NGS Transcriptomes and Enzyme Inhibitors Unravel Complexity of Picrosides Biosynthesis in Picrorhiza kurroa Royle ex. Benth

Picrorhiza kurroa is an important medicinal herb valued for iridoid glycosides, Picroside-I (P-I) and Picroside-II (P-II), which have several pharmacological activities. Genetic interventions for developing a picroside production platform would require knowledge on biosynthetic pathway and key control points, which does not exist as of today. The current study reports that geranyl pyrophosphate (GPP) moiety is mainly contributed by the non-mevalonate (MEP) route, which is further modified to P-I and P-II through phenylpropanoid and iridoid pathways, in total consisting of 41 and 35 enzymatic steps, respectively. The role of the MEP pathway was ascertained through enzyme inhibitors fosmidomycin and mevinolin along with importance of other integrating pathways using glyphosate, aminooxy acetic acid (AOA) and actinomycin D, which overall resulted in 17%-92% inhibition of P-I accumulation. Retrieval of gene sequences for enzymatic steps from NGS transcriptomes and their expression analysis vis-à-vis picrosides content in different tissues/organs showed elevated transcripts for twenty genes, which were further shortlisted to seven key genes, ISPD, DXPS, ISPE, PMK, 2HFD, EPSPS and SK, on the basis of expression analysis between high versus low picrosides content strains of P. kurroa so as to eliminate tissue type/ developmental variations in picrosides contents. The higher expression of the majority of the MEP pathway genes (ISPD, DXPS and ISPE), coupled with higher inhibition of DXPR enzyme by fosmidomycin, suggested that the MEP route contributed to the biosynthesis of P-I in P. kurroa. The outcome of the study is expected to be useful in designing a suitable genetic intervention strategy towards enhanced production of picrosides. Possible key genes contributing to picroside biosynthesis have been identified with potential implications in molecular breeding and metabolic engineering of P. kurroa.


Introduction
Picrorhiza kurroa Royle ex. Benth is an important medicinal herb valued for its hepatoprotective activity as well as other medicinal properties like anti-malarial, anti-inflammatory, anti- additional genes (PAL and COMT) of phenylpropanoid pathway. Five remaining genes of MEP and MVA pathway were cloned by Pandit et al [21]. Additionally, cloning of UGT gene of iridoid pathway was done by Bhat et al [22]. Each of these studies has shown that the corresponding enzymatic steps are involved in the biosynthesis of P-I and P-II. However, none has clarifies as to which of the MVA/MEP pathways contribute to the iridoid backbone, GPP and which genes are playing key role in contributing to the biosynthesis of P-I and P-II in P. kurroa. Multiple pathways contribute in biosynthesis of secondary metabolites, therefore it is necessary to identify the flux of individual pathways for the production of final product. Inhibitor studies can provide significant clues in ascertaining the contribution of individual biosynthetic pathways towards production of terpenoids. Palazon et al [23] showed that non-mevalonate pathway is the main source of universal terpenoid precursor isopentenyl diphosphate (IPP) for biosynthesis of taxanes in Taxus baccata. The major involvement of mevalonate pathway for shikonins biosynthesis in Arnebia euchroma has also been proved through inhibitor assays [24]. However, any such studies has not been taken up in P. kurroa for identification of major contributing pathway for picrosides among various integrating pathways.
Present work reports on ascertaining the contribution of MVA and/or MEP route in the biosynthesis of P-I through enzyme inhibitor experiments. Also, genes catalysing the enzymatic steps were mapped to iridoid branch of the picrosides biosynthetic pathway which were not known in P. kurroa. The availability of NGS transcriptomes of different P. kurroa tissues enabled the selection of appropriate paralogs for pathway genes. Expression analysis of all genes involved in the complete biosynthetic pathway was carried out in four different tissues of P. kurroa with varying contents of P-I (0.0% and 2.7%) and P-II (0.0% and 0.4%) to associate key genes involved in picrosides biosynthesis. Further, to ascertain the involvement of genes in picrosides biosynthesis as well as to eliminate the effect of tissue type/developmental stage visà-vis picrosides contents, expression analysis of pathway genes was also assesed in high versus low P-I content strains of P. kurroa.

