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1.  Development of efficient catharanthus roseus regeneration and transformation system using agrobacterium tumefaciens and hypocotyls as explants 
BMC Biotechnology  2012;12:34.
As a valuable medicinal plant, Madagascar periwinkle (Catharanthus roseus) produces many terpenoid indole alkaloids (TIAs), such as vindoline, ajamlicine, serpentine, catharanthine, vinblastine and vincristine et al. Some of them are important components of drugs treating cancer and hypertension. However, the yields of these TIAs are low in wild-type plants, and the total chemical synthesis is impractical in large scale due to high-cost and their complicated structures. The recent development of metabolic engineering strategy offers a promising solution. In order to improve the production of TIAs in C. roseus, the establishment of an efficient genetic transformation method is required.
To develop a genetic transformation method for C. roseus, Agrobacterium tumefaciens strain EHA105 was employed which harbors a binary vector pCAMBIA2301 containing a report β-glucuronidase (GUS) gene and a selectable marker neomycin phosphotransferase II gene (NTPII). The influential factors were investigated systematically and the optimal transformation condition was achieved using hypocotyls as explants, including the sonication treatment of 10 min with 80 W, A. tumefaciens infection of 30 min and co-cultivation of 2 d in 1/2 MS medium containing 100 μM acetosyringone. With a series of selection in callus, shoot and root inducing kanamycin-containing resistance media, we successfully obtained stable transgenic regeneration plants. The expression of GUS gene was confirmed by histochemistry, polymerase chain reaction, and genomic southern blot analysis. To prove the efficiency of the established genetic transformation system, the rate-limiting gene in TIAs biosynthetic pathway, DAT, which encodes deacetylvindoline-4-O-acetyltransferase, was transferred into C. roseus using this established system and 9 independent transgenic plants were obtained. The results of metabolite analysis using high performance liquid chromatography (HPLC) showed that overexpression of DAT increased the yield of vindoline in transgenic plants.
In the present study, we report an efficient Agrobacterium-mediated transformation system for C. roseus plants with 11% of transformation frequency. To our knowledge, this is the first report on the establishment of A. tumefaciens mediated transformation and regeneration of C. roseus. More importantly, the C. roseus transformation system developed in this work was confirmed in the successful transformation of C. roseus using a key gene DAT involved in TIAs biosynthetic pathway resulting in the higher accumulation of vindoline in transgenic plants.
PMCID: PMC3483169  PMID: 22748182
Catharanthus roseus; Agrobacterium tumefaciens; Deacetylvindoline-4-O-acetyltransferase; Regeneration; Vindoline
2.  The Complete Plastid Genome Sequence of Madagascar Periwinkle Catharanthus roseus (L.) G. Don: Plastid Genome Evolution, Molecular Marker Identification, and Phylogenetic Implications in Asterids 
PLoS ONE  2013;8(6):e68518.
The Madagascar periwinkle (Catharanthusroseus in the family Apocynaceae) is an important medicinal plant and is the source of several widely marketed chemotherapeutic drugs. It is also commonly grown for its ornamental values and, due to ease of infection and distinctiveness of symptoms, is often used as the host for studies on phytoplasmas, an important group of uncultivated plant pathogens. To gain insights into the characteristics of apocynaceous plastid genomes (plastomes), we used a reference-assisted approach to assemble the complete plastome of C. roseus, which could be applied to other C. roseus-related studies. The C. roseus plastome is the second completely sequenced plastome in the asterid order Gentianales. We performed comparative analyses with two other representative sequences in the same order, including the complete plastome of Coffeaarabica (from the basal Gentianales family Rubiaceae) and the nearly complete plastome of Asclepiassyriaca (Apocynaceae). The results demonstrated considerable variations in gene content and plastome organization within Apocynaceae, including the presence/absence of three essential genes (i.e., accD, clpP, and ycf1) and large size changes in non-coding regions (e.g., rps2-rpoC2 and IRb-ndhF). To find plastome markers of potential utility for Catharanthus breeding and phylogenetic analyses, we identified 41 C. roseus-specific simple sequence repeats. Furthermore, five intergenic regions with high divergence between C. roseus and three other euasterids I taxa were identified as candidate markers. To resolve the euasterids I interordinal relationships, 82 plastome genes were used for phylogenetic inference. With the addition of representatives from Apocynaceae and sampling of most other asterid orders, a sister relationship between Gentianales and Solanales is supported.
PMCID: PMC3688999  PMID: 23825699
3.  Development of SSR and gene-targeted markers for construction of a framework linkage map of Catharanthus roseus 
Annals of Botany  2011;108(2):321-336.
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.
For simple sequence repeat (SSR) isolation, a genomic library enriched for (GA)n repeats was constructed from C. roseus ‘Nirmal’ (CrN1). In addition, GTMs were also designed from 12 genes of the TIA (terpenoid indole alkaloid) pathway – the medicinally most significant pathway in C. roseus. An F2 mapping population was also generated by crossing two diverse accessions of C. roseus CrN1 (Nirmal)×CrN82 (Kew).
