Skip to main content

Advertisement

SpringerLink
Log in

De novo transcriptome analyses reveals putative pathway genes involved in biosynthesis and regulation of camptothecin in Nothapodytes nimmoniana (Graham) Mabb.

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

Key message

Comprehensive transcriptome analysis of leaf and root tissues of Nothapodytes nimmoniana unravels several putative pathway genes, transcription factors and CYPs related to camptothecin (CPT) biosynthesis. Additionally, post-transcriptional suppression by artificial microRNA (aMIR) of NnCYP76B6 (geraniol 10-hydroxylase) suggests its role in CPT biosynthesis. Tissue-specific LC-MS/MS analysis revealed the presence of secologanin as the central intermediate of MIA pathway in N. nimmoniana.

Abstract

Nothapodytes nimmoniana is a rich source of potent anticancer drug camptothecin (CPT) whose biosynthetic pathway is unresolved due to the lack of genomic and transcriptomic information. Present investigation entails deep transcriptome analysis of N. nimmoniana which led to identification of putative pathway genes and regulatory components involved in CPT biosynthesis. Using Illumina HiSeq 2500 sequencing platform a total of 31,172,889 (6.23 Gb) and 31,218,626 (6.24 Gb) raw reads were generated from leaf and root wood, respectively. These were assembled de novo into 138,183 unique contigs. Additionally, 16 cytochrome P450 transcripts related to secondary metabolism were also identified. Further, transcriptome data pool presented 1683 putative transcription factors of which transcripts corresponding to WRKY TFs were the most abundant (14.14%). A total of 2741 transcripts were differentially expressed out of which 478 contigs showed downregulation in root wood and 2263 contigs were up-regulated. Further, comparative analyses of 17 genes involved in CPT biosynthetic pathway were validated by qRT-PCR. On basis of intermediates, two distinct seco-iridoid pathways are involved in the biosynthesis of monoterpene indole alkaloids either through multiple isomers of strictosidinic acid or strictosidine. Tissue-specific LC-MS/MS analysis revealed the presence of secologanin as the central intermediate of MIA pathway in N. nimmoniana. Geraniol-10 hydroxylase (NnCYP76B6) an important enzyme in CPT biosynthesis which specifically shunts geraniol into the secologanin pathway was also cloned from the trancriptome resource. In planta transient expression of NnCYP76B6 showed a significant enhancement in mRNA transcript levels coincident with enhanced CPT accumulation. Further, artificial microRNA (aMIR) mediated downregulation of NnCYP76B6 resulted in reduction of mRNA transcript levels as well as CPT content in comparison to control. These empirical results suggest a plausible regulatory role for NnCYP76B6 in CPT biosynthesis and also establish a valuable repository for deciphering various structural, rate limiting and regulatory genes of CPT biosynthetic pathway.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • An X, Chen J, Zhang J, Liao Y, Dai L, Wang B, Liu L, Peng D (2015) Transcriptome profiling and identification of transcription factors in ramie (Boehmeria nivea L. Gaud) in response to PEG treatment, using illumina paired-end sequencing technology. Int J Mol Sci 16:3493–3511

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Anders S, Huber W (2010) Differential expression analysis for sequence count data. Genome Biol 11:R106. https://doi.org/10.1186/gb-2010-11-10-r106

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Asada K, Salim V, Masada-Atsumi S, Edmunds E, Nagatoshi M, Terasaka K, Mizukami H, De Luca V (2013) A 7-deoxyloganetic acid glucosyltransferase contributes a key step in secologanin biosynthesis in Madagascar periwinkle. Plant Cell 25:4123–4134

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bak S, Beisson F, Bishop G, Hamberger B, Höfer R, Paquette S, Werck-Reichhart D (2011) Cytochromes P450. Arabidopsis Book. https://doi.org/10.1199/tab.0144

