JXB Advance Access originally published online on May 28, 2003
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Journal of Experimental Botany, Vol. 54, No. 388, pp. 1761-1769,
July 1, 2003
© 2003 Oxford University Press
12CO2 emission from different metabolic pathways measured in illuminated and darkened C3 and C4 leaves at low, atmospheric and elevated CO2 concentration
Received 14 November 2002; Accepted 7 April 2003
CNR–Istituto di Biologia Agroambientale e Forestale, Via Salaria Km. 29,300, 00016 Monterotondo Scalo (Roma), Italy
* To whom correspondence should be addressed. Fax: +39 06 9064492. E-mail: francesco.loreto{at}ibaf.cnr.it
Abbreviations: 
*, , compensation points in the absence and in the presence of mitochondrial respiration, respectively; Pn, photosynthesis; Rd, 12CO2 emission by mitochondrial respiration in the light; Rn, 12CO2 emission by mitochondrial respiration in the dark.
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Abstract |
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The detection of 12CO2 emission from leaves in air containing 13CO2 allows simple and fast determination of the CO2 emitted by different sources, which are separated on the basis of their labelling velocity. This technique was exploited to investigate the controversial effect of CO2 concentration on mitochondrial respiration. The 12CO2 emission was measured in illuminated and darkened leaves of one C4 plant and three C3 plants maintained at low (30–50 ppm), atmospheric (350–400 ppm) and elevated (700–800 ppm) CO2 concentration. In C3 leaves, the 12CO2 emission in the light (Rd) was low at ambient CO2 and was further quenched in elevated CO2, when it was often only 20–30% of the 12CO2 emission in the dark, interpreted as the mitochondrial respiration in the dark (Rn). Rn was also reduced in elevated CO2. At low CO2, Rd was often 70–80% of Rn, and a burst of 12CO2 was observed on darkening leaves of Mentha sativa and Phragmites australis after exposure for 4 min to 13CO2 in the light. The burst was partially removed at low oxygen and was never observed in C4 leaves, suggesting that it may be caused by incomplete labelling of the photorespiratory pool at low CO2. This pool may be low in sclerophyllous leaves, as in Quercus ilex where no burst was observed. Rd was inversely associated with photosynthesis, suggesting that the Rd/Rn ratio reflects the refixation of respiratory CO2 by photosynthesizing leaves rather than the inhibition of mitochondrial respiration in the light, and that CO2 produced by mitochondrial respiration in the light is mostly emitted at low CO2, and mostly refixed at elevated CO2.. In the leaves of the C4 species Zea mays, the 12CO2 emission in the light also remained low at low CO2, suggesting efficient CO2 refixation associated with sustained photosynthesis in non-photorespiratory conditions. However, Rn was inhibited in CO2-free air, and the velocity of 12CO2 emission after darkening was inversely associated with the CO2 concentration. The emission may be modulated by the presence of post-illumination CO2 uptake deriving from temporary imbalance between C3 and C4 metabolism. These experiments suggest that this uptake lasts longer at low CO2 and that the imbalance is persistent once it has been generated by exposure to low CO2.
Key words: 13C labelling, C3 versus C4 metabolism, mitochondrial respiration, photosynthesis, photorespiration.
