Construction and analysis of EST libraries of the trans-polyisoprene producing plant, Eucommia ulmoides Oliver.

Eucommia ulmoides Oliver is one of a few woody plants capable of producing abundant quantities of trans-polyisoprene rubber in their leaves, barks, and seed coats. One cDNA library each was constructed from its outer stem tissue and inner stem tissue. They comprised a total of 27,752 expressed sequence tags (ESTs) representing 10,520 unigenes made up of 4,302 contigs and 6,218 singletons.

Efficient in vitro synthesis of cis-polyisoprenes using a thermostable cis-prenyltransferase from a hyperthermophilic archaeon Thermococcus kodakaraensis.

The Tk-idsB encoding cis-prenyltransferase which catalyzes consecutive cis-condensation of isopentenyl diphosphate to allylic diphosphate was isolated from a hyperthermophilic archaeon Thermococcus kodakaraensis, and enzymatic characteristics of the recombinant Tk-IdsB were examined. Tk-IdsB was not fully denatured even at 90 degrees C and preferably utilizes both C(10) and C(15) allylic diphosphates to yield mainly the C(60)-C(65) products. Based on structural models, single alanine-substitution mutants at Glu68, Lys109, or Leu113 were constructed, showing that all the three produced longer chains (C(65)-C(70)) than the wild-type and the substitution at 109 (K109A) was the most effective.

Expression of chlorophyllase is not induced during autumnal yellowing in Ginkgo biloba.

Autumnal tints are one of the most fascinating natural phenomena, but the molecular mechanism of chlorophyll (Chl-)degradation in deciduous trees has not been fully understood. In this study, from the leaves of Ginkgo biloba, chlorophyllase-homologous GbCLH was cloned by RT-PCR with degenerated primers. The expression of GbCLH in different yellowing stages was analyzed by Northern hybridization.

The essential fatty acid myristate causes severe growth retardation in Hpelo disruptants of the yeast Hansenula polymorpha.

Myristate (C14:0) is an essential multi-functional fatty acid in a variety of organisms. We found that C14:0, but not other fatty acids, causes severe growth retardation in Hpelo1Delta and Hpelo2Delta mutants of the yeast Hansenula polymorpha, defective in elongation of very long-chain fatty acids. While transcription of HpELO1 and HpELO2 is transiently increased by C14:0, this was not found to be responsible for the growth retardation.

Efficient synthesis of trans-polyisoprene compounds using two thermostable enzymes in an organic-aqueous dual-liquid phase system.

The trans-polyisoprene compounds are synthesized by trans-isoprenyl diphosphate synthase (IDS) with consecutive condensation of isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP). The in vitro condensation by IDS does not proceed efficiently by hydrophobic interaction between IDS and the hydrocarbon of longer products. In the present study, the enzymatic synthesis of trans-polyisoprenyl diphosphates was attempted in an organic-aqueous dual-liquid phase system with thermostable enzymes obtained from Thermococcus kodakaraensis.

Characterization and subcellular localization of chlorophyllase from Ginkgo biloba.

Chlorophyllase (Chlase) catalyzes the initial step of chlorophyll (Chl)-degradation, but the physiological significance of this reaction is still ambiguous. Common understanding of its role is that Chlase is involved in de-greening processes such as fruit ripening, leaf senescence, and flowering. But there is a possibility that Chlase is also involved in turnover and homeostasis of Chls.

An isotope effect on the comparative quantification of flavonoids by means of methylation-based stable isotope dilution coupled with capillary liquid chromatography/mass spectrometry.

Ionization suppression is a serious problem in liquid chromatography/mass spectrometry-based metabolomics, and stable isotope dilution-based comparative quantification is one of the most important methods of overcoming this problem. Herein, the use of [(13)C]-methylation-based stable isotope dilution for comparative quantification of flavonoids is demonstrated. This is in contrast to the equivalent deuterium labeling methylation method, which has an adverse isotope effect on reverse phase chromatography.

A facile transformation of Arabidopsis thaliana using ceramic supported propagation system.

Arabidopsis thaliana can be a useful model plant in the field of plant molecular biology because it is a sole dicotyledonous plant whose genome sequence has been completely determined at the present time. To develop functional genomics of A. thaliana, a large number of transgenic plants must be produced.

Introduction of the archaebacterial geranylgeranyl pyrophosphate synthase gene into Chlamydomonas reinhardtii chloroplast.

