Susumu Nakashima

Mechanism of Gene Expression of Arabidopsis Glutathione S -Transferase, AtGST1 , and AtGST11 in Response to Aluminum Stress

The gene expression of two Al-induced Arabidopsis glutathione S-transferase genes, AtGST1 and AtGST11, was analyzed to investigate the mechanism underlying the response to Al stress. An approximately 1-kb DNA fragment of the 5′-upstream region of each gene was fused to a β-glucuronidase (GUS) reporter gene (pAtGST1::GUS and pAtGST11::GUS) and introduced into Arabidopsis ecotype Landsberg erecta. The constructed transgenic lines showed a time-dependent gene expression to a different degree in the root and/or leaf by Al stress. The pAtGST1::GUS gene was induced after a short Al treatment (maximum expression after a 2-h exposure), while the pAtGST11::GUS gene was induced by a longer Al treatment (approximately 8 h for maximum expression). Since the gene expression was observed in the leaf when only the root was exposed to Al stress, a signaling system between the root and shoot was suggested in Al stress. A GUS staining experiment using an adult transgenic line carrying the pAtGST11::GUS gene supported this suggestion. Furthermore, Al treatment simultaneously with various Ca depleted conditions in root region enhanced the gene expression of the pAtGST11::GUS in the shoot region. This result suggested that the degree of Al toxicity in the root reflects the gene response of pAtGST11::GUS in the shoot via the deduced signaling system. Both transgenic lines also showed an increase of GUS activity after cold stress, heat stress, metal toxicity, and oxidative damages, suggesting a common induction mechanism in response to the tested stresses including Al stress.

Mercurial-sensitive water transport in barley roots

An isolated barley root was partitioned into the apical and basal part across the partition wall of the double-chamber osmometer. Transroot water movement was induced by subjecting the apical part to a sorbitol solution, while the basal part with the cut end was in artificial pond water. The rate of transroot osmosis was first low but enhanced by two means, infilitration of roots by pressurization and repetition of osmosis. Both effects acted additively. The radial hydraulic conductivity (Lpr) was calculated by dividing the initial flow rate with the surface area of the apical part of the root, to which sorbitol was applied, and the osmotic gradient between the apical and basal part of the root. Lpr which was first 0.02–0.04 pm s−1 Pa−1 increased up to 0.25–0.4 pm s−1 Pa−1 after enhancement. Enhancement is assumed to be caused by an increase of the area of the plasma membrane which is avallable to osmotic water movement. The increased Lpr is in the same order of magnitude as the hydraulic conductivity (Lp) of epidermal and cortical cells of barley roots obtained by Steudie and Jeschke (1983). HgCl2, a potent inhibitor of water channels, suppressed Lpr of non-infiltrated and infiltrated roots down to 17% and 8% of control values, respectively. A high sensitivity of Lpr to HgCl2 suggests that water channels constitute the most conductive pathway for osmotic radial water movement in barley roots.

A Novel Histidine-Rich CPx-ATPase from the Filamentous Cyanobacterium Oscillatoria brevis Related to Multiple-Heavy-Metal Cotolerance

A novel gene related to heavy-metal transport was cloned and identified from the filamentous cyanobacterium Oscillatoria brevis. Sequence analysis of the gene (the Bxa1 gene) showed that its product possessed high homology with heavy-metal transport CPx-ATPases. The CPC motif, which is proposed to form putative cation transduction channel, was found in the sixth transmembrane helix. However, instead of the CXXC motif that is present in the N termini of most metal transport CPx-ATPases, Bxa1 contains a unique Cys-Cys (CC) sequence element and histidine-rich motifs as a putative metal binding site. Northern blotting and real-time quantitative reverse transcription-PCR showed that expression of Bxa1 mRNA was induced in vivo by both monovalent (Cu(+) and Ag(+)) and divalent (Zn(2+) and Cd(2+)) heavy-metal ions at similar levels. Experiments on heavy-metal tolerance in Escherichia coli with recombinant Bxa1 demonstrated that Bxa1 conferred resistance to both monovalent and divalent heavy metals. This is the first report of a CPx-ATPase responsive to both monovalent and divalent heavy metals.

Metal-tolerant moss Scopelophila cataractae accumulates copper in the cell wall pectin of the protonema.

The growth kinetics in the presence of copper (Cu) of the protonema of the moss Scopelophila cataractae and the matrix polysaccharides of its cell walls have been analyzed in this study. Protonemal cells cultured in a medium containing 0.2mM CuSO(4) showed a rapid accumulation of Cu, reaching a maximum between 30 and 60d at approximately 65 micromolg(-1) DW. Uronic acids were found in similar amounts in cell walls of both control and Cu-treated cells, whereas arabinose and galactose decreased to 61-67% in the presence of Cu. Cell wall polysaccharides were determined after successive extraction with 50mM CDTA, 50mM Na(2)CO(3), 1M KOH, and 4M KOH. The pectic fractions (CDTA- and Na(2)CO(3)-soluble) decreased to 47% and the hemicellulosic fractions (1M KOH- and 4M KOH-soluble) to 86% under Cu application. Approximately 43% of the Cu taken into cell walls was released following endo-pectate lyase treatment, suggesting that two-fifths of the total Cu accumulation was tightly bound to the homogalacturonan of the cell wall pectin.

