Elicitor-mediated induction of phenylalanine ammonia lyase and tryptophan decarboxylase: Accumulation of phenols and indole alkaloids in cell suspension cultures of Catharanthus roseus

Cell suspension cultures (cell line No 615) of Catharanthus roseus cv. Little Delicata responded to elicitor treatment by accumulating monoterpenoid indole alkaloids and phenolic compounds. The excretion of phenols into the culture medium resulted from the induction of the branch-point enzyme phenylalanine ammonia lyase. The accumulation of alkaloids, however, occurred several hours earlier than the elicitor-mediated induction of tryptophan decarboxylase through which shikimate pathway intermediates are channelled into tryptamine and related indole alkaloids. The results indicate that both pathways for phenol and indole alkaloid biosynthesis responded to elicitor treatment and that no obvious causal relationship between pathways could be deduced from this study.Abbreviations PAL phenylalanine ammonia lyase - TDC tryptophan decarboxylase Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

Overexpression of a tryptophan decarboxylase cDNA inCatharanthus roseus crown gall calluses results in increased tryptamine levels but not in increased terpenoid indole alkaloid production

The enzyme tryptophan decarboxylase (TDC) (EC 4.1.1.28) catalyses a key step in the biosynthesis of terpenoid indole alkaloids inC. roseus by converting tryptophan into tryptamine. Hardly anytdc mRNA could be detected in hormone-independent callus and cell suspension cultures transformed by the oncogenic T-DNA ofAgrobacterium tumefaciens. Supply of tryptamine may therefore represent a limiting factor in the biosynthesis of alkaloids by such cultures. To investigate this possibility, chimaeric gene constructs, in which atdc cDNA is linked in the sense or antisense orientation to the cauliflower mosaic virus 35S promoter and terminator, were introduced inC. roseus cells by infecting seedlings with an oncogenicA. tumefaciens strain. In the resulting crown gall tumour calluses harbouring thetdc sense construct, an increased TDC protein level, TDC activity and tryptamine content but no significant increase in terpenoid indole alkaloid production were observed compared to empty-vector-transformed tumour calluses. In tumour calluses containing thetdc antisense construct, decreased levels of TDC activity were measured. Factors which might be responsible for the lack in increased terpenoid indole alkaloid production in thetdc cDNA overexpressing crown gall calluses are discussed.     

Tryptophan decarboxylase from Catharanthus roseus cell suspension cultures: purification,molecular and kinetic data of the homogenous protein

The purification of tryptophan decarboxylase from Catharanthus roseus (TDC, E.C.:4.1.1.27), to apparent homogeneity, is described. The enzyme represents a soluble protein with a molecular weight of 115 000±3 000, consisting of 2 identical subunits of 54 000±1 000. The pI was estimated to be 5.9 and the K_m for L-tryptophan was found to be 7.5×10^-5 M. Phenylalanine, tyrosine and DOPA were not decarboxylated by tryptophan decarboxylase from Catharanthus cells. Similar to the aromatic amino acid decarboxylase from hog kidney the enzyme does not appear to be obligatorily dependent on exogenously supplied pyridoxal phosphate, as it seems to contain a certain amount of this cofactor. The average percentage of TDC in the cells was found to be 0.002% in the growth medium while the level increased up to 0.03% when indole alkaloid biosynthesis was induced. The role of the protein as a bottleneck enzyme of indole alkaloid biosynthesis is discussed.     

Influence of phosphate on the formation of the indole alkaloids and phenolic compounds in cell suspension cultures of Catharanthus roseus I. Comparison of enzyme activities and product accumulation

In cell suspension cultures of Catharanthus roseus a rapid accumulation of secondary compounds (tryptamine, indole alkaloids, phenolics) was observed after transfer of the cells into special ‘induction’-media devoid of phosphate and other essential growth factors [11, 14]. The increase of product levels was suppressed in the presence of phosphate which was almost completely taken up from the medium and accumulated by the cells within 48 h after inoculation. The activities of tryptophan decarboxylase (TDC), the first enzyme in indole alkaloid biosynthesis, and of phenyl-alanine ammonia-lyase (PAL), the key enzyme of phenylpropanoid biosynthesis, were influenced differently by phosphate. Whereas the accumulation of phenolics and PAL activity were similarly inhibited by low concentration of phosphate, the medium-induced enhanced activity of TDC was not affected although the product pools were considerably reduced. Some consequences for the regulation of secondary metabolism will be discussed.     

