Studies of the Plant Cell Cycle in Synchronous Cultures of Catharanthus roseus Cells

Catharanthus roseus cells can be induced to divide with a highdegree of synchrony by two different methods, namely, the phosphatestarvation method and the auxin starvation method. In such synchronizedcultures, cell-cycle-dependent events characteristic of plantcells, for example, synthesis of the cell wall and related morphologicalchanges, have been the focus of fruitful investigations. Itappears that polyamines and phosphatidylinositol are involvedin the regulation of the progression of the cell cycle. Genesfor proliferating-cell nuclear antigen and mitotic cyclins,as well as several genes identified as cell-cycle-dependentgenes by screening of a library prepared from synchronized cells,have been characterized and the results indicate that featurescommon to both plant cells and animal cells are involved inthe regulation of the progression of the cell cycle. ²Present address: Department of Plant Science, Graduate Schoolof Science, The University of Tokyo, Hongo, Tokyo, 113 Japan.     

Turnover of Cell Wall Polysaccharides during the Cell Cycle in a Synchronous Culture of Catharanthus roseus

Pulse-chase experiments were done using a synchronous cultureof Catharanthus roseus in order to study cell wall turnoverduring the cell cycle. [¹⁴C]Glucose was fed for 1 h to cells35 and 49 h after the re-start of the cell cycle. Radioactivitywas then diluted with a large amount of cold glucose and chasedduring the early G1 phase after the first cell division, thetime at which an increase in the amount of cell walls mainlytook place. A pulse-chase with [¹⁴C]glucose was also made duringthe S phase when cell walls had not increased so much. Radioactivity of the EDTA-soluble (pectin) fraction decreasedduring the chase in the early G1 phase; whereas, the radioactivitiesof the other cell wall fractions, as well as extracellular polysaccharide(ECP) increased during the chase, both in the early G1 and inthe S phases. The radioactivity of uronic acid in ECP was higherin the early G1 phase than in the S phase. These results indicatethat an active turnover of pectin may take place in the earlyG1 phase after the first cell division. ¹ Present address and reprint requests: Biological Institute,Tohoku University, Sendai 980, Japan. (Received November 5, 1984; Accepted April 2, 1985)     

Phase-Specific Polypeptides and Poly(A) RNAs during the Cell Cycle in Synchronous Cultures of Catharanthus roseus Cells

This study shows an overall analysis of gene expression during the cell cycle in synchronous suspension cultures of Catharanthus roseus cells. First, the cellular cytoplasmic proteins were fractionated by two-dimensional gel electrophoresis and visualized by staining with silver. Seventeen polypeptides showed qualitative or quantitative changes during the cell cycle. Second, the rates of synthesis of cytoplasmic proteins were also investigated by autoradiography by labeling cells with [³⁵S]methionine at each phase of the cell cycle. The rates of synthesis of 13 polypeptides were found to vary during the cell cycle. The silverstained electrophoretic pattern of proteins in the G₂ phase in particular showed characteristic changes in levels of polypeptides, while the rates of synthesis of polypeptides synthesized during the G₂ phase did not show such phase-specific changes. This result suggests that posttranslational processing of polypeptides occurs during or prior to the G₂ phase. In the G₁ and S phases and during cytokinesis, several other polypeptides were specifically synthesized. Finally, the variation of mRNAs was analyzed from the autoradiograms of in vitro translation products of poly(A)⁺ RNA isolated at each phase. Three poly(A)⁺ RNAs increased in amount from the G₁ to the S phase and one poly (A)⁺ RNA increased preferentially from the G₂ phase to cytokinesis.     

The Effect of Nutrient Medium Composition on the Growth Cycle of Catharanthus roseus G. Don Cells Grown in Batch Culture

Growth of C. roseus cell suspension culture was defined in termsof dry weight, cell number, mitotic index, and packed cell volume.Removal of major nutrients from the medium was monitored asa function of culture growth. Phosphate and sucrose were theonly macronutrients completely exhausted. Utilization of thesetwo nutrients occurred parallel with increments in dry weightand cell number. Increasing the nutrient medium levels of sucroseand phosphate prolonged growth of this culture; lag and exponentialphases were extended; cell number and dry weight yield weredoubled. Dry weight assimilation was enhanced by increasingthe nutrient medium level of sucrose, whereas increments incell number were related to phosphate level. Two alkaloid fractions(fractions 1 and 2) were identified in this cell line. Fraction2 alkaloid level declined as the nutrient medium supply of nitrogenwas depleted.     

