13CO2 as a universal metabolic tracer in isotopologue perturbation experiments.
A tobacco plant was illuminated for 5h in an atmosphere containing (13)CO(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 (13)CO(2) pulse phase followed by (ii) partial catabolism of the primary photosynthetic products, and (iii) recombination of the (13)C-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 alpha-ketoglutarate as basic building blocks. The study demonstrates that pulse/chase labeling with (13)CO(2) as precursor is a powerful tool for the analysis of quantitative aspects of plant metabolism in completely unperturbed whole plants.
Römisch-Margl
Werner
W
Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstr. 4, D-85747 Garching, Germany.
Schramek
Nicholas
N
Radykewicz
Tanja
T
Ettenhuber
Christian
C
Eylert
Eva
E
Huber
Claudia
C
Römisch-Margl
Lilla
L
Schwarz
Christine
C
Dobner
Maria
M
Demmel
Norbert
N
Winzenhörlein
Bernhard
B
Bacher
Adelbert
A
Eisenreich
Wolfgang
W
D000596:Amino Acids / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D002245:Carbon Dioxide / Q000737:chemistry / Q000378:metabolism*; D002247:Carbon Isotopes; D003198:Computer Simulation; D005947:Glucose / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013058:Mass Spectrometry; D009538:Nicotine / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010788:Photosynthesis; D018515:Plant Leaves / Q000737:chemistry / Q000378:metabolism; D014026:Nicotiana / Q000737:chemistry / Q000378:metabolism*
D000596:Amino Acids; D002247:Carbon Isotopes; D002245:Carbon Dioxide; D009538:Nicotine; D005947:Glucose