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Fractionation of oxygen isotopes by root respiration: Implications for the isotopic composition of atmospheric O2
Geochimica et Cosmochimica Acta, Vol. 65, No. 11, pp. 1695–1701, 2001 Copyright © 2001 Elsevier Science Ltd Printed in the USA. All rights reserved 0016-7037/01 $20.00 ⫹ .00
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PII S0016-7037(01)00567-1
Fractionation of oxygen isotopes by root respiration: Implications for the isotopic composition of atmospheric O2 ALON ANGERT* and BOAZ LUZ The Institute of Earth Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel (Received August 1, 2000; accepted in revised form January 18, 2001)
Abstract—The ratio of 18O/16O in atmospheric oxygen depends on the isotopic composition of the substrate water used in photosynthesis and on discrimination against 18O in respiratory consumption. The current understanding of the composition of air O2 attributes the magnitude of the respiratory fractionation to biochemical mechanisms alone. Thus the discrimination against 18O is assumed as 18‰ in normal dark respiration and 25‰ to 30‰ in cyanide resistant respiration. Here we report new results on the fractionation of O2 isotopes in root respiration. The isotopic fractionation was determined from the change in ␦18O of air due to partial uptake by roots in closed containers. The discrimination in these experiments was in the range of 11.9‰ to 20.0‰ with an average of 14.5‰. This average is significantly less than the known discrimination in dark respiration. A simple diffusion-respiration model was used to explain the isotopic discrimination in roots. Available data show that O2 concentration inside roots is low due to slow diffusion. As a result, due to diffusion and biological uptake at the consumption site inside the root, the overall discrimination is small. Root respiration is an important component of the global oxygen uptake. Our new result that the discrimination against 18O is less than generally thought indicates that the mechanisms affecting ␦18O of atmospheric oxygen should be re-evaluated. Copyright © 2001 Elsevier Science Ltd In recent treatments of past variations in the Dole effect, it was assumed that global terrestrial respiration preferentially discriminates against 18O by 18‰ with respect to 16O (Beerling, 1999; Bender et al., 1994; Malaize et al., 1999). This value is based on measurements in isolated plant organs (Guy et al., 1993; Guy et al., 1989). Epstein and Zeiri (1988) and Guy et al. (1989) noted that if O2 diffusion to the consumption site is limiting, the discrimination of a system will depend not only on the discrimination in the consumption process but also on the discrimination in diffusion, and on the relative rates of consumption and diffusion. In an extreme case where diffusion is very slow in comparison to respiration, oxygen level in the consumption site is near zero, and any O2 entering is immediately respired. The discrimination in this case is the discrimination in diffusion since no discrimination in respiration takes place. Bender et al. (1994) recognized the role of diffusion limitation on oxygen isotope discrimination in their treatment of the Dole effect. They suggested that in the ocean the role of diffusion limitation is negligible because most of the respiration takes place by small organisms in which the ratio of surface area to volume is large. However, this limitation cannot be neglected in roots where O2 supply to the consumption site is slowed down by low diffusivity of root tissues (Armstrong et al., 1994; Ikeda and Nakamura, 1996). Hence, we expect the overall discrimination against 18O in root respiration to be lower than in dark respiration alone (18‰ in cyanide-sensitive respiration (Guy et al., 1989)). On the other hand, part of the respiration in roots may be through the cyanide-resistant pathway (alternative pathway) (Millar et al., 1998), in which the discrimination is 25‰ to 30‰ (Guy et al., 1993; Robinson et al., 1992), and an increase in the total discrimination is expected if this mechanism is engaged. Recognizing the importance of the Dole effect in understand-
1. INTRODUCTION
Changes in the 18O enrichment of air O2 with respect to ocean water (“Dole effect”) can be used to infer past variations in the ratio of marine to terrestrial biospheric production, related to past climatic changes (Bender et al., 1994; Malaize et al., 1999). However, to derive quantitative estimates, it is necessary to gain better understanding of the basic mechanisms affecting the isotopic composition of O2 during its photosynthetic production and respiratory consumption. There is convincing evidence that photosynthesis produces O2 of identical ␦18O as of the substrate water (Guy et al., 1993), whereas respiration preferentially removes the lighter isotope (Guy et al., 1989; Lane and Dole, 1956). As a result, atmospheric O2 becomes enriched in 18O with respect to ocean water (Dole, 1935). O2 produced by the terrestrial biosphere has higher ␦18O than ocean water because the substrate—leaf water— becomes enriched in 18O due to evapotranspiration (Dongmann, 1974). Thus increased Dole effect can be interpreted to indicate increased terrestrial/marine production and visa versa. However, quantifying the past ratio of terrestrial to marine productivity is difficult, and even the present-day Dole effect (23.5‰ with respect to SMOW (Kroopnick and Craig, 1972)) is not very well explained from the current understanding of globally important isotope fractionation mechanisms (Bender et al., 1994). Soil respiration is a major component of the global carbon cycle and roots contribute 30 to 70% of total soil respiration (Raich and Schlesinger, 1992). Therefore a substantial fraction of the global O2 consumption is accounted by root respiration with significant influence on the Dole effect.
