Tatsuhiko Kataoka

Genes encoding proteins of the cation diffusion facilitator family that confer manganese tolerance.

The yeast Saccharomyces cerevisiae expressing a cDNA library prepared from Stylosanthes hamata was screened for enhanced Mn2+ tolerance. From this screen, we identified four related cDNAs that encode membrane-bound proteins of the cation diffusion facilitator (CDF) family. One of these cDNAs (ShMTP1) was investigated in detail and found to confer Mn2+ tolerance to yeast by internal sequestration rather than by efflux of Mn2+. Expression of ShMTP1 in a range of yeast mutants suggested that it functions as a proton:Mn2+ antiporter on the membrane of an internal organelle. Similarly, when expressed in Arabidopsis, ShMTP1 conferred Mn2+ tolerance through internal sequestration. The ShMTP1 protein fused to green fluorescent protein was localized to the tonoplast of Arabidopsis cells but appeared to localize to the endoplasmic reticulum of yeast. We suggest that the ShMTP1 proteins are members of the CDF family involved in conferring Mn2+ tolerance and that at least one of these proteins (ShMTP1) confers tolerance by sequestering Mn2+ into internal organelles.

Highly sensitive analytical method for aluminum movement in soybean root through lumogallion staining

We present highly sensitive aluminum detection method in root using fluorescent lumogallion. Roots treated with 100 μM AlCl3 including 0.2 mM CaCl2 (pH 4.5) were stained for 60 min with 10 μM lumogallion fluorescence solution and fluorescence from aluminum complex in root was observed under confocal laser microscope. There was a good correlation between the intensity of fluorescence and aluminum content. When the amount of aluminum lost during each step in staining process was measured, it was found that about 10% of aluminum was lost only at staining stage. Through lumogallion staining method, aluminum accumulation especially at an early stage of aluminum treatment in root was shown. At the beginning (2 hr), aluminum began to be accumulated in root cap. After 4 hr treatment, the aluminum distribution was spread to about 3 mm from root apex in the root cap and outer cortex. When aluminum was found in the outer cortex in 3–5 mm from the root apex, the viability was tended to be decreased in the same area (6 hr). At the same time, aluminum amount in meristem was increased. However the comparison of lumogallion staining method with that of morin, which has been widely used to detect aluminum in root, the sensitivity of lumogallion method was found to be much higher.

Strategies to isolate transporters that facilitate organic anion efflux from plant roots

The efflux of organic anions from roots plays an important role in plant nutrition. The release of simple carboxylic anions such as citrate, malate and oxalate have been implicated in mechanisms of aluminium (Al) tolerance and improved acquisition of soil phosphorus. These metabolites are likely to cross cell membranes as multivalent anions and recent evidence indicates that anion-permeable channels facilitate this flow in the Al-dependent efflux of malate and citrate from wheat and maize, respectively. However, the genes encoding these anion channels, or any other protein that facilitates the release of citrate, malate or oxalate have not been isolated. This is an obstacle for the application of biotechnology to combat Al toxicity and to improve P-acquisition efficiency in plants. We discuss several strategies aimed at isolating genes that facilitate organic anion release from plant roots.

Aluminium distribution in soybean root tip for a short time Al treatment

Summary Using the lumogallion staining method which we developed (Kataoka et al. 1997a), Al movement in soybean ( Glycine max. (L.) Merr. cv. Tsurunoko) root tips treated for a short time was studied. We have indicated that the majority of Al accumulated in the root was found between 0 and 2 mm from the root apex within 2 h (Kataoka et al. 1997a, b). In the study presented here two-day seedlings of the soybean were treated with 50 μmol/L AlCl 3 (pH 4.4), including 0.2 mmol/L CaCl 2 , for 1 h, and Al accumulation in the root sections at both 1 and 2 mm apart from root apex was analyzed by a confocal laser microscopy. Although the early indicators, callose induction and the decrease of growth recovery, were not observed in the root when treated for 15 min, a trace amount of Al was already incorporated into the nucleus of cells and the middle tissue of the root. The non-toxic level of Al was more rapidly absorbed than previously thought. The initial increase of callose accumulation and the reduction of the growth recovery were found after 30 min. Therefore, the difference between Al accumulation profiles of 15 and 30 min was analyzed to find out what triggered a toxic Al effect. Increase of Al accumulation in whole root tissue was observed in the root sections, at both 1 and 2 mm from the root apex, and the greatest amount of Al was found in the cytoplasm of the outer cortex, 1 mm away from the root apex. These results are consistent with the fact that Al exclusion from root tip cells is an important mechanism of Al tolerance.

The decrease of extracted apoplast protein in soybean root tip by aluminium treatment

Aluminium effect on the mobility of apoplast protein in root tips was studied. Two-day seedlings of soybean (Glycine max. (L.) Merr. cv. Tsurunoko) were treated with 50 μM AlCl3 for 2 h. Using infiltration method, the apoplast protein in root tips was extracted with 20 or 100 mM MgCl2. When 20 mM MgCl2 was used to collect weakly bound protein to apoplast, the amount of protein extracted was reduced to be about 20 % compared with that of control and the band of 97 kDa disappeared in SDS-PAGE gel. However, the 97 kDa protein could be extracted by 100 mM MgCl2, which were used for extraction of more tightly bound protein to apoplast, and the amount was estimated to be the same as that of control. When the protein was further developed in two-dimensional electrophoresis, three spots were found between pI 6.4 and 6.5. This is the first report of an Al effect on the mobility of apoplast protein.

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.

A Study of Nutrient Recycling in a Plant - Soil System Using NAA

To understand how nutrients are recycled in a particular soil - plant system, we have analyzed the kinetics of the elements Na, Mg, K, Ca, Mn, and Br throughout the life cycle of the plant. The plant sample was collected at 6 different stages of its growth. The effect of V (100 µM) on the element recycling system was also analyzed. The amount of the elements in each tissue of the plant, root, stem, petiole, leaf, and seed, were then determined by neutron activation analysis. The total elemental uptake rapidly increased when the plant developed from the juvenile to the adult phase. More than half of the Mg and Mn in cotyledon was transported to the younger leaves before the cotyledon was shed. From the pattern of movement of each element in plant tissue we have constructed an element recycling model in the soil-plant system. Based on the model, it was found that the amount of K to be a limiting factor for the recycling activity in the system.

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.

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.