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. Homologues of genes coding for rubber particle membrane proteins that participate in the synthesis of high-molecular poly-isoprene in latex were isolated, as well as those encoding known major latex proteins (MLPs). MLPs extensively shared ESTs, indicating their abundant expression during trans-polyisoprene rubber biosynthesis. The six mevalonate pathway genes which are implicated in the synthesis of isopentenyl diphosphate (IPP), a starting material of poly-isoprene biosynthesis, were isolated, and their role in IPP biosynthesis was confirmed by functional complementation of suitable yeast mutants. Genes encoding five full-length trans-isoprenyl diphosphate synthases were also isolated, and two among those synthesized farnesyl diphosphate from IPP and dimethylallyl diphosphate, an assumed intermediate of rubber biosynthesis. This study should provide a valuable resource for further studies of rubber synthesis in E. ulmoides.

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. Tk-IdsB was applied to an organic-aqueous dual-phase system and more than 90% of the products were recovered from the organic phase when 1-butanol or 1-pentanol was overlaid. When 1-octanol was overlaid, 70% of the products were obtained from the upper organic phase. The product distributions were changed depending on the hydrophobicity of organic solvents used. Tk-IdsB was then immobilized onto silica beads to make Tk-IdsB more tolerant, showing that half-life of enzyme at 80 degrees C was prolonged by immobilization. When the immobilized Tk-IdsB was applied in the organic-aqueous dual-phase system, immobilized Tk-IdsB catalyzed consecutive condensation more efficiently than the unimmobilized one.

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. Transcription of HpFAS1 and HpFAS2 encoding fatty acid synthase is repressed by C14:0, but this repression was also not found to be responsible for growth retardation. A screen for suppressors that resulted in restored growth of the Hpelo1Delta disruptant on media containing C14:0 identified two types of suppressors. One exhibited a defect in C14:0 uptake while the other did not. Molecular genetic and genomic analysis of these suppressor mutations is anticipated to shed new light on the processes of fatty acid transport and the crucial role of C14:0 in the growth of eukaryotic organisms.

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. The conversion from DMAPP to a longer-chain product was achieved in a dual-liquid phase system, and more than 80% of the products were recovered in the organic phase. When the mutant IDS-Y81S, in which Tyr81 is replaced with Ser, was used in the dual-phase system, productivity was enhanced about four times and the ratio of the longer-chain products was increased. Co-incubation of IPP isomerase from T. kodakaraensis with IDS or IDS-Y81S enabled the direct synthesis of polyisoprenyl diphosphates from IPPs.

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. Among the de-greening processes, autumnal coloration is one of the most striking natural phenomena, but the involvement of Chlase during autumnal coloration is not clear. Previously, it was shown that Chlase activity and expression level of the Chlase gene were not increased during autumnal coloration in Ginkgo biloba, indicating that Chlase does not work specially in the de-greening processes in G. biloba. In this study, we characterized the recombinant Chlase and analyzed its subcellular localization to understand the role of the cloned Chlase of G. biloba (GbCLH). GbCLH exhibited its highest activity at pH 7.5, 40 degrees C. Kinetic analysis revealed that GbCLH hydrolyzes pheophytin (Pheo) a and Chl a more rapidly than Pheo b and Chl b. Transient expression analysis of 40 N-terminus amino acids of GbCLH fused with GFP (green fluorescent protein) and subcellular fractionation showed that GbCLH localizes within chloroplasts. Together with our previous results, property of GbCLH and its location within the chloroplasts suggest that GbCLH plays a role in the turnover and homeostasis of Chls in green leaves of G. biloba.

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. Scanning electron microscope (SEM) observation revealed that the cuticle layer of the irradiated region was completely ablated, and the cellulose microfibrils of the secondary cell wall were partially removed; furthermore, 4 days after laser treatment, the regeneration of cell wall fibrils was observed. As a model experiment, the transient expression of synthetic green fluorescent protein (sGFP) was performed by the microinjection of cauliflower mosaic virus (CMV) 35S promoter-derived sGFP gene through an "aperture" in the treated cell surface. Moreover, micron-sized fluorescent beads were successfully introduced by the same method into the onion cells indicating that this method can be used to introduce foreign materials as large as organelles.

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.

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. Complete incorporation of (15)N in folate, S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH) in T87 cells was accomplished after culturing for 21 d. Accurate comparative quantification of folate, SAM, and SAH was established by means of LC/MS using the isotopomers of the target metabolites as internal standards. The within- and between-run assay coefficients of variation for the folate, SAM, and SAH levels were all less than 8.5%. Stable isotope labeling by nitrogen source in Arabidopsis T87 cell culture provided simple, inexpensive, and accurate amino acid profiling. This interesting new protocol is valuable for the study of dynamic changes in N-compound pools in cultured cells.

