PkGPPS.SSU interacts with two PkGGPPS to form heteromeric GPPS in Picrorhiza kurrooa: Molecular insights into the picroside biosynthetic pathway.

Geranyl geranyl pyrophosphate synthase (GGPPS) is known to form an integral subunit of the heteromeric GPPS (geranyl pyrophosphate synthase) complex and catalyzes the biosynthesis of monoterpene in plants. Picrorhiza kurrooa Royle ex Benth., a medicinally important high altitude plant is known for picroside biomolecules, the monoterpenoids. However, the significance of heteromeric GPPS in P. kurrooa still remains obscure. Here, transient silencing of PkGGPPS was observed to reduce picroside-I (P-I) content by more than 60% as well as picroside-II (P-II) by more than 75%. Thus, PkGGPPS was found to be involved in the biosynthesis of P-I and P-II besides other terpenoids. To unravel the mechanism, small subunit of GPPS (PkGPPS.SSU) was identified from P. kurrooa. Protein-protein interaction studies in yeast as well as bimolecular fluorescence complementation (BiFC) in planta have indicated that large subunit of GPPS PkGPPS.LSUs (PkGGPPS1 and PkGGPPS2) and PkGPPS.SSU form a heteromeric GPPS. Presence of similar conserved domains such as light responsive motifs, low temperature responsive elements (LTRE), dehydration responsive elements (DREs), W Box and MeJA responsive elements in the promoters of PkGPPS.LSU and PkGPPS.SSU documented their involvement in picroside biosynthesis. Further, the tissue specific transcript expression analysis vis-à-vis epigenetic regulation (DNA methylation) of promoters as well as coding regions of PkGPPS.LSU and PkGPPS.SSU has strongly suggested their role in picroside biosynthesis. Taken together, the newly identified PkGPPS.SSU formed the heteromeric GPPS by interacting with PkGPPS.LSUs to synthesize P-I and P-II in P. kurrooa.

Enzymatic inhibitory activity of iridoid glycosides from Picrorrhiza kurroa against matrix metalloproteinases: Correlating in vitro targeted screening and docking.

One specific group of MMPs; gelatinases A (MMP-2) and B (MMP-9) are of precise interest in view of the development and progression of cancer. In the current work, an attempt was made to investigate the enzymatic inhibitory activity of Kutkin (KT), Kutkoside (KS), and Picroside I (PS) by inhibition assay and to further check the downregulation of the expression of mRNA levels of MMP-2 and -9. Further in silico docking studies were performed to investigate the interaction of KT, KS and PS with MMP-2 and MMP-9. The results revealed a dose dependent cytotoxic activity of the compounds under investigation and showed a significant inhibition of MMP-9 in comparison to the activity against MMP-2. In addition, a considerable decrease in expression of mRNA levels (MMP-9) was observed in KT, KS, and PS-treated MDA-MB-231 and MDA-MB-435 cancer cells as was detected by reverse transcriptase polymerase chain reaction (semi-quantitative RT-PCR). The molecular docking studies between KT, KS, PS with MMPs revealed that KT, KS, PS occupied the active site of MMP-9 and showed better binding interactions in comparison to MMP-2. The binding energies of the complexes were -7.4, -7.1 and -7.2 kJ/mol for KT, KS and PS with MMP-9, respectively and -8.9, -8.0 and -8.0 kJ/mol for KT, KS and PS with MMP-2, respectively. The findings from the in vitro studies revealed that KT, KS and PS exhibited significant anti-proliferative effects on both MDA-MB-231 and MDA-MB-435 breast cancer cells. In addition, the results of inhibition assay showed that MMP-9 activity was significantly inhibited by KT, KS and PS and the results were consistent with in silico assay.

A phenylalanine ammonia-lyase ortholog (PkPAL1) from Picrorhiza kurrooa Royle ex. Benth: molecular cloning, promoter analysis and response to biotic and abiotic elicitors.

