INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Photosynthetic CO₂ assimilation of C₃ plants discriminates against ¹³CO₂ so that organic matter is, on average, 20 depleted in ¹³C compared with atmospheric carbon dioxide (for recent review, see Brugnoli and Farquhar, 2000). Respiratory carbon fluxes in light (i.e. photorespiration and "day" respiration) are often assumed to be negligible or weakly fractionating processes. However, the carbon isotope signature of organic matter may be modified by nighttime respiration depending on the ¹³C of the evolved CO₂ because respiratory carbon lost by many plants has been shown to be within 30% to 60% of the carbon fixed through photosynthesis (Evans, 1993; Amthor, 2000).

In vitro studies using protoplasts have shown that respired CO₂ isotope composition is identical to that of the Suc supplied to the culture medium, indicating that no fractionation occurs during respiration in the dark (Lin and Ehleringer, 1997). A similar result was also obtained in long-term experiments with animals, where the isotope composition of CO₂ expired by mice (Mus musculus) reflected that of the diet (Perkins and Speakman, 2001). In contrast, it has been shown previously that CO₂ produced by respiration in the dark is 6 ¹³C enriched when compared with Suc in intact French bean (Phaseolus vulgaris) leaves (Duranceau et al., 1999). Similar results were also obtained in Nicotiana sylvestris and sunflower (Helianthus annuus), although CO₂ was less ¹³C enriched with ¹³C values of 4 and 3, respectively (Ghashghaie et al., 2001). Moreover, it has been demonstrated that the ¹³C value of CO₂ evolved in the dark decreased in sunflower leaves subjected to drought (Ghashghaie et al., 2001). Assuming carbohydrates as the main respiratory substrate, Ghashghaie and coworkers suggested that discrimination occurred during dark respiration in C₃ plants, but it varied among species and with drought conditions. When compared with total organic matter, respired CO₂ was found to be enriched in ¹³C in wheat (Triticum aestivum; Troughton et al., 1974), tomato (Lycopersicon esculentum; Park and Epstein, 1961), and Cucurbita moschata (Smith, 1971), whereas it was depleted in ¹³C in Pinus radiata and maize (Zea mays; Smith, 1971). Moreover, in sliced potato (Solanum tuberosum) tubers, it varied with time, presumably reflecting some metabolic shifts (Jacobson et al., 1970). Interestingly, there seems to be a linear relationship between the carbon isotope composition of non-lipid plant material and the fraction of lipids in plants or algae (Park and Epstein, 1961), suggesting that the isotopic composition of carbohydrates subsequently oxidized into CO₂ is a function of general carbon metabolism. Carbon isotope composition of the CO₂ evolved in the dark may reflect both the discrimination of the carbon by cell metabolism in the dark, which at least occurs during the pyruvate dehydrogenase (PDH) reaction (O'Leary, 1976; Melzer and Schmidt, 1987) and the isotope composition of the carbon source feeding the Krebs cycle.

In this work, we address the question of the metabolic origin of the carbon isotope signature of CO₂ evolved by French bean leaves in the dark. Respiratory activity was modified by changing both leaf temperature and by increasing dark period length from 1 h to several days. The carbon isotope composition of the respired CO₂ was related to the respiratory quotient (RQ) and the carbon isotope composition of carbohydrates, heat-precipitated proteins, and fatty acids.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Effect of Temperature on RQ and ¹³C of CO₂ Evolved in Darkness

Leaf respiration increased with temperature with Q₁₀ values of 2 (when leaf temperature shifted from 10°C to 20°C) and 1.5 (from 20°C to 30°C; Fig. 1A). RQ decreased from about 1.1 at 10°C to 0.8 at 30°C (Fig. 1B). The ¹³C value of the dark-respired CO₂ decreased linearly as temperature increased. The slope of the regression line was approximately 0.2 °C¹ (Fig. 1C). The ¹³C values of Suc (23.5), starch (24.7), heat-precipitated proteins (27.2 ), and lipids (33.5) are also given (Fig. 1C). For leaf temperatures ranging from 10°C to 30°C, the respired CO₂ was always ¹³C enriched compared with the analyzed metabolites.

