Roseus Cell Suspension Research Articles

Metabolic Regulation Analysis of Ajmalicine Biosynthesis Pathway in Catharanthus roseus (L.) G. Don Suspension Culture Using Nanosensor

Ajmalicine is one of the most popular antihypertensive drugs obtained from the root barks of Cathranthus roseus (L.) G. Don and Rauvolfia serpentine (L.) Benth. ex Kurz. It has also potential antimicrobial, cytotoxic, central depressant and antioxidant activities. As the demand for the alkaloid is significantly high, metabolic engineering approaches are being tried to increase its production in both homologous and heterologous systems. The metabolic engineering approach requires knowledge of the metabolic regulation of the alkaloid. For understanding the metabolic regulation, fluxomic analysis is important as it helps in understanding the flux of the alkaloid through the complicated metabolic pathway. The present study was conducted to analyse the flux analysis of the ajmalicine biosynthesis, using a genetically encoded Fluorescent Resonance Energy Transfer FRET-based nanosensor for ajmalicine (FLIP-Ajn). Here, we have silenced six important genes of terpenoid indole alkaloid (TIA), namely G10H, 10HGO, TDC, SLS, STR and SDG, through RNA-mediated gene silencing in different batches of C. roseus suspension cells, generating six silenced cell lines. Monitoring of the ajmalicine level was carried out using FLIP-Ajn in these silenced cell lines, with high spatial and temporal resolution. The study offers the rapid, high throughput real-time measurement of ajmalicine flux in response to the silenced TIA genes, thereby identifying the regulatory gene controlling the alkaloid flux in C. roseus suspension cells. We have reported that the STR gene encoding strictosidine synthase of the TIA pathway could be the regulatory gene of the ajmalicine biosynthesis.

A novel strategy for the synthesis of gold nanoparticles with Catharanthus roseus cell suspension culture

The purpose of this research was to develop a green method to synthesise gold nanoparticles (Au NPs) with cell suspension cultures of Catharanthus roseus. Reaction mixtures containing cell-free culture filtrate of C. roseus and gold III ions, which was initially pale yellow, turned wine red after 24-h incubation due to Au NP formation. UV–Vis spectra of the reaction mixtures showed a characteristic absorption peak at 546 nm. The biosynthesised NPs were predominantly spherical with 2–20 nm size; their crystalline nature was revealed by X-ray diffraction patterns. FTIR analysis indicated that Au NPs were capped by amide characteristic of plant proteins, which makes them apt for various applications.

Biotransformation of Cyclodextrine-Complexed Semisynthetic Betulin Derivatives by Plant Cells.

In this study, three semisynthetic betulonic acid-based compounds, 20(29)-dihydrolup-2-en[2,3-d]isoxazol-28-oic acid, 1-betulonoylpyrrolidine, and lupa-2,20(29)-dieno[2,3-b]pyrazin-28-oic acid, were studied in biotransformation experiments using Nicotiana tabacum and Catharanthus roseus cell suspension cultures. Biotransformation was performed using cyclodextrin to aid dissolving poorly water-soluble substrates. Several new derivatives were found, consisting of oxidized and glycosylated (pentose- and hexose-conjugated) products.

The basic helix-loop-helix transcription factor BIS2 is essential for monoterpenoid indole alkaloid production in the medicinal plant Catharanthus roseus.

Monoterpenoid indole alkaloids (MIAs) are produced as plant defence compounds. In the medicinal plant Catharanthus roseus, they comprise the anticancer compounds vinblastine and vincristine. The iridoid (monoterpenoid) pathway forms one of the two branches that feed MIA biosynthesis and its activation is regulated by the transcription factor (TF) basic helix-loop-helix (bHLH) iridoid synthesis 1 (BIS1). Here, we describe the identification and characterisation of BIS2, a jasmonate (JA)-responsive bHLH TF expressed preferentially in internal phloem-associated parenchyma cells, which transactivates promoters of iridoid biosynthesis genes and can homodimerise or form heterodimers with BIS1. Stable overexpression of BIS2 in C. roseus suspension cells and transient ectopic expression of BIS2 in C. roseus petal limbs resulted in increased transcript accumulation of methylerythritol-4-phosphate and iridoid pathway genes, but not of other MIA genes or triterpenoid genes. Transcript profiling also indicated that BIS2 expression is part of an amplification loop, as it is induced by overexpression of either BIS1 or BIS2. Accordingly, silencing of BIS2 in C. roseus suspension cells completely abolished the JA-induced upregulation of the iridoid pathway genes and subsequent MIA accumulation, despite the presence of induced BIS1, indicating that BIS2 is essential for MIA production in C. roseus.

