Betaine is an important osmoprotectant, synthesized by many plants in response to abiotic stresses. Almost all known biosynthetic pathways of betaine are two-step oxidations of choline. Recently, a biosynthetic pathway of betaine from glycine, catalyzed by two N -methyltransferase enzymes, was found. Here, the potential role of N -methyltransferase genes for betaine synthesis was examined in a freshwater cyanobacterium, Synechococcus sp. PCC 7942, and in Arabidopsis plants. It was found that the coexpression of N -methyltransferase genes in Synechococcus caused accumulation of a significant amount of betaine and conferred salt tolerance to a freshwater cyanobacterium sufficient for it to become capable of growth in seawater. Arabidopsis plants expressing N -methyltransferase genes also accumulated betaine to a high level in roots, stems, leaves, and flowers and improved seed yield under stress conditions. Betaine levels were higher than those produced by choline-oxidizing enzymes. These results demonstrate the usefulness of glycine N -methyltransferase genes for the improvement of abiotic stress tolerance in crop plants.

Transgenic tobacco plants expressing the ascorbate oxidase (AAO) gene in sense and antisense orientations, and an Arabidopsis mutant in which the T-DNA was inserted into a putative AAO gene, were used to examine the potential roles of AAO for salt-stress tolerance in plants. AAO activities in the transgenic tobacco plants expressing the gene in sense and antisense orientations were, respectively, about 16-fold and 0.2-fold of those in the wild type. Under normal growth conditions, no significant differences in phenotypes were observed, except for a delay in flowering time in the antisense plants. However, at high salinity, the percentage germination, photosynthetic activity, and seed yields were higher in antisense plants, with progressively lower levels in the wild type and the sense plants. The redox state of apoplastic ascorbate in sense plants was very low even under normal growth conditions. Upon salt stress, the redox state of symplastic and apoplastic ascorbate decreased among the three types of plants, but was lowest in the sense plants. The hydrogen peroxide contents in the symplastic and apoplastic spaces were higher in sense plants, progressively lower in the wild type, followed by the antisense plants. The Arabidopsis T-DNA inserted mutant exhibited very low ascorbate oxidase activity, and its phenotype was similar to that of antisense tobacco plants. These results suggest that the suppressed expression of apoplastic AAO under salt-stress conditions leads to a relatively low level of hydrogen peroxide accumulation and a high redox state of symplastic and apoplastic ascorbate which, in turn, permits a higher seed yield.

The salt tolerance of a freshwater cyanobacterium, Synechococcus sp. PCC 7942, transformed with genes involved in the synthesis of a Na ⁺ /H ⁺ antiporter, betaine, catalase, and a chaperone was examined. Compared with the expression of betaine, catalase, and the chaperone, the expression of the Na ⁺ /H ⁺ antiporter gene from a halotolerant cyanobacterium (ApNhaP) drastically improved the salt tolerance of the freshwater cyanobacterium. The Synechococcus cells expressing ApNhaP could grow in BG11 medium containing 0.5 M NaCl as well as in sea water, whereas those expressing betaine, catalase, and the chaperone could not grow under those conditions. The coexpression of ApNhaP with catalase or ApNhaP with catalase and betaine did not further enhance the salt tolerance of Synechococcus cells expressing ApNhaP alone when grown in BG11 medium containing 0.5 M NaCl. Interestingly, the coexpression of ApNhaP with catalase resulted in enhanced salt tolerance of cells grown in sea water. These results demonstrate a key role of sodium ion exclusion by the Na ⁺ /H ⁺ antiporter for the salt tolerance of photosynhetic organisms.

