Alkaloid production in Catharanthus roseus (L.) G. Don.

Analysis of 76 cell clones derived from one leaf of a periwinkle plant (Catharanthus roseus (L.) G. Don) showed the occurrence of Corynanthe-, Strychnos-, and Aspidosperma-type alkaloids. The majority of clones (62%) displayed compounds of all three types. Variation of the alkaloid spectra of the cell clones was low when compared to that found previously with serially subcultured callus and cell suspensions derived from different plants.NRCC # 19100     

Hydroponic culture of Catharanthus roseus (L.) G. Don and studies on seed production

Under climatic conditions of Poland, Catharanthus roseus may be grown in the open field only as annual crop, using transplants raised in a greenhouse or in a plastic tunnel. An effective method of hydroponic culture has been elaborated and tested. Herbal material obtained in hydroponics showed considerable concentration of alkaloids in different plant parts. Seeds of C. roseus may also be produced under these conditions. Characteristics of seeds as well as the effect of different methods of their storage are presented. Seeds stored in paper bags at room temperature may retain germinability for 7 years and those stored in a refrigerator, at 5°C, up to 15 years. Storage in a freezer, at -10°C, resulted in a quick and sharp reduction of germination rate.     

Possibilities and limitations of seed production of Catharanthus roseus (L.) G. Don under Polish climatic conditions

The studies were carried out at Radzików, Central Poland in the years 1982–1991, using a progeny of an Indian population. It was proved that Catharanthus roseus may be grown in Poland under field conditions as an annual crop, using plants previously raised in glass or plastic houses. However, the culture of C. roseus in the greenhouse resulted in a higher yield of herb and particularly in a better setting of fruits and seeds than under field conditions. The yield of seeds obtained under cover was several times higher than in the field. Growing of plants under cover may be justified, as greenhouses usually are not utilised in the summertime. Consequent mass selection conducted during successive seasons has lead to obtaining the lines better adapted to the conditions of a cool climate and hence more productive. Improvement of generative traits, resulting from selection, was more pronounced than improvement of the vegetative indices. The ‘yellow silique’ has been recognised as an optimal stage for harvest, as at this stage siliques could be easily separated from the stems, whereas the risk of seed shedding was still minimal. Germinability of seeds obtained from siliques harvested at this stage was high and uniform.     

Cultural conditions affect somatic embryogenesis in Catharanthus roseus L. (G.) Don

We established an efficient plant regeneration system for Catharanthus roseus L. (G.) Don through somatic embryogenesis. Embryogenic callus was induced from hypocotyl of seed germinated in vitro. Somatic embryogenesis in Catharanthus has been categorized into three distinct stages: (1) initiation and proliferation of embryo; (2) maturation, and; (3) germination or plantlet conversion. Beside plant growth regulators, various stages of embryogenesis were screened for their response to a wide variety of factors (pH, gelrite, light, sugar alcohols, polyethyleneglycol and amino acids), which affect embryogenesis. All of the tested factors had a small to marked influence on embryogeny and eventual conversion to plantlets. The plantlets were acclimatized successfully in a greenhouse. To our knowledge, this is the first report describing a detailed study of various cultural factors which regulate embryogenesis in C. roseus. The results discussed in this paper may be used in mass propagation to produce medicinal raw material, and the embryo precursor cells could be used in genetic modification programmes that aim to improve the alkaloid yield as well.     

Characterization of Alkaloid Uptake by Catharanthus roseus (L.) G. Don Protoplasts

