Roseus Cells Research Articles

UV-B-induced signaling events leading to enhanced-production of catharanthine in Catharanthus roseus cell suspension cultures.

BackgroundElicitations are considered to be an important strategy towards improved in vitro production of secondary metabolites. In cell cultures, biotic and abiotic elicitors have effectively stimulated the production of plant secondary metabolites. However, molecular basis of elicitor-signaling cascades leading to increased production of secondary metabolites of plant cell is largely unknown. Exposure of Catharanthus roseus cell suspension culture to low dose of UV-B irradiation was found to increase the amount of catharanthine and transcription of genes encoding tryptophan decarboxylase (Tdc) and strictosidine synthase (Str). In the present study, the signaling pathway mediating UV-B-induced catharanthine accumulation in C. roseus suspension cultures were investigated.ResultsHere, we investigate whether cell surface receptors, medium alkalinization, Ca2+ influx, H2O2, CDPK and MAPK play required roles in UV-B signaling leading to enhanced production of catharanthine in C. roseus cell suspension cultures. C. roseus cells were pretreated with various agonists and inhibitors of known signaling components and their effects on the accumulation of Tdc and Str transcripts as well as amount of catharanthine production were investigated by various molecular biology techniques. It has been found that the catharanthine accumulation and transcription of Tdc and Str were inhibited by 3–4 fold upon pretreatment of various inhibitors like suramin, N-acetyl cysteine, inhibitors of calcium fluxes, staurosporine etc.ConclusionOur results demonstrate that cell surface receptor(s), Ca2+ influx, medium alkalinization, CDPK, H2O2 and MAPK play significant roles in UV-B signaling leading to stimulation of Tdc and Str genes and the accumulation of catharanthine in C. roseus cell suspension cultures. Based on these findings, a model for signal transduction cascade has been proposed.

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.

Preparation of mixed alginate elicitors with high activity for the efficient production of 5′-phosphodiesterase by Catharanthus roseus cells

When various autoclaved microbial cells suspensions (exogenous elicitors) were added to Catharanthus roseus cell cultures, its growth was inhibited but 5'-phosphodiesterase (PDase) production was stimulated. The greatest effect was with autoclaved Alteromonas macleodii: the dry cell concentration decreased from 13 to 10.9 mg/ml while PDase production increased from 0.022 to 0.235 U/ml. A combination of A. macleodii (as exogenous elicitor) and 0.1%(w/v) alginate oligomers (AO: acting as both endogenous elicitor and scavenger of active oxygen species) minimized the cell growth inhibition but enhanced PDase production (0.474 U/ml) about 20 times higher than the control (no addition). The method for the preparation of mixed alginate elicitors with high activities containing exogenous elicitor (autoclaved A. macleodii), endogenous elicitor (AO), and trans-4,5-dihydroxy-2-cyclopenten-1-one was developed. The mixed alginate elicitors significantly promoted PDase production (2.67 U/ml) by C. roseus, and the productivity was increased 120-fold compared to the control without cell growth inhibition.

Biotransformation of (–)-Caryophyllene Oxide by Cell Suspension Culture of Catharanthus roseus

Abstract Catharanthus roseus cell suspension cultures were employed for the biotransformation of (-)-caryophyllene oxide (1), and four metabolites, 15-hydroxycaryophyllene oxide (2), 4β ,5α- dihydroxycaryophyll-8(13)-ene (3), 2β -hydroxycaryophyllene oxide (4), and 2-hydroxy-4,5- epoxycaryophyllan-13-ol (5) were obtained. Metabolites 4 and 5 were found to be new compounds, and their structures were deduced by different spectroscopic techniques.

Precursor limitations in methyl jasmonate-induced Catharanthus roseus cell cultures

Jasmonates enhance the expression of various genes involved in terpenoid indole alkaloid (TIA) biosynthesis in Catharanthus roseus. We applied precursor feeding to our C. roseus suspensions to determine how methyl jasmonate (MJ) alters the precursor availability for TIA biosynthesis. C. roseus suspensions were induced with MJ (100 microM) on day 6 and fed loganin (0.30 mM), tryptamine (0.15 mM), loganin plus tryptamine, or geraniol (0.1-1.0 mM) on day 7. While MJ increased ajmalicine production by 3-fold, induced cultures were still limited by terpenoid precursors. However, both induced and non-induced cultures became tryptamine-limited with excess loganin. Geraniol feeding also increased ajmalicine production in non-induced cultures. But MJ appeared to increase geraniol availability in induced cultures, due presumably to the increased expression of Dxs with MJ addition.