*Author to whom correspondence should be addressed (alon_a@ mail.com). 1695
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Fig. 1. Schematic illustration of the incubation chamber and the sampling method.
ing past global changes, it is important to provide improved parameters of O2 isotope discrimination. In the present study we derived such information from laboratory experiments with intact roots. 2. EXPERIMENTAL METHODS 2.1. Incubation of Roots Roots of Philodendron plants and wheat seedlings were incubated in closed chambers at various experimental conditions (Table 1), and the respiratory fractionation was calculated from the change in [O2] and ␦18O of O2. Most of the experiments were conducted with intact plants, and the area around the stems was sealed with Aquaseal威. In addition, the incubation chambers were submerged in water to avoid possible air leaks. In an attempt to minimize bacterial respiration in the incubation chambers, the roots were placed in a medium with minimum organic content. This medium was either sand-vermiculite mixture enriched with nutrient solution, moist air or distilled water. In the latter, changes in dissolved O2 were monitored. To keep the conditions close as possible to natural ones, and to prevent anaerobic environment, the level of O2 in all the experiments was rarely below 70% of ambient air and never below 50%. In the short-term incubations (⬃ 10 h) the specimens (either whole
plants or detached roots) were transferred from the growth medium to the incubation chamber, and were used only for a single experiment. The incubation chamber was sampled at the end of the experiment. In these short-term incubations the medium containing the roots was either moist air or distilled water. Long-term incubations were also conducted. It was thought that in long term experiments the plants would acclimatize to conditions in closed chambers. On the other hand, we could not eliminate possibly significant bacterial growth in these experiments. In the long term incubations, Philodendron roots were placed in sand-vermiculite mixture enriched in nutrient solution. The roots were then sealed for two months in an incubation chamber. Between experimental runs, the chamber headspace was flushed continuously with outside air for at least 24 h. In each run, that lasted several days, the chamber was sealed and samples were taken daily. In another long-term incubation experiment, Philodendron was grown in water for two months and O2 concentration was kept near saturation by continuous bubbling of air. Respiratory discrimination was determined by sealing the root chamber for 4 h and [O2]aq and ␦18O were measured at the beginning and the end of this incubation period. The incubation temperature was changed to induce varying respiration rates. In some of the long term experiments excessive CO2 rise was eliminated by its reaction with KOH solution (20M). The latter was held in an airtight vessel connected to the root chamber. To eliminate the possibility of admixture of photosynthetic O2 through the stem, the
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Table 1. Summary of oxygen isotope discrimination (D) in root respiration. Plant Wheat Wheat Wheat Wheat Wheat Wheat Wheat Wheat Wheat Wheat Philo.e Philo.e Philo.e Philo.e Philo.e Philo.e Philo.e Philo.e Philo.e Philo.e Philo.e Philo.e
Incubation period
Incubation medium
Temp (°C)
Light (hr/d)
KOHa
D
SEb
nc
Resp.d (% per day)
Remarks
Short Short Short Short Short Short Short Short Short Short Long Long Long Long Long Long Long Long Long Long Long Short
Moist air Moist air Moist air Moist air Moist air Moist air Moist air Moist air Water Water Sand⫹Ver.f Sand⫹Ver.f Sand⫹Ver.f Sand⫹Ver.f Sand⫹Ver.f Sand⫹Ver.f Sand⫹Ver.f Sand⫹Ver.f Sand⫹Ver.f Water Water Moist air
32 25 25 25 25 25 32 32 25 25 25 25 25 32 32 25 25 32 32 32 32 25
0 0 0 0 0 0 0 0 0 0 24 10 24 10 0 0 24 0 0 0 0 -
No No No No No No No No No Yes Yes Yes Yes Yes No No Yes No
12.6 14.0 14.0 15.7 15.6 15.0 15.4 15.2 16.0 18.5 12.3 16.7 15.9 17.2 16.3 19.0 19.0 19.9 19.2 12.3 16.0 12.6
0.30 0.15 0.16 0.40 0.13 0.30 0.53 0.22 0.08 0.21 0.07 0.19 0.06 0.08 0.10 0.23 0.12 0.04 0.02 0.30 0.22 0.12
8 4 4 4 4 4 3 2 2 2 10 6 4 2 4 8 6 6 2 2 2 6
— — — — — — — — — — 3.44 2.4 2.83 4.58 3.63 1.57 1.48 3.44 2.75 — — —
9 d old seedling 7 d old seedling 6 d old seedling 20 d old seedling 14 d old seedling 15 d old seedling 17 d old seedling 31 d old seedling 15 d old seedling 19 d old seedling 7 d in chamber 12 d in chamber 18 d in chamber 23 d in chamber 26 d in chamber 37 d in chamber 48 d in chamber 51 d in chamber 54 d in chamber 8 d in chamber 64 d in chamber Detached root
term term term term term term term term term term term term term term term term term term term term term term
a
KOH-Yes indicates CO2 removal with KOH. SE-standard error. c n-number of data that points the discrimination calculation is based on. d Resp.-respiration rate in percent per day of the initial O2 concentration. e Philo.-Philodendron. f Sand-vermiculite mixture enriched in nutrient solution. b
plants were kept in the dark during the incubation period in most of the experiments. In part of the long-term experiments, the plants were subjected to different periods of illumination per day.
