Advertisement

Biologia Plantarum

, Volume 62, Issue 2, pp 250–260 | Cite as

Activation of polyketide synthase gene promoter in Cannabis sativa by heterologous transcription factors derived from Humulus lupulus

  • G. S. Duraisamy
  • A. K. Mishra
  • T. Kocábek
  • J. MatoušekEmail author
Original Papers
  • 261 Downloads

Abstract

Cannabis sativa, an annual herbaceous plant, produce wide variety of secondary metabolites among which delta-9-tetrahydrocannabinol (THC) is the most important one. The dissection of biosynthetic pathway(s) of this compound and its regulation by transcription factors (TFs) is an important prerequisite for efficient biotechnological manipulation of its secondary metabolome. A polyketide synthase (PKS) of C. sativa catalyzes the first step of cannabinoid biosynthesis, leading to the biosynthesis of olivetolic acid. Cloning and analysis of PKS promoter based on online PLACE, PlantCARE, and Genomatix Matinspector professional databases, indicated that PKS promoter consisted of cis-elements such as TATA-box, CAAT-box, W-box, Myb-box, E-box, and P-box. Plant expression vector PKS::GUS was constructed in such a way that the ATG of the PKS gene was in the frame with the β-glucuronidase (GUS) coding region. Using a combinatorial transient GUS expression system in Nicotiana benthamania leaves, it was shown that heterologous TFs such as HlWRKY1, HlMYB3, HlWDR1 and HlbZIP1 from Humulus lupulus significantly activated PKS promoter. Moreover, Tombusvirus p19 core protein, which is known for silencing suppressor functions, acted in our combinatorial transient expression system as an enhancer of PKS promoter activity along with hop TFs. Our analyses suggested the involvement of the hop derived TFs (HlWRKY1, HlMYB3, HlWDR1 and HlbZIP1A) and p19 in the activation of PKS gene promoter, which could be used for the genetic manipulation of C. sativa to enhance the cannabinoid production.

Additional key words

β-glucuronidase bZIP1 MYB3 RT-qPCR Tombusvirus p19 core protein WDR1 WRKY1 

Abbreviations

bZIP1

basic-leucine Zipper Domain 1

CHS

chalcone synthase

GUS

β-glucuronidase

MU

4-methylumbelliferone

MYB3

myeloblastosis proto-oncogene family of R2R3

PKS

polyketide synthase

35S

35S cauliflower mosaic virus promoter

TF

transcriptional factor

TBSV

Tomato bushy stunt virus

THC

tetrahydrocannabinol

THCA

Δ9-tetrahydrocannabinolic acid

WDR1

WD repeat containing protein

WRKY1

transcription factor containing WRKYGQK motif

X-Gluc

5-bromo-4-chloro-3-indolyl-β-D-glucuronide.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Supplementary material

10535_2017_766_MOESM1_ESM.pdf (111 kb)
Supplementary material, approximately 110 KB.

