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.     

Glucosylation of benzyl alcohols by the cultured suspension cells of Nicotiana tabacum and Catharanthus roseus

The cultured cells of Nicotiana tabacum (white cells) converted regioselectively exogenous 2-, 3-, and 4-hydroxybenzyl alcohols into corresponding hydroxybenzyl-β-d-glucopyranoside. (RS)-1-Phenylethanol having chiral center in its substituent was also glucosylated to give 1-phenylethyl-β-d-glucopyranoside by the cultured cells of N. tabacum (white and green cells) and Catharanthus roseus. The glucosylation with the green cells of N. tabacum occurred enantioselectively to give the glucoside of (S)-alcohol preferentially, while the glucosylation with the white cells of N. tabacum and the C. roseus cells gave preferentially the glucoside of (R)-alcohol.     

Biosynthesis of brassinolide from castasterone in cultured cells of Catharanthus roseus

Feeding experiments with tritium- and deuterium-labeled castasterone (CS) were conducted with three cell lines of Catharanthus roseus, including crown gall cells and nontransformed cells. In all three cell lines, the conversion of CS to brassinolide (BL) was observed and unequivocally confirmed by gas chromatography/mass spectrometry (GC/MS). This is the first conclusive evidence that CS is the biosynthetic precursor of BL.Biosynthesis of brassinosteroids in Catharanthus roseus. Part II. Part I of this series: Yokota et al. (1990a).     

Biosynthesis of brassinosteroids in cultured cells of Catharanthus roseus

Precursor administration experiments with 2H-labeled 6-oxocampestanol, 6-deoxocastasterone and 6alpha-hydroxycastasterone in cultured cells of Catharanthus roseus were performed and the metabolites were analyzed by GC-MS. [2H6]Cathasterone was identified as a metabolite of [2H6]6-oxocampestanol, whereas [2H6]6alpha-hydroxycastasterone and [2H6]castasterone were identified as metabolites of [2H6]6-deoxocastasterone, and [2H6]castasterone was identified as a metabolite of [2H6]6alpha-hydroxycastasterone, indicating that 6-deoxocastasterone is converted to castasterone via 6alpha-hydroxycastasterone. In addition, 6-deoxocathasterone, a putative biosynthetic intermediate in the late C6-oxidation pathway, was identified as an endogenous brassinosteroid. These studies provide further evidence supporting our proposed biosynthetic pathways for brassinolide.     

Noninvasive NMR studies of metabolism in cultured Catharanthus roseus cells

Summary Nuclear magnetic resonance (NMR) spectroscopy provides a unique modality for the study of tissue-cultured plant cells. One of its major attractions is that it allows noninvasive studies of plant material. In addition, it can provide insight into the pH in the vacuole and cytoplasm, and into the compartmentalization of certain metabolites. In this review we show how phosphorus-31 NMR is used to study intracellular pH, phosphate uptake and storage, and energy metabolism in suspension cells of Catharanthus roseus. In addition, multinuclear NMR studies of the uptake of ammonium and the gradients of K⁺ over the membrane are discussed as well. The use of two-dimensional NMR for the study of whole cell extracts is also described. Finally, we show how nitrogen-14 and nitrogen-15 NMR are used to obtain information about the assimilation of inorganic sources in developing carrot somatic embryos. These NMR studies provide a unique insight into the metabolism of tissue-cultured plant cells.     