2.3. Calculation of Respiratory Fractionation of Oxygen Isotopes The instantaneous fractionation factor is defined as:
␣R ⫽ Rp/Rs 2.2. Sampling and Mass Spectrometry Special care was taken to avoid possible atmospheric leaks during sampling. To this end, we made sampling vessels consisting of 4 cm3 glass tubes with high vacuum valves (9 mm, Lowers Happert威). Rubber septa were fitted at the neck of each vessel, before the valve. The vessels were evacuated via a needle through the rubber septum. Similar rubber septum was inserted in the sampling port of the incubation chambers. Air was transferred to the sampling vessels by piercing both septa with a double pointed needle use for blood collection (similar to Magnusson, 1989). Ambient atmospheric pressure was maintained in the incubation chambers during sampling by replacement of the sampled volume with either water (in the short-term incubation) or N2 (in the long-term incubation) (Fig. 1). In the experiments in which roots were incubated in water, 100 cm3 water were sampled in 250 cm3 preevacuated flasks closed with a Lowers Happert威 valve. Extraction of the dissolved gases was done according to Emerson et al. (1995). The change in [O2] was calculated from the difference in the ratio of O2 to Ar determined by mass spectrometric analysis of the same sample used for isotopic analysis. Sample preparation and mass spectrometry was according to Luz et al. (1999). The results were calculated with respect to an air standard (HLA) as ␦O2/Ar and ␦18O) with precision of 0.5‰ and 0.05% respectively, where:
␦O2Ar(‰) ⫽ [(O2/Ar)HLA ⫺ 1]*103
18
(1) 16
where Rs and Rp are the isotopic ratio ( O/ O) of the oxygen substrate and the respired O2 respectively. The isotopic discrimination D is related to ␣R: D ⫽ (1⫺␣R) 䡠 103 (2)
(2)
D can be obtained from Raleigh distillation equation (after Kroopnick and Craig, 1976): lnR/R0 3 䡠10 ⫺ln f
D⫽
(3)
where R is the isotopic ratio of the substrate oxygen at the time of the sampling, and R0 is the initial ratio (R/R0 ⫽ [(␦18O)sample/103⫹1)/ (␦18O)initial/103⫹1). f is the remaining O2 fraction, and was calculated from the measured ␦O2/Ar value (this is possible because argon is inert and its concentration in the chamber remains constant throughout the experiment). The isotopic discrimination was calculated from the regression of ln R/R0 against -ln f. D is the slope of the straight regression line (Fig. 2). The regression line is forced to pass through the origin because the initial air in our experiments is atmospheric air that is used as our primary standard. Such forcing is not justified when the initial isotopic composition is unknown (Henry et al., 1999). Replacing the air in the incubation chamber while the roots where already in it, avoided the problem of tissue equilibration Henry et al. (1999) described. 3. RESULTS
and ␦18O(‰) ⫽ [(18O/16O)sample/(18O/16O)HLA ⫺ 1]*103.
Measured values of D at different experimental conditions are given in Table 1.
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Fig. 2. Relative changes in the isotopic composition of O2 (expressed as 1000*ln R/R0) as a function of the natural log of the remaining fraction (ln f). The slope of the fitted regression line gives the discrimination factor (D). SE — standard error; n — number of data points. A. An example of an experiment with Philodendron (Table 1, line 11). B. A plot showing most of the experimental results (excluding data points with 1000*ln R/R0 ⬎ 4). The solid line shown was derived from linear regression of all data points. The pooled discrimination value (14.5‰) was derived from the slope of this line. The sector between the two dashed lines shows the maximum range of discrimination in the experiments (11.9‰–20.0‰).
The discrimination in wheat seedlings incubated in moist air, was 12.3‰ to 15.6‰. Younger seedlings (6 –9 d old) had lower discrimination (12.6‰–14.0‰) than older ones (15.0‰– 15.6‰). In the younger plants, the discrimination measured in the experiments that were held at 32°C was lower (12.6‰) than the experiments that were held at 25°C (14.0‰). The discrimination in the wheat seedlings in water experiments was 16.0‰ to 18.5‰. In the long-term incubation of Philodendron grown in sand, the discrimination increased from about 12‰ at the beginning of the incubation to about 20‰ at the end. This change was not associated with the changes in temperature, light, or [CO2], although the respiration rate was correlated with the temperature. In much the same way the discrimination in Philodendron roots grown in water, changed from 12.3‰ in the beginning of the incubation period to 16.0‰ at its end, without any apparent change in the external conditions. The discrimination measured in detached Philodendron root was 12.6‰. The entire set of experimental data is shown on Figure 2b. The discrimination calculated from the regression line of the pooled data (excluding data points with 1000*ln R/R0 ⬎ 4 for statistical reasons) is 14.5‰. 4. DISCUSSION
4.1. The Measured Discrimination The discrimination measured in most (82%) of the incubation experiments as well as the averaged value for all the experiments (14.5‰) is lower than generally expected for dark respiration (⬃18‰). We can interpret this apparent discrepancy in the framework of a simple, one box, diffusion-respiration model representing the roots (Fig. 3). When diffusion is rate limiting, internal O2 concentration (Ci) is lower than am-
bient O2 concentration (Ca). Following Farquhar et al. (1982) the overall discrimination for such diffusion-respiration model is: Dtotal ⫽ Dd ⫹ (Dr ⫺ Dd)Ci/Ca
(4)
Where Dtotal is the overall discrimination, Dd is the discrimination in diffusion, and Dr is the respiratory discrimination. Since lateral diffusion in roots is thought to be in liquid-phase (Armstrong et al., 1994) and because the discrimination of diffusion in liquids is usually small (Farquhar and Lloyd, 1993) we assume that the discrimination in diffusion is zero. Substituting Dd⫽ 0, Dr⫽ 18, Dtotal⫽ 12 into Eqn. 1 yields Ci/Ca ⫽ 0.67. This means that the lowest discrimination that was measured, about 12‰, can result from limiting diffusion that lowers the internal oxygen concentration to 67% of the ambient concentration. In a study where O2 in maize roots was measured with microelectrodes, it was found that O2 level dropped by about 50% between the outer and the inner parts of the root (Armstrong et al., 1994). Hence, limiting diffusion can explain the low discrimination that we measured. However, it is important to notice that there is no real separation in roots between areas where slow diffusion takes place, and areas where respiration takes place. Nevertheless the general effect of limiting diffusion on fractionation could be inferred from this simple model. As shown in Table 1, there is some tendency for increased discrimination with age in the wheat seedling experiments. The simple model cannot account for this increase. Millar et al. (1998) found a positive correlation between the rate of cyanideresistant respiration in roots of soybean plants and age. 4 d old Soybean roots did not show any cyanide-resistant respiration,
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Fig. 3. Root model: Oxygen diffuses through the root tissues with discrimination Dd ⫽ 0‰, and is respired inside the root with discrimination Dr ⫽ 18‰. Since the diffusion into the root is slow the internal oxygen concentration becomes low (Ci ⬍ Ca), and the overall discrimination becomes less than 18‰.
