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WHOLE PLANT AND ECOPHYSIOLOGY

Measurement and Interpretation of the Oxygen Isotope Composition of Carbon Dioxide Respired by Leaves in the Dark

Environmental Biology Group and Cooperative Research Center for Greenhouse Accounting, Research School of Biological Sciences, Institute of Advanced Studies, Australian National University, Canberra, Australian Capitol Territory 2601 Australia

	ABSTRACT

TOP ABSTRACT THEORY RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
We measured the oxygen isotope composition (¹⁸O) of CO₂ respired by Ricinus communis leaves in the dark. Experiments were conducted at low CO₂ partial pressure and at normal atmospheric CO₂ partial pressure. Across both experiments, the ¹⁸O of dark-respired CO₂ (_R) ranged from 44 to 324 (Vienna Standard Mean Ocean Water scale). This seemingly implausible range of values reflects the large flux of CO₂ that diffuses into leaves, equilibrates with leaf water via the catalytic activity of carbonic anhydrase, then diffuses out of the leaf, leaving the net CO₂ efflux rate unaltered. The impact of this process on _R is modulated by the ¹⁸O difference between CO₂ inside the leaf and in the air, and by variation in the CO₂ partial pressure inside the leaf relative to that in the air. We developed theoretical equations to calculate ¹⁸O of CO₂ in leaf chloroplasts (_c), the assumed location of carbonic anhydrase activity, during dark respiration. Their application led to sensible estimates of _c, suggesting that the theory adequately accounted for the labeling of CO₂ by leaf water in excess of that expected from the net CO₂ efflux. The _c values were strongly correlated with ¹⁸O of water at the evaporative sites within leaves. We estimated that approximately 80% of CO₂ in chloroplasts had completely exchanged oxygen atoms with chloroplast water during dark respiration, whereas approximately 100% had exchanged during photosynthesis. Incorporation of the ¹⁸O of leaf dark respiration into ecosystem and global scale models of C¹⁸OO dynamics could affect model outputs and their interpretation.

The net rate of CO₂ efflux from a leaf in the dark can be thought of as the difference between two one-way diffusional fluxes, one from the atmosphere to the leaf and the other from the leaf to the atmosphere. For example, if the net respiratory CO₂ efflux (_n) is defined as _n = g_c(c_i – c_a), where g_c is the leaf conductance to CO₂, and c_i and c_a are CO₂ mole fractions in the intercellular air spaces and atmosphere, respectively, the one-way flux from leaf to atmosphere becomes g_cc_i and that from atmosphere to leaf becomes g_cc_a. The difference between _n and g_cc_i will depend on the magnitude of the CO₂ concentration difference between c_i and c_a; this difference will in turn depend on the leaf conductance to CO₂ and the CO₂ production rate inside the leaf. If the CO₂ concentration difference between c_i and c_a is very large, then the magnitude of the net CO₂ efflux will approach that of the one-way CO₂ efflux from leaf to atmosphere. However, if the CO₂ concentration inside the leaf is only a little larger than that in the atmosphere, the net CO₂ efflux from the leaf will be much smaller than the one-way CO₂ efflux from the leaf.

It has previously been recognized that one of the primary controls over the ¹⁸O of CO₂ diffusing out of leaves in the dark should be the ¹⁸O of leaf water (Flanagan et al., 1997, 1999). This is because gaseous CO₂ exchanges oxygen atoms with water during interconversion between CO₂ and bicarbonate. In plant tissues, this interconversion is catalyzed by the enzyme carbonic anhydrase. The rate constant for carbonic anhydrase is very fast, such that CO₂ diffusing out of leaves is expected to reflect nearly complete oxygen isotope exchange with leaf water. There is an equilibrium fractionation that takes place during the exchange reaction, such that at 25°C, the ¹⁸O of CO₂ will be enriched by approximately 41 compared to the ¹⁸O of water with which it has equilibrated.

In this article, we present measurements of the ¹⁸O of CO₂ respired by Ricinus communis leaves in the dark. We theorized that it should be the one-way flux of CO₂ out of a respiring leaf that is labeled with the leaf water ¹⁸O signal, rather than the net CO₂ efflux. This led us to hypothesize that the effect of a respiring leaf on the ¹⁸O of CO₂ in air passing over the leaf could be much greater than predicted by considering the net CO₂ efflux alone.

	THEORY

TOP ABSTRACT THEORY RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

Interpretation of the Oxygen Isotope Composition of Dark-Respired CO₂

Natural abundance oxygen isotope ratios are commonly expressed relative to the value of a standard:

(1)

where _x represents the proportional deviation of R_X, the ¹⁸O/¹⁶O of material X, from R_Std, the ¹⁸O/¹⁶O of a standard. Using notation, we present the following equation for the ¹⁸O of CO₂ respired by leaves in the dark (_R):

(2)

where is the proportion of CO₂ in the chloroplast that has completely exchanged oxygen atoms with chloroplast water, _e is the oxygen isotope composition of water at the evaporating sites within the leaf, _w is the equilibrium fractionation between water and CO₂, _c0 is the oxygen isotope composition of CO₂ in the chloroplast that has not exchanged oxygen atoms with chloroplast water, C_a is the ambient carbon dioxide partial pressure, C_c is the chloroplastic CO₂ partial pressure, _a is the oxygen isotope composition of ambient CO₂, and is the weighted mean isotopic discrimination against C¹⁸OO during diffusion from the chloroplast to the atmosphere. A summary of all symbols used in the text is given in Table I. A derivation of Equation 2 is presented (see "Derivation 1" in text). As described for photosynthesizing leaves by Gillon and Yakir (2000b), we make the assumption that CO₂ inside the leaf comprises a mixture of CO₂ completely equilibrated with leaf water (of proportion ) and CO₂ that has undergone no equilibration with leaf water (of proportion 1 – ). We further assume that chloroplasts are appressed against intercellular air spaces in the mesophyll cells (Evans and von Caemmerer, 1996), such that CO₂ evolved from mitochondria interacts with chloroplasts during diffusion out of the cells. Because carbonic anhydrase resides primarily in chloroplasts in C₃ leaves (Everson, 1970; Jacobson et al., 1975; Tsuzuki et al., 1985), the chloroplastic CO₂ concentration becomes the relevant parameter for modeling _R.

