Tryptophan decarboxylase from transformed roots of Catharanthus roseus

Tryptophan decarboxylase (TDC, EC 4.1.1.28) from Catharanthus roseus hairy roots was purified 80-fold. Antibodies against TDC were obtained and they recognized only one protein of 55 kDa in crude extracts from hairy root cultures. Elicitation of transformed root cultures with macerozyme yielded a marked increase in TDC activity, which was accompanied by a similar increase in the amount of immunoreactive TDC protein. These results suggest that the alkaloid accumulation, produced by elicitation, requires the synthesis of new TDC polypeptide in C. roseus root cultures and establishes important differences in the regulatory control of this enzyme in root cultures compared to developing seedlings, where the posttranslational regulation apparently plays a major role.     

Presence of guanine nucleotide-binding proteins in Catharanthus roseus transformed roots

Biochemical analysis revealed the presence of GTP-binding proteins (G-proteins) in Catharanthus roseus hairy root cultures. In a microsomal fraction, several proteins, with molecular masses of 17, 21, 38, 42, 65, and 79 kDa were substrates for ADP-ribosylation by cholera toxin. Antisera raised against a conserved amino-acid sequence (GTSNSGKSTIVKQMK) of mammalian G α subunits recognized three proteins of 42, 50, and 79 kDa. Incubation of nitrocellulose blots with [ α -³²P]-GTP also indicated the presence of several proteins (17, 21, 50, and 79 kDa) that could bind GTP. In this system, we previously identified a phosphatidylinositol 4,5-bisphosphate-phospholipase C (PLC, EC 3.1.4.11) activity. As the activation of PLC by G-proteins was described, we decided to see whether, in our system, G-protein activators, such as guanosine 5- o -(3-thiotriphosphate) (GTP Γ S) and sodium fluoride ions, were able to regulate PLC activity in C. roseus transformed roots. Our results show that these agents regulated PLC activity in an inhibitory fashion and that this effect is dose-dependent. GTP was ineffective in producing either stimulation or inhibition of PLC activity. Our results demonstrate that non-hydrolyzable guanine nucleotides and fluoride ions exert an inhibitory effect on membrane PLC activity. In summary, a set of proteins of 17, 21, 38, 42, 50, and 79 kDa present in C. roseus transformed roots possessed at least two of the three main characteristics of a GTP-binding protein, and one of these proteins may be involved in the regulation of PLC activity in C. roseus transformed roots.     

Phospholipase C activity from Catharanthus roseus transformed roots: aluminum effect

The effect of aluminum on the activity of PLC was examined in transformed roots from Catharanthus roseus (L) G. Don. When added in vitro to the reaction mixture, Al inhibited the enzymatic activity in a concentration and time-dependent fashion. This effect is very similar for both activities (soluble and membrane-associated). When roots were treated in vivo with Al 0.1 mM for short periods (0-4 h), PLC activity was also inhibited. Aluminum (1 mM) diminished root growth in approximately 50% when added on the first day of the culture cycle conditions in which PLC activity is also affected. Other enzymatic activities (NAD+-GDH, NADH-GDH, NADH-GOGAT and HMGR) were not affected when roots were treated with Al (0.1 mM) for short periods of time (1 h). Results obtained in this work suggest that the Al can affect PLC activity as a specific target. Enzymes: Phospholipase C (EC 3.1.4.10).     

Evidence of protein-tyrosine kinase activity in Catharanthus roseus roots transformed by Agrobacterium rhizogenes

Homogenate fractions (soluble and particulate) from transformed roots of Catharanthus roseus (L.) G. Don showed several phosphorylated proteins when incubated with γ-[³²P]ATP. The phosphorylation in the proteins of 55, 40, 25, 18 and 10 kDa in the particulate fraction and 63 kDa in the soluble fraction was resistant to alkali treatment. Several proteins in both fractions gave a positive signal with monoclonal antiphosphotyrosine antibodies. In-situ phosphorylation in both fractions showed several proteins that cross-reacted with the antiphosphotyrosine antibodies. Tyrosine kinase activity was detected using an exogenous substrate RR-SRC, a synthetic peptide derived from the amino acid sequence surrounding the phosphorylation site in pp60^src. This activity was inhibited by genistein, a tyrosine kinase inhibitor. These results indicate, for the first time, the presence of protein-tyrosine kinase (EC 2.7.1.112) activity in transformed plant tissues. Received: 29 March 1997 / Accepted: 21 May 1997     

