Suspension-cultured Catharanthus Roseus Cells Research Articles

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.

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.

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.

Involvement of rapid nucleotide synthesis in recovery from phosphate starvation of Catharanthus roseus cells

Growth of suspension-cultured Catharanthus roseus cells ceased during phosphate starvation, but the cells grew again upon addition of Pi even after long-term starvation. The metabolic fate of [(33)P]Pi was studied in 1-week-old stationary phase cells in ordinary culture and in 1- or 2-week-old Pi-starved cells. Immediately after administration, the most heavily labelled organic compounds are nucleotides, followed by sugar phosphates. Two weeks Pi starvation slowed down the speed of incorporation of (33)P into nucleotides. The RNA, protein, and free nucleotide content all decreased gradually during Pi starvation; however, these compounds, especially nucleotides, increased markedly in the 24 h after addition of Pi. These responses are found in all cells examined, although the total amounts of these compounds were lower in the long-term Pi-deficient cells. Of the nucleotides, a marked increase was observed in nucleoside triphosphates and UDP-glucose. The transcript level of phosphate transporter and the activities of acid phosphatase, 5'- and 3'-nucleotidase, and adenosine nucleosidase were all reduced by the addition of Pi. In contrast, the activities of adenine phosphoribosyltransferase, nicotinate phosphoribosyltransferase, and nicotinamidase, which are salvage enzymes of purine and pyridine nucleotides, were markedly increased in the Pi-fed cells. Little or no increase was observed in adenosine kinase. In the light of these results, the possible involvement of net nucleotide synthesis in the initial metabolic events of recovery from Pi deficiency are discussed.

Effect of long-term phosphate starvation on the levels and metabolism of purine nucleotides in suspension-cultured Catharanthus roseus cells

The effect of long-term phosphate (Pi) starvation of up to 3 weeks on the levels of purine nucleotides and related compounds was examined using suspension-cultured Catharanthus roseus cells. Levels of adenine and guanine nucleotides, especially ATP and GTP, were markedly reduced during Pi-starvation. There was an increase in the activity of RNase, DNase, 5′- and 3′-nucleotidases and acid phosphatase, which may participate in the hydrolysis of nucleic acids and nucleotides. Accumulation of adenosine, adenine, guanosine and guanine was observed during the long-term Pi starvation. Long-term Pi starvation markedly depressed the flux of transport of exogenously supplied [8- 14C]adenosine and [8- 14C]adenine, but these labelled compounds which were taken up by the cells were readily converted to adenine nucleotides even in Pi-starved cells, in which RNA synthesis from these precursors was significantly reduced. The activities of adenosine kinase, adenine phosphoribosyltransferase and adenosine nucleosidase were maintained at a high level in long-term Pi starved cells.

Biotransformation of triptolide and triptonide by cell suspension cultures of Catharanthus Roseus

Catharanthus roseus cell suspension cultures were used to bioconvert both triptolide (1) and triptonide (2). The same reaction path was followed in both biotransformations. Two biotransformed products were obtained and their structures identified as triptriolide (3) and 12β,13α-dihydroxytriptonide (4), respectively, from 1 and 2. Product 4 is a new compound.

Molecular cloning and characterization of a glucosyltransferase catalyzing glucosylation of curcumin in cultured Catharanthus roseus cells

Catharanthus roseus cell suspension cultures are capable of converting exogenously supplied curcumin to various glucosides. The glucosylation efficiency is enhanced by addition of methyl jasmonate (MJ) to the cultures prior to curcumin administration. Two cDNAs encoding UDP-glucosyltransferases (CaUGT1 and CaUGT2) were isolated from a cDNA library of cultured C. roseus cells, using a PCR method directed at the conserved UDP-binding domain of plant glycosyltransferases. The sequence identity between their deduced amino acid sequences was 27%. The expression of both genes was up-regulated by addition of MJ to the cell cultures although the mRNA level of CaUGT1 was much lower than that of CaUGT2. The corresponding cDNAs were expressed in Escherichia coli as fusion proteins with maltose-binding protein. The recombinant CaUGT1 exhibited no glucosylation activity with either curcumin or curcumin monoglucoside as substrate, whereas the recombinant CaUGT2 catalyzed the formation of curcumin monoglucoside from curcumin and also conversion of curcumin monoglucoside to curcumin diglucoside. The use of the recombinant CaUGT2 may provide a useful new route for the production of curcumin glucosides.