View larger version (15K): [in this window] [in a new window]   Figure 1.   Effect of temperature on leaf respiration in French bean. Respiration rate (A), RQ (B), and 13C of CO2 (circles), Suc (triangles), starch (St, squares), heat-precipitated proteins (Prot, hexagons), and lipids (Lip, diamonds; C) of intact leaves are plotted as a function of temperature. CO2 was collected in a closed system for respiration measurements and isotope analyses. Data are means of three independent replicates ± SEs. Linear regressions for the RQ and 13C value of respired CO2 give y = 1.47 102 x + 1.24 (r2 = 0.87) and y = 0.21x 16.98 (r2 = 0.91), respectively. Regressions are significant for both RQ and 13C (F = 26.30, P < 0.0068, and F = 61.16, P < 0.0002, respectively).

Effect of a Prolonged Period of Darkness

Variation of Some Leaf Metabolites in Continuous Darkness

Variation of Carbohydrate Pool in Continuous Darkness

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View larger version (18K): [in this window] [in a new window]   Figure 2.   Variation in metabolite contents in intact French bean leaves maintained in darkness for several days at different temperatures (circles, 20°C; triangles, 10°C; and squares, 30°C) and then subjected to 6 h of light at a photosynthetic photon flux density (PPFD) of about 500 µmol m2 s1 (r). Starch (A), Suc (B), and Glc (C) amounts are expressed as percentage of initial content and are plotted as a function of time.

Variation of Respiration Rate, RQ, and ¹³C-Respired CO₂ in Continuous Darkness

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View larger version (17K): [in this window] [in a new window]   Figure 3.   Variation of respiration rate in intact French bean leaves maintained in darkness for several days at different temperatures (circles, 20°C; triangles, 10°C; and squares, 30°C) and then subjected to 6 h of light at a PPFD of about 500 µmol m2 s1 (r). Leaf respiration (A), RQ (B), and 13C of respired CO2 (C) are plotted as a function of time. 13C of starch (24.7), heat-precipitated proteins (26.7), total organic matter (27.5), and lipids (33) are constant. CO2 was collected in a closed system for respiration measurements and isotope analyses. Data points were obtained on two individual plants.

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Relationship between RQ and ¹³C of CO₂

RQ and ¹³C data from "temperature" (Fig. 1, B and C) and "continuous darkness" (Fig. 3, B and C) experiments have been replotted in Figure 4 (white symbols). The relationship between RQ and ¹³C, obtained without data from the continuous darkness experiment at 10°C, is clearly linear (r² = 0.87) and gives a slope of 16.57 per RQ unit (Fig. 4A). ¹³C of respired CO₂ (from Smith, 1971; Park and Epstein, 1961) and RQ values (from James, 1953) of sunflower, wheat, castor bean (Ricinus communis), and squash (Cucurbita pepo) seedlings and leaves of tomato and pea (Pisum sativum), grown in the greenhouse at ambient temperature, have also been plotted in Figure 4A (black symbols). Data from "continuous darkness" experiments at 10°C show a nonlinear relationship (Fig. 4B).

View larger version (14K): [in this window] [in a new window]   Figure 4.   Relationship between 13C of dark-respired CO2 and the RQ. A, Data are taken from Figures 1 (diamonds) and 3 (white circles, 20°C; and squares, 30°C) and from the literature (James, 1953; Park and Epstein, 1961; Smith, 1971; black circles). The linear regression does not take into account data from the literature. The regression equation is: y = 16.57x  37.62 (r2 = 0.87). The regression is significant (F = 144.21, P < 0.0001). B, Data taken from the "continuous darkness" experiment at 10°C of Figure 3 (triangles). The solid line represents the regression line obtained in A.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

The carbon isotope signature of CO₂ produced in darkness linearly decreased when temperature increased, from around 19 at 10°C to 24 at 35°C (Fig. 1C). It also progressively decreased, from 21 to 30, when leaves were maintained in continuous darkness for several days (Fig. 3C). Under "normal" conditions (temperature less than 30°C and a usual dark period duration), the evolved CO₂ was enriched in ¹³C compared with carbohydrates, the most ¹³C-enriched metabolites. This is in agreement with previous results (Duranceau et al., 1999; Ghashghaie et al., 2001), suggesting that a fractionation occurs during dark respiration. However, at the end of a long dark period (carbohydrate starvation), respired CO₂ was ¹³C depleted even when compared with the composition of total organic matter (Fig. 3C).

