Biomedical subjects
Publications and source records attributed to Shigeo Yoshida.
When brassinosteroid (BR)-deficient mutant (det2) or wild-type (WT) seedlings were treated with brassinolide (BL), the most active BR, for 3 h, the abundance of PIN4 and PIN7 transcripts decreased, and there were fewer PIN4 and PIN7 transcripts in det2 than in the WT. This suggests that BL selectively regulates the PIN gene in a complex manner.
When exogenous chemicals allow rapid, conditional, reversible, selective, and dose-dependent control of biological functions, they act like conditional mutations, either inducing or suppressing the formation of a specific phenotype of interest. Exploration of the small molecules that induce the brassinosteroid (BR) deficient-like phenotype in Arabidopsis led us to identify brassinazole as the first candidate for a BR biosynthesis inhibitor. Brassinazole treatment reduced BR content in plant cells. Investigation of target site(s) of brassinazole revealed that the compound directly binds to the DWF4 protein, a cytochrome P450 monooxygenase that catalyzes 22-hydroxylation of the side chain of BRs. These results suggest that brassinazole is a BR biosynthesis inhibitor. There are currently at least two BR biosynthesis inhibitors that act like conditional mutations in BR biosynthesis. They allow the investigation of the functions of BRs in a variety of plant species. Application of BR biosynthesis inhibitors to a standard genetic screen to identify mutants that confer resistance to these inhibitors allowed the identification of new components working in BR signal transduction. This method has advantages over mutant screens using BR-deficient mutants as a background. Development of chemicals that induce phenotypes of interest is now emerging as a useful way to study biological systems in plants and this would be a complement to classical biochemical and genetic methods.
We characterized a rice dwarf mutant, ebisu dwarf (d2). It showed the pleiotropic abnormal phenotype similar to that of the rice brassinosteroid (BR)-insensitive mutant, d61. The dwarf phenotype of d2 was rescued by exogenous brassinolide treatment. The accumulation profile of BR intermediates in the d2 mutants confirmed that these plants are deficient in late BR biosynthesis. We cloned the D2 gene by map-based cloning. The D2 gene encoded a novel cytochrome P450 classified in CYP90D that is highly similar to the reported BR synthesis enzymes. Introduction of the wild D2 gene into d2-1 rescued the abnormal phenotype of the mutants. In feeding experiments, 3-dehydro-6-deoxoteasterone, 3-dehydroteasterone, and brassinolide effectively caused the lamina joints of the d2 plants to bend, whereas more upstream compounds did not cause bending. Based on these results, we conclude that D2/CYP90D2 catalyzes the steps from 6-deoxoteasterone to 3-dehydro-6-deoxoteasterone and from teasterone to 3-dehydroteasterone in the late BR biosynthesis pathway.
Despite numerous physiological studies addressing the interactions between brassinosteroids (BRs) and auxins, little is known about the underlying molecular mechanisms. We studied the expression of IAA5 and IAA19 in response to treatment with indole acetic acid (IAA) or brassinolide (BL), the most active BR. Exogenous IAA induced these genes quickly and transiently, whereas exogenous BL induced them gradually and continuously. We also found that a fusion of DR5, a synthetic auxin response element, with the GUS (beta-glucuronidase) gene was induced with similar kinetics to those of the IAA5 and IAA19 genes in response to both IAA and BL treatment of transgenic plants. These results suggest that the IAA genes are induced by BL, at least in part, via the activation of the auxin response element. Endogenous IAA levels per gram fresh weight did not increase when seedlings of Arabidopsis wild type (WT) or the BR-deficient mutant det2 were treated with BL. Furthermore, the levels of IAA transcripts were lower in the det2 mutant than in the WT, even though endogenous IAA levels per gram fresh weight were higher in the det2 mutant than in the WT. In conclusion, the lack of evidence for auxin-mediated activation of early auxin-inducible genes in response to BL suggests that the BR and auxin signaling pathways independently activate the transcriptional system of the IAA and DR5-GUS genes.
