Isolation of Δ5-3β-hydroxysteroid dehydrogenase involved in the biosynthetic pathway of cardenolides and its expression level under the influence of salicylic acid and methyl jasmonate elicitors in foxglove (Digitalis nervosa L)

Document Type : Research Paper

Authors

1 Assist. Prof., Department of Agronomy and Plant Breeding, University of Kurdistan, Sanandaj, I.R. Iran

2 M.Sc., Agricultural Biotechnology, University of Kurdistan, Sanandaj, I.R.Iran

3 Assoc. Prof., Department of Agronomy and Plant Breeding, University of Kurdistan, Sanandaj, I.R.Iran

Abstract

Foxglove plants are important sources of medicinal cardiac glycosides compounds such as digoxin and digitoxin, which are used for treatment of congestive heart failure and cardiac arrhythmia. Digitalis has several valuable species but the only species native to Iran is digitalis nervosa. Considering the importance of study on native plants in order to be used in pharmaceutical industry this research was carried out. One of the key genes in the biosynthesis of digitalis cardenolide in plants is 3β-HSD, which encodes the enzyme 3β-HSD with important role in early stage of the cardenolide biosynthesis pathway. Purpose of the present study was to determine nucleotide sequence of 3β-HSD, phylogenetic analyses and its expression under influence of salicylic acid and methyl jasmonate elicitors in different tissues. Results revealed that a sequence encoding the enzyme show very close affinity to Digitalis lanata. In addition expression pattern of 3β-HSD in the transcript level revealed that a significant expression level was detected in leaf tissue, and also under treatment of methyl jasmonate and salicylic acid the expression level raised. Findings of our study could be used to increase cardenolide through metabolic engineering and genetic manipulation.