Production of unnatural glucosides of curcumin with drastically enhanced water solubility by cell suspension cultures of Catharanthus roseus

Catharanthus roseus cell suspension cultures converted exogenously supplied curcumin to a series of glucosides, none of which has been found in nature so far. The efficiency of glucosylation was dependent on culture stage of the cells and medium sucrose concentration. Methyl jasmonate and salicylic acid enhanced the glucoside formation only when they were added to the cultures prior to the addition of curcumin. The glucoside yield was 2.5 μmol/g fresh weight of the cells at an optimal culture condition. The water solubility of curcumin-4′,4″- O-β- D-digentiobioside was 0.65 mmol/ml, which was 20 million-fold higher than that of curcumin.

Biotransformation of 2alpha,5alpha,10beta,14beta-tetraacetoxy-4(20),11-taxadiene by cell suspension cultures of Catharanthus roseus.

Catharanthus roseus cell suspension cultures were employed for the biotransformation of 2alpha,5alpha,10beta,14beta-tetraacetoxy-4(20),11-taxadiene ( 1), and four metabolites were obtained. Based on their physical and chemical data, the structures of the four metabolites were respectively elucidated as 10beta-hydroxy-2alpha,5alpha,14beta-triacetoxy-4(20),11-taxadiene (2), 5alpha-hydroxy-2alpha,10beta, 14beta-triacetoxy-4(20),11-taxadiene (3), 6alpha,10beta-dihydroxy-2alpha,5alpha,14beta-triacetoxy-4(20),11-taxadiene (4), and 6alpha,9alpha,10beta-trihydroxy-2alpha,5alpha,14beta-triacetoxy-4(20),11-taxadiene (5), among which 3 and 5 were characterized as new taxoids. The effects of the stages of substrate addition on the biotransformation were also investigated. The results revealed that the biotransformation rate for 1 reached 85.3 % and the yield of 2 70 % when 1 was administered during the mid-linear phase (9 - 12 th day) of the cell growth cycle. On the other hand, the yield for 4 reached the highest level of 11.8 % when 1 was added in the early linear phase (6 th day).

Effect of phytohormones on growth and alkaloid accumulation by a Catharanthus roseus cell suspension cultures fed with alkaloid precursors tryptamine and loganin

This work presents a study of the effect of different phytohormones on growth and accumulation of terpenoid indole alkaloids in a Catharanthus roseus cell suspension culture upon feeding with the precursors loganin and tryptamine. The phytohormones tested were 2,4-dichlorophenoxyacetic acid, salicylic acid, methyl jasmonate and abscisic acid. Among these only methyl jasmonate enhanced the accumulation of alkaloids. Abscisic acid did not enhance the accumulation of alkaloids but delayed the catabolism of strictosidine.

Enhanced catharanthine production in catharanthus roseus cell cultures by combined elicitor treatment in shake flasks and bioreactors.

Chemical and fungal elicitors were added to Catharanthus roseus cell suspension cultures so as to improve the production of indole alkaloids. A synergistic effect on alkaloid accumulation was observed in C. roseus cell cultures when treated with some combined elicitors of fungal preparations and chemicals. Among them, the combination of tetramethyl amminium bromide and Aspergillum niger mycelial homogenate gave the highest ajmalicine yield (63 mg l(-1)) and an improved catharanthine accumulation (17 mg l(-1)). The combined elicitors of malate and sodium alginate resulted in the highest catharanthine yield (26 mg l(-1)) and a high ajmalicine accumulation (41 mg l(-1)) in the cell cultures. Based on the synergistic effect of malate and sodium alginate, a process with enhanced catharanthine production in Catharanthus roseus cell cultures was developed in shake flasks and a bioreactor. After 10 days of culture, 25 mg l(-1), 32 mg l(-1) and 22 mg l(-1) catharanthine yield were obtained in 500-ml flasks, 1000-ml flasks and in a 20-l airlift bioreactor, respectively. Upon malate-alginate combining treatments, peroxidase, catalase and superoxide dismutase activities decreased in elicited cells but phenylalanine ammonia lyase and lipoxygenase activities increased dramatically. That suggests a typical defense responses took place in the combined elicitors-treated cell cultures. Furthermore, the combined elicitors also caused a significant increase of malondialdehyde level in cell cultures, which suggests a serious lipid peroxidation occurred in the elicited cell cultures. Comparison of these results suggests that malate and alginate combining treatment also stimulates defense responses, such as lipid peroxidation, in all C. roseus culture processes and this may mediate the indole alkaloid production via jasmonate pathway.