Alkaloid production by transformed root cultures of Catharanthus roseus

Transformed roots of Catharanthus roseus were obtained following infection of detached leaves with Agrobacterium rhizogenes. Roots would not grow in full strength Gamborg's B5 medium but would grow satisfactorily if the medium was diluted to one half strength. Little alkaloid appeared in the growth medium but root tissue contained a high level and wide variety of alkaloids. Ajmalicine, serpentine, vindolinine and catharanthine were prominent components. Vinblastine could also be detected by a combination of HPLC and radioimmunoassay, though at a level of only 0.05g/g dry weight.Abbreviations B5 Gamborg's B5 nutrient salts - LC/MS combined liquid chromatography/mass spectrometry - FW fresh weight - Kb kilobase     

Immunocytolocalization of tryptophan decarboxylase in Catharanthus roseus hairy roots

We investigated the intracellular distribution of tryptophan decarboxylase (TDC) (EC 4.1.1.28) in Catharanthus roseus hairy roots using immunofluorescence and immunogold techniques. TDC was detected by immunofluorescence localization in the cytosol and in the apoplastic region of the meristematic cells of the roots, with a slight enrichment in the epidermal cells of the root cap and in the meristematic region. In the enlargement zone, TDC was localized only in the first three layers of the cortex. In the maturation zone, the enzyme was not present. Immunogold studies confirmed that the enzyme was localized in the cytosol of the meristematic region, and intense gold labeling was found in the apoplastic zone. A protein fraction isolated from the apoplastic zone and assayed for TDC activity showed high activity.     

MAP kinase-like activity in transformed Catharanthus roseus hairy roots varies with culture conditions such as temperature and hypo-osmotic shock.

Mitogen activated protein (MAP) kinase-like activity was determined in extracts obtained from transformed Catharanthus roseus hairy roots by the ability to phosphorylate myelin basic protein (MBP). Both in solution and in gel kinase assays showed variation in activity, depending on root developmental stage. In gel kinase assays, using the extract soluble fraction, revealed a 56 kDa polypeptide with phosphorylation activity on MBP. In addition, another 75 kDa polypeptide was observed in the particulate fraction. Immunodetection with monoclonal antibodies against ERK-1, a mammalian MAP kinase, and with anti-phosphotyrosine antibodies cross-reacted with the 56 kDa polypeptide, named SMK56, from the soluble fraction, suggesting that this polypeptide could be related with members of the MAP kinase family. Antibodies against the dually phosphorylated threonine-tyrosine motif, characteristic of active forms of MAP kinases, also cross-reacted with this 56 kDa polypeptide. Changes in the levels of SMK56 were detected within the first 30 min of root exposure to low temperatures or hypo-osmotic shock, suggesting that this protein may be involved in the perception of environmental changes.     

Expression of deacetylvindoline-4-O-acetyltransferase in Catharanthus roseus hairy roots

Madagascar periwinkle [Catharanthus roseus (L.) G Don] is a pantropical plant of horticultural value that produces the powerful anticancer drugs vinblastine and vincristine that are derived from the dimerization of the monoterpenoid indole alkaloids (MIAs), vindoline and catharanthine. The present study describes the genetic engineering and expression of the terminal step of vindoline biosynthesis, deacetylvindoline-4-O-acetyltransferase (DAT) in Catharanthus roseus hairy root cultures. Biochemical analyses showed that several hairy root lines expressed high levels of DAT enzyme activity compared to control hairy root cultures expressing β-gulucuronidase activity (GUS) activity. Metabolite analysis using high performance liquid chromotagraphy established that hairy root extracts had an altered alkaloid profile with respect to hörhammericine accumulation in DAT expressing lines in comparison to control lines. Further analyses of one hairy root culture expressing high DAT activity suggested that DAT expression and accumulation of hörhammericine (9) were related. It is concluded that expression of DAT in hairy roots altered their MIA profile and suggests that further expression of vindoline pathway genes could lead to significant changes in alkaloid profiles. Evidence is provided that hörhammericine (9) accumulates via a DAT interaction with the root specific minovincinine-19-O-acetyltransferase (MAT) that inhibits the MAT mediated conversion of hörhammericine (9) into 19-O-acetyl-hörhammericine (12).     

Application of transport engineering to promote catharanthine production in Catharanthus roseus hairy roots

Plant Cell, Tissue and Organ Culture (PCTOC) - The flowering transition is a qualitative change in the life cycle of higher plants and an important turning point in the transition from vegetative...     

The expression of Terpenoid Indole Alkaloid (TIAs) pathway genes in Catharanthus roseus in response to salicylic acid treatment

Molecular Biology Reports - Vinblastine and vincristine are two important anti-cancer drugs that are synthesized by the Terpenoid Indole Alkaloids (TIAs) pathway in periwinkle (Catharanthus...     

