␤-Alanine (␤-Ala) betaine is an osmoprotective compound accumulated by most members of the highly stress-tolerant family Plumbaginaceae. Its potential role in plant tolerance to salinity and hypoxia makes its synthetic pathway an interesting target for metabolic engineering. In the Plumbaginaceae, ␤-Ala betaine is synthesized by S-adenosyl-lmethionine-dependent N-methylation of ␤-Ala via N-methyl ␤-Ala and N,N-dimethyl ␤-Ala. It was not known how many N-methyltransferases (NMTases) participate in the three N-methylations of ␤-Ala. An NMTase was purified about 1,890-fold, from Limonium latifolium leaves, using a protocol consisting of polyethylene glycol precipitation, heat treatment, anion-exchange chromatography, gel filtration, native polyacrylamide gel electrophoresis, and two substrate affinity chromatography steps. The purified NMTase was trifunctional, methylating ␤-Ala, N-methyl ␤-Ala, and N,N-dimethyl ␤-Ala. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses indicated that the native NMTase is a dimer of 43-kD subunits. The NMTase had an apparent K m of 45 m S-adenosyl-l-methionine and substrate inhibition was observed above 200 m. The apparent K m values for the methyl acceptor substrates were 5.3, 5.7, and 5.9 mm for ␤-Ala, N-methyl ␤-Ala, and N,N-dimethyl ␤-Ala, respectively. The NMTase had an isoelectric point of 5.15 and was reversibly inhibited by the thiol reagent p-hydroxymercuribenzoic acid.

Alanine betaine is an osmoprotective compound accumu-donor. Leaf extracts from -alanine betaine accumulators -Armeria maritima, L. latifolium and L. ramosissimum -had lated by most members of the plant family Plumbaginaceae. Leaf and root tissues of Limonium latifolium known to accu-detectable NMT activities while none were found in L. perezii, mulate -alanine betaine readily convert supplied -alanine to a species that does not accumulate -alanine betaine. The -alanine betaine. To identify the intermediates and the en-NMT activities were further characterized from the leaves of zymes involved in -alanine betaine synthesis, radiotracer L. latifolium. The activities had a pH optimum of 8.0, were experiments using [ 14 C] formate were employed. These studies soluble and inhibited by S-adenosyl-L-homocysteine. Extractable activities were similar from plants grown under demonstrate that -alanine betaine is synthesized from -alanine via N-methyl and N,N-dimethyl -alanines. A rapid and control and salinity stress conditions. Radiolabeling with [ 14 C] L-aspartic acid indicated that, unlike in bacteria, decarboxyla-sensitive radiometric assay was developed to measure Ntion of L-aspartic acid is not the source of -alanine in the methyltransferase (NMT) activities by using [methyl-14 C] or Plumbaginaceae.