Geranylgeranyl pyrophosphate (GGPP) synthase gene (gds) derived from a thermophilic Archae Sulfolobus acidocaldarius, was introduced into a unicellular green alga Chlamydomonas reinhardtii chloroplast. Heat treatment abolished the prenyltransferase activity of the wild strain, but the activity of the transforment remained. The transformant accumulated gds gene mRNA and translation product.

Methylamine treatment changes the allocation of carbohydrate to roots in rice plants.

Methylamine (MA), an ammonia analogue, has been used to investigate ammonia uptake. This compound competes with ammonia to be taken up and also inhibits the nitrate assimilation pathway. However, the effect of MA on plant growth is unknown.

A novel gene delivery system in plants with calcium alginate micro-beads.

We have produced micrometer-sized calcium alginate beads referred to as "bio-beads" that encapsulate plasmid DNA molecules carrying a reporter gene. In order to evaluate the efficiency of the bio-beads in mediating genetic transfection, protoplasts isolated from cultured tobacco cells (BY-2) were transfected with bio-beads containing a plasmid that carries the modified green fluorescent protein gene CaMV35S-sGFP. With the bio-beads treatment, approximately ten-fold higher GFP expression was observed after 24 h incubation compared to that with the conventional method using a naked plasmid solution.

A novel microsurgery method for intact plant tissue at the single cell level using ArF excimer laser microprojection.

A novel microsurgery technique for the partial removal of rigid cell-walls in intact plant tissue is established. Using a size-variable slit, an ArF excimer laser was microprojected on the surface of the targeted cell, and this method enabled the area- and depth-controllable processing of the cortical structure of plant cells including the cuticle and cell wall layer. In epidermal cells of all tested plants, viabilities of more than 90% were retained 24 h after irradiation.

Stable isotope dilution-based accurate comparative quantification of nitrogen-containing metabolites in Arabidopsis thaliana T87 cells using in vivo (15)N-isotope enrichment.

Stable isotope dilution-based comparative quantification of nitrogen-containing metabolites for highly sensitive and selective metabolomics was developed using liquid chromatography/mass spectrometry (LC/MS) and (15)N-isotope enrichment. We produced metabolically stable isotope-labeled Arabidopsis T87 cells by culturing with (15)N-labeled medium. We found that the growth of cells maintained in (15)N-labeled medium is very similar to the growth in normal medium, as evidenced by cell morphology, doubling time, and measurement of chlorophyll and carotenoid contents.

Structural characterization of alpha-terminal group of natural rubber. 1. Decomposition of branch-points by lipase and phosphatase treatments.

Deproteinized natural rubber latex (DPNR-latex) was treated with lipase and phosphatase in order to analyze the structure of the chain-end group (alpha-terminal). The enzymatic treatment decreased the content of long-chain fatty acid ester groups in DPNR from about 6 to 2 mol per rubber molecule. The molecular weight and intrinsic viscosity were reduced to about one-third after treatment with lipase and phosphatase.

Cloning of a cryptochrome homologue from the holoparasitic plant Orobanche minor Sm.

Orobanche minor is a non-photosynthetic root holoparasitic plant. Although it is known that photosynthesis-related genes are inactivated or have been eliminated from the plastid genomes of holoparasites, little is known about the alterations in their genes involved in the signaling networks by which light regulates photosynthesis. Cryptochromes (crys), which are blue-light receptors, appear to control both photosynthesis-related and non-photosynthetic responses to light in higher plants.

Enzymatic and structural characterization of type II isopentenyl diphosphate isomerase from hyperthermophilic archaeon Thermococcus kodakaraensis.

Enzymatic and thermodynamic characteristics of type II isopentenyl diphosphate (IPP):dimethylallyl diphosphate (DMAPP) isomerase (Tk-IDI) from Thermococcus kodakaraensis, which catalyzes the interconversion of IPP and DMAPP, were examined. FMN was tightly bound to Tk-IDI, and the enzyme required NADPH and Mg2+ for the isomerization in both directions. The melting temperature (Tm), the change of enthalpy (deltaH(m)), and the heat capacity change (deltaC(p)) of Tk-IDI were 88.

Functional analysis of two solanesyl diphosphate synthases from Arabidopsis thaliana.