The bHLH transcription factor BIS1 controls the iridoid branch of the monoterpenoid indole alkaloid pathway in Catharanthus roseus

Plants make specialized bioactive metabolites to defend themselves against attackers. The conserved control mechanisms are based on transcriptional activation of the respective plant species-specific biosynthetic pathways by the phytohormone jasmonate. Knowledge of the transcription factors involved, particularly in terpenoid biosynthesis, remains fragmentary. By transcriptome analysis and functional screens in the medicinal plant Catharanthus roseus (Madagascar periwinkle), the unique source of the monoterpenoid indole alkaloid (MIA)-type anticancer drugs vincristine and vinblastine, we identified a jasmonate-regulated basic helix-loop-helix (bHLH) transcription factor from clade IVa inducing the monoterpenoid branch of the MIA pathway. The bHLH iridoid synthesis 1 (BIS1) transcription factor transactivated the expression of all of the genes encoding the enzymes that catalyze the sequential conversion of the ubiquitous terpenoid precursor geranyl diphosphate to the iridoid loganic acid. BIS1 acted in a complementary manner to the previously characterized ethylene response factor Octadecanoid derivative-Responsive Catharanthus APETALA2-domain 3 (ORCA3) that transactivates the expression of several genes encoding the enzymes catalyzing the conversion of loganic acid to the downstream MIAs. In contrast to ORCA3, overexpression of BIS1 was sufficient to boost production of high-value iridoids and MIAs in C. roseus suspension cell cultures. Hence, BIS1 might be a metabolic engineering tool to produce sustainably high-value MIAs in C. roseus plants or cultures.

Metabolic alterations and distribution of five-carbon precursors in jasmonic acid-elicited Catharanthus roseus cell suspension cultures

In Catharanthus roseus cell cultures, the terpenoid moiety is considered as the limiting factor in terpenoid indole alkaloid (TIA) production. The pathway of terpenoidal precursors in TIA is strongly linked with other terpenoid pathways, suggesting that TIA production might be limited by competition for the five-carbon (C5) precursors. This raises the question whether the stimulation of TIA production by certain signal molecules is due to a redistribution of C5 precursors between the associated terpenoid pathways and/or to a total increase of the precursor availability. To investigate the effect of a TIA-increasing signal molecule on C5 distribution, the cell suspension cultures of C. roseus were elicited with jasmonic acid (JA) and the metabolic changes of different terpenoid pathways were evaluated targeting on TIA (monoterpenoid; C10), carotenoids (tetraterpenoid; C40), and phytosterols (triterpenoid; C30). The chromatographic analyses showed that TIA and carotenoid levels almost doubled upon JA elicitation, while phytosterol levels remained constant if compared to the controls. Apparently, both TIA and carotenoid routes benefit from an increased C5 flow in the methyl-erythritol phosphate pathway, and potential export of C5 precursors to the cytosolic terpenoid routes, e.g., towards the phytosterols is minimal. However, the relative composition of individual compounds within each group remains similar when comparing elicited and control cells. This suggests that the increased production of TIA and carotenoid upon JA elicitation is predominantly caused by an increase of the precursor availability rather than due to a redistribution of C5 precursors between the associated terpenoid pathways. Furthermore, NMR-based metabolomics analysis showed a discrimination of JA-elicited and control cells between 24 and 72 h after treatments with significant changes in levels of strictosidine, malic acid, and sucrose. This study portrays metabolic alterations upon JA elicitation and channeling of C5 precursors in different terpenoid biosynthetic pathways, which provides a knowledge platform for developing strategies to engineer fluxes in a complex biosynthetic network in order to obtain high TIA-producing C. roseus cell lines.

An active Catharanthus roseus desacetoxyvindoline-4-hydroxylase-like gene and its transcriptional regulatory profile.