(3)

where C_i is the CO₂ partial pressure in the intercellular air spaces, and C_s is that at the leaf surface. The term a_w describes the summed discrimination against C¹⁸OO during liquid-phase diffusion and dissolution (0.8); a is the discrimination during diffusion through the stomata (8.8); and a_b is the discrimination during diffusion through the leaf boundary layer (5.8). We note that Equation 3 is precisely the same as the equation given for by Farquhar and Lloyd (1993); we have simply multiplied both their numerator and denominator by –1. The equilibrium fractionation between water and CO₂ can be calculated as (Brenninkmeijer et al., 1983)

(4)

where T is leaf temperature in K.

The oxygen isotope composition of CO₂ in the chloroplast of a respiring leaf (_c) can be calculated from the following equation:

(5)

a derivation of equation 5 is presented (see "Derivation 1" in text). Equations 23 and 24 can be combined, and, after dividing through by R_Std, give

(6)

For a series of measurements made at different values of _e, _c can be calculated from Equation 5 and plotted against _e. According to Equation 6, the slope of the relationship between _c and _e (m) is then equal to (1 + _w), such that can be calculated as = m/(1 + _w). The intercept of the relationship, I, is equal to _w + _c0(1 – ), such that _c0 can be calculated as _c0 = (I – _w)/(1 – ). We note that such an analysis assumes that only _e varies across the series of measurements; thus, , _w, and _c0 are assumed invariant.

The oxygen isotope enrichment at the evaporative sites in leaves (_e) can be calculated as (Craig and Gordon, 1965; Dongmann et al., 1974; Farquhar and Lloyd, 1993)

(7)

where ⁺ is the equilibrium fractionation that occurs during the phase change from liquid to vapor, _k is the kinetic fractionation that occurs during diffusion of vapor from the leaf intercellular air space to the atmosphere, _v is the isotopic enrichment of vapor in the atmosphere, and e_a/e_i is the ratio of ambient to intercellular vapor pressures. The _e and _v are defined with respect to source water, such that _e = R_e/R_s – 1 and _v = R_v/R_s – 1, where R_e is ¹⁸O/¹⁶O of water at the evaporating sites, R_s is ¹⁸O/¹⁶O of source water, and R_v is ¹⁸O/¹⁶O of vapor in the atmosphere. The term _e can be calculated from _e as

(8)

where _s is the oxygen isotope composition of source water relative to a standard. The parameter _v in Equation 6 can be calculated from measurements of the oxygen isotope composition of vapor in the atmosphere (_v) and source water as _v = (_v – _s)/(1 + _s). The equilibrium fractionation between liquid and vapor, ⁺, can be calculated as (Bottinga and Craig, 1969)

(9)

where T is leaf temperature in K. The kinetic fractionation, _k, can be calculated as (Farquhar et al., 1989)

(10)

where r_s and r_b are the stomatal and boundary layer resistances to water vapor diffusion (m² s mol^–1), and 32 and 21 are associated fractionation factors scaled to per mil. These fractionation factors have been revised up from values of 28 and 19, respectively, based on recent measurements showing the isotope effect for diffusion of H₂¹⁸O in air to be 1.032 (Cappa et al., 2003), rather than 1.028 (Merlivat, 1978).

Measurement of the Oxygen Isotope Composition of Dark-Respired CO₂

For our first dark respiration experiment, in which air entering the leaf chamber was free of CO₂, we calculated the oxygen isotope composition of respired CO₂, _R, simply as the oxygen isotope composition of CO₂ exiting the chamber, _a. In our second dark respiration experiment, where air entering the leaf chamber had a CO₂ concentration sufficient to bring that inside the chamber close to that normally found in the atmosphere, we calculated _R with a modified form of the equation presented previously by Evans et al. (1986):

(11)

where C_a is the CO₂ partial pressure (µbar) of air within the chamber when dried, _a is ¹⁸O of CO₂ within the chamber, C_in is the CO₂ partial pressure (µbar) of dry air entering the chamber, and _in is the ¹⁸O of CO₂ entering the chamber. A derivation of Equation 11 is provided (see "Derivation 2" in text). The terms C_a and _a are measured in gas exiting the leaf chamber, due to effective stirring of air within the chamber.

Calculation of Photosynthetic Discrimination against ¹³C and ¹⁸O

For measurements in the light, we calculated carbon and oxygen isotope discrimination during photosynthesis as described by Evans et al. (1986):

(12)

where R_a is ¹³C/¹²C or ¹⁸O/¹⁶O of CO₂ within the leaf chamber, R_A is ¹³C/¹²C or ¹⁸O/¹⁶O of CO₂ removed from the chamber by photosynthesis, _a is ¹³C or ¹⁸O of CO₂ within the leaf chamber, _in is ¹³C or ¹⁸O of CO₂ entering the chamber, and is defined as C_in/(C_in – C_a), where C_in and C_a refer to CO₂ partial pressures in dry air. We calculated the oxygen isotope composition of chloroplast CO₂ during photosynthesis by rearranging the C¹⁸OO discrimination equation presented by Farquhar and Lloyd (1993):

(13)

where _A is discrimination against C¹⁸OO during photosynthesis, as defined above, and _ca is defined as (R_c/R_a) – 1, where R_c is ¹⁸O/¹⁶O of chloroplast CO₂. We then calculated _c as _c = _ca(1 + _a) + _a.

For the photosynthesis measurements that comprised our third experiment, we compared the regression approach to calculating , as described above in the theory relating to dark respiration, to the method suggested by Gillon and Yakir (2000b), whereby can be calculated separately for each individual photosynthesis measurement:

(14)

where _ea is the value of _ca expected if chloroplastic CO₂ were in full oxygen isotope equilibrium with _e. The _ea was calculated as

(15)

Equation 14 incorporates an assumption that is not applied in the regression approach to calculating that we described above for dark respiration. The assumption is that the ¹⁸O of CO₂ in the chloroplast that has not equilibrated with leaf water can be calculated from the equation R_c0 = R_a[1 – (1 – C_c/C_a)] (Gillon and Yakir, 2000b), which can be replaced, to a close approximation, by Defining _c0 in this way assumes no discrimination against C¹⁸OO by Rubisco; it also ignores any possible effect of photorespiration or day respiration on _c0.

