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Betaine is an important osmoprotectant in many plants, but its transport activity has only been demonstrated using a proline transporter from tomato, a betaine-nonaccumulating plant. In this study, two full-length and one partial transporter genes were isolated from betaine-accumulating mangrove Avicennia marina. Their homologies to betaine transporters from bacteria and betaine/4-aminobutyrate transporters from mammalian cells were low but were high to proline transporters from Arabidopsis and tomato. Two full-length transporters could complement the Na(+)-sensitive phenotype of the Escherichia coli mutant deficient in betT, putPA, proP, and proU. Both transporters could efficiently take up betaine and proline with similar affinities (K(m), 0.32-0.43 mm) and maximum velocities (1.9-3.6 nmol/min/mg of protein). The uptakes of betaine and proline were significantly inhibited by mono- and dimethylglycine but only partially inhibited by betaine aldehyde, choline, and 4-aminobutyrate. Sodium and potassium chloride markedly enhanced betaine uptake rates with optimum concentrations at 0.5 m, whereas sucrose showed only modest activation. The change of amino acids Thr(290)-Thr-Ser(292) in a putative periplasmic loop to Arg(290)-Gly-Arg(292) yielded the active transporter independent of salts, suggesting the positive charge induced a conformational change to the active form. These data clearly indicate that the betaine-accumulating mangrove contains betaine/proline transporters whose properties are distinct from betaine transporters of bacteria and mammalian cells.

The rice slender mutant (slr1-1) is caused by a single recessive mutation and results in a constitutive gibberellin (GA) response phenotype. The mutant elongates as if saturated with GAs. In this mutant, (1) elongation was unaffected by an inhibitor of GA biosynthesis, (2) GA-inducible alpha-amylase was produced by the aleurone layers without gibberellic acid application, and (3) endogenous GA content was lower than in the wild-type plant. These results indicate that the product of the SLR1 gene is an intermediate of the GA signal transduction pathway. SLR1 maps to OsGAI in rice and has significant homology with height-regulating genes, such as RHT-1Da in wheat, D8 in maize, and GAI and RGA in Arabidopsis. The GAI gene family is likely to encode transcriptional factors belonging to the GRAS gene superfamily. DNA sequence analysis revealed that the slr1-1 mutation is a single basepair deletion of the nuclear localization signal domain, resulting in a frameshift mutation that abolishes protein production. Furthermore, introduction of a 6-kb genomic DNA fragment containing the wild-type SLR1 gene into the slr1-1 mutant restored GA sensitivity to normal. These results indicate that the slr1-1 mutant is caused by a loss-of-function mutation of the SLR1 gene, which is an ortholog of GAI, RGA, RHT, and D8. We also succeeded in producing GA-insensitive dwarf rice by transforming wild-type rice with a modified SLR1 gene construct that has a 17-amino acid deletion affecting the DELLA region. Thus, we demonstrate opposite GA response phenotypes depending on the type of mutations in SLR1.

We used a transient expression system to study the mechanism by which carbohydrates repress a rice (Oryza sativa L.) alpha-amylase (EC 3.2.1.1) gene. Exogenously fed metabolizable carbohydrates are able to elicit repression of the alpha-amylase gene RAmy3D in the rice embryo, and our results indicate that repression is also triggered efficiently by endogenous carbohydrates. Glucose analogs that are taken up by plant cells but not phosphorylated by hexokinase are unable to repress the alpha-amylase gene studied, while 2-deoxyglucose, which is phosphorylable but not further metabolized, down-regulates RAmy3D promoter activity, indicating a role for hexokinase in the sugar-sensing mechanism triggering repression of the RAmy3D gene. We tested two different hexokinase inhibitors, mannoheptulose and glucosamine, but only the latter was able to relieve RAmy3D promoter activity from repression by endogenous carbohydrates. This correlates with the higher ability of glucosamine to inhibit the activity of rice hexokinases in vitro. The glucosamine-mediated relief of RAmy3D promoter activity from repression by endogenous carbohydrates does not correlate with a reduced rate of carbohydrate utilization.

The gibberellin-inducible rice alpha-amylase gene, RAmy1 A, was demonstrated to be sugar repressed in rice embryos and functional dissection of the promoter of RAmy1 A in relation of its sugar-modulated expression was performed. Gibberellin-response cis-elements of GARE (TAACAAA) and pyrimidine box (CCTTTT) were partially involved in the sugar repression.

Sixty rice varieties (Oryza sativa L.), belonging to three subspecies, japonica, indica and javanica (some japonicaXindica hybrids were included), were compared for their capacity for callus growth and plant regeneration. Tissue cultures initiated from mature seeds on Murashige and Skoog's (1962) medium with 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) were transferred to a medium containing 0.02 mg/l 2,4-D and 10 mg/l kinetin, from which plantlets were regenerated. Large variabilities in callus growth and plant regeneration potentials were revealed among the varieties tested. Most japonica varieties formed a callus that weighed more than 100 mg per seed 30 days after inoculation, and showed a relatively high regenerative potential, whereas indica varieties, japonica-indica hybrids and javanica varieties showed poor callus growth and plant regeneration, although considerable varietal variation was observed in each subspecies. The callus growth potential was not correlated with the plant regeneration potential. Histological observations revealed that the epithelium cells of the scutellum mainly proliferated to form a callus, from which shoot and root primordia were differentiated independently from each other. The shoot primordia developed into plantlets when roots were formed adventitiously. In a few cases, shoots and roots were bilaterally initiated from a single primordium.

Calli were initiated from seedling roots in rice (Oryza sativa L. var. Tadukan) and subcultured at 45-day intervals on MS medium supplemented with 2 mg/l 2,4-D. Sectors of callus which differentiated shoot meristems (green spots) under the same 2,4-D concentration were selected from the calli subcultured 90 days after initiation. The selection was continued for about 2 years. Responses to 2,4-D between original and selected lines differed considerably, although differentiation was not generally seen in rice callus in the presence of 2 mg/l 2,4-D. After 180 days, calli of the selected line differentiated into numerous shoot-bud primordia and grew out new callus tissues under 2 mg/l 2,4-D concentration; the frequency of the differentiation exceeded 90%. On the other hand, no calli of non-selected line differentiated into shootbuds under 2 mg/l 2,4-D, and the frequency of the shootbud was only about 50% under lower 2,4-D concentration (0.1 mg/l). The pattern and activity of peroxidase isozyme varied markedly between calli of the selected and non-selected lines. First, two strong peroxidase bands which show fast mobility and one intermediate peroxidase band with slow mobility were detected only in the calli of selected line. Secondly, changes in band pattern of proteins separated by SDS-PAGE were observed. In the calli of selected line, there was a loss of the polypeptide bands with molecular weight of 24 and 42 K in the selected calli, but they were clearly present in the unselected line. The appearance of new peroxidase isozyme bands and loss of polypeptide bands, change in response to auxin and increased ability for shoot bud differentiation are closely correlated to each other.