot junction and the electrode surface (4·475 x 105 s cm–3 in the example shown in Fig. 2C), is the sum of the longitudinal diffusive resistance through the microcaps (Rc), and the radial resistances (RSi) and (Rls) through the wall of the silicone rubber ‘root’ tip and the liquid shell between ‘root’ tip and the electrode, respectively. These are calculable as: Rc = Lc/Doa Axc, RSi = ri loge(ro/ri)/DSi ASi, and Rls = (Coa/Cow) x ro loge(re/ro)/Dow Asi, where Lc is the length of capillary, Doa is the diffusion coefficient of O2 in air (0·205 cm2 s–1 at 23 °C), Axc is the cross-sectional area of the capillary gas space, ri and ro are the inner and outer radii of the silicone rubber tip (0·025 and 0·05 cm), DSi is the effective O2 diffusion coefficient in the silicone rubber (taken as 4·2 x 10–6 cm2 s–1), Coa is the O2 concentration in air, Cow is the concentration of O2 in air-saturated water, re is the electrode radius (0·1125 cm) and Dow is the O2 diffusion coefficient in the liquid shell between the silicone rubber ‘root’ tip and the electrode (2·267 x 10–5 cm2 s–1 at 23 °C). Multiplying by the factor (Coa/Cow) adjusts the liquid shell resistance to account for the large concentration drop at the air–water interface. For this root, the relationship between ROL and O2 partial pressure in the root–shoot junction is shown in Fig. 2D.
To investigate the effects of photosynthesis on pressurization and ROL, cuttings were illuminated by a high-pressure sodium vapour lamp in the configuration illustrated diagrammatically in Fig. 2E. Heat loading was kept to a minimum by an intervening glass plate, cooling fan and a heat sink cooling tower fed continuously with tap water. The side opposite the light source was illuminated by the reflected light from a three-piece wrap-round mirror placed opposite the light source.
Exploring the potential for pressure development in photosynthesizing stem cuttings
Water charged with 6·9 mM CO2 was percolated through the xylem of a stem cutting for several hours from an attached reservoir such as that shown in Fig. 2B. The top and bottom ends of the cutting were then sealed using Terostat-filled aluminium caps, a weight was attached to the bottom cap and the cutting was then submerged upright in de-ionized water in a transparent acrylic tower as shown in Fig. 2F. A hole through the lid of the tower connected the head space to a compressed air source and a pressure transducer. A three-way tap was inserted in the gas line tubing that connected the compressed air source to the tower. The container was illuminated as in Fig. 2E using a 500 W high-pressure sodium vapour lamp. When the light was turned on and whenever photosynthetically induced gas bubbling from the lenticels had commenced, the air pressure of the head space was raised by a fixed amount, using the compressed air source. At first, the bubbling was only temporarily stopped by this procedure and when it recommenced the pressure was again raised. These steps were repeated until there was no more bubbling. Temperatures ranged between 21 and 23 °C.
Measuring photosynthetic rates of stem cuttings
The xylem of a stem cutting was pre-percolated with either CO2-enriched (6·9 mM) or air-saturated water before the cutting was sealed into a glass tube as shown in Fig. 2G. The bottom of the tube was fitted with a rubber bung. The tube had three side ports: the top port was fitted with a thermocouple screened from direct light, a Clark-type polarographic O2 electrode probe (YSI 5331, Yellow Springs Inc., OH), similarly screened, was sealed into the middle port, and the lower port was left open. However, the gas space around the cutting was isolated from the outside atmosphere by water that had percolated through the xylem filling the bottom of the tube and the lower port. The diameter of the lower port was such that the surface tension forces offered sufficient resistance to create a small head of water above it and the water then leaked away at the same rate as it percolated through the xylem. In this way, the gas pressure differences, and gas exchange, between the inside and outside of the tube were minimized. Again the cuttings were exposed to radiation from a high-pressure sodium vapour lamp located behind a glass screen and heat sink as shown in Fig. 2E. Reflected light from a three-piece mirror supplemented the light regime imposed on the cutting. Direct PAR was 550 µmol m–2 s–1 and the reflected PAR was approx. 350 µmol m–2 s–1.
