Stable carbon isotopes reveal dynamics of respiratory metabolism

Measurement of respiration via the rate of CO2 release is conceptually simple, but assigning the metabolic origin of respired CO2 to the appropriate biochemical pathway is extremely difficult. By comparing the stable carbon isotope composition of CO2 respired by plant organs with that of likely respiratory substrates, by consideration of potential isotope fractionations and by the judicious application of labeled substrates, three papers in this issue of New Phytologist were able to draw strong conclusions regarding the metabolism involved in leaf (Gessler et al.; pp. 374–386) and root (Bathellier et al.; pp. 387–399) respiration in model species, and leaf respiration across plant functional groups (Priault et al.; pp. 400–412). These three papers used elegant experimental methods to test a number of theories regarding the relative importance of different respiratory pathways and, as such, provide a significant advance in understanding the processes involved. ‘... leaf-respired CO2 has been found to be even more enriched than the theoretical maximum of 6‰, ...’ For some years, considerable research has been carried out on terrestrial carbon cycling because it has become evident that understanding vegetation responses to the environment are vital to mitigating and adapting to future climate change. While net fluxes of carbon into and out of ecosystems may be directly measured (for instance using eddy covariance techniques), partitioning the net flux into component fluxes is challenging. Stable isotopes are seen as a powerful tool in understanding carbon cycling, both as a tracer and as a record of processes where isotopic fractionation occurs (Yakir & Sternberg, 2000; Bowling et al., 2008). Accurate partitioning of ecosystem fluxes requires a mechanistic understanding of plant metabolism, and while photosynthetic metabolic pathways and related isotopic fractionations have been described in mechanistic models (Brugnoli & Farquhar, 2000), the same cannot be said for respiration. Not only are the relative activities of metabolic pathways involved in plant respiration poorly understood, but carbon isotope effects during plant respiration are only just becoming elucidated (Ghashghaie et al., 2003; Schnyder et al., 2003; Barbour et al., 2007; Bathellier et al., 2008). Respiration is the broad term used to describe the release of CO2, or the uptake of O2, during cellular metabolism of a carbon substrate to provide energy. CO2 is released by (1) decarboxylation of pyruvate as the first glycolytic step in the mitochondria; (2) by isocitrate dehydrogenase and (3) 2-oxoglutarate dehydrogenase in the mitochondrial Krebs cycle; (4) during the formation of ribulose 5-phosphate from glucose in the pentose phosphate pathway operating in the cytosol and plastids; and (5) during decarboxylation of malate in the mitochondria by the NAD-dependent malic enzyme. Each of these reactions may contribute to the measured CO2 flux to a greater or a lesser extent in different tissues and at different times during growth. A number of recent papers have suggested that the carbon isotope composition of respired CO2 (δ13Cr) may provide information on the relative importance of these pathways, because δ13Cr has been shown to vary between species (Werner et al., 2007) and plant organs (Klumpp et al., 2005), with environmental conditions such as light (Hymus et al., 2005) and to co-vary with the type of substrates used for respiration (Tcherkez et al., 2003). The probability of finding a 13C atom in the six different carbon positions of a hexose molecule is not equal, with atoms C3 and C4 being 2 and 6‰ more enriched, respectively, than the average for the whole molecule (Roßmann et al., 1991). These carbon atoms are released as CO2 by pyruvate dehydrogenase during glycolysis and provide a credible explanation for 13C enrichment in respired CO2 under conditions when the resulting acetyl-CoA is diverted to lipid synthesis rather than being fully consumed in the Krebs cycle (Ghashghaie et al., 2003; Tcherkez et al., 2003). However, leaf-respired CO2 has been found to be even more enriched than the theoretical maximum of 6‰, which is only achievable with atom C4 of glucose as the sole source of respired CO2. This was particularly evident during the light-enhanced dark respiration (LEDR) peak observed immediately after darkening a leaf. This extreme and transient enrichment led Barbour et al. (2007) to hypothesize that malate derived from phosphoenolpyruvate carboxylase (PEPc) activity was used for respiration during the LEDR peak. Phosphoenolpyruvate carboxylase fixes 13C-enriched carbon into position C4 in malate in the light. Some cytosolic malate is transported into the mitochondria and is decarboxylated by the NAD+-dependent malic enzyme, releasing the enriched carbon at position C4. Using measurements of δ13Cr, activities of key enzymes and metabolite concentrations, Gessler et al. (2009) were able to