Terniflora DC Research Articles

Comprehensive multi-omics analysis reveals the importance of CtCOSY in the energy metabolism and coumarin biosynthesis in Clematis terniflora DC

Coumarin synthase (COSY) is the key enzyme involved in the coumarin biosynthesis. Although it has been identified firstly in Arabidopisis thaliana, there is little known about the COSY gene function in Clematis terniflora DC. Our previous study indicated that the contents of coumarins in C. terniflora leaf were increased under ultraviolet-B radiation and dark treatments (UV-D). To reveal the function of COSY in C. terniflora and its role in response to UV-D stress, the functional characterization of CtCOSY which was identified as a BADH acyltransferase was performed. The results showed that coupled reaction of CtCOSY with 4-coumarinyl CoA-ligase in vitro to produce umbelliferone. Moreover, the functions of CtCOSY in response to UV-D stress was studied using proteomic and metabolomic techniques. Multi-omics analyses of CtCOSY-silenced C. terniflora indicated that as well as a limitation of coumarin synthesis, aspects of the energy metabolism, such as the TCA cycle, were disturbed in these plants. Under UV-D stress, CtCOSY affected the biosynthesis of phenylpropanoids in C. terniflora, resulting in the improvement of coumarin biosynthesis and affecting purine metabolism. This study provides the evidences of CtCOSY responsible for the biosynthesis of coumarin in C. terniflora and the resistance to UV-D stress.

Molecular characterization of a feruloyl-CoA 6′-hydroxylase involved in coumarin biosynthesis in Clematis terniflora DC

Coumarin is an important secondary metabolite that affects plant physiology. It is a lactone of cis-o-hydroxycinnamic acid and widely exists in medicinal plants. Clematis terniflora DC. is a plant belonging to Ranunculaceae and is rich in variety of coumarins. Feruloyl-CoA 6′-hydroxylase has been reported as a key enzyme in the formation of coumarin basic skeleton only in some common plants, however, its evidence in other species is still lacking especially for the biosynthesis of coumarins in C. terniflora. In the present study, we identified a feruloyl-CoA 6′-hydroxylase CtF6′H in C. terniflora, and functional characterization indicated that CtF6′H could hydroxylate feruloyl-CoA to 6-hydroxyferuloyl-CoA. Furthermore, the expression level of CtF6′H was differed among different tissues in C. terniflora, while under UV-B radiation, the level of CtF6′H was increased in the leaves. Biochemical characteristics and subcellular location showed that CtF6′H was mainly present in the cytosol. The crystal structure of CtF6′H was simulated by homology modeling to predict the potential residues affecting enzyme activity. This study provides the additional evidence of feruloyl-CoA 6′-hydroxylase in different plant species and enriches our understanding of biosynthetic mechanism of coumarin in C. terniflora.

Integrative Proteomic and Phosphoproteomic Analyses Revealed the Regulatory Mechanism of the Response to Ultraviolet B Stress in Clematis terniflora DC.

Clematis terniflora DC. (C. terniflora) has been used as an ancient Chinese traditional herbal medicine. The active substances in C. terniflora have been confirmed to be effective in treating diseases such as prostatitis. UV light radiation is a common environmental factor that damages plants and influences primary and secondary metabolism. Previous studies showed that ultraviolet B (UV-B) radiation followed by dark stress resulted in the accumulation of secondary metabolites in C. terniflora leaves. An in-depth understanding of how C. terniflora leaves respond to UV-B stress is crucial for improving C. terniflora value. Here, we conducted label-free proteomic and phosphoproteomic analyses to explore the protein changes under UV-B and UV-B combined with dark treatment. A total of 2839 proteins and 1638 phosphorylated proteins were identified. Integrative omics revealed that the photosynthetic system and carbohydrate balance were modulated under both stresses. The phosphoproteomic data indicated that the mitogen-activated protein kinase signaling pathway was triggered, while the abundance of phosphorylated proteins related to osmotic stress was increased under UV-B stress. Differentially abundant phosphoproteins from UV-B followed by dark treatment were mainly enriched in response to stimulus including calcium-mediated proteins. This study provides new insight into the impact of UV-B stress on C. terniflora and plant molecular resistance mechanisms through proteomic and phosphoproteomic analyses.