Epidermis is a pivotal site of at least four secondary metabolic pathways in Catharanthus roseus aerial organs

Catharanthus roseus produces a wide range of secondary metabolites, some of which present high therapeutic values such as antitumoral monoterpenoid indole alkaloids (MIAs), vinblastine and vincristine, and the hypotensive MIA, ajmalicine. We have recently shown that a complex multicellular organisation of the MIA biosynthetic pathway occurred in C. roseus aerial organs. In particular, the final steps of both the secoiridoid-monoterpene and indole pathways specifically occurred in the epidermis of leaves and petals. Chorismate is the common precursor of indole and phenylpropanoid pathways. In an attempt to better map the spatio-temporal organisation of diverse secondary metabolisms in Catharanthus roseus aerial organs, we studied the expression pattern of genes encoding enzymes of the phenylpropanoid pathway (phenylalanine ammonia-lyase [PAL, E.C. 4.3.1.5], cinnamate 4-hydroxylase [C4H, E.C. 1.14.13.11] and chalcone synthase [CHS, E.C. 2.3.1.74]). In situ hybridisation experiments revealed that CrPAL and CrC4H were specifically localised to lignifying xylem, whereas CrPAL, CrC4H and CrCHS were specifically expressed in the flavonoid-rich upper epidermis. Interestingly, these three genes were co-expressed in the epidermis (at least the upper, adaxial one) together with three MIA-related genes, indicating that single epidermis cells were capable of concomitantly producing a wide range of diverse secondary metabolites (e.g. flavonoïds, indoles, secoiridoid-monoterpenes and MIAs). These results, and data showing co-accumulation of flavonoids and alkaloids in single cells of C. roseus cell lines, indicated the spatio-temporal feasibility of putative common regulation mechanisms for the expression of these genes involved in at least four distinct secondary metabolisms.

Effect of long-term phosphate starvation on the levels and metabolism of purine nucleotides in suspension-cultured Catharanthus roseus cells

The effect of long-term phosphate (Pi) starvation of up to 3 weeks on the levels of purine nucleotides and related compounds was examined using suspension-cultured Catharanthus roseus cells. Levels of adenine and guanine nucleotides, especially ATP and GTP, were markedly reduced during Pi-starvation. There was an increase in the activity of RNase, DNase, 5′- and 3′-nucleotidases and acid phosphatase, which may participate in the hydrolysis of nucleic acids and nucleotides. Accumulation of adenosine, adenine, guanosine and guanine was observed during the long-term Pi starvation. Long-term Pi starvation markedly depressed the flux of transport of exogenously supplied [8- 14C]adenosine and [8- 14C]adenine, but these labelled compounds which were taken up by the cells were readily converted to adenine nucleotides even in Pi-starved cells, in which RNA synthesis from these precursors was significantly reduced. The activities of adenosine kinase, adenine phosphoribosyltransferase and adenosine nucleosidase were maintained at a high level in long-term Pi starved cells.

Ajmalicine production in methyl jasmonate-induced Catharanthus roseus cell cultures depends on Ca2+ level

Cytosolic Ca(2+) and jasmonate mediate signals that induce defense responses in plants. In this study, the interaction between Ca(2+) and methyl jasmonate (MJ) in modulating defense responses was investigated by monitoring ajmalicine production in Catharanthus roseus suspension cultures. C. roseus suspensions were treated with nine combinations of CaCl(2) (3, 23, and 43 mM) and MJ (0, 10, and 100 microM) on day 6 of growth. Increased Ca(2+) influx through the addition of extracellular CaCl(2) suppressed ajmalicine production in MJ-induced cultures. The highest ajmalicine production (4.75 mg/l) was observed when cells were treated with a low level of calcium (3 mM) combined with a high level of MJ (100 microM). In the presence of 3 mM CaCl(2) in the medium, the addition of Ca(2+) chelator EGTA (1, 2.5, and 5 mM) or Ca(2+) channel blocker verapamil (1, 10, and 50 muM) to MJ-induced (100 microM) cultures on day 6 also inhibited ajmalicine production at higher levels of the Ca(2+) inhibitors. Hence, ajmalicine production in MJ-induced C. roseus cultures depended on the intracellular Ca(2+) concentration and a low extracellular Ca(2+) concentration (3 mM) enhanced MJ-induced ajmalicine production.

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.