Oxygen concentrations in the gas space around the cutting were measured in the light and in the dark with the output from the polarograph monitored continuously using a flat-bed chart recorder. Temperatures in the gas space were recorded to allow correction for any temperature-induced concentration (–0·37 % °C–1) and membrane permeability coefficient (+4 % °C–1) changes to the readings. In the event, the temperatures within the chamber which averaged 22–23 °C never changed by more than 1 °C during the observation period. Net photosynthesis in the light (µmol CO2 m–2 stem surface s–1) and stem respiration in the dark (µmol O2 m–2 stem surface s–1 or µmol O2 cm–3 stem s–1) were calculated from the slopes of the recorded traces and the assumption that 1 mol of O2 evolved indicated the consumption of 1 mol of CO2 reduced, and vice versa. Dark measurements were made with the glass tube assembly wrapped in black polythene.
Effects of light, pressure, root temperature and O2 concentrations around the shoot on ROL from the adventitious roots of whole plants
Whole plants were removed from their pots, the soil gently washed from the roots and then placed with their shoots in the air and their root systems in a stagnant O2-depleted 1/4-strength Hoaglands 0·05 % agar–water medium. A 2000 mL glass measuring cylinder was first used but, later, water-jacketed glass containers were employed: for smaller root systems, this was a cylindrical vessel (i.d. = 90 mm, o.d. = 112 mm, length = 440 mm), while for large root systems it was rectangular (internal dimensions 170 x 170 x 300 mm; external dimensions 220 x 220 x 330 mm). The agar medium had previously been bubbled with O2-free N2 for 24 h during which time the only escape route from the head space was through a 1 cm long, 1 mm diameter vent. With tap water continuously circulated through the outer jacket of the vessel, the rooting medium could be reduced from room temperature (21–23 °C) to approx. 11–12 °C. The rooting medium was separated from the atmosphere by a partition consisting of either a rubber bung or a split wooden plate (10 mm thick) secured in/on the top of the vessel. The shoot and cylindrical electrode cables were secured in appropriately sized holes in the partition, the shoot being packed round with wet cotton wool to complete the seal; holes were also made for the insertion of the fibre junctions of Ag/AgCl anodes. The shoot was additionally supported by a clamp half way up the stem. The rooting medium surface was raised to the top surface of the partition and in short-term experiments was maintained at this level whenever possible. However, in longer term experiments, transpirational water loss sometimes lowered the liquid level by a few centimetres before replenishment could be effected. To enable changes to be made in the O2 regime around the shoot, a short collar-like chamber with inlet and outlet ports was fabricated from an acrylic syringe body. This was fitted around the shoot base, and sealed top and bottom with Terostat. The ports were normally left open, but occasionally were used to change the atmosphere around the shoot base from air to 100 % O2. To enable gas pressure in the secondary cortex of the stem to be raised artificially, a small disc of bark including a lenticel and subtending secondary cortex was first removed using a razor blade, and the acrylic base of a syringe needle (obliquely cut) was sealed on to the stem around the cavity using quick-setting Araldite. Narrow non-permeable plastic tubing linked the syringe needle to the pressure transducer via a three-way tap. To enable a high level of illumination (PAR 550 µmol m–2 s–1) to be applied to the plant without creating an excessive heat load, the plants were positioned as for previous experiments (Fig. 2E). Heavy-duty black polythene sheeting was applied as required to keep the submerged parts of the shoot and root in darkness.
Sleeving Pt electrodes were used to monitor continuously the ROL from the adventitious roots at approx. 2–7 mm from the tips (a) under different light regimes; (b) under different temperatures in the rooting medium; (c) under different O2 regimes around the shoot base; and (d) as gas pressures in the secondary cortex of the stem were raised artificially using compressed air applied through the acrylic connectors attached to the stem.
Anatomy
Transverse sections of stems and roots were cut either by hand or by sledge-microtome using fresh material. They were examined by light microscopy (Olympus BX40) or by fluorescence microscopy (Olympus CH with fluorescence attachment). A digital camera (Olympus Camedia 4040Zoom) was used for the photography.
