UV-B-induced signaling events leading to enhanced-production of catharanthine in Catharanthus roseuscell suspension cultures
© Ramani and Chelliah; licensee BioMed Central Ltd. 2007
Received: 13 November 2006
Accepted: 07 November 2007
Published: 07 November 2007
Elicitations 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.
Here, 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.
Our 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.
C. roseus produces terpenoid indole alkaloids (TIAs) as a part of its secondary metabolism. TIAs provide protection against microbial infection, herbivores and abiotic environmental stresses such as UV irradiation [1, 2]. Some of the TIAs are of pharmaceutical importance such as the antitumor dimeric alkaloids, vincristine and vinblastine, and the anti-hypertensive monomeric alkaloids, ajmalicine and serpentine . The anti-tumor dimeric alkaloids, which accumulate in the leaves of C. roseus, are composed of catharanthine and vindoline monomers and are exclusively found in C. roseus plants. In plants, the dimeric alkaloids and the monomer catharanthine accumulate in low amounts whereas the monomer vindoline accumulates at a relatively higher level [4, 5]. C. roseus cell cultures have been investigated as alternative means of production of terpenoid indole alkaloids, but they failed to produce vindoline . Therefore, it has been considered desirable to produce the dimers by coupling catharanthine obtained from cell cultures with vindoline obtained from the cultivated plants. The production of catharanthine by C. roseus cell cultures has been one of the most extensively explored areas of plant cell culture and is still limited due to the low yield .
Elicitations 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 . Fungal elicitors have been widely tested for elicitation of catharanthine production in various C. roseus cells [5, 9]. However, molecular basis of elicitor-signaling cascades leading to increased production of secondary metabolites of plant cell is largely unknown. It is known that receptor proteins that bind elicitors generate signals that are transmitted to the sites of gene expression via different components, such as Ca2+/ion fluxes, medium alkalinization and cytoplasmic acidification, oxidative burst, jasmonate and nitric oxide etc. . Many CDPKs and MAPKs have been identified to play a role in defense responses and also secondary metabolite production .
The effect of UV-B irradiation on expression of TIA biosynthetic genes, Tdc and Str, and catharanthine production has been reported previously in C. roseus leaves[11–13]. The transcription factor GT-1 binds to the promoter region of Tdc in vitro. The functional importance of GT-1 in the induction of Tdc expression by UV light has been demonstrated by point mutations in the GT-1 binding site . However, the molecular basis of UV-B signaling cascades leading to the induction of expression of Tdc and Str genes and the production of TIAs is largely unknown. It has been observed that the polypeptide wound signal, systemin- specific cell surface receptors initiate a signal transduction cascade upon UV-B irradiation in L. peruvianum cell suspension cultures . In the present study, the signaling pathways mediating UV-B-induced catharanthine accumulation in C. roseus suspension cultures were investigated. UV-B induced alkalinization of the culture medium, generation of hydrogen peroxide, activation of CDPK and MBPK as well as accumulation of catharanthine and stimulation of transcription of Tdc and Str genes were studied. Inhibitors of binding of ligand-cell surface receptors, protein kinases and phosphatases, calcium fluxes and H2O2 were used to dissect the UV-B signaling cascade.
Alkalinization of C. roseuscell-suspension medium in response to UV-B irradiation and its inhibition by suramin
Suramin is known to bind with cell surface components such as the systemin receptor  and interfere with the signaling events and this system is affected by UV-B irradiation in L. peruvianum cells . Since UV-B irradiation of C. roseus cells caused alkalinization of the medium, we investigated whether suramin could inhibit the UV-B-induced medium alkalinization. The results show that the UV-B-induced alkalinization was inhibited by suramin (Figure 1b). Suramin inhibited alkalinization of the growth medium for all exposure times of UV-B irradiation. Heparin, which is similar to suramin in possessing polysulfonated groups, had no effect on alkalinization of the medium induced by UV-B irradiation.