Effects of Sugars on the Glucosylation of Exogenous Hydroquinone by Catharanthus roseus Cells in Suspension Culture

The effects of sugars on the glucosylation of exogenous hydroquinone(HQ) was investigated by supplying individual sugars simultaneouslywith HQ to a suspension culture of Catharanthus roseus cells.The production of arbutin was enhanced as much as 2- to 3-foldby sucrose or glucose at concentrations of up to 6%, with theenhancement being directly dependent on the concentration ofthe sugar. The exogenously added sugar was not metabolized andremained unchanged. Sorbitol also promoted the production ofarbutin in a similar manner. Sucrose improved the viability of cells and, in cultures suppliedwith sucrose and HQ, the activity of UDP-glucose: hydroquinoneglucosyltransferase increased over a much longer period of timethan that in control cultures supplemented with HQ only. (Received December 11, 1989; Accepted March 26, 1990)     

Phosphoenolpyruvate Carboxylase from Heterotrophically Cultured Catharanthus roseus Cells

Maximum activity of phosphoenolpyruvate carboxylase (PEPC, EC4.1.1.31) was detected at the stationary phase of growth ofCatharanthus roseus cells in a heterotrophic culture. The activityof PEPC, after partial purification by fractionation with ammoniumsulphate and chromatography on Q-Sepharose, was greatly influencedby pH. The K_m of phosphoenolpyruvate (PEP) was 23 µM atpH 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 pHof the reaction mixture. By contrast, glucose-6-phosphate, fructose-1,6-bisphosphateand acetyl-CoA, known as stimulators of the activity of PEPCfrom other sources, had little or no effect on the activityof the partially purified PEPC. The possible role and mechanismof regulation of PEPC in C. roseus cells are discussed.Copyright1994, 1999 Academic Press Catharanthus roseus, Apocynaceae, Madagascar periwinkle, suspension culture, phosphoenolpyruvate carboxylase, enzyme kinetics, glycolysis     

Metabolism of Pyrimidines in Protoplasts from Cultured Catharanthus roseus Cells

The metabolism of [2-¹⁴C]thymine, [2-¹⁴C]thymidine, [2-¹⁴C]uraciland [¹⁴C]uridine was investigated in protoplasts obtained fromsuspension cultures of Catharanthus roseus. Most of the exogenouslysupplied thymine, thymidine and uracil was degraded, and salvageof these pyrimidines accounted for 5–36 per cent of thetotal amount of ¹⁴C-labelled precursors which was metabolized.However, more than 80 per cent of the labelled uridine was utilizedfor the biosynthesis of nucleotides and nucleic acids, and therest was degraded. In contrast to the results from protoplastsof sugar cane cells in suspension culture, the data indicatethat protoplasts possess a pathway for the degradation of pyrimidines,and that the overall metabolism of these pyrimidines in protoplastsis very similar to the metabolism in the intact cells. Catharanthus roseus, madagascar periwinkle, protoplasts, pyrimidine metabolism     

Adenine and Guanine Salvage in Suspension Cultured Cells of Catharanthus roseus

Changes in the activity of adenine and guanine salvage in nucleotideand nucleic acid synthesis during the growth of Catharanthusroseus were investigated. Incorporation of [8-¹⁴C]adenine intoATP and ADP and that of [8-¹⁴C]guanine into GTP and GDP increasedmarkedly in the lag phase of cell growth and then sharply decreased.The incorporation into RNA from both precursors showed a similarpattern. The role of rapid purine salvage observed in the lagphase of cell growth is discussed. Catharanthus roseus, Madagascar periwinkle, suspension culture cells, purine salvage, adenine, guanine     

Differences in the Composition of the Cell Walls of Two Morphologically Different Lines of Suspension-Cultured Catharanthus roseus Cells

