Showing papers by "Kazuki Saito published in 1996"

Subcellular localization of spinach cysteine synthase isoforms and regulation of their gene expression by nitrogen and sulfur.

Abstract: Subcellular localization and regulation of the spinach (Spinacia oleracea) cysteine synthase (O-acetyl-L-serine[thiol]-lyase, EC 4.2.99.8) isoforms (CysA, CysB, and CysC) were determined in transgenic tobacco (Nicotiana tabacum) and in spinach cell cultures. The 5[prime] regions of CysB and CysC encoding the chloroplastic (CysB-TP) and the putative mitochondrial (CysC-TP) transit peptide (TP) sequences were fused to a bacterial [beta]-glucuronidase gene (gus) and expressed in tobacco under the control of the cauliflower mosaic virus 35S promoter. Subcellular fractionation of transgenic tobacco showed transportation of [beta]-glucuronidase proteins to chloroplasts by CysB-TP and to mitochondria by CysC-TP, respectively, indicating that both presequences were sufficient to act specifically as chloroplastic and mitochondrial TPs in vivo. The mRNA expression patterns of CysA (cytoplasmic form), CysB, and CysC genes under nitrogen- and sulfur-starved conditions were characterized in spinach cells cultures. In sulfur-starved cells, only slight differences (approximately 1.2- to 1.5-fold) in the mRNA levels of CysA and CysB were observed during the short-term (0–24 h) cultivation periods compared with cells growth in Murashige-Skoog medium. However, under nitrogen and nitrogen/sulfur double-deficient stress conditions, mRNA levels of CysC increased up to 500% of the original level within 72 h.

Simultaneous Measurements of Liquid Properties Using Multichannel Shear Horizontal Surface Acoustic Wave Microsensor.

Abstract: Surface acoustic waves (SAW) with a shear horizontal polarized displacement (SH-SAW) can be used to determine mechanical and electrical properties of an adjacent liquid. We propose a new multichannel SH-SAW microsensor for liquid characterization. The SH-SAW microsensor, which is fabricated on 36° rotated Y-cut X-propagating LiTaO 3 , consists of three SAW delay lines. The propagating surfaces of two of the delay lines are metallized and electrically shorted, and the other one has a free surface which is an electrically active area. The viscosity, conductivity and permittivity of liquid are obtained simultaneously using the multichannel SH-SAW microsensor.

Tissue-specific activity of two manganese superoxide dismutase promoters in transgenic tobacco

Abstract: In eukaryotes, manganese superoxide dismutase is a nuclear-encoded protein that scavenges superoxide radicals in the mitochondrial matrix. We have isolated two manganese superoxide dismutase genes from Nicotiana plumbaginifolia L. and fused the 5' upstream regulatory region of these genes to the beta-glucuronidase reporter gene. The two gene fusions displayed a differential tissue specificity in transgenic tobacco (Nicotiana tabacum). Promoter activity of the SodA1 gene fusion was found in the pollen, middle layer, and stomium of anthers, but was usually undetectable in vegetative organs of mature plants. The SodA2 gene fusion was expressed in the leaves, stems, roots, and flowers. SodA2 promoter activity was most prominent in the vascular bundles, stomata, axillary buds, pericycle, stomium, and pollen. Histochemical analysis of succinate dehydrogenase activity suggested that the spatial expression of the two gene fusions is generally correlated with mitochondrial respiratory activity.

Transgenic fertile Scoparia dulcis L., a folk medicinal plant, conferred with a herbicide-resistant trait using an Ri binary vector.

Abstract: Transgenic herbicide-resistant Scoparia dulcis plants were obtained by using an Ri binary vector system. The chimeric bar gene encoding phosphinothricin acetyltransferase flanked by the promoter for cauliflower mosaic virus 35S RNA and the terminal sequence for nopaline synthase was introduced in the plant genome by Agrobacterium-mediated transformation by means of scratching young plants. Hairy roots resistant to bialaphos were selected and plantlets (R0) were regenerated. Progenies (S1) were obtained by self-fertilization. The transgenic state was confirmed by DNA-blot hybridization and assaying of neomycin phosphotransferase II. Expression of the bar gene in the transgenic R0 and S1 progenies was indicated by the activity of phosphinothricin acetyltransferase. Transgenic plants accumulated scopadulcic acid B, a specific secondary metabolite of S. dulcis, in amounts of 15-60% compared with that in normal plants. The transgenic plants and progenies showed resistant trait towards bialaphos and phosphinothricin. These results suggest that an Ri binary system is one of the useful tools for the transformation of medicinal plants for which a regeneration protocol has not been established.

Molecular characterization of cysteine biosynthetic enzymes in plants

Abstract: La synthese de la cysteine constitue le mecanisme majeur de l'assimilation du soufre dans les plantes. La cysteine synthase (CSase) [O-acetylserine(thiol)lyase] est responsable de l'etape finale de la synthese de la cysteine et catalyse la formation de L-cysteine a partir de l'O-acetyl-L-serine (OAS) et de SH 2 . L'OAS est fournie par la serine acetyltransferase (SATase) a partir de l'acetyl-CoA et de la serine. Il y a au moins 3 isoformes de la CSase dans les cellules des plantes, qui sont respectivement localisees dans le cytosol, les chloroplastes et les mitochondries. La localisation subcellulaire de ces isoformes a ete confirmee par des experiences in vivo en utilisant des plantes transgeniques exprimant des genes de fusion peptide de « transit »-gus. Des tabacs transgeniques exprimant, sous le controle du promoteur 35S, soit l'ADNc codant pour la CSase, soit l'ADNc codant pour la CSase associee au peptide de « transit » de la petite sous-unite de la ribulose 1,5 bisphosphate carboxylase du pois, soit l'ADNc codant pour la construction antisens de la CSase ont ete obtenus et analyses pour la modulation de la synthese de la cysteine en reponse a des stress de soufre varies. Les resultats suggerent que l'accumulation de cysteine synthase etrangere dans les chloroplastes augmente la synthese de la cysteine. Les ADNc codant pour la SATase ont ete isoles a partir des vegetaux. L'activite SATase recombinante est inhibee par la L-cysteine d'une maniere non competitive. Un complexe multienzymatique se forme chez le melon d'eau entre SATase et CSase recombinantes suggerant un channelling biosynthetique de la serine a la cysteine empechant ainsi la diffusion du substrat intermediaire, l'OAS.