Direct assessment of the metabolic origin of carbon atoms in glutamate from illuminated leaves using ¹³C-NMR

Glutamate (Glu) is the cornerstone of nitrogen assimilation and photorespiration in illuminated leaves. Despite this crucial role, our knowledge of the flux to Glu de novo synthesis is rather limited. Here, we used isotopic labelling with ¹³CO₂ and ¹³C-NMR analyses to examine the labelling pattern and the appearance of multi-labelled species of Glu molecules to trace the origin of C-atoms found in Glu. We also compared this with ¹³C-labelling patterns in Ala and Asp, which reflect citrate (and thus Glu) precursors, that is, pyruvate and oxaloacetate. Glu appeared to be less ¹³C-labelled than Asp and Ala, showing that the Glu pool was mostly formed by ‘old’ carbon atoms. There were modest differences in intramolecular ¹³C–¹³C couplings between Glu C-2 and Asp C-3, showing that oxaloacetate metabolism to Glu biosynthesis did not involve C-atom redistribution by the Krebs cycle. The apparent carbon allocation increased with carbon net photosynthesis. However, when expressed relative to CO₂ fixation, it was clearly higher at low CO₂ while it did not change in 2% O₂, as compared to standard conditions. We conclude that Glu production from current photosynthetic carbon represents a small flux that is controlled by the gaseous environment, typically upregulated at low CO₂.