Immobilization of cultured Catharanthus roseus cells using a fibreglass substratum

Summary Cultured Catharanthus roseus cells were immobilized using geometrically identical needled fibreglass mats prepared with a range of surface coatings. The phenyl (PS), polyglycol (PG), aldehyde (CHO), alkyl (CTMS), and silanol (AW) coatings, along with the untreated glass (HC) surface, produced surfaces with a range of surface tensions. The immobilization efficiency of the substratum, measured as the percentage of cells immobilized, increased with increasing substratum surface tension in the order PS < PG < CHO < CTMS < AW < HC. The dependence of immobilization efficiency on substratum surface tension can be described using a thermodynamic model of adhesion that considers the extent of plant cell adhesion to be a function of the surface tensions of the substratum, the suspending liquid, and the plant cells. In addition, this dependence also demonstrates the fundamental role of adhesion in the immobilization process involving a glass fibre matrix. However, cell entrapment processes are also implicated. The untreated glass fibre substratum (HC), which demonstrated the greatest immobilization efficiency, was used for further characterization of the immobilization strategy. Maximum inoculum biomass was determined to be approximately 1.9 g cells (fresh weight)/g substratum (dry weight) to achieve greater than 90% immobilization efficiency. The growth rate of immobilized cultures was slower than suspension cultures, probably due to mass transfer limitations. Production of the indole alkaloids, tryptamine, catharanthine, and ajmalicine, was also suppressed relative to suspension-cultured cells. These results are considered in relation to other immobilization strategies and their apparent effects on cellular processes. Offprint requests to: F. Dicosmo     

Cryopreservation of periwinkle,Catharanthus roseus cells cultured in vitro

A procedure for prolonged cryogenic storage of periwinkle cell cultures is described. Cells derived from periwinkle, Catharanthus roseus (L.) G. Don, and subcultured as suspension in 1-B5C nutrient medium have been frozen, stored in liquid nitrogen (–196°C) for 11 weeks, thawed and recultured. Maximal survival was achieved when 3–4 day-old cells precultured for 24 h in nutrient medium with 5% DMSO were frozen at slow cooling rates of 0.5 or 1°C/min prior to storage in liquid nitrogen. The only loss in viability of cells occurred subsequent to treatment with DMSO. Abbreviations: DMSO, dimethylsulfoxide; 2,4-D, 2,4-dichlorophenoxyacetic acid; TTC, triphenyltetrazolium chloride.NRCC No. 20082     

High-level production of arbutin from hydroquinone in suspension cultures of Catharanthus roseus plant cells

Summary Plant cell suspensions of Catharanthus roseus efficiently converted exogenously supplied hydroquinone (HQ) into arbutin. Arbutin productivity of the cells was strongly influenced by the growth stage of the cultivated cells and by the manner of the addition of HQ. We have developed two methods: (i) cultivating suitable cells for producing arbutin at high density; (ii) efficiently adding toxic HQ to the cells. The yield of arbutin could be increased up to 9.2 g/l (45% of cell dry weight), which is the highest yield in the field of plant biotechnology. Repeated examinations and scaling up to a 20-l jar fermentor suggested that C. roseus cells stably produce arbutin in large amounts under the established conditions. Offprint requests to: S. Inomata     

Formation of catharanthine,akuammicine and vindoline in Catharanthus roseus suspension cells

Catharanthine and akuammicine, together with ajmalicine and strictosidine, were isolated from a culture strain of Catharanthus roseus suspension cells. The biosynthetic capability of the cultured cells to produce akuammicine, catharanthine and vindoline was confirmed by feeding experiments with dl-tryptophan-[3-¹⁴C] to yield the radioactive alkaloids.     

Serpentine release by suspension cultures of Catharanthus roseus

Summary Suspension cultures ofCatharanthus roseus filtered (with or without a vacuum) and resuspended in fresh or spent medium will release serpentine into the medium. This treatment is associated with small increases in pH and conductivity of the medium. The released serpentine quickly disappears, and is probably taken up by the cells.     