Light absorption by stem tissues
To investigate the degree to which photosynthetically active radiation was absorbed by the different tissues, discs of different thickness cut from the stem surface were used. A thin coating of a clear lubricating jelly was applied to the inner surface and the disc then placed over the sensing element of a LI-COR light meter (Model LI-189). A ring of Terostat was used to prevent light leaking in though the side of the sensing unit. The light flux was measured with the stem discs held normal to the various strength of light source, and any light flux passing right through the disc was recorded.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONBACKGROUNDMATERIALS AND METHODSRESULTS AND DISCUSSIONGENERAL DISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Artificial root–stem model: effects on ROL of raised gas pressure per se, raised O2 partial pressures and partial pressurized through-flow
With the base of the model open to the atmosphere and the diffusion gradients between atmosphere and electrode surface at equilibrium, the ROL from the silicone rubber apex of the artificial root–stem model gave an electrolytic reduction current for O2 of 3·904 µA (radial O2 flux = 123·6 ng cm–2 root surface min–1). When compressed air was applied at the acrylic T-junction and the pressure raised in a stepwise manner, the ROL quickly increased and equilibrated at each stage (Fig. 3). However, each stepwise increase in flux was tiny and, even at the comparatively high pressure of 2530 Pa, ROL was only 126·5 ng cm–2 root surface min–1 (3·996 µA), just 2·35 % greater than for the original non-pressurized condition: a 0·003 % rise per 100 a. This can be accounted for almost entirely by the increased density of the gases at 2530 Pa according to the equation
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When the Terostat was removed from the basal end of the artificial stem creating a by-pass akin to leaking lenticels exposed to a gaseous head space at atmospheric pressure e.g. as in Fig. 5, the ROL increased rapidly and substantially (by 43·5 %) to reach a new equilibrium value of 177·3 cm–2 root surface min–1. This is the type of effect that we believe probably led to the increased ethane movement in the original experiments of Grosse et al. (1985
, 1985) although the pressurization in that case might have been a combination of photosynthetic O2 production, thermal transpiration and humidity-induced diffusion. In this example, the pressure was not released between stages.
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A further contrast to the minimal effect on ROL of pressurization per se is the result obtained by increasing the O2 partial pressure at the base of the model (Fig. 6): exposure to 35 % O2 increased the ROL by 46 %, and again, removing the Terostat to create a partial through-flow increased the ROL to approx. 106 % of the original diffusion-only value obtained with air around the base.
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Alder stems with artificial roots—effects of photosynthesis
Examples from two separate experiments are shown. In the first, Fig. 7, the ROL from the artificial roots was measured initially with the submerged (lower) portion of the stem in darkness and the emergent (upper) part receiving low light (approx. 30 µmol m–2 s–1 PAR) from a north-facing window. The xylem of the stem had previously been perfused with water containing 4 mM CO2. Under these conditions, the ROL was low (15 ng cm–2 min–1 in the example shown) and this indicated an O2 partial pressure of only 1·35 kPa in the secondary cortex of the stem and base of the artificial root. When the sodium vapour light was switched on to give a PAR flux of 550 µmol m–2 s–1 over the emergent part of the stem, the ROL rose to a new equilibrium value of approx. 85 ng cm–2 min–1, indicating now an O2 partial pressure of 7·5 kPa in the stem–artificial root junction. When the covers were removed, and the whole stem illuminated at 550 µmol m–2 s–1, the ROL again increased, this time to 336 ng cm–2 min–1, showing that the O2 partial pressure at the stem–artificial root junction must have risen to approx. 30 kPa (30 % O2). This could only have come about due to O2 generation, presumably by stem photosynthesis. A second artificial root attached to this stem behaved similarly, but the pressure sensors had become leaky during these manipulations and no pressure data for the stem were obtained. Earlier, however, a sensor on the upper part of the stem registered 2000 Pa (Furness Controls CPFM pressure transducer outputting to a suitably scaled chart recorder) when the whole stem was submerged and illuminated, and some bubbling from the lenticels began at approx. 1920 Pa.