Calculation of the Conductance from C_i to C_c

The CO₂ conductance from leaf intercellular air spaces to the sites of carboxylation in chloroplasts (g_i) was calculated from ¹³C discrimination measurements during photosynthesis using the method of Evans et al. (1986):

(16)

where _obs is the observed ¹³C discrimination, b is the discrimination against ¹³CO₂ during carboxylation (taken as 29), is the sum of discriminations against ¹³CO₂ during dissolution of CO₂ and liquid phase diffusion (1.8), A is the net photosynthetic rate (µmol CO₂ m^–2 s^–1), C_a is the ambient CO₂ partial pressure (µbar), _d is day respiration (µmol CO₂ m^–2 s^–1), e is the associated discrimination against ¹³CO₂, k is the carboxylation efficiency (mol m^–2 s^–1 bar^–1), _* is the CO₂ compensation point in the absence of _d (µbar), and f is the discrimination against ¹³CO₂ associated with photorespiration. The term _i represents the discrimination that would occur if g_i were infinite, and if photorespiration and day respiration did not discriminate (Farquhar et al., 1982):

(17)

where is the discrimination against ¹³CO₂ during diffusion through the boundary layer (2.8), C_s is the CO₂ partial pressure at the leaf surface, and a¹³ is the discrimination against ¹³CO₂ during diffusion through the stomata (4.4). The term ( was calculated from the slope, m¹³, of a plot of _i – _obs against A/C_a. The term g_i was then calculated as ( The value of m¹³ is independent of values assigned to f and e in Equation 16 because varying these parameters affects the intercept of the regression rather than the slope. Therefore, there is no need to assign values to f and e for calculation of g_i.

Calculation of the Oxygen Isotope Composition of Average Lamina Leaf Water

We estimated the average lamina leaf water ¹⁸O enrichment (_L) of leaves during CO₂ collections from a model relating _L to _e (Farquhar and Lloyd, 1993):

(18)

where _e is as calculated in Equation 7, and is a lamina radial Péclet number (Farquhar and Gan, 2003). The term is defined as EL/(CD), where E is transpiration rate (mol m^–2 s^–1), L is a scaled effective path length (m), C is the molar concentration of water (5.55 x 10⁴ mol m^–3), and D is the diffusivity of H₂¹⁸O in water (2.66 x 10^–9 m^–2 s^–1). In a previous experiment, we found that the scaled effective path length for R. communis, grown and measured under the same conditions as in the present experiment, was 15.0 ± 3.5 mm (mean ± 1 SD; n = 5; Cernusak et al., 2003). This mean value was used to calculate _L. The term _L was calculated as _L = _L(1 + _s) + _s. Cernusak et al. (2003) also found that the ethanol-dry ice traps on the bypass drying loop of the gas exchange system were not quite efficient enough to remove all of the water vapor from the air cycling back to the chamber. Due to fractionation during condensation of the vapor in the traps, vapor in the air returning to the chamber was slightly enriched compared to that retained in the traps. As a result, _v for the air exiting the chamber was found to be 1.2 ± 0.5 (mean ± 1 SE; n = 5). This mean value was used in calculations of _e.

Derivation 1: Equation for Predicting the ¹⁸O of Dark-Respired CO₂

We begin by writing an equation for the total CO₂ flux from the leaf interior to the atmosphere in the dark in the steady state:

(19)

where _n is the net CO₂ efflux (µmol m^–2 s^–1); g_tc is the total conductance to CO₂ from chloroplast to atmosphere (mol m^–2 s^–1); C_c and C_a are the CO₂ partial pressures in the chloroplast and atmosphere, respectively (µbar); and P is atmospheric pressure (bar). We make the assumption that, in C₃ plants, carbonic anhydrase resides primarily in the chloroplast (Everson, 1970; Jacobson et al., 1975; Tsuzuki et al., 1985) and that it is therefore the chloroplastic CO₂ concentration that should be considered when calculating the C¹⁸OO efflux from the leaf. We further assume that the chloroplasts in C₃ plants are appressed against the intercellular air spaces in the leaf and that CO₂ evolved in mitochondria interacts with chloroplasts during diffusion out of the leaf. These assumptions may need to be reassessed for application of the model to C₄ plants. Equation 19 can be written for C¹⁸OO as

(20)

where R_R is the ¹⁸O/¹⁶O of dark-respired CO₂, is the weighted mean diffusional fractionation from chloroplast to atmosphere (calculated as described in Equation 3 above), R_c is ¹⁸O/¹⁶O of chloroplastic CO₂, and R_a is ¹⁸O/¹⁶O of ambient CO₂. Equations 19 and 20 can be combined to give

(21)

Dividing Equation 21 by the ¹⁸O/¹⁶O of a standard, R_Std, and applying the relationship R_X/R_Std = _X + 1 leads to

(22)

Solving Equation 22 for _c leads to Equation 5 above:

To write an expression for predicting _R, we apply an assumption proposed by Gillon and Yakir (2000b), under which the CO₂ within the chloroplast can be divided into two pools: one pool, of proportion , has completely exchanged oxygen atoms with chloroplast water and therefore has an ¹⁸O/¹⁶O composition of R_ce; the other pool, of proportion 1 – , has not exchanged oxygen atoms with chloroplast water and retains its initial ¹⁸O/¹⁶O composition of R_c0. We note that the term R_c0 could describe a mixture of mitochondrial CO₂ and CO₂ that has diffused into the leaf from the ambient air. Therefore, we do not define R_c0 solely as a function of CO₂ diffusing into the leaf from the atmosphere, as was done previously for photosynthesis (Gillon and Yakir, 2000b). The term R_c is then written as

(23)

The term R_ce can be calculated from the equilibrium fractionation between CO₂ and water:

(24)

where R_e is ¹⁸O/¹⁶O of chloroplast water, which we assume to be equal to ¹⁸O/¹⁶O of water at the evaporative sites. Combining Equations 21, 23, and 24 leads to

(25)

Dividing through by R_Std, and substituting 1 + _w for _w, gives

(26)

Solving Equation 26 for _R leads to Equation 2 above, which is

Derivation 2: Calculating _R From Online Gas-Exchange Measurements

Under steady-state conditions, the increase in CO₂ concentration in air flowing through a gas-exchange cuvette containing a respiring leaf can be described as

(27)

where u is the flow rate through the cuvette (mol s^–1), is the area of the leaf in the cuvette (m²), C_a and C_in are CO₂ partial pressures of dry air exiting and entering the cuvette (µbar), P is atmospheric pressure (bar), and _n is the respiration rate of the leaf (µmol CO₂ m^–2 s^–1). The corresponding mass balance for C¹⁸OO can be written as

(28)

Combining Equations 27 and 28 gives

(29)

Dividing through by the isotope ratio of a standard, R_Std, and substituting from the relationship R_X/R_Std = _X + 1 gives

(30)

Canceling common terms leads to Equation 11 above, which is

We note that the equations derived in this and the previous section can also be applied in the light. Thus, for photosynthesis, the term _R in Equations 2, 5, and 11 above can simply be replaced with the term _A. The term _A relates to _A by the relationship _A = (_a – _A)/(1 + _A).

