Norcholanic acids as substrates for recombinant 3β-hydroxysteroid dehydrogenase and progesterone 5β-reductase, enzymes of the 5β-cardenolide biosynthesis

Swarnkar

et al. 2016

Sci Rep

Calotropis procera is a medicinal plant of immense importance due to its pharmaceutical active components, especially cardiac glycosides (CG). As genomic resources for this plant are limited, the genes involved in CG biosynthetic pathway remain largely unknown till date. Our study on stage and tissue specific metabolite accumulation showed that CG’s were maximally accumulated in stems of 3 month old seedlings. De novo transcriptome sequencing of same was done using high throughput Illumina HiSeq platform generating 44074 unigenes with average mean length of 1785 base pair. Around 66.6% of unigenes were annotated by using various public databases and 5324 unigenes showed significant match in the KEGG database involved in 133 different pathways of plant metabolism. Further KEGG analysis resulted in identification of 336 unigenes involved in cardenolide biosynthesis. Tissue specific expression analysis of 30 putative transcripts involved in terpenoid, steroid and cardenolide pathways showed a positive correlation between metabolite and transcript accumulation. Wound stress elevated CG levels as well the levels of the putative transcripts involved in its biosynthetic pathways. This result further validated the involvement of identified transcripts in CGs biosynthesis. The identified transcripts will lay a substantial foundation for further research on metabolic engineering and regulation of cardiac glycosides biosynthesis pathway genes.

mentioning

confidence: 99%

Transcriptome and Metabolite analysis reveal candidate genes of the cardiac glycoside biosynthetic pathway from Calotropis procera

Swarnkar

et al. 2016

Sci Rep

In Vitro Cell.Dev.Biol.-Plant

“…Cardenolide extraction and determination Cardenolides were extracted from plant and shoot samples as described by Wiegrebe and Wichtl (1993). The extracts were analyzed by HPLC according to Wichtl et al (1982) with slight modifications (Schebitz et al 2010). For HPLC analysis, a Waters 1525 Binary HPLC Pump system with a Waters 2487 Dual CTC AGA CGT AAC GCT GGT TGT A k Absorbance Detector was employed (Waters GmbH, Eschborn, Germany).…”

Section: Methodsmentioning

confidence: 99%

Cardenolide and glucosinolate accumulation in shoot cultures of Erysimum crepidifolium Rchb.

Horn

Kemmler

Kreis

et al. 2020

Self Cite

Erysimum crepidifolium Rchb. is one of the few Brassicaceae species accumulating glucosinolates as well as cardenolides. This is possibly providing a selective advantage in evolution as both compounds are part of a chemical defense system. In order to study the biosynthesis of these compounds, a regeneration protocol for E. crepidifolium using in vitro shoot cultures derived from seeds has been developed. Murashige and Skoog (MS) culture medium supplemented with various combinations of cytokinins and auxins was used. MS medium containing NAA (naphthaleneacetic acid, 0.04 mg mL−1) and BAP (6-benzylaminopurine, 0.2·10−2 mg mL−1) proved to be optimal for root formation. Plantlets developed well on modified MS medium without the use of phytohormones. About 80% of the plantlets rooted in vitro developed into intact plants after transfer to the greenhouse. Cardenolides (1.75 mg g−1 dry weight (DW)) were detected in cultured shoots on solid DDV media while glucosinolates mainly accumulated in roots where 0.025 mg g−1 FW were detected in shoots cultured on the same medium (DDV). The expression of two progesterone 5β-reductase and three Δ5-3β-hydroxysteroid dehydrogenase genes were measured in shoot cultures since the encoded enzymes are supposed to be involved in cardenolide biosynthesis. E. crepidifolium shoot cultures propagated on solid media meet the necessary requirements, i.e., clonal homogeneity, product accumulation, and gene expression, for a suitable model to study cardenolide but not glucosinolate biosynthesis.

“…However, the 3β-HSD activity seems not to be correlated with the cardenolide content as has already been demonstrated [6,7]. The recombinant pQ3β-HSD (derived from a D. lanata cDNA) exhibits 3α-, 3β-and 17β-dehydrogenase as well as 3/17-keto reductase activities and acts on a large variety of substrates including testosterone (C 19 steroids) and norcholanic acids (C 23 steroids) [18,19]. Hence, the role of 3β-HSD in cardenolide metabolism is still a matter of debate.…”

mentioning

confidence: 99%

Expression of3β‐HSDandP5βR, Genes Respectively Coding for Δ⁵-3β-Hydroxysteroid Dehydrogenase and Progesterone5β‐Reductase, in Leaves and Cell Cultures ofDigitalis lanataEHRH

Ernst

Pádua

Herl³

et al. 2010

Planta Med

Self Cite

Plants of the genus Digitalis produce 5 beta-cardenolides that are used in the therapy of cardiac insufficiency in humans. 3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD) and progesterone 5 beta-reductase (P5 betaR) are both supposed to be important enzymes in the biosynthesis of these natural products. Activity and gene expression were demonstrated for both enzymes in cardenolide-accumulating leaves of Digitalis lanata but also in cardenolide-free permanent cell suspension cultures initiated from D. lanata leaf tissue. Enzyme activities were determined and quantified by HPLC and GC-MS methods. Expression of the respective genes, namely AY585867.1 (P5betaR gene) and DQ466890.1 (3beta-HSD gene), was made evident by real-time polymerase chain reaction (qPCR) analysis. We demonstrate for the first time that the P5betaR gene, encoding an enzyme described as a key enzyme in cardenolide biosynthesis, is also expressed in cardenolide-free tissues of cardenolide-containing plants.