Hiroshi Iikura

Overproduction of recombinant Trichoderma reesei cellulases by Aspergillus oryzae and their enzymatic properties

We have established an expression system of Trichoderma reesei cellulase genes using Aspergillus oryzae as a host. In this system, the expression of T. reesei cellulase genes were regulated under the control of A. oryzae Taka-amylase promoter and the cellulase genes were highly expressed when maltose was used as a main carbon source for inducer. The production of recombinant cellulases by A. oryzae transformants reached a maximum after 3-4 days of cultivation. In some cases, proteolysis of recombinant cellulases was observed in the late stage of cultivation. The recombinant cellulases were purified and characterized. The apparent molecular weights of recombinant cellulases were more or less larger than those of native enzymes. The optimal temperatures and pHs of recombinant cellulases were 50-70 degrees C and 4-5, respectively. Among the recombinant cellulases, endoglucanase I showed broad substrate specificities and high activity when compared with the other cellulases investigated here.

Aluminum distribution and viability of plant root and cultured cells

Viability and growth recovery in response to aluminum (Al) toxicity for tobacco (Nicotina tabacum L.) BY-2 cells and soybean (Glycine max (L.) Merr. cv. Tsurunoko) roots were investigated. When tobacco BY-2 cells were treated with 100 μM AlCl3 at pH 5.0 for 15 hours, Al accumulation was observed in the cell further into nucleus. However, there was no decrease in viability and growth recovery compared with those of control cells. In the case of soybean roots, Al accumulation was observed in root cap, epidermis and outer cortex after treatment with 200 μM AlCl3 (pH 4.5) solution containing 0.2 mM CaCl2 for 2 hours through confocal laser microscopic observation. After 4 hours, Al accumulation was found in the outer cortex of the elongation zone, in which Al remained even after washing with citrate. Though no change was found in viability and growth recovery until 2 hours of treatment, after 4 hours, both viability and the growth recovery decreased drastically. The Al in cell walls after 4 hours of treatment suggested that Al forms strong bonds in cortex cells, which this resulted in the destruction of the epidermis and cortex by inhibiting the elongation of each cell. The sensitivity of Al staining method was studied further using colorimetric reagents, aluminon, hematoxylin and pyrocatechol violet, as well as fluorescence ones, lumogallion and morin. Among the reagents investigated, lumogallion and morin showed higher sensitivity in cultured cells for an optical microscopic observation. However, in the case of the confocal laser microscope, which is suitable to observe Al distribution in intact roots, the lumogallion staining method showed much higher sensitivity than that of morin.

Analysis by Prompt Gamma-Ray Method with Cold Neutrons of Boron and Other Elements in Soybean

We have employed prompt gamma-ray analysis (PGA) with cold neutrons for the analysis of boron (B) in soybean (Glycine max). Primary, first and second leaves, as well as root samples were dried, prepared as thin disks and irradiated with cold neutrons. Prompt gamma-rays emitted from the sample was measured by Ge and bismuth germanate (BGO) detectors. Boron content in root was less than 40% of that in leaves. The same sample was used to determine Al, K, Ca, and Zn content by neutron activation analysis. When two soybean cultivars, Al susceptible (Chief) and Al tolerant (Perry) were compared, the similar B distribution was observed, except in primary leaf, where B concentration in former plant was about two times higher than that of the latter.

Influence of Mercury on Soybean Plants (Glycine max L.) at Low pH

The influence of Hg on soybean plants under different pH conditions and Hg concentrations was studied. Growth inhibition by Hg was higher in roots than the upper part of the plant, but was highly dependant on pH condition. Growth inhibition of roots was observed when Hg concentration was higher than 1 mg Hg L−1 for pH 4.0 and 5 mg Hg L−1 for pH 6.0. Using 203Hg as a radioactive tracer, the amount of Hg (1 mg Hg L−1) uptake in root was found to be about 1.5 times higher at pH 4.0 than that at pH 6.0; suggesting that Hg when highly accumulated at the lower pH induced inhibition of root growth. Decreased amounts of Hg due to evaporation during the plant growth were very low, but were higher at pH 6.0 than that at pH 4.0. There was hardly any translocation of Hg from roots to the upper parts through the stem within 24 h.

Boron analysis at different stages of the cell cycle in cultured tobacco cells

The paper presents the data suggesting that boron (B) content in the cultured tobacco (Nicotiana tabacum) cells change during the stage of cell cycle. Cultured cell line, tobacco BY-2, was synchronized by treatments with aphidicolin and propyzamide. When the cells started to grow, cells were periodically collected at each stage of the cell cycle. To measure the B content, cells were dried and prepared as tablets and prompt gamma-ray analysis (PGA) with a cold neutron beam was performed. Flow cytometry (FCM) was carried out on the same cell fraction to analyze the cell cycle in more detail. There was a clear difference in B content with respect to the cell cycle stage. Boron content in the cells at G2+M phase was found to be about half of that at G0/G1.