References

  1. Alghanim, H.J., Almirall, J.R.: Development of microsatellite markers in Cannabis sativa for DNA typing and genetic relatedness analyses. - Anal. bioanal. Chem. 376: 1225–1233, 2003.CrossRefPubMedGoogle Scholar
  2. Austin, M.B., Noel, J.P.: The chalcone synthase superfamily of type III polyketide synthases. - Nat. Prod. Rep. 20: 79–110, 2003.CrossRefPubMedGoogle Scholar
  3. Bomal, C., Bedon, F., Caron, S., Mansfield, S.D., Levasseur, C., Cooke, J.E.K., Blais, S., Tremblay, L., Morency, M.J., Pavy, N., Grima-Pettenati, J., Séguin, A., MacKay, J.: Involvement of Pinus taeda MYB1 and MYB8 in phenylpropanoid metabolism and secondary cell wall biogenesis: a comparative in planta analysis. - J. exp. Bot. 59: 3925–3939, 2008.CrossRefPubMedPubMedCentralGoogle Scholar
  4. Bomal, C., Duval, I., Giguere, I., Fortin, É., Caron, S., Stewart, D., Boyle, B., Séguin, A., MacKay, J.J.: Opposite action of R2R3-MYBs from different subgroups on key genes of the shikimate and monolignol pathways in spruce. - J. exp. Bot. 65: 495–408, 2014.CrossRefPubMedGoogle Scholar
  5. Chu, M,, Desvoyes, B., Turina, M. R., Noad, R., Scholthof, H.B.: Genetic dissection of tomato bushy stunt virus p19- protein-mediated host-dependent symptom induction and systemic invasion. - Virology 266: 79–87, 2000.CrossRefPubMedGoogle Scholar
  6. Chytilová, E., Macas, J., Galbraith, D.W.: Green fluorescent protein targeted to the nucleus, a transgenic phenotype useful for studies in plant biology. - Ann. Bot. 83: 645–654, 1999.CrossRefGoogle Scholar
  7. Downer, E.J., Campbell, V.A.: Phytocannabinoids CNS cells and development: a dead issue?. - Drug Alcohol Rev. 29: 91–98, 2010.CrossRefPubMedGoogle Scholar
  8. Duraisamy, G.S., Mishra, A.K., Kocabek, T., Matoušek, J.: Identification and characterization of promoters and cisregulatory elements of genes involved in secondary metabolites production in hop (Humulus lupulus. L). - Comput. Biol. Chem. 64: 346–352, 2016.CrossRefPubMedGoogle Scholar
  9. Fior, S., Gerola, P.D.: Impact of ubiquitous inhibitor on the GUS gene reporter system: evidence from the model plants Arabidopsis, tobacco and rice and correction methods for quantitative assays of transgenic and endogenous GUS. - Plant Methods 5: 19–29, 2009.CrossRefPubMedPubMedCentralGoogle Scholar
  10. Fischbach, M.A., Walsh, C.T.: Assembly-line enzymology for polyketide and nonribosomal peptide antibiotics: logic, machinery, and mechanisms. - Chem. Rev. 106: 3468–3496, 2006.CrossRefPubMedGoogle Scholar
  11. Flores-Sanchez, I.J., Linthorst, H.J.M., Verpoorte, R.: In silicio expression analysis of PKS genes isolated from Cannabis sativa L. - Genet. mol. Biol. 33: 703–713, 2010.CrossRefPubMedPubMedCentralGoogle Scholar
  12. Flores-Sanchez, I.J., Verpoorte, R.: Secondary metabolism in cannabis. - Phytochem. Rev. 7: 615–639, 2008.CrossRefGoogle Scholar
  13. Gagne, S.J., Stout, J.M., Liu, E., Boubakir, Z., Clark, S.M., Page, J.E.: Identification of olivetolic acid cyclase from Cannabis sativa reveals a unique catalytic route to plant polyketides. - Proc. nat. Acad. Sci. USA 109: 12811–12816, 2012.CrossRefPubMedGoogle Scholar
