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Choline (Cho) is the precursor of the osmoprotectant glycine betaine and is itself an essential nutrient for humans. Metabolic engineering of Cho biosynthesis in plants could therefore enhance both their resistance to osmotic stresses (drought and salinity) and their nutritional value. The key enzyme of the plant Cho-synthesis pathway is phosphoethanolamine N-methyltransferase, which catalyzes all three of the methylations required to convert phosphoethanolamine to phosphocholine. We show here that overexpressing this enzyme in transgenic tobacco increased the levels of phosphocholine by 5-fold and free Cho by 50-fold without affecting phosphatidylcholine content or growth. Moreover, the expanded Cho pool led to a 30-fold increase in synthesis of glycine betaine via an engineered glycine betaine pathway. Supplying the transgenics with the Cho precursor ethanolamine (EA) further enhanced Cho levels even though the supplied EA was extensively catabolized. These latter results establish that there is further scope for improving Cho synthesis by engineering an increased endogenous supply of EA and suggest that this could be achieved by enhancing EA synthesis and͞or by suppressing its degradation.

). ² These authors contributed equally to this paper. SummaryAngiosperms synthesize S-methylmethionine (SMM) from methionine (Met) and S-adenosylmethionine (AdoMet) in a unique reaction catalyzed by Met S-methyltransferase (MMT). SMM serves as methyl donor for Met synthesis from homocysteine, catalyzed by homocysteine S-methyltransferase (HMT). MMT and HMT together have been proposed to constitute a futile SMM cycle that stops the free Met pool from being depleted by an overshoot in AdoMet synthesis. Arabidopsis and maize have one MMT gene, and at least three HMT genes that belong to two anciently diverged classes and encode enzymes with distinct properties and expression patterns. SMM, and presumably its cycle, must therefore have originated before dicot and monocot lineages separated. Arabidopsis leaves, roots and developing seeds all express MMT and HMTs, and can metabolize [ The SMM cycle therefore operates throughout the plant. This appears to be a general feature of angiosperms, as digital gene expression pro®les show that MMT and HMT are co-expressed in leaves, roots and reproductive tissues of maize and other species. An in silico model of the SMM cycle in mature Arabidopsis leaves was developed from radiotracer kinetic measurements and pool size data. This model indicates that the SMM cycle consumes half the AdoMet produced, and suggests that the cycle serves to stop accumulation of AdoMet, rather than to prevent depletion of free Met. Because plants lack the negative feedback loops that regulate AdoMet pool size in other eukaryotes, the SMM cycle may be the main mechanism whereby plants achieve short-term control of AdoMet level.

Previous work has shown that tobacco (Nicotiana tabacum) plants engineered to express spinach choline monooxygenase in the chloroplast accumulate very little glycine betaine (GlyBet) unless supplied with choline (Cho). We therefore used metabolic modeling in conjunction with [14 C]Cho labeling experiments and in vivo 31 P NMR analyses to define the constraints on GlyBet synthesis, and hence the processes likely to require further engineering. The [14 C]Cho doses used were large enough to markedly perturb Cho and phosphocholine pool sizes, which enabled development and testing of models with rates dynamically responsive to pool sizes, permitting estimation of the kinetic properties of Cho metabolism enzymes and transport systems in vivo. This revealed that import of Cho into the chloroplast is a major constraint on GlyBet synthesis, the import rate being approximately 100-fold lower than the rates of Cho phosphorylation and transport into the vacuole, with which import competes. Simulation studies suggested that, were the chloroplast transport limitation corrected, additional engineering interventions would still be needed to achieve levels of GlyBet as high as those in plants that accumulate GlyBet naturally. This study reveals the rigidity of the Cho metabolism network and illustrates how computer modeling can help guide rational metabolic engineering design. Daniel Koshland's recent commentary on "the era of pathway quantification" encapsulates the growing importance to basic biochemistry of mathematical tools for quantifying metabolic fluxes in vivo (1998). Moreover, advances in metabolic control theory and metabolic engineering experience show that we must quantify fluxes if we wish to understand metabolism well enough to manipulate it effectively (Fell,