Reversible interruption of xylem sap flow by oxygen depletion in the roots
At conditions of unimpeded sap flow the leaf area-specific flow rates of B. pubescens saplings (1–1.5 mmol H2O m−2 s−1) were highly consistent with flow rates derived from mature B. pendula trees (1.2 mmol H2O m−2 s−1), naturally grown in a mixed birch-pine forest (Backes 1996). Similar rates of FLA of 1.8–3.6 mmol H2O m−2 s−1 at maximum on sunny days were also reported for mature B. pendula trees (Ladefoged 1963).
In this study xylem sap flow was reversibly reduced, and even stopped, by a combined treatment of warming and oxygen depletion in the root space. Three experimental findings are of interest: (1) warming up to 32 °C caused an initial increase in FLA by 34% compared with previous rates at 17 °C, followed by an increase of stomatal conductance and photosynthesis; (2) restriction of sap flow occurred first after 36.5 h from the onset of oxygen depletion; and (3) sap flow recommenced within 20 min after re-aeration of the roots.
Because the apoplastic path of radial water transport across the root cortex towards the xylem is interrupted or at least drastically reduced at the endo- and often at the exodermis, all water molecules must pass cell membranes. Thus, the modes of water movement across membranes are of major importance for water uptake by the roots (Steudle 1997, 2000a). Aquaporins contribute considerably to the hydraulic conductivity of plant cell membranes (Maurel 1997; Steudle 1997; Schäffner 1998; Tyerman et al. 1999). Here, water supply to the shoot is under metabolic control, and becomes affected by temperature and oxygen supply. Regulation of radial water flow by aquaporins is based on opening or closing of existing aquaporins or by variation of their density in membranes (Steudle 2000b). The rapid recommencement of sap flow in B. pubescens saplings within 20 min after re-aeration points to a reversible metabolic regulation of transmembrane water flow rather than an increase in aquaporine density.
Oxygen deficiency not only affects radial water transport by a changed activity of aquaporins – lack of phosphorylation de-activates water channels (Johansson et al. 1996; Kjellbom et al. 1999) – but also reduces energy-dependent active ion pumping. This affects the passage of water by changing the osmotic gradient across plasma membranes. In primary roots of Zea mays that were grown hydroponically anoxia caused a decrease of the ATP/ADP ratio by 64% after 15 h of deoxygenation (Birner & Steudle 1993). These authors stated that under anaerobiosis the reduction in energy charge of the root tissues causes a successive switching off of ion pumps located at the xylem and at the cortical plasmalemma, respectively.
The experimental procedure of oxygen depletion in the root space applied in this study provides some evidence that radial movement of water towards the xylem in roots of B. pubescens is primarily under metabolic control. The increase in temperature from 17 to 32 °C caused an overall increase in root metabolism including those processes being involved in water transport as long as oxygen availability did not limit respiratory ATP supply. The gradual depletion of oxygen in the root space must have affected root respiration and thus, the energy charge of the cells most likely decreased as well. Due to a low energy charge both the de-activation of aquaporins, and loss of active ion pumping are probably the key-processes for the gradual interruption of xylem sap flow. The fact that about 90% of the leaves of either tree could not survive the water-stress conditions induced by root hypoxia may prove that interruption of sap flow was complete. It may indicate further that water transport bypassing the root endodermis was negligible. Finally, the rapid and full recommencement of sap flow after re-aeration of the roots within 20 min, and the resulting stomatal opening of remaining leaves also provides evidence for a metabolically induced interruption of water transport which was neutralized when oxygenation set in.