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Introduction |
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In illuminated leaves, the CO2 evolved by photorespiration (C3 plants), and mitochondrial respiration through the Krebs cycle (C3 and C4 plants) has been difficult to measure by gas-exchange because of the overwhelming amount of CO2 uptake by photosynthesis. A simple technique that allows the measurement of CO2 emission formed by photorespiration and mitochondrial respiration in illuminated leaves with 13CO2-insensitive infrared gas analysers has recently been described (Loreto et al., 1999, 2001). The method exploits the different time-course of labelling by C isotopes of the photosynthetic, photorespiratory and respiratory pathways. In the past, some of these experiments have been carried out by using the radioactive 14C isotope (Ludwig and Canvin, 1971; Keerberg et al., 1983). The data collected with the 13C technique show that in C4 (maize) leaves maintained at atmospheric CO2 concentration (350–400 ppm), the 12CO2 emission by mitochondrial respiration is largely lower in illuminated leaves (Rd) than in the dark (Rn). By knowing the photosynthetic rate and the labelled (13C) and unlabelled (12C) internal CO2 concentration, it is also possible to calculate if the difference between Rd and Rn is due to refixation or to inhibition of mitochondrial respiration. At ambient CO2, this difference is mostly due to refixation of mitochondrial CO2 in photosynthesizing C4 leaves (Loreto et al., 2001). Rd was not measured in C3 leaves by Loreto et al. (1999), but the authors found that less than 22% of the CO2 evolved by photorespiration was emitted, and commented that Rd should be reduced to a similar extent with respect to Rn. A more refined version of the 13C technique, based on mass-spectrometric measurements to detect 13C and 12C isotopes simultaneously, also indicated that Rd is lower than Rn, especially in drought-stressed leaves (Haupt-Herting et al., 2001).
These results confirmed previous estimations of Rd based on the extrapolation of the linear relationship between photosynthesis and light intensity to darkness (Kok, 1948), or on the extrapolation of a family of photosynthesis response curves to different internal CO2 concentrations, obtained at different light intensities, to the point at which photosynthesis equates photorespiration (Laisk, 1977). However, there are cases in which Rd may be a substantial part of Rn. A clear inverse relationship between Rd and photosynthesis has previously been found. When photosynthesis is limited by high temperatures and low light most of the mitochondrial respiration is emitted by leaves (Loreto et al., 2001).
There are theoretical and experimental results indicating that Rd depends on the external (ambient) CO2 concentration. At low CO2, Rd should be present because the compensation points in the absence and in the presence of mitochondrial respiration (* and ) are not thought to be coincident (Peisker et al., 1995). Moreover, because of the inverse relationship between photosynthesis and Rd (Loreto et al., 2001), a high Rd at low CO2 (low photosynthesis) is expected.
After short-term exposure or growth at elevated CO2 (normally twice the atmospheric concentration), a reduction of the leaf mitochondrial respiration has often been reported (Gifford et al., 1985; Amthor et al., 1992) and is explained by the inhibitory effects of CO2 on mitochondrial enzymes (Gonzalez-Meier and Siedow, 1999). However, numerous other studies suggested that the mitochondrial respiration in the light (Kirschbaum and Farquhar, 1987) and in the dark (Amthor, 2000; Tjoelker et al., 2001) do not change in relation to CO2 increase. There are even studies showing that the respiration of leaves grown in elevated CO2 may increase, both in C3 (Lewis et al., 1999; Wang et al., 2001) and C4 plants (Maroco et al., 1999). As has recently been reported (Jahnke and Krewitt, 2002), mitochondrial respiration in the dark may be unaffected by elevated CO2 after removing methodological artefacts and after considering the possible CO2 leakage in homobaric leaves.
The objective of this study was to clarify the effect of CO2 on mitochondrial respiration in C3 and C4 plants by actually measuring Rd and Rn with the experimental technique developed by Loreto et al. (1999) in leaves exposed to low, atmospheric, and elevated CO2 concentration.
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Materials and methods |
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Plant material and experimental design
Plants of Mentha sativa and Phragmites australis (C3 metabolism, herbaceous), Quercus ilex (C3 metabolism, scherophyllous tree), and Zea mays (C4, NADP-ME metabolism) were grown in 2.0 l pots filled with commercial soil and regularly watered and fertilized to avoid water and nutrient stresses. Plants were grown in a greenhouse under day/night temperatures of 25/15 °C and without supplementing the natural light. The latest fully expanded leaf of each plant was used for gas-exchange studies.
The experiments were replicated on 3–7 leaves of different plants in C3 species, and on three leaves of maize. 12CO2 emission in the light and in the dark, and the traces of the infrared gas analyser reading are shown as single measurements. All other measurements are shown as mean ±SD.