Key Results
A new set of 314 STMS markers and 64 GTMs were developed in this study. A segregating F2 mapping population consisting of 111 F2 individuals was generated. For generating the linkage map, a set of 423 co-dominant markers (378 newly developed and 45 published earlier) were screened for polymorphism between the parental genotypes, of which 134 were identified to be polymorphic. A total of 114 markers were mapped on eight linkage groups that spanned a 632·7 cM region of the genome with an average marker distance of 5·55 cM. Further, the mechanism of hypervariability at the gene-targeted loci was investigated at the sequence level.
For the first time, a large array of STMS markers and GTMs was generated in the model medicinal plant C. roseus. Moreover, the first microsatellite marker-based linkage map was described in this study. Together, these will serve as a foundation for future genomics studies related to quantitative trait loci analysis and molecular breeding in C. roseus.
PMCID: PMC3143056  PMID: 21788377
Catharanthus roseus; sequence-tagged microsatellite sites; STMS; microsatellites; linkage map; gene-targeted markers; GTM
4.  Isolation of Catharanthus roseus (L.) G. Don Nuclei and Measurement of Rate of Tryptophan decarboxylase Gene Transcription Using Nuclear Run-On Transcription Assay 
PLoS ONE  2015;10(5):e0127892.
An accurate assessment of transcription ‘rate’ is often desired to describe the promoter activity. In plants, isolation of transcriptionally active nuclei and their subsequent use in nuclear run-on assays has been challenging and therefore limit an accurate measurement of gene transcription ‘rate’. Catharanthus roseus has emerged as a model medicinal plant as it exhibits an unsurpassed spectrum of chemodiversity, producing over 130 alkaloids through the terpenoid indole alkaloid (TIA) pathway and therefore serves as a ‘molecular hub’ to understand gene expression profiles.
The protocols presented here streamline, adapt and optimize the existing methods of nuclear run-on assay for use in C. roseus. Here, we fully describe all the steps to isolate transcriptionally active nuclei from C. roseus leaves and utilize them to perform nuclear run-on transcription assay. Nuclei isolated by this method transcribed at a level consistent with their response to external stimuli, as transcription rate of TDC gene was found to be higher in response to external stimuli i.e. when seedlings were subjected to UV-B light or to methyl jasmonate (MeJA). However, the relative transcript abundance measured parallel through qRT-PCR was found to be inconsistent with the synthesis rate indicating that some post transcriptional events might have a role in transcript stability in response to stimuli.
Our study provides an optimized, efficient and inexpensive method of isolation of intact nuclei and nuclear ‘run-on’ transcription assay to carry out in-situ measurement of gene transcription rate in Catharanthus roseus. This would be valuable in investigating the transcriptional and post transcriptional response of other TIA pathway genes in C. roseus. Isolated nuclei may also provide a resource that could be used for performing the chip assay as well as serve as the source of nuclear proteins for in-vitro EMSA studies. Moreover, nascent nuclear run-on transcript could be further subjected to RNA-Seq for global nuclear run-on assay (GNRO-Seq) for genome wide in-situ measurement of transcription rate of plant genes.
PMCID: PMC4449189  PMID: 26024519
5.  Karyotype analysis and visualization of 45S rRNA genes using fluorescence in situ hybridization in aroids (Araceae) 
Comparative Cytogenetics  2015;9(2):145-160.
Karyotype analysis and FISH mapping using 45S rDNA sequences on 6 economically important plant species Anthurium andraeanum Linden ex André, 1877, Monstera deliciosa Liebmann, 1849, Philodendron scandens Koch & Sello, 1853, Spathiphyllum wallisii Regel, 1877, Syngonium auritum (Linnaeus, 1759) Schott, 1829 and Zantedeschia elliottiana (Knight, 1890) Engler, 1915 within the monocotyledonous family Araceae (aroids) were performed. Chromosome numbers varied between 2n=2x=24 and 2n=2x=60 and the chromosome length varied between 15.77 µm and 1.87 µm. No correlation between chromosome numbers and genome sizes was observed for the studied genera. The chromosome formulas contained only metacentric and submetacentric chromosomes, except for Philodendron scandens in which also telocentric and subtelocentric chromosomes were observed. The highest degree of compaction was calculated for Spathiphyllum wallisii (66.49Mbp/µm). B-chromosome-like structures were observed in Anthurium andraeanum. Their measured size was 1.87 times smaller than the length of the shortest chromosome. After FISH experiments, two 45S rDNA sites were observed in 5 genera. Only in Zantedeschia elliottiana, 4 sites were seen. Our results showed clear cytogenetic differences among genera within Araceae, and are the first molecular cytogenetics report for these genera. These chromosome data and molecular cytogenetic information are useful in aroid breeding programmes, systematics and evolutionary studies.