Aphanothece halophytica is a halotolerant alkaliphilic cyanobacterium which can grow in media of up to 3.0 M NaCl and pH 11. This cyanobacterium can synthesize betaine from glycine by three-step methylation using S -adenosylmethionine as a methyl donor. To unveil the mechanism of betaine uptake and efflux in this alkaliphile, we isolated and characterized a betaine transporter. A gene encoding a protein (BetT _{A. halophytica} ) that belongs to the betaine-choline-carnitine transporter (BCCT) family was isolated. Although the predicted isoelectric pH of a typical BCCT family transporter, OpuD of Bacillus subtilis , is basic, 9.54, that of BetT _{A. halophytica} is acidic, 4.58. BetT _{A. halophytica} specifically catalyzed the transport of betaine. Choline, γ-aminobutyric acid, betaine aldehyde, sarcosine, dimethylglycine, and amino acids such as proline did not compete for the uptake of betaine by BetT _{A. halophytica} . Sodium markedly enhanced betaine uptake rates, whereas potassium and other cations showed no effect, suggesting that BetT _{A. halophytica} is a Na ⁺ -betaine symporter. Betaine uptake activities of BetT _{A. halophytica} were high at alkaline pH values, with the optimum pH around 9.0. Freshwater Synechococcus cells overexpressing BetT _{A. halophytica} showed NaCl-activated betaine uptake activities with enhanced salt tolerance, allowing growth in seawater supplemented with betaine. Kinetic properties of betaine uptake in Synechococcus cells overexpressing BetT _{A. halophytica} were similar to those in A. halophytica cells. These findings indicate that A. halophytica contains a Na ⁺ -betaine symporter that contributes to the salt stress tolerance at alkaline pH. BetT _{A. halophytica} is the first identified transporter for compatible solutes in cyanobacteria.

In plants, betaine is synthesized upon abiotic stress via choline oxidation, in which choline monooxygenase (CMO) is a key enzyme. Although it had been thought that betaine synthesis is well regulated to protect abiotic stress, it is shown here that an exogenous supply of precursors such as choline, serine, and glycine in the betaine-accumulating plant Amaranthus tricolor further enhances the accumulation of betaine under salt stress, but not under normal conditions. Addition of isonicotinic acid hydrazide, an inhibitor of glycine decarboxylase, inhibited the salinity-induced accumulation of betaine. Salt-induced accumulation of A. tricolor CMO (AmCMO) and betaine was much slower in roots than in leaves, and a transient accumulation of proline was observed in the roots. Antisense expression of AmCMO mRNA suppressed the salt-induced accumulation of AmCMO and betaine, but increased the level of choline ∼2– 3-fold. This indicates that betaine synthesis is highly regulated by AmCMO expression. The genomic DNA, including the upstream region (1.6 kbp), of AmCMO was isolated. Deletion analysis of the AmCMO promoter region revealed that the 410 bp fragment upstream of the translation start codon contains the sequence responsive to salt stress. These data reveal that the promoter sequence of CMO, in addition to precursor supply, is important for the accumulation of betaine in the betaine-accumulating plant A. tricolor.

Aphanothece halophytica is a halotolerant alkaliphilic cyanobacterium which can grow at NaCl concentrations up to 3.0 M and at pH values up to 11. The genome sequence revealed that the cyanobacterium Synechocystis sp. strain PCC 6803 contains five putative Na ⁺ /H ⁺ antiporters, two of which are homologous to NhaP of Pseudomonas aeruginosa and three of which are homologous to NapA of Enterococcus hirae . The physiological and functional properties of NapA-type antiporters are largely unknown. One of NapA-type antiporters in Synechocystis sp. strain PCC 6803 has been proposed to be essential for the survival of this organism. In this study, we examined the isolation and characterization of the homologous gene in Aphanothece halophytica . Two genes encoding polypeptides of the same size, designated Ap- napA1 - 1 and Ap- napA1 - 2 , were isolated. Ap-NapA1-1 exhibited a higher level of homology to the Synechocystis ortholog (Syn-NapA1) than Ap-NapA1-2 exhibited. Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1 complemented the salt-sensitive phenotypes of an Escherichia coli mutant and exhibited strongly pH-dependent Na ⁺ /H ⁺ and Li ⁺ /H ⁺ exchange activities (the highest activities were at alkaline pH), although the activities of Ap-NapA1-2 were significantly lower than the activities of the other polypeptides. Only one these polypeptides, Ap-NapA1-2, complemented a K ⁺ uptake-deficient E. coli mutant and exhibited K ⁺ uptake activity. Mutagenesis experiments suggested the importance of Glu129, Asp225, and Asp226 in the putative transmembrane segment and Glu142 in the loop region for the activity. Overexpression of Ap-NapA1-1 in the freshwater cyanobacterium Synechococcus sp. strain PCC 7942 enhanced the salt tolerance of cells, especially at alkaline pH. These findings indicate that A. halophytica has two NapA1-type antiporters which exhibit different ion specificities and play an important role in salt tolerance at alkaline pH.