Norimoto Murai

High-frequency generation of transgenic tobacco plants after modified leaf disk cocultivation withAgrobacterium tumefaciens

A modified protocol for theAgrobacterium tumefaciens-mediated transformation of tobacco (Nicotina tabacum L.) leaf disks was developed for greater recovery of transgenic plants. Modifications include transformation ofAgrobacterium by a freeze-thaw procedure, initial cocultivation of leaf disks andAgrobacterium under vacuum, subsequent growth with nurse cells for one week, rooting of shoots in medium lacking carbenicillin, longer, growth in rooting medium, and a shortened “hardening” step. By this procedure, an average of 1.3 kanamycin-resistant calli were obtained per leaf disk, and 38% of, the callus cultures used were regenerated to produce 133 independently transformed tobacco plants.

The Bean Seed Storage Protein [beta]-Phaseolin Is Synthesized, Processed, and Accumulated in the Vacuolar Type-II Protein Bodies of Transgenic Rice Endosperm.

The seed storage protein [beta]-phaseolin of the common bean (Phaseolus vulgaris L.) was expressed in the endosperm of transgenic rice (Oryza sativa L.) plants. The 5.1- or 1.8-kb promoter fragment of the rice seed storage protein glutelin Gt1 gene was fused transcriptionally to either the genomic or cDNA coding sequence of the [beta]-phaseolin gene. The highest quantity of phaseolin estimated by enzyme-linked immunosorbent assay was 4.0% of the total endosperm protein in the transgenic rice seeds. The phaseolin trait was segregated as a single dominant trait with a positive gene dosage effect and was stably inherited through three successive generations. Both phaseolin genomic and cDNA coding sequences were used to synthesize four isoforms of mature phaseolin protein with apparent molecular masses of 51, 48, 47, and 45 kD. Enzyme deglycosylation experiments indicated that the 51-kD form contains high-mannose N-glycans; the 48- and 47-kD forms have further modified N-glycans; and the 45-kD form is a nonglycosylated protein. Immunolabeling studies using light and electron microscopy demonstrated that phaseolin accumulates primarily in the vacuolar type-II protein bodies located at the periphery of the endosperm near the aleurone layer. We discuss the implications of these results on nutritional improvement of rice grains.

A novel basic region/helix-loop-helix protein binds to a G-box motif CACGTG of the bean seed storage protein β-phaseolin gene

Expression of the bean seed storage protein fl-phaseolin is under strict developmental control primarily at the level of transcription. A G-box motif CACGTG has been shown to be a major positive &-acting element of the /3-phuseolit1 gene and to be recognized by a DNA binding protein from bean seed nuclei. To understand the molecular nature of the G-box-binding protein, we screened a cDNA expression library from immature bean seed and identified a positive clone based on binding activity of the proteins expressed in Escherichia coli to an oligodeoxyribonucleotide probe. DNA sequence analysis showed that the cDNA for the phaseolin G-box-binding protein (PGl) encodes a basic regionhelix-loop-h&x (bHLH) domain hear the carboxyl terminus. Sequence comparison with other known plant bHLH proteins such as maize R/B and Antirrhinum DEL suggests that PGl represents a new member of bHLH protein family in plants. Electrophoresis mobility-shift and DNase I footprinting assays demonstrated that PGl protein binds preferentially to the G-box (CACGTG) and not to other two phaseolin E-box motifs (CACCTG and CATATG). Expression of PGl RNA was detzble in all stages of seed development as well as in flower, roxand leaf. Wediscuss the implications of the bHLH protein for transcriptional regulation of the /3-phaseolin gene.

High frequency generation of fertile transgenic rice plants after PEG-mediated protoplast transformation

An efficient method for the transformation and regeneration of fertile transgenic rice (Oryza sativa L.) plants is presented. In this protocol seed calli from the varietiesNipponbare andTaipei 309 were used to produce rice suspension cultures in General Medium. Protoplasts were isolated from suspension cells (8 × 106 protoplasts perg fresh weight), then were incubated with sterile DNA in the presence of MaMg solution, followed by addition of PEG to a final concentration of 25%. A hygromycin phosphotransferase (hph) gene under the plant transcriptional regulatory signals was used as a selectable marker gene. Hygromycin-resistant colonies were selected in the presence of 95 μM hygromycin B with apparent frequencies of 2×10−4 and 5×10−4 forNipponbare andTaipei 309, respectively. Plantlets were regenerated from resistant colonies in Murashige and Skoog plant regeneration medium. Among 628 transgenic plants grown to maturity in the greenhouse, two-thirds bore viable seeds.

The pea seed storage protein legumin was synthesized, processed, and accumulated stably in transgenic rice endosperm

Abstract To improve the amino acid composition of rice grain, the pea seed storage protein legumin was expressed in the endosperm of transgenic rice plants. Genomic or cDNA coding sequence of the pea legumin gene (LegA, including the 3′-downstream region) was placed under transcriptional control of either a 1.8 or 5.1 kb promoter of the rice seed storage protein glutelin gene (Gt1). The 5.1 kb promoter conferred a higher level of legumin accumulation in mature seeds than the 1.8 kb promoter, while the choice of genomic or cDNA coding sequence did not affect expression level significantly. Segregation analysis of RO seeds suggests a monogenic Mendelian inheritance, indicating a single locus or closely linked loci of gene insertion. Western blot analysis indicated that the legumin precursor is cleaved endoproteolytically into the acidic and basic subunits in the rice endosperm as is in its native pea cotyledon. We expect a significant improvement in the nutritional quality of rice seed generated by a high level expression of legumin.

