Japonica Cells Research Articles

A resource of single-cell gene expression profiles in a planarian Dugesia japonica.

The freshwater planarian Dugesia japonica maintains an abundant heterogeneous cell population called neoblasts, which include adult pluripotent stem cells. Thus, it is an excellent model organism for stem cell and regeneration research. Recently, many single-cell RNA sequencing (scRNA-seq) databases of several model organisms, including other planarian species, have become publicly available; these are powerful and useful resources to search for gene expression in various tissues and cells. However, the only scRNA-seq dataset for D. japonica has been limited by the number of genes detected. Herein, we collected D. japonica cells, and conducted an scRNA-seq analysis. A novel, automatic, iterative cell clustering strategy produced a dataset of 3,404 cells, which could be classified into 63 cell types based on gene expression profiles. We introduced two examples for utilizing the scRNA-seq dataset in this study using D. japonica. First, the dataset provided results consistent with previous studies as well as novel functionally relevant insights, that is, the expression of DjMTA and DjP2X-A genes in neoblasts that give rise to differentiated cells. Second, we conducted an integrative analysis of the scRNA-seq dataset and time-course bulk RNA-seq of irradiated animals, demonstrating that the dataset can help interpret differentially expressed genes captured via bulk RNA-seq. Using the R package "Seurat" and GSE223927, researchers can easily access and utilize this dataset.

Structural and Physical Properties of Alginate Pretreated by High-Pressure Homogenization.

To develop a high-efficient extraction method, we investigated the use of high-pressure homogenization (HPH) as a novel pretreatment technology for the extraction of sodium alginate (SA) from Laminaria japonica. After the single-factor experiment, the results demonstrated that under the conditions of 100 MPa HPH pressure, 4 cycles, pH 6.0, and 0.5% EDTA for 3.0 h, the optimized extraction yield of HPH reached 34%. To further clarify the effect on the structural properties of HPH-extracted SA, we conducted comprehensive analysis using SEM, FTIR, MRS, NMR, XRD, TGA, and a T-AOC assay. Our findings revealed that HPH pretreatment significantly disrupted the structure of L. japonica cells and reduced their crystallinity to 76.27%. Furthermore, the antioxidant activity of HPH-extracted SA reached 0.02942 mgVceq∙mg-1. Therefore, the HPH pretreatment method is a potential strategy for the extraction of alginate.

Pseudoalteromonas piscicida X-8 causes bleaching disease in farmed Saccharina japonica

Saccharina japonica is a commercially farmed seaweed of global importance. However, disease occurrence during different stages of cultivation can result in substantial economic losses. Identification of the causative agents of disease remains a significant bottleneck to the large scale cultivation of S. japonica. In this study, an aerobic heterotrophic, flagellated, rod-shaped Gram-negative bacterial strain X-8 was isolated from the bleaching diseased S. japonica sporelings. Pathogenecity of strain X-8 was tested by re-infection assay. The ultrastructural changes of infected S. japonica cells by strain X-8 indicated that chloroplasts were the first organelle responding to X-8 infection with deformed structure and later followed by fragmented nucleus. However, the ultra-structure of mitochondria and cell wall remained intact during the re-infection. Based on 16S rRNA gene sequence, morphological and biochemical characteristics, strain X-8 was designated as Pseudoalteromonas piscicida X-8. The pathogenicity of P. piscicida X-8 was identified by Koch's Postulate under laboratory conditions. Our results will not only help to establish a stable experimental model between the pathogenic bacteria and the host S. japonica to further elucidate the virulence mechanisms, but will also provide information for disease management to effectively prevent and mitigate the occurrence of bleaching disease of S. japonica at nursery stage.