Active brassinosteroids, such as brassinolide (BL) and castasterone, are growth promoting plant hormones. An Arabidopsis cytochrome p450 monooxygenase encoded by CYP72B1 has been implicated in brassinosteroid catabolism as well as photomorphogenesis. We expressed CYP72B1 in yeast, coupled with brassinosteroid feeding, and established the biochemical function to be the hydroxylation of BL and castasterone, to give 26-hydroxybrassinolide and 26-hydroxycastasterone, respectively. Brassinosteroid feeding experiments with wild-type Arabidopsis, a CYP72B1 null mutant, and a CYP72B1 overexpression line demonstrated that carbon 26 hydroxylation of active brassinosteroids is an endogenous function of CYP72B1. Seedling growth assays demonstrated that 26-hydroxybrassinolide is an inactive brassinosteroid. Genetic and physiological analysis of the hypocotyl response to exogenous BL and varying intensities of white and monochromatic light suggested that CYP72B1 modulates photomorphogenesis primarily through far-red light and to a lesser extent through blue- and red-light pathways. CYP72B1 transcript accumulation in dark-grown seedlings was organ specific and down-regulated after 1 h of illumination in dim white, red, and blue light, but not far-red light. CYP72B1 translational fusions with the beta-glucuronidase reporter gene demonstrated that protein levels increased in the hypocotyl elongation zone when shifted from the dark to far-red light, but not blue or red light. We propose a model in which Arabidopsis seedling development switches from dark-grown development (skotomorphogenesis) to light-grown development (photomorphogenesis) in part by rapid modulation of brassinosteroid sensitivity and levels. CYP72B1 provides an intersection between the light and brassinosteroid pathways mainly by far-red-light-dependent modulation of brassinosteroid levels.
Limited information is available concerning the interactions between the brassinosteroid (BR) and auxin signaling pathways. The expression pattern of the SAUR-AC1 gene, an early auxin-inducible gene in Arabidopsis, was studied in response to brassinolide (BL), in the presence of a BR-biosynthesis inhibitor, in a BR-deficient mutant, and in combination with auxin. The results suggested that the SAUR-AC1 gene is regulated by BRs independently of auxin levels, and that it is important in BR-mediated elongation. The axr1 (auxin insensitive 1) mutant was less sensitive to BL-induced elongation and BL-induced SAUR-AC1 expression, suggesting that a ubiquitin ligase-mediated system is involved in BR-mediated elongation.
Nitrile hydratase (NHase) is a non-heme iron or non-corrin cobalt enzyme having two post-translationally modified ligand residues, cysteine-sulfinic acid (alphaCys112-SO(2)H) and -sulfenic acid (alphaCys114-SOH). We studied the interaction between Fe-type NHase and isobutyronitrile (iso-BN) which had been reported as a competitive inhibitor with a K(i) value of 5 microM. From detailed kinetic studies of the inhibitory effect of iso-BN on Fe-type NHase, we found that authentic iso-BN was hydrated normally and that the impurity present in commercially available iso-BN inhibited NHase activity strongly. The inhibitory compound induced significant changes in the UV-vis absorption spectrum of NHase, suggesting its interaction with the iron center. This compound was purified by using reversed-phase HPLC and identified as 2-cyano-2-propyl hydroperoxide (Cpx) by (1)H and PFG-HMBC NMR spectroscopy. Upon addition of a stoichiometric amount of Cpx, NHase was irreversibly inactivated, probably by the oxidation of alphaCys114-SOH to Cys-SO(2)H. This result suggests that the -SOH structure of alphaCys114 is essential for the catalytic activity. The oxygen atom in Cys-SO(2)H is confirmed to come from the solvent H(2)O. The oxidized NHase was found to induce the UV-vis absorption spectral changes by addition of Cpx, suggesting that Cpx strongly interacted with iron(III) in the oxidized NHase to form a stable complex. Thus, Cpx functions as a novel irreversible inhibitor for NHase.
PURPOSE: Crystallins are expressed at high levels in lens fiber cells. Recent studies have revealed that several members of the alpha, beta, and gamma-crystallin family are also distributed in many non-lens tissues, though at lower levels. We observed that the use of retinal RNA as target for both custom I-Gene microarrays and Affymetrix GeneChips revealed significant expression of many crystallin genes. This prompted us to undertake a comprehensive investigation to delineate the baseline expression of crystallin genes in the adult mouse retina. METHODS: Quantitative RT-PCR was carried out using gene specific primers (derived from the mouse genomic sequence) for each crystallin gene. Immunofluorescence studies using frozen sections of the mouse retinas were performed with crystallin-specific antibodies. Retinal lysates were analyzed by immunoblotting using antibodies specific to alphaA and alphaB crystallins and those produced against total beta-crystallin and gamma-crystallin fractions of bovine lenses. RESULTS: Microarray analysis followed by quantitative RT-PCR revealed that mouse retinal cells express transcripts for 20 different members of the crystallin gene family; these are alphaA, alphaA-INS, alphaA-nov1, alphaB, betaA1, betaA2, betaA3, betaA4, betaB1, betaB2, betaB3, gammaA, gammaC, gammaD, gammaE, gammaF, gammaS, mu, zeta, and lambda-crystallin. The gene products of alphaA, alphaB, beta-, and gamma-crystallins are detected in the outer and inner nuclear layers of the retina by immunofluorescence analysis. In addition, alpha and beta-crystallins are detected in the photoreceptor inner segments. Retinal expression of these proteins was further confirmed by immunoblot analysis. Interestingly, our studies also showed a significant animal-to-animal variation in the expression level of some of the crystallins. CONCLUSIONS: Our results establish the expression of many crystallins in the adult mouse retina. Detection of crystallins in the retinal nuclear layers, though surprising, is consistent with their proposed role in cell survival and genomic stability. We suggest that crystallins play vital functions in protecting retinal neurons from damage by environmental and/or metabolic stress.
