Journal of Experimental Botany, Vol. 53, No. 368, pp. 559-563,
March 1, 2002
© 2002 Oxford University Press
A new method to determine the oxygen concentration inside the sapwood of trees
Institut für Botanik, Universität für Bodenkultur Wien, Gregor Mendel Str. 33, A-1180 Wien, Austria
Received 19 February 2001; Accepted 28 September 2001
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Abstract |
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Research into the short-term fluctuations of oxygen concentrations in tree stems has been hampered by the difficulty of measuring oxygen inside tissues. A new method, which is based on fluorescence quenching of a ruthenium complex in the presence of oxygen, has been applied to measure changes of oxygen concentration in the sapwood of trees. During a field day-course oxygen increased with the radiation load and fell during the night (in Fagus orientalis from 20.3% in the afternoon to 17.5% in the morning next day). In a greenhouse experiment the sapwood oxygen concentration of Laurus nobilis could be influenced by flooding the root system. The very fast response, high resolution (better than 0.1%), easy calibration, and dependence only on oxygen and temperature make the technique well suited for measurements of oxygen concentrations in the sapwood.
Key words: Fibre optic oxygen probe, oxygen concentration, sapwood, transpiration.
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Introduction |
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Oxygen supply to plant tissues sometimes becomes difficult, especially for aquatic plants and for subterranean organs on water-logged soil. A number of anatomical responses and physiological adaptations increase the anoxia resistance in such oxygen-depleted soils (Vartapetian and Jackson, 1997
). Far less investigated is the oxygen status of massive above-ground organs, such as tree stems.
There are two possible pathways for supplying O2 to the living cells in the sapwood: radially by diffusion through phloem and cambium, and vertically with the transpiration stream from the soil through the roots. Oxygen often cannot diffuse in the gas phase across the cambium, since in most species this meristem is free of intercellular spaces (Hook et al., 1972). Furthermore, an active cambium and its growing daughter cells are likely to consume most of the O2 arriving from the outside in the liquid phase, so that oxygen required for the respiration of living xylem cells must be obtained from the transpiration stream (Bailey, 1913; Hook et al., 1972) in equilibrium with xylem air spaces (von Höhnel, 1879; Haberlandt, 1918). On well-aerated soils, transpiration may play a vital role in supplying oxygen to xylem and inner cambial zones during the growing season (Hook et al., 1972; Shain and Graham Mackay, 1973; Eklund, 2000).
The respiration of different regions of the sapwood and cambium zones of Pinus radiata D. Don. were shown to be different for different seasons (Shain and Graham Mackay, 1973). Eklund found that, in winter, the oxygen levels in the sapwood of Picea abies (L.) Karst. were close to ambient free air (20%), but after the onset of cambial growth decreased to values below 5% in summer (Eklund, 2000). When the growth rate declined at the end of summer, oxygen concentration started to increase, returning to almost ambient in autumn. In all species investigated (Picea abies [Eklund, 1990; Eklund et al., 1998], Quercus robur L., Acer platanoides L. [Eklund, 1993], and Acacia mearsii de Willd. [Carrodus and Triffett, 1975]) a decrease to very low oxygen levels during the period of intensive growth has been reported. Eklund furthermore found that the oxygen concentration in the sapwood of Picea abies decreased under drought, when transpiration was reduced (Eklund, 2000), adding strength to the hypothesis by Hook et al. of oxygen supply via the transpiration stream (Hook et al., 1972).
Techniques for sampling gas from sapwood are problematic for two main reasons (Carrodus and Triffett, 1975): First they depend on the withdrawal of gas samples through a syringe under vacuum from a system where the volume of dead spaces is significant and leaks are very difficult to detect, and second they are not useful for sampling well-defined regions in the stem. Furthermore, vacuum sampling may lead to errors because of the neglect of gas retention by the tissues and of dissolved gas passing from the tissues to the gas sample (Kenten, 1956). The most recent method has been to drill through the bark and to position the open end of a steel tube 10 mm inside the cambium, exposing 1 cm2 of wood to the space in the tube. This gas phase is sampled every 2 weeks and the composition determined using gas chromatography and mass spectrometry (Eklund 1990, 1993, 2000; Eklund et al., 1992). Apart from the above shortcomings, these methods are very laborious and time-consuming and do not permit the registration of short-term oxygen fluctuations. This paper describes a new method for determining oxygen concentrations in sapwood, with an optical sensor based on fluorescence quenching which does not require the extraction of gas and allows changes in oxygen concentrations to be monitored continuously. The observations of short-term fluctuations in oxygen concentration provide evidence for the possible role of transpiration in the supply of oxygen to the sapwood.
