九连小檗悬浮培养细胞生理生化特性的研究——Ⅲ.药根碱累积与细胞生长的关系

本文叙述了九连小檗植物细胞悬浮培养过程中,药根碱的累积和细胞生长与培养液中可溶性糖转化的关系。实验表明细胞培养过程中培养液的可溶性糖逐渐消耗,细胞生物量和药根碱量都逐渐增加,且细胞生长与药根碱累积的曲线几乎是平行的。然细胞生长速度较快,其生长速率曲线的峰形较尖陡。药根碱累积速度较慢,延续时间较长,其累积速率曲线的峰形较平缓。根据糖的消耗与细胞生物量增长和药根碱累积的关系,计算出蔗糖——细胞转化率为59%,蔗糖——药根碱转化率为3.3%。     

Cell-pool tryptophan phases in ergot alkaloid fermentation

Three cell-pool tryptophan phases are recorded as characteristics of the alkaloid fermentation byClaviceps paspali grown on a simple defined medium without tryptophan. Within the early phase designated “tryptophan down” the alkaloid-biosynthetic activity of the mycelium attains the maximum, protein synthesis is reduced and extracellular proteases are formed. Cell-pool tryptophan level (b) drops, tryptophan synthetase activity (c) intensifies and sums of logb+logc after different time intervals remain constant. In the subsequent “tryptophan up” phase tryptophan level (b) increases, alkaloid yield (a) becomes a function of time and reaches the top level still tolerable by tryptophan synthetase. The difference of the logb—logc is constant. The tryptophan synthetase diminishes its activity simultaneously with the alkaloid-biosynthetic activity of the mycelium. The district between the “tryptophan down” and “tryptophan up” phase is an especially promising target for the investigation of the regulation of alkaloid formation and continuous fermentation of these compounds. During the third, i.e. “tryptophan over” phase, cell-pool tryptophan accumulates and attains a concentration exerting a negative effect on the alkaloid biosynthesis.     

Stability of alkaloid production in cell suspension cultures of Tabernaemontana divaricata during long-term subculture

Two cell lines of Tabernaemontana divaricata cell suspension culture with different growth and alkaloid production profiles were transferred to the same medium. During 30 subcultures the changes in growth and alkaloid production were followed and compared to those of the original cell lines. The presence of NAA and BAP in the medium resulted in an increase of biomass and alkaloid yield. The effect on the growth proved to be stable during these 30 subcultures. Alkaloid production showed a maximum in the 4th subculture after the change of the medium, and stabilized on a higher level than found in the original cell lines. During some growth cycles also the activities of tryptophan decarboxylase (TDC), strictosidine synthase (SSS), and phenylalanineammonia-lyase (PAL) were measured. In both the original cell lines and the derived cell lines, growth and alkaloid production proved to be stable all through the experiment, although the derived cell lines had a period of adaptation to the new medium with increased productivity.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - BAP benzylaminopurine - DW dry weight - TDC tryptophan decarboxylase - SSS strictosidine synthase - PAL phenylalanineammonia-lyase - PAT phenylalanineammonia-transaminase     

Proteome analysis of the medicinal plant <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

A proteomic approach is undertaken aiming at the identification of novel proteins involved in the alkaloid biosynthesis of Catharanthus roseus. The C. roseus cell suspension culture A11 accumulates the terpenoid indole alkaloids strictosidine, ajmalicine and vindolinine. Cells were grown for 21 days, and alkaloid accumulation was monitored during this period. After a rapid increase between day 3 and day 6, the alkaloid content reached a maximum on day 16. Systematic analysis of the proteome was performed by two-dimensional polyacrylamide gel electrophoresis. After day 3, the proteome started to change with an increasing number of protein spots. On day 13, the proteome changed back to roughly the same as at the start of the growth cycle. 88 protein spots were selected for identification by mass spectrometry (MALDI-MS/MS). Of these, 58 were identified, including two isoforms of strictosidine synthase (EC 4.3.3.2), which catalyzes the formation of strictosidine in the alkaloid biosynthesis; tryptophan synthase (EC 4.1.1.28), which is needed for the supply of the alkaloid precursor tryptamine; 12-oxophytodienoate reductase, which is indirectly involved in the alkaloid biosynthesis as it catalyzes the last step in the biosynthesis of the regulator jasmonic acid. Unique sequences were found, which may also relate to unidentified biosynthetic proteins.     