Results
Mapping genes to enzymatic steps of iridoid branch of the pathway The first seven enzymatic steps GS, G10H, 10HGO (10HD), IS, MC, CPM, UGT of iridoid pathway have recently been identified during this study in medicinal plant species such as P. kurroa, Catharanthus roseus and Gardenia jasmonides [22,25,26] while rest of the steps were unknown. The eighth step in the pathway is conversion of boschnaloside to 8-epideoxy loganic acid which involves hydroxylation/oxidation reaction. This enzymatic step is most probably catalysed by aldehyde dehydrogenase (ALD). The ninth step is conversion of 8-epideoxy loganic acid to mussaenosidic acid, which is catalysed by an hydroxylase enzyme. The similar reaction is catalyzed by flavanone 3-dioxygenase/hydoxylase (F3D). The tenth step is conversion of mussaenosidic acid to deoxygeniposidic acid, which is catalyzed by dehydratase enzyme. We searched for all the dehydratases present in P. kurroa transcriptomes and looked for their involvement in secondary metabolism. 2-hydroxyisoflavanone dehydratase (2HFD) was the only dehydratase involved in secondary metabolites biosynthesis, therefore, it was predicted to be catalysing a similar reaction in P. kurroa. The eleventh step is conversion of deoxygeniposidic acid to geniposidic acid, which involves hydroxylation reaction. The similar reaction is catalyzed by deacetoxycephalosporin-C hydroxylase. The twelth step is conversion of geniposidic acid to bartsioside, which is catalyzed by a decarboxylase enzyme. Two decarboxylases, uroporphyrinogen decarboxylase (UPD) and udp-glucuronic acid decarboxylase (UGD) were identified from P. kurroa transcriptomes. Uroporphyrinogen decarboxylase catalyzes the decarboxylation of uroporphyrinogen to coproporphyrinogen. Similar chemical reaction is involved in the conversion of geniposidic acid to bartsioside. The thirteenth step is the conversion of bartsioside to aucubin and this reaction is similar to ninth step, which is being catalyzed by a similar enzyme flavanone 3-dioxygenase/hydroxylase (F3D). Fourteenth step is conversion of aucubin to catalpol which is catalyzed by an epoxidase or monooxygenase enzyme. We searched for all the monooxygenases and epoxidases in P. kurroa transcriptomes and identified two enzymes squalene monooxygenase (SQM) and squalene epoxidase (SQE) involved in secondary metabolism but squalene epoxidase did not show significant expression w.r.t. P-I or P-II contents. The last step in the formation of P-I and P-II from catalpol involves acyl group transfer reaction and we predicted this reaction to be catalyzed by anthocyanin acyltransferase (ACT). The complete biosynthetic pathway with corresponding enzymatic steps is shown in Fig 1. Identification of appropriate paralogs/isoforms NGS transcriptomes were generated from different P. kurroa tissues varying in picrosides content mentioned in our previous work [27]. The assembly statistics for transcriptome datasets is given in S1 Table. Mining of transcriptomes for picrosides biosynthetic pathway genes showed the existence of multiple paralogs of these genes. The appropriate paralog for each gene was identified and selected for expression analysis using the methodology described in methods section. The paralogs selected for pathway genes in different shoot transcriptomes of P. kurroa are given in S2 Table. Expression analysis of P-I and P-II biosynthetic pathway genes vis-à-vis picrosides content The results are in continuation to our previous work which reported the expression status of 15 genes of MEP and MVA pathway in different tissues of P. kurroa [21]. Expression analysis of remaining genes of the biosynthetic pathway (15 genes from iridoid pathway and 12 from phenylpropanoid pathway) was done in four tissues varying for P-I and P-II contents. Majority of the genes showed higher expression