Small-flowered clematis collections of the Stavropol Botanical Garden

Relevance. The biological features were studied, the average dates of the beginning, end and duration of the phenological phases of growth and development of species, forms and varieties of the genus Clematis (Clematis L.) were established. The assortment of small-flowered clematis for cultivation in the soil and climatic conditions of the Stavropol upland, characterized by high decorative properties, resistance to drought, pests and diseases, is highlighted.Methods. The object of research is small-flowered species, forms and varieties of the genus Clematis L., cultivated in the collection of the Stavropol Botanical Garden. The studies were conducted in 2012–2021 according to the methods of variety study and evaluation of the decorative qualities of clematis species and varieties.Results. The dates of the beginning and end of the growth of shoots are determined. According to the duration of shoot growth, all taxa are divided into groups: with a short growth period (38–51 days), medium (60–79 days); long (90–126 days). According to the timing of the beginning of flowering, the studied taxa are classified into five groups, most of them are included in the groups of early summer and summer flowering. According to the duration of flowering, three groups are distinguished — with short flowering, up to 30 days (C. jubata Bsch., C. recta L., C. recta f. atropurpurea, C. terniflora DC.); medium, from 41 to 60 days (С. fusca Turcz., C. ‘Bryzgi Morja’, C. integrifolia L., C. orientalis L., C. serratifolia Rehder, C. tangutica (Maxim.) Korsh., C. virginiana L., C. viticella L., C. viticella f. rosea); long-lasting, more than 60 days (C. ‘Fargesioides’, C. heracleifolia DC., C. hexapetala DC.).

Mitochondrial proteomic analysis reveals the regulation of energy metabolism and reactive oxygen species production in Clematis terniflora DC. leaves under high-level UV-B radiation followed by dark treatment

Clematis terniflora DC. is an important medicinal plant from the family Ranunculaceae. A previous study has shown that active ingredients in C. terniflora, such as flavonoids and coumarins, are increased under ultraviolet B radiation (UV-B) and dark treatment and that the numbers of genes related to the tricarboxylic acid cycle and mitochondrial electron transport chain (mETC) are changed. To uncover the mechanism of the response to UV-B radiation and dark treatment in C. terniflora, mitochondrial proteomics was performed. The results showed that proteins related to photorespiration, mitochondrial membrane permeability, the tricarboxylic acid cycle, and the mETC mainly showed differential expression profiles. Moreover, the increase in alternative oxidase indicated that another oxygen-consuming respiratory pathway in plant mitochondria was induced to minimize mitochondrial reactive oxygen species production. These results suggested that respiration and mitochondrial membrane permeability were deeply influenced to avoid energy consumption and maintain energy balance under UV-B radiation and dark treatment in C. terniflora leaf mitochondria. Furthermore, oxidative phosphorylation was able to regulate intracellular oxygen balance to resist oxidative stress. This study improves understanding of the function of mitochondria in response to UV-B radiation and dark treatment in C. terniflora. SignificanceC. terniflora was an important traditional Chinese medicine for anti-inflammatory. Previous study showed that the contents of coumarins which were the main active ingredient in C. terniflora were induced by UV-B radiation and dark treatment. In the present study, to uncover the regulatory mechanism of metabolic changes in C. terniflora, mitochondrial proteomics analysis of leaves was performed. The results showed that photorespiration and oxidative phosphorylation pathways were influenced under UV-B radiation and dark treatment. Mitochondria in C. terniflora leaf played a crucial role in energy mechanism and regulation of cellular oxidation-reduction to maintain cell homeostasis under UV-B radiation followed with dark treatment.

Taxonomic identity of Korean endemic Clematis taeguensis Y. N. Lee (Ranunculales: Ranunculaceae)