Newly discovered role of the heterotrophic nanoflagellate Katablepharis japonica, a predator of toxic or harmful dinoflagellates and raphidophytes

Heterotrophic nanoflagellates are ubiquitous and known to be major predators of bacteria. The feeding of free-living heterotrophic nanoflagellates on phytoplankton is poorly understood, although these two components usually co-exist. To investigate the feeding and ecological roles of major heterotrophic nanoflagellates Katablepharis spp., the feeding ability of Katablepharis japonica on bacteria and phytoplankton species and the type of the prey that K. japonica can feed on were explored. Furthermore, the growth and ingestion rates of K. japonica on the dinoflagellate Akashiwo sanguinea—a suitable algal prey item—heterotrophic bacteria, and the cyanobacteria Synechococcus sp., as a function of prey concentration were determined. Among the prey tested, K. japonica ingested heterotrophic bacteria, Synechococcus sp., the prasinophyte Pyramimonas sp., the cryptophytes Rhodomonas salina and Teleaulax sp., the raphidophytes Heterosigma akashiwo and Chattonella ovata, the dinoflagellates Heterocapsa rotundata, Amphidinium carterae, Prorocentrum donghaiense, Alexandrium minutum, Cochlodinium polykrikoides, Gymnodinium catenatum, A. sanguinea, Coolia malayensis, and the ciliate Mesodinium rubrum, however, it did not feed on the dinoflagellates Alexandrium catenella, Gambierdiscus caribaeus, Heterocapsa triquetra, Lingulodinium polyedra, Prorocentrum cordatum, P. micans, and Scrippsiella acuminata and the diatom Skeletonema costatum. Many K. japonica cells attacked and ingested a prey cell together after pecking and rupturing the surface of the prey cell and then uptaking the materials that emerged from the ruptured cell surface. Cells of A. sanguinea supported positive growth of K. japonica, but neither heterotrophic bacteria nor Synechococcus sp. supported growth. The maximum specific growth rate of K. japonica on A. sanguinea was 1.01 d−1. In addition, the maximum ingestion rate of K. japonica for A. sanguinea was 0.13ngC predator−1d−1 (0.06 cells predator−1d−1). The maximum ingestion rate of K. japonica for heterotrophic bacteria was 0.019ngC predator−1d−1 (266 bacteria predator−1d−1), and the highest ingestion rate of K. japonica for Synechococcus sp. at the given prey concentrations of up to ca. 107 cells ml−1 was 0.01ngC predator−1d−1 (48 Synechococcus predator−1d−1). The maximum daily carbon acquisition from A. sanguinea, heterotrophic bacteria, and Synechococcus sp. were 307, 43, and 22%, respectively, of the body carbon of the predator. Thus, low ingestion rates of K. japonica on heterotrophic bacteria and Synechococcus sp. may be responsible for the lack of growth. The results of the present study clearly show that K. japonica is a predator of diverse phytoplankton, including toxic or harmful algae, and may also affect the dynamics of red tides caused by these prey species.

Protective Effects of Tormentic Acid, a Major Component of Suspension Cultures of Eriobotrya japonica Cells, on Acetaminophen-Induced Hepatotoxicity in Mice.

An acetaminophen (APAP) overdose can cause hepatotoxicity and lead to fatal liver damage. The hepatoprotective effects of tormentic acid (TA) on acetaminophen (APAP)-induced liver damage were investigated in mice. TA was intraperitoneally (i.p.) administered for six days prior to APAP administration. Pretreatment with TA prevented the elevation of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (T-Bil), total cholesterol (TC), triacylglycerol (TG), and liver lipid peroxide levels in APAP-treated mice and markedly reduced APAP-induced histological alterations in liver tissues. Additionally, TA attenuated the APAP-induced production of nitric oxide (NO), reactive oxygen species (ROS), tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), and IL-6. Furthermore, the Western blot analysis showed that TA blocked the protein expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), as well as the inhibition of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) activation in APAP-injured liver tissues. TA also retained the superoxidase dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) in the liver. These results suggest that the hepatoprotective effects of TA may be related to its anti-inflammatory effect by decreasing thiobarbituric acid reactive substances (TBARS), iNOS, COX-2, TNF-α, IL-1β, and IL-6, and inhibiting NF-κB and MAPK activation. Antioxidative properties were also observed, as shown by heme oxygenase-1 (HO-1) induction in the liver, and decreases in lipid peroxides and ROS. Therefore, TA may be a potential therapeutic candidate for the prevention of APAP-induced liver injury by inhibiting oxidative stress and inflammation.