Glycosylation of capsaicin and 8-nordihydrocapsaicin by cultured cells of Catharanthus roseus

The glycosylation of capsaicin and 8-nordihydrocapsaicin was investigated using cultured cells of Catharanthus roseus. In addition to capsaicin 4-O-beta-d-glucopyranoside (170 microg/g fr. wt of cells), the biotransformation products, capsaicin 4-O-(6-O-beta-D-xylopyranosyl)-beta-D-glucopyranoside (116 microg/g fr. wt of cells) and capsaicin 4-O-(6-O-alpha-L-arabinopyranosyl)-beta-D-glucopyranoside (83 microg/g fr. wt of cells), were isolated from the cell suspension after three days of incubation with capsaicin. Two other compounds, 8-nordihydrocapsaicin 4-O-(6-O-beta-D-xylopyranosyl)-beta-D-glucopyranoside (171 microg/g fr. wt of cells) and 8-nordihydrocapsaicin 4-O-(6-O-alpha-L-arabinopyranosyl)-beta-D-glucopyranoside (122 microg/g fr. wt of cells), together with the known 8-nordihydrocapsaicin 4-O-beta-D-glucopyranoside (204 microg/g fr. wt of cells) were also isolated from the cell suspension after incubation with 8-nordihydrocapsaicin.     

Biosynthesis of brassinolide from teasterone via typhasterol and castasterone in cultured cells of Catharanthus roseus

The biosynthesis of brassinolide (BL) in crown gall and nontransformed cells of Catharanthus roseus in which BL, castasterone (CS), typhasterol (TY), and teasterone (TE) are endogenous was investigated using deuterated TY and TE as substrates. The metabolites were analyzed by gas chromatography-mass spectrometry (GC-MS) and/or GC-selected ion monitoring (SIM). It was found that these cells converted TY to CS and BL, as well as TE to TY and CS. Because the pathway from CS to BL in the cells has already been confirmed, a biosynthetic sequence of TE TY CS BL was established. Reversible conversion between TE and TY was observed.Biosynthesis of brassinosteroids in Catharanthus roseus. Part III. Part II of this series: Suzuki et al. (1993).     

Effect of nitric oxide on catharanthine production and growth of Catharanthus 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.     

Dissimilation of methionine in cell suspension cultures from Catharanthus roseus L.

Plant cell suspension cultures from Catharanthus roseus were investigated for their capability to dissimilate methionine or its analogs in order to reutilize the sulphane group for cysteine biosynthesis. Three steps have been described as prerequisites of this process: (a) oxidative degradation by the amino-acid oxidase of methionine giving rise to methanethiol production; (b) demethylation by methyltransferases leading to homocysteine and S-methylmethionine (c) replacement of the homocysteine sulphane sulphur by alkylthiol yielding methionine and free hydrogen sulphide. A reversal of the cystathionine pathway as a source of cysteine was ruled out because the cells lack cystathionine γ-lyase. The absence of this enzyme is compensated by the S-alkyl exchange of homocysteine with methylmercaptan. Hydrogen sulphide thus liberated is used for de novo synthesis of cysteine. The complete pathway can be catalyzed by the constitutive set of enzymes present in the higher plant.     

Subcellular localization of tryptophan decarboxylase,strictosidine synthase and strictosidine glucosidase in suspension cultured cells of Catharanthus roseus and Tabernaemontana divaricata

The subcellular localization of tryptophan decarboxylase, strictosidine synthase and strictosidine glucosidase in suspension cultured cells of Catharanthus roseus (L.) G. Don and Tabernaemontana divaricata (L.) R. Br. ex Roem. et Schult, was investigated. It was found that tryptophan decarboxylase is an extra-vacuolar enzyme, whereas strictosidine synthase is active inside the vacuole. Strong indications were obtained for the localization of strictosidine glucosidase on the outside of the tonoplast. The results suggest that tryptamine is transported into the vacuole where it is condensed with secologanin to form strictosidine, and that strictosidine passes the tonoplast and is subsequently hydrolysed outside the vacuole.Abbreviations AM -mannosidase - EDTA ethylenediaminetetraacetic acid - Hepes N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid - HPLC highperformance liquid chromatography - MDH malate dehydrogenase - SG strictosidine glucosidase - SSS strictosidine synthase - TDC tryptophan decarboxylase     

Detection of mRNAs correlated with proliferation of cells in suspension cultures of Catharanthus roseus