Clematis taeguensis Y. N. Lee (Ranunculaceae) was first collected from Beommul-dong, Daegu-si and described by Lee (1982) as endemic to Korea. Morphologically, it resembles C. terniflora DC. var. terniflora, C. terniflora var. mandshurica (Rupr.) Ohwi, and C. brachyura Maxim; thus, it is often misidentified and has therefore not been included in domestic flora or research articles after being published as a new species. It was erroneously considered synonymous with C. serratifolia Rehder in sect. Meclatis. In addition, recurring difficulties have also occurred in recognizing its distribution sites. The present study therefore aimed to resolve the taxonomic controversy concerning C. taeguensis and redefine the taxonomy of this species and identify its distribution patterns using comprehensive external morphology and molecular analysis and confirmatory collections. Unlike other related taxa in sect. Clematis, C. taeguensis lacks a shoot remnant in the winter season and has abaxial and marginal hairs on the sepal, yellow to brown style hairs, and a persistent style is more than 1.9 cm long. It also has a limited distribution in the central (Gangwon: Jungseon, Yeongwol; Chungbuk: Jecheon, Yeongdong) and southern (Gyeongbuk: Daegu, Bonghwa; Gyeongnam: Changnyeong) parts of the Korean Peninsula. Molecular analysis via cpDNA sequencing confirmed that C. taeguensis formed its own group. Hence, C. taeguensis should be regarded as a separate species endemic to South Korea.

A metabolomic strategy revealed the role of JA and SA balance in Clematis terniflora DC. Response to UVB radiation and dark.

Clematis terniflora DC. is a valuable resource with potential high pharmaceutical value. Proteomic, transcriptomic and metabolomic analyses of C. terniflora that has been exposed to high levels of UVB irradiation and dark conditions (HUVB + D) have revealed the mechanisms underlying its medicinal potential. However, the signal transduction pathways and the mechanisms of regulation for the accumulation of secondary metabolites remain unclear. In this study, we show that the jasmonic acid (JA) and salicylic acid (SA) signals were activated in C. terniflora in response to HUVB + D. Metabolomic analysis demonstrated that the perturbation in JA and SA balance led to additional reallocation of carbon and nitrogen resources. Evaluating the fold change ratios of differentially changed metabolites proved that JA signal enhanced the transformation of nitrogen to carbon through the 4-aminobutyric acid (GABA) shunt pathway, which increased the carbon reserve to be utilized in the production of secondary metabolites. However, SA signal induced the synthesis of proline, while avoiding the accumulation of secondary metabolites. Over all, the results indicate that the co-increase of JA and SA reconstructed the dynamic stability of transformation from nitrogen to carbon, which effectively enhanced the oxidative defense to HUVB + D in C. terniflora by increasing the secondary metabolites.

Enhanced metabolic process to indole alkaloids in Clematis terniflora DC. after exposure to high level of UV-B irradiation followed by the dark.

BackgroundIndole alkaloids, which characteristically contain an indole nucleus, have pharmaceutical potential in a diverse range of applications. UV-B can elicit the accumulation of indole alkaloids. The indole alkaloid (6-hydroxyl-1H-indol-3-yl) carboxylic acid methyl ester with cytotoxic activity was found to accumulate in Clematis terniflora DC. leaves after exposure to high level of UV-B irradiation and the dark. However, a more in-depth analysis of the process behind this response has not yet been performed. Therefore, an integrated approach involving metabolomic, proteomic, and transcriptomic analyses is essential to detail the biosynthetic mechanisms of the regulation of indole alkaloid under binary stress.ResultsIndole alkaloid (6-hydroxyl-1H-indol-3-yl) carboxylic acid methyl ester was found to increase 7-fold in C. terniflora leaves post-treatment with high level of UV-B irradiation followed by an incubation in the dark compared with pre-treatment. Analysis by proteomics and metabolomics indicates a decrease in photosynthesis and carbohydrate metabolism, respectively. By contrast, amino acid metabolism was activated by this binary stress, and, specifically, the genes involved in the metabolic pathway converting shikimate to L-tryptophan were concurrently upregulated. Metabolites involved in indole biosynthesis (shikimate metabolic) pathway were anthranilate, indole, and L-tryptophan, which increased 2-, 441-, and 1-fold, respectively. In addition, there was an increase of 2- and 9-fold in L-serine deaminase (L-SD) and L-tryptophan synthase activity in C. terniflora leaves after exposure to high level of UV-B irradiation and the dark.Conclusions(6-hydroxyl-1H-indol-3-yl) carboxylic acid methyl ester was found to increase in response to high level of UV-B irradiation followed by an incubation in the dark, implying that indole alkaloid biosynthesis was activated in C. terniflora leaves. Analysis of perturbations in metabolism in these leaves demonstrated that amino acid metabolism was specifically activated by this binary stress. In addition, an enhancement in serine level and L-SD activity was noted, which likely leads to an accumulation of pyruvate that, in turn, supplies shikimate metabolic pathway. The genes, metabolites, and L-tryptophan synthase activity that are involved in the metabolic pathway leading from shikimate to L-tryptophan all increased under the experimental binary stress, resulting in an enhancement of indole biosynthesis (shikimate metabolic) pathway. Therefore, the metabolic process to indole alkaloids in C. terniflora was enhanced after exposure to high level of UV-B irradiation followed by the dark.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0920-3) contains supplementary material, which is available to authorized users.