The role of xylem sap flow for the oxygen status of the sapwood
In June, the sapwood of B. pubescens saplings showed a high oxygen status during unimpeded sap flow. At standard temperature and pressure (T = 20 °C, P = 100 kPa) daily maxima of [O2] of up to 245 µmol O2 L−1 occurred. Such values are in line with those reported for different tree species when the growing season begins (Table 1). Even the daily minimum of [O2] was high (about 210 µmol O2 L−1), whereas in spring, in mature B. pendula the minimum value dropped to 70 µmol O2 L−1 (Gansert et al. 2001). Similar minima were also reported for Picea abies, Quercus robur or Acer platanoides during the growing season in July and August (Table 1). Although the diurnal span of concentration of dissolved oxygen in young B. pubescens was less distinct than in mature B. pendula, the daily course with an early morning maximum and minimum in the late afternoon was basically the same. However, due to constant temperature conditions in the climate chamber, the nocturnal rise in [O2], typically observed under natural conditions, was missing.
The gradual reduction of sap flow provided a quantitative assessment of its effect on oxygen concentrations and flow rates in the sapwood of B. pubescens saplings. Because the day/night temperature regime variation was nearly constant over the 3 d period from day 6–8 of the experiment, the decrease in daytime [O2] from 75% (188 µmol O2 L−1) at unimpeded sap flow to a minimum of 30% (77 µmol O2 L−1) when sap flow was interrupted could not be attributed to an increase in respiratory oxygen consumption. Rather, this provides evidence that sap flow accounts for some 60% of the oxygen concentration in the sapwood. Undoubtedly, sap flow rate, oxygen loading of xylem sap and oxygen withdrawal from it by oxidative respiration of living cells of the shoot, are factors which synergistically influence the extent of oxygen available from the aqueous transport path. The observed oxygen depletion down to 77 µmol O2 L−1 when sap flow ceased is comparable with the low value of 42 µmol O2 L−1 observed in B. pendula during flushing; namely when sap flow was negligible (Gansert et al. 2001). From wood anatomical studies of B. pendula and B. pubescens (Braun 1970; Grosser 1977; Schweingruber 1978) it can be assumed that, with the exception of multiseriate rays, the parenchymatous tissues of birch sapwood have little or no contact with the intercellular gas-space continuum. The drastic reduction of [O2] in B. pubescens caused by interruption of sap flow supports this assumption. Investigations on oxygen supply to the sapwood of Olea europaea saplings also indicated that about 80% of the oxygen concentration in the sapwood was delivered by xylem sap flow (Mancuso & Marras 2003). Interestingly, a similar pattern of gradual oxygen depletion in the sapwood of O. europaea was observed with the strongest hypoxia on the third day of root anaerobiosis.
However, radial gaseous diffusion of atmospheric oxygen through lenticels and the cambium into the sapwood cannot be left out of consideration. For example, a nocturnal increase of [O2] could be measured on days 7 and 8 of the experiment, irrespective of sap flow being reduced to half the rate of previous nights or even completely blocked (Figs 4d & 5). Under these conditions radial gaseous diffusion of oxygen into the sapwood accounted for 0.2–0.5 nmol O2 L−1 s−1. For comparison, participation of sap flow after re-aeration accounted for a specific oxygenation rate of 2 nmol O2 L−1 s−1 per mmol H2O m−2 LA s−1 transpired, which is one order of magnitude higher than the diffusion component of oxygen transport. Taking into account the anatomical features mentioned above, and experimental findings that both CO2 efflux from stems of different birch species such as B. pendula or B. ermanii (Levy et al. 1999; Gansert unpubl.), and O2 exchange across the stem surface (see below) are affected by sap flow, the cambium of birch can hardly be seen as an impervious sheath. Rather, it appears to allow the passage of gases preferentially through the intercellular gas-spaces of the rays (Larson 1994).
