In response to Tcherkez (2010) I clarify first, to what extent relative flux changes in the respiratory pathways can cause significant variations in the isotopic ratios of respired CO2 (δ13Cres); second, why positional 13C-pyruvate labelling experiments do provide information on these flux changes; and third, that ecologically relevant information can be gained from these types of experiments.
Tcherkez (2010) challenges that large changes in respired δ13CO2 originate from fragmentation fractionation, that is, from the metabolic branching point after the decarboxylation of pyruvate (by pyruvate dehydrogenase, PDH), when the remaining acetyl-CoA can either enter the Krebs cycle (KC or TCA cycle) or be converted into secondary metabolism. First, his numeric example seems somewhat inconsistent with the data reported by Rossmann et al. (1991), who give −21‰ at C-1 and −27‰ (not −25‰ as in Tcherkez, 2010) at C-2 and C-3 positions of pyruvate (the overall glucose molecule was −25‰). Therefore, the theoretical maximum isotopic variation between 0 or 100% commitment to KC decarboxylation is 4‰ (either −21‰ of C-1, or −25‰ of the full molecule). However, in vivo the commitment into KC decarboxylation may vary. The KC is an important source of amino acid biosynthesis, providing carbon skeletons for glutamic acid and aspartic acid, the latter being very common with large quantities required for protein synthesis and turnover (Hayes, 2001). If pyruvate is not fully respired, both equilibrium and kinetic isotope effects occur in the KC, leading to a depletion of both respired CO2 and the organic acids (Tcherkez & Farquhar, 2005). As pointed out by Tcherkez (2010), neither a low nor a high commitment into KC decarboxylation results in large changes in δ13Cres. However, at intermediate mixing ratios, δ13Cres can decrease below that of the source δ13C signature. For example, when 50% of the acetyl-CoA is respired in the KC, fractionation by the citrate synthase (intrinsic fractionation of −23‰; Tcherkez & Farquhar, 2005) results in an effective isotope effect of −11.6‰. The overall reaction with 50% flux into the KC then yields (−21‰ × 100 + (−27‰ −11.6‰) × 50 − 27‰ × 50)/200 = −26.9‰. Moreover, α-ketoglutarate dehydrogenase may also fractionate (−23‰; Tcherkez & Farquhar, 2005) because the reaction is incomplete as a result of the diversion to amino acid synthesis (notably the most 13C-enriched amino acids, Hayes, 2001). In the above example, δ13Cres would be −29.8‰. Furthermore, PDH may also fractionate if the reaction is incomplete, which would further deplete the C-2 position of pyruvate (e.g. 4.5‰ depletion for a 75% commitment to the PDH, Melzer & Schmidt, 1987). Thus, relative carbon flux changes through PDH and the KC may theoretically induce much larger δ13Cres variations (> 9‰) than suggested by Tcherkez (2010). However, little is known on these processes in vivo.
Nevertheless, changes in the relative decarboxylation rates by PDH or the KC (i.e. the PDH/KC ratio) may not entirely explain the observed dynamics in δ13Cres because the observed range in δ13Cres can be larger and δ13Cres can become more positive than −21‰ (e.g. a diurnal variation of δ13Cres of −26.7 to −18.3‰ in Quercus ilex and of −29.9 to −15.1‰ in Halimium sp.; Werner et al., 2009; F. Wegener et al., unpublished).
Markedly positive δ13CO2 ratios may evolve from rapid decarboxylation of a malate pool after darkening, which may accumulate during the light, resulting in a short postillumination burst of CO2 (known as light enhanced dark respiration (LEDR), Barbour et al., 2007; Gessler et al., 2009). Malate is 13C-enriched in the C-4 position, which is fixed by phosphoenol pyruvate carboxylase. However, the malic enzyme is most certainly associated with an isotope effect. If we assume a dynamic Rayleigh process (see the equation in Gessler et al., 2009), δ13Cres would be more depleted immediately after darkening while become more enriched as the malate pool declines (see Fig. 5 in Werner et al., 2009). This is inverse to the observed dynamics in δ13Cres during light–dark transitions (Werner et al., 2007, 2009; Barbour et al., 2007).
Hence, to date, these mechanisms cannot entirely explain the temporal dynamics in δ13Cres and probably multiple processes are superimposed (Werner et al., 2009). Other pathways, such as the pentose-phosphate pathway, may also be involved (Bathellier et al., 2009). There are still many uncertainties in fractionation processes during dynamic metabolic fluxes, and isotopic mass-balance calculations alone will not clarify these issues. Our knowledge on the isotopic fractionation and intermolecular distribution of carbon isotopes is based on sparse measurements of one or two plant species (or even yeast). Hence, experiments addressing diurnal dynamics in metabolic fluxes and isotopic signatures in different plant functional groups are needed to identify the underlying processes.
