SDS-PAGE profile of purified recombinant proteins.

<p>SDS-PAGE (10%) of purified recombinant proteins from <i>E.coli</i> BL-21 transformed with pGEX-<i>WsCPR1</i> and pGEX-<i>WsCPR2</i>. Lane 1; Standard protein markers, Lane 2&3; Cell lysate (CL) of WsCPR1 and WsCPR2 expressing cells remained after incubation with GST-beads, Lane 4; Purified recombinant GST-fused WsCPR1, Lane 5; Purified recombinant GST fused WsCPR2, Lane 6; Purified WsCPR1 after removal of GST using thrombin and Lane 7; Purified CPR2 after removal of GST.</p

Phylogenetic analysis of deduced amino acid sequences.

<p>Phylogeny of <i>WsCPRs</i> was inferred using the Neighbor-joining method using MEGA 5 software. A total of 33 protein sequences used for analysis were from following plant species: <i>Withania somnifera</i> (WsCPR1: HM036710, WsCPR2: GU808569), <i>Petunia hybrida</i> (CPR1: DQ099544, CPR2: DQ099545); <i>Petroselinum crispum</i> (CPR1: AF024635, CPR2: AF024634); <i>Gossipium hirsutum</i> (CPR1: FJ719368, CPR2: FJ719369); <i>Populus trichocarpa</i> (CPR1: XM_002307300, CPR2: XM_002329600, CPR3: AF302498); <i>Arabidopsis thaliana</i> (CPR1: X66016, CPR2: X66017); <i>Capsicum annuum</i> (EU616557); <i>Vigna radiata</i> (P37116); <i>Vicia sativa</i> (Z26252); <i>Stevia rebaudiana</i> (DQ269454); <i>Ricinus communis</i> (XM_002514003); <i>Pisum sativum</i> (AF002698); <i>Picrorhiza kurroo</i>a (JN968968); <i>Artemisia annua</i> (EF104642); <i>Papaver somniferum</i> (U67185); <i>Taxus cuspidate</i> (AY571340); <i>Taxus chinensis</i> (AY959320); <i>Perilla frutescens</i> (GQ120439); <i>Ophiorrhiza pumila</i> (AB086169); <i>Medicago truncatula</i> (XM_003610061); <i>Lotus japonicas</i> (AB433810); <i>Catharanthus roseus</i> (Q05001); <i>Centaurium erythraea</i> (AY596976); <i>Zea mays</i> (CAC83301); <i>Triticum aestivum</i> (AGC27711) and <i>Eschscholzia californica</i> (U67186). All CPRs were grouped into two clusters where the WsCPR1 and WsCPR2 confined to their corresponding cluster like other CPRs.</p

Tissue-specific real-time expression analysis.

<p>Quantitative assessment of the expression of <i>WsCPR1</i> and <i>WsCPR2</i> in different tissues of <i>Withania somnifera.</i> Data were compared and analysed with analysis of variance (<i>ANOVA</i>). Values are means, with standard errors indicated by bars, representing three independent biological samples, each with three technical replicates. Differences were scored as statistical significance at *<i>p</i><0.05 and **<i>p</i><0.01 levels.</p

Time course effect of elicitor treatments on withanolides accumulation.

<p>10A: Effect of methyl jasmonate (MeJA) treatment on withanolides accumulation at different time intervals. HPLC analysis demonstrated the change in three key withanolides of withanolide A (WS-1), withanone (WS-2) and withaferin A (WS-3) at 6, 12, 24 and 48 h after treatments of micro-shoots with 0.1 mM MeJA. WS-3 was observed to be enhanced more with respect to WS-1 while WS-2 was detected in sample harvested after 48 h. All values obtained were means of triplicate with standard errors. Time course accumulation of WS-1 and WS-3 was statistically significant at <i>p</i><0.01 level. 10B: Effect of salicylic acid (SA) on withanolide accumulation at different time interval. The WS-3 level was also up-regulated in salicylic acid treated samples but WS-1 was enhanced more in comparison to methyl jasmonate (MeJA) treated samples. All values obtained were means of triplicate with standard errors. Time course accumulation of WS-1 and WS-3 was statistically significant at <i>p</i><0.001 level.</p

Three dimensional models and conserved residue prediction for WsCPR1 and WsCPR2.

