Cultured Catharanthus Roseus Cells Research Articles

Enzymatic resolution of ( RS)-1-phenylalkyl β- d-glucosides to ( R)-1-phenylalkyl β-primeverosides and ( S)-1-phenylalkyl β- d-glucosides via plant xylosyltransferase

The stereoselective xylosylation of ( RS)-1-phenylalkyl β- d-glucosides was investigated using plant xylosyltransferase isolated from cultured Catharanthus roseus cells. Enzymatic xylosylation of ( RS)-1-phenylethyl β- d-glucoside afforded ( R)-1-phenylethyl β-primeveroside and ( S)-1-phenylethyl β- d-glucoside. The ( R)-selective xylosylation of ( RS)-1-phenylbutyl β- d-glucoside also occurred to give ( R)-1-phenylbutyl β-primeveroside and recovered ( S)-1-phenylbutyl β- d-glucoside.

Metabolic Conversion of Castasterone and Brassinolide into Their Glucosides in Higher Plants

Castasterone (CS) and brassinolide (BL) were administered to mung bean (Vigna radiata) explants, Arabidopsis thaliana seedlings, and cultured Catharanthus roseus cells, and the glucosylated metabolites were analyzed using LC/MS/MS. In mung bean and C. roseus, CS-2-O-glucoside (CS-2G), -3-O-glucoside (CS-3G), -22-O-glucoside (CS-22G), and -23-O-glucoside (CS-23G) were identified as metabolites of CS, whereas BL-2G, BL-3G, and BL-23G were identified as metabolites of BL. In A. thaliana, CS and BL were converted into their respective 2-O- and 23-O-glucosides. Of the metabolites identified with BL and CS administration, BL-23G was the predominant metabolite in mung bean and A. thaliana, whereas the 3-O-glucoside of BL was abundant in C. roseus. This is the first report of the metabolic conversion of CS into CS-2G, CS-3G, CS-22G, and CS-23G, and of BL into BL-2G and BL-3G. Our results indicate that the glucosylation profiles of BL and CS vary with plant species, and that the glucosylation of CS is rather limited quantitatively, compared with that of BL.

Area Expansivity Moduli of Regenerating Plant Protoplast Cell Walls Exposed to Shear Flows

To control the elasticity of the plant cell wall, protoplasts isolated from cultured Catharanthus roseus cells were regenerated in shear flows of 115 s-1 (high shear) and 19.2 s-1 (low shear, as a control). The surface area expansivity modulus and the surface breaking strength of these regenerating protoplasts were measured by a micropipette aspiration technique. Cell wall synthesis was also measured using a cell wall-specific fluorescent dye. High shear exposure for 3 h doubled both the surface area modulus and breaking strength observed under low shear, significantly decreased cell wall synthesis, and roughly quadrupled the moduli of the cell wall. Based on the cell wall synthesis data, we estimated the three-dimensional modulus of the cell wall to be 4.1±1.2 GPa for the high shear, and 0.35±0.2 GPa for the low shear condition, using the surface area expansivity modulus divided by the cell wall thickness, which is identical with the Young's modulus divided by 2(1-σ), where σ is Poisson's ratio. We concluded that high shear exposure considerably strengthens the newly synthesized cell wall.

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.

Sequential solubilization of proteins precipitated with trichloroacetic acid in acetone from cultured Catharanthus roseus cells yields 52% more spots after two-dimensional electrophoresis.

Sample preparation is still the most critical step in two-dimensional gel electrophoresis (2-DE), and needs to be optimized for each type of sample. To analyze the proteome of the medicinal plant Catharanthus roseus, we developed and evaluated a sequential solubilization procedure for the solubilization of proteins after precipitation in trichloroacetic acid and acetone. The procedure includes solubilization with a conventional urea buffer followed by a stronger solubilizing buffer containing thiourea. The sequential solubilization of the precipitated proteins results in very different spot patterns following 2-DE. The number of protein spots which could be detected in both samples of the sequential solubilization was only about 10% of the total number of spots. Compared to solubilization in a single step, the total number of spots that could be detected in the sequential solubilization procedure was increased by 52%. The method described is simple and is applicable to different types of plant tissue.

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.

