Toshinori Abe

Enhanced saline stress resistance in threonine and methionine overproducing mutant cell line from protoplast culture of rice (Oryza sativa L.)

Summary This study was designed to examine whether saline stress resistance was enhanced in mutant in which free amino acids were overproduced. We selected a mutant cell line (RLTA-2) that overproduced threonine and methionine, and a NaCl resistant cell line (RNaCI-1) through protoplast culture treated with ethylmethane-sulfonate (EMS) with addition of inhibitory concentrations of 1 mM each of lysine plus threonine (1 mM LT) and 200 mM NaCl, respectively. Concentrations of threonine, methionine and the total free amino acids changed largely, being 13.8, 6.2 and 1.6 times higher in RLTA-2 than in the wild type, respectively. Proline concentration increased slightly in RLTA-2 when it was subcultured on the medium supplemented with 200 mM NaCl (RLTA-2-200), i.e. ca. 1.4 times in RLTA-2, while a remarkable increase of proline concentration was observed in RNaCI-1, i.e. 11.5 times as compared with the wild type. Furthermore, calcium accumulation was 3 to 5 times in RLTA-2, but not in RNaCl-1, regardless of NaCl concentration. The selected RLTA-2 calli grew better than the wild type under high concentration of NaCl, but the growth rate of RLTA-2 and RNaCl-1 calli was almost similar above 150 mM NaCl concentration. Almost all RLTA-2 calli survived on N6 medium supplemented with 200 mM NaCl. The capacity to maintain the low level of K + and the high level of Ca ++ in the RLTA-2 cell line under the high level of Na + was highly correlated with free amino acid level as compatible cytoplasmic solute and nitrogen source in cytoplasm. The present results indicate the enhancement of saline resistance in the mutant cell line in which free amino acids are overproduced. The relationship of free amino acid accumulation to enhanced saline resistance is discussed.

Large deletions in chloroplast DNA of rice calli after long-term culture

Summary Alteration in chloroplast DNA (ctDNA) in long-term cultures of calli derived from rice seeds were investigated using a Bam HI-1 fragment of rice ctDNA as a probe. Among 10 callus lines cultured for 36 months, two lines (T36-1 and T36-2) were found to have large deletions (76 kb and 63 kb, respectively) in the chloroplast genome, while none of the same callus lines cultured for 6 months showed any ctDNA alteration. When T36-1 and T36-2 were cultured on MS maintenance medium for an additional six months (becoming T42-1 and T42-2, respectively), the ctDNA of T42-2 underwent a further deletion of 0.5 kb. Maps of deleted ctDNA molecules were constructed using fifteen hybridization probes. Based on the restriction maps of the deleted ctDNAs, the T36-1 and T42-1 callus lines lost two large inverted repeats, together with the intervening small single copy region. The T36-2 and T42-2 callus lines lost the rbcL gene and most of the large single copy region. The only region that these two ctDNA molecules have in common is an approximately 18 kb region that includes various psb and trn genes. These results suggest that this region of the rice chloroplast genome contains at least one functional replication origin of rice ctDNA. Regenerated plantlets were obtained from long-term cultured calli of T36-2, indicating that the deletion of a part of the ctDNA did not affect shoot differentiation from the calli. Southern hybridization patterns of the regenerated plantlets were almost the same as those of the callus lines. The mechanisms responsible for deletion of ctDNA in the callus cells are discussed.

Lysine Accumulation in Transgenic Tobacco Expressing Dihydrodipicolinate Synthase of Escherichia coli

