Biomedical subjects

Shigeo Yoshida

Publications and source records attributed to Shigeo Yoshida.

122 records · Page 7Linked to original sources

PubMed2002

Loss-of-function of a rice brassinosteroid biosynthetic enzyme, C-6 oxidase, prevents the organized arrangement and polar elongation of cells in the leaves and stem.

Molecular genetic and physiological studies on brassinosteroid (BR)-related mutants of dicot plants have revealed that BRs play important roles in normal plant growth and development. However, little is known about the function of BR in monocots (grasses), except for the phenotypic analysis of a rice mutant partially insensitive to BR signaling. To investigate the function of BR in monocots, we identified and characterized BR-deficient mutants of rice, BR-deficient dwarf1 (brd1). The brd1 mutants showed a range of abnormalities in organ development and growth, the most striking of which were defects in the elongation of the stem and leaves. Light microscopic observations revealed that this abnormality was primarily owing to a failure in the organization and polar elongation of the leaf and stem cells. The accumulation profile of BR compounds in the brd1 mutants suggested that these plants may be deficient in the activity of BR C-6 oxidase. Therefore, we cloned a rice gene, OsDWARF, which has a high sequence similarity to the tomato C-6 oxidase gene, DWARF. Introduction of the wild-type OsDWARF gene into brd1 rescued the abnormal phenotype of the mutants. The OsDWARF gene was expressed at a low level in all of the examined tissues, with preferential expression in the leaf sheath, and the expression was negatively regulated by brassinolide treatment. On the basis of these findings, we discuss the biological function of BRs in rice plants.

Alleles↗

PubMed2002

The HAT2 gene, a member of the HD-Zip gene family, isolated as an auxin inducible gene by DNA microarray screening, affects auxin response in Arabidopsis.

The plant hormone, auxin, regulates many aspects of growth and development. Despite its importance, the molecular mechanisms underlying the action of auxin are largely unknown. To gain a more comprehensive understanding of the primary responses to auxin, we analyzed the expression of genes in Arabidopsis seedlings treated with indole-3-acetic acid (IAA) for 15 min. We identified a single gene that is downregulated early, and 29 genes that are upregulated early. Several types of typical transcription factors are identified as early upregulated genes, suggesting that auxin signals are mediated by a master set of diverse transcriptional regulators. Of the genes that responded to auxin, the expression of the homeobox gene, HAT2, was induced rapidly. Furthermore, we show that the expression of HAT2 is induced by auxin, but not by other phytohormones. To analyze the function of HAT2 in the plant's response to auxin, we generated 35S::HAT2 transgenic plants. These produced long hypocotyls, epinastic cotyledons, long petioles, and small leaves, which are characteristic of the phenotypes of the auxin-overproducing mutants, superroot1 (sur1) and superroot2 (sur2). On the other hand, 35S::HAT2 plants showed reduced lateral root elongation, and reduced auxin sensitivity compared to wild-type plants. Together with the results of RNA blotting and biochemical analyses, these findings suggest that HAT2 plays opposite roles in the shoot and root tissues in regulating auxin-mediated morphogenesis.

Amino Acid Sequence↗

PubMed2002

A trans-zeatin riboside in root xylem sap negatively regulates adventitious root formation on cucumber hypocotyls.

Shoot cultures of cucumber were used to analyse the roles of root-derived substances in adventitious root formation on hypocotyl tissues. Xylem sap collected from the roots of squash had a strong inhibitory effect on the formation of hypocotyl adventitious roots. Double-solvent extraction followed by fractionation with both normal and reverse phase column chromatographies and analysis by liquid chromatography/tandem mass spectrometry identified trans-zeatin riboside (ZR) as the primary suppressor of adventitious root formation. ZR was the predominant cytokinin present in the xylem sap, occurring at a concentration of 2x10(-8 )M. Application of ZR at concentrations from 3.16x10(-9) M effected inhibition of adventitious root formation. These results suggest that ZR transported from roots via xylem sap may act as an endogenous suppressor of hypocotyl adventitious root formation in planta.

