ABC transporters mined through comparative transcriptomics associate with organ-specific accumulation of picrosides in a medicinal herb, Picrorhiza kurroa.

Picrorhiza kurroa Royle ex Benth is a valuable medicinal herb of North-Western Himalayas due to presence of two major bioactive compounds, picroside-I and picroside-II used in the preparation of several hepatoprotective herbal drugs. These compounds accumulate in stolons/rhizomes; however, biosynthesized in different organs, viz., picroside-I in shoots and picroside-II in roots. As of today, no information exists on what transporters are transporting these metabolites from shoots and roots to the final storage organ, stolon, which ultimately transforms into rhizome. The ATP-binding cassette (ABC) transporters are reported to transport majority of secondary metabolites, including terpenoids in plants, therefore, we mined P. kurroa transcriptomes to identify and shortlist potential candidates. A total of 99 ABC transporter-encoding transcripts were identified in 3 differential transcriptomes, PKSS (shoots), PKSTS (stolons), and PKSR (roots) of P. kurroa, based on in silico comparative analysis and transcript abundance. 15 of these transcripts were further validated for their association using qRT-PCR in shoots, roots and stolon tissues in P. kurroa accessions varying for picroside-I and picroside-II contents. Organ-specific expression analysis revealed that PkABCA1, PkABCG1, and PkABCB5 had comparatively elevated expression in shoots; PkABCB2 and PkABCC2 in roots; PkABCB3 and PkABCC1 in stolon tissues of P. kurroa. Co-expression network analysis using ABC genes as hubs further unravelled important interactions with additional components of biosynthetic machinery. Our study has provided leads, first to our knowledge as of today, on putative ABC transporters possibly involved in long distance and local transport of picrosides in P. kurroa organs, thus opening avenues for designing a suitable genetic intervention strategy.

Capturing acyltransferase(s) transforming final step in the biosynthesis of a major Iridoid Glycoside, (Picroside-II) in a Himalayan Medicinal Herb, Picrorhiza kurroa.

BACKGROUND: Picrorhiza kurroa has been reported as an age-old ayurvedic hepato-protection to treat hepatic disorders due to the presence of iridoids such as picroside-II (P-II), picroside-I, and kutkoside. The acylation of catalpol and vanilloyl coenzyme A by acyltransferases (ATs) is critical step in P-II biosynthesis. Since accumulation of P-II occurs only in roots, rhizomes and stolons in comparison to leaves uprooting of this critically endangered herb has been the only source of this compound. Recently, we reported that P-II acylation likely happen in roots, while stolons serve as the vital P-II storage compartment. Therefore, developing an alternate engineered platform for P-II biosynthesis require identification of P-II specific AT/s. METHODS AND RESULTS: In that direction, egg-NOG function annotated 815 ATs from de novo RNA sequencing of tissue culture based 'shoots-only' system and nursery grown shoots, roots, and stolons varying in P-II content, were cross-compared in silico to arrive at ATs sequences unique and/or common to stolons and roots. Verification for organ and accession-wise upregulation in gene expression of these ATs by qRT-PCR has shortlisted six putative 'P-II-forming' ATs. Further, six-frame translation, ab initio protein structure modelling and protein-ligand molecular docking of these ATs signified one MBOAT domain containing AT with preferential binding to the vanillic acid CoA thiol ester as well as with P-II, implying that this could be potential AT decorating final structure of P-II. CONCLUSIONS: Organ-wise comparative transcriptome mining coupled with reverse transcription real time qRT-PCR and protein-ligand docking led to the identification of an acyltransferases, contributing to the final structure of P-II.

The first draft genome of Picrorhiza kurrooa, an endangered medicinal herb from Himalayas.

Picrorhiza kurrooa is an endangered medicinal herb which is distributed across the Himalayan region at an altitude between 3000-5000 m above mean sea level. The medicinal properties of P. kurrooa are attributed to monoterpenoid picrosides present in leaf, rhizome and root of the plant. However, no genomic information is currently available for P. kurrooa, which limits our understanding about its molecular systems and associated responses. The present study brings the first assembled draft genome of P. kurrooa by using 227 Gb of raw data generated by Illumina and PacBio RS II sequencing platforms. The assembled genome has a size of n = ~ 1.7 Gb with 12,924 scaffolds. Four pronged assembly quality validations studies, including experimentally reported ESTs mapping and directed sequencing of the assembled contigs, confirmed high reliability of the assembly. About 76% of the genome is covered by complex repeats alone. Annotation revealed 24,798 protein coding and 9789 non-coding genes. Using the assembled genome, a total of 710 miRNAs were discovered, many of which were found responsible for molecular response against temperature changes. The miRNAs and targets were validated experimentally. The availability of draft genome sequence will aid in genetic improvement and conservation of P. kurrooa. Also, this study provided an efficient approach for assembling complex genomes while dealing with repeats when regular assemblers failed to progress due to repeats.