Picrorhiza kurrooa Royle ex Benth. is a highly reputed medicinal herb utilised in the preparation of a number of herbal drug formulations, principally due to the presence of novel monoterpene iridoid glycosides kenned as picrosides. Phenylalanine ammonia-lyase catalyses an important rate-limiting step in phenylpropanoid pathway and supplies precursors like cinnamic acid, vanillic acid, ferulic acid, etc., to a variety of secondary metabolites including picrosides. The imperilled status of P. kurrooa coupled with lack of information regarding biogenesis of picrosides necessitates deciphering the biosynthetic pathway for picrosides. In the present study, a PAL gene, designated PkPAL1 was isolated from P. kurrooa. The cDNA is 2312 bp in length, consisting of an ORF of 2142 bp encoding for a 713 amino acid protein having a predicted molecular weight of 77.66 kDa and an isoelectric point of pH 6.82. qRT-PCR analysis of various tissues of P. kurrooa showed that PkPAL1 transcript levels were highest in the leaves, consistent with picroside accumulation pattern. Using Genome walking, a 718 bp promoter region was also isolated resulting in identification of distinct cis-regulatory elements including TGA-element, TGACG-motif, CGTCA-motif, etc. qRT-PCR indicated up-regulation of PkPAL1 by methyl jasmonate, salicylic acid, 2,4-dicholorophenoxy acetic acid and UV-B elicitations that corroborated positively with the identified cis-elements within the promoter region. Moreover, altitude was found to have a positive effect on the PkPAL1 transcript levels, driving the expression of PkPAL1 abundantly. Based on docking analysis, we identified eight residues as potentially essential for substrate binding in PkPAL1.

An inducible NADPH-cytochrome P450 reductase from Picrorhiza kurrooa - an imperative redox partner of cytochrome P450 enzymes.

Picrorhiza kurrooa synthesizes a large array of pharmacologically important monoterpenoid iridoid glycosides called picrosides. Although chemical profile and pharmacological activities of P. kurrooa have been extensively studied, limited attempts have been made to decipher the biosynthetic route and to identify the key regulatory genes involved in picroside biosynthesis. In the present study, NADPH-cytochrome P450 reductase, a key enzyme involved in electron transfer to cytochrome P450s was identified from P. kurrooa. The full length cDNA (2679 bp) contained an open reading frame of 2133 bp, corresponding to 710 amino acids. PkCPR was heterologously expressed in Escherichia coli and the kinetic parameters of the recombinant enzyme were determined. Specific activity, V max and K m of PkCPR were found to be 5.8 ± 0.05 µmol min(-1) mg(-1), 8.1 ± 0.12 µmol min(-1) mg(-1) and 7.8 µM, respectively. PkCPR was found to be spatially regulated at transcript level, being maximally expressed in leaf tissues. Altitude was found to have a positive effect on the picroside concentration and the picroside content positively correlated with the PkCPR transcript levels in samples collected at varied altitudes. Further, transcript profiling under methyl jasmonate, salicylic acid, 2,4-dicholorophenoxy acetic acid and UV-B elicitations displayed differential transcriptional regulation of PkCPR that fully corroborated with the identified cis-elements within the PkCPR promoter. Expression of PkCPR was inducible by UV-B and phytohormone elicitation, indicating that the PkCPR is possibly related to defence reactions, including biosynthesis of secondary metabolites. Present study is so far the only report of identification and functional characterization of CPR ortholog from P. kurrooa.

Molecular characterization of UGT94F2 and UGT86C4, two glycosyltransferases from Picrorhiza kurrooa: comparative structural insight and evaluation of substrate recognition.