BackgroundDesacetoxyvindoline-4-hydroxylase is a key enzyme in the biosynthesis of vindoline, the important intermediate leading to vinblastine and vincristine in Catharanthus roseus.ResultsA d4h-like gene has been isolated from C. roseus C20hi cells based on an EST sequence from the Suppression Subtractive Hybridization cDNA library. The full length cDNA of d4h-like was 1427 bp encoding 372 amino acids. It had 66% identities and 80% positives with d4h at the amino acid level. It belonged to 2-oxoglutarate dependent oxygenase superfamily as d4h did. Real-time quantitative PCR analysis revealed that d4h-like was expressed high in roots, flowers and C20hi cells, very low in leaves and stems. Methyl jasmonate could significantly increase the accumulation of d4h-like transcripts. 2,4-D inhibited its expression. An approximate 2,910 bp of 5′-promoter region of d4h-like was obtained, fused to GUS reporter gene and analyzed with fluorescence quantitative assays using transient expression in C. roseus cell suspensions, indicating that d4h-like promoter could drive GUS gene expression in vivo.ConclusionThese results suggest that d4h-like is closely related with d4h in the genetic evolution but with different transcriptional expression profiles. It may be revolved in the hormone-independency of C20hi cells.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-55-29) contains supplementary material, which is available to authorized users.

Analysis of metabolites in the terpenoid pathway of Catharanthus roseus cell suspensions

In Catharanthus roseus cell cultures, the monoterpenoid pathway has been shown to be a limiting factor in terpenoid indole alkaloid (TIA) production. This could be due to competition at the level of isopentenyl diphosphate::dimethylallyl diphosphate (C5) which leads to the biosynthesis of different terpenoid groups. For future engineering of the terpenoid pathway, chemical characterization of C. roseus cell cultures is a necessity. Therefore, in this study nine C. roseus cell suspension lines were characterized by analyzing the levels of the major terpenoids derived from different biosynthetic pathways which may compete for the same precursors; TIA (monoterpenoid, C10), carotenoids (tetraterpenoid, C40), and sterols (triterpenoid, C30). Among the cell lines, CRPP (S) was the most promising TIA-producing cell line which provided more TIA [24 μmol g−1 dry weight (DW)] than carotenoids (15 μmol g−1 DW) and sterols (2 μmol g−1 DW). However, when considering the distribution of the isopentenyl-precursor (C5), the carotenoids which assemble from 8× C5 represent twofold more C5-units (122 μmol g−1 DW) than the TIA in this cell line. In the CRPP (G), A12A2 (G), and A12A2 (S) cell lines, the C5 distribution was predominant toward carotenoid biosynthesis as well, resulting in a relatively high accumulation of carotenoids. The geranylgeranyl diphosphate (C20) pathway toward carotenoid production is therefore considered competitive toward TIA biosynthesis. For channeling more precursors to the TIA, the branch point for C10 and C20 seems an interesting target for metabolic engineering. Using principal component analysis of the chromatographic data, we characterized the cell lines chemically based on their metabolite levels. The information on the metabolic composition of C. roseus cell cultures is useful for developing strategies to engineer the metabolic pathways and for selection of cell lines for future studies.

Impact of artemisinic acid on the growth and catharanthine production in Catharanthus roseus cultured cells

In this paper, we investigate the effects of artemisinic acid on the growth and alkaloids production of Catharanthus roseus cell cultures. For the first time, we have shown that artemisinic acid could promote the production of catharanthine in C. roseus suspension cell cultures. The highest total catharanthine yield (0.584 mg/flask) was obtained with the addition of 60 mg·L-1 artemisinic acid after 96 h, which was 4.95 fold of that in ethanol treated group (0.118 mg/flask). In order to elucidate the underlying mechanism, transcriptional levels of geraniol 10-hydroxylase (G10H), strictosidine β-D-glucosidase (SGD), tryptophan decarboxylase (TDC) and strictosidine synthase (STR) were measured by RT-PCR. The results indicated that artemisinic acid may be able to up-regulate G10H,SGD and TDC expressions without affecting STR expression. In addition, growth of C. roseus suspension cells was inhibited at high concentrations (more than 20 mg·L-1) of artemisinic acid. No vindoline was observed in C. roseus suspension cell cultures. Key words: Catharanthus roseus, cultured cells, catharanthine, vindoline, artemisinic acid, gene expression.