Gas-exchange studies
Conventional gas exchange studies: A 4.9 cm2 circular portion of the leaf was enclosed in a gas-exchange cuvette and illuminated with a KL1500 LCD (Schott, Wiesbaden, Germany) light source using a round fibre glass illuminator (Schott) at a light intensity of 1000 µmol m–2 s–1. At this light intensity, roughly equal to the maximal intensity experienced during growth, leaf photosynthesis was not limited by illumination and Rubisco was fully activated by light. The cuvette was thermostated by water circulating inside the cuvette body, and the leaf temperature was set at 30 °C and measured with a thermocouple appressed to the adaxial leaf side. The leaf disc was exposed to a flow of 500 ml min–1 of synthetic air (N2, O2 and CO2) formed and adjusted with mass flow controllers (Brooks Instruments B.V. series 5800, Veenendaal, The Netherlands). The O2 concentration was set at 20% or 2% to induce photorespiratory and non-photorespiratory conditions in C3 plants. Three CO2 concentrations were used in the experiment carried out with C3 plants: the compensation point (the CO2 at which no gas-exchange between the leaf and air was detected, i.e. between 30 and 50 ppm), atmospheric (350–400 ppm), and elevated (700–750 ppm). In the C4 plant Zea mays, the compensation point was set by operating with CO2-free air, and five more CO2 concentrations (50, 100, 400, 700, 800 ppm) were used. The N2 and O2 mixture was humidified by bubbling it in water and condensing part of the humidity in a water bath set at a temperature lower than the leaf temperature. The relative humidity was maintained at about 40% and the vapour pressure difference between the leaf and air was maintained below 20 mbar. The absolute CO2 concentration in the cuvette was measured with an infrared gas analyser (Gas-hound, Li-Cor, Lincoln, USA) while CO2 and H2O exchanges between the leaf and air were measured with a differential infrared gas analyser (Li 6262, Li-Cor).
Labelling experiments and determination of Rd and Rn: 12CO2 emission in a 13CO2 atmosphere was measured as explained in detail by Loreto et al. (1999, 2001). Briefly, the method exploits the facts that the absolute infrared gas analyser has a very low sensitivity to 13CO2, and that 12CO2 sources are labelled with different time-courses, photorespiration (C3 plants) or bundle sheath leakage (C4 plants) being labelled more rapidly (within a few seconds) than mitochondrial respiration. (>10 min). Each measurement consisted of four steps, as shown the inset in Fig. 2: (1) a CO2-free gas was passed through the infrared gas analyser Gas-hound to zero the instrument; (2) air with the set 13CO2 concentration was passed through the Gas-hound bypassing the leaf cuvette, this read the actual sensitivity of the Gas-hound to 13CO2; (3) the air containing 12CO2 flowing in the cuvette with the illuminated leaf was instantaneously substituted by the air containing the same concentration of CO2, but entirely as 13CO2, and the 12CO2 emitted by the leaf was recorded at steady-state with the Gas-hound; (4) the leaf was darkened and the 12CO2 burst (where measurable) and steady-state emission after darkening were recorded. The emission of 12CO2 was recorded in illuminated leaves for 240 s after switching to 13CO2, and in darkened leaves for 180 s. In a previous experiment (Loreto et al., 2001), the emissions at the end of these two periods were interpreted as the CO2 emitted from mitochondrial respiration in the light and in the dark, respectively. Measurements of Rn could be carried out after 90 s (as in Loreto et al., 1999), but at low CO2 this may not discriminate Rn from the CO2 burst which is probably of photorespiratory origin (see Results and discussion). Thus, in this work Rn measurements were taken when the burst (if any) was elapsed. Measurements required corrections to take into account the remaining sensitivity of the infrared gas analyser to 13CO2, and the CO2 drawdown by photosynthesis which lowers the CO2 concentration sampled by the infrared gas analyser with respect to a reference sample, as detailed in Loreto et al. (1999, 2001). The actual recorded trace during a typical experiment is shown in Fig. 2a inset, while traces shown in Figs 2a and 3a are drawn after corrections. To calibrate the technique, measurements of 12CO2 release into a 13CO2 atmosphere were compared with measurements of CO2 emission by conventional gas exchange without the C isotope in dark-adapted leaves. As expected, and also previously reported (Loreto et al., 2001), the two values were similar with a r2 >0.90 (data not shown). Measurements at CO2 concentrations different from atmospheric may be affected by artefacts caused by technical or biological CO2 leakage (Jahnke and Krewitt, 2002). In this system, a leakage of 12C into a 13C atmosphere would delay considerably the attainment of a steady-state 12CO2 concentration. Since a steady-state 12CO2 concentration was reached with a very similar time-course in all experiments and with different plant materials, it is believed that no artefacts due to CO2 leakage were present.