  14. Gao, Z., Liu, C., Zhang, Y., Li, Y., Yi, K., Zhao, X., Cui, M.L.: The promoter structure differentiation of a MYB transcription factor RLC1 causes red leaf coloration in empire red leaf cotton under light. - PLoS ONE 8: e77891, 2013.CrossRefPubMedPubMedCentralGoogle Scholar
  15. Giacoppo, S., Mandolino, G., Galuppo, M., Bramanti, P., Mazzon, E.: Cannabinoids: new promising agents in the treatment of neurological diseases. - Molecules 19: 18781–18816, 2014.CrossRefPubMedGoogle Scholar
  16. Guo, H.X., Kan, Y.C., Liu, W.Q.: Differential expression of miRNAs in response to topping in flue-cured tobacco (Nicotiana tabacum) roots. - PLoS ONE 6: 1–15, 2011.Google Scholar
  17. Haney, M., Gunderson, E.W., Rabkin, J., Hart, C.L., Vosburg, S.K., Comer, S.D., Foltin, R.W.: Dronabinol and marijuana in HIV-positive marijuana smokers. Caloric intake, mood, and sleep. - J. Acquir. Immune Defic. Syndr. 45: 545–554, 2007.CrossRefPubMedGoogle Scholar
  18. Hearne, P.Q., Knorr, D.A., Hillman, B.I., Morris, T.J.: The complete genome structure and synthesis of infectious RNA from clones of Tomato bushy stunt virus. - Virology 177: 141–151, 1990.CrossRefPubMedGoogle Scholar
  19. Hellens, R.P., Moreau, C., Lin-Wang, K., Schwinn, K.E., Thomson, S.J., Fiers, M., Frew, T.J., Murray, S.R., Hofer, J.M.I., Jacobs, J.M.E.: Identification of Mendel’s white flower character. - PLoS ONE 5: 1–8, 2010.CrossRefGoogle Scholar
  20. Hichri, I., Barrieu, F., Bogs, J., Kappel, C., Delrot, S., Lauvergeat, V.: Recent advances in the transcriptional regulation of the flavonoid biosynthetic pathway. - J. exp. Bot. 62: 2465–2483, 2011.CrossRefPubMedGoogle Scholar
  21. Hillig, K.W.: Genetic evidence for speciation in Cannabis (Cannabaceae). - Genet. Resour. Crop Evol. 52: 161–182, 2005.CrossRefGoogle Scholar
  22. Huda, K.M.K., Banu, M.S.A., Pathi, K.M., Tuteja, N.: Reproductive organ and vascular specific promoter of the rice plasma membrane Ca2+ ATPase mediates environmental stress responses in plants. - PLoS ONE 8: e57803, 2013.CrossRefPubMedPubMedCentralGoogle Scholar
  23. Jefferson, R.A., Kavanagh, T.A., Bevan, M.W.: GUS fusion: β- glucuronidase as a sensitive and versatile gene fusion marker in higher plants. - EMBO J. 6: 3901–3907, 1987.PubMedPubMedCentralCrossRefGoogle Scholar
  24. Langley, R.A., Kado, C.I.: Studies on Agrobacterium tumefaciens. Conditions for mutagenesis by N-methyl-N′- nitrosoguanidine and relationships of A. tumefaciens mutants to crown gall tumor induction. - Mutat. Res. 14: 277–286, 1972.CrossRefGoogle Scholar
  25. Li, F., Huang, C., Li, Z., Zhou, X.: Suppression of RNA silencing by a plant DNA virus satellite requires a host calmodulin-like protein to repress RDR6 expression. - PLoS Pathog. 10: e1003921, 2014.CrossRefPubMedPubMedCentralGoogle Scholar
  26. Livak, K.J., Schmittgen, T.D.: Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. - Methods 25: 402–408, 2001.CrossRefPubMedGoogle Scholar
  27. Mano, H., Ogasawara, F., Sato, K., Higo, H., Minobe, Y.: Isolation of a regulatory gene of anthocyanin biosynthesis in tuberous roots of purple-fleshed sweet potato. - Plant Physiol. 143: 1252–1268, 2007.CrossRefPubMedPubMedCentralGoogle Scholar
  28. Mao, K., Wang, K., Zhao, M., Tao, X.T., Klionsky, D.J.: Two MAPK-signaling pathways are required for mitophagy in Saccharomyces cerevisiae. - J. cell. Biol. 93: 755–767, 2011.CrossRefGoogle Scholar
