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    Mass production of Ajmalicine by bioreactor cultivation of hairy roots of Catharanthus roseus
    (Elsevier B.V., 2017) Thakore, Dhara; Srivastava, Ashok Kumar; Sinha, Alok Krishna
    Catharanthus roseus has been known to produce Ajmalicine which is used for treatment of circulatory disorders. Significantly low content of the drug in the natural plant necessitates development of alternative production protocols. Hairy root propagation was considered as a viable alternative. Mass cultivation of the hairy root culture was attempted in several bioreactor configurations; Bubble column, Rotating drum bioreactor, Modified Bubble column with polypropylene (PP) mesh support and Modified Bubble column with Polyurethane foam (PUF) support. Important factors like medium composition, inoculum density, illumination period and aeration rates were optimized before mass propagation of hairy roots. From the data gleaned, the cultivation in rotating drum bioreactor resulted in ajmalicine content of only 4.6 ± 0.4 mg/l. However, the ajmalicine concentration surpassed even that obtained in shake flask (with 34 ± 2.3 mg/l) in a customized bioreactor wherein the hairy roots were anchored onto a polyurethane foam, the highest reported in this scale of cultivation.
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    Transcriptome analysis of Catharanthus roseus for gene discovery and expression profiling
    (PLOS, 2014) Verma, Mohit; Ghangal, Rajesh; Sharma, Raghvendra; Sinha, Alok Krishna; Jain, Mukesh
    The medicinal plant, Catharanthus roseus, accumulates wide range of terpenoid indole alkaloids, which are well documented therapeutic agents. In this study, deep transcriptome sequencing of C. roseus was carried out to identify the pathways and enzymes (genes) involved in biosynthesis of these compounds. About 343 million reads were generated from different tissues (leaf, flower and root) of C. roseus using Illumina platform. Optimization of de novo assembly involving a two-step process resulted in a total of 59,220 unique transcripts with an average length of 1284 bp. Comprehensive functional annotation and gene ontology (GO) analysis revealed the representation of many genes involved in different biological processes and molecular functions. In total, 65% of C. roseus transcripts showed homology with sequences available in various public repositories, while remaining 35% unigenes may be considered as C. roseus specific. In silico analysis revealed presence of 11,620 genic simple sequence repeats (excluding mono-nucleotide repeats) and 1820 transcription factor encoding genes in C. roseus transcriptome. Expression analysis showed roots and leaves to be actively participating in bisindole alkaloid production with clear indication that enzymes involved in pathway of vindoline and vinblastine biosynthesis are restricted to aerial tissues. Such large-scale transcriptome study provides a rich source for understanding plant-specialized metabolism, and is expected to promote research towards production of plant-derived pharmaceuticals.
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    Catharanthus roseus mitogen-activated protein kinase 3 confers UV and heat tolerance to Saccharomyces cerevisiae
    (Taylor & Francis, 2013) Raina, Susheel Kumar; Wankhede, Dhammaprakash Pandhari; Sinha, Alok Krishna
    Catharanthus roseus is an important source of pharmaceutically important Monoterpenoid Indole Alkaloids (MIAs). Accumulation of many of the MIAs is induced in response to abiotic stresses such as wound, ultra violet (UV) irradiations, etc. Recently, we have demonstrated a possible role of CrMPK3, a C. roseus mitogen-activated protein kinase in stress-induced accumulation of a few MIAs. Here, we extend our findings using Saccharomyces cerevisiae to investigate the role of CrMPK3 in giving tolerance to abiotic stresses. Yeast cells transformed with CrMPK3 was found to show enhanced tolerance to UV and heat stress. Comparison of CrMPK3 and SLT2, a MAPK from yeast shows high-sequence identity particularly at conserved domains. Additionally, heat stress is also shown to activate a 43 kDa MAP kinase, possibly CrMPK3 in C. roseus leaves. These findings indicate the role of CrMPK3 in stress-induced MIA accumulation as well as in stress tolerance.