Transcriptomic and proteomic analyses of leaves from Clematis terniflora DC. under high level of ultraviolet-B irradiation followed by dark treatment

High level of UV-B irradiation followed by dark treatment (HUV-B+D) causes accumulation of secondary metabolites in Clematis terniflora DC. To investigate the response mechanism under HUV-B+D, transcriptomic and proteomic analyses were performed in leaves of C. terniflora. The number of genes related to tetrapyrrole synthesis, amino acid metabolism, tricarboxylic acid cycle, and mitochondrial electron transport chains was hierarchically changed in leaves of C. terniflora under HUV-B+D. Data from RNA-sequencing transcriptomics and gel-free/label-free proteomics were integrated. The genes related to biosynthesis of lignins and flavonoids/isoflavonoids were significantly upregulated. Luteolin 7-O-β-D-glucosiduronic acid, rutin, and kaempferol 3-O-rutinose were accumulated. The number of differentially expressed genes (DEGs) and differentially abundant proteins (DAPs) related to protein metabolism were largely changed in posttranslational modification, ubiquitin proteasome, and ribosomal protein. The expression of NADP-dependent malic enzyme and the abundance of NADP-malate dehydrogenase were upregulated and increased, respectively. The activities of these two enzymes were also enhanced. These results suggest that the secondary metabolism pathway and tricarboxylic acid cycle might be activated in leaves of C. terniflora in response to HUV-B+D. Biological significanceThis study reported response mechanism in leaves of Clematis terniflora DC. under high level of UV-B irradiation followed by dark treatment using transcriptomic and proteomic techniques. Transcriptomic analysis revealed that the number of genes related to secondary metabolism was increased by >2 times. Phenylalanine ammonia-lyase, 4-Coumarate: CoA ligase, chalcone synthase, isoflavone reductase homolog, and cinnamoyl-CoA reductase were significantly upregulated, which suggests that the secondary metabolism pathway related to lignins and flavonoids/isoflavonoids might be activated. Genes and proteins related to protein metabolism were largely changed in postranslational modification, ubiquitin proteasome, and ribosomal protein. NADP-dependent malic enzyme and NADP-malate dehydrogenase were activated in tricarboxylic acid cycle, which suggests that tricarboxylic acid cycle might be enhanced in leaf of C. terniflora in response to HUV-B+D.

Proteomic and Metabolomic Analyses of Leaf from Clematis terniflora DC. Exposed to High-Level Ultraviolet-B Irradiation with Dark Treatment.

Clematis terniflora DC. has potential pharmaceutical value; on the contrary, high-level UV-B irradiation with dark treatment led to the accumulation of secondary metabolites. Metabolomic and proteomic analyses of leaf of C. terniflora were performed to investigate the systematic response mechanisms to high-level UV-B irradiation with dark treatment. Metabolites related to carbohydrates, fatty acids, and amino acids and/or proteins related to stress, cell wall, and amino acid metabolism were gradually increased in response to high-level UV-B irradiation with dark treatment. On the basis of cluster analysis and mapping of proteins related to amino acid metabolism, the abundances of S-adenosylmethionine synthetase and cysteine synthase as well as 1,1-diphenyl-2-picrylhydrazyl scavenging activity were gradually increased in response to high-level UV-B irradiation with dark treatment. Furthermore, the abundance of dihydrolipoyl dehydrogenase/glutamate dehydrogenase and the content of γ-aminobutyric acid were also increased following high-level UV-B irradiation with dark treatment. Taken together, these results suggest that high-level UV-B irradiation with dark treatment induces the activation of reactive oxygen species scavenging system and γ-aminobutyric acid shunt pathway in leaf of C. terniflora.

