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Hiroyuki Ohta

Publications and source records attributed to Hiroyuki Ohta.

34 records · Page 2Linked to original sources
PubMed2004

Arabidopsis type B monogalactosyldiacylglycerol synthase genes are expressed during pollen tube growth and induced by phosphate starvation.

The galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) constitute the major glycolipids of the thylakoid membranes in chloroplasts. In Arabidopsis, the formation of MGDG is catalyzed by a family of three MGDG synthases, which are encoded by two types of genes, namely type A (atMGD1) and type B (atMGD2 and atMGD3). Although the roles of the type A enzyme have been intensively investigated in several plants, little is known about the contribution of type B enzymes to MGDG synthesis in planta. From our previous analyses, unique expression profiles of the three MGDG synthase genes were revealed in various organs and developmental stages. To characterize the expression profiles in more detail, we performed histochemical analysis of these genes using beta-glucuronidase (GUS) assays in Arabidopsis. The expression of atMGD1::GUS was detected highly in all green tissues, whereas the expression of atMGD2::GUS and atMGD3::GUS was observed only in restricted parts, such as leaf tips. In addition, intense staining was detected in pollen grains of all transformants. We also detected GUS activity in the pollen tubes of atMGD2::GUS and atMGD3::GUS transformants grown in wild-type stigmas but not in atMGD1::GUS, suggesting that type B MGDG synthases may have roles during pollen germination and pollen tube growth. GUS analysis also revealed that expression of atMGD2 and atMGD3, but not atMGD1, are strongly induced during phosphate starvation, particularly in roots. Because only DGDG accumulates in roots during phosphate deprivation, type B MGDG synthases may be acting primarily to supply MGDG as a precursor for DGDG synthesis.

Arabidopsis↗
PubMed2004

ARC3, a chloroplast division factor, is a chimera of prokaryotic FtsZ and part of eukaryotic phosphatidylinositol-4-phosphate 5-kinase.

The arc3 (accumulation and replication of chloroplast) mutant of Arabidopsis thaliana has a small number of abnormally large chloroplasts in the cell, suggesting that chloroplast division is arrested in the mutant and ARC3 has an important role in the initiation of chloroplast division. To elucidate the role of ARC3, first we identified the ARC3 gene, and determined the location of ARC3 protein during chloroplast division because the localization and spatial orientation of such division factors are vital for correct chloroplast division. Sequencing analysis showed that ARC3 was a fusion of the prokaryotic FtsZ and part of the eukaryotic phosphatidylinositol-4-phosphate 5-kinase (PIP5K) genes. The PIP5K-homologous region of ARC3 had no catalytic domain but a membrane-occupation-and-recognition-nexus (MORN) repeat motif. Immunofluorescence microscopy, Western blotting analysis and in vitro chloroplast import and protease protection assays revealed that ARC3 protein was soluble, and located on the outer surface of the chloroplast in a ring-like structure at the early stage of chloroplast division. Prokaryotes have one FtsZ as a gene for division but have no ARC3 counterparts, the chimera of FtsZ and PIP5K, suggesting that the ARC3 gene might have been generated from FtsZ as another division factor during the evolution of chloroplast by endosymbiosis.

Amino Acid Motifs↗
PubMed2004

Sphingomonas oligophenolica sp. nov., a halo- and organo-sensitive oligotrophic bacterium from paddy soil that degrades phenolic acids at low concentrations.

The taxonomic position of a halo- and organo-sensitive, oligotrophic soil bacterium, strain S213(T), was investigated. Cells were Gram-negative, non-motile, strictly aerobic, yellow-pigmented rods of short to medium length on diluted nutrient broth. When 0.1-0.4 % (w/v) NaCl was added to diluted media composed of peptone and meat extract, growth was inhibited with increasing NaCl concentration and the cells became long aberrant forms. When 6 mM CaCl(2) was added, the cells grew quite normally and aberrant cells were no longer found at 0.1-0.5 % (w/v) NaCl. Chemotaxonomically, strain S213(T) contains chemical markers that indicate its assignment to the Sphingomonadaceae: the presence of ubiquinone Q-10 as the predominant respiratory quinone, C(18 : 1) and C(16 : 0) as major fatty acids, C(14 : 0) 2-OH as the major 2-hydroxy fatty acid and sphingoglycolipids. 16S rRNA gene sequence analysis indicated that strain S213(T) belongs to the genus Sphingomonas, exhibiting high sequence similarity to the 16S rRNA gene sequences of Sphingomonas mali IFO 15500(T) (98.3 %), Sphingomonas pruni IFO 15498(T) (98.0 %), Sphingomonas asaccharolytica IFO 15499(T) (97.9 %) and Sphingomonas echinoides DSM 1805(T) (97.8 %). The results of DNA-DNA hybridization experiments and its phenotypic characteristics clearly distinguished the strain from its nearest neighbours and demonstrate that strain S213(T) represents a novel Sphingomonas species, for which the name Sphingomonas oligophenolica sp. nov. is proposed. The type strain is S213(T) (=JCM 12082(T)=CIP 107926(T)).