CaaX-prenyltransferases are essential for expression of genes involvedin the early stages of monoterpenoid biosynthetic pathwayin Catharanthus roseus cells

CaaX-prenyltransferases (CaaX-PTases) catalyse the covalent attachment of isoprenyl groups to conserved cysteine residues located at the C-terminal CaaX motif of a protein substrate. This post-translational modification is required for the function and/or subcellular localization of some transcription factors and components of signal transduction and membrane trafficking machinery. CaaX-PTases, including protein farnesyltransferase (PFT) and type-I protein geranylgeranyltransferase (PGGT-I), are heterodimeric enzymes composed of a common alpha subunit and a specific beta subunit. We have established RNA interference cell lines targeting the beta subunits of PFT and PGGT-I, respectively, in the Catharanthus roseus C20D cell line, which synthesizes monoterpenoid indole alkaloids in response to auxin depletion from the culture medium. In both types of RNAi cell lines, expression of a subset of genes involved in the early stage of monoterpenoid biosynthetic pathway (ESMB genes), including the MEP pathway, is strongly decreased. The role of CaaX-PTases in ESMB gene regulation was confirmed by using the general prenyltransferase inhibitor s-perillyl alcohol (SP) and the specific PFT inhibitor Manumycin A on the wild type line. Furthermore, supplementation of SP inhibited cells with monoterpenoid intermediates downstream of the steps encoded by the ESMB genes restores monoterpenoid indole alkaloids biosynthesis. We conclude that protein targets for both PFT and PGGT-I are required for the expression of ESMB genes and monoterpenoid biosynthesis in C. roseus, this represents a non previously described role for protein prenyltransferase in plants.

De novo and salvage biosynthetic pathways of pyridine nucleotides and nicotinic acid conjugates in cultured plant cells

To estimate the operation of the de novo and salvage pathways of pyridine nucleotide synthesis, [ 3H]quinolinic acid, an intermediate of the de novo synthesis, and [ 14C]nicotinamide and [ 14C]nicotinic acid, substrates of the salvage pathways, were administered to cultured white spruce ( Picea glauca) and Madagascar periwinkle ( Catharanthus roseus) cells and overall metabolism of these radioactive compounds was examined over the culture period. In P. glauca cells, all three precursors were able to generate pyridine nucleotides (mainly NAD and NADP) and trigonelline. Supplied [ 3H]quinolinic acid was efficiently converted to pyridine nucleotides and trigonelline in both at the logarithmic and the stationary stages of cell growth, although uptake of [ 3H]quinolinic acid by P. glauca cells was very low. [ 14C]Nicotinic acid and [ 14C]nicotinamide were taken up by the cells in a relatively facile manner, and 15–20 and 32–58% of total radioactivity from these compounds was found in pyridine nucleotides (mainly NAD and NADP) and trigonelline, respectively, after 18 h incubation. In C. roseus cells, these three precursors were utilised for pyridine nucleotides, but in contrast to P. glauca, nicotinic acid glucoside, but not trigonelline, was heavily labeled. Nicotinic acid and nicotinamide were better precursors for pyridine nucleotide synthesis than quinolinic acid. In Pi-starved cells, the uptake of quinolinic acid, nicotinic acid and nicotinamide was markedly decreased. Pyridine nucleotide synthesis de novo was greatly reduced in Pi-starved C. roseus cells, while little effect was found in the salvage pathway of nicotinic acid. Pi-deficiency slightly increased the rate of nicotinic acid-glucoside synthesis from nicotinic acid and nicotinamide. From the in vitro determination of enzyme activity, it is concluded that quinolinic acid and nicotinic acid are converted to nucleotides by quinolinate phosphoribosyltransferase (2.4.2.19) and nicotinate phosphoribosyltransferase (2.4.1.11), respectively. High activity of nicotinamidase (3.5.1.19) but no detectable activity of nicotinamide phosphoribosyltransferase (2.4.2.12) suggests that nicotinamide is converted to nicotinic acid, and then salvaged by nicotinate phosphoribosyltransferase.

Nitric oxide stimulates indole alkaloid production in Catharanthus roseus cell suspension cultures through a protein kinase-dependent signal pathway

Nitric oxide (NO) mediates a signal pathway that is involved in many defense responses of plants to biotic and abiotic stresses such as the hypersensitive cell death and the activation of defense-related genes. To determine the role of NO in secondary metabolite production of plant cells, sodium nitroprusside (SNP) is utilized as the donor of NO to investigate its effect on the production of terpenoid indole alkaloids of Catharanthus roseus cell suspension cultures. The results showed that the production of ajmalicine, catharanthine, and total alkaloids of C. roseus cells treated with 10.0 mmol/L SNP were 20.1, 24.2 and 51.3 mg/L, being 1.6-, 2.9- and 1.8-fold higher than those of untreated cells respectively. SNP-induced terpenoid indole alkaloid production of the cells was blocked by NO specific scavenger 2-4-carboxypheny-l-4,4,5,5-tetramethylimidazoline-1-oxyl-3- oxide (CPITO), which indicated that the effects of the application of SNP were caused by NO released from it rather than SNP itself. SNP treatment also induced the activation of protein kinases in C. roseus cell cultures. Pretreatment of the cells with protein kinase inhibitor K-252a suppressed SNP-induced terpenoid indole alkaloid production. The results strongly suggested that NO released from SNP triggered terpenoid indole alkaloid biosynthesis of C. roseus cells through a protein kinase-dependent signal pathway.