Uridine diphosphate glycosyltransferases (UGTs) are pivotal in the process of glycosylation for decorating natural products with sugars. It is one of the versatile mechanisms in determining chemical complexity and diversity for the production of suite of pharmacologically active plant natural products. Picrorhiza kurrooa is a highly reputed medicinal herb known for its hepato-protective properties which are attributed to a novel group of iridoid glycosides known as picrosides. Although the plant is well studied in terms of its pharmacological properties, very little is known about the biosynthesis of these important secondary metabolites. In this study, we identified two family-1 glucosyltransferases from P. kurrooa. The full length cDNAs of UGT94F4 and UGT86C4 contained open reading frames of 1455 and 1422 nucleotides, encoding polypeptides of 484 and 473 amino acids respectively. UGT94F2 and UGT86C4 showed differential expression pattern in leaves, rhizomes and inflorescence. To elucidate whether the differential expression pattern of the two Picrorhiza UGTs correlate with transcriptional regulation via their promoters and to identify elements that could be recognized by known iridoid-specific transcription factors, upstream regions of each gene were isolated and scanned for putative cis-regulatory elements. Interestingly, the presence of cis-regulatory elements within the promoter regions of each gene correlated positively with their expression profiles in response to different phytohormones. HPLC analysis of picrosides extracted from different tissues and elicitor-treated samples showed a significant increase in picroside levels, corroborating well with the expression profile of UGT94F2 possibly indicating its implication in picroside biosynthesis. Using homology modeling and molecular docking studies, we provide an insight into the donor and acceptor specificities of both UGTs identified in this study. UGT94F2 was predicted to be an iridoid-specific glucosyltransferase having maximum binding affinity towards 7-deoxyloganetin while as UGT86C4 was predicted to be a kaempferol-specific glucosyltransferase. These are the first UGTs being reported from P. kurrooa.

Identification of imperative enzymes by differential protein expression in Picrorhiza kurroa under metabolite accumulating and non-accumulating conditions.

Picrorhiza kurroa, an endangered medicinal plant found in the North-Western Himalayan region has a number of medicinal properties due to the presence of metabolites picroside-I and picroside-II. It is used in various herbal formulations like Picroliv, Livokin, Picrolax, Livomap, Tefroliv etc. Review of literature revealed that no information is available as of today on the proteome analysis of Picrorhiza kurroa. Hence, we aim to analyse the difference in proteome of Picrorhiza kurroa in response to ~ 17 times higher content of picroside-I at 15°C as compared to its content at 25°C. Thus, differential protein expression was studied. Densitometry analysis of SDS-PAGE gels of samples under two differential conditions of temperature revealed the presence of distinct set of proteins under picroside-I accumulating (15°C) versus non-accumulating (25°C) conditions. Mass spectrometric analysis of these proteins using MALDI-TOF MS followed by protein identification using database search on MASCOT search engine gave interesting results. The significant proteins identified were NAD(P)H-quinone oxidoreductase subunit K, shikimate kinase, ribulose bisphosphate carboxylase small chain and fructokinase fragment. Hence, these findings throw light on the involvement of these enzymes in the crucial physiological processes of Picrorhiza kurroa and can provide an insight into the biosynthesis of picrosides and other secondary metabolites.

Expression pattern of fifteen genes of non-mevalonate (MEP) and mevalonate (MVA) pathways in different tissues of endangered medicinal herb Picrorhiza kurroa with respect to picrosides content.

Picrorhiza kurroa, has become an endangered medicinal herb due to excessive utilization, therefore it necessitates the understanding of biology and molecular basis of major chemical constituents i.e. Picroside-I (P-I) and Picroside-II (P-II). Estimation of P-I and P-II in different tissues of P. kurroa showed that shoots contain only P-I whereas P-II is present only in roots. Differential conditions with varying concentrations of P-I (0-27 µg/mg) and P-II (0-4 µg/mg) were selected. Four genes of MEP pathway; DXPS, ISPD, ISPE, MECPS and one gene of MVA pathway PMK showed elevated levels of transcripts in shoots (57-166 folds) and stolons (5-15 folds) with P-I contents 0-27 µg/mg and 2.9-19.7 µg/mg, respectively. Further HDS and DXPR genes of MEP pathway showed higher expression ~9-12 folds in roots having P-II (0-4 µg/mg). The expression of ISPH and ISPE was also high ~5 folds in roots accumulating P-II. GDPS was the only gene with high transcript level in roots (9 folds) and shoots (20 folds). Differential biosynthesis and accumulation of picrosides would assist in regulating quality of plant material for herbal drug formulations.