Physiological studies on ergot: further studies on the induction of alkaloid synthesis by tryptophan and its inhibition by phosphate

The failure of l-leucine to stimulate ergot alkaloid production in a synthetic medium indicates that the previously observed stimulation by tryptophan and tryptophan analogues does not merely represent a nutritional effect. Tryptophan, but not mevalonate or 5-methyltryptophan, is able to overcome the inhibition of alkaloid synthesis by high levels of inorganic phosphate. Therefore, high phosphate levels seem to limit the synthesis of tryptophan; they may, in addition, prevent induction of alkaloid synthesis by preventing accumulation of tryptophan. Experiments which indicate a 2- to 3-fold temporary increase of intracellular free tryptophan and a 20- to 25-fold increase of tryptophan synthetase activity during the transition period between growth and alkaloid production phase are in agreement with the previously postulated induction of alkaloid synthesis by tryptophan. The latter experiments also indicate 4- to 6-fold repression of this enzyme by tryptophan.     

Physiological status of submergedClaviceps during enzymic assembly of ergot alkaloids

Ergot alkaloids are formed only for arelatively brief time during the lifespan of the culture and under conditions of reduced proliferation. They cannot be taken for waste products of general metabolism. Ergot strains are capable of carrying out all the simple steps of the anthranilic acid—tryptophan cycles. Alkaloids influence activities of certain enzymes of primary metabolism in the ergot mycelium,e.g. tryptophan synthetase, acetyl-CoA carboxylase, citrate synthase, isocitrate lyase, and malate synthase. Ergot alkaloids do not belong to a group of physiologically inert secondary metabolites. A tentative scheme of the enzymic assembly of the ergoline nucleus is presented. The increased yield of ergoline alkaloids may be attributed to the following points: (1) Unbalnced growth of the culture. (2) Support of competition of fatty acids and alkaloid biosynthesis for acetyl-CoA. (3) Decreased activities of tricarboxylic acid and glyoxylate cycles. (4) Positive association between the rate of protein turnover and alkaloid formation. (5) Stimulation of both tryptophan synthesis and degradation via kynurenine—anthranilate. (6) Regulation of tryptophan-histidine cross-pathway. (7) Continuous control of the alkaloid level during fermentation.     

Secondary metabolite biosynthesis in cultured cells of Catharanthus roseus (L.) G. Don immobilized by adhesion to glass fibres

Summary Suspension-cultured cells of Catharanthus roseus (L.) G. Don were immobilized on glass fibre mats and cultivated in shake flasks. The highly-aggregated immobilized cells exhibited a slower growth rate and accumulated reduced levels of tryptamine and indole alkaloids, represented by catharanthine and ajmalicine, in comparison to cells in suspension. The increased total protein synthesis in immobilized cells suggests a diversion of the primary metabolic flux toward protein biosynthetic pathways and away from other growth processes. In vitro assays for the specific activity of tryptophan decarboxylase (TDC) and tryptophan synthase (TS) suggest that the decreased accumulation of tryptamine in immobilized cells was due to reduced tryptophan biosynthesis. The specific activity of TDC was similar in immobilized and suspension-cultured cells. However, the expression of TS activity in immobilized cells was reduced to less than 25% of the maximum level in suspension-cultured cells. The reduced availability of a free tryptophan pool in immobilized cells is consistent with the reduced TS activity. Reduced tryptamine accumulation, however, was not responsible for the decreased accumulation of indole alkaloids in immobilized cells. Indole alkaloid accumulation increased to a similar level in immobilized and suspension-cultured cells only after the addition of exogenous secolaganin to the culture medium. The addition of tryptophan resulted in increased accumulation of tryptamine, but had no effect on indole alkaloid levels. Reduced biosynthesis of secologanin, the monoterpenoid precursor to indole alkaloids, in immobilized cells is suggested. Immobilization does not appear to alter the activity of indole alkaloid biosynthetic enzymes in our system beyond, and including, strictosidine synthase. Offprint requests to: P. J. Facchini     