    PubMed  PubMed Central  Google Scholar 

  • Barleben L, Panjikar S, Ruppert M, Koepke J, Stöckigt J (2007) Molecular architecture of strictosidine glucosidase: the gateway to the biosynthesis of the monoterpenoid indole alkaloid family. Plant Cell 19:2886–2897

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Brown S, Clastre M, Courdavault V, O’Connor SE (2015) De novo production of the plant-derived alkaloid strictosidine in yeast. Proc Natl Acad Sci 112:3205–3210

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cárdenas-Conejo Y et al (2015) De novo transcriptome sequencing in Bixa orellana to identify genes involved in methylerythritol phosphate, carotenoid and bixin biosynthesis. BMC Genomics 16:877. https://doi.org/10.1186/s12864-015-2065-4

    Article  PubMed  PubMed Central  Google Scholar 

  • Charoonratana T, Wungsintaweekul J, Keawpradub N, Verpoorte R (2013) Molecular cloning and expression of tryptophan decarboxylase from Mitragyna speciosa. Acta Physiol Plant 35:2611–2621

    Article  CAS  Google Scholar 

  • Chen R, Liao Z, Chen M, Wang Q, Yang C, Yang Y (2008) Molecular cloning and characterization of the Strictosidine synthase Gene from Rauwolfia verticillata. Russ J Plant Physiol 55:670–675

    Article  CAS  Google Scholar 

  • Cirak C, Radusiene J, Camas N, Caliskan O, Odabas MS (2013) Changes in the contents of main secondary metabolites in two Turkish Hypericum species during plant development. Pharm Biol 51:391–399

    Article  PubMed  Google Scholar 

  • Collu G, Unver N, Peltenburg-Looman AM, van der Heijden R, Verpoorte R, Memelink J (2001) Geraniol 10-hydroxylase, a cytochrome P450 enzyme involved in terpenoid indole alkaloid biosynthesis. FEBS Lett 508:215–220

    Article  CAS  PubMed  Google Scholar 

  • Collu G, Garcia AA, van der Heijden R, Verpoorte R (2002) Activity of the cytochrome P450 enzyme geraniol 10-hydroxylase and alkaloid production in plant cell cultures. Plant Sci 162:165–172

    Article  CAS  Google Scholar 

  • Cui L et al (2015) Co-overexpression of geraniol-10-hydroxylase and strictosidine synthase improves anti-cancer drug camptothecin accumulation in Ophiorrhiza pumila. Sci Rep https://doi.org/10.1038/srep08227

    Google Scholar 

  • de Waal A, Meijer AH, Verpoorte R (1995) Strictosidine synthase from Catharanthus roseus: purification and characterization of multiple forms. Biochem J 306:571–580

    Article  PubMed  PubMed Central  Google Scholar 

  • Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fulzele DP, Satdive RK (2005) Comparison of techniques for the extraction of the anti-cancer drug camptothecin from Nothapodytes foetida. J Chromatogr A 1063:9–13

    Article  CAS  PubMed  Google Scholar 

  • Geourjon C, Deleage G (1995) SOPMA—significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Comput Appl Biosci: CABIOS 11:681–684

    CAS  PubMed  Google Scholar 

  • Govindachari T, Viswanathan N (1972) Alkaloids of Mappia foetida. Phytochemistry 11:3529–3531

    Article  CAS  Google Scholar 

  • Gowda HH, Vasudeva R, Mathachen G, Shaanker RU, Ganeshaiah K (2002) Breeding types in Nothapodytes nimmoniana Graham.: an important medicinal tree. Curr Sci 83:1077–1078

    Google Scholar 

  • Guirimand G et al (2010) Strictosidine activation in Apocynaceae: towards a nuclear time bomb? BMC Plant Biol 10:182

    PubMed  PubMed Central  Google Scholar 

  • Gupta P, Goel R, Agarwal AV, Asif MH, Sangwan NS, Sangwan RS, Trivedi PK (2015) Comparative transcriptome analysis of different chemotypes elucidates withanolide biosynthesis pathway from medicinal plant Withania somnifera. Sci Rep. https://doi.org/10.1038/srep18611