Isolation and functional analysis of the Catharanthus roseus deacetylvindoline-4-O-acetyltransferase gene promoter

Madagascar periwinkle (Catharanthus roseus) produces many therapeutically valuable terpenoid indole alkaloids (TIAs), such as vinblastine and vincristine derived from the monomers vindoline and catharanthine. Deacetylvindoline-4-O-acetyltransferase (DAT) is a key enzyme for the terminal step of vindoline biosynthesis. In this research, the DAT gene promoter was cloned, sequenced, and analyzed. An approximately 1,773 bp genomic DNA fragment of DAT promoter was obtained. Sequence analysis revealed that DAT promoter contained several potential regulatory elements which were involved in the regulation of gene expression. To investigate its function, the promoter fragments with 5' deletions and gain-of-function deletions were fused to GUS reporter gene, and their expressions were analyzed by transient expression in C. roseus cell suspensions. The regulatory activity of DAT promoter was identified with fluorescence quantitative assays. Three TGACG motifs and one inverted motif (CGTCA) between -808 and -1,086 bp in the DAT promoter were found to be involved in methyljasmonate signal transduction pathway.

Metabolic changes of salicylic acid-elicited Catharanthus roseus cell suspension cultures monitored by NMR-based metabolomics

The effect of salicylic acid (SA) on the metabolic profile of Catharanthus roseus suspension cells throughout a time course (0, 6, 12, 24, 48 and 72 h after treatment) was investigated using NMR spectroscopy and multivariate data analysis. When compared to control cell lines, SA-treated cells showed a high level of sugars (glucose and sucrose) up to 48 h after treatment, followed by a dynamic change in amino acids, phenylpropanoids, and tryptamine. Additionally, one compound—2,5-dihydroxybenzoic-5-O-glucoside—was detected solely in SA-treated cells.

Purification and Characterization of UDP-Glucose : Curcumin Glucoside 1,6-Glucosyltransferase from Catharanthus roseus Cell Suspension Cultures

Catharanthus roseus cell suspension cultures converted exogenously added curcumin to a series of curcumin glucosides that possessed drastically enhanced water solubility. A cDNA clone encoding a glucosyltransferase responsible for glucosylation of curcumin to form curcumin 4'-O-glucoside was previously isolated, and in the present study a novel sugar-sugar glycosyltransferase, UDP-glucose:curcumin glucoside glucosyltransferase (UCGGT), was purified approximately 900-fold to apparent homogeneity from cultured cells of C. roseus. The purified enzyme (0.2% activity yield) catalyzed 1,6-glucosylation of curcumin 4'-O-glucoside to yield curcumin 4'-O-gentiobioside. The molecular weight and isoelectric point were estimated to be about 50 kDa and 5.2, respectively. The enzyme showed a pH optimum between 7.5 and 7.8. Both flavonoid 3-O- and 7-O-glucosides were also preferred acceptor substrates of the enzyme, whereas little activity was shown toward simple phenolic glucosides such as arbutin and glucovanillin, cyanogenic glucoside (prunasin) or flavonoid galactoside. These results suggest that UCGGT may also function in the biosynthesis of flavonoid glycosides in planta.

UV-B-induced signaling events leading to enhanced-production of catharanthine in Catharanthus roseus cell suspension cultures.