Effect of differentiation on the regulation of indole alkaloid production in Catharanthus roseus hairy roots

In vitro cultures of hairy root derived from Catharanthus roseus accumulate higher levels of indole alkaloids than cell suspension cultures. Hairy roots were interconverted to undifferentiated cells by manipulation of the culture medium. When the concentration of micronutrients in the culture medium was five times that of Phillips and Collins (1979) medium, cell suspensions formed from the hairy roots. The alkaloid content was five times lower in the cell suspensions than in the control, but upon regeneration of the roots the alkaloid content regained its original level. The formation of cell suspensions from hairy roots was also accompanied by a reduction in tryptophan decarboxylase and the strictosidine synthase activity to less than 5% and 30%, respectively. 3-Hydroxymethylglutaryl coenzyme A reductase activity was the same in the cell suspension and in the regenerated line. Received: 12 February 1998 / Revision received: 21 May 1998 / Accepted: 5 June 1998     

Shikimate pathway activity in shake and fermenter cultures of Cinchona succirubra

Cell suspension cultures of Cinchona succirubra were cultivated in shake cultures and for the first time in airlift fermenters. Under both conditions L-tryptophan exerts a stimulatory effect on alkaloid formation. In this context the regulatory pattern of some shikimate pathway enzymes was investigated in non-supplemented and tryptophan supplemented Cinchona cell cultures. A remarkable increase of tryptophan decarboxylase (TDC) activity was observed in Cinchona cells under the influence of tryptophan. Apparently, like in some other indole alkaloid producing cell cultures, a high TDC activity is a prerequisite for alkaloid formation. Growth pattern and some enzyme activities of C. succirubra fermenter cultures at controlled and non-regulated pH levels were followed. Optimum growth and alkaloid formation were recorded under non-regulated (normal) pH conditions.Abbreviations TDC tryptophan decarboxylase - try L-tyrosine - phe L-phenylalanine - DAHP 3-deoxy-D-arabino-heptulosonic acid-7-phosphate - trp L-tryptophan - E-4-P erythrose-4-phosphate - PEP phosphoenolpyruvate - MDH malate dehydrogenase - G-6-PDH glucose-6-phosphate dehydrogenase - 6-PG-DH 6-phosphogluconate dehydrogenase - Ch-mutase chorismate mutase - AS-synthase anthranilate synthase - n.d. not determined     

Lamellar body formation precedes pulmonary surfactant apoprotein expression during embryonic mouse lung development in vivo and in vitro

Abstract. The purpose of this investigation was to determine whether lamellar inclusion body (LB) formation and surfactant apoprotein (SP-35) production are directly coordinated by temporal and positional information during development. In the present study we report a comparison between embryonic B10.A mouse lung morphogenesis and cytodifferentiation in vivo with that observed during organ culture in serumless medium. Precursor LB were first detected at embryonic day 12 (E12d), and progressively larger numbers and forms were produced during subsequent differentiation of respiratory alveolar duct epithelium. SP-35 was first detected during the canalicular period (E16.5d). Lung cultures (E12 d) showed pseudoglandular and canalicular periods of morphogenesis, and both ciliated epithelial and type II cell differentiation. Nonciliated cells produced increasing numbers of lamellar inclusion bodies throughout the culture period. SP-35 was detected at 9 days in vitro (d.i.v.). These observations indicate (i) precursor LB formation precedes SP-35 expression and is not dependent on apoprotein synthesis; (ii) E12d lung development in vitro using serumless medium proceeds at a rate equivalent to 0.5 days in vivo through 11 d.i.v.; and (iii) morphogenesis and differentiation occur in the absence of exogenous hormones and growth factors. The cell-cell interactions that play a role in morphogenesis and cell differentiation appear to be intrinsic to the developmental program for embryonic lung development and are likely to be mediated by autocrine and/or paracrine factors.     