Aerobiosis↗
PubMed2004

Gene expression profiling of the tetrapyrrole metabolic pathway in Arabidopsis with a mini-array system.

Tetrapyrrole compounds, such as chlorophylls, hemes, and phycobilins, are synthesized in many enzymatic steps. For regulation of the tetrapyrrole metabolic pathway, it is generally considered that several specific isoforms catalyzing particular enzymatic steps control the flow of tetrapyrrole intermediates by differential regulation of gene expression depending on environmental and developmental factors. However, the coordination of such regulatory steps and orchestration of the overall tetrapyrrole metabolic pathway are still poorly understood. In this study, we developed an original mini-array system, which enables the expression profiling of each gene involved in tetrapyrrole biosynthesis simultaneously with high sensitivity. With this system, we performed a transcriptome analysis of Arabidopsis seedlings in terms of the onset of greening, endogenous rhythm, and developmental control. Data presented here clearly showed that based on their expression profiles at the onset of greening, genes involved in tetrapyrrole biosynthesis can be classified into four categories, in which genes are coordinately regulated to control the biosynthesis. Moreover, genes in the same group were similarly controlled in an endogenous rhythmic manner but also by a developmental program. The physiological significance of these gene clusters is discussed.

Arabidopsis↗
PubMed2003

Digalactosyldiacylglycerol is a major glycolipid in floral organs of Petunia hybrida.

In higher plants, glycolipids such as monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are major components of chloroplast membranes in leaves. A recent study identified an isoform of MGDG synthase that is expressed specifically in floral organs, suggesting a novel function for glycolipids in flowers. To elucidate the localization and developmental changes of glycolipids and their biosynthetic activities in flowers, we carried out a series of analytical studies with Petunia hybrida. The results showed that the biosynthetic activities of galactolipid synthesis, particularly for DGDG, increased during flower development. Among the floral organs, the pistil had the highest galactolipid synthetic activity. Its specific activity for incorporation of UDP-galactose to yield galactolipids was estimated to be more than twice that of leaves, which are the major site of galactolipid synthesis in plant tissues. Analysis of lipid contents of pistils revealed that they contained higher amounts of galactolipids than other floral organs. Moreover, DGDG was more abundant than MGDG in both pistils and petals. These results show that DGDG is a major glycolipid in floral organs and that DGDG biosynthetic activity is highly upregulated in the pistils and petals of Petunia flowers.

Flowers↗
PubMed2003

Chlorophyllase as a serine hydrolase: identification of a putative catalytic triad.

Chlorophyllases (Chlases), cloned so far, contain a lipase motif with the active serine residue of the catalytic triad of triglyceride lipases. Inhibitors specific for the catalytic serine residue in serine hydrolases, which include lipases effectively inhibited the activity of the recombinant Chenopodium album Chlase (CaCLH). From this evidence we assumed that the catalytic mechanism of hydrolysis by Chlase might be similar to those of serine hydrolases that have a catalytic triad composed of serine, histidine and aspartic acid in their active site. Thus, we introduced mutations into the putative catalytic residue (Ser162) and conserved amino acid residues (histidine, aspartic acid and cysteine) to generate recombinant CaCLH mutants. The three amino acid residues (Ser162, Asp191 and His262) essential for Chlase activity were identified. These results indicate that Chlase is a serine hydrolase and, by analogy with a plausible catalytic mechanism of serine hydrolases, we proposed a mechanism for hydrolysis catalyzed by Chlase.

Amino Acid Sequence↗
PubMed2003

Light and cytokinin play a co-operative role in MGDG synthesis in greening cucumber cotyledons.