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    New evidences about strictosidine synthase (Str) regulation by salinity, cold stress and nitric oxide in Catharanthus roseus
    (Springer, 2013) Dutta, Ajaswrata; Sen, Jayanti; Deswal, Renu
    Alkaloid production in plants is altered by abiotic stressors, but the mechanism(s) are poorly understood. Present study provides novel evidences about differential regulation of strictosidine synthase (Str), the key gene of terpenoid indole alkaloid (TIA) biosynthetic pathway in response to salinity and low temperature stress in Catharanthus roseus. HPLC analysis of terpene indole alkaloids correlated with differential regulation of Str by low temperature and salinity stress. Administration of exogenous calcium and calcium channel modulator preferentially regulated Str transcript. In addition, administration of kinase and phosphatase inhibitors modulated Str expression. Involvement of nitric oxide (NO) signaling was ascertained by NO donor and nitric oxide synthase (NOS) scavenger treatments. This finding suggests co-ordinated action of calcium, protein kinases, phosphatases and nitric oxide in abiotic stress signaling for TIA biosynthetic pathway in C. roseus.
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    CrMPK3, a mitogen activated protein kinase from Catharanthus roseus and its possible role in stress induced biosynthesis of monoterpenoid indole alkaloids
    (BMC, 2012) Raina, Susheel Kumar; Wankhede, Dhammaprakash Pandhari; Jaggi, Monika; Singh, Pallavi; Jalmi, Siddhi Kashinath; Raghuram, Badmi; Sheikh, Arsheed Hussain; Sinha, Alok Krishna
    Mitogen activated protein kinase (MAPK) cascade is an important signaling cascade that operates in stress signal transduction in plants. The biologically active monoterpenoid indole alkaloids (MIA) produced in Catharanthus roseus are known to be induced under several abiotic stress conditions such as wounding, UV-B etc. However involvement of any signaling component in the accumulation of MIAs remains poorly investigated so far. Here we report isolation of a novel abiotic stress inducible Catharanthus roseus MAPK, CrMPK3 that may have role in accumulation of MIAs in response to abiotic stress.
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    Ectopic overexpression of vacuolar and apoplastic Catharanthus roseus peroxidases confers differential tolerance to salt and dehydration stress in transgenic tobacco
    (Springer Science, 2012) Kumar, Santosh; Jaggi, Monika; Sinha, Alok Krishna
    CrPrx and CrPrx1 are class III peroxidases previously cloned and characterized from Catharanthus roseus. CrPrx is known to be apoplastic in nature, while CrPrx1 is targeted to vacuoles. In order to study their role in planta, these two peroxidases were expressed in Nicotiana tabacum. The transformed plants exhibited increased peroxidase activity. Increased oxidative stress tolerance was also observed in transgenics when treated with H(2)O(2) under strong light conditions. However, differential tolerance to salt and dehydration stress was observed during germination of T1 transgenic seeds. Under these stresses, the seed germination of CrPrx-transformed plants and wild-type plants was clearly suppressed, whereas CrPrx1 transgenic lines showed improved germination. CrPrx-transformed lines exhibited better cold tolerance than CrPrx1-transformed lines. These results indicate that vacuolar peroxidase plays an important role in salt and dehydration stress over cell wall-targeted peroxidase, while cell wall-targeted peroxidase renders cold stress tolerance.