The accumulation of alkaloids by protoplasts of Catharanthus roseus (L.) G. Don var. Little Bright Eye was studied to determine the specificity of uptake and the role of ion trapping in the storage of alkaloids. Accumulation of the indole alkaloids vindoline, ajmalicine, tabersonine, and vinblastine was found to be biphasic, with an initial burst of uptake followed by a slow, prolonged phase of accumulation. The concentration and pH dependence of the initial burst of uptake for vindoline suggested that uptake occurred by simple diffusion. Uptake of nicotine was monophasic, with a half life of 5.2 minutes. The accumulation ratio (Ci/Ce) for nicotine at steady state and for the initial burst of uptake for vindoline and ajmalicine suggested that accumulation was driven by the pH gradient between the vacuole and the external assay medium. The second, sustained phase of uptake of vindoline was sensitive to inhibition by either 20 millimolar NaN₃ or 0.5 millimolar Cu²⁺. In azide-treated protoplasts, the uptake for vindoline conformed to the kinetics of simple diffusion, with a half life of 4 minutes. The second phase of uptake for ajmalicine, although sensitive to inhibition by Cu²⁺, was insensitive to inhibition by NaN₃. The biphasic uptake of the indole alkaloids was not due to any significant metabolism. It is concluded that accumulation and storage of the indole alkaloids is due only partly to ion trapping of the alkaloids by the low pH of the vacuole lumen. In the case of vindoline, there appears to be a specific energy-requiring uptake that is not seen with nicotine (which is not endogenous to Catharanthus). Accumulation of ajmalicine appears to involve both ion trapping and an azide-insensitive component, which may be due to complexation with organic counterions and phenolics.     

长春花愈伤组织的诱导及限速酶活性的研究

长春花生物碱是目前应用最有效的天然植物抗肿瘤药物,目前利用生物工程生产长春花生物碱成果甚微。通过对长春花愈伤组织的诱导条件进行研究,同时对植株各部位的色氨酸脱羧酶(TDC)、过氧化物酶(POD)进行精确测定。认为长春花愈伤组织的诱导以MS培养基、激素2,4-D 2 mg/L和6-BA 0.5 mg/L最好,生物碱的合成是植物体多器官共同参与的结果。     

Strategies for the genetic modification of the medicinal plant Catharanthus roseus (L.) G. Don

This review describes the different plant transformation techniques, including guided infection with Agrobacterium tumefaciens and A. rhizogenes, particle bombardment and protoplast fusion, that have been attempted to create transgenic Catharanthus roseus (L.) G. Don cell cultures, hairy roots and whole plants. The review also focuses on the different approaches used to manipulate and improve secondary metabolite yields in various culture systems, with special attention to the most relevant results achieved. Finally, under future perspectives, the authors propose several approaches which would likely be implemented with this species, to try to boost the accumulation of the anti-tumour agents, vinblastine and vincristine. Some comments on how the future of the genetic manipulation of medicinal plants may proceed aiming at achieving higher secondary metabolite yields are also given.     

Purification and characterization of mevalonate kinase from suspension-cultured cells of Catharanthus roseus (L.) G. Don

Mevalonate kinase was purified to homogeneity from Catharanthus roseus (L.) G. Don suspension-cultured cells. The purified enzyme had an M(r) of 104,600 and a subunit size of about 41,500. Kinetic studies indicated an ordered sequential mechanism of action, in which mevalonate was the first substrate to bind and ADP was the last product to leave the enzyme. True values for the kinetic constants were determined for mevalonate, with K(ma) = 76 microM and K(ia) = 74 microM, and for ATP, with K(mb) = 0.13 mM and K(ib) = 0. 13 mM; the true V(max) was calculated to be 138.7 nkat/mg of protein. Product inhibition was only detectable at rather high concentrations: above 0.7 mM for 5-phosphomevalonate and above 2 mM for ADP, with an ADP/ATP ratio of at least 1. Mevalonate kinase activity was shown to be strongly inhibited by farnesyl diphosphate. Farnesyl diphosphate acted as a competitive inhibitor toward ATP, with a K(i) value of 0.1 microM. Mevalonate kinase activity was dependent on the presence of divalent ions. At a concentration of 2 mM, Mg(2+) and Mn(2+) were best and equally effective in sustaining activity; compared to Mg(2+) and Mn(2+), relative activities of 35, 30, 16, 4.8, and 3.4% were detected at equimolar concentrations of Zn(2+), Fe(2+), Co(2+), Ca(2+), and Ni(2+), respectively. The pH-dependent activity profile of mevalonate kinase showed a broad pH optimum between pH 7 and 10, with a maximum at about pH 8.9.     