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Materials and methods |
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Oxygen determination
For the oxygen measurements a fibre optic oxygen sensor (FOXY-R, Ocean Optics Europe, Duiven, The Netherlands, 1 mm core diameter fibre in 1.59 mm stainless steel ferrule) was used. The probe consists of a glass fibre coated on the distal end with a thin layer of a hydrophobic sol-gel material covered with a gas-permeable membrane Teflon coating. A ruthenium complex is trapped in the sol-gel matrix, and effectively immobilized and protected from water. A blue light (475 nm) LED (LS-450, Ocean Optics Europe) excites the ruthenium complex, which fluoresces at about 600 nm. The method is based on the decrease of emission intensity (quenching) of the fluorescent dye in the presence of oxygen, which does not consume any oxygen. If quenching is entirely diffusional, the fluorescence intensity is related to the quencher concentration by one of the Stern–Volmer equations (Bacon and Demas, 1991
; Watkins et al., 1998).
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Calibration of the probe
The probe was calibrated for oxygen following the method described in the manual. A two-point calibration was done with nitrogen gas (0% O2) and air (20.9% O2) at room temperature.
A temperature drift curve was measured for each oxygen calibration (one in September and the other in November) using a thermocouple and the probe fitted through a rubber stopper in a glass tube covered with aluminium foil to prevent external radiation from reaching the sensor in a water bath, with slowly changing temperatures. For the first curve the temperature was varied from 13 °C to 22 °C and for the second from 4 °C to 34 °C. Both these calibrations were highly linear: for the first run r2=0.984 (n=36) and for the second r2=0.990 (n=104). Figure 1 shows the drift calibrations at the temperature interval of interest (from 12 °C to 24 °C). These temperature drift curves were used to correct the oxygen values measured in the sapwood. The regression lines were used to estimate the drift of the sensor from 20.9% oxygen concentration for each temperature recorded. The regression of September was used for the September measurements and the regression of November for the December measurements. The drift values, negative or positive, were added to the oxygen measurement recorded for each corresponding temperature. The difference in the two regression lines of the drift calibrations is explained by a difference of less than 1 °C in room temperature during the oxygen calibration of the system.
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) and in November (
) using a thermocouple and the probe fitted through a rubber stopper in a glass tube in a water bath, with slowly changing temperatures.
Plant material and experimental setup
Sample trees were selected in the arboretum of the University of Agricultural Sciences, Vienna. On 31 August, specimens of Fagus orientalis Lipsky (diameter at breast height, DBH, 90 cm), Carya ovata (Mill.) K. Koch (DBH 51 cm) and Larix sibirica Ledeb. (DBH 17 cm) were measured. Stems were bored at breast height through the bark with a drill of 8.5 mm diameter until the sapwood was reached and then with a 3.5 mm diameter drill a further 3 cm inside the sapwood where the oxygen sensor was positioned and sealed in place with a rubber stopper and silicone grease. Temperature was recorded with a thermocouple in a parallel, 1 mm diameter hole near the sensor. On 9 September, F. orientalis was bored in the morning at 09.30 h and the oxygen concentration, sapwood temperature and total irradiance (LI-188 radiometer, Li-Cor, Lincoln, NE) followed during the entire day until 19.00 h. Measurements were resumed early next day at 08.00 h and continued until 11.30 h. The first day was warm and completely overcast, the second clear, cold and sunny.
To test the hypothesis that soil oxygen affects the O2 concentration in the stem, an experiment in the greenhouse was designed. A 21-year-old Laurus nobilis L. tree (7.2 cm stem diameter at the base) was drilled 1.7 cm deep at 14.5 cm from the soil. The oxygen sensor was screwed into the wood with the help of a custom-made steel sealing jacket (Fig. 2) to prevent any outside oxygen from leaking in. A small hole (1.5 cm deep) was drilled nearby, where a thermocouple was positioned and sealed with silicon grease. The light in the greenhouse was increased with an OSRAM HQL 400 W lamp and a halogen photo lamp. The measurements were conducted between 18 December and 22 December. Air temperature and relative humidity were measured with an RMP45 sensor (Vaisala, Helsinki, Finland) and radiation with an 8101 pyranometer (Schenk, Vienna, Austria) and logged with a CR10X datalogger (Campbell Scientific, Logan, Utah, USA) every 15 min. Sapflow was measured on a branch, 2.7 cm in diameter, with a T639.1 stem heat balance sapflow system (EMS, Brno, Czech Republic), logged every minute and stored as 15 min averages. Oxygen content and temperature of the potted L. nobilis stem were measured under different soil conditions. On 18 December the tree was not watered. The following days the pot was repeatedly flooded with tap water and allowed to drain out freely.