in relation to picrosides content. Four genes of iridoid pathway (GS, 2HFD, DCH and SQM) and two genes of phenylpropanoid pathway (DQS and TAT) showed elevated levels of transcripts (~12-130 folds) in FGS having 2.7% P-I content w. r.t. TCS having negligible (0.01%) P-I. Two genes of iridoid pathway (F3D and ACT) and six genes of phenylpropanoid pathway (DQD, QSD, CAM, SK, EPSPS and PAL) showed~16-96 folds expression in FGR having 0.4% P-II content wr.t. TCR having no P-II content. Four genes of iridoid pathway (G10H, CPM, ALD and UPD/UGD) and two genes of phenylpropanoid pathway (CM and APD) showed~12-42 folds higher expression in FGS as well as FGR w.r.t. their tissue cultured counterparts. The difference in fold expression in field grown tissues in comparison to tissue culture grown for iridoid and phenypropanoid pathways is given in Fig  2a, 2b, 2c and 2d. The overall expression status of pathway genes w.r.t. P-I and P-II is given in Fig 3. The genes which showed above 10 folds transcript abundance in initial screening w.r.t. either P-I or P-II or both (including MEP/MVA pathway genes from our previous study) were checked for their expression status on shoot tissues of high versus low P-I content strains of P. kurroa. A high P-I content strain, PKS-1 (2.7%) and a low content strain, PKS-4 (0.3%) were used in comparative expression analysis. This resulted in the identification of seven genes, ISPD, DXPS, ISPE, PMK, 2HFD, EPSPS, SK from the complete biosynthetic pathway with~5-57 folds higher transcript abundance in high content strain compared to low content strain of P. kurroa (Fig 4). The expression of three genes of MEP pathway, DXPS, ISPE, ISPD, one gene of MVA pathway, PMK and one gene of iridoid pathway, 2HFD decreased from 57-160 folds to~5-17 folds when compared for their relative transcript abundance between strains versus  Table 1). The relative expression status of pathway genes between high versus low content strains thus provided a realistic association with the biosynthesis of picrosides.

Effect of enzyme inhibitors on biosynthesis of Picroside-I
The inhibition profile of five inhibitors was assessed at 15 th and 30 th day of culturing P. kurroa shoots by quantifying P-I content ( Fig 5) because cultured shoots are reported to accumulate only P-I [11]. Out of five inhibitors, mevinolin, fosmidomycin, glyphosate and AOA are competitive inhibitors which are highly specific for their target enzymes while actinomycin D is a transcriptional inhibitor. The best inhibitory concentrations were 5 μM for mevinolin and actinomycin D, 150 μM for fosmidomycin, 4 mM for glyphosate and AOA. Mevinolin was found to be least effective in inhibiting P-I biosynthesis as it showed maximum inhibition of only 17% and 11.6% at 15 th and 30 th day, respectively. Other inhibitors showed upto 92% inhibition in P-I content in comparison to control. All inhibitors showed higher inhibition at 30 th day in comparison to 15 th day, except mevinolin which unlike others showed higher inhibition at 15 th day. At their best inhibitory concentrations, fosmidomycin, glyphosate and AOA showed a drastic reduction of 85.3%, 87.3% and 64.6% respectively in P-I accumulation at 15 th day and reduction of 90.6%, 92.9% and 63.4%, respectively at 30 th day. Fosmidomycin, glyphosate and AOA were thus found to be potent inhibitors of P-I biosynthesis. Actinomycin D moderately inhibited P-I biosynthesis in P. kurroa as it showed 47.9% and 61.2% inhibition at 15 th and 30 th day, respectively.

Gene expression in response to inhibitors
To see the effect of inbitor treatments on target gene transcripts, expression analysis was carried out on inhibitor treated shoot tissues in comparison to untreated control for related genes    6). Actinomycin D showed somewhat higher reduction in transcripts from 1 to 17.5 folds (Fig 7).