De novo sequencing and characterization of Picrorhiza kurrooa transcriptome at two temperatures showed major transcriptome adjustments.

BACKGROUND: Picrorhiza kurrooa Royle ex Benth. is an endangered plant species of medicinal importance. The medicinal property is attributed to monoterpenoids picroside I and II, which are modulated by temperature. The transcriptome information of this species is limited with the availability of few hundreds of expressed sequence tags (ESTs) in the public databases. In order to gain insight into temperature mediated molecular changes, high throughput de novo transcriptome sequencing and analyses were carried out at 15 °C and 25 °C, the temperatures known to modulate picrosides content. RESULTS: Using paired-end (PE) Illumina sequencing technology, a total of 20,593,412 and 44,229,272 PE reads were obtained after quality filtering for 15 °C and 25 °C, respectively. Available (e.g., De-Bruijn/Eulerian graph) and in-house developed bioinformatics tools were used for assembly and annotation of transcriptome. A total of 74,336 assembled transcript sequences were obtained, with an average coverage of 76.6 and average length of 439.5. Guanine-cytosine (GC) content was observed to be 44.6%, while the transcriptome exhibited abundance of trinucleotide simple sequence repeat (SSR; 45.63%) markers.Large scale expression profiling through "read per exon kilobase per million (RPKM)", showed changes in several biological processes and metabolic pathways including cytochrome P450s (CYPs), UDP-glycosyltransferases (UGTs) and those associated with picrosides biosynthesis. RPKM data were validated by reverse transcriptase-polymerase chain reaction using a set of 19 genes, wherein 11 genes behaved in accordance with the two expression methods. CONCLUSIONS: Study generated transcriptome of P. kurrooa at two different temperatures. Large scale expression profiling through RPKM showed major transcriptome changes in response to temperature reflecting alterations in major biological processes and metabolic pathways, and provided insight of GC content and SSR markers. Analysis also identified putative CYPs and UGTs that could help in discovering the hitherto unknown genes associated with picrosides biosynthesis.

Light and temperature regulated terpene biosynthesis: hepatoprotective monoterpene picroside accumulation in Picrorhiza kurrooa.

Picrorhiza (Picrorhiza kurrooa) is an endangered medicinal plant with well-known hepatoprotective activity attributed to monoterpenoid picrosides. The present article details on regulatory genes of terpenoid metabolism, 3-hydroxy-3-methylglutaryl coenzyme A reductase (pkhmgr) and 1-deoxy-D-xylulose-5-phosphate synthase (pkdxs) from picrorhiza. Since no molecular information was available, these genes were cloned to full-length by degenerate primers and rapid amplification of cDNA ends, followed by cloning of the upstream sequences that showed the presence of core sequences for light and temperature responsiveness. Electrophoretic mobility shift assay confirmed binding of protein to these motifs. Expression of pkhmgr and pkdxs was up-regulated at 15 degrees C as compared to at 25 degrees C as well as under light as compared to dark conditions. Picrosides content exhibited the trend similar to gene expression. To rule out the possible limitation of carbon pool under dark condition, plantlets of picrorhiza were raised in vitro in Murashige and Skoog medium supplemented with 3% sucrose. Results showed similar up-regulation of both the genes and the higher picrosides content in in vitro raised plantlets in the presence of light. Data suggested the important roles played by light and temperature in regulating pkhmgr and pkdxs, and the picrosides level in picrorhiza.