Proteome analysis of the medicinal plant Catharanthus roseus

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.

Effect of nitric oxide on catharanthine production and growth ofCatharanthus roseus suspension cells

Sodium nitroprusside (SNP) was used as the donor of nitric oxide (NO) to investigate its effect on catharanthine synthesis and the growth of Catharanthus roseus suspension cells. The results showed that SNP at high concentrations (10.0 and 20.0 mmol/L) stimulated catharanthine formation of C. roseus cells, but inhibited growth of the cells. Low concentrations of SNP (0.1 and 0.5 mmol/L) enhanced the growth of C. roseus cells, but had no effect on catharanthine synthesis. The maximum total catharanthine production was achieved by the addition of 0.5 and 10.0 mmol/L SNP to the cultures at day 0 and day 10, respectively, being about threefold of the control. NO-induced catharanthine production of C. roseus cells was strongly suppressed by jasmonic acid (JA) biosynthesis inhibitor ibuprofen (IBU) and nordihydroguaiaretic (NDGA). The result suggests that the stimulatory role of NO on catharanthine production is partially JA-dependent.

Zinc Finger Proteins Act as Transcriptional Repressors of Alkaloid Biosynthesis Genes in Catharanthus roseus

In Catharanthus roseus cell suspensions, the expression of several terpenoid indole alkaloid biosynthetic genes, including two genes encoding strictosidine synthase (STR) and tryptophan decarboxylase (TDC), is coordinately induced by fungal elicitors such as yeast extract. To identify molecular mechanisms regulating the expression of these genes, a yeast one-hybrid screening was performed with an elicitor-responsive part of the TDC promoter. This screening identified three members of the Cys(2)/His(2)-type (transcription factor IIIA-type) zinc finger protein family from C. roseus, ZCT1, ZCT2, and ZCT3. These proteins bind in a sequence-specific manner to the TDC and STR promoters in vitro and repress the activity of these promoters in trans-activation assays. In addition, the ZCT proteins can repress the activating activity of APETALA2/ethylene response-factor domain transcription factors, the ORCAs, on the STR promoter. The expression of the ZCT genes is rapidly induced by yeast extract and methyljasmonate. These results suggest that the ZCT proteins act as repressors in the regulation of elicitor-induced secondary metabolism in C. roseus.

O 2− from elicitor-induced oxidative burst is necessary for triggering phenylalanine ammonia-lyase activation and catharanthine synthesis in Catharanthus roseus cell cultures

Elicitor, derived from the cell walls of Aspergillus niger, induced rapid generation of reactive oxygen intermediates (ROI), including superoxide anion (O 2 −) and hydrogen peroxide (H 2O 2), sequentially followed by phenylalanine ammonia-lyase (PAL) activation and catharanthine biosynthesis in Catharanthus roseus suspension cells. The elicitor-induced PAL activation and catharanthine biosynthesis were blocked by NAD(P)H oxidase inhibitor, diphenylene iodonium (DPI). O 2 − generated by the reaction of xanthine/xanthine oxidase (X/XO) triggered PAL activation and catharanthine biosynthesis of C. roseus cells in the absence of elicitor and reversed the inhibitory effect of DPI on elicitor-induced PAL activation and catharanthine biosynthesis. External application of H 2O 2 and catalase had no effect on PAL activity and catharanthine contents of C. roseus cells. The results demonstrated a causal relationship between elicitor-induced oxidative burst and PAL activation in C. roseus suspension cells and suggested a sequence of signaling events from ROI production to PAL activation and catharanthine synthesis. Within this sequence, O 2 − rather than H 2O 2 appeared to trigger the subsequent reactions.

Elicitor-induced nitric oxide burst is essential for triggering catharanthine synthesis in Catharanthus roseus suspension cells.