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Measurements were done in steady-state conditions, that is, when the leaf photosynthesis and stomatal conductance were steady for at least 30 min. Only in the experiment shown in Fig. 4 was this protocol not observed and measurements were carried out with a time series of 5, 15, 30, and 60 min after changing CO2 concentration in leaves previously adapted for a long period (2–4 h) at 50 or 350 ppm of CO2.
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Results |
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The sources of 12CO2 emission in the light can be different in C3 plants (Loreto et al., 1999; and following discussion), while in C4 plants the only source should be the mitochondrial respiration (Rd), the possible CO2 leakage from bundle sheath cells being very rapidly labelled (Hatch et al., 1995). By contrast, the 12CO2 emission in the dark is contributed only by mitochondrial respiration (Rn) irrespective of the plant metabolism. This can be verified by comparing the conventional gas-exchange with the 12CO2 emission (Loreto et al., 2001) and this important verification was repeated successfully (data not shown).
Photosynthesis, and 12CO2 emission in the light and in the dark showed differences between C3 plant species, being lower in the tree species (Quercus) than in the herbaceous species (Fig. 1). Irrespective of these differences, the 12CO2 emission in the light and in the dark were high at low CO2 and decreased at ambient and elevated CO2 (Fig. 1A–C, left panels). The ratio between the 12CO2 emission in the light and in the dark was also clearly higher at the compensation point than at higher CO2 (Fig. 1A–C, right panels). In Mentha and Phragmites, but not in Quercus, the lowest ratio was observed at elevated CO2.
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After darkening the leaf maintained at the compensation point, a burst of 12CO2 was observed (Fig. 2A). The CO2 burst peaked 25–30 s after darkening and was clearly detectable in the leaves of Mentha and Phragmites but not in Quercus or in the C4 species Zea. The burst was very small and almost undetectable in Mentha and Phragmites leaves exposed to atmospheric or elevated CO2 (data not shown). Both in Mentha and Phragmites the burst was remarkably higher at 20% than at 2% O2 (Fig. 2B).
In the C4 plant species Zea mays, photosynthesis at atmospheric CO2 was high (data not shown) and the 12CO2 emission in the light was very low, ranging between 10–20% of Rn (Fig. 3A). The 12CO2 emission in the dark was not affected by low CO2 concentrations but it was clearly inhibited in CO2-free air (Fig. 3A). To a lesser extent, Rn was also reduced in elevated CO2. a clear increase was also observed in the time length needed to restore Rn in leaves exposed to CO2-free air (Fig. 3A). By measuring Rn in leaves exposed to different CO2 concentrations, it was determined that the emission rate, or the velocity by which 12CO2 release increased linearly after darkening (ppm s–1) was CO2-dependent, at least at concentrations lower than atmospheric CO2 (Fig. 3B). Consequently, the time needed to reach Rn was also CO2-dependent. Rn was quickly reached at atmospheric and elevated CO2, but slowly as CO2 decreased from 200 ppm to 0 ppm (Fig. 3C).
