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Acetylation of plant metabolites fundamentally changes their volatility, solubility and activity as semiochemicals. Here we present a new technique termed dynamic 13C-pulse chasing to track the fate of C1–3 carbon atoms of pyruvate into the biosynthesis and emission of methyl acetate (MA) and CO2. 13C-labelling of MA and CO2 branch emissions respond within minutes to changes in 13C-positionally labelled pyruvate solutions fed through the transpiration stream. Strong 13C-labelling of MA emissions occurred only under pyruvate-2-13C and pyruvate-2,3-13C feeding, but not pyruvate-1-13C feeding. In contrast, strong 13CO2 emissions were only observed under pyruvate-1-13C feeding. These results demonstrate that MA (and other volatile and non-volatile metabolites) derive from the C2,3 atoms of pyruvate while the C1 atom undergoes decarboxylation. The latter is a non-mitochondrial source of CO2 in the light generally not considered in studies of CO2 sources and sinks. Within a tropical rainforest mesocosm, we also observed atmospheric concentrations of MA up to 0.6 ppbv that tracked light and temperature conditions. Moreover, signals partially attributed to MA were observed in ambient air within and above a tropical rainforest in the Amazon. Our study highlights the potential importance of acetyl coenzyme A (CoA) biosynthesis as a source of acetate esters and CO2 to the atmosphere.