    Google Scholar 

  • Hamberger B, Bak S (2013) Plant P450s as versatile drivers for evolution of species-specific chemical diversity. Philos Trans R Soc London B: Biol Sci 368:20120426

    Article  Google Scholar 

  • He L, Zhao S, Hu Z (2012) A novel cytochrome P450 gene from Catharanthus roseus cell line C 20 hi: cloning and characterization of expression. Acta Pharm Sinica B 2:250–255

    Article  CAS  Google Scholar 

  • Höfer R et al (2013) Geraniol hydroxylase and hydroxygeraniol oxidase activities of the CYP76 family of cytochrome P450 enzymes and potential for engineering the early steps of the (seco) iridoid pathway. Metab Eng 20:221–232

    Article  PubMed  Google Scholar 

  • Huang F-C, Sung P-H, Do Y-Y, Huang P-L (2012) Differential expression and functional characterization of the NADPH cytochrome P450 reductase genes from Nothapodytes foetida. Plant Sci 190:16–23

    Article  CAS  PubMed  Google Scholar 

  • Irmler S et al (2000) Indole alkaloid biosynthesis in Catharanthus roseus: new enzyme activities and identification of cytochrome P450 CYP72A1 as secologanin synthase. Plant J 24:797–804

    Article  CAS  PubMed  Google Scholar 

  • Jin J, Zhang H, Kong L, Gao G, Luo J (2013) Plant TFDB 3.0: a portal for the functional and evolutionary study of plant transcription factors. Nucleic Acids Res gkt1016

  • Kai G et al (2013) Molecular cloning and expression analysis of a gene encoding 3-hydroxy-3-methylglutaryl-CoA synthase from Camptotheca acuminata. Russ J Plant Physiol 60:131–138

    Article  CAS  Google Scholar 

  • Käll L, Krogh A, Sonnhammer EL (2007) Advantages of combined transmembrane topology and signal peptide prediction—the Phobius web server. Nucleic Acids Res 35:W429–W432

    Article  PubMed  PubMed Central  Google Scholar 

  • Kalra S, Puniya BL, Kulshreshtha D, Kumar S, Kaur J, Ramachandran S, Singh K (2013). De novo transcriptome sequencing reveals important molecular networks and metabolic pathways of the plant, Chlorophytum borivilianum. PLoS ONE, 8(12):e83336

    Article  PubMed  PubMed Central  Google Scholar 

  • Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJ (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat Proto 10:845–858

    Article  CAS  Google Scholar 

  • Kulkarni K, Raghuwanshi K, Naik R, Borkar S, Chimote V (2015) Antifungal activity of Camptothecin extracted from Mappia foetida against disease causing pathogens in pomegranate (Punica granatum L.). J Pure Appl Microbiol 9:329–334

    CAS  Google Scholar 

  • Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lattoo SK, Dhar RS, Dhar AK, Sharma PR, Agarwal SG (2006) Dynamics of essential oil biosynthesis in relation to inflorescence and glandular ontogeny in Salvia sclarea. Flavour Fragrance J 21:817–821

    Article  CAS  Google Scholar 

  • Leng X, Jia H, Sun X, Shangguan L, Mu Q, Wang B, Fang J (2015) Comparative transcriptome analysis of grapevine in response to copper stress. Sci Rep. https://doi.org/10.1038/srep17749

    Google Scholar 

  • Li CY, Leopold AL, Sander GW, Shanks JV, Zhao L, Gibson SI (2013) The ORCA2 transcription factor plays a key role in regulation of the terpenoid indole alkaloid pathway. BMC Plant Biol 13:155