A multidrug and toxic compound extrusion transporter mediates berberine accumulation into vacuoles in Coptis japonica

Plants produce a large variety of alkaloids, which have diverse chemical structures and biological activities. Many of these alkaloids accumulate in vacuoles. Although some membrane proteins on tonoplasts have been identified as alkaloid uptake transporters, few have been characterized to date, and relatively little is known about the mechanisms underlying alkaloid transport and accumulation in plant cells. Berberine is a model alkaloid. Although all genes involved in berberine biosynthesis, as well as the master regulator, have been identified, the gene responsible for the final accumulation of berberine at tonoplasts has not been determined. This study showed that a multidrug and toxic compound extrusion protein 1 (CjMATE1) may act as a berberine transporter in cultured Coptis japonica cells. CjMATE1 was found to localize at tonoplasts in C. japonica cells and, in intact plants, to be expressed preferentially in rhizomes, the site of abundant berberine accumulation. Cellular transport analysis using a yeast expression system showed that CjMATE1 could transport berberine. Expression analysis showed that RNAi suppression of CjbHLH1, a master transcription factor of the berberine biosynthetic pathway, markedly reduced the expression of CjMATE1 in a manner similar to the suppression of berberine biosynthetic genes. These results strongly suggest that CjMATE1 is the transporter that mediates berberine accumulation in vacuoles.

Characterization of the Promoter Region of Biosynthetic Enzyme Genes Involved in Berberine Biosynthesis in Coptis japonica.

The presence of alkaloids is rather specific to certain plant species. However, berberine, an isoquinoline alkaloid, is relatively broadly distributed in the plant kingdom. Thus, berberine biosynthesis has been intensively investigated, especially using Coptis japonica cell cultures. Almost all biosynthetic enzyme genes have already been characterized at the molecular level. Particularly, two transcription factors (TFs), a plant-specific WRKY-type TF, CjWRKY1, and a basic helix-loop-helix TF, CjbHLH1, were shown to comprehensively regulate berberine biosynthesis in C. japonica cells. In this study, we characterized the promoter region of some biosynthetic enzyme genes and associated cis-acting elements involved in the transcriptional regulation via two TFs. The promoter regions of three berberine biosynthetic enzyme genes (CYP80B2, 4′OMT and CYP719A1) were isolated, and their promoter activities were dissected by a transient assay involving the sequentially truncated promoter::luciferase (LUC) reporter constructs. Furthermore, transactivation activities of CjWRKY1 were determined using the truncated promoter::LUC reporter constructs or constructs with mutated cis-elements. These results suggest the involvement of a putative W-box in the regulation of biosynthetic enzyme genes. Direct binding of CjWRKY1 to the W-box DNA sequence was also confirmed by an electrophoresis mobility shift assay and by a chromatin immunoprecipitation assay. In addition, CjbHLH1 also activated transcription from truncated 4′OMT and CYP719A1 promoters independently of CjWRKY1, suggesting the involvement of a putative E-box. Unexpected transcriptional activation of biosynthetic enzyme genes via a non-W-box sequence and by CjWRKY1 as well as the possible involvement of a GCC-box in berberine biosynthesis in C. japonica are discussed.

Identification of carotenoids from the extremely halophilic archaeon Haloarcula japonica

The carotenoids produced by extremely halophilic archaeon Haloarcula japonica were extracted and identified by their chemical, chromatographic, and spectroscopic characteristics (UV-Vis and mass spectrometry). The composition (mol%) was 68.1% bacterioruberin, 22.5% monoanhydrobacterioruberin, 9.3% bisanhydrobacterioruberin, <0.1% isopentenyldehydrorhodopin, and trace amounts of lycopene and phytoene. The in vitro scavenging capacity of a carotenoid, bacterioruberin, extracted from Haloarcula japonica cells against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals was evaluated. The antioxidant capacity of bacterioruberin was much higher than that of β -carotene.