Understanding the role of 'epigenetic' changes such as DNA methylation and chromatin remodeling has now become critical in understanding many biological processes. In order to delineate the global methylation pattern in a given genomic DNA, computer software has been developed to create a virtual image of restriction landmark genomic scanning (Vi-RLGS). When using a methylation- sensitive enzyme such as NotI as the restriction landmark, the comparison between real and in silico RLGS profiles of the genome provides a methylation map of genomic NotI sites. A methylation map of the Arabidopsis genome was created that could be confirmed by a methylation-sensitive PCR assay. The method has also been applied to the mouse genome. Although a complete methylation map has not been completed, a region of methylation difference between two tissues has been tested and confirmed by bisulfite sequencing. Vi-RLGS in conjunction with real RLGS will make it possible to develop a more complete map of genomic sites that are methylated or demethylated as a consequence of normal or abnormal development.
Little is known concerning the mechanisms by which auxin and cytokinin exert their effects on proliferation and differentiation. Cyclin-dependent kinases (CDKs) are major regulators of the eukaryotic cell cycle, thus they are assumed to control cell differentiation as well as proliferation in response to phytohormone signals. Here, we overexpressed rice R2 cDNA, which encodes a CDK-activating kinase, in tobacco leaf explants by using the glucocorticoid-mediated transcriptional induction system. Transient expression of R2 during the first 7 days of culture triggered callus formation in the absence of cytokinin. This phenotype was enhanced by higher expression of R2 or coexpression of cyclin H, and suppressed by treatment with roscovitine, a CDK inhibitor. R2 expression at a later stage did not prevent cells from differentiation into roots, suggesting a restricted period for sensing CDK activities that control differentiation fate of cells during organogenesis.
Triadimefon (Bayleton), a widely used triazole-type fungicide, affects gibberellin (GA) biosynthesis and 14 alpha-demethylase in sterol biosynthesis. The present study revealed that the phenotype of Arabidopsis treated with triadimefon resembled that of a brassinosteroid (BR)-biosynthesis mutant, and that the phenotype was rescued by brassinolide (BL), the most active BR, partly rescued by GA, and fully rescued by the co-application of BL and GA, suggesting that triadimefon affects both BR and GA biosynthesis. The target sites of triadimefon were investigated using a rescue experiment, feeding triadimefon-treated Arabidopsis BR-biosynthesis intermediates, and a binding assay to expressed DWF4 protein, which is reported to be involved in the BR-biosynthesis pathway. The binding assay indicated that the dissociation constant for triadimefon was in good agreement with the activity in an in planta assay. In the triadimefon-treated Arabidopsis cells, the CPD gene in the BR-biosynthesis pathway was up-regulated, probably due to feedback regulation caused by BR deficiency. These results strongly suggest that triadimefon inhibits the reaction catalysed by DWF4 protein and induces BR deficiency in plants. As triadimefon treatment has proved to be beneficial to plants, this result suggests that BR-biosynthesis inhibitors can be applied to crops.
Brassinolide (BL), considered to be the most important brassinosteroid (BR) and playing pivotal roles in the hormonal regulation of plant growth and development, was found to induce disease resistance in plants. To study the potentialities of BL activity on stress responding systems, we analyzed its ability to induce disease resistance in tobacco and rice plants. Wild-type tobacco treated with BL exhibited enhanced resistance to the viral pathogen tobacco mosaic virus (TMV), the bacterial pathogen Pseudomonas syringae pv. tabaci (Pst), and the fungal pathogen Oidium sp. The measurement of salicylic acid (SA) in wild-type plants treated with BL and the pathogen infection assays using NahG transgenic plants indicate that BL-induced resistance does not require SA biosynthesis. BL treatment did not induce either acidic or basic pathogenesis-related (PR) gene expression, suggesting that BL-induced resistance is distinct from systemic acquired resistance (SAR) and wound-inducible disease resistance. Analysis using brassinazole 2001, a specific inhibitor for BR biosynthesis, and the measurement of BRs in TMV-infected tobacco leaves indicate that steroid hormone-mediated disease resistance (BDR) plays part in defense response in tobacco. Simultaneous activation of SAR and BDR by SAR inducers and BL, respectively, exhibited additive protective effects against TMV and Pst, indicating that there is no cross-talk between SAR- and BDR-signaling pathway downstream of BL. In addition to the enhanced resistance to a broad range of diseases in tobacco, BL induced resistance in rice to rice blast and bacterial blight diseases caused by Magnaporthe grisea and Xanthomonas oryzae pv. oryzae, respectively. Our data suggest that BDR functions in the innate immunity system of higher plants including dicotyledonous and monocotyledonous species.
