Keishi Senoo

Characterization of Mycorrhizas Formed by Glomus sp. on Roots of Hypernodulating Mutants of Lotus japonicus

Lotusjaponicus hypernodulating mutants, Ljsym78-1 and Ljsym78-2, by the arbuscular mycorrhizal fungus Glomus sp. was characterized. The mutants are defective in systemic autoregulation of nodulation and nitrate inhibition, and form an excess of nodules and lateral roots. The percent root length colonized by the arbuscular mycorrhizal fungi was significantly higher for the mutant than wild-type roots. Detailed assessment of the colonization indicated that the percentage of colonization by arbuscules was increased, but that by external hyphae, internal hyphae and vesicles was decreased, in the mutant roots compared with the wild-type. The succinate dehydrogenase activity of arbuscules, external hyphae and internal hyphae showed similar trends. In addition, the majority of individual arbuscules that formed on the mutant roots had a well-developed and seemingly tough morphology. The results suggest that mutation at the Ljsym78 locus positively stimulates the growth and activity of arbuscules, but leads to reduced growth and activity of hyphae. We report the first identification of Lotus japonicus mutants that show significantly increased arbuscule formation and termed these mutants Arb++.

Multiple Tolerance of Rhodotorula glutinis R-1 to Acid, Aluminum Ion and Manganese Ion, and Its Unusual Ability of Neutralizing Acidic Medium.

A red yeast isolated from the acidic water of Kusatsu hot spring could grow in an acidic medium of pH 1.5 and was identified as Rhodotorula glutinis. Electron microscope observations (scanning electron microscopy [SEM] and transmission electron microscopy [TEM]) showed that cell envelope became wrinkled and thick as the pH values of media became lower. The cell membrane grown at pH 1.5 was about four times as thick as that grown at pH 6.0. It was suggested that the change of cell envelope plays an important role in the acid tolerance. Cellular proteins at pH 1.5 appeared to be different from those at pH 6.0 and the amounts of phospholipids and non-phospholipids increased and decreased under low pH conditions, respectively. The acid-tolerant yeast also showed strong resistance to both aluminum and manganese ions. An acidic medium (pH 3.0) containing these ions (100 mM) was shifted to neutral pH by long-term cultivation of the red yeast, suggesting the potential of using this yeast in the bioremediation of acidic soil containing these ions at a high level.

Changes in the level of protein and activity of Cu/Zn-superoxide dismutase in zinc deficient rice plant, Oryza sativa L.

Abstract In order to clarify the physiological role of Zn in rice plants through the identification and characterization of proteins affected by Zn deficiency, changes in the peptide profile in Zn-sufficient and Zn-deficient rice meristems were examined by two-dimensional pI-SDS PAGE. The level of some peptides fluctuated under Zn deficiency. One of the peptides for which the spot intensity decreased in the profile derived from Zn-deficient rice meristems was identified as Cu/Zn super oxide dismutase (Cu/Zn-SOD) by amino-terminal sequencing. Decrease in the Cu/Zn-SOD activity and of the Cu/Zn-SOD protein content was observed based on a nitro blue tetrazolium reduction assay and immunoblotting analysis, respectively. These results indicate that Zn deficiency in rice causes a reduction of the activity of Cu/Zn-SOD through the decrease of the protein content and/or inactivation of the enzyme in the meristem.

Symbiotic efficiency and compatibility of native rhizobia in northern Thailand with different soybean cultivars. III. Laboratory experiment using native isolates from upland rainfed soybean-growing areas