The oxygen relations in the sapwood of B. pubescens saplings presented here substantiate the importance of sap flow for oxygen supply to the sapwood as has been shown earlier in the field for mature B. pendula (Gansert et al. 2001). It also supports the concept of a dual transport system that supplies wood parenchyma of trees with oxygen via radial gas diffusion and axial flow of oxygen dissolved in the xylem sap as suggested earlier (Hook et al. 1972; Gansert et al. 2001). During daytime, the xylem sap flow appears to be the major path for transport of dissolved oxygen axially through the sapwood. During night-time, when sap flow approximates zero, the gaseous path for O2 transport, driven by diffusion gradients radially through intercellular gas-spaces prevails (Gansert et al. 2001). The water-saturated cell walls of the apoplast function as the primary absorbing matrix for gaseous oxygen supplied by radial gaseous diffusion. Thus, xylem sap can be enriched with oxygen using the overall surface of the above-ground woody cormus as the absorbing area. On the other hand, xylary diffusion namely the axial transport of dissolved oxygen in the xylem sap of tracheids and vessels only by diffusion, can hardly be considered as an efficient pathway for oxygen supply from the roots to the above-ground sapwood when there is no sap flow at night. According to Fick's second law, movement of oxygen in water over a distance of 1 m driven by diffusion alone takes several years, depending on the concentration gradient, so that this pathway can only be effective over short distances smaller than 100 µm (von Willert et al. 1995).
Respiration of wood parenchyma represents an intrinsic biogenous factor which complicates quantitative estimates of diurnal oxygen supply to the sapwood from measurements of the local [O2] inside the stem. Respiratory oxygen consumption of parenchymatous tissues of the wood strongly depends on their quantity and actual metabolic conditions with respect to growth, storage or secretion activities. During the day, respiratory oxygen consumption exponentially increases with a rise in temperature, and the reverse pattern is shown at night. Thus, O2 consumption approximates the daily minimum before dawn. The oxygen status of the sapwood is therefore a resultant of oxygen consumption (O2 sink) by respiration of wood parenchyma as a function of temperature and metabolic condition, and oxygen supply (O2 source) via the aqueous and gaseous pathways. The contribution of the aqueous path in the form of xylem sap flow depends above all on canopy transpiration which is a function of light incident on the leaves and the leaf-to-air water vapour pressure deficit (VPD). Gaseous diffusion is driven by concentration, temperature and pressure gradients. Therefore, the diurnal variation of the source–sink relation of oxygen in the sapwood is differentially affected by abiotic variables. The stoichiometrical approach on the relation between CO2 efflux and O2 consumption applied here, provided an estimate of oxygen exchange across the stem surface which seems to be physico-chemically coupled with xylem sap flow. Principally, in the absence of bark photosynthesis the stem was never a net source for oxygen. However, enhanced nocturnal O2 efflux contributed to smaller O2 deficits in the cuvette atmosphere than during daytime. This observation is in line with a nocturnal increase in [O2] of xylem sap measured under field conditions in spring and summer (Gansert et al. 2001). Conversely, oxygen deficits higher than those solely due to respiratory oxygen consumption mark the existence of an increasing endogenous oxygen sink as was measured in the sapwood when sap flow was interrupted. These results may indicate that xylem sap flow not only affects the oxygen status of the sapwood itself, but also has an effect on radial oxygen transport across the stem surface. Exposure of mature stems of B. pendula to a hypoxic atmosphere and prevention of bark photosynthesis caused an oxygen efflux from the stem which was coupled with xylem sap flow (Gansert & Burgdorf, unpublished). Hence, the cambium of birch species (B. pubescens, B. pendula, and B. ermanii) is not an impervious sheath, but allows radial fluxes of O2 and CO2 into and out of the stem through the intercellular gas-space continuum.
Although Bailey (1913) clearly recognized the importance of the transpiration stream as an aqueous pathway for gases 90 years ago, the quantitative analysis of the role of xylem sap flow for endogenous source-sink relations of O2 and CO2, and gas exchange between woody plant parts and atmosphere has just begun. The causal understanding of these relations will depend on the success in quantitative differentiation between biogenous processes such as respiration and bark photosynthesis, and physico-chemical processes such as diffusion, solubility and dissociation or flow in the gaseous and aqueous phase in woody parts of arborescent plants.