Indole alkaloid accumulation and tryptophan decarboxylase activity in Catharanthus roseus cells cultured in three different media

Catharanthus roseus cells were cultured in three types of media. These media were: a low sucrose subculture medium and two high sucrose media, each of which differed in their mineral and hormonal contents. The kinetics of tryptophan decarboxylase activity and the accumulations of tryptophan, tryptamine, ajmalicine and serpentine were different in each series but no correlation between maximum enzyme activity and alkaloid contents was observed. Ajmalicine and serpentine productions were unaffected by addition of Trp to the media, whereas addition of secologanin enhanced alkaloid production. The results seem to imply that the terpenoid pathway is the limiting factor in alkaloid production in C. roseus cells.     

Tryptophan decarboxylase activity in transformed roots fromCatharanthus roseus and its relationship to tryptamine,ajmalicine, and catharanthine accumulation during the culture cycle

Summary Tryptophan decarboxylase (TDC), the enzyme that catalyzes the decarboxylation of tryptophan to trytamine, was studied in aCatharanthus roseus transformed root culture. Its activity was evaluated through the culture cycle (36 days), along with the variations in the tryptamine pool as well as the accumulation of alkaloids. Ajmalicine and catharanthine contents in the tissues increased coordinately with an increase in TDC-specific activity after 18 days of growth. No dramatic shifts were observed for the total alkaloid and tryptamine profiles.     

Alkaloid formation by habituated and tumorous cell suspension cultures of Catharanthus roseus

Habituated and tumorous Catharanthus roseus cells grown in the absence of hormones accumulated indole alkaloids. Total alkaloids and alkaloid pattern were the same when cells were cultured in medium without hormones or in alkaloid production medium with and without indole acetic acid. Treatment of cells with Pythium homogenate as elicitor did not increase total alkaloids or change the pattern of alkaloids produced. When either habituated or tumorous cells were grown in 1B5 medium after Gamborg et al (1968) containing 2,4-dichlorophenoxyacetic acid (2,4-D), their capacity to accumulate alkaloids decreased with time. The levels of tryptophan decarboxylase (TDC) and strictosidine synthase (SS) specific activities were constant throughout growth except when cells were exposed to 2,4-D in 1B5 medium, where enzyme activities declined in step with the decrease in alkaloid accumulation. Neither habituated nor tumorous cell suspension cultures accumulated vindoline, nor could they be induced to produce this alkaloid by any of the given treatments.NRCC No. 27514     

A phosphorus-31 nuclear magnetic resonance study of elicitor-mediated metabolic changes in Catharanthus roseus suspension cultures

Summary The induction of metabolic changes in suspension cultured cells of Catharanthus roseus upon elicitation has been investigated. Addition of a yeast glucan preparation to the growth medium resulted in induction of phenylalanine ammonia lyase. Phosphate uptake and metabolism of elicited cells was followed by ³¹P nuclear magnetic resonance. The uptake rate of Pi from the medium by oxygenated cells of C. roseus was reduced immediately after elicitation. Despite this reduced Pi uptake elicited cells had significantly increased amounts of ATP (twofold increase within 6 h). Cytoplasmic levels of Pi, phosphomonoesters, and Uridine Diphasphate glucose (UDP-Glc) were unaffected by eliciation. Furthermore, the cytoplasmic and vacuolar pH remained constant after addition of elicitor.     

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     

The role of pH gradients across the plasmalemma of Catharanthus roseus and its involvement in the release of alkaloids

During growth, Catharanthus roseus cells exhibit an acidification of the culture medium that may be controlled by Ca²⁺. With a view to enhance the productivity of alkaloids by plant cells, the effect of extracellular pH modifications on the excretion processes has been investigated. Ca²⁺ dependent proton pumping leads to the release of various lipophilic amine-like compounds (benzylamine, methylamine, nicotine) initially accumulated by the cells, but also facilitates the excretion of endogenous ajmalicine. Once released in the medium, these compounds are however taken up again by the cells, probably as the charged form. For the alkaloid contained in C. roseus some evidence suggests that the diffusible form comes from the cytosolic compartment and not from the storage vacuoles. This appears to be a major production limitation to the use of pH gradients in order to favour alkaloid excretion.     

Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase by wounding and methyl jasmonate

HMGR (3-hydroxy-3-methylglutaryl-coenzyme A reductase; E.C.1.1.1.34) supplies mevalonate for the synthesis of many plant primary and secondary metabolites, including the terpenoid component of indole alkaloids. Suspension cultures of Camptotheca acuminata and Catharanthus roseus, two species valued for their anticancer indole alkaloids, were treated with the elicitation signal transducer methyl jasmonate (MeJA). RNA gel blot analysis from MeJA treated cultures showed a transient suppression of HMGR mRNA, followed by an induction in HMGR message. Leaf disks from transgenic tobacco plants containing a chimeric hmgl::GUS construct were also treated with MeJA and showed a dose dependent suppression of wound-inducible GUS activity. The suppression of the wound response by MeJA was limited to the first 4 h post-wounding, after which time MeJA application had no effect. The results are discussed in relation to the differential regulation of HMGR isogenes in higher plants.Abbreviations GUS -glucuronidase - hmg gene of hmgr - HMGR 3-hydroxy-3-methylglutaryl-coenzyme A reductase - JA jasmonic acid - MeJA methyl jasmonate - MUG methylumbelliferyl--d-glucuronide - TDC tryptophan decarboxylase - SDS sodium dodecyl sulfate - SS strictosidine synthase     

Suspension cultured transgenic cells of Nicotiana tabacum expressing tryptophan decarboxylase and strictosidine synthase cDNAs from Catharanthus roseus produce strictosidine upon secologanin feeding

A transgenic cell suspension culture of Nicotiana tabacum L. `Petit Havana' SR1 was established expressing tryptophan decarboxylase and strictosidine synthase cDNA clones from Catharanthus roseus (L.) G. Don under the direction of cauliflower mosaic virus 35S promoter and nopaline synthase terminator sequences. During a growth cycle, the transgenic tobacco cells showed relatively constant tryptophan decarboxylase activity and an about two- to sixfold higher strictosidine synthase activity, enzyme activities not detectable in untransformed tobacco cells. The transgenic culture accumulated tryptamine and produced strictosidine upon feeding of secologanin, demonstrating the in vivo functionality of the two transgene-encoded enzymes. The accumulation of strictosidine, which occurred predominantly in the medium, could be enhanced by feeding both secologanin and tryptamine. No strictosidine synthase activity was detected in the medium, indicating the involvement of secologanin uptake and strictosidine release by the cells. Received: 25 February 1996 / Revision received: 16 August 1996 / Accepted: 30 September 1996     

A virus-induced gene silencing approach to understanding alkaloid metabolism in Catharanthus roseus

The anticancer agents vinblastine and vincristine are bisindole alkaloids derived from coupling vindoline and catharanthine, monoterpenoid indole alkaloids produced exclusively by the Madagascar periwinkle (Catharanthus roseus). Industrial production of vinblastine and vincristine currently relies on isolation from C. roseus leaves, a process that affords these compounds in 0.0003–0.01% yields. Metabolic engineering efforts to either improve alkaloid content or provide alternative sources of the bisindole alkaloids ultimately rely on the isolation and characterization of the genes involved. Several vindoline biosynthetic genes have been isolated, and the cellular and subcellular organization of the corresponding enzymes has been well studied. However, due to the leaf-specific localization of vindoline biosynthesis, and the lack of production of this precursor in cell suspension and hairy root cultures of C. roseus, further elucidation of this pathway demands the development of reverse genetics approaches to assay gene function in planta. The bipartite pTRV vector system is a Tobacco Rattle Virus-based virus-induced gene silencing (VIGS) platform that has provided efficient and effective means to assay gene function in diverse plant systems. A VIGS method was developed herein to investigate gene function in C. roseus plants using the pTRV vector system. The utility of this approach in understanding gene function in C. roseus leaves is demonstrated by silencing known vindoline biosynthetic genes previously characterized in vitro.     