  29. Matoušek, J., Kocábek, T., Patzak, J., Bříza, J., Siglová, K., Mishra, A.K., Duraisamy, G.S., Týcová, A., Ono, E., Krofta, K.: The "putative" role of transcription factors from HlWRKY family in the regulation of the final steps of prenylflavonid and bitter acids biosynthesis in hop (Humulus lupulus L.). - Plant mol. Biol. 92: 263–277, 2016.CrossRefPubMedGoogle Scholar
  30. Matoušek, J., Kocábek, T., Patzak, J., Füssy, Z., Procházková, J., Heyerick, A.: Combinatorial analysis of lupulin gland transcription factors from R2R3MYB, bHLH and WDR families indicates a complex regulation of chs_H1 genes essential for prenylflavonoid biosynthesis in hop (Humulus lupulus L.). - BMC Plant Biol. 12: 27, 2012.CrossRefPubMedPubMedCentralGoogle Scholar
  31. Matoušek, J., Kocabek, T., Patzak, J., Skopek, J., Maloukh, L., Heyerick, A., Fussy, Z., Roldàn-Ruiz, I., De-Keukeleire, D.: HIMyb3, a putative regulatory factor in hop (Humulus lupulus L.), shows diverse biological effects in heterologous transgenotes. - J. Agr. Food Chem. 55: 7767–7776, 2007.CrossRefGoogle Scholar
  32. Matoušek, J., Kocábek, T., Patzak, J., Stehlík, J., Füssy, Z., Krofta, K., Heyerick, A., Roldán-Ruiz, I., Maloukh, L., De-Keukeleire, D.: Cloning and molecular analysis of HlbZip1 and HlbZip2 transcription factors putatively involved in the regulation of the lupulin metabolome in hops (Humulus lupulus L.). - J. Agr. Food Chem. 58: 902–912, 2010.CrossRefGoogle Scholar
  33. Matoušek, J., Vrba, L., Škopek, J., Orctová, L., Pešina, K., Heyerick, A., Baulcombe, D., De Keukeleire, D.: Sequence analysis of a “true” chalcone synthase (chs_H1) oligofamily from hop (Humulus lupulus L.) and PAP1 activation of chs_H1 in heterologous systems. - J. Agr. Food Chem. 54: 7606–7615, 2006.CrossRefGoogle Scholar
  34. Matus, J.T., Poupin, M.J., Cañón, P., Bordeu, E., Alcalde, J.A., Arce-Johnson, P.: Isolation of WDR and bHLH genes related to flavonoid synthesis in grapevine (Vitis vinifera L.). - Plant mol. Biol. 72: 607–620, 2010.CrossRefPubMedGoogle Scholar
  35. Menke, F.L., Kang, H.G., Chen, Z., Park, J.M., Kumar, D., Klessig, D.F.J.: Tobacco transcription factor WRKY1 is phosphorylated by the MAP kinase SIPK and mediates HRlike cell death in tobacco. - Mo.l Plant-Microbe Interact. 18: 1027–1034, 2005.CrossRefGoogle Scholar
  36. Russo, E.B., Burnett, A., Hall, B., Parker, K.K.: Agonistic properties of cannabidiol at 5-HT1A receptors. - Neurochem. Res. 30: 1037–1043, 2005.CrossRefPubMedGoogle Scholar
  37. Saghai-Maroof, M.A., Soliman, K.M., Jorgensen, R.A., Allard, R.W.: Ribosomal DNA spacer-length polymorphism in barley: Mendelian inheritance, chromosomal location, and population dynamics. - Proc. natl. Acad. Sci. USA 8: 8014–8018, 1984.CrossRefGoogle Scholar
  38. Shimada, S., Otsuki, H., Sakuta, M.: Transcriptional control of anthocyanin biosynthetic genes in the Caryophyllales. - J. exp. Bot. 58: 957–967, 2006.CrossRefPubMedGoogle Scholar
  39. Silhavy, D., Molnar, A., Lucioloi, A., Szittya, G., Hornyik, C., Tavazza, M., Burgyan, J.A.: Viral protein suppresses RNA silencing and binds silencing generated, 21 to 25 nucleotide double stranded RNAs. - EMBO J. 21: 3070–3080, 2002.CrossRefPubMedPubMedCentralGoogle Scholar