Interestingly, the temperature induced decrease in ¹³C was paralleled by a decrease in RQ (Fig. 1), the ratio of CO₂ production to oxygen consumption. Because RQ values close to 1 indicate highly oxygenated substrates (e.g. carbohydrates) with a relative high level of ¹³C and RQ values near to 0.6 indicate weakly oxygenated substrates (e.g. fatty acids) with a relative low level of ¹³C, it follows that the decline in ¹³C is best explained by the isotope composition of the carbon of the products feeding the respiratory processes. RQ values lower than 0.6 may be observed when respiration is low and coupled to gluconeogenesis from lipids (Lundegardh, 1966). RQ values higher than 1 may be observed when metabolites such as malate or citrate are oxidized. Thus, it is possible that newly synthesized malate and citrate were oxidized together with carbohydrates in a dark period that immediately follows a period of photosynthesis. As shown in Table I, under such conditions, the amount of malate is substantial. ¹³C of malate has not been measured in these experiments; however, it has been shown using tobacco (Nicotiana tabacum; Jamin et al., 1997) that the ¹³C to ¹²C ratio of malate is close to that found for carbohydrates. As expected, the decline in RQ as leaf temperature increases (Fig. 1B) is consistent with a much faster decrease in starch, Suc, and Glc contents observed with increasing temperature (Fig. 2). This is presumably because of an increase of dark respiration rate and Suc loading under these conditions. As a result, leaves maintained at an elevated temperature rapidly consume respiratory carbohydrates and use proteins and fatty acids to feed respiration, and this causes a decline in the ¹³C of CO₂.

Similarly, when leaves are maintained in darkness, RQ, which progressively decreases from 1 to around 0.6 after 5 d, parallels the ¹³C decrease. Dark-induced sugar starvation (Fig. 2) is coupled to an increased contribution of fatty acid -oxidation. Amino acids may also contribute to oxidative metabolism because the isotope composition of respired CO₂ decreased to 30, which is between that of proteins (around 27) and lipids (around 33). A switch to fatty acid catabolism as a consequence of sugar starvation is well documented and has been described in tomato leaves maintained in darkness (Park and Epstein, 1961), cell suspensions of A. pseudoplatanus (Aubert et al., 1996), and maize root tips (Dieuaide-Noubhani et al., 1997).

When plotted together, the results give a linear relationship between the carbon isotope composition of CO₂ and the RQ (Fig. 4A, white symbols), thus pointing out that the variability of CO₂ signature mainly originates from substrate switching (r² = 0.87). The residual variability observed in Figure 4A may come from a natural variation in isotope signature from one plant line to another. However, there is an obvious qualitative change in dark metabolism in leaves maintained under continuous darkness at 10°C because for RQ values from 1.1 to 0.6, the ¹³C of CO₂ remained high (Fig. 4B), with a "carbohydrate signature" despite the starch, Suc, and Glc contents being very low. This might presumably indicate the occurrence of a dark metabolism involving gluconeogenesis from ¹³C-enriched molecules like amino acids, e.g. Ala and Ser (Abelson and Hoering, 1961). Variability may also be partly because of the temperature dependence of isotope effects of enzymes involved in dark metabolism such as decarboxylases. In vitro, yeast (Saccharomyces cerevisiae) PDH discriminates during CO₂ production with a positional isotope effect on the C-1 of pyruvate around 1.006 at 15°C and 1.008 at 35°C (DeNiro and Epstein, 1977). It can be assumed that temperature has the same effect on -ketoglutarate decarboxylase, which could be another fractionating enzyme because it has an enzymatic mechanism that bears much similarities with that of PDH. Further experiments are needed to investigate dark metabolism at low temperature.

When the RQ is around 1, the ¹³C value of respired CO₂ is higher than that of carbohydrates (around 24 for Suc). This raises the question of the origin of the ¹³C-enrichment of CO₂ compared with substrates oxidized through respiration. This has already been discussed in Ghashghaie et al. (2001); they emphasized that carbon atom positions C-1, C-2, C-5, and C-6 of Glc-feeding glycolysis are ¹³C depleted when compared with the C-3 and C-4 positions (Rossmann et al., 1991). This contributes to induce both a ¹³C depletion of acetyl-CoA and subsequent fatty acids, and a ¹³C enrichment of carbon dioxide produced by PDH. Moreover, PDH isolated in vitro discriminates against ¹³C during acetyl-CoA formation (DeNiro and Epstein, 1977; Melzer and Schmidt, 1987). This also contributes to ¹³C depletion in acetyl-CoA. Thus, there are two main origins of metabolic CO₂ sources: one ¹³C enriched from pyruvate decarboxylation, and another ¹³C depleted from acetyl-CoA degradation through the Krebs cycle. The imbalance between these two sources may be responsible for the prevalence of ¹³C in respired CO₂.