To study the functions of the nuclear genes involved in chloroplast development, we systematically analyzed albino and pale-green Arabidopsis thaliana mutants by using a two-component transposon system based on the Ac/Ds element of maize as a mutagen. One of the pale-green mutants, albino or pale green mutant 1 (designated as apg1), did not survive beyond the seedling stage, when germinated on soil. The chloroplasts of the apg1 plants contained decreased numbers of lamellae with reduced levels of chlorophyll. A gene encoding a 37 kDa polypeptide precursor of the chloroplast inner envelope membrane was disrupted by insertion of the Ds transposon in apg1. The 37 kDa protein had partial sequence similarity to the S-adenosylmethionine-dependent methyltransferase. The apg1 plants lacked plastoquinone (PQ), suggesting that the APG1 protein is involved in the methylation step of PQ biosynthesis, which is localized at the envelope membrane. Our results demonstrate the importance of the 37 kDa protein of the chloroplast inner envelope membrane for chloroplast development in Arabidopsis.
Abscisic acid (ABA) is a phytohormone that plays a key role as a stress signal, regulating water relations during drought conditions, by inducing stomatal closure. However, to date, no putative ABA receptor(s) has been reported at the protein sequence, gene family, or cellular localization levels. We used biotinylated ABA (bioABA) to characterize the ABA-perception sites in the stomatal guard cells of Vicia faba. Treatment with bioABA induced stomatal closure and shrinkage of guard cell protoplasts (GCPs). The ABA-perception sites were visualized by fluorescence microscopy and confocal laser scanning microscopy (CLSM), using bioABA and fluorescence-labeled avidin. Fluorescent particles were observed in patches on the surface of the GCPs. Fluorescence intensity was quantified by flow cytometry (FCM) as well as by CLSM. Binding of bioABA was inhibited by ABA in a dose-dependent manner. Pre-treatment of GCPs with proteinase K also blocked the binding of bioABA. Binding of bioABA was inhibited by RCA-7a, an ABA analog that induces stomatal closure, but not by RCA-16, which has no effect on stomatal aperture. Another ABA analog, PBI-51, inhibited ABA-induced stomatal closure. This ABA antagonist also inhibited binding of bioABA to the GCPs. These results suggest that ABA is perceived on the plasma membrane of stomatal guard cells, and that the present experimental methods constitute valuable tools for characterizing the nature of the ABA receptor(s) that perceives physiological ABA signals. These imaging studies allow us to demonstrate the spatial distribution of the ABA-perception sites. Visualization of the ABA-perception sites provides new insights into the nature of membrane-associated ABA receptor(s).
Japanese morning glory (Pharbitis nil) is a model plant characterized by a large stock of spontaneous mutants. The recessive mutant Uzukobito shows strong dwarfism with dark-green rugose leaves. The phenotype was rescued by the application of brassinolide, a bioactive brassinosteroid (BR), indicating that Uzukobito was a BR-deficient mutant. A detailed analysis of the endogenous BR levels in Uzukobito and its parental wild-type plant showed that Uzukobito had a lower level of BRs downstream of (24R)-24-methyl-5alpha-cholestan-3-one and (22S, 24R)-22-hydroxy-24-methyl-5alpha-cholestan-3-one than those in wild-type plants, while their immediate precursors (24R)-24-methylcholest-4-en-3-one and (22S, 24R)-22-hydroxy-24-methylcholest-4-en-3-one accumulated relatively more in Uzukobito. These results indicate that Uzukobito had a defect in the conversion of (24R)-24-methylcholest-4-en-3-one and (22S, 24R)-22-hydroxy-24-methylcholest-4-en-3-one to their 5alpha-reduced forms, which is catalyzed by de-etiolated2 (DET2) in Arabidopsis. The P. nil ortholog of the DET2 gene (PnDET2) was cloned and shown to have the greatest similarity to DET2 among all the putative genes in Arabidopsis. Uzukobito had one amino acid substitution from Glu62 to Val62 in the deduced amino acid sequence of PnDET2. Recombinant PnDET2 expressed in COS-7 cells was found to be a functional steroid 5alpha-reductase (S5alphaR) converting (24R)-24-methylcholest-4-en-3-one to (24R)-24-methyl-5alpha-cholestan-3-one, while PnDET2 with the mutation did not show any catalytic activity. This shows that a plant S5alphaR can convert an intrinsic substrate. All these results clearly demonstrate that the Uzukobito phenotype resulted from a mutation on PnDET2, and a morphological mutant has been characterized at the molecular level among a large stock of P. nil mutants.