	RESULTS

TOP ABSTRACT THEORY RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

Dark Respiration with CO₂ Free Air Entering the Leaf Chamber

In the first dark respiration experiment, air entering the leaf chamber was free of CO₂, and air exiting the leaf chamber had a mean CO₂ partial pressure of 47 µbar. The CO₂ exiting the leaf chamber was collected and analyzed for its isotopic composition. A summary of gas exchange parameters measured just prior to each CO₂ collection is presented in Table II. The dark respiration rates of the leaves ranged from 0.8 to 2.0 µmol CO₂ m^–2 s^–1 on a projected leaf area basis, with a mean value of 1.5. The C_a/C_i values ranged from 0.46 to 0.93, with a mean value of 0.81.

View larger version (21K): [in this window] [in a new window]   Figure 1. The 18O of chloroplast CO2 plotted against the 18O of water at evaporative sites in R. communis leaves during dark respiration. In this experiment, air entering the leaf chamber was free of CO2, and the CO2 partial pressure of air exiting the chamber averaged 47 µbar. The 18O of chloroplast CO2 was calculated from measurements of the 18O of CO2 exiting the chamber and gas exchange parameters, as described in the theory section of the main text. The broken line on the graph represents the relationship expected if chloroplast CO2 were in full oxygen isotope equilibrium with water at evaporative sites. The 18O values are presented relative to VSMOW. The 18O of irrigation water fed to the plants was –7.2.

Dark Respiration at Atmospheric CO₂ Concentration

In the second dark respiration experiment, the partial pressure of CO₂ in the air entering the leaf chamber was adjusted such that the air exiting the chamber had a partial pressure of approximately 350 µbar. Under these conditions, leaf dark respiration rates were similar to those observed in the first experiment, ranging from 1.0 to 2.0 µmol CO₂ m^–2 s^–1, with a mean value of 1.4. Stomatal conductance was lower than in the first experiment, having a mean value less than half that observed in the first experiment (Table II). This presumably reflects a response to the increased CO₂ partial pressure within the leaf chamber. Although stomatal conductance was lower, C_a/C_i values were higher than in the first experiment due to the increase in C_a; values ranged from 0.91 to 0.99, with a mean of 0.97. The ¹⁸O of CO₂ in air entering the leaf chamber was 19.1 ± 0.1 (mean ± 1 SE; n = 5). The mean ¹⁸O of CO₂ exiting the chamber was 43.5.

The most striking difference between the first and second dark respiration experiments was the difference in observed _R. The mean observed _R in the second experiment was 277, which can be compared with 52 for the first experiment (Table II). Mean values for _e, _L, and _c were similar between the two experiments (Table II). Differences between _e and _L in the second experiment were slightly less than in the first experiment, reflecting the lower transpiration rates (Table II). As in the first experiment, variation in _c was significantly correlated with variation in _e (Fig. 2 ), showing an r value of 0.95 (P < 0.0001, n = 10). It was also correlated with _L, with a slightly lower correlation coefficient (r = 0.94, P < 0.0001, n = 10). The regression slope of the relationship between _c and _e was 0.82, resulting in an estimate for of 0.79, suggesting that 79% of the CO₂ in chloroplasts had equilibrated with chloroplast water during dark respiration in the second experiment. This value is the same as the value of 0.79 estimated in the first experiment. The value of the intercept of the regression of _c on _e was 34.6, yielding an estimate for _c0 of 14.3; this value is lower than the _c0 of 36.2 estimated in the first experiment.

View larger version (21K): [in this window] [in a new window]   Figure 2. The 18O of chloroplast CO2 plotted against the 18O of water at evaporative sites in R. communis leaves during dark respiration. In this experiment, air entering the leaf chamber had an average CO2 partial pressure of 314 µbar, and air exiting the chamber had an average CO2 partial pressure of 347 µbar. The broken line on the graph represents the relationship expected if chloroplast CO2 were in full oxygen isotope equilibrium with water at the evaporative sites. The 18O values are presented relative to VSMOW. The 18O of irrigation water fed to the plants was –7.2.

A comparison of modeled _R values across both experiments with observed _R showed that modeled _R accounted for 80% of variation in observed _R. The regression line relating the two was _R(observed) = 0.72_R(modeled) + 39.5 (R² = 0.80, P < 0.0001, n = 21).

Carbon and Oxygen Isotope Discrimination during Photosynthesis

In the third experiment, R. communis leaves were placed in the leaf chamber in the light, and gas exchange and isotopic analyses were conducted. Photosynthesis rates ranged from 8.5 to 30.9 µmol CO₂ m^–2 s^–1, with a mean value of 20.4. The CO₂ partial pressure of air exiting the chamber ranged from 328 to 395 µbar, whereas the CO₂ partial pressure of incoming air ranged from 533 to 967 µbar; this gave rise to values ranging from 1.5 to 3.0. Stomatal conductance was approximately 4-fold larger in the light than in the dark at similar CO₂ partial pressure (Table II). The C_i/C_a ranged from 0.66 to 0.90, with a mean of 0.79. The ¹⁸O of CO₂ entering the leaf chamber was 19.1 ± 0.1 (mean ± 1 SE; n = 5); the ¹³C of CO₂ entering the leaf chamber was –33.1 ± 0.2 (mean ± 1 SE; n = 5). The ¹⁸O of CO₂ exiting the leaf chamber ranged from 40.1 to 45.4; the ¹³C of CO₂ exiting the chamber ranged from –25.3 to –19.3.

