The variations of δ13C in leaf metabolites (lipids, organic acids, starch and soluble sugars), leaf organic matter and CO2 respired in the dark from leaves of Nicotiana sylvestris and Helianthus annuus were investigated during a progressive drought. Under well-watered conditions, CO2 respired in the dark was 13C-enriched compared to sucrose by about 4‰ in N. sylvestris and by about 3‰ and 6‰ in two different sets of experiments in H. annuus plants. In a previous work on cotyledonary leaves of Phaseolus vulgaris, we observed a constant 13C-enrichment by about 6‰ in respired CO2 compared to sucrose, suggesting a constant fractionation during dark respiration, whatever the leaf age and relative water content. In contrast, the 13C-enrichment in respired CO2 increased in dehydrated N. sylvestris and decreased in dehydrated H. annuus in comparison with control plants. We conclude that (i) carbon isotope fractionation during dark respiration is a widespread phenomenon occurring in C3 plants, but that (ii) this fractionation is not constant and varies among species and (iii) it also varies with environmental conditions (water deficit in the present work) but differently among species. We also conclude that (iv) a discrimination during dark respiration processes occurred, releasing CO2 enriched in 13C compared to several major leaf reserves (carbohydrates, lipids and organic acids) and whole leaf organic matter.
variation in modelled discrimination at a given pi/pa relative to a reference value at pi/pa = 0·7
leaf conductance to CO2 diffusion
high performance liquid chromatography
ambient partial pressure of CO2
intercellular partial pressure of CO2
photosynthetic photon flux density
13C/12C ratio of standard VPDB
13C/12C ratio of sample
ribulose 1,5 bisphosphate carboxylase-oxygenase
leaf relative water content
vapour pressure deficit
carbon isotopic composition
Carbon isotope discrimination during leaf CO2 assimilation has been extensively studied and models have been developed (Farquhar, O'Leary & Berry 1982; Evans et al. 1986). The simple version of these models, which does not include the discrimination during respiration, has been validated for many species, suggesting that the discrimination during respiration is negligible and does not significantly modify the net discrimination during on-line measurements compared to the predicted values (for a recent review see Brugnoli & Farquhar 2000). Yet, the carbon isotope signature of plant dry matter integrates not only the discrimination during net CO2 assimilation in the light (including CO2 diffusion from the atmosphere to the chloroplasts, carboxylation, photorespiration and day respiration) but also the discrimination that could occur during the night-time respiration. Therefore, any fractionation during the night and/or the use of heavy or light substrates for dark respiration (releasing 13C-enriched or 13C-depleted CO2 compared with leaf material) should change the isotopic signature of the remaining leaf material. Moreover, when non-photosynthesizing organs are taken into account, the release of 13C-enriched or 13C-depleted CO2 will further contribute to changes in whole-plant carbon isotopic signature. Henderson, von Caemmerer & Farquhar (1992) observed in some C4 species that the discrimination determined on leaf dry matter was significantly greater than that measured on-line. Using a modelling approach, they proposed that at least a part of this difference could be explained by the fractionation during dark respiration, releasing CO2 enriched in 13C relative to the plant material. We obtained similar results on Phaseolus vulgaris (unpublished results) and Nicotiana sylvestris (Duranceau, Ghashghaie & Brugnoli 2001). In contrast, Brugnoli & Farquhar (2000) reported that whole-leaf dry matter was 13C-enriched relative to leaf sugars in Gossypium hirsutum plants. If this difference is due to carbon isotope discrimination during dark respiration as proposed by Henderson et al. (1992), the released CO2 during the night should be 13C-depleted in this species.
There are only a few contradictory data in the literature on the carbon isotope composition of CO2 respired in the dark by plants. Respiratory CO2 has been reported to be 13C-enriched (1–8‰) or 13C-depleted (1–4‰) compared to leaf or whole-plant dry matter (see Smith 1971 and review of O'Leary 1981). Recently, we observed that respired CO2 was 13C-enriched by about 6‰ in intact cotyledonary leaves of bean plants compared to leaf sucrose pool independently of leaf age and relative water content (Duranceau et al. 1999). Assuming that sucrose (or a closely linked metabolite) is used as the main substrate for respiration, we concluded that a fractionation during dark respiration occurs in this species. By contrast, Lin & Ehleringer (1997) found no fractionation during dark respiration in mesophyll protoplasts (isolated from mature leaves of C3 and C4 plants) incubated with substrates of known δ13C (fructose, glucose or sucrose).