    Article  PubMed  PubMed Central  Google Scholar 

  • Li CY, Leopold AL, Sander GW, Shanks JV, Zhao L, Gibson SI (2015) CrBPF1 overexpression alters transcript levels of terpenoid indole alkaloid biosynthetic and regulatory genes. Front Plant Sci. https://doi.org/10.3389/fpls.2015.00818

    Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−∆∆CT method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  • Lorence A, Nessler CL (2004) Camptothecin, over four decades of surprising findings. Phytochemistry 65:2735–2749

    Article  CAS  PubMed  Google Scholar 

  • Ma X, Panjikar S, Koepke J, Loris E, Stöckigt J (2006) The structure of Rauvolfia serpentina strictosidine synthase is a novel six-bladed β-propeller fold in plant proteins. Plant Cell 18:907–920

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Manjunatha B, Singh H, Ravikanth G, Nataraja KN, Shankar R, Kumar S, Shaanker RU (2016) Transcriptome analysis of stem wood of Nothapodytes nimmoniana (Graham) Mabb. identifies genes associated with biosynthesis of camptothecin, an anti-carcinogenic molecule. J Biosci 41:119–131

    Article  CAS  PubMed  Google Scholar 

  • Martin M (2011) Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J 17:10–12

    Article  Google Scholar 

  • Mathijssen RH, Loos WJ, Verweij J, Sparreboom A (2002) Pharmacology of topoisomerase I inhibitors irinotecan (CPT-11) and topotecan. Curr Cancer Drug Targets 2:103–123

    Article  CAS  PubMed  Google Scholar 

  • Miettinen K et al (2014) The seco-iridoid pathway from Catharanthus roseus. Nat Commun. https://doi.org/10.1038/ncomms4606

    PubMed  PubMed Central  Google Scholar 

  • Misra P et al (2010) Modulation of transcriptome and metabolome by At MYB12 transcriptome factor leads to insect tolerance. Plant Physiol 152:2258–2268

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Namdeo A, Sharma A (2012) HPLC analysis of camptothecin content in various parts of Nothapodytes foetida collected on different periods. Asian Pac J Trop Biomed 2:389–393

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Namdeo A, Priya T, Bhosale B (2012) Micropropagation and production of camptothecin form in vitro plants of Ophiorrhiza mungos. Asian Pac J Trop Biomed. https://doi.org/10.1016/S2221

    PubMed  PubMed Central  Google Scholar 

  • Niu QW, lin SS, Rayes JL, Chen KC, Wu HW, Yeh SD, Chua NH (2006) Expression of artificial microRNAs in transient Arabidopsis thaliana confers virus resistance. Nat Biotechnol 24:1420–1428

    Article  CAS  PubMed  Google Scholar 

  • O’Connor SE, Maresh JJ (2006) Chemistry and biology of monoterpene indole alkaloid biosynthesis. Nat Prod Rep 23:532–547

    Article  PubMed  Google Scholar 

  • Padmanabha B et al (2006) Patterns of accumulation of camptothecin, an anti-cancer alkaloid in Nothapodytes nimmoniana Graham., in the Western Ghats, India: implications for identifying high-yielding sources of the alkaloid. Curr Sci 90:95–100

    CAS  Google Scholar 

  • Pan Q et al (2012) Overexpression of ORCA3 and G10H in Catharanthus roseus plants regulated alkaloid biosynthesis and metabolism revealed by NMR-metabolomics. PLoS ONE. https://doi.org/10.1371/journal.pone.0043038

    Google Scholar 

  • Pandit S, Shitiz K, Sood H, Chauhan RS (2013) Differential biosynthesis and accumulation of picrosides in an endangered medicinal herb Picrorhiza kurroa. J Plant Biochem Biotechnol 22:335–342

    Article  CAS  Google Scholar 

  • Parra R, Paredes MA, Sanchez-Calle IM, Gomez-Jimenez MC (2013) Comparative transcriptional profiling analysis of olive ripe-fruit pericarp and abscission zone tissues shows expression differences and distinct patterns of transcriptional regulation. BMC Genomics. https://doi.org/10.1186/1471-2164-14-866