The mean observed oxygen isotope discrimination during photosynthesis (_A) was 44.6; the range is given in Table II. The _c values for the photosynthesis experiment were somewhat higher than for the dark respiration experiment at similar CO₂ concentration, presumably reflecting a higher proportion of chloroplast CO₂ equilibrated with chloroplast water (i.e. higher ). Differences between _e and _L were larger in the photosynthesis experiment than in the dark respiration experiments, reflecting the higher transpiration rates (Table II). Variation in _c was significantly correlated with variation in _e (r = 0.97, P < 0.0001, n = 8), as shown in Figure 3 . The _c was also correlated with _L (r = 0.91, P = 0.001, n = 8), but the correlation was not as strong as with _e. The slope of the relationship between _c and _e was 1.31; using Equation 6, this indicates a value for of 1.25. However, this slope estimate was strongly influenced by one outlying data point; this datum is identified by an arrow in Figure 3. If this outlying datum is excluded from the analysis, the slope of the relationship between _c and _e becomes 1.11, yielding an estimate for of 1.06. The individual values calculated according to the method of Gillon and Yakir (2000b) ranged from 0.93 to 1.24, with a mean value of 1.02. If the outlying data point identified with the arrow in Figure 3 is excluded, these individual estimates ranged from 0.93 to 1.06, with a mean of 0.99. Because the values were very close to 1.0, we did not estimate a _c0 value for the photosynthesis experiment.

View larger version (21K): [in this window] [in a new window]   Figure 3. The 18O of chloroplast CO2 plotted against the 18O of water at evaporative sites in R. communis leaves during photosynthesis. In this experiment, air entering the leaf chamber had an average CO2 partial pressure of 833 µbar, and air exiting the chamber had an average CO2 partial pressure of 363 µbar. Irradiance varied from 300 to 800 µmol PAR m–2 s–1, and chamber air temperature varied between 25 and 30°C. The 18O of chloroplast CO2 was calculated as described in the theory section of the main text. The broken line on the graph represents the relationship expected if chloroplast CO2 were in full oxygen isotope equilibrium with water at the evaporative sites. The 18O values are presented relative to VSMOW. The 18O of irrigation water fed to the plants was –7.2. The arrow on the graph indicates an outlying datum that strongly influenced the slope of the regression between c and e. Excluding this datum resulted in a slope between c and e of 1.10.

	DISCUSSION

TOP ABSTRACT THEORY RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
The most important result of this study is that we have shown that it is the one-way CO₂ efflux from a respiring leaf that is labeled with the leaf water ¹⁸O signal, rather than the net CO₂ efflux. The one-way efflux can be calculated as g_tcC_c/P, where g_tc is the total conductance to CO₂ from chloroplast to atmosphere (mol m^–2 s^–1), and P is atmospheric pressure (bar). In our second dark respiration experiment, where C_a averaged 347 µbar, values for g_tcC_c/P ranged from 7.4 to 50.1 µmol CO₂ m^–2 s^–1, whereas the net respiratory efflux, _n, ranged from 1.0 to 2.0 µmol CO₂ m^–2 s^–1; the ratio of g_tcC_c/P to _n averaged 16.9. Thus, in cases where the CO₂ diffusing out of a respiring leaf has a ¹⁸O different from CO₂ in canopy air, the effect of _R on _a could be significantly underestimated if one assumes that only the net CO₂ efflux is influenced by the isotopic composition of leaf water. The analogous requirement for considering one-way CO₂ fluxes when calculating the effect of photosynthesizing leaves on ¹⁸O of atmospheric CO₂ was discerned by Farquhar et al. (1993).

Previous attempts to model the effect of leaf dark respiration on the ¹⁸O of CO₂ in canopy air have considered only the net respiratory CO₂ efflux. We will refer to this method as the net flux model. In the net flux model, _R is calculated as _R = _e + _w – a, where a is usually taken as 8.8. The C¹⁸OO isoflux is then calculated as the product of _n and _R. For the purposes of this discussion, we define an isoflux as the product of a net CO₂ flux and its ¹⁸O. The net flux model has been used to interpret nighttime measurements of ¹⁸O in canopy CO₂ (Flanagan et al., 1997, 1999; Mortazavi and Chanton, 2002; Bowling et al., 2003a, 2003b) and in global simulations of ¹⁸O dynamics in atmospheric CO₂ (Cuntz et al., 2003a, 2003b). Earlier global studies did not differentiate leaf respiration from soil respiration, and thus did not define _R for leaves (Farquhar et al., 1993; Ciais et al., 1997). A slightly different version of the net flux model, with a modified term for diffusional fractionation, has also been applied at the leaf level (Yakir et al., 1994; Yakir, 1998). If we apply the net flux model to data from our second experiment, where C_a was near that found in the atmosphere, predicted values for _R range from 42 to 61. These values can be compared to observed _R values ranging from 233 to 324. Thus, in the second dark respiration experiment, the net flux model underestimated the observed _R by 180 to 266. Note that these observed _R values are effective values that result when one treats the modification of ¹⁸O of CO₂ in air passing over the leaf as if it resulted from the net CO₂ efflux alone. Thus, using these observed _R values, the C¹⁸OO isoflux is still calculated as _nR, and the large difference between _n and g_tcC_c/P becomes manifested in the _R term.

If we apply the net flux model to our first experiment, where air entering the leaf chamber was free of CO₂, it predicts _R values ranging from 36 to 60. Observed _R values in this experiment ranged from 44 to 59, in good agreement with predictions from the net flux model. The difference in the performance of the net flux model between the first and second dark respiration experiments can be effectively understood by examining an alternative formulation of Equation 2. If the definition of _c from Equation 6 is substituted into Equation 2, and the term () in the denominator of Equation 2 is assumed equal to unity, Equation 2 can be rewritten as

(31)

Equation 31 is informative in that terms I and II on the right side are analogous to the net flux model; the difference is that in Equation 31 _c is defined as in Equation 6, whereas in the net flux model _c is defined as _e + _w. Term III on the right side of Equation 31 reflects the proportion of CO₂ that diffuses into the leaf and equilibrates with leaf water, then diffuses out of the leaf, thereby altering the isotopic composition of CO₂ in the leaf chamber while leaving the net CO₂ efflux rate unaltered. This process is analogous to the invasion effect that has been described for soil respiration (Tans, 1998; Miller et al., 1999; Stern et al., 2001). In the first dark respiration experiment, where air entering the leaf chamber was free of CO₂, this process was also occurring, but had a much smaller impact on _R than in the second experiment. This is because (_c – _a) was small in the first experiment, having a mean value of 1.8; in contrast, (_c – _a) in the second experiment had a mean value of 7.9. Additionally, [C_a/(C_c – C_a)] was much smaller in the first experiment than in the second, having a mean value of 4.8 in the former versus 47.7 in the latter. As a result, the mean value for term III in Equation 31, which can be thought of as the invasion term, was 5.2 for the first dark respiration experiment, and 234 for the second dark respiration experiment.