Elicitor prepared from the cell walls of Penicillium citrinum induced multiple responses in Catharanthus roseus suspension cells, including rapid generation of nitric oxide (NO), sequentially followed by enhancement of catharanthine production by C. roseus cells. Elicitor-induced catharanthine biosynthesis was blocked by NO-specific scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and nitric oxide synthase (NOS) inhibitor S,S'-1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea (PBITU). PBITU also strongly inhibited elicitor-induced NO generation by C. roseus suspension cells. The inhibiting effect of PBITU on elicitor-induced catharanthine production was reversed by external application of NO via the NO-donor sodium nitroprusside. The results strongly suggested that NO, generated by NOS or NOS-like enzymes in C. roseus suspension cells when treated with the fungal elicitor, was essential for triggering catharanthine synthesis.

Effects of salt on lipid peroxidation and antioxidant enzyme activities of Catharanthus roseus suspension cells

Cells of Catharanthus roseus (L.) G. Don were maintained and sub-cultured in the absence of NaCl (control) or were sub-cultured for 7 days in the presence of 50 mM (“50”) or 100 mM (“100”) NaCl, or were maintained and sub-cultured for 8 months in the continuous presence of 50 mM NaCl (“50T” or salt-adapted cells). Exposure of cells to salt treatment reduced growth in a concentration-dependent manner. Salt-treated cells strongly accumulated Na + and, to a lesser extent, Cl −. However, no significant change in Cl − accumulation was observed in 50T cells. Salt progressively increased superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities whereas a strong increase in catalase (CAT) activity was only obtained in 50 and 50T cells. SOD activity was lower in 50T than in 50 cells, whereas the opposite was observed for APX activity. After native polyacrylamide gel electrophoresis (PAGE) analysis, five SOD isoenzymes were detected and the increase in SOD activity observed in salt-treated cells seemed to be mainly due to isoenzymes (SOD 1, 2 and 3). Three APX isoenzymes (APX 1, 2 and 3) were detected markedly, especially in salt-adapted cells. Salt strongly induced CAT 2 isoenzyme in 50 cells and CAT 1 isoenzyme in 50T cells. These data suggest that salt treatment provoked an oxidative stress in C. roseus cells, as shown by the increase in lipid peroxidation, in spite of the induction of antioxidant enzymes. This increase in lipid peroxidation was paralleled by a rise in lipoxygenase (LOX) activity. Increases in antioxidant activities could also be a response to the cellular damage provoked by NaCl. Probably, this increase could not stop the deleterious effects of salt, but reduced stress severity thus allowing cell growth to occur.

Cloning of a Gene Encoding a Putative Ethylene Receptor in Catharanthus roseus and its Expression in Plant and Cell Cultures

A full length cDNA (designated CrETR1) was isolated by polymerase chain reaction amplification of a cDNA library from periwinkle (Catharanthus roseus) cell cultures. CrETR1 cDNA encodes a polypeptide of 740 amino acids with a predicted molecular mass of 82 kDa. The deduced protein contains a hydrophobic ethylene-binding transmembrane region, a GAF domain, a third domain homologous to the histidine protein kinase domain of the prokaryotic two-component systems, and a fourth carboxyl-terminal domain homologous to the receiver domain of the response regulators, as found in the A. thaliana ethylene receptor ETR1. CrETR1 transcripts are strongly accumulated in petals and ovaries of C. roseus young plants whereas no significant changes are detected in cell cultures submitted to various stress or hormonal (including ethylene) treatments. The amount of the monoterpene indole alkaloid ajmalicine in the cells treated by ethylene is reduced after addition of inhibitors of histidine kinases showing a possible involvement of CrETR1 protein in the ethylene-related signalling pathway leading to alkaloid biosynthesis enhancement in C. roseus cell cultures.

Activation of the oxidative burst by yeast elicitor in Catharanthus roseus cells occurs independently of the activation of genes involved in alkaloid biosynthesis

In Catharanthus roseus cell suspensions, expression of several terpenoid indole alkaloid (TIA) biosynthetic genes, including those encoding strictosidine synthase and tryptophan decarboxylase, is coordinately induced by fungal elicitors such as yeast extract (YE). This induction is mediated by several signaling steps including the biosynthesis of jasmonic acid, and the activation of the jasmonic acid-responsive ORCA transcription factors. We investigated a possible role of reactive oxygen species (ROS) as a second messenger in this system. YE was shown to activate the production of ROS, which was dependent on protein phosphorylation and calcium influx. However, ROS generation was neither necessary for the induction of genes involved in TIA biosynthesis by YE nor by itself sufficient to induce these genes. Therefore, we conclude that activation of the oxidative burst by YE occurs independently of the activation of genes involved in TIA biosynthesis.