    PubMed  PubMed Central  Google Scholar 

  • Pasquali G, Porto DD, Fett-Neto AG (2006) Metabolic engineering of cell cultures versus whole plant complexity in production of bioactive monoterpene indole alkaloids: recent progress related to old dilemma. J Biosci Bioeng 101:287–296

    Article  CAS  PubMed  Google Scholar 

  • Peebles CA, Hughes EH, Shanks JV, San K-Y (2009) Transcriptional response of the terpenoid indole alkaloid pathway to the overexpression of ORCA3 along with jasmonic acid elicitation of Catharanthus roseus hairy roots over time. Metabolic Eng 11:76–86

    Article  CAS  Google Scholar 

  • Pommier Y (2009) DNA topoisomerase I inhibitors: chemistry, biology, and interfacial inhibition. Chem Rev 109:2894–2902

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Priel E, Showalter SD, Blair DG (1991) Inhibition of Human Immunodeficiency Virus (HTV-1) replication in vitro by noncytotoxic doses of camptothecin, a topoisomerase I inhibitor. AIDS Res Hum Retroviruses 7:65–72

    CAS  PubMed  Google Scholar 

  • Qiu JL, Fiil BK, Petersen K, Nielsen HB, Botanga CJ, Thorgrimsen S, Palma K, Suarez-Rodriguez MC, Sandbech-Clausen S, Lichota J (2008) Arabidopsis MAP kinase 4 regulates gene expression through transcription factor release in the nucleus. EMBO J 27:2214–2221

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Qu X, Pu X, Chen F, Yang Y, Yang L, Zhang G, Luo Y (2015) Molecular cloning, heterologous expression, and functional characterization of an NADPH-Cytochrome P450 reductase gene from Camptotheca acuminata, a camptothecin-producing plant. PLoS ONE 10:e0135397. https://doi.org/10.1371/journal.pone.0135397

    Article  PubMed  PubMed Central  Google Scholar 

  • Ramesha B et al (2008) Prospecting for camptothecines from Nothapodytes nimmoniana in the Western Ghats, South India: identification of high-yielding sources of camptothecin and new families of camptothecines. J Chromatogr Sci 46:362–368

    Article  CAS  PubMed  Google Scholar 

  • Riano-Pachon DM, Ruzicic S, Dreyer I, Mueller-Roeber B (2007) PlnTFDB: an integrative plant transcription factor database. BMC Bioinform 8:42

    Article  Google Scholar 

  • Sadre R et al (2016) Metabolite diversity in alkaloid biosynthesis: a multilane (Diastereomer) highway for camptothecin synthesis in Camptotheca acuminata. Plant Cell 28:1926–1944

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425

    CAS  PubMed  Google Scholar 

  • Salim V, Yu F, Altarejos J, Luca V (2013) Virus-induced gene silencing identifies Catharanthus roseus 7-deoxyloganic acid-7-hydroxylase, a step in iridoid and monoterpene indole alkaloid biosynthesis. Plant J 76:754–765

    Article  CAS  PubMed  Google Scholar 

  • Schmidt A, Gershenzon J (2008) Cloning and characterization of two different types of geranyl diphosphate synthases from Norway spruce (Picea abies). Phytochemistry 69:49–57

    Article  CAS  PubMed  Google Scholar 

  • Shivaprakash KN, Ramesha BT, Shaanker RU, Dayanandan S, Ravikanth G (2014) Genetic structure, diversity and long term viability of a medicinal plant, Nothapodytes nimmoniana Graham. (Icacinaceae), in protected and non-protected areas in the Western Ghats biodiversity hotspot. PLoS ONE 9:e112769. https://doi.org/10.1371/journal.pone.0112769