Interaction of spermine with a signal transduction pathway involving phospholipase C, during the growth of Catharanthus roseus transformed roots

In Cantharanthus roseus transformed roots, the application of methylglyoxal bis(guanylhydrazone) (MGBG), an inhibitor of S -adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50), inhibited the root growth in a dose-dependent manner with a DL₅₀ of about 300 µm. Spermidine and spermine (Spm) levels and SAMDC and phospholipase C (PLC; EC 3.1.4.3) activities were reduced in the presence of the inhibitor. The inhibition was reversed by the addition of Spm. Radioactivity from [¹⁴C]Spm was detected in an immunoprecipitated fraction with an antibody anti-PLC-δ. To our knowledge, this is the first direct evidence that demonstrates an interaction of Spm with the signal transduction cascade phosphoinositide-Ca²⁺.     

Characterization of an inducible promoter system in Catharanthus roseus hairy roots

Transgenic hairy root cultures of Catharanthus roseus were established with a glucocorticoid-inducible promoter controlling the expression of green fluorescent protein (GFP), and GFP expression was characterized. The inducible system shows a tightly controlled, reversible, and dosage-dependent response to the glucocorticoid dexamethasone in C. roseus hairy roots. Full induction was noted after 12-18 h in the mature regions of the root tips and after 6 h in the meristem tissue. Upon removal of the inducing agent, GFP expression declined to undetectable levels in the mature tissues after 24 h and in the meristem after 48 h. Although no dosage-dependent response was noted in the meristem region, such a response was apparent in the mature region of the tip and verified by quantitative GFP analysis. The inducible promoter system allowed quantitative control of GFP expression between 0.01 and 10 microM dexamethasone with saturation occurring at higher levels. Using GFP as a model system allowed demonstration of the ability to control temporal and quantitative gene expression with the glucocorticoid-inducible promoter in transgenic C. roseus hairy roots.     

Characterization of an ethanol-inducible promoter system in Catharanthus roseus hairy roots

Efforts to engineer Catharanthus roseus hairy roots to produce commercially significant amounts of valuable compounds, such as the terpenoid indole alkaloids vinblastine and vincristine, require the development of tools to study the effects of overexpressing key metabolic and regulatory genes. The use of inducible promoters allows researchers to control the timing and level of expression of genes of interest. In addition, use of inducible promoters allows researchers to use a single transgenic line as both the control and experimental line, minimizing the problems associated with clonal variation. We have previously characterized the use of a glucocorticoid-inducible promoter system to study the effects of gene overexpression within the terpenoid indole alkaloid pathway on metabolite production. Here the feasibility of using an ethanol-inducible promoter within C. roseus hairy roots is reported. This ethanol-inducible promoter is highly sensitive to ethanol concentration with a concentration of 0.005% ethanol causing a 6-fold increase in CAT reporter activity after 24 h of induction. The ethanol-inducible CAT activity increased 24-fold over a 72-h induction period with 0.5% ethanol.     

Long-term instability of alkaloid production by stably transformed cell lines of Catharanthus roseus

The productivity of several transgenic cell lines of Catharanthus roseus was monitored over a period of 30 months. The transgenic cultures were obtained by Agrobacterium-mediated transformation of leaf explants with constructs containing recombinant versions of the endogenous Str and Tdc genes, which, respectively, encode strictosidine synthase (STR) and tryptophan decarboxylase (TDC). The expression of these transgenes and the -glucuronidase marker gene were also measured periodically, at the enzymatic level, during this time. Cultures were maintained in selective medium containing either hygromycin or kanamycin and showed GUS activity in the presence of X-gluc, indicating that they carried functional transgenes. The activities of STR and TDC varied greatly over time, occasionally falling to levels not significantly different from those of non-transgenic cultures, and showed susceptibility to the composition of the culture medium. Despite maintaining their transgenic character, the cell lines gradually lost the ability to accumulate terpenoid indole alkaloids (TIAs). The diversity of alkaloids produced was also negatively affected by long-term subculture. We conclude that a strategy of indirect selection, such as the use of antibiotic-resistance genes, is insufficient to maintain the concerted expression of TIA-pathway elements necessary for high productivity.     

