Transcriptomic Analysis of Kalopanax septemlobus and Characterization of KsBAS, CYP716A94 and CYP72A397 Genes Involved in Hederagenin Saponin Biosynthesis.[Pubmed: 29186583]

Kalopanax septemlobus, commonly named the castor aralia tree, is a highly valued woody medicinal tree belonging to the family Araliaceae. Kalopanax septemlobus contains approximately 15 triterpenoid saponins primarily constituted of hederagenin aglycones. Hederagenin is a representative precursor for hemolytic saponin in plants. In the present study, transcriptome analysis was performed to discover genes involved in hederagenin saponin biosynthesis in K. septemlobus. De novo assembly generated 82,698 unique sequences, including 17,747 contigs and 64,951 singletons, following 454 pyrosequencing. Oxidosqualene cyclases (OSCs) are enzymes that catalyze the formation of diverse triterpene skeletons from 2,3-oxidosqualene. Heterologous expression of an OSC sequence in yeast revealed that KsBAS is a β-amyrin synthase gene. Cytochrome P450 genes (CYPs) make up a supergene family in the plant genome and play a key role in the biosynthesis of sapogenin aglycones. In total, 95 contigs and 110 singletons annotated as CYPs were obtained by sequencing the K. septemlobus transcriptome. By heterologous expression in yeast, we found that CYP716A94 was β-amyrin 28-oxidase involved in oleanolic acid production from β-amyrin, and CYP72A397 was oleanolic acid 23-hydroxylase involved in hederagenin production from oleanolic acid. Engineered yeast co-expressing KsBAS, CYP716A94 and CYP72A397 produced hederagenin. Kalopanax septemlobus CYP72A397 is a novel CYP enzyme that synthesizes hederagenin aglycone from oleanolic acid as a single product. In conclusion, we characterized three genes participating in sequential steps for hederagenin biosynthesis from β-amyrin, which are likely to play a major role in hederagenin saponin biosynthesis in K. septemlobus.

Differences in drought- and freeze-induced embolisms in deciduous ring-porous plant species in Japan.[Pubmed: 27376942]

Deciduous ring-porous species in Japan shed all of their leaves under severe water stress before large vessels in earlywood are embolized, and embolization take place during winter. Water in deciduous ring-porous species is mainly conducted upward via large earlywood vessels of the current year. Water columns in large vessels are vulnerable to drought-induced and freeze stress-induced embolisms. Although a vulnerability curve can be created to estimate the hydraulic capacity of plants, it remains unclear why the loss of conductivity in potted plants of ring-porous species does not reach 100 % under severe drought stress. In this study, two deciduous ring-porous species in Japan (Kalopanax septemlobus and Toxicodendron trichocarpum) were used to explain the species-specific pattern in the water-conducting pathway of the stem. We monitored and visualized the spatial distribution of xylem embolisms in the stem of K. septemlobus saplings under drought stress and freeze stress using compact magnetic resonance imaging and cryo-scanning microscopy. In addition, we evaluated the water ascent in the stems of both species using a dye uptake method. Although embolisms of large vessels were observed under drought stress and in winter, all leaves were dropped to avoid fatal water loss after embolization of some large vessels. In contrast, all large vessels were embolized in winter. Larger-diameter vessels of latewood in T. trichocarpum tended to function in trees growing in the warm temperate zone. Thus, our results suggest that the unclear curve may be derived from a discrepancy between leaf water potential and actual water potential in the xylem under severe drought stress. The frequency of xylem embolisms in deciduous ring-porous species in Japan mainly depends on the number of freeze-thaw cycles.

[Study on ecological environment and accompanying plants' community characteristics study of wild Panax japonicus in Enshi].[Pubmed: 28891605]

