Notes on Isotope fractionation, Biosynthesis and Respiration

CO2 evolved during daytime leaf respiration is often observed to be enriched in 13C by up to 10 per mil relative to bulk leaf carbon or leaf sugars (Park
and Epstein 1961, Hsu and Smith 1972, Duranceau et al. 1999, Tcherkez et al. 2003, Xu et al. 2004, Hymus et al. 2005, Prater et al. 2005, Tu and Dawson 2005). This is surprising since it is generally thought that there is no fractionation during respiration and the CO2 evolved during respiration is typically assumed to equal that of the substrate being respired (e.g. Lin and Ehleringer 1997). This apparent discrepancy may be explained by considering whether substrates are being formed or degraded, and whether leaves are in the light or the dark (Park and Epstein 1961, Ghashghaie et al. 2003). There are two main sources of metabolic CO2, both of which occur in the mitochondria; one is enriched in 13C from pyruvate decarboxylation during the PDH reaction, and the other is depleted in 13C from degredation of acetyl-CoA in the Krebs cycle (Tcherkez et al. 2003). That is, the PDH reaction produces both 13C-enriched CO2 and 13C-depleted acetyl-CoA, and the fate of acetyl-CoA (degredation via the Krebs cycle or incorporation into compounds such as lipids) ultimately determines the net amount of 13C evolved as CO2. When leaves are illuminated the Krebs cycle is significantly reduced (Tcherkez et al. 2005), leaving the 13C-enriched CO2 evolved during the PDH reaction as the dominant source of CO2. This dominance of the PDH reaction in the light along with a reduction in Krebs cycle suggests that acetyl-CoA is used for purposes other than respiration (Tcherkez et al. 2005). This other purpose is likely fatty acid synthesis which is well-established as a light-dependent process in leaves (Stumpf et al. 1967, Browse et al. 1981, Sauer and Heise 1983, Liedvogel and Bauerle 1986, Roughan and Ohlrogge 1996). Fatty acid synthesis appears to be coordinated with photosynthesis by way of electron transport during the light reactions which generates the redox potential needed to activate acetyl-CoA carboxylase (ACCase), a key enzyme that regulates the rate of fatty acid synthesis (Sasaki et al. 1997). Thus, respiration in the light appears to be dominated by anabolic reactions that lead to the formation of fatty acids (Ohlrogge and Jaworski 1977) and 13C-enriched CO2 (Park and Epstein 1961), rather than catabolic reactions that provide energy via the Krebs cycle. In contrast, respiration in the dark is dominated by catabolic reactions that provide energy and have little or no net fractionation against 13C relative to the substrate (e.g. Lin and Ehleringer 1997).

            If the enrichment in 13C of CO2 during daytime respiration results from the predominance of CO2 produced during the PDH reaction (Park and Epstein 1961, Ghashghaie et al. 2003), then this should be reflected in the amounts of O2 consumed and CO2 evolved during respiration. The ratio CO2/O2 or the respiratory quotient (RQ) should be near 1 when carbohydrates are being degraded or during fatty acid synthesis, and RQ should be near 0.6 when fatty acids are being degraded (Tcherkez et al. 2003). Another and potentially more flexible and quantitative measure is the diverted reductant utilization rate, DRUR = 4*(O2+CO2) (Willms et al. 1999). DRUR can be calculated based on net fluxes of O2 and CO2 during both light and dark conditions and therefore should provide a more useful measure of biosynthesis metabolism (Willms et al. 1999). DRUR provides a measure of the rate of synthesis of biomass that is more reduced per unit carbon than glucose (in photosynthesizing tissue) or than the substrate of metabolism (in respiring tissue). That is, DRUR can inform whether the products of metabolism in the light (e.g. fatty acids) are more reduced than carbohydrates, and if the rate of biosynthesis is greater in the light than the dark. While precise measurements of O2 are required, recent advances in differential O2 analyzers
(Willms et al. 1997, Qubit Systems) now make this approach more feasible and cost effective than 13C-labeling studies. Using this approach, simultaneous measurements O2 and CO2 exchange rates could help identify the underlying metabolic processes responsible for variations in the 13C-enrichment of leaf respired CO2.

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