    Article  PubMed  PubMed Central  Google Scholar 

  • Simkin AJ et al (2013) Characterization of the plastidial geraniol synthase from Madagascar periwinkle which initiates the monoterpenoid branch of the alkaloid pathway in internal phloem associated parenchyma. Phytochemistry 85:36–43

    Article  CAS  PubMed  Google Scholar 

  • Suhas S, Ramesha B, Ravikanth G, Gunaga RP, Vasudeva R, Ganeshaiah K, Shaanker RU (2007) Chemical profiling of Nothapodytes nimmoniana populations in the Western Ghats, India for anti-cancer compound, camptothecin. Curr Sci 92:1142–1147

    CAS  Google Scholar 

  • Suma HK et al (2014) Pyrenacantha volubilis Wight, (Icacinaceae) a rich source of camptothecine and its derivatives, from the Coromandel Coast forests of India. Fitoterapia 97:105–110

    Article  CAS  PubMed  Google Scholar 

  • Sun Y et al (2011) Pyrosequencing of the Camptotheca acuminata transcriptome reveals putative genes involved in camptothecin biosynthesis and transport. BMC Genomics 12:533

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Swarbreck SM et al (2011) Analysis of leaf and root transcriptomes of soil-grown Avena barbata plants. Plant Cell Physiol 52:317–332

    Article  CAS  PubMed  Google Scholar 

  • Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tsuchida Y, Shitara T (2005) Topotecan and irinotecan in the treatment of pediatric solid tumors. Curr Pediatr Rev 1:55–61

    Article  CAS  Google Scholar 

  • Upadhyay AK et al (2015) Genome sequencing of herb Tulsi (Ocimum tenuiflorum) unravels key genes behind its strong medicinal properties. BMC Plant Biol. https://doi.org/10.1186/s12870-015-0562-x

    PubMed  PubMed Central  Google Scholar 

  • Vinogradov AE (2003) DNA helix: the importance of being GC-rich. Nucleic Acids Res 31:1838–1844

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wagner GJ, Kroumova AB (2008) The use of RNAi to elucidate and manipulate secondary metabolite synthesis in plants. In: Current perspectives in microRNAs (miRNA). Springer, Dordrecht, pp 431–459

    Chapter  Google Scholar 

  • Wu SF et al (2008) Camptothecinoids from the seeds of Taiwanese Nothapodytes foetida. Molecules 13:1361–1371

    Article  CAS  PubMed  Google Scholar 

  • Yamazaki M et al (2013) Coupling deep transcriptome analysis with untargeted metabolic profiling in Ophiorrhiza pumila to further the understanding of the biosynthesis of the anti-cancer alkaloid camptothecin and anthraquinones. Plant Cell Physiol 54:686–696

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yang CQ, Fang X, Wu XM, Mao YB, Wang LJ, Chen XY (2012) Transcriptional regulation of plant secondary metabolism. J Integr Plant Biol 54:703–712

    Article  CAS  PubMed  Google Scholar 

  • Yerkes N, Wu JX, McCoy E, Galan MC, Chen S, O’Connor SE (2008) Substrate specificity and diastereoselectivity of strictosidine glucosidase, a key enzyme in monoterpene indole alkaloid biosynthesis. Bioorg Med Chem Lett 18:3095–3098

    Article  CAS  PubMed  Google Scholar 

  • Zhu X, Zeng X, Sun C, Chen S (2014) Biosynthetic pathway of terpenoid indole alkaloids in Catharanthus roseus. Front Med 8:285–293

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

Authors are thankful to Prabhu Dutt at CSIR-IIIM, Jammu for facilitating HPLC analysis. GAR and SAP are thankful to UGC for providing Senior Research Fellowships. AS thankfully acknowledges the DST-INSPIRE Senior Research Fellowship. We also appreciate the support provided by three of our former research fellows, Satiander Rana, Wajid W. Bhat and Niha Dhar in initial phase of the project. This manuscript represents Institutional Communication Number IIIM/2174/2017.