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In the second experiment, the pressure sensors functioned well but the ROL data could only be collected digitally from one root because of electrical interference. However, ROL from the second root was monitored on a chart recorder and the data (not shown) followed the same pattern as for the digitally monitored root. The cutting was pre-percolated with de-ionized water containing CO2 at 6·9 mM. When the lower (submerged) portion of the twig was in darkness, and the upper (emergent) part of the shoot was receiving only north light, the ROL from the artificial roots was barely detectable (Fig. 8A). When the emergent part of the twig was illuminated (550 µmol m–2 s–1 PAR), there was a 17 min delay before there was any obvious effect on the ROL; it then rose and by 45 min had reached a quasi-equilibrium value of approx. 15 ng cm–2 min–1. Gas pressures in both the upper and lower sections of the twig were initially negative but began to rise as soon as the upper portion of the stem was illuminated. Their ultimate equilibration at between 2·5 and 3·0 kPa coincided approximately with the equilibration in ROL. Pressure in the emergent portion was greater than in the darkened submerged stem, and this could indicate some release of pressure between the two sampling points. When the covers were removed and the lower portion of the stem was also illuminated, the pressures began to rise once more and very quickly became coincident top and bottom. Unfortunately, however, the pressure recording for the upper section went off-scale at approx. 4·0 kPa and no data were available from this sensor until the pressures declined later in the experiment. The second pressure rise was approximately the same as the first (approx. 2·5 kPa) but the rise in ROL was very much greater in the second phase, equilibrating at approx. 390 ng cm–2 min–1 (Fig. 8A). This ROL value equates with a stem–artificial root junction O2 partial pressure of 32 kPa and indicates, as in experiment 1, that photosynthesis, not pressurization, was the major contributor to root aeration. We attribute the supplementary large rise in ROL to the additional photosynthetic generation and accumulation of O2 in the lower portion of the stem where its escape is hindered because it cannot readily diffuse into the water. In diffusion terms, the lower portion of the stem would therefore have become the effective O2 source and the atmosphere around the emergent shoot the principal sink. In the first stage of the experiment, the O2 generated in the upper part of the stem probably fed the lower stem section and the root by diffusion only and, judging by the almost undetectable initial ROL, the ‘effective resistance’, i.e. synergism between diffusive resistance and stem O2 consumption, was very substantial. In the second stage of this second experiment, the pressures generated did cause some obvious convective flow, because, at approx. 4·5 kPa gas bubbles began to escape from the submerged part of the stem. However, since resistance to escape will have been greater below than above the water line, it seems reasonable to assume that if there was any directional component to convective flow within the stem at this stage, it would have been directed upwards from the lower to the upper part.
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Neither pressurization nor the high values of ROL were sustained indefinitely in this experiment. After 90 min, the pressures began to fall. Shortly afterwards, this was followed by a steady decline in ROL (Fig. 8B) which continued until the lights were switched off. Bubbling was very much reduced when the internal pressures reached 4·0 kPa. When the lights were switched off, the pressure differential between the upper and lower stem sections became evident again and the pressures fell rapidly, eventually becoming negative again as at the start of the experiment. The decline in ROL also accelerated after the lights were switched off but took 20 min longer to reach its initial value and was still appreciable when the pressures had already become negative. This again indicates a lack of dependence between ROL and pressurization per se. The finally negative pressures might indicate internal stem temperatures lower than ambient resulting in an outwardly directed thermo-osmosis (Idso, 1981
; Steinburg, 1996). We are uncertain as to why pressurization and ROL were not sustained at their peak values in the light and did not investigate this further. Several reasons could be suggested: (a) CO2 availability may have begun to decline since the twigs were not re-charged with CO2-enriched water during the experiment; (b) O2 enrichment and declining CO2 may have increased photorespiration; (c) respiratory demand in the shoot might have been stimulated by substrate accumulation; or (d) substrate accumulation might have reduced photosynthesis by a negative feedback. With whole plants and adventitious roots, the ROL did not show this decline if the submerged parts were maintained at 12 °C (Figs 15 and 16).
Alder stems with artificial roots—effects of artificially applied pressure
Following on from the previous experiment, ROL was monitored as pressure from a compressed air source was applied artificially to the upper section of stem via the feed tube previously used to sense internal pressures. No pressures could be recorded from the lower stem section because of a leaky joint. The results are shown in Fig. 9. Again it is evident how little pressurization itself affects root aeration: an increase in ROL only became really obvious when the pressures had exceeded 7 kPa and a continuous bubbling from lenticels on the stem near the junction with the artificial root had begun (Fig. 9B). However, after the stem was illuminated above and below the water line to stimulate photosynthesis, ROL increased very rapidly (Fig. 9A).
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Photosynthetic rates of stem cuttings
With CO2-enriched water (6·9 µM) percolating through the xylem, the net photosynthetic rates for the stem cuttings (measured as O2 output) averaged 1·28 µmol m–2 s–1 at a light flux of 550 µmol m–2 s–1 PAR, while dark respiration measured as O2 uptake averaged 2·46 µmol m–2 s–1 (Table 1). Without CO2 enrichment, i.e. when only de-ionized water in equilibrium with air was percolated through the xylem, net photosynthesis was only marginally positive (data not shown).