Relative carbon fluxes involved in the respiratory pathway may change depending on the cell's metabolic status. For example, acetyl-CoA (light carbons) may be used for anabolic purposes (e.g. fatty acids synthesis) when carbohydrates are degraded (RQ around 1 and ¹³C-enriched CO₂ around 21). In contrast, acetyl-CoA is produced by -oxidation of fatty acids when lipids are degraded (RQ around 0.6 and ¹³C-depleted CO₂ around 30).

The isotope composition of CO₂ produced by respiratory metabolism taking into account heterogeneous isotope distribution in Glc (Rossmann et al., 1991) is shown in Figure 5 (values in parentheses). Enzymatic isotope effects have not been used for calculations because of uncertainty in their values measured in vitro only (PDH) or because they are unknown (isocitrate dehydrogenase and -ketoglutarate dehydrogenase). If dark carbon fixation (through phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, and carbamylphosphate synthase) and CO₂ production through the pentose phosphate cycle are neglected, the isotope composition of dark respired CO₂ should be close to 21 (mean value of C-3 and C-4 in Glc) when pyruvate dehydrogenation predominates and close to 27 (mean value of C-1, C-2, C-5, and C-6 in Glc) when the Krebs cycle is coupled to -oxidation of fatty acids. This interval fits well the observed variation range of evolved CO₂ ¹³C values (between 20 and 30).

View larger version (22K): [in this window] [in a new window]   Figure 5.   Carbon metabolism in darkness in relation to 13C of leaf-respired CO2. 13C values of metabolites are those measured in French bean leaves. 13C of CO2 in brackets are derived from positional 13C values in natural Glc as given by Rossmann et al. (1991). PEPC, Phosphoenolpyruvate carboxylase; KC, Krebs cycle. The symbol "?" points out the uncertainty about the amplitude of the PEPC reaction in darkness.

It is concluded that the isotope signature of respired CO₂ in C₃ plants is not constant and is determined mainly by: (a) the carbon source used for respiration, i.e. the relative metabolic activities in the cell (glycolysis, -oxidation, and gluconeogenesis); (b) the non-statistical carbon isotope distribution in Glc; and (c) the possible isotope effects of respiratory enzymes. Although all the fractionating (enzymatic) steps involved in respiration are not well known, the observed ¹³C enrichment in CO₂ compared with substrate does suggest that there is a "fractionation" during dark respiration (Duranceau et al., 1999).

Moreover, our data show that the ¹³C of dark-respired CO₂ is modulated by environmental conditions such as temperature. Presumably, respiration during nighttime, particularly at low temperature when CO₂ is strongly ¹³C enriched, may have a significant ¹³C-depleting effect on the remaining leaf organic material. Some studies are now needed on other plant organs to determine if the carbon isotope composition of dark-respired CO₂ is similar at the whole-plant level.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Plant Material

French beans (Phaseolus vulgaris L. cv Contender, Vilmorin, La Verpillière, France) were grown from seed in 1-L pots of potting mix in a greenhouse. Minimum PPFD during a 16-h photoperiod was maintained at approximately 500 µmol m² s¹ at leaf level by supplementary lighting from high-pressure sodium lamps. Temperature and the leaf-to-air vapor pressure deficit were maintained at approximately 25.5°C/18.5°C and 1.2/1.4 kPa day/night, respectively. Carbon isotope composition (¹³C) of CO₂ in greenhouse air was 9.5 ± 0.3. All experiments were carried out on the first trifoliar mature leaf. The harvested leaves were immediately frozen in liquid nitrogen, lyophilized, and powdered.

Carbon Isotope Analysis and Gas Exchange Measurements

Dark-respired CO₂ was analyzed on-line with a closed system directly coupled to an elemental analyzer NA-1500 (Carlo-Erba, Milan) through a 15-mL loop. After placing intact leaves in the respiration chamber, CO₂ was removed with soda lime columns; when the CO₂ level remained stable, flow from the soda lime columns was switched to the loop and carbon dioxide was accumulated. Molar fractions of respiratory CO₂ were measured with an infrared gas analyzer (Finor, Maihak, Germany) placed in the closed system. When CO₂ reached around 300 µL L¹, the loop was shunted and the gas inside was introduced into the elemental analyzer with helium for GC. The connection valve between the elemental analyzer and the isotope ratio mass spectrometer (VG Optima, Micromass, Villeurbanne, France) was opened when the CO₂ peak emerged from the elemental analyzer. Isotope analysis of metabolites and total organic matter was conducted using the same elemental analyzer and isotope ratio mass spectrometer. Carbon isotope compositions were calculated as deviations of the carbon isotope ratio (¹³C to ¹²C, called R) from international standards (Pee Dee Belemnite) according to Farquhar et al. (1982): ¹³C = 10³ [(R_sample  R_standard)/R_standard].