Discussion
Through this study MEP pathway has been identified as the major contributor of geranyl pyrophosphate (GPP), the iridoid backbone for picrosides biosynthesis in P. kurroa. After GPP biosynthesis, iridoid and phenylpropanoid pathways play equally important role in picrosides biosynthesis by providing catalpol and cinnamate/vanillate moieties, respectively. The importance of iridoid and phenylpropanoid pathways has been justified by precursor feeding studies in P. kurroa, where exogenous feeding of catalpol and cinnamic acid showed that both pathways act in conjunction for increased production of P-I in P. kurroa [Kumar et al. 2015, unpublished]. Current study is an initial endeavor towards deciphering the complete biosynthetic pathway including corresponding enzymatic steps for picrosides biosynthesis in P. kurroa. The identification of enzymatic steps of iridoid branch of the pathway, not known earlier has been done, however functional characterization is required to support their proposed functions in P. kurroa. The availability of NGS transcriptomes of different P. kurroa tissues enabled the selection of appropriate paralogs for pathway genes as multiple paralogs were present for these genes. The higher expression of gene transcripts for corresponding enzymes vis-à-vis P-I and P-II contents and their involvement in secondary metabolism in other plant species suggested their possible role in catalysing the required enzymatic reactions in picrosides biosynthesis. Aldehyde dehydrogenase is reported to be involved in the biosynthesis of ferulic acid and sinapic acid in Arabidopsis thaliana [28]. Flavanone 3-dioxygenase/hydoxylase (F3D) is involved in flavanoids biosynthesis in many plant species like Ginkgo biloba, Camellia sinensis [29,30]. 2-hydroxyisoflavanone dehydratase is involved in isoflavone biosynthesis in Lotus japonica [31]. Deacetoxycephalosporin-C hydroxylase is involved in cephalosporin biosynthesis [32]. Uroporphyrinogen decarboxylase is associated with the activity of enzymes involved in tetrapyrrole biosynthesis and pathogen defense response in Nicotiana tabacum [33,34]. Squalene epoxidase/monooxygenase is involved in ginsenoside biosynthesis in Panax ginseng [35,36] and triterpenoids in Uncaria tomentosa [37]. Anthocyanin acyltransferases catalyze regiospecific acyl transfer from acyl-CoA to the sugar moiety of anthocyanins [38] and are involved in anthocyanin biosynthesis in plant species [39]. The identified enzymes belong to cytochrome P450 family which possess broad substrate specificity i.e. same enzyme can bind to different substrates but enzyme kinetics vary considerably from one substrate to another. Therefore, these enzymes inspite of preferably using other substrates can also catalyse the similar reactions for picrosides biosynthesis using pathway intermediates as their substrates.
The expression analysis revealed that most of the genes of picrosides biosynthetic pathway had relatively higher expression in field grown tissues of P. kurroa containing higher amounts of P-I or P-II contents compared to tissue culture grown plants with negligible or no picrosides content at all. Genes, GS, G10H, CPM, ALD, F3D, 2HFD, DCH, UPD/UGD, SQM, ACT from iridoid pathway and DQS, DQD, QSD, SK, EPSPS, CM, APD, TAT, PAL from shikimic acid/ phenylpropanoid pathway showed significantly higher folds expression vis-à-vis picrosides content. Various studies have reported expression of multiple genes of biosynthetic pathways to be positively correlated with the terpenoid biosynthesis; shikonins in Arnebia euchroma [24], artemisinin in Artemisia annua [40], MIAs in Catharanthus roseus [41], flavonoids biosynthesis in Fagopyrum species [42], lignin biosynthesis in Arabidopsis thaliana [43]. Geraniol synthase (GS) is an important enzyme which initiates monoterpenoid branch of the pathway in Catharanthus roseus [44] and G10H is reported to be a rate-limiting enzyme for biosynthesis of TIAs in Catharanthus roseus [45] alongwith its importance in iridoid monoterpenoid swertiamarin biosynthesis in Swertia mussotii [46]. EPSPS, SK