Equation 31 can be used to highlight the conditions under which large departures in _R from values predicted by the net flux model can be expected at the ecosystem level under natural conditions. For example, if _c is very similar to _a, term III will be small. Additionally, if stomata are tightly closed, [C_a/(C_c – C_a)] will be small, and term III will also be small. Thus, the largest departures in _R from the predictions of the net flux model should occur when there is a relatively large difference between _c and _a, and when stomata are relatively open, such that [C_a/(C_c – C_a)] is large. The approximation in Equation 31 that () equals unity introduces a very small bias into calculations with this equation; however, this bias is less than 1% and is therefore negligible. Thus, Equation 31, in combination with Equation 6, can be used in place of Equation 2, if so desired.

Photosynthesis enriches the atmosphere in C¹⁸OO due to exchange of CO₂ with evaporatively enriched leaf water in the chloroplast, whereas soil respiration is generally thought of as depleting the atmosphere in C¹⁸OO, because soil CO₂ exchanges with water in soil that has generally not been enriched by evaporation (Flanagan and Ehleringer, 1998). In this study, we have observed that leaf dark respiration is capable of enriching air passing over a leaf in C¹⁸OO to as great an extent as photosynthesis. The mean ¹⁸O value of CO₂ exiting the leaf chamber in the respiration measurements at atmospheric CO₂ partial pressure was 43.5; the mean value for photosynthesis measurements at similar C_a was 42.3. The ¹⁸O of incoming CO₂ in both experiments was 19.1, and flow rates through the chamber were similar between the two experiments. Thus, dark respiration had as marked an effect as photosynthesis on the ¹⁸O of CO₂ passing over the leaves, even though the net exchange of CO₂ between the leaf and ambient air is roughly an order of magnitude less, and in the opposite direction, during dark respiration.

The effect of both photosynthesis and respiration on ¹⁸O of CO₂ in canopy air is partly controlled by the isotopic composition of leaf water. In natural systems, nighttime leaf water ¹⁸O is typically intermediate between daytime leaf water ¹⁸O and the ¹⁸O of source water (Dongmann et al., 1974; Förstel, 1978; Zundel et al., 1978; Förstel and Hützen, 1983; Flanagan and Ehleringer, 1991; Flanagan et al., 1993, 1999; Cernusak et al., 2002; Mortazavi and Chanton, 2002). We therefore expect nighttime leaf respiration to impart a C¹⁸OO signal on the atmosphere that is intermediate between the soil respiration signal and the photosynthesis signal.

Accurate prediction of the oxygen isotope composition of leaf water is important for interpreting vegetation effects on ¹⁸O of atmospheric CO₂. Equation 7 can be used to calculate _e under steady state conditions. However, leaf water ¹⁸O is unlikely to be at steady state at night (Flanagan and Ehleringer, 1991; Harwood et al., 1998; Cernusak et al., 2002). Cernusak et al. (2002) applied a non-steady state equation for ¹⁸O in leaf water, derived by G.D. Farquhar and L.A. Cernusak (unpublished theory), and found good agreement between predicted and observed nighttime values. The combination of the non-steady state leaf water equation and the model that we have provided here for _R should allow reasonable predictions to be made of the impact of leaf dark respiration on ¹⁸O of atmospheric CO₂.

Stomatal conductance will be an important parameter in the prediction of both _e and _R during the night. However, little attention has been paid historically to nighttime stomatal conductance. Snyder et al. (2003) recently observed nighttime stomatal conductance to water vapor ranging from 10 to 150 mmol m^–2 s^–1 for 17 plant species in the western United States. However, a mechanistic framework for interpreting such variation does not currently exist. Further investigation into the patterns and processes controlling nighttime stomatal conductance will lead to more accurate prediction of nighttime _e and _R. We note that the mean stomatal conductance that we observed in the dark for R. communis at normal atmospheric CO₂ concentration was 130 mmol m^–2 s^–1 (Table II), near the high end of values observed by Snyder et al. (2003) at night in the field. Our measurements were made during the day, and it is likely that stomatal conductance was influenced by circadian rhythms, causing it to be higher than it would be in the dark at night.

The mean value of for the photosynthesis experiment calculated by the method described by Gillon and Yakir (2000b) was very close to 1.0. If the outlying data point, indicated by an arrow in Figure 3, was excluded from the analysis, the regression method resulted in a similar estimate of 1.06. Thus, both calculations suggested values close to unity for photosynthesizing R. communis leaves. A quick examination of Figure 3 shows that observed _c estimates lie very close to those expected for full equilibrium, with the exception of the one outlier, which is several per mil above the value expected for full equilibrium. We are unable to find a satisfactory explanation for why this particular datum should differ so markedly from the others. Results have been reported for a number of other C₃ species in which the CO₂ diffusing out of photosynthesizing leaves appeared to be very close to full equilibrium with _e (Farquhar et al., 1993; Gillon and Yakir, 2001). Interestingly, the values that we observed during dark respiration in R. communis were lower than those observed during photosynthesis, having values close to 0.80. Further research is necessary to determine the cause of this apparent discrepancy between in the light and in the dark.

Gillon and Yakir (2000b) suggested that during photosynthesis _c0, the ¹⁸O of CO₂ in the chloroplast not equilibrated with chloroplast water, can be calculated, to a close approximation, as _c0 = _a – (1 – C_c/C_a). This definition assumes no discrimination against C¹⁸OO by Rubisco during photosynthesis, and neglects any influence of photorespiration or day respiration on _c0. The latter statement is tantamount to saying that CO₂ evolved from the mitochondria in the light has the same oxygen isotope composition as CO₂ in the chloroplast. In that case, any addition of mitochondrial CO₂ will have no impact upon the ¹⁸O of chloroplast CO₂. The photosynthesis data set that we collected for R. communis did not allow us to test these assumptions because was very close to 1.0; thus, the _c0 signal was completely washed out by the activity of carbonic anhydrase.