but 17 d old roots had almost 50% of their respiration through the cyanide-resistant pathway. We thus suggest that the increasing D values with age may indicate increasing rate of cyanideresistant respiration (with D ⫽ 25–30‰). Another possible explanation for the increased discrimination with age is that the root tissues become more diffusive. The younger wheat seedlings had lower discrimination at higher temperatures. The diffusion respiration model can explain this temperature dependence. According to the known dependency of root respiration on temperature (Boone et al., 1998; Raich and Schlesinger, 1992) the 7°C temperature rise should have doubled the respiration rate. As a result of the higher respiration rate, the internal O2 concentration decreases and causes lower overall discrimination. However, respiration rate cannot explain all the variations in our experiments. Longitudinal diffusion of photosynthetic oxygen from leaves through stems is known to be an important source of oxygen to roots in wetland plants, as well as in non-wetland ones (Armstrong et al., 1994; De Willigen and Van Noordwijk, 1989). The ␦18O of this photosynthetically produced oxygen is much lower than of ambient air. Transport of such O2 through the stem and the roots to the incubation chamber can be a significant source of error. To eliminate possible errors due to this mechanism, the leaves were kept in the dark in most of the experiments. In other experiments we deliberately exposed the leaves to different periods of illumination per day. We expected that if longitudinal O2 transport occurred, an experiment with longer periods of illumination would have a weaker apparent discrimination. The absence of this correspondence in our results suggests, that the measured discrimination was not affected by O2 transport along the roots. This conclusion is reinforced by the detached root experiment, in which longitudinal O2 transport is completely ruled out. The discrimination in the detached root experiment case was at the lower end of the range obtained in our experiments (12.6‰).
In the experiments with the Philodendron planted in sand, there was some tendency for increased discrimination with the period that the plant was in the incubation chamber, but there was no correlation with temperature and respiration rate. We suggest two possible explanations for the time-discrimination trend: 1. The relative rate of cyanide-resistant may have increased with time. 2. Bacterial growth on decomposing roots or near live ones increased the ratio of bacterial to root respiration. In this case the value obtained in the first experiment (12.3‰) should represent the true discrimination of the root system. As discussed above, long-term experiments may suffer from problems related to long closure of the plants in the chamber. Short-term experiments, on the other hand, are free from these problems but may suffer from possible effect of plant transfer to the incubation chamber. Conducting both types of experiments helps to reduce the experimental uncertainty. Moreover, the fact that the discriminations measured were lower than the known value for dark respiration in 90% of the short-term experiments and 64% of the long-term experiments seems to strengthen our conclusion on the importance of slow diffusion. Our experiments also showed that the effect of cyanide-resistant respiration was not as strong as that of slow diffusion, as the overall discrimination was much lower than that of cyanideresistant respiration (25‰–30‰) in all experimental runs. 4.2. Implication for the Dole Effect The discrimination calculated from the pooled experimental data can not, of course, truly represent the average discrimination in global root respiration, with its variety of species and
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environmental conditions. However this value (14.5‰) indicates that the value used so far (18‰) is too high and neglects the importance of diffusion limitations. Diffusion limitation for oxygen is found not only in roots but also in soil aggregates (Sexstone et al., 1985; Zausig et al., 1993), hence we expect that the overall discrimination in soil respiration to be low. Indeed, by analysis of the ␦18O and [O2] profiles in heavy and light soils we determined an average discrimination of 12 ⫾ 1‰ (the details will be published elsewhere). To arrive at a representative global figure for soil discrimination it is necessary to conduct more field studies in soils of tropical rain forests and temperate forests as well as in other globally important ecosystems. Until such studies are carried out, the value used in models of the Dole effect for the discrimination in root respiration should be lower than 18‰ used so far. In a review on the regulating factors of the Dole effect (Bender et al., 1994) the present-day Dole effect was calculated as 20.8‰, which is 2.7‰ less than the observed value (23.5‰). In this calculation, it was assumed that the mean global ␦18O of leaf water—the substrate for photosynthesis on land—is about ⬃4‰ higher than the ␦18O of ocean water. It was suggested (Bender et al., 1994) that the discrepancy between the observed and calculated Dole effect might be resolved by higher ␦18O values of leaf water (⬃8‰). However, there is no observational evidence to support this suggestion. Our present finding increases the discrepancy even further since it significantly lowers the discrimination value for root respiration. Recently, it has been shown that cyanide resistant respiration is widespread among diverse marine phytoplankton (Eriksen and Lewitus, 1999). Moreover, Lewitus and Kana (1995) demonstrated that this respiration was quantitatively important in illuminated cultures. It is thus possible that the oceanic discrimination against 18O was underestimated. In this case, the discrepancy mentioned above may be resolved. 5. CONCLUSIONS
The measured discrimination of root respiration against 18O ranges from of 11.9‰ to 20.0‰, with an average value of 14.5‰. This average is significantly lower than in the process of dark-respiration (⬃18‰), and should be taken into account in global models of the Dole effect. Theoretical considerations, supported by known oxygen gradients in roots, show that this relatively low discrimination can be explained by slow diffusion through the root that limits the oxygen concentration in the consumption site. Acknowledgments—We thank D. Yakir for his advice on the experimental setup and for providing plants for our experiments. The help of E. Barkan in all aspects related to isotopic measurements is greatly appreciated. This research has been supported by a grant from the Israel Science Foundation. Associate editor: S. M. F. Sheppard REFERENCES Armstrong W., Strange M. E., Cringle S., and Beckett P. M. (1994) Microelectrode and modeling study of oxygen distribution in roots. Ann. of Botany London 74(3), 287–299.
