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

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.

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

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.

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.

    Article  PubMed  CAS  Google Scholar 

  2. Austin, M.B., Noel, J.P.: The chalcone synthase superfamily of type III polyketide synthases. - Nat. Prod. Rep. 20: 79–110, 2003.

    Article  PubMed  CAS  Google 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.

    Article  PubMed  PubMed Central  CAS  Google 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.

    Article  PubMed  CAS  Google 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.

    Article  PubMed  CAS  Google 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.

    Article  Google Scholar 

  7. Downer, E.J., Campbell, V.A.: Phytocannabinoids CNS cells and development: a dead issue?. - Drug Alcohol Rev. 29: 91–98, 2010.

    Article  PubMed  Google 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.

    Article  PubMed  CAS  Google 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.

    Article  PubMed  PubMed Central  CAS  Google 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.

    Article  PubMed  CAS  Google 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.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Flores-Sanchez, I.J., Verpoorte, R.: Secondary metabolism in cannabis. - Phytochem. Rev. 7: 615–639, 2008.

    Article  CAS  Google 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.

    Article  PubMed  Google 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.

    Article  PubMed  PubMed Central  CAS  Google 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.

    Article  PubMed  CAS  Google 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.

    Article  PubMed  CAS  Google 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.

    Article  PubMed  CAS  Google 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.

    Article  CAS  Google 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.

    Article  PubMed  CAS  Google Scholar 

  21. Hillig, K.W.: Genetic evidence for speciation in Cannabis (Cannabaceae). - Genet. Resour. Crop Evol. 52: 161–182, 2005.

    Article  CAS  Google 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.

    Article  PubMed  PubMed Central  CAS  Google 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.

    PubMed  PubMed Central  CAS  Article  Google 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.

    Article  CAS  Google 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.

    Article  PubMed  PubMed Central  CAS  Google 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.

    Article  PubMed  CAS  Google 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.

    Article  PubMed  PubMed Central  CAS  Google 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.

    Article  CAS  Google 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.

    Article  PubMed  CAS  Google 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.

    Article  PubMed  PubMed Central  CAS  Google 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.

    Article  CAS  Google 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.

    Article  CAS  Google 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.

    Article  CAS  Google 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.

    Article  PubMed  CAS  Google 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.

    Article  CAS  Google 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.

    Article  PubMed  CAS  Google 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.

    Article  Google Scholar 

  38. Shimada, S., Otsuki, H., Sakuta, M.: Transcriptional control of anthocyanin biosynthetic genes in the Caryophyllales. - J. exp. Bot. 58: 957–967, 2006.

    Article  PubMed  CAS  Google 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.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. Small, E., Cronquist, A.: A practical and natural taxonomy for Cannabis. - Taxon 25: 405–435, 1976.

    Article  Google 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.

    Article  PubMed  PubMed Central  CAS  Google 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.

    Article  PubMed  PubMed Central  CAS  Google 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.

    Article  PubMed  CAS  Google 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.

    Article  Google 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.

    Article  PubMed  CAS  Google 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.

    Google 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.

    Article  PubMed  CAS  Google 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.

    Article  PubMed  CAS  Google 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.

    Article  PubMed  CAS  Google 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.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

Author information

Affiliations

Authors

Corresponding author

Correspondence to J. Matoušek.

Additional information

Acknowledgements: The work was supported by Czech Science Foundation project (GACR 13-03037S) and by Institutional support RVO: 60077344. The first two authors equally contributed to this work.

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Duraisamy, G.S., Mishra, A.K., Kocábek, T. et al. Activation of polyketide synthase gene promoter in Cannabis sativa by heterologous transcription factors derived from Humulus lupulus. Biol Plant 62, 250–260 (2018). https://doi.org/10.1007/s10535-017-0766-z

Download citation

Additional key words

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