and PAL are also reported to be important regulatory enzymes of shikimate/phenylpropanoid pathway [47][48][49]. In initial analysis, multiple genes showed higher expression for P-I or P-II or both. But the differential expression in field grown tissues in comparison to tissue cultured plants might be due to tissue type or environmental variations. Therefore, to ascertain whether the elevated levels of transcripts of pathway genes are only affecting the biosynthesis of picrosides uniquely, the expression status of genes was further studied between shoots of P. kurroa genotypes that were varying for Picroside-I. High and low Picroside-I content genotypes of P. kurroa (one each) grown for 3 years in the controlled environment in greenhouse were used for comparative expression analysis so as to reflect genetic differences contributing to the increase or decrease of gene transcripts rather than tissue type or developmental stage. It was observed that most of the genes did not show significant difference in expression between high versus low content strains, thereby suggesting that all the genes which showed higher expression initially might be contributing to the biosynthesis of secondary metabolites other than picrosides. This additional analysis shortlisted seven genes, PMK, ISPE, 2HFD, DXPS, ISPD, EPSPS and SK with major contribution in picrosides biosynthesis. Among these seven genes, four of MEP/MVA pathway ISPD, DXPS, ISPE and PMK are reported to be positively correlated with picrosides content in P. kurroa [21] and DXPS is a well known regulatory enzyme of MEP pathway [50,51]. EPSPS is a key regulatory gene of shikimate pathway [46] which is associated with herbicide tolerance [52,53] and biosynthesis of secondary metabolites [54,55]. SK is an important regulatory gene in secondary metabolism as it has been suggested that plant SKs act as regulatory points for the shikimate pathway [48]. It has been reported that 2-hydroxyisoflavanone dehydratase plays a key role in the regulation of isoflavone biosynthesis as its overexpression resulted in accumulation of daidzein and genistein in Lotus japonicas [31]. Majority of these key genes belonged to MEP pathway. Also higher expression of two genes of shikimic acid/phenylpropanoid pathway and one gene of iridoid pathway, among seven genes, highlights the importance of these modules of the biosynthetic pathway. This suggests that each module of the pathway is important in contributing to the structures of parental compounds, P-I and P-II in P. kurroa.
Five inhibitors targeting important enzymatic steps of the picrosides biosynthetic pathway were selected to assess their effect on picrosides accumulation. The inhibitor concentrations were chosen based on previous reports [56][57][58][59], [23]. Mevinolin and fosmidomycin are highly specific inhibitors of MVA (HMGR) and MEP (DXPR) pathways, respectively [60,61]. These two inhibitors were selected to rule out whether MVA and/or MEP pathway contributes in picrosides biosynthesis. Fosmidomycin produced drastic inhibition of upto 90.6% in P-I accumulation whereas mevinolin resulted in slight (17%) inhibition. This suggested that the MEP pathway plays major role in the production of GPP, the precursor for iridoid backbone biosynthesis. Picrosides are monoterpenoids and our results are in accordance with the previous findings that monoterpenoids have non-mevalonate (plastidial) origin and monoterpenoid synthases are localized to plastids [62]. It has also been reported by Eisenreich et al. [63] that MEP pathway is a predominant contributor for monoterpenoid biosynthesis, however, crosstalk occurs between two pathways [64]. Our previous reports also suggested the predominant role of MEP pathway in picrosides biosynthesis as majority of the MEP pathway genes were highly expressed in relation to picrosides content [21]. Sood and Chauhan [11] have also highlighted the importance of plastids (chloroplasts) by showing that the biosynthesis of Picroside-I occurs only in in vitro cultured leaf and stem segments but not in undifferentiated callus cultures.