Agrobacterium-mediated transformation of monocot and dicot plants using the NCR promoter derived from soybean chlorotic mottle virus

Abstract The NCR promoter (PNCR) from soybean chlorotic mottle virus (SoyCMV) was used to express the selectable marker, neomycin phosphotransferase (nptII) gene, in Agrobacterium-mediated transformation of both monocot (rice) and dicot (tobacco) plants. A multi-cloning site for insertion of a gene of interest into the binary vector pTN is located proximal to the right border region of T-DNA. When chimeric genes under the control of other strong promoters were located in a head-to-head orientation to the PNCR-nptII gene, kanamycin-resistant tobacco shoots were generated more efficiently than when using the original pTN vectors. This suggests that the enhancer-like sequences in the promoters adjacent to PNCR may promote expression of the PNCR-nptII gene.

Strategies for selecting mutation sites for methionine enhancement in the bean seed storage protein phaseolin

The complete three-dimensional structure of the bean seed storage protein phaseolin was generated from α-carbon coordinates by using molecular mechanic calculations. This structure was used as a template to simulate modifications aimed at increasing the methionine content of phaseolin. A hydrophilic, methionine-rich looping insert sequence was designed. Simulated mutagenesis shows that the insert might be accommodated in turn and loop regions of the protein, but not within an α-helix. Methionine content was also increased by the replacement of hydrophobic amino acids with methionine in the central core β-barrels of the phaseolin protein. Calculations indicated that methionine can effectively replace conserved or variant leucine, isolecuine, and valine residues. However, alanine residues were much more sensitive to substitution, and demonstrated high variability in the effects of methionine replacement. Introduction of multiple substitutions in the barrel interior demonstrated that the replaced residues could interact favorably to relieve local perturbations caused by individual substitutions. Molecular dynamics simulations were also utilized to study the structural organization of phaseolin. The calculations indicate that there are extensive packing interactions between the major domains of phaseolin, which have important implications for protein folding and stability. Since the proposed mutant proteins can be produced and studied, the results presented here provide an ideal test to determine if there is a correlation between the effects obtained by computer simulation and the effects of the mutations on the protein structure expressedin vivo.

Efficient plant regeneration from rice protoplasts in general medium

SummaryWe established an efficient and reproducible procedure for protoplast propagation and fertile plant regeneration of rice (Oryza sativa L.) cultivars Nipponbare and Taipei 309. Selection of scutellum-derived secondary calli, the use of General medium and nurse culture were all found to be critical in the procedure. When 5 basal media (Murashige and Skoog, RY-2, modified R2, Amino Acid and General media) were compared, suspension callus growth rate, protoplast yield and plating efficiency were all about 30% higher in General medium than in the second-best R2 medium. Only one month was required to develop suspension cultures for protoplast isolation using General medium. A plating efficiency as high as 17% and a plant regeneration frequency of 67% were achieved by the improved procedure. Agronomic traits of protoplast- and seed-derived plants were found to be similar.

Induction of two alcohol dehydrogenase polypeptides in rice roots during anaerobiosis

Abstract In vivo pulse labeling of rice roots exposed to anaerobiosis shows that new proteins are synthesized while the synthesis of other proteins ceases as a result of the stress. Alcohol dehydrogenase (ADH) activity in the rice roots gradually increased over a 24-h period, leveling off subsequently. An antiserum against ADH was used to show that ADH is present at low levels in aerobically grown roots, and that two molecular forms of ADH, differing in molecular weight, are synthesized de novo when the roots are stressed. The rise in ADH activity under a lack of oxygen does not involve the conversion of an inactive protein into active enzyme.

Functional analysis of Gossypium hirsutum cellulose synthase catalytic subunit 4 promoter in transgenic Arabidopsis and cotton tissues

Gossypium hirsutum cellulose synthase catalytic subunit 4 (GhCesA4) plays an important role in cellulose biosynthesis during cotton fiber development. The transcript levels of GhCesA4 are significantly up-regulated as secondary cell wall cellulose is produced in developing cotton fibers. To understand the molecular mechanisms involved in transcriptional regulation of GhCesA4, β-glucuronidase (GUS) activity regulated by a GhCesA4 promoter (-2574/+56) or progressively deleted promoters were determined in both cotton tissues and transgenic Arabidopsis. The spatial regulation of GhCesA4 expression was similar between cotton tissues and transgenic Arabidopsis. GUS activity regulated by the GhCesA4 promoter (-2574/+56) was found in trichomes and root vascular tissues in both cotton and transgenic Arabidopsis. The -2574/-1824 region was responsible for up-regulation of GhCesA4 expression in trichomes and root vascular tissues in transgenic Arabidopsis. The -1824/-1355 region negatively regulated GhCesA4 expression in most Arabidopsis vascular tissues. For vascular expression in stems and leaves, the -898/-693 region was required. The -693/-320 region of the GhCesA4 promoter was necessary for basal expression of GhCesA4 in cotton roots as well as Arabidopsis roots. Exogenous phytohormonal treatments on transgenic Arabidopsis revealed that phytohormones may be involved in the differential regulation of GhCesA4 during cotton fiber development.