Role of endogenous tryptophan during submerged fermentation of ergot alkaloids

Shake flask cultures ofClaviceps paspali (Stev. et Hall) andClaviceps purpurea (Fr.) Tul. on simple synthetic medium have been studied. Both strains grown in the absence of added tryptophan accumulate extra endogenous tryptophan. A certain concentration of cell-pool tryptophan is needed to promote alkaloid synthesis. Alkaloid production commences while tryptophan synthetase activity is increasing. In the alkloid-producing phase cell-pool tryptophan shows a single minimum while the change in level of cell-protein tryptophan is negligible. Alkaloid formation is suggested to reflect a regulatory device to keep endogenous tryptophan balanced. By adding amitrole the alkaloid spectrum is changed. The tryptophan-histidine cross-pathway probably serves a useful function inthe biosynthesis of ergot alkaloids.     

Substrate-associated macromolecules promote cytodifferentiation of BC3H1 myogenic cells.

Differentiated mouse BC3H1 myogenic cells secrete substrate-associated macro-molecules (SAM) which restrict the proliferation of undifferentiated cells and promote both cell shape changes and expression of predominantly the vascular smooth muscle (VSM)-specific isoform of the contractile protein alpha-actin. While we previously reported that high cell density was required for stimulating maximal expression of VSM alpha-actin in BC3H1 cells (Strauch and Reeser: Journal of Biological Chemistry 264:8345-8355, 1989), the permissive effect of SAM on myoblast cytodifferentiation was not at all dependent on the formation of cell to cell contacts. This observation suggests that biogenesis of an extracellular matrix rather than the formation of physical contacts between cells may be the rate-limiting step for induction of VSM alpha-actin expression at high cell density. The biologically active moieties in SAM that promote cytodifferentiation also are expressed by mouse embryonic fibroblast cell lines and are distinctly different from a class of adheron-like macromolecules released by differentiated BC3H1 myocytes directly into the culture medium. While SAM was cell growth restrictive, reconstituted particulate material (RPM) prepared from myocyte-conditioned medium promoted the adhesion and proliferation of growth-arrested myoblasts. SAM and RPM are composed of different polypeptide subunits which collectively may establish microenvironmental conditions that are permissive for BC3H1 myogenic cell differentiation.     

Changes in the Synthesis of RNA and Protein during Tracheary Element Differentiation in Single Cells Isolated from the Mesophyll of Zinnia elegans

Cycloheximide (CH) prevented tracheary element (TE) differentiationand cell division in a culture of single cells isolated fromthe mesophyll of Zinnia elegans at the concentrations whichinhibited incorporation of [¹⁴C]-leucine into protein. Whenthe cells were pulse-treated with this inhibitor for 12 h atvarious times of culture, TE formation was inhibited most stronglyby the treatments made between 24 and 60 h of culture. Incorporationof [¹⁴C]-leucine into protein showed a high level during thisperiod. The inhibitory effect of actinomycin D (Act-D) on TEdifferentiation was also marked when it was administered from24 to 60 h of culture when incorporation of [¹⁴C]-uridine intonucleic acid was at a high level. These results indicate thatRNA and protein syntheses are prerequisites for cytodifferentiationto TE and that the syntheses between 24 and 60 h of cultureare closely associated with cytodifferentiation. Studies of qualitative changes in proteins using two-dimensionalelectrophoresis revealed that approximately 400 polypeptidesextracted from [³⁵S]-methionine-labeled cells could be reproduciblyresolved and that most of them were synthesized in both differentiatingand non-differentiating cells. During TE differentiation, however,the synthesis of two polypeptides was shut off and two otherpolypeptides were newly synthesized between 48 and 60 h of culture,preceding the morphological changes. The relationship betweenTE differentiation and the synthesis of RNA and protein is discussed. (Received November 20, 1982; Accepted February 18, 1983)     