  40. Small, E., Cronquist, A.: A practical and natural taxonomy for Cannabis. - Taxon 25: 405–435, 1976.CrossRefGoogle Scholar
  41. Solís-Ramos, L., Nahuath-Dzib, S., Andrade-Torres, A., Barredo-Pool, F., González-Estrada, T., De la Serna, E.: Indirect somatic embryogenesis and morphohistological analysis in Capsicum chinense. - Biológia 65: 504–511, 2010.Google Scholar
  42. Stracke, R., Ishihara, H., Huep, G., Barsch, A., Mehrtens, F., Niehaus, K., Weisshaar, B.: Differential regulation of closely related R2R3-MYP transcription factors controls flavonol accumulation in different parts of the Arabidopsis thaliana seedling. - Plant J. 50: 660–677, 2007.CrossRefPubMedPubMedCentralGoogle Scholar
  43. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S.: MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. - Mol. Biol. Evol. 28: 2731–2739, 2011.CrossRefPubMedPubMedCentralGoogle Scholar
  44. Taura, F., Sirikantaramas, S., Shoyama, Y., Yoshikai, K., Shoyama, Y., Morimoto, S.: Cannabidiolic-acid synthase, the chemotype-determining enzyme in the fiber-type Cannabis sativa. - FEBS Lett. 581: 2929–2934, 2007.CrossRefPubMedGoogle Scholar
  45. Van Bakel, H., Stout, J.M., Cote, A., Tallon, C.M., Sharpe, A.G., Hughes, T.R., Page, J.E.: The draft genome and transcriptome of Cannabis sativa. - Genome Biol. 12: 1–17, 2011.CrossRefGoogle Scholar
  46. Várallyay, E., Oláh, E., Havelda, Z.: Independent parallel functions of p19 plant viral suppressor of RNA silencing required for effective suppressor activity. - Nucl. Acids Res. 42: 599–608, 2014.CrossRefPubMedGoogle Scholar
  47. Venter, M., Botha, F.C.: Synthetic promoter engineering. - In: Pua, E.C., Davey, M.R. (ed.): Plant Development and. Biological and Biotechnological Perspectives. Vol. 1. Pp. 393–414. Springer-Verlag, Berlin - Heidelberg 2010.CrossRefGoogle Scholar
  48. Voinnet, O., Rivas, S., Mestre, P., Baulcombe, D.: An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. - Plant J. 33: 949–956, 2003.CrossRefPubMedGoogle Scholar
  49. Yang, Z., Patra, B., Li, R., Pattanaik, S., Yuan, L.: Promoter analysis reveals cis regulatory motifs associated with the expression of the WRKY transcription factor CrWRKY1 in Catharanthus roseus. - Planta 238: 1039–1049, 2013.CrossRefPubMedGoogle Scholar
  50. Yoshida, T., Fujita, Y., Sayama, H., Kidokoro, S., Maruyama, K., Mizoi, J., Shinozaki, K., Yamaguchi-Shinozaki, K.: AREB1, AREB2, and ABF3 are master transcription factors that cooperatively regulate ABRE-dependent ABA signaling involved in drought stress tolerance and require ABA for full activation. - Plant J. 61: 672–685, 2010.CrossRefPubMedGoogle Scholar
  51. Zhang, B., Wang, O., Qin, J., Liu, S., Sun, S., Liu, H., Kuang, J., Jiang, G., Zhang, W.: cis-acting elements and trans-acting factors in the transcriptional regulation of Raf kinase inhibitory protein expression. - PLoS ONE 8: e83097, 2013.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© The Institute of Experimental Botany 2017

Authors and Affiliations

  • G. S. Duraisamy
    • 1
  • A. K. Mishra
    • 1
  • T. Kocábek
    • 1
  • J. Matoušek
    • 1
    Email author
  1. 1.Institute of Plant Molecular BiologyBiology Centre of the Academy of Sciences of the Czech RepublicČeské BudějoviceCzech Republic

Personalised recommendations