13CO 2 as a universal metabolic tracer in isotopologue perturbation experiments

Abstract

A tobacco plant was illuminated for 5 h in an atmosphere containing 13CO 2 and then maintained for 10 days under standard greenhouse conditions. Nicotine, glucose, and amino acids from proteins were isolated chromatographically. Isotopologue abundances of isolated metabolites were determined quantitatively by NMR spectroscopy and mass spectrometry. The observed non-stochastic isotopologue patterns indicate (i) formation of multiply labeled photosynthetic carbohydrates during the 13CO 2 pulse phase followed by (ii) partial catabolism of the primary photosynthetic products, and (iii) recombination of the 13C-labeled fragments with unlabeled intermediary metabolites during the chase period. The detected and simulated isotopologue profiles of glucose and amino acids reflect carbon partitioning that is dominated by the Calvin cycle and glycolysis/glucogenesis. Retrobiosynthetic analysis of the nicotine pattern is in line with its known formation from nicotinic acid and putrescine via aspartate, glyceraldehyde phosphate and α-ketoglutarate as basic building blocks. The study demonstrates that pulse/chase labeling with 13CO 2 as precursor is a powerful tool for the analysis of quantitative aspects of plant metabolism in completely unperturbed whole plants.