Accumulation of loganin and secologanin in vacuoles from suspension cultured Catharanthus roseus cells

The distribution of the iridoids loganin and secologanin at the subcellular level was studied in secologanin accumulating Catharanthus roseus cells. Vacuoles were isolated from protoplasts by an osmotic shock treatment, followed by flotation centrifugation. The iridoid and alkaloid contents of cells, protoplasts and vacuoles were analysed by HPLC. Secologanin was found to be stored exclusively in the vacuoles. In loganin fed cells, loganin accumulated in the vacuoles while vacuolar secologanin contents increased, indicating that the conversion of loganin to secologanin occurs in the vacuoles. However, feedings of loganin to isolated protoplasts and vacuoles did not result in increased secologanin levels. Utilizing these strategies we attempted to substantiate the possible vacuolar conversion of loganin to secologanin.

Purification of xyloglucan endotransglycosylase based on affinity sorption of the active glycosyl-enzyme intermediate complex to cellulose.

Xyloglucan endotransglycosylase (XET) catalyzes the cleavage of xyloglucan (XG) molecules by a transglycosylation mechanism involving two steps: (a) endocleavage of the beta-(1,4)-linked polyglucosyl backbone of the xyloglucan molecule with formation of a glycosyl-enzyme intermediate; (b) transfer of the glycosyl residue from the intermediate to the C-4 position of the nonreducing end glucosyl unit of another molecule of XG or an XG-derived oligosaccharide with liberation of the enzyme (Z. Sulová et al., 1998, Biochem. J. 330, 1475-1480). The formation of a relatively stable active complex of XET with XG and the tendency of xyloglucan to bind tightly via hydrogen bonds to cellulose were exploited in the present method of purification of XET. Crude extracts from nasturtium (Tropaeolum majus) cotyledons and other plant sources containing the enzyme were mixed with XG in order to form the XET:XG complex, which was applied onto cellulose. Unadsorbed proteins were removed by washing and the XET was released from the adsorbed XET:XG complex by transglycosylation of its glycosyl moiety to added XG-derived oligosaccharides. The described procedure resulted in an over 100-fold increase in specific activity of XET in a single step. Further purification of the enzyme to homogeneity was achieved by gel-permeation chromatography on Bio-Gel P30. Similar procedure could be used for purification of XET from other plant sources, such as lentil (Lens culinaris) seeds, pea (Pisum sativum) epicotyls, and supernatant of suspension-cultured Catharanthus roseus cells.

Involvement of the octadecanoid pathway and protein phosphorylation in fungal elicitor-induced expression of terpenoid indole alkaloid biosynthetic genes in catharanthus roseus

Two key genes in terpenoid indole alkaloid biosynthesis, Tdc and Str, encoding tryptophan decarboxylase and strictosidine synthase, respectively, are coordinately induced by fungal elicitors in suspension-cultured Catharanthus roseus cells. We have studied the roles of the jasmonate biosynthetic pathway and of protein phosphorylation in signal transduction initiated by a partially purified elicitor from yeast extract. In addition to activating Tdc and Str gene expression, the elicitor also induced the biosynthesis of jasmonic acid. The jasmonate precursor alpha-linolenic acid or methyl jasmonate (MeJA) itself induced Tdc and Str gene expression when added exogenously. Diethyldithiocarbamic acid, an inhibitor of jasmonate biosynthesis, blocked both the elicitor-induced formation of jasmonic acid and the activation of terpenoid indole alkaloid biosynthetic genes. The protein kinase inhibitor K-252a abolished both elicitor-induced jasmonate biosynthesis and MeJA-induced Tdc and Str gene expression. Analysis of the expression of Str promoter/gusA fusions in transgenic C. roseus cells showed that the elicitor and MeJA act at the transcriptional level. These results demonstrate that the jasmonate biosynthetic pathway is an integral part of the elicitor-triggered signal transduction pathway that results in the coordinate expression of the Tdc and Str genes and that protein kinases act both upstream and downstream of jasmonates.

Effects of alkaloid precursor feeding and elicitation on the accumulation of secologanin in a Catharanthus roseus cell suspension culture

In a Catharanthus roseus cell line accumulating secologanin, time-course studies on the uptake of loganin and the in vivo conversion to secologanin were performed. Four-day-old cells converted 100% of the fed loganin to secologanin within 24 hours, showing that this step is unlikely to be limiting for alkaloid accumulation. Thirteen-day-old cells also took up loganin, but only about 25% was recovered as secologanin. A saturation in the uptake of loganin and in the conversion of loganin into secologanin was observed after feeding increasing amounts of loganin. Elicitation by cellulase and pectinase decreased the cellular contents of secologanin and strictosidine whereas it increased the tryptamine content. In addition, the uptake of loganin in elicited cells was blocked. In vitro assays with protein extracts of elicited Catharanthus roseus cells indicated the activation of secologanin degrading enzyme(s). Feeding of tryptophan did not result in any increase in alkaloid contents, despite its complete uptake. Tryptamine feeding led to increased strictosidine contents, but ajmalicine levels remained unchanged.