Adenosine↗

PubMed2002

Isolation and expression of a novel starch-storing cell-specific gene containing the KH RNA binding domain from tobacco-cultured cells BY-2.

In cultured Bright Yellow-2 tobacco (Nicotiana tabacum) cells, the depletion of 2,4-dichlorophenoxyacetic acid (2,4-D) in the culture medium induces amyloplast development. This differentiation also includes a decrease in cell multiplication, and an increase in cell size. These changes were primarily triggered by the depletion of 2,4-D, and accelerated by the addition of benzyladenine (BA). Three cDNAs were identified whose transcript levels are specifically increased during differentiation of starch-storing cells using the differential display method, and designated as starch-storing cell induced genes (SCI genes). One of these cDNAs, SCI2 encodes a 285 amino acids long protein with a KH RNA-binding domain. A database search revealed that this protein showed similarity to respective domains of mammalian quaking proteins. 2,4-D addition, which can convert starch-storing cells into dividing cells, to starch-storing BY-2 cells, immediately decreases the SCI2 transcript level, suggesting that SCI2 may have some role in starch-storing cell differentiation in BY-2 cells.

2,4-Dichlorophenoxyacetic Acid↗

PubMed2002

Chloroisonicotinamide derivative induces a broad range of disease resistance in rice and tobacco.

Systemic acquired resistance (SAR) is a potent innate immunity system in plants that is effective against a broad range of pathogens. SAR in dicotyledonous plants such as tobacco and Arabidopsis has been partially elucidated and is mediated by salicylic acid (SA). However, the SAR mechanism of monocotyledonous rice plants remains to be clarified, although some similarities between SAR mechanisms in both types have been reported. Here we have characterized N-cyanomethyl-2-chloroisonicotinamide (NCI) as an effective SAR inducer in both plant species. Soil drench application of NCI induces a broad range of disease resistance in tobacco and rice and, more specifically, PR gene expression in tobacco. Both SA measurements in wild-type NCI-treated tobacco and pathogenic infection studies using NahG transgenic tobacco plants indicate that NCI-induced resistance enhancement does not require SA. Therefore, it is suggested that NCI induces SAR by triggering signaling at the same level as or downstream of SA accumulation as do both benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester and 2,6-dichloroisonicotinic acid. The fact that all of these chemicals are effective in rice and tobacco suggests that several common components function in disease resistance in both plant species.

Bacteria↗

PubMed2002

Amyloplast formation in cultured tobacco BY-2 cells requires a high cytokinin content.

When cytokinin-autonomous tobacco BY-2 cell cultures are transferred into 2,4-dichlorophenoxyacetic acid (2,4-D)-deprived medium, amyloplast development is initiated. Using this in vitro amyloplast-inducing system, the role of cytokinins in amyloplast formation was investigated. We show that addition of lovastatin, an inhibitor of mevalonate synthesis, to amyloplast-inducing medium reduced starch accumulation. Microscopic observation also revealed that lovastatin treatment decreased starch deposition; however, the overall morphologies of cells and plastids were less affected than control cell cultures. In addition, lovastatin lowered the transcription level of the ADP-glucose pyrophosphorylase small subunit (AgpS) gene. Application of mevalonate or zeatin dramatically restored the decrease in starch deposition, and restored AgpS mRNA accumulation. Moreover, addition of other molecules with cytokinin activity, such as adenine- and phenylurea-type compounds, restored starch accumulation and AgpS transcript levels, whereas other isopentenyl pyrophosphate-derived phytohormones did not. Liquid chromatography-mass spectrometry/mass spectrometry quantification of endogenous cytokinins revealed that endogenous cytokinins increased when BY-2 cells were transferred into 2,4-D-deprived medium from conventional medium containing 2,4-D. In addition, lovastatin treatment decreased endogenous cytokinins to some extent when cultured under 2,4-D-deprived conditions. Our results suggest that both 2,4-D deprivation and an increase in endogenous cytokinins have important roles in accelerating the changes in plastid morphology, starch accumulation, and AgpS gene expression.