UV-B-induced H2O2 production and involvement of protein kinases in UV-B-induced H2O2production
Activation of protein kinases in response to UV-B irradiation in C. roseussuspension cell cultures
Many protein kinases are known to respond to both biotic and abiotic stresses. Two kinases, MAPKs and CDPKs, have been implicated to play pivotal roles in response to diverse stimuli [17, 20]. Previous studies have demonstrated that C. roseus cells also respond to UV-B irradiation by expressing biosynthetic genes and production of TIAs . To establish a functional link between these processes, we first examined the possible activation of MAPK and CDPK in cells irradiated with UV-B. MBP is known to be a conventional MAPK substrate and MAPK homologs also have MBP kinase activity . To determine if a MAPK is associated with the UV-B signaling the activation of MBP kinase was investigated
To further characterize the MBPK activity induced by UV-B, immunoprecipitation and in-gel kinase assays were used. The protein extracts were incubated with anti-phosphotyrosine monoclonal antibody and immunoprecipitated with protein A-agarose. The immunoprecipitated proteins were separated on a SDS-polyacrylamide gel containing MBP as a substrate and MBPK activity was assayed in the gel in the presence of 32P- ATP. As shown in Figure 3c, a 49 kDa protein kinase was again detected in the immunoprecipitate from UV-B-irradiated cells. Co-incubation with phosphotyrosine prevented immunoprecipitation of the 49 kDa protein kinase with anti-phosphotyrosine antibody, but co-incubation with phosphothreonine did not. These results indicate that only phosphotyrosine and not phosphothreonine could act as a competitor during immunoprecipitation, showing that MBP phosphorylating kinase was specifically phosphorylated on a tyrosine residue. Till date MAPK are the only known plant kinases to be phosphorylated on tyrosine residues.
UV-B-induced MBPK and CDPK activities, Tdc and Strgene expression and catharanthine accumulation are inhibited by suramin
Role of Ca2+ in UV-B induced responses in C. roseuscells
Role of protein phosphorylation in UV-B induced responses in C. roseuscells
Several studies have demonstrated the involvement of signal components, such as receptors, Ca2+ influx, medium alkalinization, oxidative burst, and protein kinases and phosphatases in responses to elicitors for enhanced production of secondary metabolites via increased transcription of relevant genes . It has been shown earlier in C. roseus that the abiotic elicitor UV-B induces the formation of dimeric TIAs, and Tdc and Str mRNA accumulation . There is also evidence that nuclear factor GT-1 function in the regulation of Tdc gene expression by UV light in C. roseus . However, the UV-B signaling pathway that regulates activity of transcription factor GT-1 leading to Tdc gene expression is still obscure. In the present study, we present evidence for involvement of a putative receptor(s), calcium, reactive oxygen species, Ca2+-dependent protein kinase, and a putative MAPK in UV-B signaling and transcriptional activation of Tdc and Str genes and catharanthine biosynthesis in C. roseus cells.
Based on suramin interference with the binding of systemin to its cell surface receptor and UV-B responses in L. peruvianum cells  we used suramin to assess the involvement of a cell surface receptor in UV-B-induced expression of TIA biosynthetic genes. The results shown in Figure 1, 2 and 5 show that the UV-B-induced medium alkalinization, ROS production, CDPK and MBPK activities, Tdc and Str gene expression, and accumulation of catharanthine were all inhibited by suramin. Suramin per se is not known to affect medium alkalinization directly but acts via a receptor . This suggested that suramin acts upstream of the afore-mentioned UV-B-induced responses and the UV-B-induced TIA biosynthesis. The inhibitory effect of suramin on the UV-B responses supports role of a putative cell surface receptor in UV-B signal pathway for the enhancement of Tdc and Str mRNA and catharanthine accumulation in the C. roseus cells.
We used a Ca2+ chelator; EGTA, and Ca2+ channel blocker, verapamil to investigate the role of Ca2+ in UV-B induced responses. Both the treatments blocked the UV-B-induced stimulation of MBPK and CDPK activities and the UV-B-induced accumulation of Tdc and Str mRNAs, and catharanthine. Because EGTA and verapamil are unlikely to enter cells, and verapamil blocks the Ca2+ channels localized in the plasma membrane [28, 29], our data indicate that the influx of Ca2+ from extracellular medium is required for the transduction of the UV-B signal, and that UV-B may influence the activity of the Ca2+ channels. Our study does not rule out the possibility of mobilization of calcium from intracellular compartments such as endoplasmic reticulum, golgi body and vacuole. Ca2+ signaling involves parallel and/or sequential use of different sources of Ca2+ and different channels in different sub-cellular locations. It was demonstrated in tobacco cells that hypo-osmotic shock stimulates Ca2+ influxes in a sequential manner, deriving first from external and then internal Ca2+ stores and that these influxes are mediated by Ca2+ channels . Thus, the present study provides evidence that Ca2+ serves as a second messenger in UV-B signal transduction involving activation of genes involved in TIA biosynthesis.
