Nobushige Nakazawa

OsHMA3, a P1B‐type of ATPase affects root‐to‐shoot cadmium translocation in rice by mediating efflux into vacuoles

• The cadmium (Cd) over-accumulating rice (Oryza sativa) cv Cho-Ko-Koku was previously shown to have an enhanced rate of root-to-shoot Cd translocation. This trait is controlled by a single recessive allele located at qCdT7. • In this study, using positional cloning and transgenic strategies, heavy metal ATPase 3 (OsHMA3) was identified as the gene that controls root-to-shoot Cd translocation rates. The subcellular localization and Cd-transporting activity of the gene products were also investigated. • The allele of OsHMA3 that confers high root-to-shoot Cd translocation rates (OsHMA3mc) encodes a defective P(1B) -ATPase transporter. OsHMA3 fused to green fluorescent protein was localized to vacuolar membranes in plants and yeast. An OsHMA3 transgene complemented Cd sensitivity in a yeast mutant that lacks the ability to transport Cd into vacuoles. By contrast, OsHMA3mc did not complement the Cd sensitivity of this yeast mutant, indicating that the OsHMA3mc transport function was lost. • We propose that the root cell cytoplasm of Cd-overaccumulating rice plants has more Cd available for loading into the xylem as a result of the lack of OsHMA3-mediated transportation of Cd to the vacuoles. This defect results in Cd translocation to the shoots in higher concentrations. These data demonstrate the importance of vacuolar sequestration for Cd accumulation in rice.

Mutations in Rice (Oryza sativa) Heavy Metal ATPase 2 (OsHMA2) Restrict the Translocation of Zinc and Cadmium

Widespread soil contamination with heavy metals has fostered the need for plant breeders to develop new crops that do not accumulate heavy metals. Metal-transporting transmembrane proteins that transport heavy metals across the plant plasma membrane are key targets for developing these new crops. Oryza sativa heavy metal ATPase 3 (OsHMA3) is known to be a useful gene for limiting cadmium (Cd) accumulation in rice. OsHMA2 is a close homolog of OsHMA3, but the function of OsHMA2 is unknown. To gain insight into the function of OsHMA2, we analyzed three Tos17 insertion mutants. The translocation ratios of zinc (Zn) and Cd were clearly lower in all mutants than in the wild type, suggesting that OsHMA2 is a major transporter of Zn and Cd from roots to shoots. By comparing each allele in the OsHMA2 protein structure and measuring the Cd translocation ratio, we identified the C-terminal region as essential for Cd translocation into shoots. Two alleles were identified as good material for breeding rice that does not contain Cd in the grain but does contain some Zn, and that grows normally.

Partial restoration of sporulation defect in sake yeasts, kyokai no. 7 and no. 9, by increased dosage of the IME1 gene

Abstract Sake yeast (Saccharomyces cerevisiae) strains, Kyokai no. 7 and no. 9 sporulate little if at all. To determine the reason for their poor sporulation, we analyzed transcripts of the IME1 and IME2 (inducer of meiosis), and RME1 (regulator of meiosis) genes. Kyokai no. 7 produced transcripts of IME1 and IME2 at low levels and their levels were not induced, but rather repressed, in sporulation conditions. Introduction of the cloned wild-type IME1 gene ligated to a multicopy plasmid into these cells partially restored their sporulation frequency in sporulation conditions. However, on dissection of asci no germination of spores was observed. A double transformant of Kyokai no. 7 with IME1 and IME2 plasmids did not show greater increase in sporulation frequency than a single transformant with the IME1 plasmid. The IME1 transcription level of the IME1 transformant cells of Kyokai no. 7 was the same as that of the wild-type a/ α diploid cells, but the RME1 transcription level of these cells was severely repressed. Their level of IME2 transcription was much lower than that in the wild-type a/ α diploid cells. An IME2-lacZ fusion gene, bearing a 1.1-kbp IME2 promoter region and ligated to a multicopy plasmid was not expressed in Kyokai no. 7 cells or in a/a cells. The IME2-lacZ fusion gene was, however, expressed in these cells by further introduction of a multicopy plasmid bearing the IME1 gene. Thus, we conclude that Kyokai no. 7 might have defects in the IME1 gene and its expression, and weakness in regulatory circuits for starvation signals and/or in the sporulation program.