In the cases where a difference between 13C content of respired CO2 and potential substrates and/or bulk organic matter was observed, the individual process leading to fractionation could not be identified. Yet, fractionation may be expected during dark respiration because of (i) non-uniform 13C-distribution within the hexose molecules, as reported by Rossmann, Butzenlechner & Schmidt (1991) and Gleixner & Schmidt (1997); and (ii) isotope effects during the pyruvate dehydrogenase (PDH) reaction (De Niro & Epstein 1977; Jordan, Kuo & Monse 1978; Melzer & Schmidt 1987). Effect (i) can lead to 13C-enriched respiratory CO2; effect (ii) can result in 13C-depleted respiratory CO2. The magnitude of the expected effect will depend on the fraction of carbon diverted from the Krebs cycle for biosynthesis of secondary metabolites and on the limiting step in the PDH catalysed reaction. Similarly, Ivlev, Bykova & Igamberdiev (1996) observed, on mitochondrial preparations isolated from photosynthetic tissues of different C3 plants, a huge variation (−8 to +16‰) among species in the 13C content of photorespired CO2 compared to the substrate glycine. They proposed that this variation depends, as in other enzymatic reactions, on the limiting step of the glycine decarboxylation reaction. Other potential causes of fractionation, such as discrimination during transmembrane transport leading to different 13C signatures of metabolites in different cellular compartments, should also be taken into consideration.
Accordingly, the 13C-enrichment (or 13C-depletion) in respiratory CO2 relative to carbohydrates are expected to be variable among different species and to change with environmental conditions as well as relative activities of different metabolic pathways. This could explain the contradictory data reported in the literature concerning the carbon isotope composition of dark-respired CO2.
The potential changes of isotopic signatures in plant organic matter due to respiratory processes are of relevance for the use of 13C in ecosystem studies. The isotopic composition of carbon exchange fluxes can be used to distinguish between oceanic and terrestrial CO2 fluxes, to study CO2 recycling in ecosystems and to decompose net fluxes into gross photosynthetic and respiratory fluxes in combination with 18O studies at the ecosystem level (Yakir & Sternberg 2000). The models applied in these studies often build on the assumption that the signature of organic matter is determined by the signature of photosynthetic products, i.e. respiratory metabolism does not alter the signature of organic matter left behind.
Thus, it is essential to explore whether discrimination during plant respiration is a widespread phenomenon and if there are relevant temporal variations in this discrimination. In our previous work, on bean cotyledonary leaves, the 13C-enrichment in respired CO2 compared to leaf sucrose pool was surprisingly constant during leaf ageing and plant dehydration (Duranceau et al. 1999). This constant fractionation during dark respiration is in contrast with the above considerations. If such a large isotopic fractionation occurs in other C3 plants, this should be taken into consideration in the whole-plant and ecosystem scale studies.
The main objectives of the present work were to examine: (i) whether the fractionation during dark respiration occurs in other C3 species; (ii) if yes, whether this fractionation is similar to that observed in P. vulgaris under similar conditions or if it is different in other C3 species as hypothesized above; and (iii) if this fractionation is constant during a dehydration cycle as was the case for P. vulgaris. Indeed, dehydration was suggested to change the carbohydrate pool sizes among individual plants, probably changing the relative metabolite fluxes and thus the fractionation during dark respiratory metabolism. In our previous work, sucrose (or closely linked substances) was supposed to be the main substrate for respiration and the discrimination was calculated as the 13C-enrichment in respired CO2 relative to the leaf sucrose pool. Another objective of the present work was therefore to examine (iv) whether the respired CO2 is also 13C-enriched compared to metabolites other than carbohydrates.
Therefore, we investigated the carbon isotope composition of major leaf metabolites (starch, soluble sugars, organic acids, lipids), whole-leaf dry matter and CO2 respired in the dark from the leaves of two other C3 species (Nicotiana sylvestris and Helianthus annuus) during plant dehydration. In order to compare the results of the present work with those observed on bean leaves, the experiments were conducted under the same conditions.
MATERIALS AND METHODS
Seeds of Nicotiana sylvestris (fertile botanical line of the Institut des Tabacs, Seita, Bergerac, France) and Helianthus annuus (cv Rigasol, Cargill, Sauzet, France) were sown in small pots, before transplanting into 4 L, 16-cm-diameter, plastic pots containing a peat-based compost. Plants were grown in a controlled-environment chamber in which the temperature was 21 and 17 °C during the day and night, respectively. The photosynthetic photon flux density (PPFD) at plant level was around 220 μmol m−2 s−1 during the 14 h photoperiod. Initially, plants were watered every 2 d to field capacity (30 d period for H. annuus, 60 d period for N. sylvestris). Drought was then induced, for both species, by withholding water from the pots. The dehydration cycle was around 20 d for both N. sylvestris and H. annuus plants. All measurements were made on the fifth or sixth leaf from the top of the plants. In both species, different plants were used for each day of the experiment.