Some characteristics of tryptophan uptake in Claviceps species

Tryptophan serves as a precursor for the biosynthesis of alkaloids in the ergot fungus, Claviceps purpurea (Fries) Tulasne, and also is believed to act as an inducer of the enzymes necessary for alkaloid production. The characteristics of the transport system responsible for the accumulation of tryptophan in ergot mycelium were investigated, with the goal of clarifying the complex relationships among tryptophan uptake, size of the free intracellular pool of tryptophan, and alkaloid production. The characteristics of tryptophan uptake were studied by pulse feeding radioactively labeled tryptophan to cultures of Claviceps species, strain SD-58, which represented a variety of ages and nutritional states. Tryptophan accumulation in strain SD-58 is mediated by an energy-requiring system which exhibits specificity for neutral aromatic and aliphatic l-amino acids, is pH and temperature dependent, and shows saturation at high substrate concentrations. Tryptophan transport is a function of the intracellular concentration of free tryptophan, the nitrogen deficiency of the mycelium, the rate of growth, and alkaloid production, which were measured in Claviceps strain SD-58 growth in several culture media, some of which promoted alkaloid production and some of which did not. The results indicate that the initial velocity of tryptophan transport is not directly related to alkaloid production.     

Effects of Sequential Exposure to Auxin and Cytokinin on Xylogenesis in Cultured Explants of Jerusalem Artichoke (Helianthus tuberosus L.)

During the course of a 4-d culture period, explants of Jerusalemartichoke tuber were exposed to auxin (0.2 mg 1^–1 2, 4-dichlorophenoxyaceticacid), and cytokinin (5.0 mg 1^–1 benzyl-amino purine),under a range of sequential regimes, to study the influenceof each hormone on tracheary element formation. The resultsindicate that auxin was necessary early in the culture periodand was primarily involved in cell proliferation. Cytokininstimulated xylogenesis when present late in the culture period,concomitant with the phase of cytodifferentiation, but not whenrestricted to the early period. The implications for a sustainedperiod of commitment to differentiation are discussed. Xylem differentiation, Jerusalem artichoke, auxin, cytokinin, tissue culture     

Effect of phosphate on ergot alkaloid synthesis inAspergillus fumigatus

High concentration of inorganic phosphate in the culture medium ofAspergillus fumigatus inhibited ergot alkaloid synthesis. Addition ofl-tryptophan but not mevalonate or 5-methyltryptophan to the above culture restored the alkaloid synthesis to the level found in normal cultures. The decrease in alkaloid synthesis in the fungus accompanies an increase in cell mass, cellular protein and sterol content. Aspartate aminotransferase and alanine aminotransferase activities were significantly increased in the high-phosphate culture. Part of the work was presented at the seminar on “Enzymatic Methods in Mycology” organised by the Czechoslovak Microbiological Society in Brno, Czechoslovakia, in June 1975.     

Induction of ajmalicine formation and related enzyme activities in Catharanthus roseus cells: effect of inoculum density

In Catharanthus roseus cell cultures the time courses of four enzyme activities, tryptophan decarboxylase (TDC), strictosidine synthase (SSS), geraniol-10-hydroxylase (G10H) and anthranilate synthase (AS), and alkaloid accumulation were compared under two different culture conditions (low-inoculum density and high-inoculum density on induction medium) and a control on growth medium. In growth medium a transient increase in TDC activity was first observed after which G10H reached its maximum activity; only tryptamine accumulated, no ajmalicine could be detected. Apparently, a concerted induction of enzyme activities is required for ajmalicine formation. Cells inoculated in induction medium showed such a concerted induction of AS, TDC and G10H activities. After 30 days the low-density culture had accumulated six times more ajmalicine (in moles/g) than the high-density culture. Thus, increase in biomass concentration (high-density cultures) did not enhance the total alkaloid production. The major differences observed in enzyme levels between high-and low-density cultures were in the AS and TDC activities, which were two to three times higher in the low-density culture, indicating that there is a positive correlation between ajmalicine formation and AS and TDC activities.Biotechnology Delft Leiden, Project Group Plant Cell Biotechnology Correspondence to: R. Verpoorte     

Physiological study of ergot: induction of alkaloid synthesis by tryptophan at the enzymatic level.

