Biomedical subjects
Publications and source records attributed to Shinjiro Yamaguchi.
Rice anthers contain high concentrations of gibberellins A(4) and A(7). To understand their physiological roles, we examined the site of their biosynthesis by analyzing the expression pattern of a gene (OsCPS) encoding coparyl diphosphate synthase in developing rice flowers. Expression was apparent in the anthers 1-2 days before flowering, and CPS mRNA accumulated in the maturing pollen.
The plant growth hormone gibberellin (GA) is important for many aspects of plant growth and development. Although most genes encoding enzymes at each step of the GA biosynthetic pathway have been cloned, their regulation is less well understood. To assess how up-regulation of early steps affects the biosynthetic pathway overall, we have examined transgenic Arabidopsis plants that overexpress either AtCPS or AtKS or both. These genes encode the enzymes ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase, which catalyze the first two committed steps in GA biosynthesis. We find that both CPS and CPS/ent-kaurene synthase overexpressors have greatly increased levels of the early intermediates ent-kaurene and ent-kaurenoic acid, but a lesser increase of later metabolites. These overexpression lines do not exhibit any GA overdose morphology and have wild-type levels of bioactive GAs. Our data show that CPS is limiting for ent-kaurene production and suggest that conversion of ent-kaurenoic acid to GA12 by ent-kaurenoic acid oxidase may be an important rate-limiting step for production of bioactive GA. These results demonstrate the ability of plants to maintain GA homeostasis despite large changes in accumulation of early intermediates in the biosynthetic pathway.
The hormone-mediated control of plant growth and development involves both synthesis and response. Previous studies have shown that gibberellin (GA) plays an essential role in Arabidopsis seed germination. To learn how GA stimulates seed germination, we performed comprehensive analyses of GA biosynthesis and response using gas chromatography-mass spectrometry and oligonucleotide-based DNA microarray analysis. In addition, spatial correlations between GA biosynthesis and response were assessed by in situ hybridization. We identified a number of transcripts, the abundance of which is modulated upon exposure to exogenous GA. A subset of these GA-regulated genes was expressed in accordance with an increase in endogenous active GA levels, which occurs just before radicle emergence. The GA-responsive genes identified include those responsible for synthesis, transport, and signaling of other hormones, suggesting the presence of uncharacterized crosstalk between GA and other hormones. In situ hybridization analysis demonstrated that the expression of GA-responsive genes is not restricted to the predicted site of GA biosynthesis, suggesting that GA itself, or GA signals, is transmitted across different cell types during Arabidopsis seed germination.
A GA biosynthesis inhibitor, uniconazole, caused many shrunken embryos when it was supplied to cultured carrot (Daucus carota L.) cells at the induction of somatic embryos. The abnormality was prevented by exogenous GA(1) or GA(4). To analyze the status of GA biosynthesis during somatic embryogenesis, expression patterns of newly isolated genes encoding GA biosynthetic enzymes, two GA 20-oxidases, three GA 3-oxidases, and two GA 2-oxidases were observed by using a semi-quantitative reverse-transcription-polymerase chain reaction with gene-specific primers. Transcript levels of GA 20-oxidases and GA 2-oxidases did not change greatly during development of the somatic embryo. On the other hand, drastic changes were found in three GA 3-oxidase genes. Strikingly, expression of a GA 3-oxidase gene, DcGA3ox2, was elevated once in somatic embryogenesis, but not in the non-induced suspension cells. The enzymatic functions of these gene products were also confirmed using recombinant proteins expressed in Escherichia coli. Our results indicate that GA biosynthesis is required for carrot somatic embryogenesis.