The plant architecture was different for the two species studied. The N. sylvestris plants had short stems and short internodes and the leaves completely covered the soil surface in the pots, probably reducing both the evaporation from the soil and the interception of light by the lower leaves, whereas the stems of the H. annuus plants were high, the soil surface was exposed to the air in the culture room, enhancing the evaporation, and the leaves were well exposed to the light.
Two sets of experiments were conducted on H. annuus plants and one set on N. sylvestris. The first set of the experiments was conducted simultaneously on both species (in February/March) but the second set was made only on H. annuus (in September/October). Growth and experimental conditions were the same for both sets of experiments and for both species except for the relative humidity, which could be variable depending on the seasons. Relative humidity in the culture room was not controlled. The carbon isotope composition of the air in the culture room was not continually monitored but air sampling from time to time gave an average value of about −8‰.
Net CO2 uptake
Leaf net CO2 assimilation was measured using a portable open gas-exchange system (Li 6400, LI-COR Inc., Lincoln, NE, USA). The assimilation chamber allowed measurements to be made on a leaf area of about 6 cm2. Air flow rate was 500 μmol s−1 and leaf boundary-layer conductance for water vapour was 2·84 mol m−2 s−1. The leaf was illuminated using a 6400–02 LED (light emitting diode) light source. The leaf gas-exchange parameters were calculated according to the Li 6400 software. The values of stomatal conductance to water vapour, calculated by the Li 6400 software, were then divided by 1·6 to obtain the stomatal conductance for CO2. Leaf net CO2 assimilation measurements were carried out in the morning after 4–6 h of the photoperiod. They were started after net CO2 uptake and leaf transpiration reached a steady-state rate under a limiting PPFD of 220 μmol m−2 s−1 (saturating PPFD was about 600 μmol m−2 s−1 for N. sylvestris and 1000 μmol m−2 s−1 for H. annuus) in air containing 350 μmol mol−1 CO2. Leaf temperature during the experiment was 22 ± 0·5 °C. Vapour pressure deficit was 0·9 ± 0·3 kPa. Respiration was measured after a 10 min dark period following the leaf net CO2 assimilation measurement.
Respired CO2 sampling
After 8 h of light in the growth chamber, intact leaf-respired CO2 was collected during a period of 2 h in the dark. A tightly closed system, as previously described in Duranceau et al. (1999), was used with two modifications: (i) the infrared gas analyser used here was a FINOR (Maihak, Montmorency, France); and (ii) the magnesium perchlorate column was replaced by an ethanol–liquid nitrogen water trap (−25 ± 5 °C).
Extraction of starch and water-soluble fraction
Leaf discs were harvested on the same plant before and after the respired-CO2 sampling period. Sixteen leaf discs (0·5 cm2 each disc) were harvested in the mid-afternoon (8 h into the photoperiod) on the leaf below the one used for respired CO2 sampling. A 4 cm2 leaf disc was used on the same leaf for RWC determination. The rest of the leaf was oven-dried and ground and 1 mg was used for isotope analysis of organic matter. For the first set of experiments in both species, 16 leaf discs were also harvested after 2 h of darkness, on the leaf used for respired-CO2 sampling. They were rapidly frozen in liquid nitrogen and stored at −80 °C until sugar extraction. For the second set of experiments on H. annuus, the leaf discs were harvested only before dark respiratory-CO2 sampling.
Extraction was carried out on the 16 leaf discs, harvested before and after dark respiration. Four leaf discs (2 cm2) sampled on the same leaf and at the same time were ground in 300 μL distilled water using a chilled pestle. The re-sulting extract was treated as previously described in Duranceau et al. (1999). The supernatant, containing the water-soluble fraction, was stored at −20 °C for further purification of soluble sugars and of organic acids by high performance liquid chromatography (HPLC). The white pellet, containing the starch and cellular residues of four leaf discs sampled before respiration, was suspended with 2 × 500 μL 6N HCl. Two HCl-suspended pellets were pooled (eight leaf discs, 4 cm2) and maintained for 1 h at 5 °C. The resulting pool was then centrifuged for 10 min at 4500 g and then 80% (v/v) methanol was added to the supernatant containing the HCl and soluble starch, and the sample was maintained at 5 °C overnight to precipitate starch. After 10 min of centrifugation at 12 000 g, the precipitate containing starch was isolated and desiccated. The starch powder obtained was stored for carbon isotope analysis.