Brassinosteroid biosynthesis

Biosynthetic pathways of brassinolide from campesterol was demonstrated by studies using cultured Catharanthus roseus cells. Brassinolide is biosynthesized through two pathways, early C6-oxidation pathway and late C6-oxidation pathway, branching off at the conversion of campestanol. Recent characterization of brassinosteroid-deficient mutants of Arabidopsis, pea and tomato confirmed that the pathways operate in wide variety of plant species. Biochemical and molecular genetic studies of the mutants are providing important knowledge on genes and enzymes involved in brassinosteroid biosynthesis. The established biosynthetic pathways of brassinosteroids and the regulation of biosynthesis including up-to-date findings are introduced in this review.

Noninvasive NMR studies of metabolism in cultured Catharanthus roseus cells

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.

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.

Purification and characterization of multiple forms of phosphatidylinositol-specific phospholipases D from suspension cultured Catharanthus roseus cells

Two soluble and two membrane bound phosphatidylinositol (PI) specific phospholipases D (EC 3.1.4.4) were identified, partially purified and characterized from Catharanthus roseus suspension cultured cells. In order to compare the chromatographic properties of the four enzymes, column chromatography was performed under the same conditions for all four enzymes. The soluble enzymes have apparent molecular weights of 132 kD and 48 kD, the plasma membrane bound enzymes of 132 kD and 58 kD. Based on their origin and their molecular weight the types were designated S132, S48, PM132 and PM58, respectively. Isoelectric points of the native type S132, S48, PM132, PM58 phospholipases D were 4.89, 4.89, 4.6 and 4.8. After ammonium sulphate precipitation in the presence of Triton X-100, the cytosolic enzymes precipitated, whereas the plasma membrane localized enzymes floated up. In the presence of Triton X-100 all enzymes do not depend on divalent cations. Maximum activity is obtained at a Triton X-100 PI ratio of 8:1 and half maximum activity at a ratio of 14:1 to 20:1. Using Triton X-100 PI mixed micelles, the 48 and 58 kD enzymes depended on the surface concentration of PI as well as on the bulk concentration of PI, whereas the 132 kD proteins depended nearly exclusively on the surface concentration of PI. A detailed analysis of the 48 kD and 58 kD phospholipases show that they very closely follow the surface dilution kinetic model as proposed by Deems et al. (R.A. Deems, B.R. Eaton, E.A. Dennis. J. Biol. Chem., 250 (1975) 9013–9020). The interfacial Michaelis constants ( K m B) for phosphatidylinositol were 0.017 mol fraction for the 48 kD cytosolic protein and 0.062 for the 58 kD membrane-bound phospholipase, respectively. As has been reported for microsomal PI-specific phospholipases D (A. Becher, J.B. Wissing, K.G. Wagner. Plant Sci., 97 (1994) 143–151) all these enzymes do not display any transphosphatidylation activity. The formation of diphosphatidylglycerol was also not observed. Further Mg 2+ or Ca 2+ are not needed for activity, EDTA, EGTA and DTPA show no effect. The pH optimum is 5.25 for the low molecular weight and 6.5 for the high molecular weight isoforms. Half maximum activity is observed at pH 4.2 and 6.2 for the smaller enzymes and at pH 4.5 and 7.5 for the larger enzymes.

Properties of phosphatidylinositol 4-kinase I from suspension cultured Catharanthus roseus cells

A phosphatidylinositol (PI) 4-kinase (ATP:phosphatidylinositol phosphotransferase, EC 2.7.1.67) was extracted from plasma membranes, obtained from suspension cultured Catharanthus roseus cells, and partially purified. An apparent molecular weight of about 500 000 was determined by gel filtration in the presence of Triton X-100 and a p I value of 5.8 by isoelectric focusing. Kinetic parameters and the reaction mechanism ere studied at low Triton concentration; K m values for ATP and PI were found as 62 and 35 μM, respectively. The equilibrium constant for the PI kinase reaction was found to be 110, i.e. on the side of the products. The kinase catalyses a sequential Bi-Bi reaction; isotope exchange patterns were consistent with a random substrate binding and an ordered product release, with PIP leaving the enzyme before ADP. These findings are similar to the mechanism found in the case of PI kinases from baker's yeast.