Lignostilbene-alpha,beta-dioxygenase (LSD, EC 1.13.11.43) is involved in oxidative cleavage of the central double bond of lignostilbene to form the corresponding aldehydes by a mechanism similar to those of 9-cis-epoxycarotenoid dioxygenase and beta-carotene 15,15'-dioxygenase, key enzymes in abscisic acid biosynthesis and vitamin A biosynthesis, respectively. In this study, several N-benzylideneanilines and amine were synthesized and examined for their efficacy as inhibitors of LSD. N-(4-Hydroxybenzylidene)-3-methoxyaniline was found to be a potent inhibitor with IC50 = 0.3 microM and N-(4-hydroxybenzyl)-3-methoxyaniline was also active with IC50 = 10 microM. The information obtained from the structure-activity relationships study here can aid in discovering inhibitors of both abscisic acid and vitamin A biosynthesis.
Brassinosteroids (BRs) are steroidal phytohormones that are essential for many processes in plant growth and development, such as cell expansion, vascular differentiation, and responses to stress. The effects of BRs on cell division are unclear, as attested by contradictory published results. To determine the effect of BRs on cell division, the tobacco (Nicotiana tabacum) BY-2 cell line, which is a widely-used model system in plant cell biology, was used. It was found that brassinolide (BL) promoted cell division only during the early phase of culture and in the absence of auxin (2,4-D). This promotion of cell division was confirmed by RNA gel blot analyses using cell-cycle-related gene probes. At later stages in the culturing periods of BL-supplied and 2,4-D-supplied BY-2 cells, differences in cell multiplication and cell-cycle-related gene expression were observed. Moreover, the BL-treated BY-2 cells had morphological differences from the 2,4-D-treated cells. To determine whether suppressed organellar DNA replication limited this promotion of cell division during the early culture phase, this replication was examined and it was found that BL treatment had no effect on activating organellar (plastid- and mitochondrial-) DNA synthesis. As preferential organellar DNA synthesis, which is activated by 2,4-D, is necessary during successive cell divisions in BY-2 cells, these data suggest that the mechanism of the promotion of cell division by BL treatment is distinct from that regulated by the balance of auxin and cytokinin.
Corneal neovascularization develops in several pathologic conditions, but its underlying mechanisms remain elusive. We used a mouse inflammatory corneal model (corneas cauterized with silver nitrate) and assessed the role of monocyte/macrophage-attracting factors, macrophage chemotactic protein-1 (MCP-1), and a proinflammatory cytokine, IL-1beta, on macrophage recruitment and neovascularization. Both MCP-1, IL-1beta protein, and mRNA levels increased markedly 12 hours after the chemical cauterization. In situ hybridization showed that MCP-1 was located in corneal epithelial cells, and IL-1beta was located in corneal epithelial cells and infiltrating inflammatory cells. In addition, double staining of corneas with antibodies specific for monocytes/macrophages and IL-1beta revealed that IL-1beta was found in infiltrating monocytes/macrophages at Day 2 after cauterization. Both IL-1beta and MCP-1 induced neovascularization in a rat cornea model, and the cauterization-induced corneal neovascularization was partially inhibited by subconjunctival injection of anti-IL-1beta or anti-MCP-1. Coadministration of two antibodies inhibited corneal neovascularization slightly more than that by the administration of each. In contrast, administration of the anti-MCP-1 or anti-IL-1beta showed minimal inhibition of basic fibroblast growth factor-driven corneal neovascularization by mouse cornea assay. Cauterized corneas treated with anti-MCP-1 antibody had significantly fewer monocytes/macrophages than control. These results indicate the existence of distinct monocyte/macrophage-involved angiogenic pathways in mouse cornea, in which MCP-1 released from corneal epithelial cells attracts monocytes/macrophages into the cornea, where they release IL-1beta leading to inflammatory neovascularization. In addition, the IL-1beta and MCP-1 released from the corneal epithelial cells may directly induce corneal neovascularization.