The current research investigated the regulation of monogalactosyldiacylglycerol (MGDG) biosynthesis, catalyzed by MGDG synthase (MGD) (UDP-galactose:1,2-diacylglycerol 3-beta-D-galactosyltransferase; EC 2.4.1.46), during chloroplast development in cucumbers (Cucumis sativus L. cv. Aonagajibai). In etiolated seedlings, white light induced a transient increase in MGD mRNA, followed by a subsequent increase in enzyme activity. MGDG, digalactosyldiacylglycerol (DGDG), and linolenic acid (18 : 3) of both MGDG and DGDG accumulated in a light-dependent manner. Early light-dependent induction of MGD protein was also identified in isolated chloroplasts. When cotyledons were detached from seedlings, these light-induced changes diminished. However, when a synthetic cytokinin, benzyladenine, was added to the detached cotyledons, a transient increase in MGD mRNA and a linear increase in the enzyme activity were induced even in the dark. Galactolipids subsequently accumulated to some extent and 18 : 3 content also increased. MGDG fully accumulated in detached cotyledons with co-treatment of light and a cytokinin. Red light (>600 nm) and far-red light (>700 nm) both induced an increase in MGD mRNA and enzyme activity but far-red light did not induce an accumulation of MGDG. These results suggest that (1). galactolipid biosynthesis is regulated by the cooperation of light and a cytokinin; (2). the accumulation of MGDG requires cytokinin in addition to light; (3). a red light (600-700 nm) dependent factor is necessary for the maximal galactolipid accumulation in addition to increase in MGD transcript and activity.

Adenine↗
PubMed2003

Functional analysis of isoforms of NADPH: protochlorophyllide oxidoreductase (POR), PORB and PORC, in Arabidopsis thaliana.

NADPH:protochlorophyllide oxidoreductase (POR) catalyzes the light-dependent reduction of protochlorophyllide. To elucidate the physiological function of three differentially regulated POR isoforms (PORA, PORB and PORC) in Arabidopsis thaliana, we isolated T-DNA tagged null mutants of porB and porC. The mature seedlings of the mutants had normal photosynthetic competencies, showing that PORB and PORC are interchangeable and functionally redundant in developed plants. In etiolated seedlings, only porB showed a reduction in the photoactive protochlorophyllide and the size of prolamellar bodies (PLBs), indicating that PORB, as well as PORA, functioned in PLB assembly and photoactive protochlorophyllide formation in etiolated seedlings. When illuminated, the etiolated porB seedling was able to green to a similar extent as the wild type, whereas the greening was significantly reduced under low light conditions. During greening, high light irradiation increased the level of PORC protein, and the greening of porC was repressed under high light conditions. The porB, but not porC, etiolated seedling was more sensitive to the far-red block of greening than the wild type, which is caused by depletion of endogenous POR proteins resulting in photo-oxidative damage. These results suggest that, at the onset of greening, PLBs are important for efficient capture of light energy for photoconversion under various light conditions, and PORC, which is induced by high light irradiation, contributes to photoprotection during greening of the etiolated seedlings.

Arabidopsis↗
PubMed2003

Sphingobium amiense sp. nov., a novel nonylphenol-degrading bacterium isolated from a river sediment.

A nonylphenol-degrading bacterial strain (YT(T)) was isolated previously from a river sediment sample obtained in Ami-machi, Ibaraki, Japan, and identified as a Sphingomonas species. In this study, the taxonomic relationship between strain YT(T), a recently described nonylphenol-degrading strain, Sphingomonas cloacae, and Sphingobium yanoikuyae, which is phylogenetically related, was examined. Their phenotypic characteristics were compared and levels of DNA-DNA relatedness between these strains were determined. Based on the results of physiological and biochemical tests and DNA-DNA hybridization, it is proposed that strain YT(T) (=IAM 15006(T)=JCM 11777(T)=CIP 107839(T)) represents a novel species of the genus Sphingobium, Sphingobium amiense sp. nov.

Alphaproteobacteria↗
PubMed2002

Identification of two differentially regulated isoforms of protochlorophyllide oxidoreductase (POR) from tobacco revealed a wide variety of light- and development-dependent regulations of POR gene expression among angiosperms.