However, this was not the case for dark respiration, during which was approximately 0.80. The method of Gillon and Yakir (2000b) leads to mean _c0 values for the first and second dark respiration experiments of 55.2 and 43.7, respectively. These values can be compared to the mean _c0 values generated by the regression method of 30.8 and 14.3, respectively. Although the regression method makes no a priori assumptions about the controls on _c0, we caution against over-interpretation of these latter values for the following reason: the regression analysis, as summarized in Equation 6, assumes no variation in and _c0 among individual measurements in each experiment. The _a values varied among measurements according to how the leaf was modifying the ¹⁸O of CO₂ in the leaf chamber. Therefore, to the extent that _c0 is controlled by _a, _c0 could also have varied among individual measurements.

Nonetheless, the large variation between _c0 calculated as suggested by Gillon and Yakir (2000b) and the apparent _c0 values observed in the dark respiration experiments warrants some discussion. There are three possible sources for the oxygen in CO₂ evolved in mitochondria during either dark respiration or photosynthesis: atmospheric O₂, organic oxygen from respiratory substrates, and oxygen from leaf water. Atmospheric O₂ has a ¹⁸O near 23.5 (VSMOW scale), and discrimination against ¹⁸OO during respiration in plant tissues ranges from about 17 to 26 (Guy et al., 1992). We would therefore expect the ¹⁸O of respiratory CO₂ deriving its oxygen atoms from O₂ to be in the range of 0 to 5. Assuming the O₂ tank used in our experiments had a ¹⁸O similar to atmospheric O₂, this range of values would apply. Organic oxygen in phloem sap sugars of the R. communis plants that we studied had a mean ¹⁸O of 27.5 ± 0.6 (mean ± 1 SD; n = 10). Generally, this oxygen pool is expected to have a ¹⁸O enriched by 27 compared to _L at the time of photosynthesis (Cernusak et al., 2003). Oxygen atoms derived from water during respiratory reactions would also be expected to be enriched by 27 compared to the ¹⁸O of the water source. The difference between the ¹⁸O of CO₂ derived from any of these three sources and that of CO₂ diffusing into the leaf from the atmosphere, prior to equilibration with leaf water, would depend on _a and, in the case of organic oxygen and oxygen from water, _L. However, it seems likely that under most circumstances the effect of incomplete equilibration between CO₂ evolved from mitochondria and leaf water would be to decrease _c0 below the value predicted by the formulation given by Gillon and Yakir (2000b). More experiments like those conducted by Yakir et al. (1994) would be helpful for resolving this issue.

Farquhar and Lloyd (1993) discussed the departure of _c from that predicted for equilibrium with _e during photosynthesis in terms of the ratio of the rate of carboxylation by Rubisco to the rate of CO₂ hydration by carbonic anhydrase. This ratio was termed . A simplified non-equilibrium equation for discrimination against C¹⁸OO during photosynthesis, neglecting the possible effects of photorespiration and day respiration, was presented as (Farquhar and Lloyd, 1993)

(32)

where b¹⁸ is discrimination against C¹⁸OO by Rubisco. Using this equation, and assuming b¹⁸ = 0, we calculated a mean value for our photosynthesis measurements of –0.002 ± 0.009 (mean ± 1 SD; n = 8); if the outlier in Figure 3 is excluded, the mean value becomes 0.001 ± 0.006 (mean ± 1 SD; n = 7). These values can be compared to a mean value calculated for Phaseolus vulgaris of 0.025 (Flanagan et al., 1994). Thus, the values that we observed for R. communis were somewhat smaller than those observed previously for P. vulgaris. These values can be compared to a theoretical prediction for of approximately 0.05 (Cowan, 1986).

In our calculations we have assumed that the ¹⁸O of chloroplast water is equivalent to _e. One might expect chloroplast water to be slightly less enriched than _e due to the Péclet effect (Farquhar and Lloyd, 1993), which describes the interplay between advection of water toward the evaporative sites and diffusion of heavy isotopes away from the evaporative sites. We found that correlations between _c and _e were generally stronger than between _c and _L. This agrees with previous results (Flanagan et al., 1994), and suggests that _e is a more relevant parameter for predicting ¹⁸O of CO₂ diffusing out of leaves than _L.

Gillon and Yakir (2000a) suggested that the CO₂ partial pressure at the chloroplast surface (C_cs) is a more appropriate parameter for predicting discrimination against C¹⁸OO during photosynthesis than that at the sites of carboxylation by Rubisco (C_c). They reconstructed C_cs by combining measurements of C¹⁸OO discrimination and carbonic anhydrase activity. We did not measure carbonic anhydrase activity directly, and so could not modify our calculations to take into account C_cs. In cases where the total resistance from the chloroplast to the atmosphere in the dark is dominated by the stomatal resistance, use of C_cs in place of C_c will likely not alter predictions of _R to a very large extent. However, if stomata are relatively open and (_c – _a) is large, such that the invasion term in Equation 31 is large, a variation between C_c and C_cs of as little as 2 µbar could have a significant effect on predicted _R. In such cases it may prove helpful to use C_cs in place of C_c, if possible.

Farquhar et al. (1993) found that a globally averaged leaf water ¹⁸O of 4.4 satisfactorily balanced the global budget for ¹⁸O of atmospheric CO₂. In the most recent study of the global budget for ¹⁸O of atmospheric O₂, a globally averaged leaf water ¹⁸O of between 6.1 and 6.8 was estimated (Hoffmann et al., 2004). Gillon and Yakir (2001) suggested that the globally averaged leaf water ¹⁸O could be as much as 3 more than the estimate of Farquhar et al. (1993), in agreement with the requirement for balancing the Dole effect (global ¹⁸OO budget); the global C¹⁸OO budget could then be maintained by incomplete equilibration of chloroplast CO₂ with chloroplast water (i.e. < 1). They estimated a globally averaged of 0.80. The results presented in this study provide an additional reason that the apparent leaf water signals required to balance the global C¹⁸OO and ¹⁸OO budgets should not be expected to resolve into a single value. The apparent leaf water signal relevant to the global ¹⁸O budget for O₂ is the average daytime leaf water ¹⁸O, weighted by diurnal (daytime) variation in photosynthetic oxygen evolution rates. In contrast, the apparent leaf water signal relevant to the global ¹⁸O budget for CO₂ is the 24-h average leaf water ¹⁸O, weighted by diel (day and night) variation in g_tcC_c/P. Thus, the apparent leaf water ¹⁸O signals relevant to the global C¹⁸OO and ¹⁸OO budgets are fundamentally different.