BackgroundElicitations are considered to be an important strategy towards improved in vitro production of secondary metabolites. In cell cultures, biotic and abiotic elicitors have effectively stimulated the production of plant secondary metabolites. However, molecular basis of elicitor-signaling cascades leading to increased production of secondary metabolites of plant cell is largely unknown. Exposure of Catharanthus roseus cell suspension culture to low dose of UV-B irradiation was found to increase the amount of catharanthine and transcription of genes encoding tryptophan decarboxylase (Tdc) and strictosidine synthase (Str). In the present study, the signaling pathway mediating UV-B-induced catharanthine accumulation in C. roseus suspension cultures were investigated.ResultsHere, we investigate whether cell surface receptors, medium alkalinization, Ca2+ influx, H2O2, CDPK and MAPK play required roles in UV-B signaling leading to enhanced production of catharanthine in C. roseus cell suspension cultures. C. roseus cells were pretreated with various agonists and inhibitors of known signaling components and their effects on the accumulation of Tdc and Str transcripts as well as amount of catharanthine production were investigated by various molecular biology techniques. It has been found that the catharanthine accumulation and transcription of Tdc and Str were inhibited by 3–4 fold upon pretreatment of various inhibitors like suramin, N-acetyl cysteine, inhibitors of calcium fluxes, staurosporine etc.ConclusionOur results demonstrate that cell surface receptor(s), Ca2+ influx, medium alkalinization, CDPK, H2O2 and MAPK play significant roles in UV-B signaling leading to stimulation of Tdc and Str genes and the accumulation of catharanthine in C. roseus cell suspension cultures. Based on these findings, a model for signal transduction cascade has been proposed.

Biotransformation of (–)-Caryophyllene Oxide by Cell Suspension Culture of Catharanthus roseus

Abstract Catharanthus roseus cell suspension cultures were employed for the biotransformation of (-)-caryophyllene oxide (1), and four metabolites, 15-hydroxycaryophyllene oxide (2), 4β ,5α- dihydroxycaryophyll-8(13)-ene (3), 2β -hydroxycaryophyllene oxide (4), and 2-hydroxy-4,5- epoxycaryophyllan-13-ol (5) were obtained. Metabolites 4 and 5 were found to be new compounds, and their structures were deduced by different spectroscopic techniques.

Proteome analysis of the medicinal plant Catharanthus roseus

A proteomic approach is undertaken aiming at the identification of novel proteins involved in the alkaloid biosynthesis of Catharanthus roseus. The C. roseus cell suspension culture A11 accumulates the terpenoid indole alkaloids strictosidine, ajmalicine and vindolinine. Cells were grown for 21 days, and alkaloid accumulation was monitored during this period. After a rapid increase between day 3 and day 6, the alkaloid content reached a maximum on day 16. Systematic analysis of the proteome was performed by two-dimensional polyacrylamide gel electrophoresis. After day 3, the proteome started to change with an increasing number of protein spots. On day 13, the proteome changed back to roughly the same as at the start of the growth cycle. 88 protein spots were selected for identification by mass spectrometry (MALDI-MS/MS). Of these, 58 were identified, including two isoforms of strictosidine synthase (EC 4.3.3.2), which catalyzes the formation of strictosidine in the alkaloid biosynthesis; tryptophan synthase (EC 4.1.1.28), which is needed for the supply of the alkaloid precursor tryptamine; 12-oxophytodienoate reductase, which is indirectly involved in the alkaloid biosynthesis as it catalyzes the last step in the biosynthesis of the regulator jasmonic acid. Unique sequences were found, which may also relate to unidentified biosynthetic proteins.

Effect of nitric oxide on catharanthine production and growth ofCatharanthus roseus suspension cells

Sodium nitroprusside (SNP) was used as the donor of nitric oxide (NO) to investigate its effect on catharanthine synthesis and the growth of Catharanthus roseus suspension cells. The results showed that SNP at high concentrations (10.0 and 20.0 mmol/L) stimulated catharanthine formation of C. roseus cells, but inhibited growth of the cells. Low concentrations of SNP (0.1 and 0.5 mmol/L) enhanced the growth of C. roseus cells, but had no effect on catharanthine synthesis. The maximum total catharanthine production was achieved by the addition of 0.5 and 10.0 mmol/L SNP to the cultures at day 0 and day 10, respectively, being about threefold of the control. NO-induced catharanthine production of C. roseus cells was strongly suppressed by jasmonic acid (JA) biosynthesis inhibitor ibuprofen (IBU) and nordihydroguaiaretic (NDGA). The result suggests that the stimulatory role of NO on catharanthine production is partially JA-dependent.

