- Top of page
- MATERIALS AND METHODS
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.
- Top of page
- MATERIALS AND METHODS
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.