Determination of biomass composition of Catharanthus roseus hairy roots for metabolic flux analysis

Metabolic flux analysis is a powerful diagnostic tool in metabolic engineering, and determination of biomass composition is indispensable to accurate flux evaluation. We report the elemental and biomolecular composition of Catharanthus roseus hairy roots, a pharmaceutically significant plant system and an important metabolic engineering target. The molecular formula of the organic material in the hairy roots was C12.0H22.7N0.4O7.6 during mid-exponential growth. The abundances of lipids, lignin, cellulose, hemicellulose, starch, protein, proteinogenic amino acids, mineral ash, and moisture in the biomass were quantified. Analysis of water-soluble components of the biomass with 1-D 13C and 2-D [1H,1H] correlation (COSY) NMR spectroscopy revealed that the water-soluble components were composed almost entirely of -glucans. Agropine, a frequently reported hairy root biomass component, was not detected. Our measurements of the biomass components quantified 83.6 +/- 9.3% (w/w) of the biomass. Together with previously reported abundances of indole alkaloids, we accounted for at least 85.9 +/- 11.6% (w/w) of the carbon in the biomass, which enabled the precise determination of 12 biomass synthesis fluxes.     

The specific responses to mechanical wound in leaves and roots of Catharanthus roseus seedlings by metabolomics

The mechanical wound is one of the unavoidable threats to survival of plants. More researchers focus on the effect of mechanical wound to the over-ground tissues. And the effects of wound to roots were frequently ignored, although it is an important organ for plant growth. In our studies, the metabolomics study was performed to reveal the mechanical wound effects in Catharanthus roseus on roots and leaves by combining gas chromatography-mass spectrometer (GC-MS), liquid chromatograph-mass spectrometer (LC-MS) and statistical analyses. The metabolic response of TIAs and PCs in plants to wound was most active at 0.5 h after treatment via Q value analysis. At this time point, then significantly responsive primary metabolites and specific secondary compounds (TIAs and PCs) were screened by PLS-DA score plot. In this case, the treatments of CK, LT (wound to leaves) and RT (wound to roots) were clearly distinguished. The targeted compounds include 8 sugars, 4 TIAs and 12 PCs and they displayed specific responses to CK, LT and RT treatments. Under RT group, plants invest more resources on the local responses using TIAs and the color reactions to regulate wound close using PCs. Whereas, LT group might lay emphasis on systemic responses via TIAs induced by SA (salicylic acid) and gallic acid. Our studies provide some basic data for further investigations of the defensive mechanism on roots treated by mechanical wound.     

Effects of buffered media upon growth and alkaloid production of Catharanthus roseus hairy roots

The influence of buffered media upon the growth and alkaloid productivity of Catharanthus roseus hairy root culture was examined. As expected, the buffers minimized shifts in the pH of the media and had slightly negative effects upon growth. The growth of the hairy roots remained optimal in unbuffered media. The specific yield of lochnericine was significantly lower in response to the addition of buffers, while tabersonine was significantly higher. In contrast, the specific yields of ajmalicine, serpentine, and horhammericine remained unchanged.     

Alkaloid metabolism in wounded Catharanthus roseus seedlings.

The effect of mechanical wounding on alkaloid metabolism was analyzed in Catharanthus roseus seedlings. Wounding induced an increase in ajmalicine accumulation, whereas catharanthine remained unaffected. A positive dual effect on vindoline was noticed. Short and mid-term effects were detected between 12 and 24 h after mechanical damage was inflicted, and apparently resulted from the accelerated transformation of the tabersonine intermediaries to vindoline. Long-term effects involved a generalized increase in carbon flux towards alkaloid synthesis. Exposure to ethylene (1 ppm) produced similar results to those observed in wounded seedlings, suggesting that it might be mediating the wound-induced increase in alkaloid synthesis. No synergistic or additive effects were observed when wounded seedlings were exposed to ethylene or jasmonate.     

Role of the non-mevalonate pathway in indole alkaloid production by Catharanthus roseus hairy roots

The 1-deoxy-D-xylulose-5-phosphate (DXP) pathway (non-mevalonate pathway) leading to terpenoids via isopentenyl diphosphate (IPP) has been shown to occur in most bacteria and in all higher plants. Treatment with the antibiotic fosmidomycin, a specific inhibitor of DXP reductoisomerase, considerably inhibited the accumulation of the alkaloids ajmalicine, tabersonine, and lochnericine by Catharanthus roseus hairy root cultures in the exponential growth phase. However, fosmidomycin did not significantly affect alkaloid levels in stationary phase hairy root cultures. Feeding with 1-deoxy-D-xylulose, 10-hydroxygeraniol, or loganin resulted in significant increases in alkaloid production by exponential phase hairy root cultures. These results suggest that the DXP pathway is a major provider of carbon for the monoterpenoid pathway leading to the formation of indole alkaloids in C. roseus hairy roots in the exponential phase.