2,4-Dichlorophenoxyacetic Acid↗

PubMed2002

An early C-22 oxidation branch in the brassinosteroid biosynthetic pathway.

The natural occurrence of 22-hydroxylated steroids in cultured Catharanthus roseus cells and in Arabidopsis seedlings was investigated. Using full-scan gas chromatography-mass spectrometry analysis, (22S)-22-hydroxycampesterol (22-OHCR), (22S,24R)-22-hydroxyergost-4-en-3-one (22-OH-4-en-3-one), (22S,24R)-22-hydroxy-5alpha-ergostan-3-one (22-OH-3-one), 6-deoxocathasterone (6-deoxoCT), 3-epi-6-deoxoCT, 28-nor-22-OHCR, 28-nor-22-OH-4-en-3-one, 28-nor-22-OH-3-one, 28-nor-6-deoxoCT, and 3-epi-28-nor-6-deoxoCT were identified. Metabolic experiments with deuterium-labeled 22-OHCR were performed in cultured C. roseus cells and Arabidopsis seedlings (wild type and det2), and the metabolites were analyzed by gas chromatography-mass spectrometry. In both C. roseus cells and wild-type Arabidopsis seedlings, [(2)H(6)]22-OH-4-en-3-one, [(2)H(6)]22-OH-3-one, [(2)H(6)]6-deoxoCT, and [(2)H(6)]3-epi-6-deoxoCT were identified as metabolites of [(2)H(6)]22-OHCR, whereas the major metabolite in det2 seedlings was [(2)H(6)]22-OH-4-en-3-one. Analysis of endogenous levels of these brassinosteroids revealed that det2 accumulates 22-OH-4-en-3-one. The levels of downstream compounds were remarkably reduced compared with the wild type. Exogenously applied 22-OH-3-one and 6-deoxoCT were found to rescue det2 mutant phenotypes, whereas 22-OHCR and 22-OH-4-en-3-one did not. These results substantiate the existence of a new subpathway (22-OHCR --> 22-OH-4-en-3-one --> 22-OH-3-one --> 6-deoxoCT) and reveal that the det2 mutant is defective in the conversion of 22-OH-4-en-3-one to 22-OH-3-one, which leads to brassinolide biosynthesis.

Arabidopsis↗

PubMed2002

Arabidopsis brassinosteroid-insensitive dwarf12 mutants are semidominant and defective in a glycogen synthase kinase 3beta-like kinase.

Mutants defective in the biosynthesis or signaling of brassinosteroids (BRs), plant steroid hormones, display dwarfism. Loss-of-function mutants for the gene encoding the plasma membrane-located BR receptor BRI1 are resistant to exogenous application of BRs, and characterization of this protein has contributed significantly to the understanding of BR signaling. We have isolated two new BR-insensitive mutants (dwarf12-1D and dwf12-2D) after screening Arabidopsis ethyl methanesulfonate mutant populations. dwf12 mutants displayed the characteristic morphology of previously reported BR dwarfs including short stature, short round leaves, infertility, and abnormal de-etiolation. In addition, dwf12 mutants exhibited several unique phenotypes, including severe downward curling of the leaves. Genetic analysis indicates that the two mutations are semidominant in that heterozygous plants show a semidwarf phenotype whose height is intermediate between wild-type and homozygous mutant plants. Unlike BR biosynthetic mutants, dwf12 plants were not rescued by high doses of exogenously applied BRs. Like bri1 mutants, dwf12 plants accumulated castasterone and brassinolide, 43- and 15-fold higher, respectively, providing further evidence that DWF12 is a component of the BR signaling pathway that includes BRI1. Map-based cloning of the DWF12 gene revealed that DWF12 belongs to a member of the glycogen synthase kinase 3beta family. Unlike human glycogen synthase kinase 3beta, DWF12 lacks the conserved serine-9 residue in the auto-inhibitory N terminus. In addition, dwf12-1D and dwf12-2D encode changes in consecutive glutamate residues in a highly conserved TREE domain. Together with previous reports that both bin2 and ucu1 mutants contain mutations in this TREE domain, this provides evidence that the TREE domain is of critical importance for proper function of DWF12/BIN2/UCU1 in BR signal transduction pathways.