Impact of hypoxia on the growth and alkaloid accumulation in <Emphasis Type="Italic">Catharanthus roseus</Emphasis> cell suspension

The Madagascar periwinkle (Catharanthus roseus) produces numerous indole alkaloids, several of which have an important pharmaceutical uses such as ajmalicine, vinblastine and vincristine. The relationship between hypoxia and ajmalicine production in a cell suspension culture of C. roseus were investigated during the cycle of cell culture, in correlation with the effects on growth. The results show that the lack of oxygenation in C20D cells provokes a very strong inhibition in accumulation of the alkaloids and of other possible substances. Moreover, the addition of loganin, a metabolic intermediate of the biosynthetic pathway, in the culture medium of cells subjected to hypoxia restored the alkaloid production. Also, the results showed that the addition of benzyladenine (BA) to the culture medium increased the ajmalicine production and that the inhibitory effect of hypoxia was almost absent in these conditions. Therefore, it could be suggested that BA can without doubt decrease the effects of the hypoxia and increase the ajmalicine production in periwinkle cell suspensions.     

Improvement of indole alkaloid production in Catharanthus roseus cell cultures by osmotic shock

Catharanthine production in Catharanthus roseussuspension cell cultures was increased by about 4-fold to 28 mg l^–1, 23 mg l^–1and 24 mg l^–1by adding sodium alginate, mannitol or polyvinyl pyrrolidone, respectively. Sodium alginate and polyvinyl pyrrolidone also enhanced ajmalicine production to 28 mg l^–1and 31 mg l^–1, respectively. Up to 55–70% of the total alkaloids were released into the medium. These treatments could stimulate higher alkaloid production in C. roseuscell cultures than NaCl and KCl stresses. The possible mechanisms for these treatment effects are discussed.     

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     

Elicitor-mediated induction of isochorismate synthase and accumulation of 2,3-dihydroxy benzoic acid in Catharanthus roseus cell suspension and shoot cultures

After elicitation, cell suspension cultures of Catharanthus roseus accumulate phenolic compounds. The major phenolic compound produced was isolated and identified as 2,3-dihydroxybenzoic acid (DHBA). The accumulation of this compound is a rapid response to the addition of elicitor; within 6 h after the addition of elicitor, DHBA concentration reached 6.3 mg/l cell suspension. DHBA was not detected in non-elicited cells. The formation of DHBA in elicited cells was correlated with the induction of the enzyme isochorismate synthase (ICS). Shoot cultures of C. roseus also presented a strong induction of ICS after elicitation. Due to its biological activity, DHBA could play a role in the defence mechanism of C. roseus.     

Increased production of serotonin by suspension and root cultures ofPeganum harmala transformed with a tryptophan decarboxylase cDNA clone fromCatharanthus roseus

Cell suspension and root cultures ofPeganum harmala were established expressing a tryptophan decarboxylase cDNA clone fromCatharanthus roseus under the control of the cauliflower mosaic virus (CaMV) 35S promoter and terminator sequences. The tryptophan decarboxylase activity of some of the transgenic lines was greatly enhanced (25–40 pkat/mg protein) as compared to control cultures (1–5 pkat per mg protein) and remained high during the growth cycle. While the levels of tryptamine, the product of the reaction catalysed by tryptophan decarboxylase, were unchanged in the transgenic lines, their serotonin contents were enhanced up to 10-fold, reaching levels of 1.5 to 2% dry mass. Thus, tryptamine produced by the engineered reaction was apparently immediately used for enhanced serotonin biosynthesis. The yields of serotonin in transgenic lines overexpressing tryptophan decarboxylase activity were further enhanced to 3–5% dry mass by feedingl-tryptophan, while no or only minor effects were seen when control cultures were fed. These data demonstrate that the production of a plant secondary metabolite can be enhanced greatly via genetic manipulation of the level of activity of the rate-limiting enzyme. The amounts of -carboline alkaloids, the other tryptamine-derived metabolites ofP. harmala, in contrast, were not affected by the overproduction of tryptamine. The information needed for successfully predicting manipulations that enhance production of a secondary metabolite is discussed.     

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