Zinc Finger Proteins Act as Transcriptional Repressors of Alkaloid Biosynthesis Genes in Catharanthus roseus

In Catharanthus roseus cell suspensions, the expression of several terpenoid indole alkaloid biosynthetic genes, including two genes encoding strictosidine synthase (STR) and tryptophan decarboxylase (TDC), is coordinately induced by fungal elicitors such as yeast extract. To identify molecular mechanisms regulating the expression of these genes, a yeast one-hybrid screening was performed with an elicitor-responsive part of the TDC promoter. This screening identified three members of the Cys(2)/His(2)-type (transcription factor IIIA-type) zinc finger protein family from C. roseus, ZCT1, ZCT2, and ZCT3. These proteins bind in a sequence-specific manner to the TDC and STR promoters in vitro and repress the activity of these promoters in trans-activation assays. In addition, the ZCT proteins can repress the activating activity of APETALA2/ethylene response-factor domain transcription factors, the ORCAs, on the STR promoter. The expression of the ZCT genes is rapidly induced by yeast extract and methyljasmonate. These results suggest that the ZCT proteins act as repressors in the regulation of elicitor-induced secondary metabolism in C. roseus.

O 2− from elicitor-induced oxidative burst is necessary for triggering phenylalanine ammonia-lyase activation and catharanthine synthesis in Catharanthus roseus cell cultures

Elicitor, derived from the cell walls of Aspergillus niger, induced rapid generation of reactive oxygen intermediates (ROI), including superoxide anion (O 2 −) and hydrogen peroxide (H 2O 2), sequentially followed by phenylalanine ammonia-lyase (PAL) activation and catharanthine biosynthesis in Catharanthus roseus suspension cells. The elicitor-induced PAL activation and catharanthine biosynthesis were blocked by NAD(P)H oxidase inhibitor, diphenylene iodonium (DPI). O 2 − generated by the reaction of xanthine/xanthine oxidase (X/XO) triggered PAL activation and catharanthine biosynthesis of C. roseus cells in the absence of elicitor and reversed the inhibitory effect of DPI on elicitor-induced PAL activation and catharanthine biosynthesis. External application of H 2O 2 and catalase had no effect on PAL activity and catharanthine contents of C. roseus cells. The results demonstrated a causal relationship between elicitor-induced oxidative burst and PAL activation in C. roseus suspension cells and suggested a sequence of signaling events from ROI production to PAL activation and catharanthine synthesis. Within this sequence, O 2 − rather than H 2O 2 appeared to trigger the subsequent reactions.

Elicitor-induced nitric oxide burst is essential for triggering catharanthine synthesis in Catharanthus roseus suspension cells.

Elicitor prepared from the cell walls of Penicillium citrinum induced multiple responses in Catharanthus roseus suspension cells, including rapid generation of nitric oxide (NO), sequentially followed by enhancement of catharanthine production by C. roseus cells. Elicitor-induced catharanthine biosynthesis was blocked by NO-specific scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and nitric oxide synthase (NOS) inhibitor S,S'-1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea (PBITU). PBITU also strongly inhibited elicitor-induced NO generation by C. roseus suspension cells. The inhibiting effect of PBITU on elicitor-induced catharanthine production was reversed by external application of NO via the NO-donor sodium nitroprusside. The results strongly suggested that NO, generated by NOS or NOS-like enzymes in C. roseus suspension cells when treated with the fungal elicitor, was essential for triggering catharanthine synthesis.

Activation of the oxidative burst by yeast elicitor in Catharanthus roseus cells occurs independently of the activation of genes involved in alkaloid biosynthesis

In Catharanthus roseus cell suspensions, expression of several terpenoid indole alkaloid (TIA) biosynthetic genes, including those encoding strictosidine synthase and tryptophan decarboxylase, is coordinately induced by fungal elicitors such as yeast extract (YE). This induction is mediated by several signaling steps including the biosynthesis of jasmonic acid, and the activation of the jasmonic acid-responsive ORCA transcription factors. We investigated a possible role of reactive oxygen species (ROS) as a second messenger in this system. YE was shown to activate the production of ROS, which was dependent on protein phosphorylation and calcium influx. However, ROS generation was neither necessary for the induction of genes involved in TIA biosynthesis by YE nor by itself sufficient to induce these genes. Therefore, we conclude that activation of the oxidative burst by YE occurs independently of the activation of genes involved in TIA biosynthesis.