Tcherkez (2010) further questions the methodological aspects of positional labelling experiments: first, marker uptake; second, influence of transpiration rates; and third, dilution effects.
Indeed, 13C-labelling experiments are very sensitive to experimental conditions such as incubation time, uptake and timing of the experiment. Nevertheless, if conducted under clearly defined conditions, positional 13C-labelling experiments deliver very reproducible results.
- •Differences in the absorption rates between molecules labelled at different intermolecular positions (i.e. 13C-1 or 13C-3 pyruvate) are very unlikely because the 13C solutions are fed through the petiole and are passively taken up through the transpiration stream of the leaf.
- •Equal uptake (transpiration) rates are required, which can be achieved for a single species grown under controlled conditions in growth chambers but may prevent a direct comparison being made between species. Indeed, the changes within a species over time, between the decarboxylation by PDH and the KC, are relevant. Moreover, the patterns of diurnal changes in the PDH : KC ratio are comparable between different species. By contrast, using the dark-adapted state as a reference (when transpiration is generally reduced), as suggested by Tcherkez (2010), must be carried out with care because natural δ13Cres rapidly declines in the first 1–2 h after darkening (Werner et al., 2009), and sometimes even throughout the entire night (Sun et al., 2009; Unger et al., 2010). Therefore, caution is needed in respect to timing of the measurements (i.e. the time of light : dark exposure before measurements are made).
- •A potential dilution effect of the 13C label within the endogenous pyruvate evolved by the malic enzyme would not affect the measured PDH : KC ratio for a species at a given time. Furthermore, an increase in the endogenous pyruvate pool during photosynthesis would cause an increasing dilution effect (i.e. a decreasing δ13Cres) over time, which contrasts with our labelling results, revealing constant KC activity in both leaves and roots in different species (Priault et al., 2009; F. Wegener et al., unpublished). Moreover, a threefold diurnal increase in carbon flow through the PDH occurred in Mediterranean shrubs, with a large diurnal increase in natural δ13Cres (Priault et al., 2009).
I therefore conclude that the concerns raised by Tcherkez (2010) will have little effect on the robustness of the measured PDH : KC ratios in labelling experiments, whereas highly relevant ecological information on the internal carbon flow may be gained.
Tcherkez (2010) has questioned whether metabolic fluxes matter for interpreting isotopic respiratory signals. In my opinion, the relevant ecological question is rather: can isotopic respiratory signals trace changes in the metabolic fluxes, thus providing a marker of carbon allocation? In fact, the magnitude of diurnal variation in δ13Cres is correlated to factors determining carbon allocation, such as light intensity, instantaneous and/or cumulative carbon assimilation rate, as well as growth status (Hymus et al., 2005; Prater et al., 2006; Gessler et al., 2009; Priault et al., 2009).
Furthermore, a little more caution is needed to avoid incorrect generalizations regarding differences between plant functional groups: woody plants do not generally produce more enriched δ13Cres than fast-growing herbs. Indeed, in most species δ13Cres is close to the isotopic signal of the respiratory substrate in early morning hours (Werner et al., 2009). Differences appear during the course of the day. A marked diurnal increase in δ13Cres occurs in many Mediterranean woody evergreens, semi-malacophyllous shrubs as well as fast-growing aromatic herbs, while some temperate woody species do not show a large diurnal variation in δ13Cres (Priault et al., 2009). This suggests that a common function among this group may be a high investment into secondary metabolism, such as defence components, photoprotection or aromatic (volatile) compounds. The substrate hypothesis of Tcherkez et al. (2003) (a change in the respiratory substrate from lignin to sugars) seems unlikely to explain the observed diurnal course in δ13Cres. In ecological terms, an increasing investment of acetyl-CoA into secondary metabolism with increasing carbon supply (i.e. when sugar pools are filled and respiratory demand is met) seems much more reasonable (Hymus et al., 2005). One potential hypothesis of a diel dynamic process could be isoprene synthesis (Priault et al., 2009). Overall, if δ13Cres dynamics trace carbon-allocation patterns in plants it may provide a powerful tool for ecological research.
Certainly there are still many unknowns regarding isotope effects in the metabolic pathways and many issues remain to be resolved. Part of the discrepancy is probably a matter of perspective between a purely mechanistic viewpoint based on fractionation effects and fluxomics or an ecological perspective analysing dynamic fractionation processes related to, for example, species-specific carbon allocation pattern, source/sink strength or environmental conditions. In my opinion both approaches are indispensible for gaining relevant new insights and advancing the field.