Hiroshi Tohoyama

Different characteristics of roots in the cadmium-tolerance and Cd-binding complex formation between mono- and dicotyledonous plants

Effects of Cd2+ on growth and Cd-binding complex formation in roots were examined with various seedlings of mono- and dicotyledonous plants. Maize, oat, barley and rice exhibited the greater tolerance to Cd2+ (100 μM) than did azuki bean, cucumber, lettuce, pea, radish, sesame and tomato (10–30 μM). Azuki bean was the most sensitive to Cd2+ (<10 μM). Under these Cd-treatments, cereal roots accumulated Cd2+ in the cytoplasmic fractions and transported Cd2+ into the same fractions of shoot tissues, to larger extents than did dicotyledonous roots. Cereal roots synthesized a Cd-binding complex containing phytochelatins in the cytoplasmic fractions, depending upon Cd2+ concentrations applied (30–100 μM). Such a complex was not detected from the same fractions of dicotyledonous roots treated with Cd2+. These results suggest that the Cd-binding complex formation has an important role in the tolerance of cereal roots against Cd2+.

Nickel resistance mechanisms in yeasts and other fungi

SummaryThis review describes nickel toxicity and nickel resistance mechanisms in fungi. Nickel toxicity in fungi is influenced by environmental factors such as pH, temperature and the existence of organic matter and other ions. We describe resistance mechanisms in nickel-resistant mutants of yeasts and filamentous fungi which were obtained by exposure to a mutagen or by successive culture in media containing increasing concentrations of nickel ion. Nickel resistance may involve: (1) inactivation of nickel toxicity by the production of extracellular nickel-chelating substances such as glutathione; (2) reduced nickel accumulation, probably by modification of a magnesium transport system; (3) sequestration of nickel into a vacuole associated with free histidine and involving Ni-insensitivity of vacuolar membrane H+-ATPase.

Changes in the levels of phytochelatins and related metal-binding peptides in chickpea seedlings exposed to arsenic and different heavy metal ions

Phytochelatin-related peptides were analyzed in chickpea plants exposed to six different heavy-metal ions. Cadmium and arsenic stimulated phytochelatin and homophytochelatin synthesis in roots but other metals did not. These metals, however, caused an overall increase in the precursors, glutathione, homoglutathione and cysteine. These changes may be different biochemical indexes for heavy-metal contamination.

The cadmium-resistant gene, CAD2, which is a mutated putative copper-transporter gene (PCA1), controls the intracellular cadmium-level in the yeast S. cerevisiae

Abstract Yeast cells carrying the CAD2 gene exhibit a resistance to cadmium. We cloned this gene and demonstrated that it was a mutated form derived from the gene of a putative copper-transporting ATPase (PCA1). By site-directed mutagenesis, it appeared that the mutation conferring cadmium resistance was a R970G-substitution in the C-terminal region of Pca1 protein. The intracellular cadmium level of cells carrying CAD2 was lower than that of cells carrying either PCA1 or Δcad2. Furthermore, cells with overexpression of CAD2 showed a much lower intracellular cadmium level than that of cells with a single-copy CAD2. From these results, we conclude that the Cad2 protein controls the intracellular cadmium level through an enhanced cadmium efflux system.

Production of metallothionein in copper- and cadmium-resistant strains ofSaccharomyces cerevisiae

SummaryCertain mutants of the yeastSaccharomyces cerevisiae show copper or cadmium resistance. Both copper- and cadmium-resistant strains produce the same metallothionein with 53 amino acid residues which causes metal detoxification by chelating copper or cadmium. The metal detoxification role is the only known function of the metallothionein in yeast. The MT is encoded by theCUP1 gene on chromosome VIII which is expressed by induction with metals. TheCUP1 is amplified to 3–14 copies with 2 kb-tandem-repeat units in the metal-resistant strains, whereas the wild-type strain contains only a single copy of theCUP1. Although transcription ofCUP1 is inducible by metals, the ACE1 protein serves a dual function as a sensor for copper and an inducer forCUP1 transcription in the copper-resistant strain. In the cadmium-resistant strain, the heat-shock factor having a point mutation may be the regulator forCUP1 transcription. Therefore, it has been clarified that production of MT in yeast in controlled by two systems, the amplification ofCUP1 and its transcriptional regulation.