Soluble sugars and organic acid purification
The water-soluble fraction obtained as described above was filtered (filter HV 0·45 μm type, Nihon Millipore Kogyo K.K, Japan). Two hundred microlitres of filtered extract per leaf was injected into a Sugar-Pak1 column (6·5 mm diameter and 300 mm length, Waters, Milford, MA, USA) as previously described in Duranceau et al. (1999). The sucrose, glucose and fructose peaks were collected for isotopic analysis. For each leaf, the first peak appearing on the chromatogram before sucrose (containing the organic acid pool), was collected and freeze-dried for further purification.
The fractions containing organic acids were dissolved with 200 μL of distilled-water. Microlitres of this extract per leaf was then injected into an Ionpak KC 811HQ column (7·8 mm diameter and 300 mm length; Showa Denko, New York, USA) (KC811 pre-column 6 mm × 50 mm). The peaks were evidenced using a refractometer (RI detector, Iota Model; Jobin Yvon, Paris, France). The flow rate was maintained at 1 mL min−1 and the temperature of the column at 40 °C, the pressure being ca. 4 MPa. The mobile phase used was 5 mM perchloric acid. In N. sylvestris extracts, α-ketoglutaric, citric and malic acids were detected and in H. annuus, only α-ketoglutaric and malic acids. Each detected acid was collected for isotope analysis when it was present in sufficient amounts.
Similar analysis (extraction and purification) using analytical-grade soluble sugar and organic acid solutions showed that no significant fractionation occurred during their extraction or during their passage through the HPLC columns (data not shown).
Total lipid fraction was extracted from pieces of leaves harvested before and after dark respiration as described by Blight & Dyer (1959). Leaf pieces were boiled (100 °C) for 5 min to inhibit lipase activity. They were then ground in a methanol–chloroform mixture (v/v) and one volume of distilled water was added to separate the two phases. The light phase containing the water-soluble fraction was removed and the heavy phase containing the total lipid fraction dissolved in chloroform was dried before isotopic analysis.
where RS and RVPDB are the molar abundance ratios of carbon isotopes,13C/12C, of the sample and the standard VPDB, respectively. The δ13C of respired CO2 was determined using a stable isotope ratio mass spectrometer (VG Optima; Fison, Villeurbane, France) with high precision (±0·2‰). Isotope abundance of samples was compared with that of a working standard reference gas having a δ13C of −35·4‰ with respect to VPDB.
The predicted discrimination during photosynthesis has been calculated using the model of Farquhar et al. (1982):
where a (= 4·4‰) is the fractionation due to diffusion of CO2 from ambient air into the leaf, b (= 28‰) the net fractionation during carboxylation by both Rubisco and PEPc and pi/pa the ratio of intercellular to atmospheric partial pressures of CO2. The model is an approximation to a more complete model of photosynthetic discrimination under the assumptions that the discrimination that may occur due to leaf internal resistances, during photorespiration and mitochondrial respiration is negligibly small.
The δ13C of carbohydrates (starch and sucrose) and organic matter, purified as described above, were determined using a continuous flow ANCA-MS (Roboprep on-line Dumas combustion and Tracermass MS; Europa Scientific Ltd, Crewe, UK). Working standard (leucine/citric acid mixture) with a known δ13C of −24·9 with respect to VPDB was periodically analysed to test the variation in isotopic composition due to sample combustion. The standard deviation (SD) obtained from periodical analyses using this mixture was around 0·4‰. Organic acid and lipid samples were combusted in a Dumas-combustion elemental analyser (Model Na-1500; Carlo Erba, Milan, Italy). The CO2 released from this combustion was purified in cryogenic traps and then analysed by a dual-inlet isotope ratio mass spectrometer (VG Isotech, Middlewich, UK). Control of combustion during analysis was achieved using a working standard (sucrose) having a δ13C of −25·09‰. The SD obtained from periodical analyses using this working standard was around 0·06‰.
Leaf water status and leaf carbohydrate contents
Under well-watered (control) conditions, leaf RWC remained high (around 90% for N. sylvestris and around 85% for H. annuus) until the end of the experiments (Fig. 1a & d, closed symbols). Under unwatered conditions (open symbols), RWC decreased slowly in N. sylvestris, reaching values around 60% at the end of the dehydration cycle, but decreased more rapidly in H. annuus, reaching values of less than 40%. This difference in the dehydration rate between the two species was not due to a difference in their leaf surface, which was around 0·35 m2 per plant for both species, but was probably due to stomatal conductance and stomatal sensitivity. Helianthus annuus showed lower stomatal conductance at equal RWC (see Fig, 2). The observed difference in dehydration rate may also be due to the plant architecture (see Material and Methods). In all figures, the data corresponding to the first set of the experiments (conducted simultaneously on both species) are represented by circles and those corresponding to the second set, which were made only on H. annuus plants, are represented by squares.