The paper is aimed to study the distribution, population density, soil conditions and community characteristics of accompanying plants' in Enshi sub-regional different areas, with a typical habitats investigation method. The results showed that the wild Panax japonicus mainly distributed in moist places under the forests, by streams, or secondary forests of high grass, within east longitude 29°-30°, north latitude 108°-110°and about 1 000-15 00 meters above sea level. The soils were mainly tide soil and humus with yellow-brown soil, yellow soil and red soil, and the humus thickness was5-30 centimeter, pH 6.0-6.8, the moisture content of 16.8%-24.2%, soil bulk density of 1.39-2.12. Its geographical vegetation types were mainly evergreen coniferous forest, evergreen-deciduous mixture broad leaved forest and evergreen coniferous forest mixed deciduous broad-leaved forest, including three levels community structure of arbors, shrubs and herbaceous; Its accompanying plants reached 86 families, 118 genera, 134 species of seed plants, the arbors included 15 families, 21 genera, 26 species and the dominant species community mainly Pinaceae such as Pinus massoniana, P. tabuliformis, P. henryi and Taxodiaceae such as Cunninghamia lanceolata, Cryptomeria fortunei etc. The shrubs included 39 families, 54 genera, 62 species with the dominant species such as Camellia oleifera, Kalopanax septemlobus, Akebia trifoliata, Trachycarpusfortunei, Rhamnus globosa, Smilax corbularia and so on. The herbaceous included 32 families, 43 genera, 46 species, and Ferns such as the black-footed Dryopteris, Dryopteris crassirhizom, Coniogramme affinis, Polystichum tripteron, Adiantum pedatum, Lunathyrium acrostichoides, Woodsia ilvensis and Woodwardia japonica were dominant species. The cover layer covered a large number of lichens and mosses. The wild P. japonicus can be found among the P. massoniana, P. tabuliformis, P. henryi, lichens and mosses. These may indicate that the wild P. japonicusin Enshi requires higher demands on the ecological environment, its accompanying plants are mainly the tree layer-shrub layer-herb layer, and vertical structure is obvious. The study provides a basis for domestication and conservation of P. japonicus resources.

Ethanol extract of Kalopanax septemlobus leaf inhibits HepG2 human hepatocellular carcinoma cell proliferation via inducing cell cycle arrest at G1 phase.[Pubmed: 27086152]

To investigate the effects of an ethanol extract of Kalopanax septemlobus (Thunb.) Koidz. leaf (EEKS) on cell proliferation in human hepatocellular carcinoma cells and its mechanisms of action.

Ethanol extract of Kalopanax septemlobus leaf induces caspase-dependent apoptosis associated with activation of AMPK in human hepatocellular carcinoma cells.[Pubmed: 26549816]

The Kalopanax septemlobus leaf (Thunb.) Koidz. has been used as a traditional medicine herb for the treatment of various human diseases for hundreds of years. In this study, we investigated the mechanism underlying the inhibitory effects of an ethanol extract of K. septemlobus leaf (EEKS) on proliferation of HepG2 hepatocellular carcinoma cells. For this study, cell viability and apoptosis were evaluated using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, DAPI (4,6-diamidino-2-phenylindole) staining, agarose gel electrophoresis, and flow cytometry. Measurements of the mitochondrial membrane potential (MMP), caspase activity assays and western blots were conducted to determine whether HepG2 cell death occurred by apoptosis. Treatment of HepG2 cells with EEKS concentration-dependently reduced cell survival while significantly increasing the ratio of apoptotic cells. EEKS treatment increased the levels of the death receptors (DRs), DR4 and DR5, and activated caspases, as well as promoting proteolytic degradation of poly(ADP-ribose)-polymerase associated with the downregulation of protein expression of members of the inhibitor of apoptosis protein family. Treatment with EEKS also caused truncation of Bid, translocation of pro-apoptotic Bax to the mitochondria, and loss of mitochondrial membrane permeabilization, thereby inducing the release of cytochrome c into the cytosol. However, treatment of HepG2 cells with a pan-caspase inhibitor reversed EEKS-induced apoptosis and growth suppression, indicating that EEKS appears to induce apoptosis though a caspase-dependent mechanism involving both intrinsic and extrinsic apoptotic pathways. In addition, the phosphorylation level of AMP-activated protein kinase (AMPK) was elevated when cells were exposed to EEKS. A specific inhibitor for AMPK attenuated the EEKS-induced activation of caspases, and consequently prevented the EEKS-induced apoptosis and reduction in cell viability. Overall, our findings suggest that EEKS inhibits the growth of HepG2 cells by inducing AMPK-mediated caspase-dependent apoptosis, suggesting the potential therapeutic application of EEKS in the treatment or prevention of cancers.

Potential of Kalopanax septemlobus leaf extract in synthesis of silver nanoparticles for selective inhibition of specific bacterial strain in mixed culture.[Pubmed: 25085530]