	CONCLUSION

TOP ABSTRACT THEORY RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
We observed a very large variation in the ¹⁸O of CO₂ respired by leaves in the dark, with observed values ranging from 44 to as high as 324. We have shown that this large range of _R values can be satisfactorily explained by taking into account the flux of CO₂ that enters the leaf, equilibrates with leaf water, and diffuses out of the leaf without affecting the net CO₂ efflux. Incorporation of the correct expression for ¹⁸O of leaf dark respiration into ecosystem and global scales models of C¹⁸OO dynamics could affect model outputs and their interpretation.

	MATERIALS AND METHODS

TOP ABSTRACT THEORY RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

Plant Material and Gas Exchange Measurements

Ricinus communis plants were grown from seeds in 10-L pots for 8 to 12 weeks in a temperature and humidity controlled glasshouse. Growth conditions were essentially the same as those described by Cernusak et al. (2003). Daytime temperature and humidity were 27°C ± 2°C and 40% ± 10%, respectively. Nighttime temperature was 20°C, with the same humidity as during the day. Measurements were made on fully expanded leaves of plants that were approximately 1 m tall. Projected areas of measured leaves ranged from approximately 400 to 800 cm². The configuration of the gas exchange system was recently described (Cernusak et al., 2003). The through-flow rate of air in the leaf chamber was approximately 3 L min^–1. Chamber air cycled continuously through a bypass drying loop to remove water vapor. The flow rate through the bypass drying loop was varied between 5 and 45 L min^–1 to achieve different vapor pressures within the chamber, and therefore different values of e_a/e_i, and consequently of _e. Air entering the leaf chamber was generated by mixing 79% dry nitrogen with 21% dry oxygen using two mass flow controllers. Carbon dioxide was added to this air stream from a cylinder of 10% CO₂ in air. Leaf temperature was measured with eight thermocouples arrayed across the underside of the leaf, and the average of these measurements used in gas-exchange and isotopic calculations. Gas-exchange calculations were performed according to the equations of Caemmerer and Farquhar (1981).

After gas exchange conditions in the leaf chamber stabilized for a time period judged long enough for leaf water to reach isotopic steady state, CO₂ was cryogenically trapped from air exiting the chamber, as described previously (Evans et al., 1986; Caemmerer and Evans, 1991). Trapping continued until approximately 50 µmol of CO₂ was obtained. The time period sufficient for leaf water to reach isotopic steady state was assumed to be three times the residence time of lamina leaf water (Förstel, 1978). The residence time of lamina leaf water was calculated as W/g_tw_i, where W is the lamina water concentration (mol m^–2), g_t is the total conductance of boundary layer plus stomata to water vapor (mol m^–2 s^–1), and w_i is the mole fraction of water vapor in the leaf intercellular air spaces (mol mol^–1). The term W was determined to be 6.3 ± 0.4 mol m^–2 (mean ± 1 SD) from measurements of the difference between fresh weight and dry weight for one leaf from each of five plants. This mean value of W was assumed for all leaves in the experiment; g_t and w_i were calculated continuously for each leaf being measured. Time periods calculated in this way for leaf water to reach isotopic steady state after a step change in humidity ranged from approximately 0.5 to 3.5 h.

Three experiments were conducted, two in the dark and one in the light. In the first dark experiment, air entering the leaf chamber was free of CO₂. All CO₂ in the air exiting the chamber was therefore derived from the leaf. Measurements were conducted on one leaf from each of five plants. Each leaf was subject to two or three different chamber vapor pressures, and CO₂ collected after gas exchange had stabilized for the requisite amount of time at each vapor pressure. Chamber air temperature was maintained at approximately 30°C. The second dark experiment was similar to the first, but differed in that CO₂ was added to the air entering the chamber, such that the partial pressure within the chamber was approximately 350 µbar. The third experiment was in the light. Irradiance varied between 300 and 800 µmol PAR m^–2 s^–1, and chamber air temperature varied between 25°C and 30°C. The CO₂ partial pressure within the chamber was approximately 350 µbar.

Isotope Measurements

The carbon and oxygen isotope composition of CO₂ exiting the leaf chamber was determined on an Isoprime mass spectrometer (Micromass, Manchester, UK) operating in dual inlet mode. Repeated analyses of the same gas sample generally showed a precision of better than 0.1 (1 SD, n = 10) for ¹³C and ¹⁸O. The carbon and oxygen isotopic composition of the gas used as a reference for the dual inlet measurements was calibrated against standard gases supplied by the International Atomic Energy Agency (Vienna). Oxygen isotope ratios in this paper are presented relative to VSMOW; carbon isotope ratios are presented relative to the Vienna Pee Dee Belemnite standard (VPDB). The oxygen isotope composition of irrigation water fed to the plants was determined with an Isochrom mass spectrometer (Micromass) operating in continuous flow mode (Farquhar et al., 1997). The water samples were pyrolyzed in a custom-built furnace at 1,300°C prior to entering the mass spectrometer. Precision of analyses, based on repeated measurements of a laboratory standard water sample, was 0.3 (1 SD, n = 10). The ¹⁸O of the irrigation water was found to be –7.2 ± 0.2 (mean ± 1 SE; n = 6).

We assumed that the only source of N₂O in the leaf chamber was the compressed air that the CO₂ was mixed into, and that the concentration of N₂O in this air was 300 nmol mol^–1. The CO₂ concentration was 10%, giving a ratio of N₂O to CO₂ of 3 x 10^–6. This ratio could have been doubled during photosynthesis measurements, when the CO₂ concentration exiting the chamber was as little as one-half that entering it, giving a ratio of 6 x 10^–6. Using the empirical equations of Mook and van der Hoek (1983), this ratio of N₂O to CO₂ would result in measurement biases of 0.002 for both ¹³C and ¹⁸O. This bias was considered negligible, and no attempt was made to account for contamination of CO₂ samples by N₂O.

	ACKNOWLEDGMENTS

 
We thank Matthias Cuntz, Roger Gifford, Ricardo Marenco, and Francesco Ripullone for helpful comments on earlier versions of the manuscript.

Received February 9, 2004; returned for revision April 29, 2004; accepted May 3, 2004.

	FOOTNOTES

 
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.040758.

^* Corresponding author; e-mail lucas.cernusak{at}cdu.edu.au; fax 61–8–8946–6847.

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