Figure 1.
A schematic diagram of the proposed picroside pathway in Picrorhiza kurrooa.
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 via catalpol. Adapted from [62], [63].
Figure 2.
Phylogenetic tree of deduced amino acid sequences of UGT86C4 and UGT94F2.
Phylogeny of UGTs was inferred using neighbour-joining method of the MEGA 5.1 software. A total of 20 protein sequences used for analysis were from following plant species: Picrorhiza kurrooa UGT94F2 (JQ996409), Picrorhiza kurrooa UGT86C4 (JQ996408), Arabidopsis thaliana UDP-glycosyltransferase 86A1 (AAM91353), Linum usitatissimum UDP-glycosyltransferase 1 (AFJ53011), Dianthus caryophyllus UDP-glucosyltransferase (BAF75888), Lycium barbarum UDP-glucose:glucosyltransferase (BAG80548), Gardenia jasminoides UDP-glucose glucosyltransferase (BAK55737) Catharanthus roseus UDP-glucose iridoid glucosyltransferase (BAK55749), Medicago truncatula cytokinin-O-glucosyltransferase (XP_003615832), Veronica persica flavonoid glycosyltransferase UGT94F1 (BAI44133), Gardenia jasminoides UDP-glucose glucosyltransferase (BAM28984), Catharanthus roseus UDP-glucose:flavonoid glucoside 1,6-glucosyltransferase (BAH80312), Panax notoginseng UDP-glycosyltransferase (AED99884), Sesamum indicum UDP-glucose:sesaminol 2′-O-glucoside-O-glucosyltransferase (BAF99027), Bupleurum chinense glycosyltransferase UGT4 (AFK79036), Zea mays anthocyanidin 3-O-glucosyltransferase (ACG26485), Ginkgo biloba UDP-glucose:flavonoid glucosyltransferase (AEQ33588), Triticum urartu flavanone 7-O-glucoside 2′′-O-beta-L-rhamnosyltransferas (EMS55799), Medicago truncatula anthocyanidin 3-O-glucosyltransferase (AES75297), Vitis vinifera UDP-glycosyltransferase 86A1(XP_002276858).
Figure 3.
Three dimensional models and conserved residue prediction for UGT86C4 and UGT94F2.
A and B: Ribbon display of the 3-D structures of UGT86C4 and UGT94F2 as predicted by PHYRE2 web server, using crystal structure Arabidopsis thaliana UGT72B1 (Protein Data Bank (PDB) Id. 2VCH-A) as template for modeling of both the proteins. N and C-terminal domains are shown. Donor and acceptor sites are also shown. C and D: Predicted ligand binding sites as predicted by 3DLigandSite web server. E and F: Evolutionary conserved residue analysis of UGT86C4 and UGT94F2 were performed using Consurf, an empirical Bayesian inference based web server. Residue conservation from variable to conserved is shown in blue (1) to violet (9). The residues involved in binding of the donor moieties are shown in the centre of the structures.
Figure 4.
Diagram showing (A) kaempferol (B) naringenin (C) apigenin (D) 7-deoxyloganetin (E) 7-deoxyloganetic acid and (F) iridotrial docked into the proposed binding pockets of UGT86C4.
Figure 5.
Diagram showing (A) kaempferol (B) naringenin (C) apigenin (D) 7-deoxyloganetin (E) 7-deoxyloganetic acid and (F) iridotrial docked into the proposed binding pockets of UGT94F2.
Figure 6.
2-D representation of interactions of UGT86C4 and UGT94F2.
2-D representation of the interaction figure (in pink) of kaempferol with UGT86C4 (A), and 2-D interaction figure of 7-deoxyloganetin with UGT94F2 (B).
Table 1.
Binding affinity of ligand data set on modeled structures of UGT86C4 and UGT94F2.
Figure 7.
Nucleotide sequences of Picrorhiza UGT gene promoters.
Nucleotide sequences of the UGT86C4 (A) and UGT94F2 (B) gene promoters. Numbering starts from the predicted transcription start site (dark green shaded). The putative core promoter consensus sequences and the motifs with significant similarity to the previously identified cis-acting elements are shaded and the names are given.
Table 2.
Putative cis-acting regulatory elements identified in the promoter of UGT86C4 and UGT94F2, using PLACE (http://www.dna.affrc.go.jp/PLACE) and PlantCare databases (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).
Figure 8.
Tissue-specific real-time expression analysis and time-course effect of elicitor treatments on UGT86C4 and UGT94F2.
Tissue-specific expression of UGT86C4 and UGT94F2 (A), time-course effect of methyl jasmonate (MeJA) (B), salicylic acid (SA) (C) and 2, 4-dichlorophenoxyacetic acid (2,4-D) (D) on the expression of UGT86C4 and UGT94F2. Data were compared and analysed with analysis of variance (ANOVA) test. 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 the *P<0.05 and **P<0.01 levels.
Figure 9.
Tissue-specific and time-course effect of elicitor treatments on picroside accumulation.
HPLC chromatogram of standard (picroside I and picroside II) at 270 nm (A). HPLC chromatogram of leaf sample showing peaks of picroside I and picroside II (B). Tissue-specific accumulation of picrosides (PK-I and PK-II) in leaves, inflorescence and rhizomes of P. kurrooa (C). Effect of methyl jasmonate (MeJA) (B), salicylic acid (SA) (C) and 2, 4-dichlorophenoxyacetic acid (2, 4-D) (D) treatments on picrosides accumulation at different time intervals (D). HPLC analysis demonstrated the change in two key picrosides, PK-I and PK-II at 12 and 24 h after treatments of mature leaves. All values obtained were means of triplicate with standard errors. Time course accumulation of PK-I and PK-II was statistically significant at p<0.001 level.