In anesthesized rats, the effects of electrical stimulation (ES) to the saphenous nerve on the microcirculation of the gracilis muscle were assessed through the measurement of two different hemodynamic parameters: (a). the muscle blood flow (MBF) using a laser Doppler flowmeter; and (b). the changes in diameter of the muscle arterioles observed directly using an intravital microscope system. Ipsilateral ES (5 V, 20 Hz, for 30 s) produced increases in MBF and mean arterial pressure (47+/-10% and 18+/-5%) over the baseline, while no significant changes in MBF were observed in the contralateral muscle. Neither selective nor simultaneous alpha- and beta-adrenergic blockade altered the increases in MBF induced by ipsilateral ES. The arteriolar diameter was found to increase by 38.9+/-5% following ipsilateral ES. This response in diameter was abolished after the topical application of a calcitonin gene-related peptide receptor antagonist (CGRP(8-37)). Contralateral ES produced a decrease in arteriolar diameter by 26+/-14%. Thus, ipsilateral nerve ES produced vasodilative responses in the muscle accompanied by increases in MBF independently of the sympathetic activity. Furthermore, CGRP was found directly involved in the reflex neural regulation of the muscle microcirculation, which suggests the participation of an axon reflex mechanism.
Gibberellins (GAs) are diterpene plant hormones essential for many developmental processes. Although the GA biosynthesis pathway has been well studied, our knowledge on its early stage is still limited. There are two possible routes for the biosynthesis of isoprenoids leading to GAs, the mevalonate (MVA) pathway in the cytosol and the methylerythritol phosphate (MEP) pathway in plastids. To distinguish these possibilities, metabolites from each isoprenoid pathway were selectively labeled with (13)C in Arabidopsis seedlings. Efficient (13)C-labeling was achieved by blocking the endogenous pathway chemically or genetically during the feed of a (13)C-labeled precursor specific to the MVA or MEP pathways. Gas chromatography-mass spectrometry analyses demonstrated that both MVA and MEP pathways can contribute to the biosyntheses of GAs and campesterol, a cytosolic sterol, in Arabidopsis seedlings. While GAs are predominantly synthesized through the MEP pathway, the MVA pathway plays a major role in the biosynthesis of campesterol. Consistent with some crossover between the two pathways, phenotypic defects caused by the block of the MVA and MEP pathways were partially rescued by exogenous application of the MEP and MVA precursors, respectively. We also provide evidence to suggest that the MVA pathway still contributes to GA biosynthesis when this pathway is limiting.
Recent reports have focused on the mechanisms of the action of electro-acupuncture stimulation (EAS) in the regulation of blood flow to different tissues. In the knee joint, blood flow is known to be modulated mainly by sympathetic postganglionic fibers, but recently the release or induction of nitric oxide (NO) synthesis in response to electrical stimulation has also been suggested. Therefore, a direct observation of the microcirculation is needed to further understand the mechanism by which blood flow is regulated by somatic afferent stimulation. In the present study, the effects of EAS to the vastus medialis muscle on systemic hemodynamics and the knee joint microcirculation were observed in vivo using a real-time confocal laser-scanning microscope system (CLMS). Electrical stimulation (5 mA, 0.5 ms, 5 Hz) was applied for 30 min using a pair of acupuncture needles introduced into the vastus medialis muscle. To clarify a plausible involvement of NO in the responses to EAS, the stimulus was applied either in the presence or absence of N(omega)-nitro-L-arginine methyl ester (L-NAME). Stimulation to either the muscle or the skin of the thigh after blockade of neuromuscular transmission was performed to determine the involvement of muscle contraction during EAS treatment. The changes in mean arterial pressure (MAP) and diameter of the arterioles supplying the knee joint were monitored continuously until 60 min poststimulus. Significant and persistent increases in arteriolar diameter by 26 +/- 6% and MAP by 17 +/- 2%, respectively, were observed after EAS to the muscle. Electro-acupuncture to the vastus medialis in the presence of L-NAME produced a strong decrease in diameter of the knee joint arterioles by -38 +/- 14% under the baseline with a simultaneous increase of 35 +/- 5% in MAP. EAS to the skin did not produce changes in arteriolar diameter while a slight increase in MAP by 12 +/- 6% over the baseline occurred after the stimulus. EAS to the muscle after neuromuscular blockade did not produce significant changes in diameter, while an increase in MAP by 24 +/- 8% was still observed, which facts suggest that the muscle contraction is required to produce vasodilatation. These responses suggest that a dynamic balance between the autonomic nervous system and the release of NO is the primary mechanism mediating the EAS effects on knee joint microcirculation.