Leaf temperature in the chamber was measured with a thermocouple and controlled with a water bath. The RQ was calculated from the ratio of carbon production [v(CO₂)] to oxygen consumption [v(O₂)]: RQ = v(CO₂)/v(O₂). The CO₂ production in darkness was measured with the infrared gas analyzer as described above. Oxygen consumption of leaf discs from one leaflet of the same leaf was measured with an oxygen electrode (Hansatech, King's Lynn, UK).

"Temperature" experiments were started after a light period of 8 to 10 h. The ¹³C value of CO₂ and respiration rate were measured at 20°C before shifting leaf temperature to either 10°C or 30°C. The duration of this experiment was approximately 2 h. For each temperature, after ¹³C measurement, some leaf blades were sampled for metabolite analysis. "Continuous darkness" experiments were done at different temperatures (5 d at 20°C and 30°C and 14 d at 10°C) and started after a light period of 10 to 12 h. For each temperature, ¹³C of CO₂ was continuously measured on the same leaf. Leaves of other plants maintained under similar conditions were sampled for metabolite analysis.

Metabolite Extraction and Quantification

The starch and Suc extraction procedures were taken from Duranceau et al. (1999). In brief, 50 mg of leaf powder was suspended with 1 mL of distilled water in an Eppendorf tube (Eppendorf Scientific, Hamburg, Germany). After centrifugation, starch was extracted from the pellet by HCl solubilization. Soluble proteins of the supernatant were heat denatured and precipitated, and soluble sugars of the protein-less extract were separated by HPLC. In most samples, Glc and Fru contents were low and only Suc was isotopically analyzed. After lyophylization, purified metabolites were suspended in distilled water and transferred to tin capsules (Courtage Analyze Service, Mont Saint-Aignan, France) for isotope analysis.

The lipid extraction procedure was carried out as in Deléens et al. (1984). Fifty milligrams of leaf powder was placed in glass tubes with 2 mL of hot ethanol (70°C) during 3 min and then cooled on ice. Two milliliters of chloroform was added and after 15 min at 0°C, 2 mL of distilled water was added and the tubes centrifuged at 2,000 rpm for 10 min at 15°C. The lower phase was collected with a Pasteur pipette and transferred to a new glass tube. Chloroform was evaporated at 60°C under a nonoxidizing atmosphere (N₂). Fatty acids were methyl esterified with 2 mL of methanol-BF₃; 0.5 mL of water and 3 mL of pentane were then added for chlorophyll/lipid separation. The upper phase was transferred to another glass tube and pentane was evaporated at 50°C with an N₂ stream. Methyl-esters were dissolved in 1.5 mL of methanolic sodium hydroxide (0.5 mol L¹ NaOH in methanol). After 1 h at 40°C, pH was neutralized with 0.2 mL of HCl (6 mol L¹), and free fatty acids were separated with 2 mL of pentane. The resulting pentane phase was transferred to a glass tube. After pentane evaporation, fatty acids were dissolved in 70 µL of pentane and transferred to tin capsules for isotope analysis.

GC-MS

For metabolite identification and quantification, samples were prepared for GC-MS analysis, as described in Bleton et al. (1996). In brief, 2 mg of leaf powder was suspended in 0.5 mL of methanolysis reagent (methanol-acetyl chloride-HCl) at 80°C during 24 h. After centrifugation, pH was neutralized with pyridine, and methanol was evaporated using an N₂ stream. Trimethylsilylation reagent (0.5 mL), composed of hexamethyldisilazane and trimethylchlorosilane (Hydrox-Sil Regis, Interchim, Montluçon, France), was then added and the glass tube maintained at 80°C for 2 h. After vacuum evaporation, samples were suspended in hexane for GC-MS analysis. GC-MS was conducted with a gas chromatograph 5890 A (Hewlett-Packard, Palo Alto, CA) coupled to a mass spectrometer Incos 50 quadrupole (Finnigan, San Jose, CA). Because of differential response coefficients of the gas chromatograph detector, signals were normalized to an internal standard molecule introduced to the samples (C₁₉ methyl ester) allowing a relative quantification of metabolites.