Our results also show that UV-B activated the generation of ROS in C. roseus cells (Figure 2a). The generation of ROS via an oxidative burst was shown to be induced by variety of elicitors, such as yeast elicitor on tobacco [31, 32], chitin oligosaccharides in tomato , fungal oligosaccharides in red clover roots , and fungal elicitors in spruce  and parsley cell suspensions . Using NAC, Ca2+ channel blocker and broad range of kinase inhibitor staurosporine, we showed that protein phosphorylation and an increase in intracellular calcium levels are required for the UV-B induced activation of ROS production. The MAPK cascade inhibitors however had no effect on the production of ROS indicating the ROS production occurs upstream of MAPK cascade activation. The most likely source of UV-B-induced ROS production in C. roseus is a membrane-bound NADPH oxidase complex, which uses molecular oxygen to make superoxide . In Arabidopsis suspension cells, a homologue of the catalytic subunit of the mammalian NADPH oxidase complex was shown to be responsive for ROS accumulation in response to bacterial protein elicitor harpin . It has been shown that protein phosphorylation is needed for the production of ROS in potato tubers, spruce and tobacco cells . The inhibitory effects of the protein kinase inhibitor staurosporine and Ca2+ channel blockers on UV-B-induced ROS production in the C. roseus cells (Figure 2b) support the fact that a calcium-dependent protein kinase is involved in the UV-B induction of ROS production. There are a few reports that CDPK activates NADPH oxidase [40–43]. It remains to be determined whether UV-B-induced ROS are generated via induction of a NADPH oxidase activity by CDPK.
The phosphorylation and dephosphorylation of proteins have been thought to play a key role in the transduction of elicitor signals in plant cells. The data shown here indicated that irradiation of C. roseus cells with UV-B light strongly activates a 49 kDa putative MAPK and the activation of the 49 kDa putative MAPK in response to UV-B was associated with tyrosine phosphorylation on the kinase, a distinguishing feature of the large family of MAPK. We conclude that UV-B-activated 49 kDa putative MAPK is likely a member of the MAPK family. Our results (Figure 4) also suggest the involvement of Ca2+-dependent protein kinase (s) or Ca-CaM (calmodulin)-dependent protein kinase (s) in the UV-B response. MAP kinases, members of a group of serine/threonine protein kinases are important transducers of intracellular signals via protein phosphorylation that is initiated by various extracellular stimuli, and they are involved in proliferation, differentiation and responses to stress in animal and yeast cells . Another notable aspect of this study is that staurosporine that has been used as an effective inhibitor of various protein kinases, completely inhibited both MAPK-like and CDPK activities (Figure 7a and 7b). It is noteworthy that pretreatments of specific synthetic inhibitors of MAPKs prevented stimulation of the UV-B-induced MAPK-like enzyme activity; however, no effects are observed for the CDPK activity (Figure 7a and 7b) suggesting that the activation of CDPK was relatively early as compared to the activation of putative MAPK. These data place MAPK downstream intermediaries in the cellular responses mediating catharanthine biosynthesis in response to UV-B and position CDPK upstream of MAPK. UV-B-mediated Tdc/Str gene transcription appeared dependent on activation of putative MAPK as well as CDPK pathway. The activity of a MAPK in cells is controlled through phosphorylation activation by its upstream kinases, MAPKK and MAPKKK, and dephosphorylation inactivation by its negative regulator, MAPK phosphatase/s. In this study, we showed that the UV-B-induced MAPK-like activity could be inhibited by PD98059, an inhibitor of ERKK (MAPKK), which similar to animal cells has no role to play in UV-B signaling. The results obtained using phosphatase inhibitors and NAC should be interpreted with caution because these inhibitors are not specific. NAC, for example is both a free radicle scavenger and phosphatase thiol group protector . Phosphatase inhibitors, on the other hand, can affect the viability of cells at higher concentrations or can mediate an over all up-regulation in the kinase activities . The reason we can attribute to absence of up-regulation in any of the UV-B-induced downstream activities in phosphatase inhibitors treated cells could probably due to the aberrational or toxic effect of these compounds on the entire cell homeostasis. In fact, treatment of cells with the inhibitors orthovandate or NAF alone activated many different kinases as assayed by MBP and H IIIS in gel phosphorylation assays (data not shown). The Tdc and Str activity and catharanthine accumulation in orthovanadate or NAF alone treated cells were again comparable to the UV-B alone treated cells (data not shown) demonstrating either imbalancing effects on cell homeostasis or that down-regulation of phosphatases alone are not the only event involved in the up regulation of the TIA pathway and other mechanisms do exist in regulation of TIA biosynthesis.