Differences in the composition of cell walls of two morphologicallydifferent lines (A and B) of suspension-cultured Catharanthusroseus cells, which have the same origin, were investigated.The cells of strain A are nearly spherical, while those of strainB are cylindrical. In strain A, the amount of cell wall pergram fresh weight of cells increased during the logarithmicphase. In strain B, the amount of cell wall per cell decreasedduring the logarithmic phase. The level of matrix polysaccharides increased markedly duringthe logarithmic phase in strain A. The amount of cellulose incell wall was relatively larger in strain B than in strain A.The following differences in sugar composition between the twostrains were observed: (a) there was an increase in the relativelevels of 4-linked galactose in the EDTA-soluble fraction andof 3-linked glucose in the 5% KOH-soluble fraction during thelogarithmic phase in strain A; (b) there were significantlyhigher levels of arabinose, probably derived from 2,5- and/or3,5-linked arabinan, in the EDTA-soluble fraction and in theextracellular polysaccharides in strain B; (c) there were decreasesin the relative amounts of some kinds of sugar, probably thosederived from xyloglucan, during the stationary phase in strainB. (Received March 31, 1989; Accepted October 12, 1989)     

Comparison of Cell Growth and Alkaloid Production of Catharanthus roseus Cells Cultured in Shake Flask and in Bioreactor

The cell growth and alkaloid production of Catharanthus roseus (L.) G. Don cells cultured in the shake flasks with different volumes and in the stirred tank bioreactor (10 L) were compared. Cell dry weight and alkaloid production showed no significant difference in the small volume scale-up shake flasks. When more broths were added to a certain volume in the shake flask, both cell weight and alkaloid production were decreased. The maximum cell dry weight was similar between the cell cultures in the shake flask and the bioreactor, but the alkaloid production of cells was much less in the bioreactor. Gas regime and shear stress were recognized to be the main factors contributing the important effect on alkaloid production during the scale-up processes.     

Long-Term Phosphate Starvation and Respiratory Metabolism in Suspension-Cultured Catharanthus roseus Cells

In order to clarify the metabolic adaptation of respiratorypathways in plants to limited levels of P_i, the effects of long-termstarvation of P_i on the activities of various enzymes relatedto respiratory metabolism were examined in suspension-culturedCatharanthus roseus cells. When the activities were expressedas units per g fresh weight, only those of phosphoenolpyruvate-hydrolyzing(PEP-hydrolyzing) enzyme (which may possibly be equivalent tothe acid phosphatase activity derived from vacuoles) and PEPcarboxylase were higher in the Pⁱ-starved cells than in controlcells. Activities of other enzymes in the Pⁱ-starved cells werelower than or similar to those of the control cells. Time-coursestudies indicated that PEP-hydrolyzing activity was inducibleby starvation of Pⁱ. However, in contrast to the results reportedby Duff et al. [(1989a) Plant Physiol. 90: 1275.], fluctuationsin the activity of PP¹:fructose-6-phosphate 1-phosphotransferaseduring starvation of Pⁱ were similar to those in levels of phosphofructokinaseand 6-phosphogluconate dehydrogenase. These data suggest thatthe concept of the phosphate starvation-inducible ‘bypasses’,which are engineered via the coarse control (i.e., induction)of specified enzymes and were proposed initially by Duff etal. in Brassica nigra cells, is not directly applicable to Catharanthusroseus cells in suspension. Tracer experiments using [U-¹⁴C]glutamineindicated that a significant proportion of respiratory substratescould be supplied from the enlarged pool of amino acids duringstarvation of Pⁱ. These assumptions are supported by the observedfluctuations in levels of free amino acids and of protein inP¹-fed and P¹-deficient Catharanthus roseus cells. ¹Part 41 in the series ‘Metabolic Regulation in PlantCell Cultrue’ ²Present Address: Morinaga Mild Industry, 5-1-83, Higashihara,Zamma-shi, Kanagawa, 228 Japan     

Isolation of Genes that Are Preferentially Expressed at the G(1)/S Boundary during the Cell Cycle in Synchronized Cultures of Catharanthus roseus Cells