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. The Huggins' k' constant of the enzyme-treated DPNR showed the formation of linear rubber molecules. The molecular weight distribution of DPNR changed apparently after treatment with lipase and phosphatase. (1)H NMR spectrum of rubber obtained from DPNR-latex showed small signals due to monophosphate, di-phosphate and phospholipids at the alpha-terminus. Treatment of DPNR-latex with lipase and phosphatase decreased the relative intensity of the (1)H NMR signals corresponding to phospholipids, whereas no change was observed for the signals due to mono- and diphosphates. The residual mono- and diphosphate signals as well as some phospholipid signals after lipase and phosphatase treatments indicate that mono- and diphosphate groups are directly linked at the alpha-terminus with the modified structure, expected by aggregation or linking with phospholipid molecules.

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 88.0 degrees C, 444 kJ mol(-1), and 13.2 kJ mol(-1) K(-1), respectively, indicating that Tk-IDI is fairly thermostable. Kinetic parameters dramatically changed when the temperature crossed 80 degrees C even though its native overall structure was stably maintained up to 90 degrees C, suggesting that local conformational change would occur around 80 degrees C. This speculation was supported by the result of the circular dichroism analysis that showed the shift of the alpha-helical content occurred at 80 degrees C.

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. Because we are interested in to what extent a cry-mediated light signaling network remains in the holoparasites, we cloned CRY homologous cDNA from O. minor (OmCRY1) and used real-time RT-PCR to compare its expression under natural daylight and darkness. We found that the OmCRY1 has a high degree of homology with CRY1 s from photosynthetic plants. Expression of the OmCRY1 gene was higher in plants grown in the dark than that in the plants grown under natural daylight. This is the first report of the gene expression of a blue-light receptor in non-photosynthetic plants.

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., Biochem. J., 370, 679-686 (2003)). In this study, we cloned a second SPP synthase (At-SPS2) gene from A. thaliana and characterized the recombinant protein. Kinetic analysis indicated that At-SPS2 prefers geranylgeranyl diphosphate to farnesyl diphosphate as the allylic substrate. Several of its features, including the substrate preference, were similar to those of At-SPS1. These data indicate that At-SPS1 and At-SPS2 share their basic catalytic machinery. Moreover, analysis of the subcellular localization by the transient expression of green fluorescent protein-fusion proteins showed that At-SPS2 is transported into chloroplasts, whereas At-SPS1 is likely to be localized in the endoplasmic reticulum in the A. thaliana cells. It is known that the ubiquinone side-chain originates from isopentenyl diphosphate derived from the cytosolic mevalonate pathway, while the plastoquinone side-chain is synthesized from isopentenyl diphosphate derived from the plastidial methylerythritol phosphate pathway. Based on this information, we propose that At-SPS1 contributes to the biosynthesis of the ubiquinone side-chain and that At-SPS2 supplies the precursor of the plastoquinone side-chain in A. thaliana.

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. Biochem., 67, 2674-2677 (2003)]. This simple system should be useful for functional analysis of genes involved in fundamental cellular processes.

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. japonica protoplasts by polyethylene glycol-mediated transformation, and their effects were monitored by reverse transcription-polymerase chain reaction. Substantial silencing of SMT gene expression was obtained by the introduction of these SMT dsRNAs. A significant reduction in SMT protein levels was also observed. The potentials of this transient RNAi system to evaluate the functions of biosynthetic genes in Coptis alkaloid research are discussed.

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. The OmPHYA contains a conserved chromophore attachment cysteine at position 323. Although OmPHYA shows high sequence identity with other PHYAs in green plants, 13 amino acid substitutions located in both the N and C-terminal domains are observed (a total of 26 amino acids). OmPHYA is encoded by a single gene within the O. minor genome. The abundance of the OmPHYA transcript as well as nuclear translocation of OmphyA occurs in a light-dependent manner.

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. The kinetic parameters obtained at 70 and 90 degrees C demonstrated that the rise of the temperature elevates the k(0) value for the C(10) allylic substrate to more than those for the C(5) and C(15) allylic substrates. These data suggest that Tk-IdsA contributes to adjust the membrane composition to the cell growth temperature by modulating its substrate and product specificities. Mutation study indicated that the aromatic side chain of Tyr-81 acts as a steric hindrance to terminate the chain elongation and defines the final product length.

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 42.9 kDa. The deduced amino acid sequence of L. chrysorrheus FPS showed about 50% identity with those of other fungi and yeasts as well as plants. We expressed the cDNA of L. chrysorrheus FPS in Escherichia coli as a glutathione-S-transferase (GST)-fusion protein. The purified obtained protein showed FPS activity in which geranyl diphosphate (GPP) served as primary substrate, with a 2.4-fold higher k(cat)/K(m) value for GPP than for dimethylallyl diphosphate (DMAPP).

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.

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.

Flower color modulations of Torenia hybrida by downregulation of chalcone synthase genes with RNA interference.

Suppression of biosynthetic genes involved in flower color formation is an important approach for obtaining target flower colors. Here we report that flower color of the garden plant Torenia hybrida was successfully modulated by RNA interference (RNAi) against a gene of chalcone synthase (CHS), a key enzyme for anthocyanin and flavonoid biosynthesis. By using each of the coding region and the 3'-untranslated region of the CHS mRNA as an RNAi target, exhaustive and gene-specific gene silencing were successfully induced, and the original blue flower color was modulated to white and pale colors, respectively. Our results indicate that RNAi is quite useful for modulations of flower colors of commercially important garden plants.