Phosphoenolpyruvate Carboxylase from Heterotrophically Cultured Catharanthus roseus Cells

Maximum activity of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) was detected at the stationary phase of growth of Catharanthus roseus cells in a heterotrophic culture. The activity of PEPC, after partial purification by fractionation with ammonium sulphate and chromatography on Q-Sepharose, was greatly influenced by pH. The Km of phosphoenolpyruvate (PEP) was 23 μM at pH 8·0 and 45 μM at pH 7·4. Malate, aspartate, citrate, ATP, pyrophosphate and Pi acted as inhibitors of PEPC, but the extent of inhibition varied in each case with the pH of the reaction mixture. By contrast, glucose-6-phosphate, fructose-1,6-bisphosphate and acetyl-CoA, known as stimulators of the activity of PEPC from other sources, had little or no effect on the activity of the partially purified PEPC. The possible role and mechanism of regulation of PEPC in C. roseus cells are discussed.

Ajimalicine production with cultured Catharanthus roseus cells: Stimulation of productivity by enhanced release of metabolites from cells. —Monograph—

Ajimalicine production with cultured Catharanthus roseus cells: Stimulation of productivity by enhanced release of metabolites from cells. —Monograph—

Phosphatidylinositol specific isoenzymes of phospholipase D from Catharanthus roseus. Purification and characterization

Microsomal membranes from suspension cultured Catharanthus roseus cells were found to contain phospholipase D activity (EC 3.1.4.4) towards phosphatidylinositol (PI). After extraction with buffer containing Triton X-100, two isoenzymes with apparent molecular weights of 50 000 and 125 000 were partially purified. The enzymes accepted only PI as substrate and transphosphatidylation activity could not be detected. Whereas several phospholipids were slightly inhibitory, phosphatidyl glycerol had stimulatory properties. The enzymes required divalent cations for activity, Ca 2+ and Mg 2+ were equally effective. In the presence of deoxycholate and Ca 2+, K m values for PI were found to be in the range 20–40 μM. Although the smaller protein had a slightly higher temperature stability and a slightly lower p I value, both isoenzymes revealed equal enzymic properties. This is the first report on a plant PI-specific phospholipase D.

IMP dehydrogenase from tea leaves and suspension-cultured Catharanthus roseus cells

The maximum catalytic activities of IMP dehydrogenase (IMPDH) in young tea ( Camellia sinensis) leaves and in suspension- cultured Catharanthus roseus cells were 321 and 48 pkat g −1 fr. wt, respectively. The K m values of IMPDH that had been partially purified from tea and from C. roseus were 28 μM and 14 μM, respectively. Both enzymes were inhibited by purine nucleotides, especially GMP. The K i , value of the enzyme for GMP was 170 μM for the enzyme from tea and 67 μM for the enzyme from C. roseus. Metabolites related to the biosynthesis of caffeine had little effect on the activity of the enzyme from either source. Participation of IMPDH in the biosynthesis of caffeine is discussed.

Characterization of cytidine triphosphate:phosphatidate cytidyltransferase from suspension cultured Catharanthus roseus cells

Cytidine diphosphate-diacylglycerol (CDP-DG) synthase activity (EC 2.7.7.41) of a microsomal membrane preparation from suspension cultured Catharanthus roseus cells was characterized. The enzyme showed a pH optimum of 7.3 and apparent K m values for cytidine triphosphate (CTP) and phosphatidic acid (PA) of 170 and 80 μM, respectively. Inhibition was observed with CDP and inorganic pyrophosphate (PPi) with apparent K i values of 240 μM and about 1.5 mM, respectively. After extraction from the microsomal membranes, the enzyme was further purified with Phenyl-Sepharose chromatography, although the yield was low. CDP-DG synthase is obviously a membrane-bound protein and located both in intracellular membranes, i.e. the endoplasmic reticulum (ER), the mitochondrial and chloroplast envelopes and the plasma membrane.