Solanesyl diphosphate (SPP) is regarded as the precursor of the side-chains of both plastoquinone and ubiquinone in Arabidopsis thaliana. We previously analyzed A. thaliana SPP synthase (At-SPS1) (Hirooka et al.

Transient RNAi induction against endogenous genes in Arabidopsis protoplasts using in vitro-prepared double-stranded RNA.

RNA interference is a powerful technique for suppressing gene functions in many eukaryotes including plants. Here we show that introduction of double-stranded RNA into Arabidopsis protoplasts leads to marked silencing of endogenous genes, as observed previously for transgenes [Biosci. Biotechnol.

Isolation and characterization of a cDNA encoding phytochrome A in the non-photosynthetic parasitic plant, Orobanche minor Sm.

In this study, the isolation and characterization of a phytochrome A (PHYA) homologous cDNA (OmPHYA) in the non-photosynthetic holoparasitic plant Orobanche minor are described. The present findings provide the first report of the presence of a PHYA homolog in the holoparasite. This study found that OmPHYA is of similar size to the other PHYAs of green plants and shows 72, 77, and 77% amino acid sequence identity with PHYA in Arabidopsis, potato, and tobacco respectively.

Transient RNA silencing of scoulerine 9-O-methyltransferase expression by double stranded RNA in Coptis japonica protoplasts.

RNAi (RNA interference, RNA silencing) is a powerful tool for functional genomics, but the construction of an RNAi vector(s) and the establishment of stable transformants are time-consuming and laborious. Here we report the transient RNAi of endogenous biosynthetic genes involved in isoquinoline alkaloid biosynthesis in Coptis japonica protoplasts. Double stranded (ds) RNA fragments of various lengths prepared from several different positions of the coding sequence of scoulerine 9-O-methyltransferase (SMT) were introduced into C.

Cloning and characterization of farnesyl diphosphate synthase from the rubber-producing mushroom Lactarius chrysorrheus.

Farnesyl diphosphate is involved in rubber biosynthesis as an initiating substrate for both polyprenol and mushroom rubber. So far, we have isolated the cDNA of a farnesyl diphosphate synthase (FPS) for the first time from a rare rubber-producing mushroom, Lactarius chrysorrheus, by the degenerate RT-PCR technique based on sequence information of FPS genes from fungi and yeasts. The open reading frame was clarified to encode a protein of 381 amino acid residues with a calculated molecular weight of 42.

Temperature-dependent modulation of farnesyl diphosphate/geranylgeranyl diphosphate synthase from hyperthermophilic archaea.

Enzyme characteristics of trans-prenyl diphosphate synthase (Tk-IdsA) from Thermococcus kodakaraensis, which catalyzes the consecutive trans-condensation of isopentenyl diphosphate (C(5)) units with allylic diphosphate, were examined. Product analysis revealed that Tk-IdsA is a bifunctional enzyme, farnesyl diphosphate (FPP, C(15))/geranylgeranyl diphosphate (GGPP, C(20)) synthase, and mainly yields both C(15) and C(20). The FPP/GGPP product ratio increases with the rise of the reaction temperature.

Biosynthetic pathway for the C45 polyprenol, solanesol, in tobacco.

Feeding experiments were independently performed with [1-13C]deoxy-D-xylulose triacetate and (RS)-[2-13C]mevalonolactone in the tobacco plant. The labeling pattern for solanesol was elucidated to reveal that the isoprene moiety of solanesol would be derived from deoxy-xylulose. The result strongly suggests that tobacco solanesol is biosynthesized via the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway.

Rapid and high-resolution analysis of geometric polyprenol homologues by connected octadecylsilylated monolithic silica columns in high-performance liquid chromatography.

A Chromolith Performance octadecylsilyl (ODS) monolithic silica column (Merck) was compared with a conventional microparticulate ODS-bonded silica column in the high-performance liquid chromatography separation of natural polyprenols. A system comprising two connected monolithic columns afforded an equivalent separation at half the analysis time of the conventional method. Furthermore, ten connected columns achieved a tremendously high-resolution separation, in which the complicated series of homologous polyprenols with geometric isomerism were fully separated.

Expression of fungal pectin methylesterase in transgenic tobacco leads to alteration in cell wall metabolism and a dwarf phenotype.

A transgenic tobacco plant (Nicotiana tabacum L.) expressing a fungal pectin methylesterase (PME; EC 3.1.