The toxic effect of the marine raphidophyte Fibrocapsa japonica on larvae of the common flatfish sole (Solea solea)

Blooms of the marine microalga Fibrocapsa japonica (Raphidophyceae) are suggested to lead to the diminution of fish stocks, but the exact mechanism(s) involved in ichthyotoxicity is still unclear. In the present study fish tests were performed, using larvae of the common flat fish sole (Solea solea), to further investigate properties and variability of F. japonica toxicity. Sole larvae were directly and indirectly exposed to a density range of a Dutch Wadden Sea F. japonica strain, as well as F. japonica cell extracts, for a maximum of 7 days, during which larval mortality was monitored. Most experiments were done at 16°C, however some experiments were performed at 20°C. Furthermore, larvae were exposed to concentration ranges of the alleged toxic compounds, e.g. the brevetoxins PbTx-2 and PbTx-3, and three poly-unsaturated fatty acids (PUFAs): AA, EPA and OTA.The larvae showed highly variable responses to the F. japonica treatments. At the late exponential growth phase of F. japonica highest sole mortality was found: 100% mortality after 7 days of direct exposure. Indirect and cell extract exposures as well as direct exposure to other growth phases caused lower larval mortality (up to 40%) after the same incubation period. Temperature had a distinct effect on sole mortality, showing significantly enhanced toxicity at 20°C, compared with 16°C. Exposure to PbTx-2 showed an LC50 of 28.7ngmL−1 after 48h, while exposure to PbTx-3 caused no 100% mortality up to 10ngmL−1. Exposure to pure PUFAs showed the fastest mortality observed in all experiments, with an LC50 of <5μgmL−1 of AA, EPA or OTA after 24h of exposure.Our research suggests pronounced ichthyotoxicity in late exponential F. japonica cells, related primarily with the combination of endo- and exotoxins, with most probably haemolytic PUFAs as the main endotoxins, and other haemolysins and/or ROS as the main exotoxins. Implications are given for present and future risks in the already warming Dutch coastal waters.

Pathway engineering of benzylisoquinoline alkaloid biosynthesis in transgenic California poppy cells with ectopic expression of tetrahydroberberine oxidase from &lt;i&gt;Coptis japonica&lt;/i&gt;

S)-Tetrahydroberberine oxidase is the enzyme in the last step of berberine biosynthesis. While a previous report described the isolation of cDNA of tetrahydroberberine oxidase (THBO) from cultured Coptis japonica cells, we later found that purified THBO was heavily contaminated by triosephosphate isomerase. Here, we report the re-isolation of THBO cDNA from cultured C. japonica cells and its functional characterization in transgenic California poppy cells. A cDNA clone for (S)-tetrahydroberberine oxidase was isolated from an EST library prepared from high berberineproducing cultured C. japonica cells based on the partial amino acid sequence of the purified enzyme. Analyses of the nucleotide sequences of the cloned cDNA inserts of 1728 base pairs revealed an open reading frame that encoded a 540-amino acid polypeptide with putative 28-amino acid signal peptides and a mature polypeptide with a molecular mass of 57,748. A protein blast search also shows that CjTHBO belongs to the FAD-containing berberine bridge enzyme oxidoreductase family. Since all attempts to produce active recombinant CjTHBO in Escherichia coli and Saccharomyces cerevisiae cells failed, we tried to express CjTHBO in California poppy (Eschscholzia californica) cells. When transgenic California poppy cells that ectopically expressed CjTHBO under the control of Cauliflower mosaic virus 35S promoter were established, the transgenic cells showed small but evident new alkaloid peaks, which were scarcely detected in control cells that did not express CjTHBO. LC-MS analyses showed that these peaks were coptisine and dehydrocheilanthifoline, which were expected to be generated by the reaction of CjTHBO from pathway intermediates, i.e., cheilanthifoline and stylopine. The usefulness of CjTHBO for metabolic engineering in transgenic California poppy cells is discussed.