Efficient selection of hybrids by protoplast fusion using drug resistance markers and reporter genes in Saccharomyces cerevisiae

We have developed a selection system for hybrids by protoplast fusion using dominant selective drug resistance markers, Tn601(903) against geneticin and AUR1-C against aureobasidin A, and reporter genes, ADH1p-PHO5-ADH1t and CLN2p-CYC1-lacZ, in Saccharomyces cerevisiae. To examine the effectiveness of this system, plasmids with each marker and reporter gene were introduced into auxotrophic sake yeasts. From the resulting transformants, eight colonies were screened by protoplast fusion in combination with the drug resistance markers and the reporter genes. Among them, seven strains were judged as hybrids between parental strains by analysis of growth on a minimal medium. This selection system was applied to wine yeasts having no genetic markers. Six strains were regarded as hybrids between parental strains by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis of the MET2 gene and by karyotype analysis using a contour-clamped homogeneous electric field (CHEF). We propose that the plotoplast fusion using dominant selective geneticin- and aureobasidin A-resistance markers and reporter genes is useful for the selection of hybrids from wine yeasts, which are homothallic and have low sporulation ability.

Use of the PDR4 gene as a dominant selective marker in combination with cerulenin for prototrophic strains in Saccharomyces cerevisiae

Abstract We found that a 2.1-kbp KpnI fragment bearing the PDR4 (YAP1/PAR1/SNQ3) gene of Saccharomyces cerevisiae, conferring pleiotropic drug resistance to S. cerevisiae, confers resistance of the cells to 5 μg of cerulenin per ml when ligated into a multicopy vector and to 1 μg of cerulenin ligated into a single copy vector. This 2.1-kbp KpnI fragment lacks the C-terminal coding region of the published nucleotide sequence (Hussain, M. and Lenard, J.: Gene, 101, 149–152, 1991). This dosage-dependent cerulenin resistance induced by the 2.1-kbp KpnI fragment of PDR4 is useful as a dominant selective marker in genetic studies and in transformation of prototrophic S. cerevisiae strains. We constructed a pair of tester strains for mating-type determination by introducing the multicopy plasmid bearing the 2.1-kbp KpnI fragment into a and α haploid strains of S. cerevisiae.

A method for direct selection of mating-competent clones from mating-incompetent industrial strains of Saccharomyces cerevisiae

Abstract Industrial strains of Saccharomyces cerevisiae are generally incompetent for mating with others and hard to sporulate. Physical and functional analyses of Kyokai no. 7, a typical sake yeast, revealed that it has functional a and a mating-type genes and the mating incompetency has the regular non-mater phenotype due to a 2 and a1- a 2 repression. An effective selection method for isolation of mating-competent mutants of this strain was developed using plasmids bearing fused genes, STE6 p- PHO5 and MFa1 p- PHO5 , consisting of the promoter of STE6 , an a-specific gene, and MFa1 , an a -specific gene, respectively, connected to the coding region of PHO5 encoding repressible acid phosphatase. Mating-competent mutants having the a or a mating type were selected by detection of repressible acid phosphatase activity by specific staining of colonies. This method was applied successfully to two other sake yeasts. Hybrid yeast strains superior for sake making were constructed by crossing these mating-competent derivatives.