Amino Acid Sequence↗

PubMed2002

Microarray analysis of brassinosteroid-regulated genes in Arabidopsis.

Brassinosteroids (BRs) are steroidal plant hormones that are essential for growth and development. Although insights into the functions of BRs have been provided by recent studies of biosynthesis and sensitivity mutants, the mode of action of BRs is poorly understood. With the use of DNA microarray analysis, we identified BR-regulated genes in the wild type (WT; Columbia) of Arabidopsis and in the BR-deficient mutant, det2. BR-regulated genes generally responded more potently in the det2 mutant than in the WT, and they showed only limited response in a BR-insensitive mutant, bri1. A small group of genes showed stronger responses in the WT than in the det2. Exposure of plants to brassinolide and brassinazole, which is a specific inhibitor of BR biosynthesis, elicited opposite effects on gene expression of the identified genes. The list of BR-regulated genes is constituted of transcription factor genes including the phytochrome-interacting factor 3, auxin-related genes, P450 genes, and genes implicated in cell elongation and cell wall organization. The results presented here provide comprehensive view of the physiological functions of BRs using BR-regulated genes as molecular markers. The list of BR-regulated genes will be useful in the characterization of new mutants and new growth-regulating compounds that are associated with BR function.

Arabidopsis↗

PubMed2002

The identification of CVP1 reveals a role for sterols in vascular patterning.

Vascular cell axialization refers to the uniform alignment of vascular strands. In the Arabidopsis cotyledon vascular pattern1 (cvp1) mutant, vascular cells are not arranged in parallel files and are misshapen, suggesting that CVP1 has a role in promoting vascular cell polarity and alignment. Characterization of an allelic series of cvp1 mutations revealed additional functions of CVP1 in organ expansion and elongation. We identified CVP1 and found that it encodes STEROL METHYLTRANSFERASE2 (SMT2), an enzyme in the sterol biosynthetic pathway. SMT2 and the functionally redundant SMT3 act at a branch point in the pathway that mediates sterol and brassinosteroid levels. The SMT2 gene is expressed in a number of developing organs and is regulated by various hormones. As predicted from SMT2 enzymatic activity, the precursors to brassinosteroid are increased at the expense of sterols in cvp1 mutants, identifying a role for sterols in vascular cell polarization and axialization.

Alleles↗

PubMed2002

Chlorophyll-deficient mutants of rice demonstrated the deletion of a DNA fragment by heavy-ion irradiation.

Heavy-ion irradiation is a new method of mutation breeding to produce new cultivars. We established the application of this method in rice plants to obtain mutants. Rice seeds were irradiated by C or Ne ions (135MeV/u) with a LET (linear energy transfer) of 22.7 or 64.2 keV/microm, respectively. Chlorophyll-deficient mutants (CDM) segregated in M2 progeny were albino, pale-green, yellow or striped-leave phenotypes. The highest rate of CDM with C-ion irradiation, 7.31%, was obtained at 40 Gy among the doses examined. Ne-ion irradiation gave the highest rate, 11.6%, at 20 Gy. We used the RLGS (Restriction Landmark Genomic Scanning) method to analyze DNA deletion in an albino mutant genome. Not I-landmark RLGS profiles detected about 2000 spots in rice. We found that one of the polymorphic spots was strongly linked to the albino phenotypic mutant derived from deleting of a DNA fragment, and demonstrated the high ability to detect of polymorphic regions by the RLGS method.