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To test the hypothesis that water content in the stem affects the speed at which oxygen diffuses in the xylem, a semicircular air-dried slab of a Fagus sylvatica L. stem (158 g, 6 cm radius and 3.5 cm thickness) was infiltrated under a vacuum of 1.2 kPa absolute pressure at 25 °C for 5 d with degassed deionized water. The hydrated weight was 285 g. The oxygen sensor was inserted through the bark side in the same way as for the trees in the arboretum.
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Results |
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For the trees in the arboretum, oxygen concentrations in the newly drilled holes filled with ambient air were followed, after plugging and sealing the probe, for all the trees until the oxygen concentrations in the hole had equilibrated. The time to reach equilibrium was 575 s for F. orientalis, 545 s for C. ovata, and 408 s for L. sibirica. The final value of oxygen concentration was 13.3% for F. orientalis on 31 August and 18% on 9 September, for C. ovata 16.9% and for L. sibirica 19.0% (Fig. 3
).
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), Carya ovata (
), and the water-infiltrated Fagus sylvatica wood (
).In the arboretum, oxygen concentrations in F. orientalis (after equilibration with the surrounding wood) followed the accumulated daytime radiation and temperature changes (Fig. 4
). The values dropped during the night, becoming similar to those recorded on the morning of the first day (Fig. 4).
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In the potted Laurus nobilis in the greenhouse, the oxygen concentration decreased at first from 16.7% in the late evening to 16.1% during the night. Next day, during the first flooding, the oxygen dropped from 16.4% at the beginning to 14.4% after 5.5 h of flooding: After draining, the concentration increased to 17.7% early next day (Fig. 5
). The second flooding (Fig. 5) showed the same trend, with oxygen decreasing to 12.6% during the flooding period and after draining increasing to 16.8% during the day. At midday next day it reached 18.5%. The oxygen supplied to the branch by transpiration can be calculated from stem temperature, oxygen concentration in the drilled hole and sapflow (Fig. 5).
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The oxygen concentration of the sap in equilibrium with the air in the hole was calculated by means of Henry's law. Henry's law constants were calculated by linear interpolation of published data (Geankoplis, 1993
) for each temperature recorded. The product of the dissolved oxygen concentration, calculated via Henry's law, and the transpiration flux give the oxygen flux into the branch owed to transpiration (Fig. 5
). Integrating the oxygen flux over whole days, the total amount of oxygen transported by the transpiration stream into the 2.7 cm diameter branch was obtained: on 18 December in the afternoon 0.35 mg, on 19, 20 and 21 December 5.50 mg, 6.62 mg and 7.92 mg, respectively, and on 22 December until 14.15 h, 4.02 mg.
The degassed and infiltrated beech wood reached an equilibrium oxygen concentration of 19.5% in the hole 61 min after the sensor was sealed in place, about 10 times slower than for the tree samples in the arboretum. Measurements used the rubber stopper seal and the steel screw arrangement (Fig. 2), both with the same result, which shows that both procedures prevent leakage of outside oxygen.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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These data show that the fibre optic oxygen probe permits rapid and easy measurement compared to the methods previously used and allows short-term variations and long-term continuous changes to be registered. The very fast response, high resolution, easy calibration, and dependence of the signal only on oxygen content and temperature makes the technique well suited for measurements of oxygen concentrations in the stem and other massive plant parts and promises to become a useful tool for measuring oxygen dynamics in plants.
The equilibrium oxygen concentration values in sapwood of several species in the arboretum are similar to the values presented (Eklund, 2000) for the same time of the year. The data show the occurrence of daily changes, plausibly caused by transpiration and temperature (Fig. 4).
In the greenhouse experiment, before flooding, the variation in the stem oxygen content was small (Fig. 5). Flooding and thus reduced root oxygen availability subsequently resulted in substantial changes in the stem oxygen content, suggesting that the soil oxygen content plays a major role in the supply of oxygen to the stem. During subsequent draining, oxygen started from low values and increased even at night (Fig. 5), suggesting that diffusion also may have a role in supplying oxygen to the wood if the difference in oxygen concentration between the stem and the outside becomes large. Even though radiation and temperature were generally low, oxygen content changed and it was possible to estimate the oxygen flux for the branch. Furthermore, the data suggest a possible relationship between sapflow and changes in oxygen content because oxygen content always increased with increase of sapflow for all the days recorded (Fig. 5).
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Acknowledgments |
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We are grateful for the constructive criticism by John Grace and two anonymous reviewers on a previous draft of this work. We thank Jószef Kósa for library assistance and Elfriede Zeisl for expert laboratory work. This work was partly funded by the Austrian Science Foundation's Special Research Program 008 (Forest Ecosystem Restoration).
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Notes |
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1 To whom correspondence should be addressed. Fax: +431476544504. E-mail: adelhier{at}edv1.boku.ac.at
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