<p>4A & 4D: Cartoon display of the 3-D structures of WsCPR1 <i>and</i> WsCPR2 as predicted by Phyre² using crystal structure of <i>Rattus norvagicus</i> (PDB ID: 1J9Z) as template. 4B & 4E: Predicted ligand (shown in green) binding sites as predicted by 3DLigandSite Web Server. 4C & 4F: Conserved residue analysis of <i>WsCPR1 and WsCPR2</i> were performed using Consurf and Conseq web servers. Residue conservation from variable to conserve is shown in blue (1) to violet (9). The residues involved in substrate binding and active site are shown in the center core of the structure.</p

An overview of putative withanolide biosynthesic pathway.

<p>DXP: 1-deoxy-D-xylulose 5-phosphate, HMBDP: 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate, IPP:Isopentylpyrophosphate, DMPP: Dimethylalyl diphosphate, IPP isomerase: Isopentylpyrophosphate isomerase, FPPS: farnesyldiphosphate synthase, SQS: Squalene synthase, SQE/CPR: Squalene epoxidase/cytochrome P450 reductase, CAS: Cycloartinol synthase, SMT-1: Sterol methyl transferase/cytochrome P450 reductase, ODM/CPR: Obtusifoliol-14-demethylase/cytochrome P450 reductase. First three highlighted (yellow) steps indicating involvement of P450 monooxygenases and CPR. Single dark arrows represent one step, two or more dark arrows represent multiple steps and dashed arrow represents unknown steps. Below the pathway: chemical structure of important withanolides.</p

List of primers used in the study.

*<p>Primers marked with star were provided with the kits.</p

Southern blot analysis of genomic DNA.

<p>A&B: Southern blot analysis of total DNA using <i>WsCPR1</i> and <i>WsCPR2</i> as a probe. Total DNA (30 µg) isolated from <i>Withania somnifera</i> was digested with the indicated restriction enzymes, The digested samples were electrophoresed on 0.8% agarose gel, blotted onto nylon membrane and subjected to hybridisation using DIG-labelled ORF of <i>WsCPR1</i> and <i>WsCPR2</i> as probes. First lane contains molecular markers with indicated molecular weight on the left side.</p

NADPH-Cytochrome P450 Reductase: Molecular Cloning and Functional Characterization of Two Paralogs from <em>Withania somnifera</em> (L.) Dunal

<div><p><i>Withania somnifera</i> (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 <i>W. somnifera</i>. The full length paralogs of <i>WsCPR1</i> and <i>WsCPR2</i> 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 <i>Escherichia coli</i> as glutathione-<i>S</i>-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 <i>Withania</i> 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 <i>WsCPR2</i> in comparison to <i>WsCPR1</i>. Similar to CPRs of other plant species, <i>WsCPR1</i> was un-inducible while <i>WsCPR2</i> 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 <i>WsCPR2</i> in withanolide biosynthesis. Present investigation so far is the only report of characterization of CPR paralogs from <i>W. somnifera</i>.</p> </div

A schematic diagram of the proposed picroside pathway in <i>Picrorhiza kurrooa.</i>

<p>Picrosides are derived from geranyl diphosphate that can be synthesized both from cytoplasmic MVA and plastidic MEP pathways. CPR: cytochrome P450 reductase; G10H: geraniol 10-hydroxylase; 10 HGO: 10-hydroxygeraniol oxidoreductase; IRS: Iridoid synthase; PAL: phenylalanine ammonia-lyase; C4H: cinnamoyl 4-hydroxylase; Route I: The route leading to the biosynthesis of secologanin, Route II: The route leading to the biosynthesis to picrosides <i>via</i> catalpol. Adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073804#pone.0073804-Damtoft1" target="_blank">[62]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073804#pone.0073804-Mahmoud1" target="_blank">[63]</a>.</p