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PII S0016-7037(01)00567-1
Fractionation of oxygen isotopes by root respiration: Implications for the isotopic composition of atmospheric O2 ALON ANGERT* and BOAZ LUZ The Institute of Earth Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel (Received August 1, 2000; accepted in revised form January 18, 2001)
Abstract—The ratio of 18O/16O in atmospheric oxygen depends on the isotopic composition of the substrate water used in photosynthesis and on discrimination against 18O in respiratory consumption. The current understanding of the composition of air O2 attributes the magnitude of the respiratory fractionation to biochemical mechanisms alone. Thus the discrimination against 18O is assumed as 18‰ in normal dark respiration and 25‰ to 30‰ in cyanide resistant respiration. Here we report new results on the fractionation of O2 isotopes in root respiration. The isotopic fractionation was determined from the change in ␦18O of air due to partial uptake by roots in closed containers. The discrimination in these experiments was in the range of 11.9‰ to 20.0‰ with an average of 14.5‰. This average is significantly less than the known discrimination in dark respiration. A simple diffusion-respiration model was used to explain the isotopic discrimination in roots. Available data show that O2 concentration inside roots is low due to slow diffusion. As a result, due to diffusion and biological uptake at the consumption site inside the root, the overall discrimination is small. Root respiration is an important component of the global oxygen uptake. Our new result that the discrimination against 18O is less than generally thought indicates that the mechanisms affecting ␦18O of atmospheric oxygen should be re-evaluated. Copyright © 2001 Elsevier Science Ltd In recent treatments of past variations in the Dole effect, it was assumed that global terrestrial respiration preferentially discriminates against 18O by 18‰ with respect to 16O (Beerling, 1999; Bender et al., 1994; Malaize et al., 1999). This value is based on measurements in isolated plant organs (Guy et al., 1993; Guy et al., 1989). Epstein and Zeiri (1988) and Guy et al. (1989) noted that if O2 diffusion to the consumption site is limiting, the discrimination of a system will depend not only on the discrimination in the consumption process but also on the discrimination in diffusion, and on the relative rates of consumption and diffusion. In an extreme case where diffusion is very slow in comparison to respiration, oxygen level in the consumption site is near zero, and any O2 entering is immediately respired. The discrimination in this case is the discrimination in diffusion since no discrimination in respiration takes place. Bender et al. (1994) recognized the role of diffusion limitation on oxygen isotope discrimination in their treatment of the Dole effect. They suggested that in the ocean the role of diffusion limitation is negligible because most of the respiration takes place by small organisms in which the ratio of surface area to volume is large. However, this limitation cannot be neglected in roots where O2 supply to the consumption site is slowed down by low diffusivity of root tissues (Armstrong et al., 1994; Ikeda and Nakamura, 1996). Hence, we expect the overall discrimination against 18O in root respiration to be lower than in dark respiration alone (18‰ in cyanide-sensitive respiration (Guy et al., 1989)). On the other hand, part of the respiration in roots may be through the cyanide-resistant pathway (alternative pathway) (Millar et al., 1998), in which the discrimination is 25‰ to 30‰ (Guy et al., 1993; Robinson et al., 1992), and an increase in the total discrimination is expected if this mechanism is engaged. Recognizing the importance of the Dole effect in understand-
1. INTRODUCTION
Changes in the 18O enrichment of air O2 with respect to ocean water (“Dole effect”) can be used to infer past variations in the ratio of marine to terrestrial biospheric production, related to past climatic changes (Bender et al., 1994; Malaize et al., 1999). However, to derive quantitative estimates, it is necessary to gain better understanding of the basic mechanisms affecting the isotopic composition of O2 during its photosynthetic production and respiratory consumption. There is convincing evidence that photosynthesis produces O2 of identical ␦18O as of the substrate water (Guy et al., 1993), whereas respiration preferentially removes the lighter isotope (Guy et al., 1989; Lane and Dole, 1956). As a result, atmospheric O2 becomes enriched in 18O with respect to ocean water (Dole, 1935). O2 produced by the terrestrial biosphere has higher ␦18O than ocean water because the substrate—leaf water— becomes enriched in 18O due to evapotranspiration (Dongmann, 1974). Thus increased Dole effect can be interpreted to indicate increased terrestrial/marine production and visa versa. However, quantifying the past ratio of terrestrial to marine productivity is difficult, and even the present-day Dole effect (23.5‰ with respect to SMOW (Kroopnick and Craig, 1972)) is not very well explained from the current understanding of globally important isotope fractionation mechanisms (Bender et al., 1994). Soil respiration is a major component of the global carbon cycle and roots contribute 30 to 70% of total soil respiration (Raich and Schlesinger, 1992). Therefore a substantial fraction of the global O2 consumption is accounted by root respiration with significant influence on the Dole effect.