Funding

This work was supported by financial grant from Council of Scientific and Industrial Research (CSIR)-Indian Institute of Integrative Medicine under Major Lab Project MLP-3012(WP 7) and partly from CSIR-Network Project BSC 0108.

Author information

Author notes

  1. Arti Sharma and Shahzad A. Pandith have contributed equally.

Authors and Affiliations

  1. Plant Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu Tawi, 180001, India

    Gulzar A. Rather, Arti Sharma, Shahzad A. Pandith, Prashant Misra & Surrinder K. Lattoo

  2. Department of Botany, University of Jammu, Jammu Tawi, 180006, India

    Veenu Kaul

  3. PK-PD Toxicology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu Tawi, 180001, India

    Utpal Nandi

Authors
  1. Gulzar A. Rather
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Arti Sharma
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shahzad A. Pandith
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Veenu Kaul
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Utpal Nandi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Prashant Misra
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Surrinder K. Lattoo
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Conceived and designed the experiments: SKL, PM; Performed the experiments: GAR, AS, SAP, UN; Analyzed the data: GAR, SKL, PM, AS, SP, VK, UN; Contributed reagents/materials/analysis tools: SKL, PM; Wrote the paper: GAR, SKL, SAP, AS, PM, UN.

Corresponding authors

Correspondence to Prashant Misra or Surrinder K. Lattoo.

Ethics declarations

Conflict of interest

The authors declare that there is no conflict of interest.

Additional information

Data deposition

The sequencing data have been submitted to Sequence Read Archive (SRA-NCBI) database under the accession number of SRP063489. All other supporting data are included as files.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary file 1: Sequencing results and three dimensional model of NnCYP76B6 (DOCX 525 kb). (DOCX 524 KB)

Supplementary file 2: Assembled sequences (DOCX 36,094kb). (DOCX 36093 KB)

Supplementary file 3: BLASTX transcriptome result (XLSX 3.49 MB). (XLSX 3579 KB)

Supplementary file 4: Swiss-UniProt transcriptome result (XLSX 22.2 MB). (XLSX 22820 KB)

Supplementary file 5: Gene Ontology terms (XLSX 244 kb). (XLSX 244 KB)

Supplementary file 6: Cytochrome P450s (XLSX 83.2 kb). (XLSX 84 KB)

Supplementary file 7: LC-MS/MS analysis (DOCX 113kb). (DOCX 115 KB)

11103_2017_690_MOESM8_ESM.xlsx

Supplementary file 8: Comparative transcriptome analysis N.nimmoniana candidate genes involved in camptothecin biosynthesis (XLSX 22 kb). (XLSX 21 KB)

Supplementary file 9: Transcription factor families (XLSX 440 kb). (XLSX 441 KB)

Supplementary file 10: Differential gene expression results (TXT 15.6 MB). (TXT 15980 KB)

Supplementary file 11: Heatmap of Up-regulated genes in nascent root wood (PNG 3.43 MB). (PNG 3517 KB)

Supplementary file 12: Heatmap of downregulated genes in leaf (PNG 11.8 MB). (PNG 12172 KB)

Supplementary file 13: Primers used for amplification (DOCX 13 kb). (DOCX 14 KB)

11103_2017_690_MOESM14_ESM.docx

Supplementary file 14: Contig numbers of sequences taken from N. nimmoniana transcriptome and accession number of sequences retrieved from NCBI used for phylogenetic analysis (DOCX 16.3 kb). (DOCX 14 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Rather, G.A., Sharma, A., Pandith, S.A. et al. De novo transcriptome analyses reveals putative pathway genes involved in biosynthesis and regulation of camptothecin in Nothapodytes nimmoniana (Graham) Mabb.. Plant Mol Biol 96, 197–215 (2018). https://doi.org/10.1007/s11103-017-0690-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11103-017-0690-9

Keywords

Search

Navigation