PMCID: PMC4488963  PMID: 26140158
Araceae; B-chromosomes; chromosome formula; cytogenetics; genome size; FISH
6.  Synthesis of silver nanoparticles using leaves of Catharanthus roseus Linn. G. Don and their antiplasmodial activities 
To develop a novel approach for the green synthesis of silver nanoparticles using aqueous leaves extracts of Catharanthus roseus (C. roseus) Linn. G. Don which has been proven active against malaria parasite Plasmodium falciparum (P. falciparum).
Characterizations were determined by using ultraviolet-visible (UV-Vis) spectrophotometry, scanning electron microscopy (SEM), energy dispersive X-ray and X-ray diffraction.
SEM showed the formation of silver nanoparticles with an average size of 35–55 nm. X-ray diffraction analysis showed that the particles were crystalline in nature with face centred cubic structure of the bulk silver with the broad peaks at 32.4, 46.4 and 28.0.
It can be concluded that the leaves of C. roseus can be good source for synthesis of silver nanoparticle which shows antiplasmodial activity against P. falciparum. The important outcome of the study will be the development of value added products from medicinal plants C. roseus for biomedical and nanotechnology based industries.
PMCID: PMC3609350  PMID: 23569974
Silver nanoparticles; Catharanthus roseus; Plasmodium falciparum; Antiplasmodial activity
7.  Development of Transcriptomic Resources for Interrogating the Biosynthesis of Monoterpene Indole Alkaloids in Medicinal Plant Species 
PLoS ONE  2012;7(12):e52506.
The natural diversity of plant metabolism has long been a source for human medicines. One group of plant-derived compounds, the monoterpene indole alkaloids (MIAs), includes well-documented therapeutic agents used in the treatment of cancer (vinblastine, vincristine, camptothecin), hypertension (reserpine, ajmalicine), malaria (quinine), and as analgesics (7-hydroxymitragynine). Our understanding of the biochemical pathways that synthesize these commercially relevant compounds is incomplete due in part to a lack of molecular, genetic, and genomic resources for the identification of the genes involved in these specialized metabolic pathways. To address these limitations, we generated large-scale transcriptome sequence and expression profiles for three species of Asterids that produce medicinally important MIAs: Camptotheca acuminata, Catharanthus roseus, and Rauvolfia serpentina. Using next generation sequencing technology, we sampled the transcriptomes of these species across a diverse set of developmental tissues, and in the case of C. roseus, in cultured cells and roots following elicitor treatment. Through an iterative assembly process, we generated robust transcriptome assemblies for all three species with a substantial number of the assembled transcripts being full or near-full length. The majority of transcripts had a related sequence in either UniRef100, the Arabidopsis thaliana predicted proteome, or the Pfam protein domain database; however, we also identified transcripts that lacked similarity with entries in either database and thereby lack a known function. Representation of known genes within the MIA biosynthetic pathway was robust. As a diverse set of tissues and treatments were surveyed, expression abundances of transcripts in the three species could be estimated to reveal transcripts associated with development and response to elicitor treatment. Together, these transcriptomes and expression abundance matrices provide a rich resource for understanding plant specialized metabolism, and promotes realization of innovative production systems for plant-derived pharmaceuticals.
PMCID: PMC3530497  PMID: 23300689
8.  Chromosome Evolution and Genome Miniaturization in Minifish 
PLoS ONE  2012;7(5):e37305.
Paedocypris is a newly established genus of fish in Southeast Asia. Paedocypris is characterized by several unique features, including a tiny adult size (thus named miniature fish or minifish), fragmentary habitats of acidic peat blackwater swamps, an unusual reproduction mode and truncated development. These peculiarities lend themselves excellent for studying chromosome evolution and rapid speciation in vertebrates but also make them highly controversial for the phylogenetic position.
Methodology and Principal Findings
We have established an organ procedure to prepare chromosome spreads from tiny organs of minifish and performed a cytogenetic study on two species of the genus Paedocypris, namely P. carbunculus (Pc) and P. sp. “Singkep” (Ps). We found 30 and 34 chromosomes in diploid cells of Pc and Ps, respectively, which are unusual in teleost fishes. The diploid metaphase has 5 pairs of metacentrics and 7 pairs of subtelocentrics in Pc compared to 3 pairs of metacentrics and 11 pairs of subtelocentrics in Ps, whereas the haploid metaphase contains 5 metacentrics and 7 subtelocentrics in Pc compared to 3 metacentrics and 11 subtelocentrics Ps. Chromosome behavior in first meiosis revealed the presence of a chromosomal ring consisting of 2 metacentrics in Pc, suggesting that centric fusion rather than fission was responsible for the karyotypic evolution from Ps to Pc. Flow cytometry revealed that Pc had a 45% nuclear staining intensity relative to medaka whose genome is 700 Mb in size and contains 0.81 pg DNA. The Pc genome should have 315 Mb in length and 0.36 pg of DNA, which represent one of the smallest values in vertebrates, suggesting genome miniaturization in this organism.
