Figure 1.
An overview of putative withanolide biosynthesic pathway.
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.
Table 1.
List of primers used in the study.
Figure 2.
Multiple sequence alignment of deduced amino acid sequences of WsCPR1 and WsCPR2 with other CPR homologs using ClustalX.
CPRs used for alignment were from Withania somnifera (WsCPR1: HM036710, WsCPR2: GU808569), Petunia hybrida CPR1:DQ099544, CPR2 DQ099545); Petroselinum crispum (CPR1:AF024635, CPR2:AF024634); Gossipium hirsutum (CPR1: FJ719368, CPR2:FJ719369); Populus trichocarpa (CPR1:XM_002307300, CPR2:XM_002329600, CPR3: AF302498); Arabidopsis thaliana (CPR1:X66016, CPR2: X66017). Typical conserved membrane anchor, cytochrome P450-, FMN, FAD and NADPH-binding domains characteristic for the CPR are highlighted grey in colour and underlined.
Figure 3.
Phylogenetic analysis of deduced amino acid sequences.
Phylogeny of WsCPRs 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: Withania somnifera (WsCPR1: HM036710, WsCPR2: GU808569), Petunia hybrida (CPR1: DQ099544, CPR2: DQ099545); Petroselinum crispum (CPR1: AF024635, CPR2: AF024634); Gossipium hirsutum (CPR1: FJ719368, CPR2: FJ719369); Populus trichocarpa (CPR1: XM_002307300, CPR2: XM_002329600, CPR3: AF302498); Arabidopsis thaliana (CPR1: X66016, CPR2: X66017); Capsicum annuum (EU616557); Vigna radiata (P37116); Vicia sativa (Z26252); Stevia rebaudiana (DQ269454); Ricinus communis (XM_002514003); Pisum sativum (AF002698); Picrorhiza kurrooa (JN968968); Artemisia annua (EF104642); Papaver somniferum (U67185); Taxus cuspidate (AY571340); Taxus chinensis (AY959320); Perilla frutescens (GQ120439); Ophiorrhiza pumila (AB086169); Medicago truncatula (XM_003610061); Lotus japonicas (AB433810); Catharanthus roseus (Q05001); Centaurium erythraea (AY596976); Zea mays (CAC83301); Triticum aestivum (AGC27711) and Eschscholzia californica (U67186). All CPRs were grouped into two clusters where the WsCPR1 and WsCPR2 confined to their corresponding cluster like other CPRs.
Figure 4.
Three dimensional models and conserved residue prediction for WsCPR1 and WsCPR2.
4A & 4D: Cartoon display of the 3-D structures of WsCPR1 and WsCPR2 as predicted by Phyre2 using crystal structure of Rattus norvagicus (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 WsCPR1 and WsCPR2 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.
Figure 5.
Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) of heterologously expressed WsCPR1 and WsCPR2.
5A: SDS-PAGE (10%) pattern of proteins obtained from E.coli BL21 (DE3) transformed with pGEX-WsCPR1 and pGEX-WsCPR2. Lane 1 & 5; Standard protein markers. Lane 2 & 7; Whole cell lysate of uninduced WsCPR1 and WsCPR2, Lane 3–6 & 10–13; Cell lysate of WsCPR1 and WsCPR2 induced with 0.8 mM IPTG harvested after 2 h, 4 h, 6 h and 8 h respectively. Lane 7 & 14; Cell lysate of E. coli BL21 (DE3) cells containing the empty vector pGEX-4T2 obtained at 4 h post-induction with 0.8 mM IPTG. 5B: The SDS-PAGE (10%) profile of optimized WsCPR2 expression at 25°C. In different lanes the samples were harvested at different time periods as indicated above. The highest expression was observed with 0.8 mM IPTG after 12 h of induction.
Figure 6.
SDS-PAGE profile of purified recombinant proteins.
SDS-PAGE (10%) of purified recombinant proteins from E.coli BL-21 transformed with pGEX-WsCPR1 and pGEX-WsCPR2. 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.
Figure 7.
Southern blot analysis of genomic DNA.
A&B: Southern blot analysis of total DNA using WsCPR1 and WsCPR2 as a probe. Total DNA (30 µg) isolated from Withania somnifera 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 WsCPR1 and WsCPR2 as probes. First lane contains molecular markers with indicated molecular weight on the left side.
Table 2.
Specific activities and of kinetic constants of WsCPR1 and WsCPR2.
Figure 8.
Tissue-specific real-time expression analysis.
Quantitative assessment of the expression of WsCPR1 and WsCPR2 in different tissues of Withania somnifera. Data were compared and analysed with analysis of variance (ANOVA). 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 *p<0.05 and **p<0.01 levels.
Figure 9.
9. Time course effect of elicitor treatments on expression profiles of WsCPRs paralogs. 9A: Time courses of WsCPR1 and WsCPR2 expression in micropropagated Withania somnifera induced by methyl jasmonate (MeJA; 0.1 mM) and salicylic acid (SA; 0.1 mM) treatments. The numbers above indicate the different time points in hours. MeJA and SA were added to the micro-propagated plantlets precultured for 2 wk on Hoagland’s medium. Total RNA from each sample was used for quantitation. Actin was kept as internal control. 9B: Expression profiles obtained by densitometric quantification of band intensities. Experiments were performed in triplicate with similar results; error bars indicate ± standard deviation of the mean. IOD, integrated optical density; A.U., arbitrary units.
Figure 10.
Time course effect of elicitor treatments on withanolides accumulation.
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 p<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 p<0.001 level.