The other inhibitors, glyphosate and AOA were selected targeting the shikimate/phenylpropanoid pathway enzymes whereas actinomycin D is a common transcriptional inhibitor. Glyphosate, a broad spectrum herbicide which competitively inhibits EPSPS [65] and AOA acts as an inhibitor of PAL [58] which is an important regulatory enzyme in phenylpropanoid pathway. Shikimic acid/phenylpropanoid pathway has a major contribution in picrosides biosynthesis, thereby, providing cinnamate and vanillate moieties for P-I and P-II, respectively. The decrease in P-I biosynthesis by inhibiting shikimic acid/phenylpropanoid pathway enzymes confirms major contribution of this component of the pathway in picrosides biosynthesis in P. kurroa. Glyphosate is reported to inhibit secondary metabolites content in soyabean and buckwheat [57,66]. AOA treatment resulted in decreased accumulation of phytoalexins in banana [59] and 2-hydroxy-4-methoxybenzaldehyde in Hemidesmus indicus roots [67].
The inhibition pattern was similar for four inhibitors fosmidomycin, glyphosate, AOA and actinomycin D where higher inhibition was observed at 30 th day than 15 th day for all concentrations. Mevinolin showed opposite inhibition pattern as higher inhibition was observed at 15 th day than 30 th day. This might be due to the fact that mevinolin is not a strong inhibitor in our case. Therefore, most of it is getting used up till 15 th day, hence not showing significant inhibition at 30 th day.
The inhibitor treatment thus resulted in decrease in picrosides biosynthesis. We further looked at whether the expression of biosynthetic pathway genes, which are targets of corresponding inhibitors, is getting affected or not. Competitive inhibitors did not show significant decrease in expression of transcripts because these might be affecting the genes only at enzymatic level but are not producing any effect at the transcriptional level. It has been reported in Arabidopsis that inhibitor treatment did not show significant effect on the expression of genes involved in sterol, chlorophyll and carotenoid metabolism, thereby indicating that posttranscriptional processes might be playing an important role in regulating the flux through isoprenoid metabolic pathways [68].

Conclusion
The knowledge of complete pathway and corresponding genes would be helpful in understanding molecular basis of picrosides biosynthesis as well as planning genetic improvement strategies for enhancing picrosides content in P. kurroa. Inhibitor experiments revealed that MEP pathway is a major contributor of GPP for picrosides in P. kurroa and picrosides biosynthesis is regulated at various control points in different modules of the biosynthetic route. Therefore, common regulatory elements for multiple genes need to be identified so as to ease genetic interventions for enhancing picrosides content. This is the first report wherein possible key genes have been identified in picrosides biosynthetic pathway which after further functional validation can have potential implications in molecular breeding and metabolic engineering.

Plant material
P. kurroa plants were procured from the nursery of Himalayan Forest Research Institute, Jagatsukh, Manali, H.P., India (1,900 m altitude, 20°35.6 0 -32°6.1 0 N and 78°57.8 0 -77°33.7 0 E) and maintained in the greenhouse of Jaypee University. These plants were cultured and maintained in an optimized Murashige and Skoog (MS) medium [69] supplemented with 3 mg/L indole-3-butyric acid and 1 mg/L kinetin in a plant tissue culture chamber maintained at 25±2°C with 16 h photoperiod provided by cool white fluorescent light (3,000 lux) (Sood and Chauhan 2010). Four P. kurroa tissues with varying picrosides content growing in field conditions and tissue culture were taken for the study ( Table 2, [21]). These included field grown shoots having 2.7% P-I (FGS), tissue cultured shoots having negligible P-I content (TCS), field grown roots having 0.4% P-II content (FGR) and tissue cultured roots with no P-II content (TCR). Two strains of P. kurroa with high and low content viz. PKS-1 (P-I 2.7%) and PKS-4 (P-I 0.3%) maintained in controlled environment in green house were also taken.