Toxicity evaluation of Fibrocapsa japonica from the Northern Adriatic Sea through a chemical and toxicological approach

Since the 1990s red-tide blooms of Fibrocapsa japonica have frequently been observed in European waters; despite their recurrence also in the Adriatic coastal areas, they have never been conclusively linked to ichthyotoxic events. Since the toxicity of F. japonica is still under debate and its effects differ among the strains, in this study all the compounds previously postulated as involved in the toxic mechanism, such as brevetoxins, fatty acids and ROS, were screened for Adriatic strains. Whole algal extracts were analyzed for brevetoxin PbTx-2, which was not found, and for a qualitative and quantitative analysis of fatty acids. The fatty acid profile evidenced the presence of PUFAs, with considerable amounts of 18:4n-3, 20:4n-6, and 20:5n-3, as already found in different strains, but with a lower amount of arachidonic acid (20:4n-6). None of the PUFAs was released in the extracellular medium. Different toxicological assays ( Vibrio fischeri, Artemia sp., haemolysis of fish erythrocytes) were performed using algal cellular, extracellular or subcellular samples as well as fractions of the algal extract, in order to identify the toxic compounds. The tested cultures were found to inhibit V. fischeri bioluminescence, to affect Artemia nauplii viability and to have haemolytic effects comparable to those previously observed in different strains. The solid phase extraction (SPE) of the Adriatic F. japonica extracts resulted in a high concentration of PUFAs in two main fractions, which caused mortality in Artemia nauplii and inhibition of V. fischeri bioluminescence. Fish ( Dicentrarchus labrax) assays were also performed, reporting mortality after long exposure times and the cause of fish death was investigated. A significant increase of H 2O 2 in the tanks where sea basses were exposed to F. japonica was obtained, indicating that the presence of fish stimulates H 2O 2 production by algal cells and leading to the hypothesis of H 2O 2 involvement in F. japonica toxicity. The presence of oxidative stress in the exposed fish was confirmed by the increased malondialdehyde concentration in their gills. Overall these results indicate that since F. japonica cells can easily cling to fish gills, due to their high polysaccharide production, it is presumable that high PUFA amounts are released in loco, as a result of cell breakage, and that exert their toxicity together with ROS. This study shows that the Adriatic strains of F. japonica can be harmful to higher level organisms. Nevertheless a high cell density and a long lasting bloom are necessary to cause severe damage to fish gills or death for crustaceans.

Feeding by raphidophytes on the cyanobacterium Synechococcus sp.

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 58:181-195 (2010) - DOI: https://doi.org/10.3354/ame01354 Feeding by raphidophytes on the cyanobacterium Synechococcus sp. Hae Jin Jeong1,*, Kyeong Ah Seong2, Nam Seon Kang1, Yeong Du Yoo1, Seung Won Nam3, Jae Yeon Park4, Woongghi Shin5, Patricia M. Glibert6, Desmond Johns6 1School of Earth and Environmental Sciences, College of Natural Sciences, Seoul National University, Seoul 151-747, Republic of Korea 2Saemankeum Environmental Research Center, Kunsan National University, Kunsan 573-701, Republic of Korea 3Division of Electron Microscopic Research, Korea Basic Science Institute, 113 Gwahangno, Yuseong-gu, Daejeon 305-333, Republic of Korea 4Environment Energy Resource Institute, Advanced Institute of Convergence Technology, Suwon 443-270, Republic of Korea 5Department of Biology, Chungnam National University, Daejeon 305-764, Republic of Korea 6University of Maryland Center for Environmental Science, Horn Point Laboratory, PO Box 775, Cambridge, Maryland 21613, USA *Email: hjjeong@snu.ac.kr ABSTRACT: We investigated feeding by the raphidophytes Chattonella ovata, C. subsalsa, Fibrocapsa japonica, and Heterosigma akashiwo on the cyanobacterium Synechococcus sp. To explore whether each species is able to feed on Synechococcus sp., we carefully observed inside target grazer cells using an epifluorescence microscope and tranbsmission electron microscope (TEM). We also explored the feeding behaviors of C. ovata and H. akashiwo on Synechococcus using high-resolution video microscopy. In addition, we measured ingestion rates of C. ovata, C. subsalsa and H. akashiwo on Synechococcus sp. as a function of prey concentration. We calculated grazing coefficients by combining the field data on abundances of H. akashiwo and co-occurring Synechococcus spp. with laboratory data on ingestion rates. Both C. ovata and H. akashiwo were able to ingest single Synechococcus cells. However, neither TEM nor video microscopy showed any Synechococcus cells inside or ingested by F. japonica. One to two ingested Synechococcus cells inside the protoplasm of F. japonica cells were very rarely observed. C. ovata and H. akashiwo engulfed a single Synechococcus cell captured by the mucus excreted from mucocysts. The ingestion rates of C. ovata, C. subsalsa, or H. akashiwo on Synechococcus increased continuously with increasing prey concentration at prey concentrations ≤4 × 106 to 5.5 × 106 cells ml–1. At a given prey concentration, the highest ingestion rates of the raphidophytes on Synechococcus were 18.6 cells raphidophyte–1 h–1 for C. ovata, 20.5 cells raphidophyte–1 h–1 for C. subsalsa, and 3.9 cells raphidophyte–1 h–1 for H. akashiwo. The calculated grazing coefficients attributable to H. akashiwo on co-occurring Synechococcus spp. were up to 1.24 d–1. The results of the present study suggest that raphidophytes sometimes have a considerable grazing impact on populations of Synechococcus. KEY WORDS: Chattonella spp. · Fibrocapsa japonica · Graze · Heterosigma akashiwo · Harmful algal bloom · HAB · Ingestion · Red tide Full text in pdf format Supplementary material PreviousNextCite this article as: Jeong HJ, Seong KA, Kang NS, Yoo YD and others (2010) Feeding by raphidophytes on the cyanobacterium Synechococcus sp.. Aquat Microb Ecol 58:181-195. https://doi.org/10.3354/ame01354Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 58, No. 2. Online publication date: January 07, 2010 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2010 Inter-Research.