An in vivo and in vitro assessment of the anti-inflammatory, antinociceptive, and immunomodulatory activities of Clematis terniflora DC. extract, participation of aurantiamide acetate

AimClematis terniflora DC. has been widely used as a traditional Chinese medicine for the treatment of tonsillitis, rheumatoid arthritis, and prostatitis. Despite its widespread use in China, there are currently no studies systematically examined its therapeutic effects and mechanism of action. As such, the present study was conducted to evaluate the anti-inflammatory, antinociceptive, and immunomodulatory effects of C. terniflora DC. using rodent and cellular models. MethodsThe anti-inflammatory properties of the 70% ethanol eluted fraction of the 70% ethanol extract of C. terniflora DC. (EECTD) were evaluated using the xylene-induced ear swelling test, the carrageenan-induced edema model, and the cotton pellet granuloma method. Its antinociceptive activities were determined using both the acetic acid-induced writhing test and the hot plate assay. In parallel, we conducted an in vitro assay in LPS-induced RAW264.7 cells to examine the anti-inflammatory effects of EECTD and its purified form, aurantiamide acetate (AA) on inhibition of nitric oxide (NO) and prostaglandin E2 (PGE2) release. ResultsEECTD (300mg/kg) significantly reduced the number of writhing, extended the pain response latency, and suppressed xylene-induced ear swelling. Each EECTD treatment group also had significant inhibition of cotton granulation formation in addition to reduced carrageenan-induced paw edema. EECTD was also shown to alleviate signs of inflammation in histopathological paw sections. However, it had a less noticeable effect on mouse ear swelling in the delayed type hypersensitivity test. A purified compound was isolated from EECTD and its structure was identified as AA. In vitro experimental results showed that both EECTD and AA were able to significantly inhibit the release of pro-inflammatory cytokines NO and PGE2 on LPS-induced RAW264.7 cells. ConclusionThese results suggest that EECTD has significant anti-inflammatory and antinociceptive activities, partially related to one of the active substances identified as AA. We hypothesize that these effects are related to its ability to inhibit the production of cytokines NO and PGE2. However, further work will be needed to determine its exact mechanism of action.

The complete chloroplast genome sequence of Clematis terniflora DC. (Ranunculaceae)

Clematis terniflora DC. is an important medicinal plant used in the treatment of inflammatory symptoms related to respiratory and urinary systems. In this study, we found that the complete cp genome of C. terniflora DC. is 159,528 bp. The phylogenetic analysis of 32 taxa showed a strong sister relationship with Ranunculus macranthus, which also strongly supports the position of Ranunculales. The complete cp genome sequence of Clematis terniflora DC. reported here has the potential to advance population and phylogenetic studies of this medicinal plant.

Aurantiamide acetate suppresses the growth of malignant gliomas in vitro and in vivo by inhibiting autophagic flux.

We aim to investigate the effect of aurantiamide acetate isolated from the aerial parts of Clematis terniflora DC against gliomas. Human malignant glioma U87 and U251 cells were incubated with different concentrations (0–100 μM) of aurantiamide acetate. Aurantiamide acetate greatly decreased the cell viability in a dose- and time-dependent manner. It induced moderate mitochondrial fragmentation and the loss of mitochondrial membrane potential. No significant difference was found in the alternation of other intracellular organelles, although F-actin structure was slightly disturbed. Apparent ultrastructure alternation with increased autophagosome and autolysosome accumulation was observed in aurantiamide acetate-treated cells. The expression of LC3-II was greatly up-regulated in cells exposed to aurantiamide acetate (P < 0.05 compared with control). The cytoplasmic accumulation of autophagosomes and autolysosomes induced by aurantiamide acetate treatment was confirmed by fluorescent reporter protein labelling. Administration of chloroquine (CQ), which inhibits the fusion step of autophagosomes, further increased the accumulation of autophagosomes in the cytoplasm of U87 cells. Autophagy inhibition by 3-methyladenine, Bafilomycin A1 or CQ had no influence on aurantiamide acetate-induced cytotoxicity, whereas autophagy stimulator rapamycin significantly suppressed aurantiamide acetate-induced cell death. The anti-tumour effects of aurantiamide acetate were further evaluated in tumour-bearing nude mice. Intratumoural injection of aurantiamide acetate obviously suppressed tumour growth, and increased number of autophagic vacuoles was observed in tumour tissues of animals receiving aurantiamide acetate. Our findings suggest that aurantiamide acetate may suppress the growth of malignant gliomas by blocking autophagic flux.