*Author to whom correspondence should be addressed (alon_a@ mail.com). 1695
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Fig. 1. Schematic illustration of the incubation chamber and the sampling method.
ing past global changes, it is important to provide improved parameters of O2 isotope discrimination. In the present study we derived such information from laboratory experiments with intact roots. 2. EXPERIMENTAL METHODS 2.1. Incubation of Roots Roots of Philodendron plants and wheat seedlings were incubated in closed chambers at various experimental conditions (Table 1), and the respiratory fractionation was calculated from the change in [O2] and ␦18O of O2. Most of the experiments were conducted with intact plants, and the area around the stems was sealed with Aquaseal威. In addition, the incubation chambers were submerged in water to avoid possible air leaks. In an attempt to minimize bacterial respiration in the incubation chambers, the roots were placed in a medium with minimum organic content. This medium was either sand-vermiculite mixture enriched with nutrient solution, moist air or distilled water. In the latter, changes in dissolved O2 were monitored. To keep the conditions close as possible to natural ones, and to prevent anaerobic environment, the level of O2 in all the experiments was rarely below 70% of ambient air and never below 50%. In the short-term incubations (⬃ 10 h) the specimens (either whole
plants or detached roots) were transferred from the growth medium to the incubation chamber, and were used only for a single experiment. The incubation chamber was sampled at the end of the experiment. In these short-term incubations the medium containing the roots was either moist air or distilled water. Long-term incubations were also conducted. It was thought that in long term experiments the plants would acclimatize to conditions in closed chambers. On the other hand, we could not eliminate possibly significant bacterial growth in these experiments. In the long term incubations, Philodendron roots were placed in sand-vermiculite mixture enriched in nutrient solution. The roots were then sealed for two months in an incubation chamber. Between experimental runs, the chamber headspace was flushed continuously with outside air for at least 24 h. In each run, that lasted several days, the chamber was sealed and samples were taken daily. In another long-term incubation experiment, Philodendron was grown in water for two months and O2 concentration was kept near saturation by continuous bubbling of air. Respiratory discrimination was determined by sealing the root chamber for 4 h and [O2]aq and ␦18O were measured at the beginning and the end of this incubation period. The incubation temperature was changed to induce varying respiration rates. In some of the long term experiments excessive CO2 rise was eliminated by its reaction with KOH solution (20M). The latter was held in an airtight vessel connected to the root chamber. To eliminate the possibility of admixture of photosynthetic O2 through the stem, the
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Table 1. Summary of oxygen isotope discrimination (D) in root respiration. Plant Wheat Wheat Wheat Wheat Wheat Wheat Wheat Wheat Wheat Wheat Philo.e Philo.e Philo.e Philo.e Philo.e Philo.e Philo.e Philo.e Philo.e Philo.e Philo.e Philo.e
Incubation period
Incubation medium
Temp (°C)
Light (hr/d)
KOHa
D
SEb
nc
Resp.d (% per day)
Remarks
Short Short Short Short Short Short Short Short Short Short Long Long Long Long Long Long Long Long Long Long Long Short
Moist air Moist air Moist air Moist air Moist air Moist air Moist air Moist air Water Water Sand⫹Ver.f Sand⫹Ver.f Sand⫹Ver.f Sand⫹Ver.f Sand⫹Ver.f Sand⫹Ver.f Sand⫹Ver.f Sand⫹Ver.f Sand⫹Ver.f Water Water Moist air
32 25 25 25 25 25 32 32 25 25 25 25 25 32 32 25 25 32 32 32 32 25
0 0 0 0 0 0 0 0 0 0 24 10 24 10 0 0 24 0 0 0 0 -
No No No No No No No No No Yes Yes Yes Yes Yes No No Yes No
12.6 14.0 14.0 15.7 15.6 15.0 15.4 15.2 16.0 18.5 12.3 16.7 15.9 17.2 16.3 19.0 19.0 19.9 19.2 12.3 16.0 12.6
0.30 0.15 0.16 0.40 0.13 0.30 0.53 0.22 0.08 0.21 0.07 0.19 0.06 0.08 0.10 0.23 0.12 0.04 0.02 0.30 0.22 0.12
8 4 4 4 4 4 3 2 2 2 10 6 4 2 4 8 6 6 2 2 2 6
— — — — — — — — — — 3.44 2.4 2.83 4.58 3.63 1.57 1.48 3.44 2.75 — — —
9 d old seedling 7 d old seedling 6 d old seedling 20 d old seedling 14 d old seedling 15 d old seedling 17 d old seedling 31 d old seedling 15 d old seedling 19 d old seedling 7 d in chamber 12 d in chamber 18 d in chamber 23 d in chamber 26 d in chamber 37 d in chamber 48 d in chamber 51 d in chamber 54 d in chamber 8 d in chamber 64 d in chamber Detached root
term term term term term term term term term term term term term term term term term term term term term term
a
KOH-Yes indicates CO2 removal with KOH. SE-standard error. c n-number of data that points the discrimination calculation is based on. d Resp.-respiration rate in percent per day of the initial O2 concentration. e Philo.-Philodendron. f Sand-vermiculite mixture enriched in nutrient solution. b
plants were kept in the dark during the incubation period in most of the experiments. In part of the long-term experiments, the plants were subjected to different periods of illumination per day.