Expression of extensin genes is dependent on the stage of the cell cycle and cell proliferation in suspension-cultured Catharanthus roseus cells.

To isolate cDNAs expressed at a specific phase of the cell cycle in a higher plant, we performed differential screening of a cDNA library prepared from the S-phase cells of synchronized cultures of Catharanthus roseus. Sequence analysis shows that two of the identified cDNAs, cyc15 and cyc17, encode extensins that represent a family of cell wall hydroxyproline-rich glycoproteins. Protein sequences deduced from the two cDNAs contain the characteristic pentapeptide repeat sequence, Ser-Pro-Pro-Pro-Pro, which is commonly observed in extensins. The protein sequences also share several other extensin characteristics such as the presence of a N-terminal signal peptide and a high content of Tyr and Lys residues. When C. roseus cell suspension cultures were synchronized by phosphate starvation, the mRNAs of both cyc15 and cyc17 were transiently expressed during the S and G2 phases of the cell cycle. However, significant amounts of the mRNAs also accumulated in phosphate-starved cells arrested in the G1 phase. In asynchronous cultures, both genes were expressed during the stationary phase, when cell proliferation ceased. The observed patterns of expression suggest that the extensin genes, cyc15 and cyc17, are under two types of regulation: one that depends on the stage of the cell cycle and another that is induced during the growth arrest. Thus, the products of these genes may function both during the progression through the cell cycle and in the strengthening of the cell wall after cell division.

Metabolism of diacylglycerol pyrophosphate by suspension cultured Catharanthus roseus cells : Identification and characterization of diacylglycerol pyrophosphate phosphatase in plants

Diacylglycerol pyrophosphate (DGPP) is a novel phospholipid found in plants that is the product of the reaction catalyzed by phosphatidate kinase. In this study we examined the metabolism of DGPP in Catharanthus roseus cells. Metabolic labeling studies showed that DGPP was rapidly metabolized to phosphatidate and P i. The enzyme catalyzing this reaction was named DGPP phosphatase. DGPP phosphatase activity was 20 times more active than phosphatidate kinase activity. Microsomal and soluble isoforms of DGPP phosphatase were found in C. roseus cells. The microsomal enzyme was primarily associated with intracellular membranes. The pH optima of the microsomal and soluble DGPP phosphatases were 4.5 and 3.5, respectively. Both activities were independent of a divalent cation requirement and were insensitive to EDTA and N-ethylmaleimide. The DGPP phosphatase activities were inhibited by NaF, MgCl 2, MnCl 2, and sphingosine. The dependence of the microsomal and soluble DGPP phosphatase activities on DGPP were dose-dependent with maximum activities obtained at 100 μM (3 mol %) and 600 μM (20 mol %), respectively, using a Triton X-100/DGPP mixed micellar substrate. Pyrophosphate, p-nitrophenylphosphate, and adenosine diphosphate (ADP) were not substrates for the DGPP phosphatase enzymes. DGPP phosphatase activity was also found in a wide range of organisms including Escherichia coli, Saccharomyces cerevisiae, rat, pig, and cattle.

Identification with a photoaffinity reagent of a tonoplast protein involved in vacuolar malate transport of Catharanthus roseus

SummaryThe effect of N‐(4‐azido‐salicylyl) aspartic acid (AzSA), a photolysable analogue of malate, was tested on the malate transport activity of tonoplast vesicles isolated from Catharanthus roseus cell suspension cultures. AzSA inhibited malate uptake in a competitive manner with a Kti of 1.7 millimolar. When iodinated, the malate analogue was found to be still photolysable and a competitive inhibitor of malate uptake. Photolysis of 125I‐labelled AzSA in the presence of purified tonoplast vesicles led to label incorporation into several polypeptides after analysis by gel electrophoresis. Only one polypeptide, with an apparent molecular mass of 37 kDa, was totally protected by the inclusion of 50 millimolar malate, the original substrate, in the photolysis medium. The labelled polypeptide is therefore apparently a specific malate‐binding protein. Diethylpyrocarbonate (DEPC), a very potent inhibitor of malate transport acting at the active site of the transporter, also protected the 37 kDa polypeptide from labelling. Citrate and, to a lesser extent, quinate afforded protection from labelling whilst other organic acids or aspartic acid (100 millimolar) did not. These photoprotection results are in good agreement with the data concerning the specificity of malate transport across the tonoplast. Polyclonal antibodies against the 37 kDa polypeptide strongly inhibited malate uptake both in tonoplast vesicles and in isolated vacuoles. These results suggest the involvement of the 37 kDa polypeptide in vacuolar malate transport.