Haemolytic activity within the species Fibrocapsa japonica (Raphidophyceae)

Strong haemolytic activity was observed for extracts of 15 Fibrocapsa japonica strains collected from different global regions. The EC 50 values ranged between 0.4 × 10 4 and 1.9 × 10 4 F. japonica cells ml −1. The relationship between the haemolytic activity observed in the cell extracts and the amount and nature of polyunsaturated fatty acids (PUFAs) was investigated. Between 7% and 89% of the haemolytic activity could be attributed to the amount and toxicity of the PUFAs C18:4n3 (OTA), C20:5n3 (EPA), and C20:4n6 (AA). However, the higher the haemolytic activity of a strain the more it appeared to be caused by haemolytic compounds other than the PUFA's. The EC 50 of the extracts with the highest haemolytic activity strongly depended on light intensity and was in line with observations made on Chattonella marina. In the cell extracts of the strains studied brevetoxins were always below detection limits. For strains where 20–35% of the haemolytic activity was not caused by PUFAs, the concentration of these compounds was close to LC 50 values reported for fish. Overall the haemolytic activity of the F. japonica strains was in the range of known harmful microalgae. Therefore, PUFAs in combination with potent light dependent haemolytic compounds could be a major cause of ichthyotoxicity observed during F. japonica blooms.

Isolation of Herbicide-Resistant 4-Hydroxyphenylpyruvate Dioxygenase from CulturedCoptis japonicaCells

4-Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the formation of homogentisate from 4-hydroxyphenylpyruvate and O(2). In plants, HPPD has been identified as a molecular target for herbicides. We report the isolation and characterization of a cDNA encoding a HPPD from cultured Coptis japonica cells. Recombinant CjHPPD showed significantly higher half-maximum inhibitory concentration (IC(50)) values for the HPPD-inhibiting herbicide destosyl pyrazolate than other plant HPPDs.

Molecular Cloning and Characterization of CYP80G2, a Cytochrome P450 That Catalyzes an Intramolecular C–C Phenol Coupling of (S)-Reticuline in Magnoflorine Biosynthesis, from Cultured Coptis japonica Cells