The variation of starch content in both treatments was similar for both species. At the beginning of the experiments, starch content was low (Fig. 1b & e) but progressively increased with plant ageing in control plants and stabilized around 2·5 g m−2, whereas in dehydrated plants it continuously decreased, reaching very low values in both species (Fig. 1b & e). The variation of sucrose content, however, was quite different for the two species. It increased in H. annuus for both treatments, reaching high values, but decreased in N. sylvestris plants during the second half of the experiment in both control and dehydrated plants (Fig. 1c & f).
Leaf gas exchange
Under control conditions and for the first set of experiments (Fig. 2b & e), stomatal conductance was the same in both species (about 100–200 mmol m−2 s−1). It was higher for the second set of experiments in H. annuus (Fig. 2e). Assimilation remained high during the experiments in control plants for both species (Fig. 2a & d). For the first set of experiments, it was higher in H. annuus (about 12 μmol m−2 s−1) than in N. sylvestris (8–10 μmol m−2 s−1). Under control conditions, leaf respiration rate varied between 2·2 and 1·2 μmol CO2 m−2 s−1 for N. sylvestris and between 1·6 and 0·8 for H. annuus plants (Figs 2c & 1f, respectively).
Under unwatered conditions, gc and A decreased with decreasing RWC, reaching very low values at RWC <80% for N. sylvestris and at RWC <65% for H. annuus. The decrease in A was more rapid in N. sylvestris than in H. annuus. Respiration data were more scattered in H. annuus (Fig. 2f), but it was clearly shown that the two species behaved differently during dehydration. Respiration decreased with dehydration in both species, but more rapidly in N. sylvestris than in H. annuus, as was the case for photosynthesis. In N. sylvestris, it reached 0·5 μmol CO2 m−2 s−1 at RWC = 60% (Fig. 2c), but in H. annuus, it reached around 1 μmol CO2 m−2 s−1 for similar RWC and then increased again at RWC <60%, reaching high values of more than 2 μmol CO2 m−2 s−1 (Fig. 2f).
Carbon isotope composition of sucrose
The variation of carbon isotope composition of sucrose is shown in Fig. 3 as a function of RWC. Under control conditions, sucrose showed a greater 13C-depletion in H. annuus (−27·5 to −33‰) compared to N. sylvestris (−23 to −28‰). The 13C-depletion in sucrose was more marked for the second set than for the first set of experiments in H. annuus. Two phases can be observed during the dehydration cycle; firstly, an increase in 13C-content and then a stabilization of sucrose δ13C around −22‰ in the first set of experiments in both species (Fig. 3a & b) and around −24‰ for the second set in H. annuus (Fig. 3b). For the second set of experiments in H. annuus, the δ13C values were lower in both well-watered and unwatered plants compared to the first set.
Variation of pi/pa and the expected discrimination
The variation of pi/pa is shown as a function of leaf net CO2 assimilation (Fig. 4). Under control conditions that correspond to high leaf photosynthesis, pi/pa varied between 0·8 and 0·7 for both species during the first set of experiments and between 0·9 and 0·8 for the second set in H. annuus. It decreased during dehydration, reaching minimal values around 0·3 (first set) and around 0·5 (second set), and then increased again at the end of the dehydration cycle. The high pi/pa values corresponding to the very low leaf photosynthesis rates (A around the CO2 compensation point) at the end of the dehydration cycle are not shown, because of the uncertainties in the estimation of pi/pa under such conditions. As already described by Duranceau et al. (1999), for each value of pi/pa, a corresponding variation of the expected discrimination (dδ) relative to a reference value (pi/pa = 0·7) was calculated (see also the legend of Fig. 4). Decreases in pi/pa from 0·7 to around 0·3 in both species for the first set of experiments, and from 0·9 to around 0·5 in the second set for H. annuus, resulted in a reduction of photosynthetic discrimination by about 8‰.
An expected value of δ13C for the recent photosynthetic products was calculated for each value of pi/pa based on Farquhar's model (Eqn 2) and taking into account the δ13C of the air in the culture room, which was around −8‰. Again, measurements with high pi/pa values corresponding to the very low leaf photosynthesis were not taken for these calculations. In H. annuus, the observed values of sucrose δ13C were similar to the expected values (Fig. 5): the data scattered around the 1 : 1 relationship for both treatments and for both sets of experiments (Fig. 5b). By contrast, in N. sylvestris, the sucrose of well-watered plants was always isotopically heavier than the expected values (Fig. 5a).