Gene expression correlated with proliferation was investigated in Catharanthus roseus (L.) G. Don. cells. Polyadenylated RNAs were isolated from cells in proliferative states or in nonproliferative states and the variations in the population and levels of mRNA were analyzed by in vitro translation followed by separation of the corresponding polypeptides by two-dimensional gel electrophoresis. Levels of two mRNAs increased in the actively cycling cells, while they were hardly detected in cells in stationary phase or in cells arrested by starvation of phosphate, sucrose or nitrogen. The molecular masses of the translated products of these two mRNAs were 53 and 60 kDa. No mRNAs were specifically synthesized in common among the various cultures of cells whose growth was arrested by nutrient starvation.     

Establishment of synchrony by starvation and readdition of auxin in suspension cultures of Catharanthus roseus cells

A system of synchronous cell division was established by starvation of auxin and its readdition to suspension cultures of cells of Catharanthus roseus L. cv. Little-Pinky. When cells in the stationary phase were transferred to fresh medium free of 2,4-dichlorophenoxyacetic acid (2,4-D), cells were arrested preferentially at the G₁ phase. After cells had been cultured for 2 days in medium without 2,4-D, readdition of 2,4-D induced the synchronous division of cells. In this system, 70–80% of cells divided synchronously within 3 to 4h, and the mitotic index increased sharply in parallel with the increase in cell number. Active synthesis of DNA was demonstrated by measurements of incorporation of [³H]-thymidine into the DNA fraction. The induction of cell division by the addition of 2,4-D was inhibited by treating cells with analogues of auxin, such as 2,4,6-trichlorophenoxyacetic acid and p-chlorophenoxyisobutyric acid.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DAPI 4,6-diamidino-2-phenylindole - IAA indole-3-acetic acid - MS Murashige & Skoog - NAA -naphthalenacetic acid - PCIB p-chlorophenoxyisobutyric acid - 2,4,6-T 2,4,6-trichlorophenoxyacetic acid     

Physiological aspects of surface-immobilized Catharanthus roseus cells

Summary Growth and alkaloid production of surface-immobilized C. roseus cells were studied in a 2-1 bioreactor. Media designed to maximize cell growth or alkaloid production were employed. Nitrate and carbohydrate consumption rates as well as growth rates and biomass yields of immobilized cultures were equal or somewhat lower than for cell suspension cultures. Respiration rate (O₂ consumption and CO₂ production rates) of immobilized C. roseus cell cultures was obtained by on-line analysis of inlet and outlet gas composition using a mass spectrometer. Respiration rate increased during the growth phase and decreased once the nitrogen or the carbon source was depleted from the medium. The respiration rate of immobilized C. roseus cells resembled rates reported in the literature for suspension cultures. Offprint requests to: Denis Rho     

Regulation of cytoplasmic pH under extreme acid conditions in suspension cultured cells of Catharanthus roseus: a possible role of inorganic phosphate

Changes in cytoplasmic pH of suspension-cultured cells of Catharanthus roseus under extreme acid conditions were measured with the pH-dependent fluorescence dye; 2',7'-bis-(2-carboxyethyl)-5 (and-6) carboxyfluorescein (-acetoxymethylester) (BCECF). When cells were treated with 1 mM HCl (pH 3 solution), the cytoplasmic pH first decreased then returned to the original level. Treatment with 10 mM HCl (pH 2 solution) acidified the cytoplasm to a greater extent, and the acidification continued at a constant level throughout the measurement. Treatment with a pH 2 solution resulted in a gradual decrease of the malate content, indicating the operation of biochemical pH regulation mechanism. The pH 2 treatment also caused a sudden decrease of the intracellular level of Pi. The cellular content of total phosphorus did not change during the acidification. The Pi was converted to the organic phosphate form. The ATP level was not increased by the pH 2 treatment, but slightly decreased. The role of Pi, which might be functioning as a regulatory factor of cytoplasmic pH, a non-competitive inhibitor of the H+-pumps of both the plasma membrane and tonoplast is discussed.