To investigate further the observed CO2-dependence of the velocity by which Rn was restored after darkening, Rd and Rn were measured 5–60 min after switching from 350 ppm to 50 ppm or from 50 ppm to 350 ppm. A reduction of CO2 caused a very rapid reduction of the emission rate (Fig. 4A) and a consequent increase of the time needed to reach Rn (Fig. 4B). Five minutes after the switch to 50 ppm the values were already very similar to those found in acclimated leaves (Fig. 3) and did not change further. On the other hand, the increase of CO2 from 50 ppm to 350 ppm did not increase the emission rate (Fig. 4A). Only a moderate reduction of the time to reach Rn was observed (Fig. 4B). This reduction, occurring during the first 20 min after the switch, and not mimicked by the concurrent increase in the emission rate, indicates a reduction of Rn following the switch from low to atmospheric CO2.
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Rd and Rn at low CO2 in C3 plants
These results indicate a clear inverse relationship between the 12CO2 emission in the light and CO2 concentration (Fig. 1). The reason why the 12CO2 emission in the light increases at low CO2 is not straightforward. In C4 plants, an increase of Rd at low light and in other conditions depressing photosynthesis was measured (Loreto et al., 2001). An increase of Rd at low light was also estimated with indirect methods (Atkin et al., 1998). The simplest explanation is that, similar to what occurs at low light and high temperature, at low CO2 Rubisco uptake is unable to refix respiratory CO2 that is, therefore, released into the atmosphere. This is not necessarily due to a reduction of Rubisco activation state but to the limitation set by the low substrate (Sharkey, 1989).
Interestingly, the 12CO2 emission in the light often exceeded that in the dark in Phragmites maintained at the compensation point. A stimulation of mitochondrial respiration by light has been occasionally reported (Peisker et al., 1995). It may be due to the contribution of heterotrophic tissues releasing CO2 (Peisker and Apel, 1980). If mitochondrial respiration of photosynthesizing cells is inhibited by light, as hypothesized by Peisker and Apel (1980), Rd may, in fact, be vastly contributed by respiration of heterotrophic cells. In Phragmites the occurrence of heterotrophic cells may be particularly high because of the open structure of the plant, favouring gas-exchanges in anoxic environments (Antonielli et al., 2002).
Another interesting phenomenon observed at low CO2 is the burst of 12CO2 released by leaves of herbaceous species when darkened (Fig. 2). The burst peaked 30 s after darkening, was 50% lower at 2% O2 than at 20% O2, and was never observed in the C4 plant species. These features suggest that the photorespiratory post-illumination burst (PIB) was monitored (Sharkey, 1988; Atkin et al., 1998). PIB should be due to glycine metabolism (Decker, 1955) that should have been totally and very rapidly labelled (Loreto et al., 1999). But, as already indicated by Ludwig and Canvin (1971), the time to label photorespiratory CO2 increases at low CO2, probably because the photorespiratory pool is still large while the flux of C isotope (13C or 14C) is low. The burst of 12CO2 was detected when leaves had photosynthesized for 4 min in air containing 13CO2 (Fig. 2A). In certain cases the burst was inducible more than once by alternating cycles of illumination and darkening (data not shown). It is therefore likely that photorespiration of herbaceous leaves is driven by a large carbon pool. The high level of unlabelled photorespiratory carbon also could explain why the 12CO2 emission in the light was higher than that in the dark in Phragmites. By contrast, since the burst in Quercus was not observed, it is suggested that the photorespiratory pool of this tree species is of limited size.