δ13C of respiratory CO2 in comparison with δ13C of sucrose
The variations in δ13C of CO2 respired in the dark compared with δ13C of the leaf sucrose pool are shown in Fig. 6. In N. sylvestris, when both treatments were considered together, a linear relationship between δ13C of CO2 respired in the dark and δ13C of sucrose was obtained with a shift compared to the 1 : 1 relationship by about 5‰. This 13C-enrichment in respired CO2 compared with sucrose was about 6·5‰ for dehydrated but only 4‰ for control N. sylvestris when average values were considered separately for each treatment (see also Fig. 7a). In well-watered H. annuus, the respired CO2 was also 13C-enriched compared to sucrose by about 3‰ for the first set (Fig. 7b) and by about 6‰ for the second set of the experiments (Fig. 6b). In contrast, in dehydrated H. annuus for both sets of the experiments, the respiratory CO2 (δ13C = −23·05‰± 0·63) was only slightly 13C-enriched (1·4‰) compared to the leaf sucrose pool (δ13C = −24·43‰± 0·53) (Fig. 6b).
As with the sucrose carbon pool, respired CO2 was 13C-enriched in unwatered compared with control plants. This difference was more pronounced for N. sylvestris than for H. annuus (Fig. 7). In dehydrated N. sylvestris leaves, the sucrose pool was 13C-depleted (by about 2·8‰± 0·7) after 2 h of dark respiration compared with the values taken before the dark respiration period (Fig. 6a), but this 13C-depletion after 2 h of dark respiration was less marked (only about 1·2‰± 0·6) in unwatered H. annuus (Fig. 6b). In control plants and for both species (Fig. 6), there was no significant difference between the sucrose δ13C determined before and after the dark respiration, probably because of a high recent carbohydrate reserve in these plants.
Carbon isotope composition of leaf metabolites
The mean carbon isotope composition of total lipid fraction, organic acids (α-ketoglutaric, citric and malic acids), starch and soluble sugars (glucose, fructose and sucrose) are shown in Fig. 7. In order to compare the two species under similar conditions, only the results of the first set of experiments are considered for the calculation of these average values. For calculation of the mean δ13C values of dehydrated plants, only leaves at RWC < 85% were taken into account. In both treatments and for both species, lipids were the most 13C-depleted metabolites and respired CO2 was the isotopically heaviest component. This was also the case for dehydrated H. annuus for which however, the δ13C value of soluble sugars was not significantly different from that of respired CO2, as shown before in Fig. 6b.
As expected, leaf metabolites and leaf total dry matter were 13C-enriched in dehydrated compared to control plants for both species. This 13C-enrichment in dehydrated plants was larger for soluble sugars than for other metabolites. In control plants, sucrose was 13C-enriched compared with glucose and fructose (Fig. 7a) and showed the δ13C value closest to that of respiratory CO2. In dehydrated plants, δ13C of all soluble sugars was closer to that of respiratory CO2 (Fig. 7b) than δ13C of the other metabolites. The 13C-enrichment in soluble sugars due to dehydration was more pronounced in H. annuus than in N. sylvestris whereas in organic acids, it was more marked in N. sylvestris than in H. annuus. This 13C-enrichment in N. sylvestris was larger in malic acid than in other organic acids. Whole leaf organic matter was 13C-depleted relative to leaf carbohydrates in dehydrated plants, and relative to sucrose and respiratory CO2 in all cases (Fig. 7a).
The total lipid fraction was 13C-enriched in N. sylvestris compared with H. annuus for both control (about −33 versus −34‰) and dehydrated plants (about −31 versus −33‰). The change in 13C-enrichment in lipids due to dehydration in N. sylvestris (about 2‰) was twice as large as that in H. annuus (less than 1‰).