Our data demonstrate that gross chromosome rearrangements and genome miniaturization have accompanied the evolution of Paedocypris fishes. Our data also place Paedocypris outside currently described taxa of the Cypriniformes.
PMCID: PMC3353929  PMID: 22615970
9.  Analyses of Catharanthus roseus and Arabidopsis thaliana WRKY transcription factors reveal involvement in jasmonate signaling 
BMC Genomics  2014;15(1):502.
To combat infection to biotic stress plants elicit the biosynthesis of numerous natural products, many of which are valuable pharmaceutical compounds. Jasmonate is a central regulator of defense response to pathogens and accumulation of specialized metabolites. Catharanthus roseus produces a large number of terpenoid indole alkaloids (TIAs) and is an excellent model for understanding the regulation of this class of valuable compounds. Recent work illustrates a possible role for the Catharanthus WRKY transcription factors (TFs) in regulating TIA biosynthesis. In Arabidopsis and other plants, the WRKY TF family is also shown to play important role in controlling tolerance to biotic and abiotic stresses, as well as secondary metabolism.
Here, we describe the WRKY TF families in response to jasmonate in Arabidopsis and Catharanthus. Publically available Arabidopsis microarrays revealed at least 30% (22 of 72) of WRKY TFs respond to jasmonate treatments. Microarray analysis identified at least six jasmonate responsive Arabidopsis WRKY genes (AtWRKY7, AtWRKY20, AtWRKY26, AtWRKY45, AtWRKY48, and AtWRKY72) that have not been previously reported. The Catharanthus WRKY TF family is comprised of at least 48 members. Phylogenetic clustering reveals 11 group I, 32 group II, and 5 group III WRKY TFs. Furthermore, we found that at least 25% (12 of 48) were jasmonate responsive, and 75% (9 of 12) of the jasmonate responsive CrWRKYs are orthologs of AtWRKYs known to be regulated by jasmonate.
Overall, the CrWRKY family, ascertained from transcriptome sequences, contains approximately 75% of the number of WRKYs found in other sequenced asterid species (pepper, tomato, potato, and bladderwort). Microarray and transcriptomic data indicate that expression of WRKY TFs in Arabidopsis and Catharanthus are under tight spatio-temporal and developmental control, and potentially have a significant role in jasmonate signaling. Profiling of CrWRKY expression in response to jasmonate treatment revealed potential associations with secondary metabolism. This study provides a foundation for further characterization of WRKY TFs in jasmonate responses and regulation of natural product biosynthesis.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-502) contains supplementary material, which is available to authorized users.
PMCID: PMC4099484  PMID: 24950738
Catharanthus roseus; Terpenoid indole alkaloid; Transcriptome; Secondary metabolism; WRKY transcription factors
10.  UV-B induced transcript accumulation of DAHP synthase in suspension-cultured Catharanthus roseus cells 
The enzyme 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase (EC catalyzes the first committed step in the shikimate pathway of tryptophan synthesis, an important precursor for the production of terpenoid indole alkaloids (TIAs). A full-length cDNA encoding nuclear coded chloroplast-specific DAHP synthase transcript was isolated from a Catharanthus roseus cDNA library. This had high sequence similarity with other members of plant DAHP synthase family. This transcript accumulated in suspension cultured C. roseus cells on ultraviolet (UV-B) irradiation. Pretreatment of C.roseus cells with variety of agents such as suramin, N-acetyl cysteine, and inhibitors of calcium fluxes and protein kinases and MAP kinase prevented this effect of UV-B irriadiation. These data further show that the essential components of the signaling pathway involved in accumulation DAHP synthase transcript in C. roseus cells include suramin-sensitive cell surface receptor, staurosporine-sensitive protein kinase and MAP kinase.
PMCID: PMC2930624  PMID: 20704760
11.  Exploiting EST databases for the mining and characterization of short sequence repeat (SSR) markers in Catharanthus roseus L. 
Bioinformation  2011;5(9):378-381.