Summary The dihydrodipicolinate synthase (DHDPS; EC 4.2.1.52) gene from E. coli was introduced into tobacco to examine the ability and role of chimeric DHDPS in the biosynthesis of lysine in higher plants. Southern blotting and genetic analysis of six transformed tobacco plants showed that they contained on an average one to three copies. Seeds at T 1 generation of K 11 T-9 showed a 3 resistant: 1 sensitive segregation ratio in the lysine-resistance test with addition of 2 mM lysine. The DHDPS purified from the heterozygous and homozygous K 11 T-9 required ca. 1 mM and ca. 2 mM lysine for 50 % inhibition of enzyme activity, respectively, but only 20 μM lysine in wild type tobacco. The DHDPS activity in the heteroand homozygous K 11 T-9 plant leaves increased ca. 34- and 59-fold as compared with wild type tobacco. The significant increase in the level of free lysine was observed in heterozygous K 11 T-9 (25-fold) and homozygous K 11 T-9 (55-fold) as compared with wild type plants. In transgenic plants, a highly significant correlation was found between the content of free lysine and the level of DHDPS activity. The accumulation levels of lysine were sufficiently high to inhibit the activity of the lysine-sensitive aspartate kinase (AK; EC 2.7.2.4) in vivo , i.e. 49 % and 68 % in heterozygous and homozygous K 11 T-9 plants, respectively, as compared with wild type plants. However, the inhibition of AK activity was not influenced by lysine accumulation in all of the transformed plants, although threonine and methionine contents decreased in homozygous K 11 T-9. The present results showed an important role of the E. coli DHDPS gene in biosynthesis of lysine in higher plants, and introduction of the lysine-insensitive DHDPS gene may confer agronomically useful levels of essential amino acid concentrations in a number of crops and vegetable species.

Asymmetric protoplast fusion between sweet potato and its relatives, and plant regeneration

Experiments were conducted to asymmetrically fuse protoplasts from sweet potato (Ipomoea batatas L. Lam.) and its wild relativesI. trifida Don. andI. lacunosa L. Protoplasts of sweet potato were treated with iodoacetamide, whereas those ofI. trifida Don. andI. lacunosa L. were irradiated with X-rays. The asymmetric protoplast fusion was carried out by the electrofusion method and by polyethylene glycol treatment. Electrically-fused protoplasts initiated cell division, and then formed calli earlier than the polyethylene glycol-fused protoplasts. Plant regeneration occurred only in electrofused calli, suggesting that polyethylene glycol had some toxic effect on plant regeneration ability. Analysis of peroxidase isozymes confirmed the interspecific hybrid characteristics of both the fusion-derived calli and regenerated plants.

Characterization and partial amino acid sequence of a high molecular weight protein from rice seed endosperm: Homology to pyruvate orthophosphate dikinase

Summary Endosperm protein of immature rice ( Oryza sativa L.) seed was characterized by two-dimensional gel electrophoresis. A promiment spot corresponding to a protein of about 94 kD and isoelectric point of 6.0 was observed. The 94 kD protein was electroblotted onto a PVDF membrane and the N-terminal amino acid sequence was determined. Proteins of the internal amino acid sequences of the 94 kD protein were determined by sequence analyses of peptides obtained by Cleveland peptide mapping. The N-terminal and internal amino acid sequences, when compared with those in a protein data base, showed that the 94 kD protein was significantly similar to the PPDK of maize. There were no intradisulfide bonds or N-linked oligosaccharide chains in the molecule. The PPDK from maize leaves cross-reacted with antiserum raised against the 94 kD protein of rice seed endosperm. These results suggest that the 94 kD protein of rice seed endosperm is structurally homologous with PPDK.

Plant regeneration from stem and petiole protoplasts of sweet potato (Ipomoea batatas) and its wild relative, I. lacunosa

A protoplast-to-plant regeneration system has been established for sweet potato (Ipomoea batatas (L.) Lam.) and its wild relative, I. lacunosa L. Viable protoplasts, isolated from preplasmolyzed stems and petioles of in vitro-grown plants, were cultured on liquid MS (Murashige & Skoog 1962) medium that supported cell division and colony formation. Embryogenic calli of sweet potato were induced on agar-solidified MS medium supplemented with 3% (w/v) sucrose, 50 mg l-1 casamino acids, 0.2–0.5 mg l-1 2,4-d, 1.0 mg l-1 kinetin and 1.0 mg l-1 ABA. On average, 3 plants were regenerated from a single sweet potato callus subcultured on semi-solid MS medium containing 3% (w/v) sucrose, 800 mg l-1 glutamine, 2.0 mg l-1 BA or 1.0 mg l-1 kinetin and 1.0 mg l-1 GA3. Embryogenic calli of I. lacunosa L. were initiated on semi-solid MS medium containing 0.2–0.5 mg l-1 IAA and 1.0–2.0 mg l-1 BA. An average of 5 plants was regenerated from a single sweet potato callus subcultured on semi-solid MS medium containing 0.5 or 1.0 mg l-1 GA3.

