Enhanced synthesis of choline and glycine betaine in transgenic tobacco plants that overexpress phosphoethanolamine N-methyltransferase.

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 and/or by suppressing its degradation.

The S-methylmethionine cycle in angiosperms: ubiquity, antiquity and activity.

Angiosperms 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 [35S]Met to [35S]SMM and vice versa. The SMM cycle therefore operates throughout the plant. This appears to be a general feature of angiosperms, as digital gene expression profiles 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.

Choline import into chloroplasts limits glycine betaine synthesis in tobacco: analysis of plants engineered with a chloroplastic or a cytosolic pathway.

The biosynthesis of the osmoprotectant glycine betaine (GlyBet) is a target for metabolic engineering to enhance stress resistance in crops. Certain plants synthesize GlyBet in chloroplasts via a two-step oxidation of choline (Cho). In previous work, a chloroplastic GlyBet synthesis pathway was inserted into tobacco (which lacks GlyBet) by expressing spinach choline monooxygenase (CMO). The transformants had low CMO enzyme activity, and produced little GlyBet (less than or = 70 nmol g(-1) fresh wt). In this study, transformants with up to 100-fold higher CMO activity showed no further increase in GlyBet. In contrast, tobacco expressing a cytosolic GlyBet synthesis pathway accumulated significantly more GlyBet (430 nmol g(-1) fresh wt), suggesting that subcellular localization influences pathway flux. Modeling of the labeling kinetics of Cho metabolites observed when [14C]Cho was supplied to engineered plants demonstrated that Cho import into chloroplasts indeed limits the flux to GlyBet in the chloroplastic pathway. A high-activity Cho transporter in the chloroplast envelope may therefore be an integral part of the GlyBet synthesis pathway in species that accumulate GlyBet naturally, and hence a target for future engineering.

cDNA cloning of phosphoethanolamine N-methyltransferase from spinach by complementation in Schizosaccharomyces pombe and characterization of the recombinant enzyme.

The N-methylation of phosphoethanolamine is the committing step in choline biogenesis in plants and is catalyzed by S-adenosyl-L-methionine:phosphoethanolamine N-methyltransferase (PEAMT, EC ). A spinach PEAMT cDNA was isolated by functional complementation of a Schizosaccharomyces pombe cho2(-) mutant and was shown to encode a protein with PEAMT activity and without ethanolamine- or phosphatidylethanolamine N-methyltransferase activity. The PEAMT cDNA specifies a 494-residue polypeptide comprising two similar, tandem methyltransferase domains, implying that PEAMT arose by gene duplication and fusion. Data base searches suggested that PEAMTs with the same tandem structure are widespread among flowering plants. Size exclusion chromatography of the recombinant enzyme indicates that it exists as a monomer. PEAMT catalyzes not only the first N-methylation of phosphoethanolamine but also the two subsequent N-methylations, yielding phosphocholine. Monomethyl- and dimethylphosphoethanolamine are detected as reaction intermediates. A truncated PEAMT lacking the C-terminal methyltransferase domain catalyzes only the first methylation. Phosphocholine inhibits both the wild type and the truncated enzyme, although the latter is less sensitive. Salinization of spinach plants increases PEAMT mRNA abundance and enzyme activity in leaves by about 10-fold, consistent with the high demand in stressed plants for choline to support glycine betaine synthesis.

Characterization and functional expression of cDNAs encoding methionine-sensitive and -insensitive homocysteine S-methyltransferases from Arabidopsis.

Plants synthesize S-methylmethionine (SMM) from S-adenosylmethionine (AdoMet), and methionine (Met) by a unique reaction and, like other organisms, use SMM as a methyl donor for Met synthesis from homocysteine (Hcy). These reactions comprise the SMM cycle. Two Arabidopsis cDNAs specifying enzymes that mediate the SMM --> Met reaction (SMM:Hcy S-methyltransferase, HMT) were identified by homology and authenticated by complementing an Escherichia coli yagD mutant and by detecting HMT activity in complemented cells. Gel blot analyses indicate that these enzymes, AtHMT-1 and -2, are encoded by single copy genes. The deduced polypeptides are similar in size (36 kDa), share a zinc-binding motif, lack obvious targeting sequences, and are 55% identical to each other. The recombinant enzymes exist as monomers. AtHMT-1 and -2 both utilize l-SMM or (S,S)-AdoMet as a methyl donor in vitro and have higher affinities for SMM. Both enzymes also use either methyl donor in vivo because both restore the ability to utilize AdoMet or SMM to a yeast HMT mutant. However, AtHMT-1 is strongly inhibited by Met, whereas AtHMT-2 is not, a difference that could be crucial to the control of flux through the HMT reaction and the SMM cycle. Plant HMT is known to transfer the pro-R methyl group of SMM. This enabled us to use recombinant AtHMT-1 to establish that the other enzyme of the SMM cycle, AdoMet:Met S-methyltransferase, introduces the pro-S methyl group. These opposing stereoselectivities suggest a way to measure in vivo flux through the SMM cycle.