A cDNA library was screened for genes that may be involved in the progression of the cell cycle of cells of higher plants. The Catharanthus roseus L. (G) Don. cells were synchronized by the double phosphate starvation method, and a λgt11 cDNA library was prepared using poly(A)⁺ RNA from cells in the S phase of the cell cycle. Two independent sequences, cyc02 and cyc07, were identified by differential screening. The levels of cyc02 and cyc07 mRNAs increased dramatically, but transiently, at the G₁/S boundary of the cell cycle. High levels of cyc02 mRNA, but not of cyc07 mRNA, were also present in cells arrested at the G₁ phase by phosphate starvation. In an asynchronous batch culture, cyc02 and cyc07 mRNAs accumulated transiently at different stages of the growth cycle, cyc02 mRNA early in the stationary phase, and cyc07 mRNA in the midlogarithmic phase. When the proliferation of cells was arrested by nutrient starvation, i.e. by sucrose or nitrogen starvation, the relative amounts of the cyc02 and cyc07 mRNAs decreased. These results indicate that cyc02 and cyc07 contain nucleotide sequences from growth-related genes. The analysis of nucleotide sequence of cyc02 shows that the predicted product of this gene is basic and is composed of 101 amino acids. No significant homology to other known proteins was detected.     

土壤农杆菌转化的长春花冠瘿细胞培养

以土壤农杆菌C₅₈诱导的长春花冠瘿组织与从长春花茎、叶外植体诱导的愈伤组织进行比较,发现冠瘿组织在生长、总吲哚生物碱含量及药用成份阿吗碱含量等方面都优于愈伤组织。测定了光照、温度、蔗糖浓度及外加L－色氨酸前体等,对长春花冠瘿细胞的生长、总生物碱及阿吗碱含量的影响,为长春花冠瘿细胞培养生产吲哚生物碱的实际应用研究提供理论依据。     

长春花悬浮培养细胞对天麻素的生物转化

应用长春花(Catharanthus roseus (L.) G. Don)悬浮细胞培养体系对天麻素进行了生物转化反应研究.经过8 d培养形成一个转化产物,应用光谱方法鉴定转化产物的结构为对羟基苯甲醇,为天麻素水解后形成的甙元.     

Uptake and Metabolism of Sugars by Suspension Cultured Catharanthus roseus Cells

The uptake and metabolism of sugars by suspension-cultured Catharanthusroseus cells were investigated. Substantially all the sucrosein the culture medium was hydrolyzed to glucose and fructosebefore being taken up by the cells. The activity of invertasebound to cell walls, determined in situ, was high at the earlystage of culture. Glucose was more easily taken up by the cellsthan was fructose. Tracer experiments using [U-¹⁴C]glucose and[U-¹⁴C]fructose indicated that glucose is a better precursorfor respiration than fructose, while fructose is preferentiallyutilized for the synthesis of sucrose, especially in the earlyphase of cell growth. Possible metabolic routes of sugar insuspension-cultured Catharanthus roseus cells are discussedin the context of these results. Catharanthus roseus, Madagascar periwinkle, suspension culture, sucrose, glucose, fructose, metabolism, glycolysis     

Cell and tissue cultures of Catharanthus roseus: A literature survey

The literature concerning the regulation and the biosynthesis of secondary metabolites in cell and tissue cultures of Catharanthus roseus is reviewed. The aim of this review is to summarise the progress achieved since the previous review of this subject from 1988 to December 1993. Several factors influencing the production of indole alkaloids are discussed. Special attention is given to large-scale cultivation methods. Some economic considerations on the production of ajmalicine are also discussed.     

Uptake and Metabolism of Sugars by Suspension cultured Catharanthus roseus Cells

p. 186, right column line 11 ‘27.2 KBq’ change to‘37.2 MBq’ p. 187, left column line 8 ‘27.2 KBq’ change to‘37.2 MBq’ line 10 ‘13.6 KBq’ change to ‘18.6 MBq’ line 11 ‘13.6 KBq’ change to ‘18.6 MBq’ line 21 ‘89%’ change to ‘80%’ right column line 24 ‘CLC-NH₄’ change to ‘CLC-NH₂’ P. 189, Table 1 appeared incorrectly: it should appear as indicated.     

营养和环境因子对长春花激素自养型细胞生长和阿玛碱生成的影响

在摇瓶中用液体培养基培养长春花（Catharanthusroseus（L．）G．Don）激素自养型细胞系C20hi,比较了不同初始糖浓度、接种量、初始pH值、光照时间、光质和摇床转速对该培养细胞生长、阿玛碱积累和释放的影响。结果表明,此细胞系具有较强的环境耐受性;一定范围内初始糖浓度增加,有利于细胞生长和生物碱生成;最适的接种量是60gFW／L;一定范围内改变培养基pH值,对生物碱生成没有显著影响;照光后生物碱生成量下降,红光较蓝光更有利于生物碱生成;最适的摇床转速是120r／min。