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When subjected to salt stress or drought, some vascular plants such as barley respond with an increased accumulation of the osmoprotectant glycine betaine (betaine), being the last step of betaine synthesis catalyzed by betaine aldehyde dehydrogenase (BADH). We report here cloning and characterization of BADH cDNA from barley, a monocot, and the expression pattern of a BADH transcript. An open reading frame of 1515 bp encoded a protein which showed high homology to BADH enzymes present in other plants (spinach and sugar-beet) and in Escherichia coli. Transgenic tobacco plants harboring the clone expressed high levels of both BADH protein and its enzymatic activity. Northern blot analyses indicated that BADH mRNA levels increased almost 8-fold and 2-fold, respectively, in leaves and roots of barley plants grown in high-salt conditions, and that these levels decreased upon release of the stress, whereas they did not decrease under continuous salt stress. BADH transcripts also accumulate in response to water stress or drought, indicating a common response of the plant to osmotic changes that affect its water status. The addition of abscisic acid (ABA) to plants during growth also increased the levels of BADH transcripts dramatically, although the response was delayed when compared to that found for salt-stressed plants. Removal of plant roots before transferring the plants to high-salt conditions reduced only slightly the accumulation of BADH transcripts in the leaves.

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.cyanobacteria ͉ methyltransferase ͉ osmoprotectant ͉ stress resistance T oday, Ϸ20% of the world's cultivated land and nearly half of all irrigated lands are affected by high salinity (1). High salinity causes ion imbalance and hyperosmotic stress in plants. Organisms that thrive in hypersaline environments possess specific mechanisms for the adjustment of their internal osmotic status. One such mechanism is the ability to accumulate low-molecular-weight organic-compatible solutes such as sugars, some amino acids, and quaternary ammonium compounds (2-4). Glycine betaine (N,N,Ntrimethylglycine, hereafter betaine) is a major osmolyte (2-4). Another mechanism for adaptation to high salinity is the exclusion of the Na ϩ ion from sodium-sensitive sites (5). Genetic engineering techniques have been applied to improve the salt tolerance of plants (6-13). Considerable success has been demonstrated by manipulating the Na ϩ ͞H ϩ antiporter genes (6-8). By contrast, the genetic engineering of betaine synthesis has been hampered by low accumulation levels of betaine (9-13). 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 (14), choline dehydrogenase (CDH) in animals and bacteria (15,16), and choline oxidase in some bacteria (11,17). The second step is catalyzed by NAD ϩ -dependent betaine aldehyde dehydrogenase in all organisms (15,18,19), although in some bacteria, CDH and choline oxidase also catalyze the second step (15-17). Hitherto, all attempts at betaine synthesis have been carried out by using choline-oxidizing enzymes (9-13). The supply and transport of betaine precursors such as choline, ethanolamine, and serine to plastids may be of importance, because these precursors have been suggested to be limiting (12,13).Recently, we showed that a halotolerant cyanobacter...

SummaryBetaine aldehyde dehydrogenase (BADH) catalyzes the last step in the plant biosynthetic pathway that leads to glycinebetaine. Rice plants (Oryza sativa L.}, albeit considered a typical non-glycinebetaine accumulating species, have been found to express this enzyme at low levels. This observation evokes an interest in phylogenic evolution of the enzyme in the plant kingdom. It is reported here that rice plants possess the ability to take up exogenously added betaine aldehyde through the roots and convert it to glycinebetaine, resulting in an enhanced salttolerance of the plants. A gene encoding a putative BADH from the rice genome was also cloned and sequenced. The gene was found to contain 14 introns, and the overall nucleotide sequence of the coding region is c. 78% identical to that of the barley BADH cDNA. Cloning of a partial BADH cDNA from rice was accomplished by reverse transcription-polymerase chain reaction (RT-PCR). The nucleotide sequence of the cloned fragment was found to be identical to the corresponding exon regions of the rice genomic BADH gene. The deduced amino acid sequences of rice and barley BADH both contain a C-terminal tripeptide SKL, a signal known to target preproteins to microbodies. This localization was confirmed by an immuno-gold labeling study of transgenic tobacco harboring barley cDNA, which showed BADH protein inside peroxisomes. Northern blot analysis revealed that the level of BADH mRNA is salt-inducible.

Glycine betaine (N,N,N-trimethylglycine) is an important osmoprotectant and is synthesized in response to abiotic stresses. Although almost all known biosynthetic pathways of betaine are two-step oxidation of choline, here we isolated two N-methyltransferase genes from a halotolerant cyanobacterium Aphanothece halophytica. The most known biosynthetic pathways of betaine are the two-step oxidation of choline. Many bacteria, plants, and animals accumulate glycine betaine (here after betaine) under abiotic stress conditions (1-3). In these organisms, it was shown that betaine is synthesized by two steps, choline 3 betaine aldehyde 3 glycine betaine. The enzyme involved in the second step seems to be the same in plants, animals, and bacteria, namely NAD ϩ -dependent betaine-aldehyde dehydrogenase (4 -6). By contrast, different enzymes are involved for the first step. In plants, it was catalyzed by a novel Rieske-type iron-sulfur enzyme choline monooxygenase (7,8). In animals and many bacteria, the first step is catalyzed by membranebound choline dehydrogenase or soluble choline oxidase (9 -11). In some bacteria, choline dehydrogenase and choline oxidase also catalyze the second oxidation step (9 -11).It was suggested that betaine might be synthesized from glycine by a series of methylation reactions in archaebacterium Methanohalophilus portucalensis (12) and anaerobic phototrophic sulfur bacterium Ectothiorhodospira halochloris (13). Betaine synthesis from simple carbon sources has also been suggested in aerobic heterotrophic eubacterium Actinopolyspora halophila (13) and halotolerant cyanobacterium of Aphanothece halophytica (14). Recently, the methyltransferase genes that are involved in betaine synthesis have been isolated from E. halochloris and A. halophila (15). Two methyltransferase genes were involved in E. halochloris. One of gene products catalyzed the methylation reactions of glycine and sarcosine to sarcosine and dimethylglycine, respectively (EcGSMT), 1 whereas the other one catalyzed the methylations of sarcosine and dimethylglycine to dimethylglycine and betaine, respectively (EcSDMT) (15,16). By contrast, one ORF was found in A. halophila of which the N-and C-terminal parts had homologous sequences to those of EcGSMT and EcSDMT, respectively (15). The functionality of A. halophila methyltransferase was not well shown due to the formation of cell pellet when expressed in Escherichia coli.Glycine N-methyltransferase (GMT) catalyzing the methylation of glycine to sarcosine is known in mammalian cells although the enzymes catalyzing the further methylation steps do not occur (17,18). The homology of amino acid sequences between mammalian GMT and EcGSMT was low. No homologous sequences to those of EcGSMT, EcSDMT, and A. halophila methyltransferase could be found. Therefore, it was interesting to examine whether betaine is synthesized from glycine by three-step methylation reactions in other organisms.