Radiotracer and computer modeling evidence that phospho-base methylation is the main route of choline synthesis in tobacco

Among flowering plants, the synthesis of choline (Cho) from ethanolamine (E A) can potentially occur via three parallel, interconnected pathways involv ing methylation of free bases, phospho-bases, or phosphatidyl-bases. We inv estigated which pathways operate in tobacco (Nicotiana tabacum L.) because previous work has shown that the endogenous Cho supply limits accumulation of glycine betaine in transgenic tobacco plants engineered to convert Cho t o glycine betaine. The kinetics of metabolite labeling were monitored in le af discs supplied with [P-33]phospho-EA, [P-33]phospho-monomethylethanolami ne, or [C-14]formate, and the data were subjected to computer modeling. Bec ause partial hydrolysis of phospho-bases occurred in the apoplast, modeling of phospho-base metabolism required consideration of the re-entry of [33P] phosphate into the network. Modeling of [C-14]formate metabolism required c onsideration of the labeling of the EA and methyl moieties of Cho. Results supported the following conclusions: (a) The first methylation step occurs solely at the phospho-base level; (b) the second and third methylations occ ur mainly (83%-92% and 65%-85%, respectively) at the phospho-base level, wi th the remainder occurring at the phosphatidyl-base level; and (c) free Cho originates predominantly from phosphatidylcholine rather than from phospho -Cho. This study illustrates how computer modeling of radiotracer data, in conjunction with information on chemical pool sizes, can provide a coherent , quantitative picture of fluxes within a complex metabolic network.