demonstrate that the enriched δ13Cr obtained during LEDR is indeed a result of the rapid decarboxylation of malate. They estimated that about 22% of the CO2 emitted during the LEDR peak is obtained through the decarboxylation of malate. After the LEDR peak in pre-illuminated leaves, leaf-respired CO2 is often still enriched compared with likely respiratory substrates. The degree of this enrichment varies considerably between species and, in some experiments, diurnally. For example, Priault et al. (2009) found that δ13Cr in the slow-growing woody shrub Halimium halifolium was increasingly enriched during the day, reaching a peak of −21‰ just before dusk and a minimum of −29‰ predawn. By contrast, the fast-growing herb Oxalis triangularis showed a constant δ13Cr of −30‰ during the day, which decreased, by only 2‰, to −32‰ predawn. By applying pyruvate labeled with 13C, in position C1 or in both positions C2 and C3, to these contrasting species, Priault et al. were able to determine that slow-growing woody plants had greatly reduced carbon flow through the Krebs cycle, relative to pyruvate dehydrogenase activity, compared with fast-growing herbaceous plants. In slow-growing plants, a higher proportion of carbon is diverted from the Krebs cycle into the secondary metabolism. This fits well with theoretical predictions made by Bowling et al. (2008), that CO2 respired by leaves of slow-growing plants will be 2.6‰ more enriched than that respired by leaves of fast-growing plants (given hexose substrates of the same isotope composition), as a result of differences in the balance between maintenance and growth respiration between the functional groups. This estimate is based on the higher levels of secondary metabolites found in slow-growing plants and the fact that differences in chemical composition have accompanying isotope effects. While carbon respired by leaves is generally enriched in 13C, root-respired CO2 is often slightly depleted compared with likely respiratory substrates. A number of hypotheses have been advanced to explain this difference. The pentose phosphate pathway is thought to account for about one-quarter of root-respired CO2, but pyruvate dehydrogenase and the Krebs cycle are also thought to contribute significantly to CO2 respired by roots (Dieuaide-Noubhani et al., 1995). As described in a previous paragraph, the CO2 released by pyruvate dehydrogenase will probably be enriched in 13C, but the Krebs cycle in all probability releases depleted CO2, as does the pentose phosphate pathway. Anaplerotic carbon fluxes, through PEPc, may also be significant in heterotrophic tissues. Using measurements at both natural abundance and with 13C-labeled substrates, Bathellier et al. (2009) were able to estimate that in Phaseolus vulgaris roots, 50% of respired CO2 comes from the Krebs cycle, 28% comes from pyruvate dehydrogenase and 22% comes from glucuronic acid decarboxylase, after 10 h in the dark. These proportions shifted, after the plant had been in the dark for 4 days, to 57, 28 and 15%, respectively. After 4 days in the dark, the Krebs cycle was increasingly fed by lipids, rather than by carbohydrates, but these changes did not result in variation in overall δ13Cr under natural abundance conditions. Similarly, PEPc activity was thought to have only a small overall effect on δ13Cr in P. vulgaris. However, in Ricinus communis stems and roots, Gessler et al. (2009) found that the PEPc activity was very high in roots, and that the depletion of δ13Cr could be explained by the refixation of CO2 by PEPc. Further species differences in the relative allocation of carbon to the different pathways in roots and stems may become apparent as more species are studied. Taken together, these three papers highlight that understanding δ13Cr is a complex, yet tractable, endeavor. It is now clear that one must take a systems approach to understand the balance among the pentose phosphate pathway, pyruvate dehydrogenase activity, the Krebs Cycle and PEPc activity because they vary by tissue, species and environmental conditions. Future studies should take advantage of the labelling and nuclear magnetic resonance (NMR) techniques. developed in the laboratory of Ghashghaie & Tcherkez, to elucidate, in more detail, the metabolic processes contributing to respiration under a wider range of environmental and growth conditions and in a wider range of species. This will be particularly important for root respiration, where metabolic understanding is in its infancy. More experiments also need to be conducted in natural ecosystems at natural abundance to start to describe environmental, species, temporal and spatial variation in δ13Cr. The recent development of field-based laser-absorption spectroscopy will rapidly advance data collection and understanding in this area. The potential for using techniques involving stable isotopes to help understand terrestrial carbon cycling has been recognized for many years, and it is time for this potential to be realized.