Haruo Saruyama

Over-expression of a small heat shock protein, sHSP17.7, confers both heat tolerance and UV-B resistance to rice plants

Exposure of rice (Oryza sativa L.) seedlings to a high temperature (42 °C) for 24 h resulted in a significant increase in resistance to UV-B damage. UV-B resistance was enhanced in parallel with the period of heat treatment. sHSP17.7 was isolated from heated rice seedlings, and the influence of rice sHSP17.7 expression on the viability of E. coli under heat-shock conditions was assessed. After heating, the survival rate of sHSP17.7 cells was 2-fold higher than that of the control cells. The molecular chaperone activity of sHSP17.7 was investigated using catalase as a substrate. Recombinant sHSP17.7 had heat-stable chaperone properties that were capable of protecting stressed catalase from precipitation. sHSP17.7 was overexpressed in the rice cultivar ‘Hoshinoyume’, by Agrobacterium-mediated transformation, under the control of a CaMV 35S promoter. Transgenic rice plants with increased levels of sHSP17.7 protein exhibited significantly increased thermotolerance compared to untransformed control plants. The level of increased thermotolerance was correlated with the level of increased sHSP17.7 protein in the transgenic plants. The transgenic rice plant with the highest constitutive expression of sHSP17.7 had significantly greater resistance to UV-B stress than untransformed control plants. Increase in the degree of resistance of transgenic plants to UV-B was accompanied by an increase in production of sHSP17.7 protein.

Relationship between the Dough Quality and Content of Specific Glutenin Proteins in Wheat Mill Streams, and Its Application to Making Flour Suitable for Instant Chinese Noodles

The content of specific proteins such as high-molecular-weight glutenin subunits HMW-GS 5+10 and low-molecular-weight glutenin subunits LMW-GS KS2 in wheat mill streams of extra-strong Kachikei 33 wheat was quantified by SDS–PAGE and 2D-PAGE. The mill streams showed varied quantities of HMW-GS 5+10 (0.077 to 2.007 mg/g of mill stream), LMW-GS KS2 (0.018 to 0.586 mg/g of mill stream) and total protein (9.42% to 18.98%). The contents of these specific proteins in the mill streams were significantly correlated with the SDS sedimentation volume and the mixing properties, which are respective indices of specific loaf volume and dough strength. The contents of these specific glutenin proteins in the mill streams were therefore found to be significantly important for improving the dough quality suitable for bread and Chinese noodles. Accordingly, we present here the application of this information to the development of an effective method for producing mill streams with high quality and yield that are suitable for instant Chinese noodles.

Isolation of ribosomal subunits from an extremely halophilic archaebacterium Halobacterium halobium by hydrophobic interaction chromatography

A new method was developed for a simple, rapid, and effective preparation of ribosomal subunits from the extremely halophilic archaebacterium Halobacterium halobium using hydrophobic interaction chromatography on phenyl-Sepharose CL-4B. One milliliter of swollen gel matrix (total bed volume) bound up to 15 A260 units of 70 S ribosomes. By a stepwise reduction of the ionic strength first 50 S and then 30 S subunits were solubilized and differentially eluted. The pooled fractions containing 50 S and 30 S subunits, respectively, were adjusted to higher ionic strength and concentrated by ultrafiltration. The yield of purified (30 S + 50 S) subunits was up to 60% of the input of 70 S ribosomes. Poly(U)-dependent polyphenylalanine synthesis assay demonstrated that the subunits were as active as native 70 S ribosomes. 30 S and 50 S subunits of nonhalophilic Escherichia coli, however, were not isolated separately by the application of this method.

Cloning and characterization of a cDNA encoding catalase in wheat.

We determined the nucleotide sequence of a cDNA encoding the catalase (CAT) isolated from wheat (Triticum aestivum L.). The deduced amino acid sequence suggests that this wheat catalase isozyme shared higher amino acid homology with group I CATs of barley CAT-1, rice CAT B and maize CAT-1 and CAT-2 but lower homology with group II CATs of barley CAT-2, rice CAT A and maize CAT-3. Both group I and II specific sequences of Ser-Arg-Leu and Ser-Ser-Ser, respectively considered as peroxisomal targeting signals were found first in monocot plant of wheat. Functionally important amino acids at active center and heme-binding sites detected in all other plant catalase were conserved.

Wheat Catalase Expressed in Transgenic Rice Plants Can Improve Tolerance Against Low Temperature Injury

We isolated wheat catalase (CAT: EC 1.11.1.6) cDNA and introduced it into rice (Oryza sativa L.). Some of 56 transgenic regenerated rice plants were confirmed to express the wheat catalase by the native PAGE with catalase activity staining. The wheat catalase detected in leaf, root, anther, seed germ and seed endosperm suggested that wheat catalase under 35S CaMV promoter is expressed in these tissues. In the transgenic rice plants, the catalase activities in leaf at 25 °C and at 5 °C were increased from 2 to 5 fold and 4 to 15 fold respectively, compared to that of non-transgenic rice. The transgenic rice CT 2-6-4, which showed the highest catalase activity among the transgenic rice plants indicated an increased resistance to low temperature stress. These results were obtained by comparing the damage in leaves of withering (curling) due to chilling at 5 °C. This chilling treatment resulted in the decrease of catalase activities in both types of plant, but the transgenic plants indicated higher catalase activities retained in the leaves than those of non-transgenic ones. The transgenic CT 2-6-4 also contained lower concentration of hydrogen peroxide in leaves than the control rice plant. During the chilling, the H2O2 concentration of non-transgenic rice increased, but the increase was depressed in the transgenic rice. Therefore, enhanced activities of catalase in genetically engineered rice plants lead to the effective detoxification of H2O2, which improves tolerance against low temperature injury.