Changes in α-amylase activity during plant regeneration from rice calli

Summary Changes in α-amylase activity in rice calli during organogenesis were investigated in 5 rice varieties that exhibit different abilities for plant regeneration. During organ differentiation in rice callus tissues, samples were taken at 5-day intervals up to 35 days and α-amylase (EC. 3.2.1.1) activities were measured. The activity in regenerative calli began to increase 15 to 20 days after transfer to the regeneration medium and were elevated 7–11 fold during the culture period, while the calli transferred to callus maintenance medium did not increase and maintained a stable state. The increase in α-amylase activity in regenerative calli was more rapid in the calli that showed higher regenerative abilities (Sasanishiki, Tadukan and Tetep) than in the calli that showed lower regenerative abilities (Fujisaka 5 and Nipponbare). Correlation coefficients between frequencies of organ differentiation and α-amylase activity in the calli 35 days after transfer to the regeneration medium was highly significant. Expression of a rice α-amylase gene ( RAmylA ) in callus tissues during organ differentiation was examined by Northern blot and Western blot analyses. Messenger RNA from the RAmylA gene, which is a main transcript in germinating rice seeds, was highly expressed in the regenerating calli 15 days after transfer. The translated product from the mRNA of the RAmylA gene and α-amylase isozyme band I (1A and 1B) were highly expressed 20 days after transfer, especially in the regeneration medium.

Identification and Microsequence Analysis of High Molecular Weight Proteins in Endosperm of the Rice Seed

Summary Several high molecular weight (HMW) proteins (40 to 96 kD) extracted from endosperm of rice seed were separated by 20-PAGE, and visualized by CBB and silver staining. Molecular weight and pi standard markers were used to identify molecular weights and isoelectric focusing points of each protein, respectively. The proteins were electroblotted onto a PVDF membrane, and the partial amino acid sequences of HMW proteins were determined by automatic Edman degradation. The N-terminal sequences of six of these proteins (having molecular weights of about 74, 71, 60, 57, 51, and 43 kD) were determined, while the N-terminal of six other proteins (having molecular weights of 96, 69, 67, 54, 45, and 42 kD) could not be sequenced due to blockage of the N-terminus. Some internal amino acid sequences were also determined. Structural homology searches resulted in identification of 6 HMW proteins (enzymes), namely, starch debranching enzyme precursor, malate dehydrogenase, phosphoglycerate mutase, enolase, xylulose, and UDP-glucose pyrophosphorylase. In addition, three of the proteins appeared to be related to proteins in the heat shock family, i.e. chloroplast stroma protein (70 kD), HSP protein (70 kD), and chloroplast chaperonin (60 kD). Three proteins (60, 43 and 42 kD) were found to have no homology with those of known plant and animal proteins. All of these proteins were found to have a single polypeptide chain without intermolecular disulfide bonding.

Accumulation of gamma-aminobutyrate (GABA) caused by heat-drying and expression of related genes in immature vegetable soybean (edamame)

We studied the effects of drying of immature seeds of vegetable soybean (Glycine max L. Merrill) on the accumulation of gamma-aminobutyrate (GABA) in the seeds. GABA accumulated after heat-drying, with the maximum at 40°C. The GABA content (447.5 mg/100 g DW) increased to more than 5 times the value in untreated seeds (79.6 mg/100 g DW). In contrast, the glutamate content decreased rapidly to 1/3 the level in the untreated seeds. The GABA content increased early in the heat-drying treatment: after 30 min, it had increased to 1.5 times the value in the untreated seeds. GABA did not accumulate in the vacuum-drying treatment. Among genes related to the GABA shunt, the gene for glutamate decarboxylase (EC 4.1.1.15), which catalyzes the decarboxylation of glutamate to produce GABA, showed relatively high expression, decreasing to only 70% of the value in untreated seeds even after 4 h of treatment. In contrast, expression of the genes for two catabolic mitochondrial enzymes, GABA transaminase (GABA-T; EC 2.6.1.19) and succinate semialdehyde dehydrogenase (SSADH; EC 1.2.1.16), decreased rapidly during heat-drying. These results suggest that the accumulated GABA was not metabolized rapidly by GABA-T and SSADH and therefore remained at high levels.