Alkaloid production in Catharanthus roseus (L.) G. Don cell cultures. XIII. Effects of bioregulators on indole alkaloid biosynthesis

A study on the effect of various bioregulators on the biosynthesis of ajmalicine (8) and catharanthine (9) in plant tissue cultures of Catharanthus roseus is described. It is shown that 1,1-dimethylpiperidine bromide (3) and 2-diethylaminoethyl-3,4-dimethylphenylether (7) are effective in increasing these alkaloid levels in the cell line PRL #200. Such studies may prove beneficial in larger scale experiments designed for the production of these alkaloids.     

Peroxidase and the biosynthesis of terpenoid indole alkaloids in the medicinal plant Catharanthus roseus (L.) G. Don

The leaves of Catharanthus roseus (L.) G. Don produce the first natural drugs used in cancer therapy – the dimeric terpenoid indole alkaloids vinblastine and vincristine. The study of C. roseus further revealed two other terpenoid indole alkaloids with important pharmacological activity: ajmalicine, used as an antihypertensive, and serpentine, used as sedative. The biosynthetic pathway of the medicinal alkaloids has been investigated in much detail and a number of steps are now well characterized at the enzyme and gene level and, recently, several regulatory genes have also been isolated and characterized. Since early studies of the biosynthesis of vinblastine, during the 1970s and 1980s, the dimerization reaction has attracted much attention due to its possible regulatory importance and potential application for the semi synthetic production of the dimeric alkaloids. After initial, inconclusive work suggesting the involvement of peroxidase-like enzymes, the search for the dimerization enzyme in leaf tissue detected a single dimerization activity credited to the single class III plant peroxidase present in the leaves of the plant – the basic isoenzyme CRPRX1. The enzyme was purified to homogeneity, the respective cDNA and genomic sequences were characterized, and a channeling mechanism was proposed for the peroxidase-mediated-vacuolar synthesis of the first dimeric alkaloid intermediate, α-3′,4′-anhydrovinblastine. On the other hand, the oxidation of ajmalicine into serpentine has been attributed to basic peroxidase isoenzymes localized in the vacuole of C. roseus cells. An overview of the work implying class III plant peroxidases in the biosynthesis of terpenoid indole alkaloids in C. roseus is presented here. Abbreviations: CRPRX1 –Catharanthus roseus peroxidase 1; DAB – diaminobenzidine; IEF – isoelectric focusing; UV – ultraviolet.     

The development of a single-stage growth and indole alkaloid production medium for Catharanthus roseus (L.) G. Don suspension cultures

By modification of a standard Murashige and Skoog plant tissue culture maintenance medium, a system has been developed for Catharanthus roseus cell suspension cultures in which both growth and indole alkaloid accumulation can occur in a single-stage culture of 14–21 days. Precise optimization of the medium depends upon the cell line under investigation, but the inclusion of lactose as the carbohydrate source, and NAA and kinetin as growth regulators, will generally increase yields of the indole alkaloid catharanthine. Treatment of cells growing in this optimized medium with agents that stimulate the accumulation of secondary metabolites both increases the yield of catharanthine and reduces the time required for production. We believe that this process could be useful for the commercial production of plant secondary metabolites.     

Characterization of a monoterpene hydroxylase from cell suspension cultures of Catharanthus roseus (L.) G. Don

Conditions have been established for the optimization of the specific activity of a membrane-bound monoterpene hydroxylase from cell suspension cultures of Catharanthus roseus. In time course studies, the hydroxylase and NADPH-cytochrome c reductase exhibited maximal activities 18–20 days after inoculation, i.e., during early stationary phase. By late stationary phase, enzyme activity had declined. In contrast an enzyme of primary metabolism achieved optimal specific activity by the 12th day and remained constant through day 26, synchronous with general growth. Effects of nutritional and hormonal factors on the specific activity of the hydroxylase and cell growth were evaluated. Inhibitors of hydroxylase activity were also assessed in vitro. A soluble form of the monoterpene hydroxylase has been detected in cultured cells possibly affording a useful source of this enzyme for further purification.     