NADPH-protochlorophyllide oxidoreductase (POR) catalyzes the light-dependent reduction of protochlorophyllide a in the chlorophyll biosynthetic pathway. Here, we identified two distinct POR cDNAs from tobacco. Both POR isoforms are encoded by a respective single copy gene in tobacco genome. The overall deduced amino acid sequences of two tobacco cDNAs, designated here POR1 and POR2, displayed significant identities ( approximately 75%), but showed different patterns of light and developmental regulation. In contrast to the previously isolated POR isoforms of Arabidopsis thaliana and barley, the expression of both tobacco POR isoforms were not negatively regulated by light and persisted in matured green tissues. Furthermore, the expression of both genes appeared to be regulated by a diurnal regulation. These results show a wide variety of light- and development-dependent regulations of POR gene expression among angiosperms. Furthermore, phylogenetic analysis including tobacco revealed that POR gene family is differentially represented by angiosperms, most of which is probably caused by independent gene duplication in individual plant. Present results imply a modification of the previous concept that chlorophyll biosynthesis and chloroplast differentiation in angiosperms are ubiquitously controlled by unique functions of two POR isoforms.

Journal Article↗
PubMed2001

Two types of ferrochelatase in photosynthetic and nonphotosynthetic tissues of cucumber: their difference in phylogeny, gene expression, and localization.

Ferrochelatase catalyzes the insertion of Fe(2+) into protoporphyrin IX to generate protoheme. In higher plants, there is evidence for two isoforms of this enzyme that fulfill different roles. Here, we describe the isolation of a second ferrochelatase cDNA from cucumber (CsFeC2) that was less similar to a previously isolated isoform (CsFeC1) than it was to some ferrochelatases from other higher plants. In in vitro import experiments, the two cucumber isoforms showed characteristics similar to their respective ferrochelatase counterparts of Arabidopsis thaliana. The C-terminal region of CsFeC2 but not CsFeC1 contained a conserved motif found in light-harvesting chlorophyll proteins, and CsFeC2 belonged to a phylogenetic group of plant ferrochelatases containing this conserved motif. We demonstrate that CsFeC2 was localized predominantly in thylakoid membranes as an intrinsic protein, and forming complexes probably with the C-terminal conserved motif, but a minor portion was also detected in envelope membranes. CsFeC2 mRNA was detected in all tissues and was light-responsive in cotyledons, whereas CsFeC1 mRNA was detected in nonphotosynthetic tissues and was not light-responsive. Interestingly, tissue-, light-, and cycloheximide-dependent expressions of the two isoforms of ferrochelatase were similar to those of two glutamyl-tRNA reductase isoforms involved in the early step of tetrapyrrole biosynthesis, suggesting the existence of distinctly controlled tetrapyrrole biosynthetic pathways in photosynthetic and nonphotosynthetic tissues.

Aldehyde Oxidoreductases↗
PubMed2001

Fermentable-sugar-level-dependent regulation of leukotoxin synthesis in a variably toxic strain of Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans, a Gram-negative periodontopathic bacterium, produces a leukotoxin belonging to the RTX family. The production of leukotoxin varies greatly among different strains of this species and under different culture conditions. A toxin-production-variable strain, 301-b, stably produces significant amounts of leukotoxin in anaerobic fructose-limited chemostat cultures, but does not do so in the presence of excess fructose. This communication describes the cloning and sequencing of the leukotoxin promoter region from 301-b, showing that this strain has a promoter region similar to that from strain 652, a moderately toxic strain. Northern blot analysis using a leukotoxin gene probe demonstrated that change in toxin production in response to the level of external fructose was due to alteration in the transcriptional level of the leukotoxin gene. Pulsing of fructose into the fructose-limited chemostat culture remarkably reduced the intracellular cAMP level from 40 pmol (mg dry wt cells)(-1) to 3.1 pmol (mg dry wt cells)(-1), which was restored when the culture was returned to fructose-limited conditions. Further, it was found that addition of external cAMP to the culture with excess fructose resulted in an apparent recovery of leukotoxin production. Taken together, these findings indicate that a cAMP-dependent mechanism, possibly a catabolite-repression-like system, may be involved in the regulation of leukotoxin production in this bacterium.

Aggregatibacter actinomycetemcomitans↗
PubMed2001

Energy metabolism of Actinobacillus actinomycetemcomitans during anaerobic and microaerobic growth in low- and high-potassium continuous culture.