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Although the net photosynthesis value for the stem is relatively low when compared with those attainable by the leaves of some herbaceous species, it is not far below rates cited for leaf photosynthesis in some deciduous tree species (Larcher, 1969
). It is interesting to note that the chlorophyll content of young aspen twigs can almost reach that of the leaves, can increase with age and develop lower chlorophyll a/b ratios indicative of becoming shade adapted (Aschan et al., 2001).
The potential for pressure development in photosynthesizing stem cuttings
When cuttings pre-charged with CO2-enriched water (6·9 mM) were submerged and illuminated, gas bubbles began to emerge and escape from the lenticels. We did not measure the O2 concentration of the bubbles, but it is clear from the artificial root ROL measurements (Figs 7 and 8) and the photosynthetic measurements that the gas bubbles would have been O2 enriched. Applied pressures were raised in 2–4 kPa steps. Such steps were sufficient to temporarily stop the bubbling and, if and when bubbling resumed, the pressure was raised again. The pressure required permanently to stop bubbling varied between cuttings but was always >15 kPa and <30 kPa and was reached within approx. 35 min, with the fastest rate of pressurization being 2 kPa min–1. We are uncertain as to why the cutting failed to pressurize beyond 25 kPa but it would seem to indicate that photosynthesis had either slowed substantially or stopped. It is quite likely that the O2 concentrations within the cutting reached levels high enough to have an inhibitory effect on photosynthesis.
The rapid development of high internal pressures can readily be explained in terms of the net rates of photosynthetic O2 output into what is a relatively small volume of gas space in the stem. The gas space is principally in the secondary cortex. The latter is approx. 200 µm thick and, from transverse sections, we estimate it to have a fractional porosity of 0·1. On this basis, the volume of internal gas space in the cutting would have been approx. 0·18 cm3.
Chloroplast distribution and light penetration of alder bark and wood
Despite the frequently brown appearance of the stems, transverse sections of stems viewed by light and fluorescence microscopy revealed the presence of chloroplasts in the secondary cortex, secondary phloem, the ray tissues of the stele and even the medulla (Fig. 10A–E). The distribution of starch grains and chloroplasts in the stele seemed to be coincident.
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We deduced from this that light must indeed be capable of penetrating even to the centre of 3-year-old alder stems and, to try to obtain some quantitative estimate of this, bark peels of different thickness and halved stem segments were affixed to the PAR sensor and the light flux penetration measured. The results are shown in Table 2.
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Most of the light (90 %) was absorbed in the periderm (i.e. phellem, phellogen and secondary cortex), and much of the remainder in the secondary phloem. Although the methodology was crude, it showed that some light did reach even into the medulla since a fully darkened sensor gave a zero reading. It is conceivable also that the results will under-estimate the flux reaching the chloroplasts deep within the stem since the sensor averages the flux over the sensor disc whereas it is most likely that the cell walls will function as a fibre-optic system.
Effects of light, pressure, root temperature and O2 concentrations around the shoot on ROL from the adventitious roots of whole plants
Several whole plants were monitored in a variety of ways and experimental assemblies. Experiments on two plants will be described.
Plant 1
The plant shown in Fig. 10F was monitored with its roots in the vessel shown and later in a water-jacketed container to cool the root system. A pressure delivery connector was affixed to the stem at a point 5 cm above the supporting bung (and agar surface) and just above an acrylic sleeve for applying O2 to the stem base. Two roots (13·5 and 12·5 cm) were fitted with cylindrical Pt electrodes and the whole assembly was positioned relative to the sodium vapour light, mirror and north-facing window as in Fig. 2E.
The first observations were made with the roots and shoots at room temperature (i.e. 22 °C). With only north light (50 µmol m–2 s–1 PAR) incident on the shoot above the water line and the submerged parts in darkness, no ROL was detected (Fig. 11A). Increasing the light flux to the emergent shoot to 550 µmol m–2 s–1 had no detectable effect on the ROL from the roots but, when the covers were removed, exposing the submerged parts to light, the ROL soon increased and rose rapidly and levelled out at approx. 12 ng cm–2 min–1 (Fig. 11A). Although the plot indicates a very significant rise, it should be realized that 12 ng cm–2 min–1 is a relatively low value of ROL for a wetland plant root. For a rice root of this length, a value of 80–100 ng cm–2 min–1 would not be exceptional. With alder, however, although the roots were at least partially aerenchymatous (Fig. 10H, I), their apical parts were not and their point of origin was 6 cm below the water table and the secondary cortex of the submerged stem was not aerenchymatous.