Periwinkle (Catharanthus roseus L.) (Family: Apocyanaceae) is a ornamental plants with great medicinal properties. Although it is represented by seven species, little work has been carried out on its genetic characterization due to non-availability of reliable molecular markers. Simple sequence repeats (SSRs) have been widely applied as molecular markers in genetic studies. With the rapid increase in the deposition of nucleotide sequences in the public databases and advent of bioinformatics tools, it has become a cost effective and fast approach to scan for microsatellite repeats and exploit the possibility of converting it into potential genetic markers. Expressed sequence tags (EST's) from Catharanthus roseus were used for the screening of Class I (hyper variable) simple sequence repeats (SSR's). A total of 502 microsatellite repeats were detected from 21730 EST sequences of turmeric after redundancy elimination. The average density of Class I SSRs account to 1 SSR per 10.21 kb of EST. Mononucleotides was the most abundant class of microsatellite motifs. It accounted for 44.02% of the total, followed by the trinucleotide (26.09%) and dinucleotide repeats (14.34%). Among all the repeat motifs, (A/T)n accounted for the highest Proportion (36.25%) followed by (AAG)n. These detected SSRs can be used to design primers that have functional importance and should also facilitate the analysis of genetic diversity, variability, linkage mapping and evolutionary relationships in plants especially medicinal plants.
PMCID: PMC3044425  PMID: 21383904
Catharanthus roseus; Expresses sequence tags; short sequence repeats; SSR Locator
12.  The ORCA2 transcription factor plays a key role in regulation of the terpenoid indole alkaloid pathway 
BMC Plant Biology  2013;13:155.
The terpenoid indole alkaloid (TIA) pathway leads to the production of pharmaceutically important drugs, such as the anticancer compounds vinblastine and vincristine. Unfortunately, these drugs are produced in trace amounts, causing them to be very costly. To increase production of these drugs, an improved understanding of the TIA regulatory pathway is needed. Towards this end, transgenic Catharanthus roseus hairy roots that overexpress the ORCA2 TIA transcriptional activator were generated and characterized.
Transcriptional profiling experiments revealed that overexpression of ORCA2 results in altered expression of key genes from the indole and terpenoid pathways, which produce precursors for the TIA pathway, and from the TIA pathway itself. In addition, metabolite-profiling experiments revealed that overexpression of ORCA2 significantly affects the levels of several TIA metabolites. ORCA2 overexpression also causes significant increases in transcript levels of several TIA regulators, including TIA transcriptional repressors.
Results presented here indicate that ORCA2 plays a critical role in regulation of TIA metabolism. ORCA2 regulates expression of key genes from both feeder pathways, as well as the genes (STR and SGD) encoding the enzymes that catalyze the first two steps in TIA biosynthesis. ORCA2 may play an especially important role in regulation of the downstream branches of the TIA pathway, as it regulates four out of five genes characterized from this part of the pathway. Regulation of TIA transcriptional repressors by ORCA2 may provide a mechanism whereby increases in TIA metabolite levels in response to external stimuli are transient and limited in magnitude.
PMCID: PMC3851283  PMID: 24099172
Terpenoid indole alkaloids; ORCA2; Catharanthus roseus; Hairy root cultures
13.  A virus-induced gene silencing approach to understanding alkaloid metabolism in Catharanthus roseus 
Phytochemistry  2011;72(16):1969-1977.
The anticancer agents vinblastine and vincristine are bisindole alkaloids derived from coupling vindoline and catharanthine, monoterpenoid indole alkaloids produced exclusively by Madagascar periwinkle (Catharanthus roseus) plants. 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 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. We have developed a VIGS method 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.
PMCID: PMC3435519  PMID: 21802100
Catharanthus roseus; Apocynaceae; Madagascar periwinkle; virus-induced gene silencing; alkaloid; vindoline; N-methyltransferase
14.  Antihyperglycemic activity of Catharanthus roseus leaf powder in streptozotocin-induced diabetic rats 
Pharmacognosy Research  2010;2(3):195-201.
Catharanthus roseus Linn (Apocynaceae), is a traditional medicinal plant used to control diabetes, in various regions of the world. In this study we evaluated the possible antidiabetic and hypolipidemic effect of C. roseus (Catharanthus roseus) leaf powder in diabetic rats. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ, 55 mg/kg body wt) to male Wistar rats. The animals were divided into four groups: Control, control-treated, diabetic, and diabetic-treated group. Diabetic-treated and control-treated rats were treated with C. roseus leaf powder suspension in 2 ml distilled water, orally (100 mg/kg body weight/day/60 days). In diabetic rats (D-group) the plasma glucose was increased and the plasma insulin was decreased gradually. In the diabetic-treated group lowering of plasma glucose and an increase in plasma insulin were observed after 15 days and by the end of the experimental period the plasma glucose had almost reached the normal level, but insulin had not. The significant enhancement in plasma total cholesterol, triglycerides, LDL and VLDL-cholesterol, and the atherogenic index of diabetic rats were normalized in diabetic-treated rats. Decreased hepatic and muscle glycogen content and alterations in the activities of enzymes of glucose metabolism (glycogen phosphorylase, hexokinase, phosphofructokinase, pyruvate kinase, and glucose-6-phosphate dehydrogenase), as observed in the diabetic control rats, were prevented with C. roseus administration. Our results demonstrated that C. roseus with its antidiabetic and hypolipidemic properties could be a potential herbal medicine in treating diabetes.