Cytochrome P450s (P450) play a key role in oxidative reactions in plant secondary metabolism. Some of them, which catalyze unique reactions other than the standard hydroxylation, increase the structural diversity of plant secondary metabolites. In isoquinoline alkaloid biosyntheses, several unique P450 reactions have been reported, such as methylenedioxy bridge formation, intramolecular C-C phenol-coupling and intermolecular C-O phenol-coupling reactions. We report here the isolation and characterization of a C-C phenol-coupling P450 cDNA (CYP80G2) from an expressed sequence tag library of cultured Coptis japonica cells. Structural analysis showed that CYP80G2 had high amino acid sequence similarity to Berberis stolonifera CYP80A1, an intermolecular C-O phenol-coupling P450 involved in berbamunine biosynthesis. Heterologous expression in yeast indicated that CYP80G2 had intramolecular C-C phenol-coupling activity to produce (S)-corytuberine (aporphine-type) from (S)-reticuline (benzylisoquinoline type). Despite this intriguing reaction, recombinant CYP80G2 showed typical P450 properties: its C-C phenol-coupling reaction required NADPH and oxygen and was inhibited by a typical P450 inhibitor. Based on a detailed substrate-specificity analysis, this unique reaction mechanism and substrate recognition were discussed. CYP80G2 may be involved in magnoflorine biosynthesis in C. japonica, based on the fact that recombinant C. japonica S-adenosyl-L-methionine:coclaurine N-methyltransferase could convert (S)-corytuberine to magnoflorine.

Evidence for inhibition of bacterial luminescence by allelochemicals from Fibrocapsa japonica (Raphidophyceae), and the role of light and microalgal growth rate

The marine microalga Fibrocapsa japon- ica Toriumi and Takano (Raphidophyceae) produces haemolysins, neurotoxins and reactive oxygen spe- cies (ROS). To quantify potential effects of such bioactive compounds on surrounding organisms the marine bacterium Vibrio fischeri was exposed to F. japonica culture samples. Inhibition of V. fischeri 's natural luminescence, indicative of impaired metab- olism, was related to the number of F. japonica cells added. The effect was fast, within 15 min. It was caused by one, possibly several, excreted substances that were less active after heating. Freezing of culture supernatant partly inactivated these substances, but ROS-scavenging enzymes had no effect. Light enhanced the V. fischeri luminescence inhibition in two ways. The direct effect of light on the action of F. japonica luminescence inhibiter(s) could be de- scribed by a saturation curve with maximum effect above 20 mmol photons m � 2 s � 1 . Light also had an indirect effect: biomass production, dependent on light availability, was closely related to the amount of inhibiting compound(s) produced by the alga. Algal growth rate, rather than its cell density, determined the bacterial luminescence inhibition per F. japonica cell, resulting in a 5-fold stronger inhibition at maximum growth rates compared to cells that barely grew during the stationary growth phase. The bioas- say with F. japonica and V. fischeri has allowed quantification of the negative effects on bacteria in the microalgal microenvironment. The results pre- sented here suggest that at favourable growth condi- tions F. japonica releases bioactive compounds that improve its competitive abilities.

Functional Analysis of Norcoclaurine Synthase in Coptis japonica

(S)-Norcoclaurine is the entry compound in benzylisoquinoline alkaloid biosynthesis and is produced by the condensation of dopamine and 4-hydroxyphenylacetaldehyde (4-HPAA) by norcoclaurine synthase (NCS) (EC 4.2.1.78). Although cDNA of the pathogenesis-related (PR) 10 family, the translation product of which catalyzes NCS reaction, has been isolated from Thalictrum flavum, its detailed enzymological properties have not yet been characterized. We report here that a distinct cDNA isolated from Coptis japonica (CjNCS1) also catalyzed NCS reaction as well as a PR10 homologue of C. japonica (CjPR10A). Both recombinant proteins stereo-specifically produced (S)-norcoclaurine by the condensation of dopamine and 4-HPAA. Because a CjNCS1 cDNA that encoded 352 amino acids showed sequence similarity to 2-oxoglutarate-dependent dioxygenases of plant origin, we characterized the properties of the native enzyme. Sequence analysis indicated that CjNCS1 only contained a Fe(2+)-binding site and lacked the 2-oxoglutarate-binding domain. In fact, NCS reaction of native NCS isolated from cultured C. japonica cells did not depend on 2-oxoglutarate or oxygen, but did require ferrous ion. On the other hand, CjPR10A showed no specific motif. The addition of o-phenanthroline inhibited NCS reaction of both native NCS and recombinant CjNCS1, but not that of CjPR10A. In addition, native NCS and recombinant CjNCS1 accepted phenylacetaldehyde and 3,4-dihydroxyphenylacetaldehyde, as well as 4-HPAA, for condensation with dopamine, whereas recombinant CjPR10A could use 4-hydroxyphenylpyruvate and pyruvate in addition to the above aldehydes. These results suggested that CjNCS1 is the major NCS in C. japonica, whereas native NCS extracted from cultured C. japonica cells was more active and formed a larger complex compared with recombinant CjNCS1.

