Plants accumulate a variety of osmoprotectants that improve their ability to combat abiotic stresses. Among them, betaine appears to play an important role in conferring resistance to stresses. Betaine is synthesized via either choline oxidation or glycine methylation. An increased betaine level in transgenic plants is one of the potential strategies to generate stress-tolerant crop plants. Here, we showed that an exogenous supply of serine or glycine to a halotolerant cyanobacterium Aphanothece halophytica, which synthesizes betaine from glycine by a three-step methylation, elevated intracellular accumulation of betaine under salt stress. The gene encoding 3-phosphoglycerate dehydrogenase (PGDH), which catalyzes the first step of the phosphorylated pathway of serine biosynthesis, was isolated from A. halophytica. Expression of the Aphanothece PGDH gene in Escherichia coli caused an increase in levels of betaine as well as glycine and serine. Expression of the Aphanothece PGDH gene in Arabidopsis plants, in which the betaine synthetic pathway was introduced via glycine methylation, further increased betaine levels and improved the stress tolerance. These results demonstrate that PGDH enhances the levels of betaine by providing the precursor serine for both choline oxidation and glycine methylation pathways.Salinity is a major problem affecting world overall agricultural production. Approximately 20% of the cultivated land worldwide is impaired by high salinity which causes ion imbalance and hyperosmotic stress in plants (1). Plants have evolved various strategies to cope with salinity, including the accumulation of low molecular weight organic compatible solutes (osmoprotectants) such as sugars, some amino acids, and quaternary ammonium compounds (2-4). Glycine betaine (N,N,N-trimethylglycine, hereafter betaine) is a major osmoprotectant to protect plants from high salinity (2-4). Most known biosynthetic pathways of betaine include a two-step oxidation of choline: choline 3 betaine aldehyde 3 betaine. The first step is catalyzed by choline monooxygenase (CMO) in plants (5), choline dehydrogenase (CDH) in animals and bacteria (6, 7), and choline oxidase in some bacteria (8, 9). The second step is catalyzed by betaine aldehyde dehydrogenase (BADH) in all organisms (6, 10, 11). Introducing the betaine synthetic pathway into betaine non-accumulating plants has been applied to improve salt tolerance of plants (8,(12)(13)(14)(15). However, the accumulation levels of betaine in the transformed plants are relatively low. Supplying betaine precursors such as choline, ethanolamine, and serine to the transformed plants enhance betaine accumulation. Thus, the availability of betaine precursors limits the betaine biosynthesis (15, 16 -18).Recently, we showed that a halotolerant cyanobacterium, Aphanothece halophytica (A. halophytica), synthesizes betaine from glycine by a three-step methylation, which is catalyzed by two N-methyltransferases (ApGSMT and ApDMT). ApGSMT is responsible for the two-step methylation reactions of ...