PMCID: PMC3141314  PMID: 21808566
Anti Catharanthus roseus; plasma insulin; plasma lipids; STZ-induced diabetes
15.  Building a model: developing genomic resources for common milkweed (Asclepias syriaca) with low coverage genome sequencing 
BMC Genomics  2011;12:211.
Milkweeds (Asclepias L.) have been extensively investigated in diverse areas of evolutionary biology and ecology; however, there are few genetic resources available to facilitate and compliment these studies. This study explored how low coverage genome sequencing of the common milkweed (Asclepias syriaca L.) could be useful in characterizing the genome of a plant without prior genomic information and for development of genomic resources as a step toward further developing A. syriaca as a model in ecology and evolution.
A 0.5× genome of A. syriaca was produced using Illumina sequencing. A virtually complete chloroplast genome of 158,598 bp was assembled, revealing few repeats and loss of three genes: accD, clpP, and ycf1. A nearly complete rDNA cistron (18S-5.8S-26S; 7,541 bp) and 5S rDNA (120 bp) sequence were obtained. Assessment of polymorphism revealed that the rDNA cistron and 5S rDNA had 0.3% and 26.7% polymorphic sites, respectively. A partial mitochondrial genome sequence (130,764 bp), with identical gene content to tobacco, was also assembled. An initial characterization of repeat content indicated that Ty1/copia-like retroelements are the most common repeat type in the milkweed genome. At least one A. syriaca microread hit 88% of Catharanthus roseus (Apocynaceae) unigenes (median coverage of 0.29×) and 66% of single copy orthologs (COSII) in asterids (median coverage of 0.14×). From this partial characterization of the A. syriaca genome, markers for population genetics (microsatellites) and phylogenetics (low-copy nuclear genes) studies were developed.
The results highlight the promise of next generation sequencing for development of genomic resources for any organism. Low coverage genome sequencing allows characterization of the high copy fraction of the genome and exploration of the low copy fraction of the genome, which facilitate the development of molecular tools for further study of a target species and its relatives. This study represents a first step in the development of a community resource for further study of plant-insect co-evolution, anti-herbivore defense, floral developmental genetics, reproductive biology, chemical evolution, population genetics, and comparative genomics using milkweeds, and A. syriaca in particular, as ecological and evolutionary models.
PMCID: PMC3116503  PMID: 21542930
16.  Ornamental Exterior versus Therapeutic Interior of Madagascar Periwinkle (Catharanthus roseus): The Two Faces of a Versatile Herb 
The Scientific World Journal  2015;2015:982412.
Catharanthus roseus (L.) known as Madagascar periwinkle (MP) is a legendary medicinal plant mostly because of possessing two invaluable antitumor terpenoid indole alkaloids (TIAs), vincristine and vinblastine. The plant has also high aesthetic value as an evergreen ornamental that yields prolific blooms of splendid colors. The plant possesses yet another unique characteristic as an amiable experimental host for the maintenance of the smallest bacteria found on earth, the phytoplasmas and spiroplasmas, and serves as a model for their study. Botanical information with respect to synonyms, vernacular names, cultivars, floral morphology, and reproduction adds to understanding of the plant while the geography and ecology of periwinkle illustrate the organism's ubiquity. Good agronomic practices ensure generous propagation of healthy plants that serve as a source of bioactive compounds and multitudinous horticultural applications. The correlation between genetic diversity, variants, and TIA production exists. MP is afflicted with a whole range of diseases that have to be properly managed. The ethnobotanical significance of MP is exemplified by its international usage as a traditional remedy for abundant ailments and not only for cancer. TIAs are present only in micro quantities in the plant and are highly poisonous per se rendering a challenge for researchers to increase yield and reduce toxicity.
PMCID: PMC4312627  PMID: 25667940
17.  Transcriptome Analysis of Catharanthus roseus for Gene Discovery and Expression Profiling 
PLoS ONE  2014;9(7):e103583.
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.
PMCID: PMC4114786  PMID: 25072156
18.  Evaluation of the Nutritive and Organoleptic Values of Food Products Developed by Incorporated Catharanthus roseus (Sadabahar) Fresh Leaves Explore Their Hypoglycemic Potential 
The Scientific World Journal  2014;2014:304120.
Diabetes becomes a real problem of public health in developing countries, where its prevalence is increasing steadily. Diabetes mellitus can be found in almost every population in the world. Since the Ayurvedic practice started in India, plants are being used in the cure of diseases. Although the Catharanthus roseus have been used for their alleged health benefits and avail their hypoglycemic effect, used as medicine by diabetics. Medicinal plants have rarely been incorporated in food preparations. To fill these lacunae, food products were prepared by using Catharanthus roseus (Sadabahar) fresh leaves with hypoglycemic properties. Commonly consumed recipes in India are prepared for diabetic patients and were developed at different levels at 3 g, 4 g, and 6 g per serving. Food product development and their acceptability appraisal through organoleptic evaluation were carried out by semitrained panel comprising 15 trained panelists from the department of Food Science and Nutrition, Banasthali University. Seven products were developed by incorporating Catharanthus roseus fresh leaves. Nine point hedonic scale was used as a medium to know about the product acceptability at various variances. All products are moderately acceptable at different concentrations except product fare “6 g” which was more acceptable than the standard. Among the three variations of incorporating the Catharanthus roseus (Sadabahar) Leaves, 3 g variation is more acceptable than other variations.