Identification of a low vindoline accumulating cultivar of Catharanthus roseus (L.) G. Don by alkaloid and enzymatic profiling

The Madagascar periwinkle [Catharanthus roseus (L.) G. Don] is a commercially important horticultural flower species and is the only source of the monoterpenoid indole alkaloids (MIAs), vinblastine and vincristine, key pharmaceutical compounds used to combat a number of different cancers. The present study uses high performance liquid chromatography for metabolic profiling of the MIAs extracted from seedlings and young leaves of 50 different flowering cultivars of C. roseus to show that, except for a single low vindoline cultivar (Vinca Mediterranean DP Orchid), they accumulate similar levels of MIAs. Further enzymatic studies with extracts from young leaves and from developing seedlings show that the low vindoline cultivar has a 10-fold lower tabersonine-16-hydroxylase activity than those of C. roseus cv. Little Delicata. It is concluded that rapid metabolic and more selective enzymatic profiling of Catharanthus mutants could be useful for the identification of a range of altered MIA biosynthesis lines.     

Temporal and Spatial Heterogeneity in the Accumulation of Anthocyanins in Cell Cultures of Catharanthus roseus (L.) G.Don.

The accumulation of anthocyanins, a group of pigmented secondarymetabolites, in cell cultures of the Madagascar periwinkle Catharanthusroseus has been investigated. In these cultures it was foundthat anthocyanin accumulation was restricted to the post-divisionphase of the culture growth cycle, during which the culturesbecame deep purple in colour. As a result of anthocyanin visibilityit has been possible to ascertain that accumulation of thesemetabolites occurred in only a small proportion of the cellpopulation. Approximately 10% of cells regularly accumulateddetectable levels. Considerable variation within this ‘productive’population was observed and using a standard integrating microdensitometerit has been possible to quantify directly this heterogeneityand compare it with data obtained from whole plants. Analysishas revealed that the variation in both intracellular anthocyanincontent and concentration in cell cultures was much greaterthan that observed within tissues of mature plants. Significantdifferences in mean values were however found between the wholeplant tissues. The relevance of this temporal and spatial heterogeneityobserved in vitro to our understanding of the control of secondarymetabolite accumulation and to the potential use of tissue culturesystems as a means to produce these compounds is discussed. Key words: Heterogeneity, anthocyanins, cell culture     

Development of STMS markers from the medicinal plant Madagascar periwinkle [Catharanthus roseus (L.) G. Don.]

We report the isolation and characterization of the first set of sequence‐tagged microsatellites sites (STMS) markers in Catharanthus roseus, a plant with a vast range of medicinal uses. The microsatellite loci were cloned from an enriched library constructed using degenerate primers. Based on the microsatellite motifs, seven STMS primer pairs were designed. They were used to amplify 32 accessions of C. roseus and one accession of Catharanthus trichophyllus. The primers amplified an average of 3.86 alleles per locus. The observed heterozygosity ranged from 0.2903 to 0.9688 with an average of 0.7511. The STMS markers of C. roseus also amplified corresponding loci in a related species (C. trichophyllus) suggesting conservation of the loci across the genus. These markers will prove useful for genetic diversity analysis and linkage map construction in C. roseus.     

Preparation of sealed tonoplast and plasma-membrane vesicles from Catharanthus roseus (L.) G. Don. cells by free-flow electrophoresis