Identification of unknown enzymatic steps
The genes catalysing the enzymatic steps of iridoid pathway were identified following the methodology described below. Initially the enzyme classes were predicted on the basis of chemical reactions/group transfer involved in the conversion of one metabolite to the next. All possible enzymes catalysing similar reactions were searched in the KEGG database. The enzymes were then matched in P. kurroa transcriptomes using BLAST and shortlisted on the basis of significant similarity. Next, the enzymes were shortlisted based on their functionality i.e. involved in secondary metabolites biosynthesis. The enzymes were then selected on the basis of abundance (FPKM) of enzyme transcripts in transcriptomes and finally supported through qRT-PCR in different tissues of P. kurroa with varying contents of P-I and P-II. The nucleotide sequences for phenylpropanoid and iridoid pathway genes were extracted from whole genome transcriptomes of P. kurroa and primers were designed using online tool Primer3.

RNA isolation and cDNA synthesis
Total RNA was isolated from P. kurroa tissues by using RaFlex™ total RNA isolation kit (GeNei™) by following manufacturer's instructions. The quality of RNA was checked by 1% (w/ v) ethidium bromide-stained agarose gel. RNA was quantified in a NanoDrop spectrophotometer (Thermo Scientific) by measuring absorbance at 260nm and 280nm and 1μg RNA was used for cDNA synthesis. cDNA was synthesized using Verso cDNA synthesis kit (Thermo Scientific) according to manufacturer's protocol. Concentration of each cDNA sample was adjusted to 100ng/μl for qRT-PCR.

Identification of appropriate paralogs/isoforms for pathway genes in transcriptomes
NGS transcriptomes for P. kurroa were generated and analysed using the methodology represented in S1 Fig Multiple paralogs of the genes were present in P. kurroa transcriptomes, therefore appropriate paralogs/isoforms were identified for each gene. For selection of suitable paralogs of genes, initially multiple sequence alignment of the selected transcripts using Clus-talW was carried out to see the homology within paralogs for each gene. Further, shortlisting of transcripts was done through BLAST analysis by looking for highest similarity with functionally characterized sequences (retrieved from the NCBI databases which were functionally characterized in the same plant or in different plant species). Thereafter, the transcripts were selected on the basis of transcript abundance (FPKM values), in different transcriptomes generated from P. kurroa tissues varying in picrosides contents. The selected paralogs were further validated through qRT-PCR analysis under differential conditions of picrosides biosynthesis.
Expression analysis using quantitative real time PCR (qRT-PCR) P. kurroa tissues with varying P-I and P-II contents were selected for correlating the expression status of pathway genes with picrosides content. Equal cDNA quantities (100ng) of each sample were taken. Gene specific primers were checked initially on cDNA for amplification of single product using standard PCR. Quantitative real time PCR was performed using using gene specific primers (Table 3) in triplicate on a CFX96 system (Bio-Rad Laboratories; Hercules CA) with the iScript one step RT PCR kit (Bio-rad). The PCR protocol was as follows: denaturation for 3 min at 95°C, followed by 39 cycles each of denaturation for 10 s at 95°C, annealing for 30 s at 50-65°C, and elongation for 20 s at 72°C. In final step melt curve analysis was done at 65-95°C with 0.5°C increment at each 0.05 s to verify amplification of a single product. Two housekeeping genes, 26s rRNA and GAPDH were used as internal controls for normalization. Genes with significantly high expression w.r.t. P-I and P-II content were checked for their expression status in high versus low picrosides content strains (one each) of P. kurroa to validate/ascertain the genes playing significant role in picrosides biosynthesis. Finally, the expression status of target genes in response to treatment with their specific inhibitors was also checked on in vitro cultured shoots of P. kurroa w.r.t. their untreated controls. . Two controls were also used, one having only agar gelled MS media and hormones another having equivalent concentration of DMSO as used for dissolving mevinolin and actinomycin D. After inoculation cultures were incubated at 15±2°C. Shoots were harvested at 15 th and 30 th day for quantification of picrosides. Estimation of picrosides was done using RP-HPLC following the method described by Sood and Chauhan (2010). The experiment was done in triplicates.