PMCID: PMC3982472  PMID: 24790561
19.  CrMPK3, a mitogen activated protein kinase from Catharanthus roseus and its possible role in stress induced biosynthesis of monoterpenoid indole alkaloids 
BMC Plant Biology  2012;12:134.
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.
CrMPK3 expressed in bacterial system is an active kinase as it showed auto-phosphorylation and phosphorylation of Myelin Basic Protein. CrMPK3 though localized in cytoplasm, moves to nucleus upon wounding. Wounding, UV treatment and MeJA application on C. roseus leaves resulted in the transcript accumulation of CrMPK3 as well as activation of MAPK in C. roseus leaves. Immuno-precipitation followed by immunoblot analysis revealed that wounding, UV treatment and methyl jasmonate (MeJA) activate CrMPK3. Transient over-expression of CrMPK3 in C. roseus leaf tissue showed enhanced expression of key MIA biosynthesis pathway genes and also accumulation of specific MIAs.
Results from our study suggest a possible involvement of CrMPK3 in abiotic stress signal transduction towards regulation of transcripts of key MIA biosynthetic pathway genes, regulators and accumulation of major MIAs.
PMCID: PMC3487899  PMID: 22871174
Catharanthus roseus; Methyl jasmonate; Mitogen activated protein kinase; Monoterpenoid indole alkaloid; Secondary metabolism
20.  Strictosidine activation in Apocynaceae: towards a "nuclear time bomb"? 
BMC Plant Biology  2010;10:182.
The first two enzymatic steps of monoterpene indole alkaloid (MIA) biosynthetic pathway are catalysed by strictosidine synthase (STR) that condensates tryptamine and secologanin to form strictosidine and by strictosidine β-D-glucosidase (SGD) that subsequently hydrolyses the glucose moiety of strictosidine. The resulting unstable aglycon is rapidly converted into a highly reactive dialdehyde, from which more than 2,000 MIAs are derived. Many studies were conducted to elucidate the biosynthesis and regulation of pharmacologically valuable MIAs such as vinblastine and vincristine in Catharanthus roseus or ajmaline in Rauvolfia serpentina. However, very few reports focused on the MIA physiological functions.
In this study we showed that a strictosidine pool existed in planta and that the strictosidine deglucosylation product(s) was (were) specifically responsible for in vitro protein cross-linking and precipitation suggesting a potential role for strictosidine activation in plant defence. The spatial feasibility of such an activation process was evaluated in planta. On the one hand, in situ hybridisation studies showed that CrSTR and CrSGD were coexpressed in the epidermal first barrier of C. roseus aerial organs. However, a combination of GFP-imaging, bimolecular fluorescence complementation and electromobility shift-zymogram experiments revealed that STR from both C. roseus and R. serpentina were localised to the vacuole whereas SGD from both species were shown to accumulate as highly stable supramolecular aggregates within the nucleus. Deletion and fusion studies allowed us to identify and to demonstrate the functionality of CrSTR and CrSGD targeting sequences.
A spatial model was drawn to explain the role of the subcellular sequestration of STR and SGD to control the MIA metabolic flux under normal physiological conditions. The model also illustrates the possible mechanism of massive activation of the strictosidine vacuolar pool upon enzyme-substrate reunion occurring during potential herbivore feeding constituting a so-called "nuclear time bomb" in reference to the "mustard oil bomb" commonly used to describe the myrosinase-glucosinolate defence system in Brassicaceae.
PMCID: PMC3095312  PMID: 20723215
21.  UV-B-induced signaling events leading to enhanced-production of catharanthine in Catharanthus roseus cell suspension cultures 
BMC Plant Biology  2007;7:61.
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.
PMCID: PMC2213653  PMID: 17988378
22.  Karyotype analysis of Panax ginseng C.A.Meyer, 1843 (Araliaceae) based on rDNA loci and DAPI band distribution 
Comparative Cytogenetics  2012;6(4):425-441.