The enhancement of ergot alkaloid production by tryptophan and its analogues in both normal and high-phosphate cultures is more directly related to increased dimethylallyltryptophan (DMAT) synthetase activity rather than to a lack of regulation of the tryptophan biosynthetic enzymes. Thiotryptophan [beta-(1-benzo-thien-3-yl)-alanine] is rather ineffective in the end product regulation of tryptophan biosynthesis, whereas tryptophan and 5-methyltryptophan are potent effectors. The presence of increased levels of DMAT synthetase in ergot cultures supplemented with tryptophan or thiotryptophan, and to a lesser extent with 5-methyltryptophan, suggests that the induction effect involves de novo synthesis of the enzyme. Thiotryptophan and tryptophan but not 5-methyltryptophan can overcome the block of alkaloid synthesis by inorganic phosphate. The results with thiotryptophan indicate that the phosphate effect cannot be explained merely on the basis of a block of tryptophan synthesis.     

Vindoline formation in shoot cultures of Catharanthus roseus is synchronously activated with morphogenesis through the last biosynthetic step

Background and Aims

The Madagascar periwinkle (Catharanthus roseus) produces the monoterpenoid alkaloid vindoline, which requires a specialized cell organization present only in the aerial tissues. Vindoline content can be affected by photoperiod and this effect seems to be associated with the morphogenetic capacity of branches; this association formed the basis of the study reported here.

Methods

Vindoline-producing in vitro shoot cultures were exposed either to continuous light or a 16-h photoperiod regime. New plantlet formation and alkaloid biosynthesis were analysed throughout a culture cycle.

Key Results

In cultures under the photoperiod, the formation of new plantlets occurred in a more synchronized fashion as compared to those under continuous light. The accumulation of vindoline in cultures under the photoperiod occurred in co-ordination with plantlet formation, in constrast to cultures under continuous light, and coincided with a peak of activity of deacetylvindoline acetyl CoA acetyltransferase (DAT), the enzyme that catalyses the last step in vindoline biosynthesis. When new plantlet formation was blocked in cultures under the photoperiod by treatment with phytoregulators, vindoline synthesis was also reduced via an effect on DAT activity. No association between plantlet formation and other biosynthetic enzymes, such as tryptophan decarboxylase (TDC) and deacetoxyvindoline 4-hydroxylase (D4H), was found. Effects of light treatment on vindoline synthesis were not mediated by ORCA-3 proteins (which are involved in the induction of alkaloid synthesis in response to elicitation), suggesting that the presence of a different set of regulatory proteins.

Conclusions

The data suggest that vindoline biosynthesis is associated with morphogenesis in shoot cultures of C. roseus.Key words: Catharanthus roseus, deacetylvindoline acetyl CoA acetyltransferase, DAT, in vitro shoot cultures, morphogenesis, vindoline     

Distribution of small Rho GTPases in the developing rat submandibular gland

During the rat submandibular gland (SMG) development, organogenesis and cytodifferentiation depend on the actin cytoskeleton, which is regulated by small Rho GTPases. These proteins link cell surface receptors to pathways that regulate cell motility, polarity, gene expression, vesicular trafficking, proliferation and apoptosis. The aim of this study was to evaluate, by immunohistochemistry, the distribution pattern of RhoA, RhoB, RhoC, Rac1 and Cdc42 during cytodifferentiation of the rat SMG and in male adults. All GTPases were found in epithelial and mesenchymal tissues throughout gland development. Rac1 appeared to be important for parenchyma expansion at the beginning of cytodifferentiation, while RhoC, Cdc42 and the inactive phosphorylated form of Rac1 seemed associated with lumen formation and cell polarization in terminal tubules. RhoA and RhoB labeling was evident throughout development. All GTPases were differentially expressed in the adult gland, suggesting that they play specific roles during differentiation and function of the rat SMG.