Cadmium-binding protein in a cadmium-resistant strain of Saccharomyces cerevisiae

A Cd-binding protein in the Cd2+-resistant strain 301N of Saccharomyces cerevisiae was induced by administration to 0.5 mM CdSO4. The protein was purified by a gel-permeation and subsequent ion-exchange column chromatographies. The purified Cd-binding protein had the characteristics of metallothioneins: (1) low molecular weight (9.0 kDa), (2) high Cd content (63 micrograms/mg protein), (3) amino-acid composition rich in cysteine (18%), basic and acidic amino acids and free from aromatic amino acids, and (4) an absorption shoulder at near 250 nm. Acid pH or EDTA treatments abolished 250 nm absorption of the Cd-binding protein, and the formed apoprotein was capable of binding Cd2+, Cu2+ and Zn2+, respectively. Heat treatment (75 degrees C) little affected the ultraviolet absorption or sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of Cd-binding protein. These results suggest that metallothionein generally found in animals also occurs in Cd-adapted yeast cells and thus has a role in its Cd-resistance.

A possible role of histidine in a nickel resistant mechanism of Saccharomyces cerevisiae

When a nickel resistant strain N08 of S. cerevisiae was grown in a Ni-supplemented medium, approximately 70% of the nickel is distributed in the soluble fraction. The soluble fraction was chromatographed on Sephadex G-10 and the fraction contained both nickel and large amounts of histidine. When cells were grown in medium containing various combinations of nickel and magnesium and which exhibited approximately 50% growth inhibition, a molar ratio of intracellular histidine and nickel contents remained constant at 1.2-1.4, indicating that the increase in histidine content is correlated with nickel accumulation. The wild type strain 0605-S6, however, exhibits no increase in histidine content when grown in a Ni-supplemented medium, and, therefore, a nickel-resistant mechanism of yeast appears to be the formation of histidine-nickel complexes.

The gene for cadmium metallothionein from a cadmium-resistant yeast appears to be identical to CUP 1 in a copper-resistant strain

SummaryA cadmium-resistant strain of Saccharomyces cerevisiae produces a cadmium metallothionein with the same characteristics as the copper metallothionein that is encoded by CUP 1 in a copper-resistant strain. The structural gene for metallothionein from the cadmium-resistant strain resembles CUP 1 in terms of the fragmentation patterns generated by restriction enzymes. Furthermore, the gene may be amplified as 2.0 kb repeating units in both the cadmium-resistant and the copperresistant strains. However, transformants with a plasmid that carried the metallothionein gene from the cadmiumresistant strain were resistant to copper but not to cadmium. It appears that the same metallothionein gene, CUP 1, is amplified in both cadmium- and copper-resistant yeasts. However, the mechanism for the cadmiumspecific inducibility of the gene may be restricted to the cadmium-resistant strain.

Resistance to cadmium is under the control of the CAD2 gene in the yeast Saccharomyces cerevisiae

SummaryA cadmium-resistant strain, X3382-3A, which is able to grow in a medium containing 0.2 mM cadmium sulfate, was picked out from our laboratory stock strains of Saccharomyces cerevisiae. The cadmium resistance of this strain is controlled by a single dominant nuclear gene, denoted as CAD2. The locus of CAD2 was mapped by gene linkage to a site 15.5 centimorgans to the right of the his7 locus on the right arm of chromosome II. The cadmium resistance of the strain carrying CAD2 was evaluated for its properties of cadmium uptake, cadmium distribution and cadmium-metallothionein formation, in comparison with those of some other strains. The results suggest that the novel type of cadmium resistance controlled by CAD2 does not involve production of a cadmiumm-metallothionein.

Contributions of cell wall and metal-binding peptide to Cd- and Cu-tolerances in suspension-cultured cells of tomato

Possible roles of cell wall and cytoplasmic peptides in the tolerance of cells to Cu2+ and Cd2+ ions were studied in suspension-cultured cells of tomato (Lycopersicon esculentum L. cv. Palace). Cu2+ and Cd2+ ions inhibited growth of wild type cells at concentrations more than 100 and 200 μM, respectively. Tomato cells readily developed tolerance to Cd2+ ions up to 1 mM but not to Cu2+ ions, after repeated subculturings in the presence of the respective ions. Such a metal-specific adaptation of cells was not due to the difference in the total uptakes between Cd2+ and Cu2+ ions by cells. Wild-type cells accumulated Cd2+ preferentially into the cytoplasmic peptide fraction and Cu2+ into the cell-wall fraction, when grown under the subtoxic metal conditions. Under excess metal conditions, Cd-tolerant cells produced greater amounts of Cd-binding peptides in the cytoplasm and retained lesser amounts of Cd2+ ions in the cell wall than did wild-type cells. In contrast, tomato cells grown in the presence of Cu2+ ions synthesized no detectable amounts of Cu-binding peptides in the cytoplasm and retained most of the Cu2+ in the cell-wall fraction, irrespective of cell lines.These results suggested that the cytoplasmic peptides rather than cell wall properties have a primary role in the response of tomato cells to excess metal environments.