Silver nanoparticles (AgNPs) were synthesised using Kalopanax septemlobus plant leaf extracts. UV-visible spectrophotometric, Fourier-transform infrared, electron dispersive X-ray spectroscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses confirmed synthesis of AgNPs. TEM micrographs revealed presence of well-dispersed AgNPs predominantly of small size and different shapes with an average particle size of 30.8 nm. Antimicrobial susceptibility tests of AgNP treatments revealed variability in sensitivity of bacteria Bacillus cereus and Saccharophagus degradans under study. Minimum inhibitory concentration (MIC) values of the AgNPs for B. cereus and S. degradans were found to be 30 and 10 μg/mL, respectively. The mixed culture of B. cereus and S. degradans treated with AgNPs at 10 μg/mL showed increase in growth with time, suggesting survival of bacteria in liquid media. The plating of mixed culture before AgNP treatment showed presence of both bacteria, but 24-h-old mixed culture treated with AgNPs at the concentration of 10 μg/mL showed presence of B. cereus colonies. SEM micrographs revealed damage to S. degradans cells but no effect on B. cereus cells after AgNP treatment. Confocal microscopic observations of AgNP-treated mixed cultures by Nile blue A staining indicated intact polyhydroxyalkanoates producing flourescent cells of B. cereus but damage and deformities in S. degradans cells. This study suggests that AgNPs can selectively inhibit growth of S. degradans and retain B. cereus at MIC of S. degradans. This report is a case study for selective inhibition of one bacteria and growth of the other in a culture using plant-synthesized silver nanoparticles.

New resinicolous Chaenothecopsis species from China.[Pubmed: 24891410]

Four new resinicolous species of Chaenothecopsis are described from China: Chaenothecopsis perforata from exudate of Rhus chinensis (Anacardiaceae), C. pallida from exudate of Ailanthus altissima (Simaroubaceae), C. resinophila from exudate of Kalopanax septemlobus (Araliaceae) and C. hunanensis from resin of Pinus massoniana (Pinaceae). All the new species are compared with previously described resinicolous mycocalicioid taxa, and several new features in these species are presented. The newly described species cannot always be distinguished by any single character, but they all possess unique combinations of morphological, chemical and ecological features. Several aspects in the ecology and evolution of boreal and tropical resinicolous species are briefly discussed.

Cryopreservation of Kalopanax septemlobus embryogenic callus using vitrification and droplet-vitrification.[Pubmed: 23224373]

A cryopreservation protocol has been developed for embryogenic callus cultures of castor aralia (Kalopanax septemlobus), a deciduous tree which is widely used in oriental medicine and in landscape design. Three preculture treatments, four loading and six vitrification solutions were tested in a vitrification procedure. Preculture of embryogenic callus (EC) with high sucrose concentrations (up to 0.7 M) showed no effect on regrowth after cryopreservation. Loading for 20 min at ambient temperature improved regrowth of cryopreserved EC by 70-75 percent compared with non-loaded samples, regardless of the composition of the loading solution. Among vitrification solutions, the highest regrowth of 95-100 percent after cryopreservation was obtained after incubation of EC in a vitrification solution A3-80 percent comprising (w/v) 33.3 percent glycerol + 13.3 percent DMSO + 13.3 percent EG + 20.1 percent sucrose for 40 min at 0°C. Profiling of crystallization and recrystallization events using differential scanning calorimetry (DSC) confirmed that freezing injury was minimized in samples after loading and cryoprotection with this vitrification solution. Unlike many other papers, the droplet-vitrification protocol did not produce higher post-cryopreservation regrowth of Kalopanax EC, compared with the vitrification procedure. When samples are sufficiently cryoprotected during VS treatment, vitrification using cryovials may be preferred, since droplet-vitrification is more complex and requires skilled personnel. Cryopreserved callus grew rapidly and produced numerous somatic embryos, which developed similarly to embryos obtained from non-cryopreserved samples.

Climate oscillation during the Quaternary associated with landscape heterogeneity promoted allopatric lineage divergence of a temperate tree Kalopanax septemlobus (Araliaceae) in East Asia.[Pubmed: 22646502]

We investigated the biogeographic history of Kalopanax septemlobus, one of the most widespread temperate tree species in East Asia, using a combined phylogeographic and palaeodistribution modelling approach. Range-wide genetic differentiation at nuclear microsatellites (G'(ST) = 0.709; 2205 samples genotyped at five loci) and chloroplast DNA (G(ST) = 0.697; 576 samples sequenced for 2055 bp at three fragments) was high. A major phylogeographic break in Central China corresponded with those of other temperate species and the spatial delineation of the two temperate forest subkingdoms of East Asia, consistent with the forests having been isolated within both East and West China for multiple glacial-interglacial cycles. Evidence for multiple glacial refugia was found in most of its current range in China, South Japan and the southernmost part of the Korean Peninsula. In contrast, lineage admixture and absence of private alleles and haplotypes in Hokkaido and the northern Korean Peninsula support a postglacial origin of northernmost populations. Although palaeodistribution modelling predicted suitable climate across a land-bridge extending from South Japan to East China during the Last Glacial Maximum, the genetic differentiation of regional populations indicated a limited role of the exposed sea floor as a dispersal corridor at that time. Overall, this study provides evidence that differential impacts of Quaternary climate oscillation associated with landscape heterogeneity have shaped the genetic structure of a wide-ranging temperate tree in East Asia.