Diffusional limitation and levels of discrimination
Under control conditions, the initial difference observed in leaf sucrose δ13C between the two species (Fig. 3), indicated a higher discrimination during CO2 assimilation in H. annuus in comparison with N. sylvestris. Lauteri, Brugnoli & Spaccino (1993) have already reported such a high discrimination (up to 23‰) in H. annuus compared to other C3 species. This difference in discrimination and thus in sucrose δ13C observed between the two species could be due to a possible difference in their mesophyll (conductance to CO2 diffusion from the substomatal cavities to the carboxylation sites) rather than stomatal conductance. Indeed, stomatal conductance (Fig. 2b & e) and thus pi/pa (Fig. 4) were similar for both species under control conditions in the first set of experiments. The difference in sucrose δ13C observed between the two sets of experiments in H. annuus can be explained by the observed difference in stomatal conductance (Fig. 2e) and thus in pi/pa (Fig. 4). Accordingly, the observed values of sucrose δ13C were almost similar to the expected ones calculated using Farquhar's model for both sets of experiments in H. annuus (Fig. 5), although in N. sylvestris, isotopically heavier sucrose than expected (according to Eqn 2) indicates a substantial draw down of CO2 between the substomatal cavities and Rubisco catalytic sites due to a lower mesophyll conductance. This is in close agreement with data in the literature. Indeed, Brugnoli et al. (1998) found a high mesophyll conductance to CO2 for different genotypes of H. annuus (up to 0·6 mmol m−2 s−1) whereas Duranceau et al. (2001) estimated low values for the same genotype of N. sylvestris as used in the current experiment (0·2 mmol m−2 s−1). Similar low mesophyll conductance has also been reported by von Caemmerer & Evans (1991) on N. tabacum.
13C enrichment in leaf metabolites during dehydration
As expected, the leaf sugars became heavier with plant dehydration in both species. This 13C-enrichment in leaf sucrose during the dehydration cycle can be attributed to the drought-induced decrease in stomatal conductance, decreasing pi/pa and thus decreasing discrimination by Rubisco. Indeed, the observed variation in pi/pa during dehydration globally explains the observed variation in sucrose δ13C (about 6−8‰) in both species (Figs 3 & 4). These results are in agreement with those previously obtained for Phaseolus vulgaris (Duranceau et al. 1999), indicating that the known relationship between the discrimination and the leaf conductance to CO2 diffusion (both stomatal and mesophyll conductances) largely explains the observed variation in the 13C signature in leaf sucrose pool among species and with dehydration.
As expected, the decrease in discrimination by Rubisco in dehydrated plants compared to the controls lead to a higher shift in 13C content of soluble sugars than of starch or of other fractions. The isotopic signature of starch, for example, shows a weaker dependence on the signature of recently assimilated substances, because it consists of a mixture of molecules synthesized over a longer time period. Nevertheless, starch, the other metabolites (organic acids and lipids) and even the total dry matter were also marked (but only slightly) by the recent photo-assimilates synthesized during dehydration (13C-enriched). This 13C-enrichment was larger in the malic acid of N. sylvestris than in other organic acids, probably due to a possible increase in the anaplerotic pathway in this species under drought conditions.
The two species showed different responses to water deficit in the changes of isotopic signature of carbohydrates relative to the changes of signature of other metabolites. In dehydrated N. sylvestris, organic acids and lipids showed a higher shift in the isotopic signature towards less negative values than in H. annuus. This suggests that recent photo-assimilates (13C-enriched) represented by sucrose were metabolized and used for synthesizing other metabolites (even the secondary metabolites such as lipids) in this species. The smaller shift in the signature of organic acids and lipids in dehydrated H. annuus together with a higher shift in carbohydrate signatures suggests that recently formed assimilates were utilized to a lower degree for synthesis of organic acids and lipids.
The δ13C values and the general pattern observed for the metabolites studied (carbohydrates, organic acids and lipids) are consistent with data reported in the literature (see the reviews of Schmidt & Gleixner 1998 and Brugnoli & Farquhar 2000). Under control conditions (Fig. 7), leaf carbohydrates and organic acids were the most 13C-enriched metabolites whereas a huge 13C-depletion in lipids even compared to leaf total dry matter was observed for both species. This 13C-depletion in lipids compared with carbohydrates can be explained by both the non-uniform 13C-distribution within the hexose molecules and the carbon isotope effect on PDH reaction (see Gleixner & Schmidt 1997 and introduction).