It has been earlier reported that yeast elicitor (YE) in C. roseus activates the octadecanoid pathway; leading to an increase in jasmonic acid (JA) levels via the activation of calcium influx and protein phosphorylation cascades . JA induces the expression of the ORCA3 gene via post-translational modification which further interacts with the Tdc promoter and the YE and JA-responsive RV fragment of the Str promoter enhancing the gene expression [46–48]. YE reportedly also induce the expression of the zinc finger proteins, which by binding to specific elements within the promoter regions of Tdc and Str can repress its gene expression . Similarly YE-induced CrBPF1 expression has been reported to be putatively involved in the regulation of STR via interaction with the BA region . It would be interesting to understand whether the UV-B and YE-induced TIA pathway share common elements in signal transduction and also if UV-B utilizes any of the transcriptional initiators or repressors induced by YE in initiating the TIA pathway.
2', 7'- DCFH-DA, EGTA, heparin, histone IIIS, N-acetyl cysteine, phosphothreonine, phosphotyrosine, sodium fluoride, sodium orthovanadate and verapamil were purchased from Sigma Chemical Company, St. Louis, USA. Sodium β-glycerophosphate and sodium fluoride were from Hi-media Laboratories, India. Catharanthine and vindoline were obtained from Shanghai kangai biologicals, China. Staurosporine and suramin were obtained from MP Biomedicals, Germany. Monoclonal antibodies to phospho-serine and phospho-tyrosine, complete protease inhibitor cocktail and myelin basic protein were purchased from Upstate laboratories, U.S.A. SB 203580 (P38 inhibitor), PD 98059 (ERKK inhibitor) and SB 600125 (JNK inhibitor) were a kind gift from Prof. Anjali Karande, I.I.Sc, Bangalore.
Cell culture and treatments of cells with UV-B and chemicals
Compounds used as agonists and antagonists to elucidate UV-B signal transduction pathway in Catharanthus roseus cultured cells
Working concentration (stock solution)
0.2 and 2 mM (0.2 M in water)
Calcium chelator 
10 and 100 mM (10 M in water)
Scavenger of reactive oxygen species and protects thiol group of phosphatases from inactivation 
1 and 10 mM (1 M in water)
Inhibitor of serine-threonine phosphatases 
1 and 10 mM (1 M in water)
Inhibitor of tyrosine phosphatases 
10 and 100 nM (10 μM in ethanol)
Broad range inhibitor of serine-threonine kinases
0.1 and 1 mM (0.1 M in water)
Inhibits binding of growth factors to their receptors 
0.5 and 5 μM (0.5 mM in ethanol)
L-type calcium channel blocker [28, 29]
PD 98059 [2-(2-amino-3-methoxyphenyl) -oxanapthalen-4-one]
5 uM (0.5 mM in DMSO)
ERKK inhibitor 
SB 203580 [4-(4-flurop henyl)-2-(4-pyridyl) 1H imidazole]
70 nM (7 μM in DMSO)
P38 MAPK inhibitor 
SB 600125 ( anthrax-[1,9-cd]-6(2H)-one]
40 nM (4 μM in DMSO)
JNK inhibitor 
Medium alkalinization response (AR) assay
To determine the UV-B-induced medium alkalinization, pH of the culture medium was measured from 0 to 120 min after 5 min of irradiation. UV-B-induced medium alkalinization response (AR) was calculated as the difference in pH between the untreated controls and the respective UV-B irradiated samples as described .
