Experimental evidence of phosphoenolpyruvate resynthesis from pyruvate in illuminated leaves

Day respiration is the cornerstone of nitrogen assimilation since it provides carbon skeletons to primary metabolism for glutamate (Glu) and glutamine synthesis. However, recent studies have suggested that the tricarboxylic acid pathway is rate limiting and mitochondrial pyruvate dehydrogenation is partly inhibited in the light. Pyruvate may serve as a carbon source for amino acid (e.g. alanine) or fatty acid synthesis, but pyruvate metabolism is not well documented, and neither is the possible resynthesis of phosphoenolpyruvate (PEP). Here, we examined the capacity of pyruvate to convert back to PEP using ¹³C and ²H labeling in illuminated cocklebur (Xanthium strumarium) leaves.We show that the intramolecular labeling pattern in Glu, 2-oxoglutarate, and malate after ¹³C-3-pyruvate feeding was consistent with ¹³C redistribution from PEP via the PEPcarboxylase reaction. Furthermore, the deuterium loss in Glu after ²H₃-¹³C-3-pyruvate feeding suggests that conversion to PEP and back to pyruvate washed out ²H atoms to the solvent. Our results demonstrate that in cocklebur leaves, PEP resynthesis occurred as a flux from pyruvate, approximately 0.5‰ of the net CO₂ assimilation rate. This is likely to involve pyruvate inorganic phosphate dikinase asnd the fundamental importance of this flux for PEP and inorganic phosphate homeostasis is discussed.