As an alternative to this explanation, the 12CO2 burst may be released from the heterotrophic cells or from the mitochondrial respiration of photosynthesizing cells simultaneously with photorespiratory CO2, once the leaf is darkened. There may also be a part of photorespiratory CO2 that does not come from glycine but from previously stored carbon and is therefore unlabelled by 13C even after 4 min. There are previous reports indicating both a sudden change of mitochondrial respiration in darkened leaves (Azcon-Bieto and Osmond, 1983), and the use of stored photosynthates in photorespiration (Xiong et al., 1998). In considering these, the former source of 12CO2 should be O2-independent while the latter source should be O2-dependent. Since it was found that at low O2 the burst was considerably reduced but still present, both sources may contribute to the phenomenon. The burst was clearly observed only at low CO2. Thus, these sources of respiratory (mitochondrial, photorespiratory, heterotrophic), unlabelled CO2, must be suppressed at atmospheric CO2.
If photorespiration is not completely labelled at low CO2, then the 12CO2 emission in the light is contributed by both photorespiration and mitochondrial respiration. In order to distinguish these two pools one should wait until the photorespiration is labelled. If the extent of the burst is taken as an indication of the depletion of the photorespiratory pool, then it seems that up to 40% of the 12CO2 emission in the light, as measured according to this study’s protocol (i.e. before darkening the leaf), is contributed by photorespiration at the time of the measurement, while the remaining part should be contributed by mitochondrial respiration. Even after subtracting the contribution of photorespiration, the 12CO2 emission in the light attributable to mitochondrial respiration at the compensation point remains a relevant part of Rn in C3 species. Thus the compensation point including the release of mitochondrial respiration () is higher than that occurring in the absence of mitochondrial respiration (*).
Previous results indicated that the 12CO2 emission in the light is far lower than that in the dark (Rn) at atmospheric CO2 concentration, especially in C4 plants (Loreto et al., 2001). This finding is substantially confirmed in C3 plants by the results shown in Fig. 1. Under atmospheric CO2, no burst was detected upon darkening, and the 12CO2 emission in the light should be totally attributed to Rd. The ratio between the 12CO2 emission in the light and in the dark therefore estimates the ratio Rd/Rn.
Rd and Rn at elevated CO2 in C3 plants
The effect of elevated CO2 on mitochondrial respiration is controversial, despite the fact that there is both biochemical and physiological evidence showing that Rn might be inhibited at elevated CO2 (see references quoted in the introduction). It is confirmed that Rn is generally lower at elevated than at ambient CO2 and it is reported that Rd is often a lower fraction of Rn at elevated than at ambient CO2. As explained previously, it is considered that these measurements were not affected by technical and biological causes of CO2 leakage, as a rapid and steady-state 12CO2 concentration was attained in 13CO2 atmosphere. It should also be mentioned that the grass-like and probably homobaric Phragmites leaves showed the lowest inhibition of Rn at elevated CO2, contrary to what would be expected in the presence of biological CO2 leakage through leaves with this anatomical feature (Jahnke and Krewitt, 2002). Thus, elevated CO2 may cause the inhibition of mitochondrial respiration in the dark but, most importantly, may reduce the mitochondrial respiration in the light (Fig. 1). In fact, the difference between Rd and Rn may be explained by the extensive refixation of respiratory CO2 in a condition (elevated CO2) that stimulates photosynthesis (Loreto et al., 2001), and decreases stomatal conductance (about 30% on average on the three C3 species, data not shown), therefore restricting CO2 exchange from the leaf to the atmosphere. Photosynthesis increased and the Rd/Rn ratio decreased significantly in Mentha and Phragmites but not in Quercus after exposure to elevated CO2 (Fig. 1), confirming the possibility that respiratory CO2 be efficiently refixed at elevated CO2.
Refixation of mitochondrial respiration can be estimated by calculating the internal concentration of 12CO2, as previously shown for leaves at ambient CO2 (Loreto et al., 2001). This may not be feasible at CO2 different from ambient. For instance, the reliable calculation of refixation would be prevented by the observed photorespiration contribution at low CO2. Other sources of CO2, unrelated to photorespiration and mitochondrial respiration but probably depending on decarboxylation of PEP metabolites (Keerberg et al., 1983), may be active at elevated CO2 (Laisk and Sumberg, 1994). The contribution of these sources to the 12CO2 emission in the light and in the dark needs to be determined as it may significantly change the actual amount of Rd and Rn and, consequently, the calculation of CO2 refixation. Measurements under conditions minimizing CO2 refixation (Bauwe et al., 1987) could elucidate this point in the future.