Highly purified tonoplast and plasmamembrane vesicles were isolated from microsomes of Catharanthus roseus (L.) G. Don. by preparative free-flow electrophoresis. The relative amounts of tonoplast and plasma-membrane vesicles in the total microsomes varied with the pH of the grinding medium. The most electronegative fractions were identified as tonoplast using nitrate-inhibited, azide-resistant Mg²⁺-ATPase and pyrophosphatase activities as enzyme markers. The least electronegative fractions were identified as plasma membrane using glucan-synthase-II and UDPG:sterolglucosyl-transferase activities as enzyme markers. Other membrane markers, latent inosine-5-diphosphatase (Golgi), NADPH-cytochrome-c reductase (ER) and cytochrome-c oxidase (mitochondria) were recovered in the fractions intermediate between tonoplast and plasma membrane and did not contaminate either the tonoplast or the plasma-membrane fractions. In the course of searching for a reliable marker for tonoplast, the pyrophosphatase activity was found to be essentially associated with the tonoplast fractions purified by free-flow electrophoresis from C. roseus and other plant materials. The degree of sealing of the tonoplast and plasmamembrane vesicles was probed by their ability to pump protons (measurements of quinacrine quenching) and to generate a membrane potential (absorption spectroscopy of Oxonol VI). A critical evaluation of vesicles sidedness is presented.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - Con A concanavalin A - Cyt cytochrome - LysoPC lysophosphatidylcholine - Pi orthophoshate - PP_iase pyrophosphatase - IDPase inosine-5-diphosphatase We thank Pr. Robert Dargent and André Moisan (Laboratoire de Cryptogamie, Toulouse, France) for use of their electron-microscope facilities. This work was supported by the Centre National de la Recherche Scientifique and by a grant Dynamique du fonctionnement de la vacuole from the Ministère de la Recherche et de la Technologie.     

Regulation of product synthesis in cell cultures of Catharanthus roseus (L) G. Don

Cell suspension cultures of the Madagascan Periwinkle Catharanthus roseus (L) G. Don were maintained on Gamborg's B5 medium and their growth monitored by measuring cellular fresh and dry weight, cell number and mitotic activity. Samples of cells of different ages and physiological states were subcultured onto an alkaloid production medium and their rates of growth and alkaloid accumulation measured over a period of 30–45 days. In two experiments the rate of biomass accumulation was directly related to the rate of cellular serpentine accumulation. Possible mechanisms underlying this phenomenon are discussed in relation to the properties of cells comprising the inocula.     

Fatty-acid composition and biosynthesis in cell suspension cultures of Glycine max (L.) Merr., Catharanthus roseus G. Don and Nicotiana tabacum L.

The fatty-acid composition of C. roseus and N. tabacum cell suspension cultures was unaffected by subculture on Wood and Braun, Murashige and Skoog, or Gamborg B5C media. However, placing the cultures — which were normally grown at 25° C — at 15° C reduced growth but resulted in enhanced formation of oleic and linolenic acids in C. roseus cultures and increased levels of linoleic and linolenic acids in cultures of G. max and N. tabacum, respectively. The incorporation of [¹⁴C]acetate into [¹⁴C]linoleic acid was more rapid in N. tabacum cells than in G. max cells, but was very poor in C. roseus where the [¹⁴C] label was distributed mainly between palmitic and oleic acids.     

Phosphate uptake across the tonoplast of intact vacuoles isolated from suspension-cultured cells of Catharanthus roseus (L.) G. Don

 Transport of inorganic orthophosphate (Pi) across the tonoplast membrane was studied using intact vacuoles isolated from suspension-cultured cells of Catharanthus roseus. Orthophosphate uptake was strongly stimulated in the presence of Mg-ATP and Mg-pyrophosphate and inhibited by bafilomycin and concanamycin which are potent inhibitors of the vacuolar H⁺-ATPase. These results indicated that the build-up of an electrochemical gradient by the H⁺ pumps was essential for the uptake of Pi. Potassium thiocyanate, which dissipates the membrane potential across the tonoplast, strongly inhibited the Mg-ATP-stimulated uptake of Pi, while only a weak inhibition was observed in the presence of NH₄Cl, which dissipates the pH gradient. These results indicate that, as observed for other anions like malate or chloride, the electrical component is the driving force of Pi uptake, whereas the ΔpH plays only a minor role. Possible competitive inhibitors of Pi, MoO²⁻ ₄, VO³⁻ ₄ and CrO²⁻ ₄ were tested. Among them, CrO²⁻ ₄ strongly inhibited Pi uptake into the vacuoles. Various inhibitors of anion transport were also tested. Only 4,4-diisothiocyanostilbene-2,2′-disulfonic acid strongly inhibited Pi uptake into the vacuoles. The function of the vacuolar Pi transporters for cytoplasmic Pi homeostasis is discussed. Received: 20 September 1999 / Accepted: 28 January 2000