Abstract
Withania somnifera (L.) Dunal, a highly reputed medicinal plant, synthesizes a large array of steroidal lactone triterpenoids called withanolides. Although its chemical profile and pharmacological activities have been studied extensively during the last two decades, limited attempts have been made to decipher the biosynthetic route and identification of key regulatory genes involved in withanolide biosynthesis. Cytochrome P450 reductase is the most imperative redox partner of multiple P450s involved in primary and secondary metabolite biosynthesis. We describe here the cloning and characterization of two paralogs of cytochrome P450 reductase from W. somnifera. The full length paralogs of WsCPR1 and WsCPR2 have open reading frames of 2058 and 2142 bp encoding 685 and 713 amino acid residues, respectively. Phylogenetic analysis demonstrated that grouping of dual CPRs was in accordance with class I and class II of eudicotyledon CPRs. The corresponding coding sequences were expressed in Escherichia coli as glutathione-S-transferase fusion proteins, purified and characterized. Recombinant proteins of both the paralogs were purified with their intact membrane anchor regions and it is hitherto unreported for other CPRs which have been purified from microsomal fraction. Southern blot analysis suggested that two divergent isoforms of CPR exist independently in Withania genome. Quantitative real-time PCR analysis indicated that both genes were widely expressed in leaves, stalks, roots, flowers and berries with higher expression level of WsCPR2 in comparison to WsCPR1. Similar to CPRs of other plant species, WsCPR1 was un-inducible while WsCPR2 transcript level increased in a time-dependent manner after elicitor treatments. High performance liquid chromatography of withanolides extracted from elicitor-treated samples showed a significant increase in two of the key withanolides, withanolide A and withaferin A, possibly indicating the role of WsCPR2 in withanolide biosynthesis. Present investigation so far is the only report of characterization of CPR paralogs from W. somnifera.
Highlights
Withania somnifera (L.) Dunal commonly known as ashwagandha or winter cherry, belongs to family Solanaceae
Plant P450s catalyse a diverse array of reactions during secondary metabolite synthesis which require involvement of their redox partner, NADPH:cytochrome P450 reductase [40]
Full length cDNAs of WsCPR1 and WsCPR2 genes were obtained from the leaf tissue of WS-3 rich chemo-variant by degenerate PCR and RACE methods (NCBI GenBank Acc No WsCPR1: HM036710 and WsCPR2: GU808569)
Summary
Withania somnifera (L.) Dunal commonly known as ashwagandha or winter cherry, belongs to family Solanaceae. Its versatile reproductive strategy of mixed mating and robust chemotypical variability enables it to thrive under marginal and xeric habitats with a wide geographic distribution [1,2]. It is a multipurpose medicinal plant, synthesizing large array of bioactive secondary metabolites. Most of the biological activities are attributed to naturally occurring C-28 steroidal lactone triterpenoids built on an intact or rearranged ergostane framework, in which C-22 and C-28 are oxidised to form a 6-membered lactone ring These steroidal triterpenoids known as withanolides have a structural resemblance with active constituents of Panax ginseng called gingosides
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