Ginseng has long been considered a valuable plant owing to its medicinal properties; however, genomic information based on chromosome characterization and physical mapping of cytogenetic markers has been very limited. Dual-color FISH karyotype and DAPI banding analyses of Panax ginseng C. A. Meyer, 1843 were conducted using 5S and 45S rDNA probes. The somatic chromosome complement was 2n=48 with lengths from 3.3 μm to 6.3 μm. The karyotype was composed of 12 metacentric, 9 submetacentric, and 3 subtelocentric pairs. The 5S rDNA probe localized to the intercalary region of the short arm of pair 11, while the 45S rDNA was located at the secondary constriction of the subtelocentric satellited chromosome 14. DAPI bands were clearly observed for most chromosomes, with various signal intensities and chromosomal distributions that consequently improved chromosome identification. As a result, all 24 chromosomes could be distinguished and numbers were assigned to each chromosome for the first time. The results presented here will be useful for the on-going ginseng genome sequencing and further molecular-cytogenetic studies and breeding programs of ginseng.
PMCID: PMC3834566  PMID: 24260682
Panax ginseng; FISH; 5SrDNA; 45S rDNA; DAPI band; Araliaceae
23.  Induction and Flow Cytometry Identification of Tetraploids from Seed-Derived Explants through Colchicine Treatments in Catharanthus roseus (L.) G. Don 
The tetraploid plants of Catharanthus roseus (L.) G. Don was obtained by colchicine induction from seeds explants, and the ploidy of the plants was identified by flow cytometry. The optimal treatment is 0.2% colchicine solution treated for 24 hours, and the induction rate reaches up to 30%. Comparing with morphological characteristics and growth habits between tetraploids and the control, we found that tetraploids of C. roseus had larger stoma and more branches and leaves. HPLC analysis showed tetraploidization could increase the contents of terpenoid indole alkaloids in C. roseus. Thus, tetraploidization could be used to produce higher alkaloids lines for commercial use. QRT-PCR results showed that the expression of enzymes involved in terpenoid indole alkaloids biosynthesis pathway had increased in the tetraploid plants. To our knowledge, this was the first paper to explore the secondary metabolism in autotetraploid C. roseus induced by colchicine.
PMCID: PMC3110335  PMID: 21660143
24.  Overexpression of ORCA3 and G10H in Catharanthus roseus Plants Regulated Alkaloid Biosynthesis and Metabolism Revealed by NMR-Metabolomics 
PLoS ONE  2012;7(8):e43038.
In order to improve the production of the anticancer dimeric indole alkaloids in Catharanthuse roseus, much research has been dedicated to culturing cell lines, hairy roots, and efforts to elucidate the regulation of the monoterpenoid indole alkaloid (MIA) biosynthesis. In this study, the ORCA3 (Octadecanoid-derivative Responsive Catharanthus AP2-domain) gene alone or integrated with the G10H (geraniol 10-hydroxylase) gene were first introduced into C. roseus plants. Transgenic C. roseus plants overexpressing ORCA3 alone (OR lines), or co-overexpressing G10H and ORCA3 (GO lines) were obtained by genetic modification. ORCA3 overexpression induced an increase of AS, TDC, STR and D4H transcripts but did not affect CRMYC2 and G10H transcription. G10H transcripts showed a significant increase under G10H and ORCA3 co-overexpression. ORCA3 and G10H overexpression significantly increased the accumulation of strictosidine, vindoline, catharanthine and ajmalicine but had limited effects on anhydrovinblastine and vinblastine levels. NMR-based metabolomics confirmed the higher accumulation of monomeric indole alkaloids in OR and GO lines. Multivariate data analysis of 1H NMR spectra showed change of amino acid, organic acid, sugar and phenylpropanoid levels in both OR and GO lines compared to the controls. The result indicated that enhancement of MIA biosynthesis by ORCA3 and G10H overexpression might affect other metabolic pathways in the plant metabolism of C. roseus.
PMCID: PMC3423439  PMID: 22916202
25.  Effect of Chromium on Antioxidant Potential of Catharanthus roseus Varieties and Production of Their Anticancer Alkaloids: Vincristine and Vinblastine 
BioMed Research International  2014;2014:934182.
Catharanthus roseus (L.) G. Don, a medicinal plant, has a very important place in the traditional as well as modern pharmaceutical industry. Two common varieties of this plant rosea and alba are named so because of pink and white coloured flowers, respectively. This plant comprises of about 130 terpenoid indole alkaloids and two of them, vincristine and vinblastine, are common anticancer drugs. The effect of chromium (Cr) on enzymatic and non-enzymatic antioxidant components and on secondary metabolites vincristine and vinblastine was studied under pot culture conditions of both varieties of C. roseus. Antioxidant responses of these varieties were analyzed under 0, 10, 50, and 100 μM chromium (Cr) level in order to investigate the plant's protective mechanisms against Cr induced oxidative stress. The results indicated that Cr affects all the studied parameters and decreases growth performance. However, vincristine and vinblastine contents were increased under Cr stress. Results are quite encouraging, as this plant shows good antioxidant potential and increased the level of active constituents under Cr stress.
PMCID: PMC3966348  PMID: 24734252

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