2.3. Calculation of Respiratory Fractionation of Oxygen Isotopes The instantaneous fractionation factor is defined as:
␣R ⫽ Rp/Rs 2.2. Sampling and Mass Spectrometry Special care was taken to avoid possible atmospheric leaks during sampling. To this end, we made sampling vessels consisting of 4 cm3 glass tubes with high vacuum valves (9 mm, Lowers Happert威). Rubber septa were fitted at the neck of each vessel, before the valve. The vessels were evacuated via a needle through the rubber septum. Similar rubber septum was inserted in the sampling port of the incubation chambers. Air was transferred to the sampling vessels by piercing both septa with a double pointed needle use for blood collection (similar to Magnusson, 1989). Ambient atmospheric pressure was maintained in the incubation chambers during sampling by replacement of the sampled volume with either water (in the short-term incubation) or N2 (in the long-term incubation) (Fig. 1). In the experiments in which roots were incubated in water, 100 cm3 water were sampled in 250 cm3 preevacuated flasks closed with a Lowers Happert威 valve. Extraction of the dissolved gases was done according to Emerson et al. (1995). The change in [O2] was calculated from the difference in the ratio of O2 to Ar determined by mass spectrometric analysis of the same sample used for isotopic analysis. Sample preparation and mass spectrometry was according to Luz et al. (1999). The results were calculated with respect to an air standard (HLA) as ␦O2/Ar and ␦18O) with precision of 0.5‰ and 0.05% respectively, where:
␦O2Ar(‰) ⫽ [(O2/Ar)HLA ⫺ 1]*103
18
(1) 16
where Rs and Rp are the isotopic ratio ( O/ O) of the oxygen substrate and the respired O2 respectively. The isotopic discrimination D is related to ␣R: D ⫽ (1⫺␣R) 䡠 103 (2)
(2)
D can be obtained from Raleigh distillation equation (after Kroopnick and Craig, 1976): lnR/R0 3 䡠10 ⫺ln f
D⫽
(3)
where R is the isotopic ratio of the substrate oxygen at the time of the sampling, and R0 is the initial ratio (R/R0 ⫽ [(␦18O)sample/103⫹1)/ (␦18O)initial/103⫹1). f is the remaining O2 fraction, and was calculated from the measured ␦O2/Ar value (this is possible because argon is inert and its concentration in the chamber remains constant throughout the experiment). The isotopic discrimination was calculated from the regression of ln R/R0 against -ln f. D is the slope of the straight regression line (Fig. 2). The regression line is forced to pass through the origin because the initial air in our experiments is atmospheric air that is used as our primary standard. Such forcing is not justified when the initial isotopic composition is unknown (Henry et al., 1999). Replacing the air in the incubation chamber while the roots where already in it, avoided the problem of tissue equilibration Henry et al. (1999) described. 3. RESULTS
and ␦18O(‰) ⫽ [(18O/16O)sample/(18O/16O)HLA ⫺ 1]*103.
Measured values of D at different experimental conditions are given in Table 1.
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Fig. 2. Relative changes in the isotopic composition of O2 (expressed as 1000*ln R/R0) as a function of the natural log of the remaining fraction (ln f). The slope of the fitted regression line gives the discrimination factor (D). SE — standard error; n — number of data points. A. An example of an experiment with Philodendron (Table 1, line 11). B. A plot showing most of the experimental results (excluding data points with 1000*ln R/R0 ⬎ 4). The solid line shown was derived from linear regression of all data points. The pooled discrimination value (14.5‰) was derived from the slope of this line. The sector between the two dashed lines shows the maximum range of discrimination in the experiments (11.9‰–20.0‰).
The discrimination in wheat seedlings incubated in moist air, was 12.3‰ to 15.6‰. Younger seedlings (6 –9 d old) had lower discrimination (12.6‰–14.0‰) than older ones (15.0‰– 15.6‰). In the younger plants, the discrimination measured in the experiments that were held at 32°C was lower (12.6‰) than the experiments that were held at 25°C (14.0‰). The discrimination in the wheat seedlings in water experiments was 16.0‰ to 18.5‰. In the long-term incubation of Philodendron grown in sand, the discrimination increased from about 12‰ at the beginning of the incubation to about 20‰ at the end. This change was not associated with the changes in temperature, light, or [CO2], although the respiration rate was correlated with the temperature. In much the same way the discrimination in Philodendron roots grown in water, changed from 12.3‰ in the beginning of the incubation period to 16.0‰ at its end, without any apparent change in the external conditions. The discrimination measured in detached Philodendron root was 12.6‰. The entire set of experimental data is shown on Figure 2b. The discrimination calculated from the regression line of the pooled data (excluding data points with 1000*ln R/R0 ⬎ 4 for statistical reasons) is 14.5‰. 4. DISCUSSION
4.1. The Measured Discrimination The discrimination measured in most (82%) of the incubation experiments as well as the averaged value for all the experiments (14.5‰) is lower than generally expected for dark respiration (⬃18‰). We can interpret this apparent discrepancy in the framework of a simple, one box, diffusion-respiration model representing the roots (Fig. 3). When diffusion is rate limiting, internal O2 concentration (Ci) is lower than am-
bient O2 concentration (Ca). Following Farquhar et al. (1982) the overall discrimination for such diffusion-respiration model is: Dtotal ⫽ Dd ⫹ (Dr ⫺ Dd)Ci/Ca
(4)
Where Dtotal is the overall discrimination, Dd is the discrimination in diffusion, and Dr is the respiratory discrimination. Since lateral diffusion in roots is thought to be in liquid-phase (Armstrong et al., 1994) and because the discrimination of diffusion in liquids is usually small (Farquhar and Lloyd, 1993) we assume that the discrimination in diffusion is zero. Substituting Dd⫽ 0, Dr⫽ 18, Dtotal⫽ 12 into Eqn. 1 yields Ci/Ca ⫽ 0.67. This means that the lowest discrimination that was measured, about 12‰, can result from limiting diffusion that lowers the internal oxygen concentration to 67% of the ambient concentration. In a study where O2 in maize roots was measured with microelectrodes, it was found that O2 level dropped by about 50% between the outer and the inner parts of the root (Armstrong et al., 1994). Hence, limiting diffusion can explain the low discrimination that we measured. However, it is important to notice that there is no real separation in roots between areas where slow diffusion takes place, and areas where respiration takes place. Nevertheless the general effect of limiting diffusion on fractionation could be inferred from this simple model. As shown in Table 1, there is some tendency for increased discrimination with age in the wheat seedling experiments. The simple model cannot account for this increase. Millar et al. (1998) found a positive correlation between the rate of cyanideresistant respiration in roots of soybean plants and age. 4 d old Soybean roots did not show any cyanide-resistant respiration,
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Fig. 3. Root model: Oxygen diffuses through the root tissues with discrimination Dd ⫽ 0‰, and is respired inside the root with discrimination Dr ⫽ 18‰. Since the diffusion into the root is slow the internal oxygen concentration becomes low (Ci ⬍ Ca), and the overall discrimination becomes less than 18‰.