A new indole alkaloidal glucoside from the aerial parts of Clematis terniflora DC.

A new indole alkaloidal glucoside together with three known compounds aurantiamide acetate (2), eleutheroside E (3) and 1-O-caffeoyl-β-d-glucopyranoside (4) has been isolated from ethanol extract of the aerial parts of Clematis terniflora DC. On the basis of their spectroscopic and chemical evidence, the new compound was elucidated as (6-O-β-d-glucopyranosyl-1H-indol-3-yl) carboxylic acid methyl ester (1). Compounds 1 and 3 showed significant cytotoxicity against human ECA-109.

In vivo study of four preparative extracts of Clematis terniflora DC. for antinociceptive activity and anti-inflammatory activity in rat model of carrageenan-induced chronic non-bacterial prostatitis

The present study was conducted to evaluate the anti-inflammatory and antinociceptive activities of Clematis terniflora DC. extracts and fractions and to further support its traditional use as Chinese folk medicine in treatment of urinary infections, especially the disease of prostatitis. The antinociceptive activity of its water extract (WE), 70% ethanol extract (EE), water eluted part of EE from AB-8 macroporous resin (WEPMR) and 70% ethanol eluted part of EE from AB-8 macroporous resin (EEPMR) was conducted using mice writhing test with different doses. Then the anti-inflammatory activity of the four parts was evaluated on rat models of carrageenan-induced chronic non-bacterial prostatitis (CNP). Preliminary study was taken to determine the phytochemical compositions of the four preparative extracts. Significant writhing inhibitory effect was found with EE at small (7.5 g/kg body wt.), moderate (15 g/kg body wt.) and large (30 g/kg body wt.) doses (doses here are presented as crude herbs) as well as EEPMR at moderate and large doses by oral administration (OA) (p≤0.01). Data from prostatic index, lecithin microsome density and white blood cell level showed that moderate dose of EE and EEPMR both had significant (p≤0.05 or p≤0.01) inhibition effect on carrageenan-induced inflammation in rat prostate. The HPLC analytical results showed that flavonoids were the main active compounds in WE, EE and EEPMR. And most flavonoids were accumulated into the part of EEPMR by AB-8 macroporous resin leaving only few compounds in WEPMR. No acute toxicity was identified in oral administration of the four parts at a dose of 100g/kg body wt. The results described here suggest that extracts of the aerial part of Clematis terniflora DC. might be of therapeutic interest in the treatment of prostatitis.

センニンソウ（Ｃｌｅｍａｔｉｓ ｔｅｒｎｉｆｌｏｒａ ＤＣ．）の開花に及ぼす温度，日長ならびにジベレリン施与の影響

Vegetative growth and flowering of Clematis terniflora DC. were favored by day/night temperatures of 24°/19°C and 30°/25°C under natural photoperiods of above 14-hr daylight from May through July. However, at 17°/12°C, the shoot growth was significantly delayed and no flower buds were differentiated.Even though plants were grown under favorable temperatures, shoot tips became necrotic when grown under an 8-hr day for one month during May or July. Flower buds were differentiated under natural photoperiods and 16-hr photoperiod, but not under 8-hr or 12-hr photoperiods. Buds require about one month under long days for flower induction to take place provided the shoot reached a minimum critical size which also requires about a month after budbreak. After differentiation occurred, continued exposure of plants to 16-hr days delayed flower bud development, bud break, and bloom dates compared to plants transferred to 8-hr days.Under natural photoperiods, sprays of 100 and 1, 000 ppm GA hastened both vegetative growth and flowering but decreased the number of flowers as compared to the non-sprayed control plants. Eight applications of GA at 15-day intervals compared to one spray treatment decreased the number of flowers markedly and more than half of the flowers produced were abnormal ones with degenerated anthers and undeveloped pistils. Moreover, among normal and abnormal flowers, there were found some flowers with more than 4 sepals and/or sepaloid stamens.