Abstract Symbiotic efficiency of 50 native bradyrhizobium isolates from northern Thailand with 12 soybean cultivars of Asian, Nigerian, or US origin was evaluated under laboratory conditions. The isolates were obtained from fields in upland rainfed areas of northern Thailand where soybeans had been traditionally cultivated. Twelve soybean cultivars of Asian or US origin were grown in a nitrogen-free nutrient solution with inoculation of each of the isolates. The symbiotic efficiency of the isolates was evaluated based on the total amount of nitrogen accumulation by host plants in comparison with uninoculated control plants or plants inoculated with Bradyrhizobium japonicum USDA110. Almost all the isolates were able to nodulate all the soybean cultivars. Compared with control soybean cultivars grown in a nitrogen-free nutrient solution (- N - R), forty-four isolates induced a high nitrogenfixing ability (E category) in association with at least one soybean cultivar, Coc Chumhat. More than half of the tested isolates induced effective nitrogen fixation in this category in association with almost all the tested soybean cultivars (except for Peking (46%) and Bossier (20%)). Unique character of the tested isolates was that most of them were effective in association with a relatively wide range of soybean cultivars, i.e. “promiscuous.” This character was not observed in the native isolates from irrigated areas in our previous study. Three soybean cultivars, Coc Chumhat, CMU0001, and IITA medium, showed a high symbiotic performance (E + e category of the isolates) with most of the tested isolates (90, 84, and 78%, respectively), suggesting their “promiscuous” character. From the results of our three experiments (one field and two laboratory experiments), it was concluded that: 1) the native rhizobial populations in the traditional soybean-growing area of northern Thailand showed a high level of effectiveness and effectiveness in association with a wide range of soybean genotypes, 2) when a proper soybean cultivar is used, adequate amount of nitrogen fixation for soybean would be expected from these native rhizobial populations, and 3) various symbiotic characteristics of the native rhizobial populations obtained from field and laboratory experiments provided useful background information for the justification of soybean cultivation and the need for inoculation in northern Thailand.

Basic subsite theory assumptions may not be applicable to hydrolysis of cellooligosaccharides by almond β-glucosidase

Steady-state kinetic parameters for the hydrolysis of cellooligosaccharides by almond beta-glucosidase were evaluated at pH 5.0 and 25 degrees C in relation to the subsite theory (K. Hiromi, Biochem. Biophys. Res. Commun., 40, 1-6, 1970). The value of k0/Km decreased monotonously with increasing degree of polymerization (DP) of the substrates (DP = 2-6). Also, the Km and k0 values for cellotriose were smaller than those for cellobiose. These DP dependencies differ from those of most amylases and glucosidases studied so far, to which the subsite theory has been successfully applied. The subsite parameters could not be consistently obtained, which suggests that one or both of the two basic assumptions of the subsite theory might not be applicable to the hydrolysis of cellooligosaccharides by the enzyme. That is, the intrinsic rate of the hydrolysis may depend on the DP and/or there may be interaction between subsites for binding the glucose residues of a substrate.

Simple method of genomic DNA extraction from Rhizobia in dried nodules of Phaseolus vulgaris for strain differentiation by PCR-based DNA fingerprinting

Abstract Repetitive DNA peR fingerprinting of bacterial genomic DNA is a useful tool for typing and differentiation of rhizobial strains. The method was reported to be suitable for strain differentiation of Rhizobia present in individual root nodules of some leguminous plants without the need for isolation and cultivation of the strains, in which rhizobial genomic DNA was extracted directly from each fresh or frozen nodule. We developed a new protocol of rhizobial genomic DNA extraction/purification from dried nodules of Phaseolus vulgaris for generating repetitive DNA peR fingerprints of Rhizobia present in the nodules. The simplified protocol consists of only three major steps, heat extraction of genomic DNA from rhizobial cells prepared from dried nodules, ethanol precipitation of the DNA and Sephadex G-50 column purification of the DNA, and generated fingerprints with good quality for differentiation of Rhizobia strains. The protocol will be useful to examine the nodule occupancy of inoculated rhizobial strains in field experiments.

