The intramolecular ¹³C-distribution in ethanol reveals the influence of the CO₂-fixation pathway and environmental conditions on the site-specific ¹³C variation in glucose

Efforts to understand the cause of ¹²C versus ¹³C isotope fractionation in plants during photosynthesis and post-photosynthetic metabolism are frustrated by the lack of data on the intramolecular ¹³C-distribution in metabolites and its variation with environmental conditions. We have exploited isotopic carbon-13 nuclear magnetic resonance (¹³C NMR) spectrometry to measure the positional isotope composition (δ¹³C_i, ‰) in ethanol samples from different origins: European wines, liquors and sugars from C₃, C₄ and crassulacean acid metabolism (CAM) plants. In C₃-ethanol samples, the methylene group was always ¹³C-enriched (∼2‰) relative to the methyl group. In wines, this pattern was correlated with both air temperature and δ¹⁸O of wine water, indicating that water vapour deficit may be a critical defining factor. Furthermore, in C₄-ethanol, the reverse relationship was observed (methylene-C relatively ¹³C-depleted), supporting the concept that photorespiration is the key metabolic process leading to the ¹³C distribution in C₃-ethanol. By contrast, in CAM-ethanol, the isotopic pattern was similar to but stronger than C₃-ethanol, with a relative ¹³C-enrichment in the methylene-C of up to 13‰. Plausible causes of this ¹³C-pattern are briefly discussed. As the intramolecular δ¹³C_i-values in ethanol reflect that in source glucose, our data point out the crucial impact on the ratio of metabolic pathways sustaining glucose synthesis.