but 17 d old roots had almost 50% of their respiration through the cyanide-resistant pathway. We thus suggest that the increasing D values with age may indicate increasing rate of cyanideresistant respiration (with D ⫽ 25–30‰). Another possible explanation for the increased discrimination with age is that the root tissues become more diffusive. The younger wheat seedlings had lower discrimination at higher temperatures. The diffusion respiration model can explain this temperature dependence. According to the known dependency of root respiration on temperature (Boone et al., 1998; Raich and Schlesinger, 1992) the 7°C temperature rise should have doubled the respiration rate. As a result of the higher respiration rate, the internal O2 concentration decreases and causes lower overall discrimination. However, respiration rate cannot explain all the variations in our experiments. Longitudinal diffusion of photosynthetic oxygen from leaves through stems is known to be an important source of oxygen to roots in wetland plants, as well as in non-wetland ones (Armstrong et al., 1994; De Willigen and Van Noordwijk, 1989). The ␦18O of this photosynthetically produced oxygen is much lower than of ambient air. Transport of such O2 through the stem and the roots to the incubation chamber can be a significant source of error. To eliminate possible errors due to this mechanism, the leaves were kept in the dark in most of the experiments. In other experiments we deliberately exposed the leaves to different periods of illumination per day. We expected that if longitudinal O2 transport occurred, an experiment with longer periods of illumination would have a weaker apparent discrimination. The absence of this correspondence in our results suggests, that the measured discrimination was not affected by O2 transport along the roots. This conclusion is reinforced by the detached root experiment, in which longitudinal O2 transport is completely ruled out. The discrimination in the detached root experiment case was at the lower end of the range obtained in our experiments (12.6‰).
In the experiments with the Philodendron planted in sand, there was some tendency for increased discrimination with the period that the plant was in the incubation chamber, but there was no correlation with temperature and respiration rate. We suggest two possible explanations for the time-discrimination trend: 1. The relative rate of cyanide-resistant may have increased with time. 2. Bacterial growth on decomposing roots or near live ones increased the ratio of bacterial to root respiration. In this case the value obtained in the first experiment (12.3‰) should represent the true discrimination of the root system. As discussed above, long-term experiments may suffer from problems related to long closure of the plants in the chamber. Short-term experiments, on the other hand, are free from these problems but may suffer from possible effect of plant transfer to the incubation chamber. Conducting both types of experiments helps to reduce the experimental uncertainty. Moreover, the fact that the discriminations measured were lower than the known value for dark respiration in 90% of the short-term experiments and 64% of the long-term experiments seems to strengthen our conclusion on the importance of slow diffusion. Our experiments also showed that the effect of cyanide-resistant respiration was not as strong as that of slow diffusion, as the overall discrimination was much lower than that of cyanideresistant respiration (25‰–30‰) in all experimental runs. 4.2. Implication for the Dole Effect The discrimination calculated from the pooled experimental data can not, of course, truly represent the average discrimination in global root respiration, with its variety of species and
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environmental conditions. However this value (14.5‰) indicates that the value used so far (18‰) is too high and neglects the importance of diffusion limitations. Diffusion limitation for oxygen is found not only in roots but also in soil aggregates (Sexstone et al., 1985; Zausig et al., 1993), hence we expect that the overall discrimination in soil respiration to be low. Indeed, by analysis of the ␦18O and [O2] profiles in heavy and light soils we determined an average discrimination of 12 ⫾ 1‰ (the details will be published elsewhere). To arrive at a representative global figure for soil discrimination it is necessary to conduct more field studies in soils of tropical rain forests and temperate forests as well as in other globally important ecosystems. Until such studies are carried out, the value used in models of the Dole effect for the discrimination in root respiration should be lower than 18‰ used so far. In a review on the regulating factors of the Dole effect (Bender et al., 1994) the present-day Dole effect was calculated as 20.8‰, which is 2.7‰ less than the observed value (23.5‰). In this calculation, it was assumed that the mean global ␦18O of leaf water—the substrate for photosynthesis on land—is about ⬃4‰ higher than the ␦18O of ocean water. It was suggested (Bender et al., 1994) that the discrepancy between the observed and calculated Dole effect might be resolved by higher ␦18O values of leaf water (⬃8‰). However, there is no observational evidence to support this suggestion. Our present finding increases the discrepancy even further since it significantly lowers the discrimination value for root respiration. Recently, it has been shown that cyanide resistant respiration is widespread among diverse marine phytoplankton (Eriksen and Lewitus, 1999). Moreover, Lewitus and Kana (1995) demonstrated that this respiration was quantitatively important in illuminated cultures. It is thus possible that the oceanic discrimination against 18O was underestimated. In this case, the discrepancy mentioned above may be resolved. 5. CONCLUSIONS
The measured discrimination of root respiration against 18O ranges from of 11.9‰ to 20.0‰, with an average value of 14.5‰. This average is significantly lower than in the process of dark-respiration (⬃18‰), and should be taken into account in global models of the Dole effect. Theoretical considerations, supported by known oxygen gradients in roots, show that this relatively low discrimination can be explained by slow diffusion through the root that limits the oxygen concentration in the consumption site. Acknowledgments—We thank D. Yakir for his advice on the experimental setup and for providing plants for our experiments. The help of E. Barkan in all aspects related to isotopic measurements is greatly appreciated. This research has been supported by a grant from the Israel Science Foundation. Associate editor: S. M. F. Sheppard REFERENCES Armstrong W., Strange M. E., Cringle S., and Beckett P. M. (1994) Microelectrode and modeling study of oxygen distribution in roots. Ann. of Botany London 74(3), 287–299.
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