Measurement of H2O2production
H2O2 production was measured using cell permeable fluorescent probe 2', 7'-dichlorodihydroflurescein diacetate (DCFH-DA) by monitoring the increase in fluorescence by oxidation of DCFH to DCF (dichlorofluorescein) as described by Pauw et al. . The 2.5 μM DCFH-DA was added to the cell suspension cultures immediately after UV-B irradiation. After UV-B irradiation for different time periods, the increase in intracellular H2O2 levels was measured by monitoring the increase in fluorescence after 15 min with 488-nm excitation and 525-nm emission wavelengths in a luminescence spectrometer (Perkin Elmer LS50B). To identify the events that inhibit the UV-B induced H2O2 production, various inhibitors were added for 10 min prior to 5 min-UV-B radiation.
Preparation of the cell extract
Treated cell suspensions were collected by centrifugation, frozen separately in liquid nitrogen, and stored at -80°C until further use. Samples were thawed to 4°C and ultrasonicated (30 % amplitude, 15 pulses) in a buffer containing 50 mM HEPES-KOH pH 7.6, 2 mM DTT, 1 mM EDTA, 1 mM EGTA, 20 mM β-glycerophosphate, 20 % glycerol, 1 mM Na3VO4, 1 mM NaF and one tablet of complete protease inhibitors (Upstate) per 50 ml of buffer solution (EDTA and EGTA were excluded for calcium dependant kinase assays). Homogenates were centrifuged at 12,000 rpm at 4°C for 25 min. The supernatant was used immediately as a source of total soluble proteins to determine the activities of CDPK and MAPK. The total protein in the supernatant was estimated by the method of Bradford  using BSA as a standard.
Protein kinase assays
Total soluble proteins extracted from C. roseus cells were assayed for CDPK and MBPK substrate phosphorylation activities according to the method of Putnam-Evans et al.  with slight modifications. Equal amounts of protein were taken and reactions were carried out in a total reaction volume of 30 μl kinase assay buffer (25 mM Tris pH 7.5, 5 mM MgCl2, 1 mM EGTA, 1 mM DTT and 2 μCi γ32P ATP for MAPK assay or in a buffer containing 25 mM Tris pH 7.5, 200 μM CaCl2, 10 mM MgCl2 and 2 μCi γ32P ATP for CDPK assay) for 30 min at room temperature. Substrate phosphorylation assays were done by adding 50 μg of myelin basic protein (MBP) or histone IIIS (HIIIS), respectively, to the same reaction buffer as mentioned above. The reaction was terminated by addition of electrophoresis sample loading buffer. After electrophoresis on 12 % SDS-polyacrylamide gels, the phosphorylated MBP and HIIIS were visualized by autoradiography.
CDPK and MBPK activities were determined by in-gel kinase assays using histone IIIS and myelin basic protein as substrates, respectively as described previously .
For immune complex kinase activity assays, MBPK and CDPK were immunoprecipitated using monoclonal anti-phosphotyrosine antibody and monoclonal anti-phosphoserine antibody, respectively as described by Stratmann and Ryan . For immunoprecipitation, soluble proteins (200 μg) that had been made up to a total volume of 100 μl with immunoprecipitation buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 1 mM NaF, 10 mM β-glycer0phosphate, 1 % [w/v] Triton X- 100, 2 mM DTT and one tablet of complete protease inhibitors per 50 ml of buffer solution) were incubated in a 1.5 ml eppendorf tube with 5 μg of monoclonal anti-phosphotyrosine or anti-phosphoserine antibody for 2 h at 4°C. For CDPK assay the same immunoprecipitation buffer was used without EDTA and EGTA. For reactions with competitor phosphoaminoacids, antibodies were preincubated for 30 min at room temperature with 1 mM of the phosphoaminoacid. Approximately 25 μl packed volume of recombinant protein A, immobilized on agarose, was added, and incubation continued for another 2 h at 4°C. The immunoprecipitated MBPK and CDPK were pelleted by centrifugation at 12,000 g for 10 min and washed two times with immunoprecipitation buffer. The samples were boiled for 2 min and separated by electrophoresis on 10 % SDS gels with MBP or H IIIS, respectively and in-gel kinase assays were done as described above.