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From the earlier observations on the stem cuttings, we attribute the rise in ROL chiefly to photosynthetic O2 production in the submerged parts of the stem. However, ROL soon declined and, after approx. 45 min, had almost halved in value. When the light was switched off, and despite some north light reaching the submerged parts, the ROL became undetectable once more. A second exposure to the high light regime also produced a rapid rise in ROL (Fig. 11B) but the peak value was lower than before and again the ROL soon began to decline once more. On this occasion, the light remained on for longer and the ROL had fallen by 80 % before the light was switched off. As with the stem cuttings and their artificial roots, we are still uncertain as to what caused the decline in ROL. It could have been that there was insufficient internal CO2 to sustain photosynthesis, and so for a third and fourth light dark cycle we charged the agar around the basal submerged region of the stem with 20 mM CO2. This did not have any obvious enhancing effect on the ROL (Fig. 11C) but it may have been insufficient to raise internal CO2 levels. ROL could also have declined for other reasons, e.g. an increase in stem and/or root respiration. This could have been brought about by an increase in root/stem temperatures or by increased availability of respiratory substrate arising from stem photosynthesis. The development of a barrier to O2 loss from the root is also a possibility. However, the following manipulations subsequently performed on the same plant and monitoring the same roots do not seem to indicate the existence of much of a barrier.
When O2 was circulated through the acrylic collar on the emergent stem base, an increase in ROL was detectable within 3 min (Fig. 12) and after 48 min it had reached 42 ng cm–2 min–1 and was still rising. When the emergent shoot and the submerged basal portion of stem were illuminated, the ROL rose more rapidly and at approx. 80 ng cm–2 min–1 had still not fully equilibrated when the experiment was halted. The rather slow equilibration when the O2 was applied may have been a function of such a large and complex root system.
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Lowering the temperature of the submerged parts also had a very significant effect on ROL. The same plant was transferred to a container with a cooling water jacket and ROL was measured with the emergent stem and the basal 5 m of submerged stem receiving only north light. The data show (Fig. 13) that the ROL from the apex of an 18 cm long root (root–shoot junction at 5 cm below the water line) was initially undetectable at 22 °C. However, once the medium began to cool, ROL soon became detectable. By the time that the whole medium had reached approx. 12 °C (after 140 min), the ROL had begun to equilibrate at approx. 13–14 ng cm–2 min–1. Subsequently, the medium was allowed to return to room temperature (22 °C) and ROL became undetectable once more (Fig. 13 inset).
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Since the cooling of the root system had produced detectable ROL without the need for high intensity illumination to emergent or submerged sections of stem, and since A. glutinosa normally inhabits rather cold wet soils, subsequent experiments were all performed with the rooting medium held at 11–12 °C. First, using the same plant and the roots at 12 °C, the effect on ROL of artificial pressurization of the stem was examined. The results are shown in Fig. 14. Unfortunately, the submerged sensor point had failed so that only the applied pressure could be recorded. However, it was clear from the bubbling that took place from the basal parts of the submerged stem that pressurization within the stem, and presumably also the root, was substantial. As with the artificial stem–root model, and the stem cuttings with artificial roots, pressurization per se had little effect on ROL. The increase in applied pressure from 6 kPa to approx. 23 kPa only raised the ROL from the living root by 4 % (i.e. from 17·2 to 17·9 ng cm–2 min–1). This is an even smaller rise than was predictable based on the perceived density change due to pressurization, but this was probably due to some pressure leakage between the application point and the root base; hypertrophied lenticels may have contributed to this. Nevertheless, compared with the increases in ROL that were recorded on exposure (a) to light, (b) to O2 around the emergent shoot base and (c) to cooling, the effect of pressurization per se is almost undetectable.
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Subsequently, with no pressure applied to the stem, exposure to the sodium vapour light raised the ROL to 39 ng cm–2 min–1 and, unlike previous experiments where roots were at room temperature, ROL did not subsequently decline until the lights were switched off again (data not shown). This observation was repeated over two daily cycles (results not shown). Internal pressures recorded within the emergent stem did not exceed 26 Pa in the light. This is a much lower figure than obtained with the cuttings, but whole plants are more likely to have imperfections such as dead buds and small branch scars which are more leaky than the phellogen, and this would reduce the ability for pressurization in the secondary cortex. They also have hypertrophied lenticels above the water line that may be more leak