Bowman–Birk Proteinase Inhibitor Confers Heavy Metal and Multiple Drug Tolerance in Yeast

Cultured Coptis japonica cells show tolerance to various toxic compounds. By yeast functional screening of cadmium (Cd) plates with its cDNA library, we isolated a gene encoding Bowman-Birk proteinase inhibitor (CjBBI). The yeast transformant of CjBBI showed multiple tolerance to various drugs adding to Cd, and revealed reduced Cd accumulation in cells. Preferential organs for Cjbbi expression were aerial parts of intact plants, and the subcellular localization of CjBBI was shown, using its green fluorescent protein fusion, to be the apoplast. Induction of Cjbbi expression by Cd treatment suggested that CjBBI was responsible for the tolerance to Cd observed in C. japonica cells.

Identification of a WRKY Protein as a Transcriptional Regulator of Benzylisoquinoline Alkaloid Biosynthesis in Coptis japonica

Selected cultured Coptis japonica cells produce a large amount of the benzylisoquinoline alkaloid berberine. Previous studies have suggested that berberine productivity is controlled at the transcript level of biosynthetic genes. We have identified a regulator of transcription in berberine biosynthesis using functional genomics with a transient RNA interference (RNAi) and overexpression of the candidate gene. The 24 primary candidate clones were selected from 1,014 expressed sequence tags (ESTs) that were obtained from a C. japonica cell line producing high levels of berberine. Further characterization of the expression profiles of these ESTs suggested that five ESTs would be good candidates as regulators of berberine production. A newly developed transient RNAi system with C. japonica protoplasts indicated that double-stranded RNA of an EST clone significantly reduced the level of transcripts of 3'-hydroxy N-methylcoclaurine 4'-O-methyltransferase. Sequence analysis showed that this EST encoded a group-II WRKY, and we named it CjWRKY1. When the effects of double-stranded RNA of the CjWRKY1 gene were examined in detail, a marked reduction in the transcripts of all genes involved in berberine biosynthesis was detected, whereas little effect was found in the transcript levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and chorismate mutase (CM) that are associated with primary metabolism. Ectopic expression of CjWRKY1 cDNA in C. japonica protoplasts clearly increased the level of transcripts of all berberine biosynthetic genes examined compared with control treatment, whereas the levels of GAPDH and CM were not affected. The functional role of CjWRKY1 as a specific and comprehensive regulator of berberine biosynthesis is discussed.

Chemical characterisation of three haemolytic compounds from the microalgal species Fibrocapsa japonica (Raphidophyceae)

The molecular structures of the three main haemolytic compounds (Fj1, Fj2 and Fj3) isolated from the ichthyotoxic microalgal species Fibrocapsa japonica have been investigated by NMR, LC–ESI–MS, ESI–MS–MS, IR, GC–MS and GC–HRMS methods. They are polyunsaturated fatty acids which we identified as: 6,9,12,15-octadecatetraenoic acid (OTA, C18:4ω3), 5,8,11,14,17-eicosapentaenoic acid (EPA, C20:5ω3) and 5,8,11,14-eicosatetraenoic acid (arachidonic acid AA, C20:4ω6). The identity of the latter two was confirmed on the basis of commercial standards (C20:5ω3 and C20:4ω6). Both displayed the same spectroscopic and chromatographic characteristics as Fj2 and Fj3 and had a similar strong haemolytic effect. We propose that when F. japonica cells accumulate in fish gills during blooms these compounds could be the cause of icthyotoxicity.