Characterization of Phosphate Uptake by Suspension Cultured Catharanthus roseus Cells

Summary The mechanism of uptake of inorganic phosphate was studied with suspension cultured Catharanthus roseus cells. The kinetics showed both a low and a high affinity phase with Km values in the μM and mM range, respectively. Experiments with different inhibitors suggested that the high affinity phosphate transport is driven by the proton motive force at the plasma membrane and probably includes a protonphosphate symport. The low affinity transport revealed properties different from those of the high affinity transport; its relation to phosphate transport at the tonoplast membrane is discussed. The high affinity phase of Pi uptake, studied with cells from different phases of the growth cycle, revealed little changes in the Km values, which were between 2 to 5 μM, whereas the Vmax values strongly depended on the growth stage with values up to 70 nmol min−1 g−1 FW.

Diacylglycerol Kinase from Suspension Cultured Plant Cells

Diacylglycerol kinase (adenosine 5'-triphosphate:1,2-diacylglycerol 3-phosphotransferase, EC 2.7.1.107), purified from suspension cultured Catharanthus roseus cells (J Wissing, S Heim, KG Wagner [1989] Plant Physiol 90: 1546-1551), was further characterized and its subcellular location was investigated. The enzyme revealed a complex dependency on lipids and surfactants; its activity was stimulated by certain phospholipids, with phosphatidylinositol and phosphatidylglycerol as the most effective species, and by deoxycholate. In the presence of Triton X-100, used for its purification, a biphasic dependency upon diacylglycerol was observed and the apparent Michaelis constant values for diacylglycerol decreased with decreasing Triton concentration. The enzyme accepted both adenosine 5'-triphosphate and guanosine 5'-triphosphate as substrate and showed rather low apparent inhibition constant values for all nucleoside diphosphates tested. Diacylglycerol kinase is an intrinsic membrane protein and no activity was found in the cytosol. An investigation of different cellular membrane fractions confirmed its location in the plasma membrane.

Plasma membrane preparations from suspension cultured plant cells contain the enzymes for the recycling of phosphatidic acid and diacylglycerol

The subcellular distribution of phosphatidylinositol(PI) kinase (EC 2.7.1.67), PI-specific phospholipase C (EC 3.1.4.3) and D, diacylglycerol (DG) kinase (EC 2.7.1.107), CTP:phosphatidic acid (PA) cytidyltransferase (EC 2.7.7.41) and PI-synthase (EC 2.7.8.11) in membrane fractions from suspension cultured Catharanthus roseus cells was investigated. Whereas activities of phospholipase C and D could be detected both in the cytosol and membrane fractions, all the other enzymes were bound to membranes. PI-kinase, membrane-bound phospholipase C and D, DG-kinase and PI-synthase had their highest specific activities in the plasma membrane enriched fraction, whereas cytidyltransferase had comparable activities both in the plasma membrane fraction and in the fractions derived from internal membranes. These data suggest that the plant plasma membrane might be autonomous with respect of the recycling of lipid cleavage products obtained by the action of phospholipase C and D.

Rapid changes in the enzyme activities and metabolites of the phosphatidylinositol-cycle upon induction by growth substances of auxin-starved suspension cultured Catharanthus roseus cells

Suspension cultured Catharanthus roseus cells grown with 2,4-dichlorophenoxyacetic acid (2,4-D) were starved for this growth substance for 3 days, thereafter cell proliferation was induced by addition of 2,4-D, kinetin, or α-naphthaleneacetic acid (α-NAA) and kinetin. In one set of experiments changes of 3H-inositol-labeled phosphoinositides and inositol phosphates were determined in a time interval of 15 min after induction. In a second set of experiments the activities of phosphatidylinositol(PI)-kinase and diacylglycerol (DG)-kinase were followed by the use of [γ- 32P]ATP. Induction by 2,4-D revealed a very fast (maximum 1 min after addition of the growth substance) increase in Ins1,4P 2, decrease in PIP and increase in PI-kinase activity. Addition of α-NAA and kinetin resulted in a very fast decrease of PI and PIP (1 min after induction) and a concomitant decrease in the activities of PI-kinase and DG-kinase, whereas slightly slower (2 min after induction) but very strong increases in Ins1P 1 and Ins1,4P 2 were observed. Although obtained with an artificial in vitro system, these results demonstrate that the activities of the PI-cycle are responsive towards phytohormones.