Rd and Rn at different CO2 in C4 plants
In Zea mays leaves, the 12CO2 emission in the light should only be caused by mitochondrial respiration since photorespiration is suppressed by the C4 metabolism and CO2 leakage from bundle sheath cells is rapidly labelled (Hatch et al., 1995). Rd of Zea mays leaves is particularly low (Loreto et al., 2001) and, contrary to what was observed in C3 plants, did not increase at low CO2 (Fig. 3). A photosynthesis rate of 8–10 µmol m–2 s–1 was measured in these leaves at low CO2 (data not shown). It is therefore likely that even at low CO2 the respiratory CO2 is prevalently refixed and does not exit maize leaves. The extensive refixation of mitochondrial respiration by maize leaves at atmospheric CO2 was clear in a previous experiment (Loreto et al., 2001). When leaves were exposed to CO2-free air, however, both Rd and Rn were even lower than in the presence of CO2, suggesting that mitochondrial respiration is inhibited under these conditions (Fig. 3). It was also observed that exposure to CO2-free conditions reduced the velocity by which Rn was reached in the dark. By repeating the experiment at CO2 increasing from 0 ppm to 800 ppm, or decreasing from 800 ppm to 0 ppm, it was observed that Rn is reached quickly at atmospheric or elevated CO2, but that this velocity is dependent on CO2 at CO2 lower than atmospheric. To interpret this finding it is suggested that in Zea mays the 12CO2 emission in the dark is modulated by the presence of the post-illumination CO2 uptake which has been described in darkened C4 NADP-ME leaves as a consequence of continuing CO2 fixation by RuBP in the presence of PEP limitations (Laisk and Edwards, 1997a). The post-illumination uptake is longer at low CO2 than at ambient CO2 (Laisk and Edwards, 1997a) and this, in turn, causes a longer CO2 refixation which was observed as a delay in the onset of Rn.
To determine how quickly the CO2 concentration can affect the post-illumination uptake and, in turn, Rn rate, the emission rate was measured in darkened leaves as well as measuring the time needed to reach Rn shortly after the switch from 350 ppm to 50 ppm or from 50 ppm to 350 ppm of CO2 (Fig. 4). The emission rate decreased and the time to reach Rn increased in concert immediately after the switch from 350 ppm to 50 ppm, indicating that the post-illumination uptake rapidly changes (increases) when CO2 is decreased. When CO2 is increased in leaves previously adapted to 50 ppm, on the other hand, the emission rate remained low, irrespective of the time taken to measure Rn at 350 ppm. This is interpreted as an indication that the post-illumination uptake is not rapidly reversed, probably because of the slow regeneration of the C4 cycle (Laisk and Edwards, 1997b).
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Conclusions |
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12CO2 detection with 13CO2-insensitive gas analysers showed that in C3 plants both Rn and Rd vary with external CO2 concentration. Rn was clearly inhibited at elevated CO2. The Rd/Rn ratio was inversely associated with photosynthesis, which may indicate efficient refixation of CO2 produced by mitochondrial respiration at elevated CO2 but not at low CO2. Other sources of 12CO2 were detected, such as post-illlumination photorespiratory bursts at low CO2.
In C4 plants, Rd was low and Rn was similar at all CO2 concentrations, probably indicating extensive CO2 refixation in the light. In CO2-free air Rn was inhibited. CO2 concentration modulated the rate of CO2 emission upon darkening, indicating the post-illumination uptake of CO2, particularly persistent at low CO2.
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Acknowledgements |
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Mr Domenico Tricoli helped with gas-exchange measurements. Dr Giorgio Di Marco is gratefully acknowledged for the stimulating discussions and valuable suggestions.
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References |
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