Actinobacillus actinomycetemcomitans, a member of the gamma subclass of the Proteobacteria, has been implicated as the agent responsible for human periodontitis. In this study, A. actinomycetemcomitans 301-b was grown in fructose-limited chemostat cultures under anaerobic [redox potential (E(h))<-400 mV] and microaerobic (E(h)= -200 mV) conditions to characterize its energy metabolism. Effects of K(+) and Na(+) on growth and metabolism were also examined. In a control medium containing 5.2 mM K(+) and 24 mM Na(+), the molar growth yield on fructose (Y(fructose)) of microaerobic cultures was 1.3 times higher than the yield of anaerobic cultures at D < or =0.10 h(-1), but the difference in the Y(fructose) between microaerobic and anaerobic cultures decreased at D< or =0.10 h(-1). When the ATP yield from fermentation was estimated from the amounts of fructose consumed and acetate formed, the value of the microaerobic culture (2.49 mol ATP produced per mol fructose consumed) was lower than the anaerobic value [3.13 mol ATP (mol fructose)(-1)]. Therefore, ATP production from fermentation could not account for the increase in the Y(fructose) at D > 0.10 h(-1) and thus additional ATP was expected to be generated via respiration. Assuming that the Y(ATP) (g cells formed per mol ATP synthesized) was similar between anaerobic and microaerobic cultures, the estimated ATP yield from respiration was between 1.2 and 2.0 mol ATP (mol fructose)(-1) below D=0.10 h(-1) and decreased to 0.3 mol ATP (mol fructose)(-1) when D was increased to 0.19 h(-1). Such growth-rate-dependent decreases in the Y(fructose) and the estimated ATP production from respiration were also observed in a high-Na(+) (5.2 mM K(+) and 106 mM Na(+)) culture but not in a high-K(+) (81 mM K(+) and 24 mM Na(+)) culture. In the high-K(+) culture, the microaerobic Y(fructose) was 1.4-2.0 times higher than the anaerobic value and the respiration-derived ATP yield was estimated to be between 1.2 and 1.9 mol ATP (mol fructose)(-1) over a wide range of dilution rate. These results suggest that higher concentrations of extracellular K(+) are required for the respiration to occur in rapidly growing cells of A. actinomycetemcomitans.

Actinobacillus Infections↗
PubMed1996

The relationships between leukotoxin production, growth rate and the bicarbonate concentration in a toxin-production-variable strain of Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans, a Gram-negative periodontopathic bacterium, produces a leukotoxin belonging to the RTX family. The production of leukotoxin varies greatly among different strains of this species. In this paper the effects of growth rate and bicarbonate on the leukotoxin production by a toxin-production-variable strain (301-b) during growth in a chemostat were examined. When the bacterium was grown in anaerobic fructose-limited chemostat cultures (pH 7.0 and 37 degrees C) at dilution rates (D) ranging from 0.04 to 0.20 h-1 in the absence and presence of 10 mM bicarbonate, it produced leukotoxin as a cluster of two polypeptides (M(r) 113,000 and 120,000) and complexed with nucleic acids on the bacterial cell surface. The relationship between leukotoxin production and specific growth rate was analysed by plotting the specific rate of leukotoxin production [qLT, in microgram (mg dry wt)-1 h-1] against D. The plots were approximated to the linear relationships qLT = 2.7D-0.058 and qLT = 9.3D-0.407 without and with bicarbonate, respectively. These relationships suggest that the apparent leukotoxin production is a result of both growth-rate-dependent production and growth-rate-independent decomposition. The cellular leukotoxin level was also followed after the change from chemostat to batch culture in the same fermenter. In batch culture leukotoxin production stopped immediately and the cellular toxin level rapidly decreased, suggesting toxin decomposition. From the slopes of the approximated linear relationships between qLT and D, a theoretical maximum leukotoxin yield (YLT) was estimated as 2.7 and 9.3 micrograms (mg dry wt)-1 in the absence and presence of 10 mM bicarbonate, respectively. The increased YLT value in the cultures containing bicarbonate indicated that the addition stimulated the efficiency of leukotoxin synthesis up to about threefold. Further increases of bicarbonate concentration to between 20 and 40 mM had no effect on the total leukotoxin production, but the amount of extracellular leukotoxin increased with higher bicarbonate concentrations.

Actinobacillus Infections↗