Enhanced Growth and nodule occupancy of red kidney bean and soybean inoculated with soil aggregate-based inoculant

Abstract Volcanic ash soil, which is widely distributed in Japan, contains a large amount of well-structured soil aggregates. By using these aggregates as carrier materials, we prepared (brady)rhizobial inoculants for red kidney bean (Phaseolus vulgaris) and soybean (Glycine max). Autoclaved soil aggregates were inoculated with Rhizobium tropici CIATS99R or Bradyrhizobium japonicum USDA110R, incubated for 15 or 21 d at 30°C, slowly air-dried at 20°C to prepare the aggregate-based inoculants, and stored at various temperatures. The populations of CIATS99R and USDA110R in the aggregate-based inoculants were maintained during several months of storage at 20°C. When the aggregate-based inoculants were mixed with soil, CIATS99R and USDA110R cells showed a remarkably improved survival in soils compared with those mixed with soil without carrier material. The effect of the aggregate-based inoculants on the growth of red kidney bean and soybean was examined in pot experiments. By placing a small amount of the inoculant just beneath the seeds at the time of sowing, plant growth was significantly enhanced compared with the use of traditional peat-based inoculant. In addition, nodule formation on the upper part of soybean roots and nodule occupancy by the inoculated strain were remarkably enhanced by the aggregate-based inoculant. It is suggested that soil aggregates might be suitable carrier materials for preparing cheap and effective (brady)rhizobial inoculants.

Distribution of a bacterium (γ-1,2,3,4,5,6-hexachlorocyclohexane-decomposing Sphingomonas paucimobilis) among soil aggregates

Abstract A great variety of microorganisms lives in soil with a heterogeneous structure. Identification and characterization of microhabitats for survival of individual soil microorganisms are important not only for gaining a better understanding of soil microbial ecology, but also for controling the biological functions of soil such as increase in nitrogen fixation, prevention of soil-borne diseases, and microbial degradation of hazardous waste materials.

Free radical injury in rice leaf under Zn deficiency

Abstract Under Zn deficiency, the Cu/Zn-SOD protein content and activity are reduced in rice meristem and the Zn-deficient rice plant is prone to photo-oxidative stress. In the present study, the generation and effect of free radical in rice leaf were examined under Zn-deficient conditions. The SOD activity was depressed and the content of thiobarbituric acid-reactive substances, an index of lipid peroxidation, increased in the rice leaves grown under Zn-deficient conditions. The amplitude of the ESR signal increased in the Zn-deficient leaves. When the oxidative stress level increased by paraquat treatment, the bleaching of the pigment associated with the destruction of chlorophyll in leaves was observed in Zn-deficient leaves. The increase in the amplitude of the ESR signal and the bleaching of pigment by paraquat treatment occurred before any Zn deficiency symptoms appeared in the plants cultured without Zn. Among the Zn nutrition problems of rice plants, one predominant factor which causes Zn deficiency symptoms may be the failure of O2 - scavenging due to the shortage of Cu/ZnSOD. Therefore, when the oxidative stress level is likely to be high, the Zn level has to be sufficient in rice plants.

Improved survival of nutrient-starved cells of Rhizobium tropici CIAT899 in acid soil associated with high Al3+ and Mn2+ contents

Abstract The effect of nutrient starvation in Rhizobium tropici strain CIAT899 on stress resistance to Al3+ and Mn2+ and on survival in acid soil associated with high Al3+ and Mn2+ contents was examined. In the liquid culture medium, after starvation by the exhaustion of carbon, nitrogen, or phosphorus nutrients, CIAT899 survived at least 5 months with a slight loss of viability, indicating its ability to survive under starvation conditions. Upon re-suspension into a low phosphorus medium containing a high concentration of Al3+ (50 µM) or Mn2+ (500 µM), the carbon-starved cells showed an increased tolerance to Al3+ and Mn2+ compared with the non-starved cells. When inoculated to acid soils associated with high Al3+ and Mn2+ contents, the carbon-starved cells of CIAT899 showed an enhanced survival ability compared with the non-starved cells. These results suggest the advantage of the use of carbon-starved cells of Rhizobium as an inoculum to leguminous plants, especially in acid soil associated with high Al3+ and Mn2+ contents, in order to increase the nodulation by the inoculated strain through enhanced survival in soil.