Mass mating method in combination with G418- and aureobasidin a-resistance markers for efficient selection of hybrids from homothallic strains in Saccharomyces cerevisiae

We have developed a mass mating method using the spore suspensions of homothallic yeasts of Saccharomyces cerevisiae in combination with dominant selective drug resistance markers, Tn601(903) against geneticin and AUR1-C against aureobasidin A for the selection of the hybrids. To examine the effectiveness of these markers in the mass mating method, each marker was introduced into a homothallic wine yeast. Using a mixed culture of spore suspensions from the resultant transformants, many hybrids were screened by the drug resistance markers. This method is more practical than the spore-to-spore mating method because it does not require the use of a micromanipulator and many hybrids are obtained at one time. The resultant hybrids could be utilized for industrial brewing because plasmids, which are used to confer resistance markers, are easily eliminated from the hybrids by cultivation in a medium without drugs. We propose that the mass mating method using spore suspensions in combination with dominant selective geneticin- and aureobasidin A-resistance markers is useful for the selection of hybrids from industrial homothallic yeasts.

Cln3 blocks IME1 transcription and the Ime1-Ume6 interaction to cause the sporulation incompetence in a sake yeast, Kyokai no. 7

Industrial yeasts, including a sake yeast Kyokai no. 7 (K7), are generally unable to sporulate. In K7 (Saccharomyces cerevisiae) cells, IME1 transcription was not induced under sporulation conditions, and K7 cells partially restored sporulation ability when transformed with a multicopy plasmid bearing IME1. However, the mechanisms of sporulation incompetence in industrial yeasts are poorly understood. We demonstrated that the deletion of the G1 cyclin CLN3, a key activator of the cell cycle, allows K7 cells to induce IME1 transcription and sporulate under sporulation conditions. In K7 cells, CLN3 mRNA and protein were not down-regulated despite sporulation conditions. Moreover, using a two-hybrid assay, we found that Ime1-Ume6 interaction was promoted in Cln3-deficient K7 cells. Thus, Cln3 is involved in the mechanism underlying sporulation incompetence by inhibiting IME1 transcription and the Ime1-Ume6 interaction. Based on these findings, we hypothesize that the absence of transmission of nutrient starvation signals to CLN3 leads to sporulation incompetence in K7 cells.

Immunosuppressive drug rapamycin restores sporulation competence in industrial yeasts

Industrial yeasts, including a sake yeast strain Kyokai no. 7 (K7), are generally unable to sporulate. Previously, we have reported that in K7 (Saccharomyces cerevisiae) cells, deletion of the G1 cyclin gene CLN3, a key activator of the cell cycle, allows the cells to induce IME1 transcription and sporulate under sporulation conditions. Here we show that treatment with the immunosuppressive drug rapamycin also restores sporulation competence in K7 cells. Moreover, sporulation was observed after rapamycin treatment in other industrial yeasts, namely bottom fermenting yeast strains and a wine yeast strain, which are not able to sporulate under normal sporulation conditions. These findings suggest that activation of TORC1 under sporulation conditions leads to sporulation incompetence in these yeasts. Thus, rapamycin treatment will be useful to restore sporulation competence in industrial yeasts.

TORC1 activity is partially reduced under nitrogen starvation conditions in sake yeast Kyokai no. 7, Saccharomyces cerevisiae

Industrial yeasts are generally unable to sporulate but treatment with the immunosuppressive drug rapamycin restores this ability in a sake yeast strain Kyokai no. 7 (K7), Saccharomyces cerevisiae. This finding suggests that TORC1 is active under sporulation conditions. Here, using a reporter gene assay, Northern and Western blots, we tried to gain insight into how TORC1 function under nitrogen starvation conditions in K7 cells. Similarly to a laboratory strain, RPS26A transcription was repressed and Npr1 was dephosphorylated in K7 cells, indicative of the expected loss of TORC1 function under nitrogen starvation. The expression of nitrogen catabolite repression-sensitive genes, however, was not induced, the level of Cln3 remained constant, and autophagy was more slowly induced than in a laboratory strain, all suggestive of active TORC1. We conclude that TORC1 activity is partially reduced under nitrogen starvation conditions in K7 cells.