Keywords

Main Subjects


-      Âzadbakht, M. and Ghasemi Dehkordi, N., 2001. Identification of cardiac glycosides of Iranian Digitalis species by HPLC. The Journal of Mazandaran Univercity of Medical Sciences, 11: 25-31.
-      Dattner, A,M., 2003. From medical herbalism to phytotherapy in dermatology: back to the future. Dermatologic Therapy, 16: 106–113.
-      Ernst, M., de Pádua, R.M., Herl, V., Müller-Uri, F. and Kreis, W., 2010. Expression of 3β- HSD und P5βR, genes encoding for Δ5-3β-hydroxisteroid dehydrogenase (3β-HSD) and progesterone 5β-reductase (P5βR) in leaves and cell cultures of Digitalis lanata Ehrh. Planta Medicalogy, 76: 923-927
-      Finsterbusch, A., Lindemann, P., Grimm, R., Eckerskorn, C. and Luckner, M., 1999. Delta(5)-3beta-hydroxysteroid dehydrogenase from Digitalis lanata Ehrh. - a multifunctional enzyme in steroid metabolism? . Planta, 209(4): 478-86.
-      Fong, H., 2002. Integration of herbal medicine into modern medical practices: issues and prospect. Integrative Cancer Therapies, 3: 287-293.
-      Fordyce, J.A. and Malcolm, S.B., 2000. Specialist weevil, Rhyssomatus lineaticollis, does not spatially avoid cardenolide defenses of common milkweed by ovipositing into pith tissue. Journal of Chemical Ecology, 26(12): 2857-2874.
-      Fraire-Velázquez, S., Rodríguez-Guerra, R. and Sánchez-Calderón, L., 2011. Abiotic and biotic stress response crosstalk in plants. 3–26. In: Shanker, A.K. and Venkateswarlu, B., (Eds). Abiotic stress response in plants- physiological, biochemical and genetic perspectives. In Tech, Rijeka, 358p.
-      Fujii, Y., Ikeda,Y. and Yamazaki, M., 1994. High-performance liquid chromatography determination of lanatosides in Digitalis lutea and Digitalis ambigua leaves. Journal Liquid Chromatogram, 17: 4451-4461.
-      Heinrich, M., Barnes, J., Gibbons, S. and Williamson, E.M., 2004. Fundamentals of Pharmacognosy and Phytotherapy. Edinburgh, 336pp.
-      Herl, V., Frankenstein, J., Meitinger, N., Müller-Ur,i F. and Kreis, W., 2007. ∆5-3β- hydroxysteroid dehydrogenase (3β-HSD) from Digitalis lanata. Heterologous expression and characterization of the recombinant enzyme. Planta Medicalogy, 73: 704-710.
-      Hoelz, H., Kreis, W., Haug, B., and Reinhard, E., 1992. Storage of cardiac glycosides in vacuoles of Digitalis lanata mesophyll cells. Phytochemistry, 31: 1167-71.
-      Huseini, H., Larijani, B., Radjabipour, B. and Mohsin, R., 2006. The Efficacy of Silybum marianum (L.) Gaertn. (Silymarin) in the Treatment of Type II Diabetes: A Randomized, Double-blind, Placebo-controlled, Clinical Trial. Phytotherapy Research, 20: 1036–1039.
-      Kreis, W., Hensel, A. and Stuhlemmer, U., 1998. Cardenolide Biosynthesis in Foxglove. Planta Medicalogy, 64: 491-99.
-      Kreis, W., 2007. Δ5-3β-hydroxysteroid dehydrogenase (3β-HSD) from Digitalis lanata. Heterologous expression and characterisation of the recombinant enzyme. Planta Medicalogy, 73: 704-710.
-      Lin, C.C.,Yang, C.C., Phua, D.H., Deng, J.F. and Lu, L.H., 2010. An outbreak of foxglove leaf poisoning. Journal of the Chinese Medical Association, 73:97–100.
-      Lopez-Lazaro, M., Palma De La Pena, N., Pastor N., Martin-Cordero, C., Navarro, E., Cortes, F. and Ayuso, MJ., 2003. Anti-tumour activity of Digitalis purpurea L. subsp. heywoodii. Planta Medicalogy, 69: 701-704.
-      Maffe, S., Cucchi, L., Zenone, F., Bertoncelli, C., Beldi, F., Colombo, M.L., Bielli, M., Paino, A.M., Parravicini, U,, Paffoni, P., Dellavesa, P., Perucca, A., Pardo, N.F., Signorotti, F., Didino, C. and Zanetta, M., 2009. Digitalis must be banished from the table: a rare case of acute accidental Digitalis intoxication of a whole family. Journal of Cardiovascular Medicine, 10:727–732.
-      Mohammed, A., Yücesan, B., Demir-Ordu, Ö., Cihangir, C., Eker, I., Kreis, W. and Gürel, E., 2015. In vitro regeneration and cardenolide determination of an endemic foxglove, Digitalis cariensis (Aegean Foxglove). In Vitro Cellular and Developmental Biology - Plant, 51: 438-444.
-      Munkert, J., Ernst, M., Muller-Uri, F. and Kreis, W., 2014. Identification and stress-induced expression of three 3b-hydroxysteroid dehydrogenases from Erysimum crepidifolium Rchb. and their putative role in cardenolide biosynthesis. Phytochemistry, 100: 26–33.
-      Newman, R., Yang, P. and Pawlus, A., 2008. Cardiac glycosides as novel cancer therapeutic agents. Molecular Physiology, 8: 36-49.
-      Paschold, A., Halitschke, R. and Baldwin, I.T., 2007. Co(i)-ordinating defenses: NaCOI1 mediates herbivore induced resistance in Nicotiana attenuata and reveals the role of herbivore movement in avoiding defenses. The Plant Journal, 51: 79-91.
-      Perez-Bermudez, P., Garcia, A.A., Tunon, I. and Gavidia, I., 2010. Digitalis purpurea P5 beta R2, encoding steroid 5 beta-reductase, is a novel defense-related gene involved in cardenolide biosynthesis. New Phytologist, 185: 687–700.
-      Piotr, C. and Sacchi, N., 2006. The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on. Nature Protocols, 1: 581-585.
-      Popova, L., Pancheva T. and Uzunova, A., 1997. Salicylic acid: properties, biosynthesis and physiological role. Bulgarian Journal of Plant Physiology, 23: 85–93.
-      Pu, G.B., Dong-Ming, M., Chen, J.L., Ma, L.Q., Wang, H., Li, G.F. and Ye, H.C., 2009. Salicylic acid activates artemisinin biosynthesis in Artemisia annua L. Plant Cell Reports, 28:1127–1135.
-      Reymond, P. and Farmer, E.E., 1998. Jasmonate and salicylate as global signals for defense gene expression. Current Opinion in Plant Biology, 1(5): 404–411.
-      Rosen, S. and Skaletsky, H., 2000. Primer3 on the WWW for general users and for biologist programmers. 365-386. In: Misener, S. and Krawetz, S.A., (Eds). Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, New Jersey, 460p.
-      Salem, ML. and Hossain, MS., 2000. Protective effect of black seed oil from Nigella sativa against murine cytomegalovirus infection. International Journal of Immunopharmacology, 22(9): 729-740.
-      Samuelson, G., 2004. Drugs of Natural Origin, A textbook of Pharmacognosy. Swedish Pharmaceutical Press, Stockholm, 620pp.
-      Seidel, S., Kreis, W. and Reinhard, E., 1990. Δ5-3β-hydroxysteroid dehydrogenase Δ5/Δ4-ketosteroid isomerase (3β-HSD), a possible enzyme of cardiac glycoside biosynthesis, in cell cultures and plants of Digitalis lanata Ehrh. Plant Cell Report, 8: 621-24.
-      Smith, J.L., De Moraes, C.M. and Mescher, M.C., 2009. Jasmonate- and salicylate-mediated plant defense responses to insect herbivores, pathogens and parasitic plants. Pest Managment Science, 65: 497–503.
-      Sun, J., Xiao, J., Wang, X., Yuan, X. and Zhao, B., 2012. Improved cardenolide production in Calotropis gigantea hairy roots using mechanical wounding and elicitation. Biotechnology Letters, 34(3): 563-569.
-      Tamura, K., Dudley, J., Nei, M. and Kumar, S., 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution, 24: 1596-1599.
-      Thompson, J.D., Higgins, D.G. and Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22(22): 4673-80.
-      Trease, G.E. and Evans, W.C., 1998. Trease and Evans Pharmacognosy. Saunders Ltd., Lodon, 616pp.
-      Tripathi, L. and Tripathi, J. N., 2003. Role of biotechnology in medicinal plants. Tropical Journal of Pharmaceutical Research, 2(2): 243-253.
-      Warren, B., 2005. Digitalis purpurea. The American Journal of Cardiology, 95(4): 544.
-      Wu, B., Li, Y., Yan, H., Ma, Y., Luo, H., Yuan, L., Chen, S. and Lu, S., 2012. Comprehensive transcriptome analysis reveals novel genes involved in cardiac glycoside biosynthesis and mlncRNAs associated with secondary metabolism and stress response in Digitalis purpurea. BMC Genomics, 13:15.
-      Yeh, J., Hunag, W., Kan, S. and Wang, P., 2001. Inhibitory effects of Digitalis on the proliferation of androgen dependent and independent prostate cancer cells. Urology, 166: 1937-1942.
Zhao, J., Davis, L. and Verpoorte, R., 2005. Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnology Advances, 23(4): 283–333.