Cucumber (Cucumis sativus) hypocotyls were cut to one-half of their diameter transversely, and morphological and histochemical analyses of the process of tissue reunion in the cortex were performed. Cell division in the cortex commenced 3 d after cutting, and the cortex was nearly fully united within 7 d. 4',6-Diamidino-2-phenylindole staining and 5-bromo-2'-deoxyuridine labeling experiments indicate that nDNA synthesis occurred during this process. In addition, specific accumulation of pectic substances was observed in the cell wall of attached cells in the reunion region of the cortex. Cell division during tissue reunion was strongly inhibited when the cotyledon was removed. This inhibition was reversed by applying gibberellin (GA, 10(-4) M GA3) to the apical tip of the cotyledon-less plant. Supporting this observation, cell division in the cortex was inhibited by treatment of the cotyledon with 10(-4) M uniconazole-P (an inhibitor of GA biosynthesis), and this inhibition was also reversed by simultaneous application of GA. In contrast to the essential role of cotyledon, normal tissue reunion in cut hypocotyls was still observed when the shoot apex was removed. The requirement of GA for tissue reunion in cut hypocotyls was also evident in the GA-deficient gib-1 mutant of tomato (Lycopersicon esculentum). Our results suggest that GA, possibly produced in cotyledons, is essential for cell division in reuniting cortex of cut hypocotyls.
The effects of electrical stimulation of the dorsal skin area on the mesenteric arterioles were investigated in anesthetized rats by the use of an intravital microscope-television system. Changes in the diameter of the mesenteric precapillary arterioles (10-40 microm in diameter) were measured with an image processor. Blood flow velocity in the mesenteric precapillary arterioles was monitored by the dual sensor method developed by the authors. Electrical stimulation was performed through two platinum electrodes placed at the right dorsal Th5-12 level skin area by the use of an electrical stimulator (0.2 ms, 20 Hz). Continuous stimulation lasting for 30 s (1-10 mA) and intermittent stimulation lasting for 10 min (3 mA) were applied. The pressor response following the depressor response was induced by a stimulus current above 8 mA. The decrease in mesenteric blood flow velocity was induced by stimulus current above 10 mA. These responses were abolished by lidocaine injection into the subcutaneous area where the electrodes were attached. No significant change in arteriolar diameter or heart rate were induced by the stimulation for 30 s. Electrical stimulation of the skin for 10 min evoked a decrease in the diameter of arterioles (-3.4 +/- 2%, p < 0.01, n = 12). In the adrenalectomized group, electrical stimulation of the skin for 10 min elicited a slight increase in the diameter (1.1 +/- 0.5%, n = 6). It is therefore suggested that electrical stimulation of the skin for 30 s reflexly evoked decreases in MAP and in blood flow velocity, and that the constriction of the mesenteric precapillary arterioles induced by the stimulation for 10 min was mediated by humoral adrenaline and noradrenaline released by somato-adrenal medullary reflex.
Bioactive gibberellins (GAs) promote seed germination in a number of plant species. In dicots, such as tomato and Arabidopsis, de novo GA biosynthesis after seed imbibition is essential for germination. Light is a crucial environmental cue determining seed germination in some species. The red (R) and far-red light photoreceptor phytochrome regulates GA biosynthesis in germinating lettuce and Arabidopsis seeds. This effect of light is, at least in part, targeted to mRNA abundance of GA 3-oxidase, which catalyzes the final biosynthetic step to produce bioactive GAs. The R-inducible GA 3-oxidase genes are predominantly expressed in the hypocotyl of Arabidopsis embryos. This predicted location of GA biosynthesis appears to correlate with the photosensitive site determined by using R micro-beam in lettuce seeds. The GA-deficient non-germinating mutants have been useful for studying how GA stimulates seed germination. In tomato, GA promotes the growth potential of the embryo and weakens the structures surrounding the embryo. Endo-b-mannanase, which is produced specifically in the micropylar endosperm in a GA-dependent manner, may be responsible for breaking down the endosperm cell walls to assist germination. Recently, a role for GA in overcoming the resistance imposed by the seed coat was also suggested in Arabidopsis from work with a range of seed coat mutants. Towards understanding the GA signaling pathway, GA response mutants have been isolated and characterized, some of which are affected in GA-stimulated seed germination.