Determination of ultramicro amounts of selenium by gas chromatography

4-Chloro-o-phenylenediamine reacts with selenous acid at pH 0-1 to form 5-chloropiaselenol, which is then extracted into 1 ml of toluene. The toluene layer is injected into an electron-capture detector gas Chromatograph. From the peak height selenium is determined, the minimum amount detectable being about 0.04 mug.

A metallothionein and CPx-ATPase handle heavy-metal tolerance in the filamentous cyanobacterium Oscillatoria brevis

A metallothionein (BmtA) and a CPx‐ATPase (Bxa1) have been identified and characterized from the cyanobacterium Oscillatoria brevis. Both bmtA and bxa1 expression can be markedly induced in vivo by Zn2+ or Cd2+. Over‐expression of bmtA or bxa1 in Escherichia coli enhances Zn2+ and Cd2+ tolerance in the transformant. Dynamic studies on the expression of two genes showed that the maximum expression of bxa1 induced by Zn2+ and Cd2+ was much quicker than that of bmtA, suggesting distinct physiological roles of metallothionein and CPx‐ATPase in the handling of surplus metal.

Spectrophotometric determination of nitrite in natural waters by flow injection analysis

Abstract The method described is suitable for the determination of low μg l−1 levels of nitrite in waters. Nitrite diazotises p-aminoacetophenone and the product is coupled with m-phenylenediamine at 30°C. The limit of detection is 0.2 μg l−1 for sample injections of 650 μl. The sampling rate is about 30 h−1 and the relative standard deviation is ≦ 1.3%.

Determination of sulphate in natural water by flow-injection analysis

ZusammenfassungAls Reagens dient Dimethylsulfonazo-III. Die Störung durch Calcium bei dieser Bestimmung wird durch eine Kationenaustauschersäule eliminiert, die direkt nach dem Probeinjektions-Ventil angeordnet ist. Zur Erhöhung der Empfindlichkeit und Reproduzierbarkeit ist es notwendig, die Trägerlösung mit Bariumsulfat zu sättigen sowie bei Nichtbenutzung des Systems den Reaktionsteil mit Ethanol-Wasser (1∶1) zu füllen. Mit dem so modifizierten Verfahren ergaben sich Standardabweichungen von 0,94–1,2% für 6–10 mg/l Sulfat, die Nachweisgrenze liegt bei 0,2 mg/l. Die Eichkurve ist bis 14 mg/l linear. Mg2+, NH4+, Na+, K+, Cl−, NO3−, PO43−, HCO3−und SiO32−stören in den normalerweise vorkommenden Konzentrationen nicht.SummarySulphate was determined in natural water samples by flow-injection analysis using dimethylsulphonazo-III as reagent. The interference by Ca was eliminated by a cation-exchanger column inserted directly after the sample injection valve. In order to ensure high sensitivity and reproducibility it was necessary to saturate the carrier solution with barium sulphate and to fill the reaction coil with ethanol-water (1∶1) when not in use. Standard deviations were 0.94–1.2% for 6–10mg/l sulphate. The limit of detection was about 0.2mg/l. The calibration graph was linear up to 14mg/l Mg2+, NH4+, Na+, K+, Cl−, NO3−, PO43−, HCO3−and SiO32−did not interfere in the normally occurring concentrations.

The separation of tellurium(IV) from water and sea water by flotation with hydrated iron(III) oxide

Abstract A flotation separation is described for sub-microgram levels of tellurium(IV) from 1-1 samples of water and sea water. Tellurium(IV) is coprecipitated with hydrated iron(III) oxide at pH 8–9. The precipitate is floated with the aid of surfactant solutions and small nitrogen bubbles, separated and dissolved in dilute hydrochloric acid. Tellurium is then converted to hydrogen telluride with sodium tetrahydroborate and measured by atomic absorption spectrometry. Recovery of added tellurium (0.4 and 0.8 μg l −1 ) was about 83%. The time required for the preconcentration is 30 rain per sample, including 15 rain stirring.

Simultaneous Preconcentration of Copper, Nickel and Cobalt In Water By Coprecipitation-Flotation For Electrothermal Atomic Absorption Spectrometry

Abstract A method is described for the simultaneous flotation separation and determination of μg-levels of copper (II), nickel (II) and cobalt (II) in water. Copper, nickel and cobalt in a 1000-ml sample of water are coprecipitated with hydrated zirconium oxide at pH 9.1±0.1. The precipitate is floated with the aid of a surfactant solution and small air bubbles, separated and dissolved in dilute hydrochloric acid. The contents of these elements are determined by electrothermal atomic absorption spectrophotometry. The method is applied to the determination of low μg/1 levels of copper, nickel and cobalt in fresh water.