RNA isolation and RT-PCR analysis
Total RNA from cells of C. roseus was isolated using the Qiazol reagent (Qiagen Inc. Germany) following the manufacturer's instructions. The RNA samples were quantified by spectrophotometry at 260 and 280 nM (A260/A280 ~2.0; A260 = 40 μg RNA/ml) and visual inspection in agarose gels. DNA was removed from total RNA samples by treatment with RNase-free DNase I. Reverse transcription was carried out in a 20 μl reaction containing 1 μg of total RNA, 5 μg oligo d(T)16–18 primer, MuMLV reverse transcriptase (40 U), RNasin (20 U), 0.5 mM dNTPs and MuMLV reverse transcriptase reaction buffer (250 mM Tris-HCl, pH 8.3, 250 mM KCl, 20 mM MgCl2 and 50 mM DTT) at 37°C for 1 h, and terminated by heating at 70°C for 10 min. After the RT reaction, the cDNA was subjected to PCR reactions. The following pairs for primers were used: 5'-TGTAGCCATGTCCAATTCTCCAGT-3', as the forward primer and 5'-ATAAACTCGTCCCGTCGAGTTAAG-3', as the reverse primer for tryptophan decraboxylase (Tdc M25151), 5'-TAAATCCATGATGGCAGTTTTCTT-3', as the forward primer and 5'-ACCCACAGAGCTATGGAAGAGAC-3', as the reverse primer for strictosidine synthase (Str X61932). One μl of the RT reaction was used for PCR in 20 μl containing 0.4 U of Taq DNA polymerase (Fermentas), 0.1 mM dNTP (Fermentas), 200 μM of each dNTP and 100 pM of each primer in a 1× reaction buffer. Reactions were amplified for a total of 15 cycles on the Minicycler (MJ Research PTC-150) using 94°C for denaturation (1 min), 55°C for annealing for Tdc and Str and 52°C for annealing for Rps9 (1 min) and 72°C for extension (1 min), following a further 5 min extension. The RT-PCR products were separated by electrophoresis on 1 % agarose gels, stained with ethidium bromide, and photographed under UV light using Alpha Imager 2200 (Alpha Innotech Corporation, San Leandro, CA). RT-PCR analysis of ribosomal protein 9 (Rps9) was used as control to check RNA integrity and accuracy of loading. The primers were: Rps9-forwad 5'-TTAGTCTTGTTCGAGTTCATTTTGTAT-3', and Rps9-reverse 5'-GAGCAAATTAACTCAATTGATAATTAAC-3', (Rps9, AJ749993). The RT-PCR products of the expected sizes 1.5, 1.2 and 0.63 kb respectively was obtained for Tdc, Str and Rps9 and their identity confirmed by sequencing.
Quantification of catharanthine by HPLC analysis
The extraction of terpenoid indole alkaloids and quantification of catharanthine using HPLC were according to Schripseme and Verpoorte . The amount of catharanthine was finally reported as mg g -1 DW (dry weight) cells.
Calcium dependent protein kinase
2', 7'- dichlorofluoresceine diacetate
Ethylene glycol bis(2-aminoethylether)- N,N,N'N'-tetraacetic acid
Extracellular regulated kinase kinase
High pressure liquid chromatography
- H IIIS:
Mitogen activated protein kinase
Mitogen activated protein kinase kinase
Mitogen activated protein kinase kinase kinase
Myelin basic protein
Murashige and Skoog medium
- PD 98059:
Reactive oxygen species
Reverse transcription and polymerase chain reaction
- SB 203580:
4-(4-fluorophenyl)-2-(4-methyl sulphinylphenyl-5-(4-pyridyl) 1H imidazole
- SB 600125:
- STR/Str Strictosidine synthase; Sur:
Tryptophan decarboxylase: TIA: Terpenoid indole alkaloid pathway
Ultraviolet B radiation
difference in pH between control and treated
The work was supported by grants from Indian Council of Medical Research, and Department of Biotechnology (Genomics Initiative at Indian Institute of Science, Bangalore, Government of India). We are grateful to Prof. Anjali Karande for providing the MAPK inhibitors, Prof. Ramesh Maheshwari, Prof. Ramasarma and Prof. Sunil Podder for critical reading of the manuscript. We also thank Dr. Nanda Devi for the help rendered in revising the manuscript. S.R is a recipient of a Research Fellowship from CSIR.
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