Respired CO2 is enriched in 13C
In both species, respired CO2 was 13C-enriched compared to all the leaf metabolites analysed and even compared to sucrose, the most 13C-enriched metabolite. This result is in agreement with that already reported on P. vulgaris for which the 13C-enrichment in respired CO2 compared to leaf sucrose pool (i.e. fractionation during dark respiration) was constant (around 6‰), whatever the leaf age and the leaf relative water content (Duranceau et al. 1999). The present work showed, however, that this discrimination varied not only among species but also with drought. Respired CO2 was 13C-enriched in comparison with sucrose by about 4, 3 and 6‰ on average in well-watered N. sylvestris, H. annuus (first set) and H. annuus (second set), respectively (Figs 6 & 7). The discrimination by 4‰ in N. sylvestris is nearly identical to the results obtained by on-line measurements on the same variety by Duranceau et al. (2001), who also found a discrimination during dark respiration of around 3‰. Contrary to what has been observed in P. vulgaris, the isotopic fractionation during dark respiration increased in dehydrated N. sylvestris and decreased in dehydrated H. annuus in comparison to the control plants. The 13C-enrichment in respired CO2 relative to the sucrose pool was around 6·5‰ in dehydrated N. sylvestris whereas in dehydrated H. annuus (in both sets of experiments), the average δ13C value of respired CO2 was found to be only slightly (1·4‰) different from that of the leaf sucrose pool (see Figs 6b & 7b).
We conclude that (i) carbon isotope fractionation during dark respiration is a widespread phenomenon occurring in C3 plants, but that (ii) this fractionation is not constant and varies among species, and (iii) also varies with environmental conditions (water deficit in the present work) but differently among species (constant in P. vulgaris, increased in N. sylvestris and decreased in H. annuus under drought compared with control conditions).
The discrimination here was calculated as the difference between signatures of sucrose and respired CO2 assuming that sucrose is representative of the main substrates of respiration. Yet, as we do not know whether another metabolite with a substantially different signature was used in mitochondrial respiration or the isotopic signature of the substrates is variable between the cellular compartments, the specific discriminating step can not be identified. However, respiratory CO2 was isotopically heavier than lipids, carbohydrates and organic acids. All these components are connected to respiratory metabolism and although CO2 released during dark respiration was enriched in 13C, whole-leaf dry matter was depleted in 13C relative to sucrose. Therefore, it can be concluded that (iv) a discrimination during dark respiration processes occurred, releasing 13C-enriched CO2 compared to several major leaf reserves (carbohydrates, lipids and organic acids) and whole-leaf organic matter as well.
Such a large 13C-enrichment in respired CO2 during the night-time should result in 13C-depletion in the remaining leaf material. According to the model developed by Henderson et al. (1992), the respiratory carbon loss relative to the total plant carbon budget and the isotopic signature of the released CO2 during the night-time should be taken into consideration when interpreting the carbon isotope composition of total leaf material. At the current stage, it is not yet possible to derive general rules for the incorporation of the observed fractionation during dark respiration into ecosystem and global scale studies (models) because of the great variability observed among species and also with drought. Furthermore, the present work was carried out under low-light conditions. The leaves had a low carbon gain and thus probably a limited exportation rate. The photo-assimilates were therefore preferentially used for synthesis of other metabolites in these leaves, increasing the fractionation during dark respiration. Under higher photon flux densities in natural conditions, an increased exportation rate of the photo-assimilates will result in a high energy demand (Bouma et al. 1995), ensured by a high respiration rate. This should lead to the consumption of carbon entering the Krebs cycle rather than divertion for synthesis of other metabolites. According to the pattern developed in the introduction, the fractionation during dark respiration is expected to decrease under such high-light conditions. A low fractionation observed in dehydrated H. annuus might also be explained by the high respiration rate observed in these plants. High respiration rates are observed although the signature of secondary metabolites does shift only moderately towards the signature of photosynthetic products under drought. This can be interpreted as an indication of close to complete oxidation of respiratory substrates in the Krebs cycle. If the non-statistical distribution of 13C in sugars is the dominant cause of potential discrimination this should result in zero discrimination. This hypothesis awaits further experimental proof.
More investigations (comparison between trees and herbaceous plants, C3 and C4 plants, photosynthetic and non-photosynthetic organs) under varying environmental conditions are needed in order to better define the conditions in which the fractionation during dark respiration occurs. Further studies are also needed in order to distinguish between different factors that potentially change the apparent discrimination during dark respiration and thus explain the observed variability between species: (i) changes in time in the activity of metabolic pathways and relative amounts of net synthesis of different leaf components; (ii) export of carbohydrates of different signature than leaf organic mass; (iii) variation in photosynthetic discrimination in the course of time; (iv) ratio of the amount of CO2 respired during the night to CO2 fixed by net photosynthesis and related changes in the energy requirements for carbohydrate export out of the leaves.
We are grateful to Professor Jim Ehleringer, Professor Gerhard Schleser, Dr Gerd Gleixner and an anonymous reviewer for helpful comments and remarks on the manuscript. We also thank Charlie Scrimgeour and Luciano Spaccino for carbon isotope analysis and Jacqueline Liébert and Dr Antoine Trémolière for their assistance during carbohydrate and lipid extractions, respectively.