Carbon↗

PubMed2002

Resistance to protoporphyrinogen oxidase-inhibiting compound S23142 from overproduction of mitochondrial protoporphyrinogen oxidase by gene amplification in photomixotrophic tobacco cells.

Tobacco YZI-IS cells exhibit a 150-fold greater resistance to the protoporphyrinogen oxidase (Protox)-inhibiting compound, S23142, from wild-type tobacco cells. To investigate the mechanism for this S23142 resistance, the protein level, enzymatic activity, and sensitivity to S23142 in two Protox isoenzymes (plastidal and mitochondrial forms) were examined. The level of mitochondrial Protox protein was greater, and its activity 5-times higher, in YZI-IS cells than in wild-type cells. Furthermore, the apparent IC50 value of S23142 was about 20 nM, which is 20-fold higher than that observed in wild-type cells. In contrast, no differences were found in the plastidal Protox protein level, activity or its inhibition by S23142 between YZI-1S and wild-type cells. A southern blot analysis revealed that the mitochondrial Protox gene had been significantly amplified in the YZI-1S cells. These results suggest that the S23142 resistance of YZI-1S cells was due to the overproduction of mitochondrial Protox by gene amplification.

Dose-Response Relationship, Drug↗

PubMed2002

Microarray analysis of gene expression in the aging human retina.

PURPOSE: To develop gene expression profiles of young and elderly human retinas and identify candidate genes for aging-associated retinal diseases. METHODS: Gene microarray slides containing 2400 human genes (primarily neuronal) were hybridized to biotin or dinitrophenyl (DNP)-labeled target cDNAs that were synthesized using total RNAs from young (13-14 years) and elderly (62-74 years) human retinas. Hybridization signals were visualized with cyanine (Cy)-5 or Cy-3 fluorescent reporter molecules, and the fluorescence intensities of the images were analyzed by computer. Northern blot analysis and real-time quantitative reverse transcription PCR (qRT-PCR) were performed to validate the microarray results. RESULTS: Of the 2400 genes represented on the microarray slides, more than 50% hybridized to the retinal cDNA targets. Expression of a majority of these genes was not altered during aging; nonetheless, changes in the expression of 24 genes were detected between young and elderly retinas. These genes could be clustered into four categories: energy metabolism, stress response, cell growth, and neuronal transmission/signaling. Northern blot analysis and qRT-PCR results confirmed the changes in expression of 8 of 10 genes examined. CONCLUSIONS: Using commercially available slide microarrays, the authors show that aging of the human retina is associated with changes in patterns of gene expression. This analysis suggests that pathways involved in stress response and energy metabolism play key roles in retinal aging. These studies demonstrate the utility of gene microarrays in identifying global patterns of retinal gene expression and lay the foundation for future studies defining the genetic basis of aging-associated retinal diseases, such as age-related macular degeneration.

Adolescent↗

PubMedDate not supplied

An analysis of BIGH3 mutations in patients with corneal dystrophies in the Kyushu district of Japan.

PURPOSE: To assess the involvement of BIGH3 in corneal dystrophies (CD) with an autosomal dominant trait, in patients referred to a hospital in the Kyushu district of Japan. METHODS: Forty-five CD patients from 44 families were studied. Genomic DNA was extracted from peripheral blood, and exons 4 and 12 of the BIGH3 gene were amplified by polymerase chain reaction followed by direct sequencing. RESULTS: In exon 4, an R124H mutation associated with Avellino corneal dystrophy (ACD) was found in 39/44 families (86.4%) and an R124C mutation associated with lattice corneal dystrophy type 1 (LCD1) was detected in 2/44 families (4.5%). In exon 12, an R555W mutation associated with granular corneal dystrophy (GCD) was detected in 4/44 families (9.1%). CONCLUSIONS: Codons R124 and R555 of the BIGH3 gene represent mutational hotspots in the genomes of Japanese patients with autosomal-dominant CD.

Adolescent↗