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    Detection and mapping of quantitative trait loci for the contents of the terpenoid indole alkaloids, vindoline and catharanthine, in the leaves of Catharanthus roseus using bulk segregant analysis
    (The Journal of Horticultural Science and Biotechnology, 2012) Chaudhary, S; Pandey, R; Tripathi, B.N.; Goel, R; Kumar, S
    A mapping population consisting of 191 F2:7 recombinant inbred lines (RILs) of Catharanthus roseus was used for segregation analysis of DNA markers, leading to the construction of a genetic linkage map of 174 DNA markers placed on eight linkage groups (LG). These markers were present at an average inter-marker distance of 10.2 cM. Leaves harvested from cultivated C. roseus plants are the main source of vindoline (V) and catharanthine (C), which are chemically condensed during commercial synthesis of the widely-used anti-cancer drugs, vincristine and vinblastine. To detect and map the major genes/quantitative trait loci (QTL) that control the accumulation of V and C in C. roseus leaves, a bulk segregant analysis approach was used. The above mapping population of RILs was phenotyped for their leaf alkaloid profiles and the distributions of V and C contents were used to assemble two pairs of contrasting bulks of RILs, one each for V and C accumulation in extremely low or extremely high amounts. Primers for the 174 already- mapped markers were PCR amplified on the bulks. Among the markers that discriminated between the high-C and low-C bulks, and the high-V and low-V bulks, those that amplified all members of their specific bulk were identified. This led to the detection of two QTLs (V1 and V2) and their co-segregating/linked DNA markers for high-V content, and one QTL (C1) and its linked DNA markers for high-C content. These three QTLs were then mapped. V1 and V2 were observed to lie on LG1, and C1 was on LG6. The QTLs V1 and V2 had large additive effects. These, and C1, appear to be suitable for marker assisted selection.
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    Development of SSR and gene-targeted markers for construction of a framework linkage map of Catharanthus roseus
    (Oxford University Press, 2011) Shokeen, Bhumika; Choudhary, Shalu; Sethy, Niroj Kumar; Bhatia, Sabhyata
    BACKGROUND AND AIMS: Catharanthus roseus is a plant of great medicinal importance, yet inadequate knowledge of its genome structure and the unavailability of genomic resources have been major impediments in the development of improved varieties. The aims of this study were to develop co-dominant sequence-tagged microsatellite sites (STMS) and gene-targeted markers (GTMs) and utilize them for the construction of a framework intraspecific linkage map of C. roseus.
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    Cloning and characterization of two new Class III peroxidase genes from Catharanthus roseus
    (Elsevier, 2011) Kumar, Santosh; Jaggi, Monika; Taneja, Jyoti; Sinha, Alok Krishna
    We report here the cloning and characterization of two new class III peroxidase genes, CrPrx3 and CrPrx4 from Catharanthus roseus. The full length cDNA of CrPrx3 is 1233 bp long encoding 330 amino acid residues. The CrPrx4 cDNA contains an ORF of 1055 bp, and encodes for 318 amino acids. Hypothetical 3-D structure model of CrPrx3 and CrPrx4 detected the presence of two Ca(2+) ion binding sites at proximal and distal end, and a heme group coordinate at central position. CrPrx3 and CrPrx4 are present in single copy number in C. roseus. Confocal scanning microscopy and phylogenetic analysis showed that CrPrx3 and CrPrx4 are apoplastic in nature. Phylogenetic analysis revealed that both genes have diverse expression patterns in a variety of plant tissues. Quantitative real-time PCR confirmed their maximum expression in stem tissues followed by flower tissue. The expression levels of CrPrx3 and CrPrx4 were down-regulated under salt and dehydration stress, respectively.
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    Overexpression of an apoplastic peroxidase gene CrPrx in transgenic hairy root lines of Catharanthus roseus
    (Springer Science, 2011) Jaggi, Monika; Kumar, Santosh; Sinha, Alok Krishna
    Peroxidases are a family of isoenzymes found in all higher plants and are known to be involved in a broad range of physiological processes. However, very little information is available concerning their role in Catharanthus roseus. The present study describes the impact of both overexpression and suppression of a peroxidase gene, CrPrx in C. roseus transgenic hairy root lines. Real-time PCR analysis in 35S-CrPrx and CrPrx-RNAi transgenic lines indicated differential transcript profile for peroxidases as well as for genes and regulators involved in MIA (monoterpenoid indole alkaloid) pathway of C. roseus. Comparative analysis revealed that MIA pathway genes showing elevated levels of expression in 35S-CrPrx transgenic lines showed a significant reduction in their transcript level in CrPrx-RNAi transgenic lines. Metabolite analysis detected higher levels of ajmalicine and serpentine accumulation in overexpressed lines. It was observed that all overexpressed transgenic lines produced more amount of H(2)O(2). These results indicate a role of CrPrx gene in the regulation of MIA pathway genes and regulators, thus affecting the production of specific alkaloids.