Transcriptome profiling reveal key hub genes in co-expression networks involved in Iridoid glycosides biosynthetic machinery in Picrorhiza kurroa.

Picrorhiza kurroa is a medicinal herb rich in hepatoprotective iridoid glycosides, picroside-I (P-I) and picroside-II (P-II). The biosynthetic machinery of picrosides is poorly understood, therefore, 'no-direction' gene co-expression networks were used to extract linked/closed and separated interactions in terpenoid glycosides-specific sub-networks. Transcriptomes generated from different organs, varying for P-I and P-II contents such as shoots grown at 15 and 25 °C and nursery-grown shoots, stolons, and roots resulted in 47,726, 44,958, 40,117, 66,979, and 55,578 annotated transcripts, respectively. Occurrence of 2810 ± 136 nodes and 15,626 ± 696 edges in these networks indicated intense, co-expressed, closed loop interactions. Either deregulation/inhibition of abscisic acid (ABA) biosynthesis/signaling or constitutive degradation of ABA resulted in organ-specific accumulation of P-I and P-II. Biosynthesis, condensation and glucosylation of isoprene units may occur in shoots, roots or stolons; but addition of phenylpropanoid moiety and further modification/s of the iridoid backbone occurs mainly inside vacuoles in roots.

Complexity of gene paralogues resolved in biosynthetic pathway of hepatoprotective iridoid glycosides in a medicinal herb, Picrorhiza kurroa through differential NGS transcriptomes.

Picrorhiza kurroa is a medicinal herb with diverse pharmacological applications due to the presence of iridoid glycosides, picroside-I (P-I), and picroside-II (P-II), among others. Any genetic improvement in this medicinal herb can only be undertaken if the biosynthetic pathway genes are correctly identified. Our previous studies have deciphered biosynthetic pathways for P-I and P-II, however, the occurrence of multiple copies of genes has been a stumbling block in their usage. Therefore, a methodological strategy was designed to identify and prioritize paralogues of pathway genes associated with contents of P-I and P-II. We used differential transcriptomes varying for P-I and P-II contents in different tissues of P. kurroa. All transcripts for a particular pathway gene were identified, clustered based on multiple sequence alignment to notify as a representative of the same gene (≥ 99% sequence identity) or a paralogue of the same gene. Further, individual paralogues were tested for their expression level via qRT-PCR in tissue-specific manner. In total 44 paralogues in 14 key genes have been identified out of which 19 gene paralogues showed the highest expression pattern via qRT-PCR. Overall analysis shortlisted 6 gene paralogues, PKHMGR3, PKPAL2, PKDXPS1, PK4CL2, PKG10H2 and PKIS2 that might be playing role in the biosynthesis of P-I and P-II, however, their functional analysis need to be further validated either through gene silencing or over-expression. The usefulness of this approach can be expanded to other non-model plant species for which transcriptome resources have been generated.

Physiological and genomic evidence supports the role of Serratia quinivorans PKL:12 as a biopriming agent for the biohardening of micropropagated Picrorhiza kurroa plantlets in cold regions.

The medicinal herb, Picrorhiza kurroa Royle ex Benth has become endangered because of indiscriminate over-harvesting. Although micropropagation has been attempted for mass propagation of the plant, survival of in vitro plantlets under green house/open field poses a major challenge. Biopriming of micropropagated plantlets with plant growth-promoting rhizobacteria (PGPR) are among the successful methods to combat this problem. Serratia quinivorans PKL:12 was the best-characterized PGPR from rhizospheric soil of P. kurroa as it increased the vegetative growth and survival of the micropropagated plantlets most effectively. Complete genome (5.29 Mb) predicted genes encoding proteins for cold adaptation and plant growth-promoting traits in PKL:12. Antibiotic and biosynthetic gene cluster prediction supported PKL:12 as a potential biocontrol agent. Comparative genomics revealed 226 unique genes with few genes associated with plant growth-promoting potential. Physiological and genomic evidence supports S. quinivorans PKL:12 as a potential agent for bio-hardening of micropropagated P. kurroa plantlets in cold regions.

Growth Kinetics, Metabolites Production and Expression Profiling of Picrosides Biosynthetic Pathway Genes in Friable Callus Culture of Picrorhiza kurroa Royle ex Benth.

The rising demand for picrosides commercially and over-exploitation of Picrorhiza kurroa from natural habitat has to initiate alternative strategies for sustainable production of metabolites. In the present research, wild leaf explant of P. kurroa was used to produce friable callus under different culture condition, i.e., dark and light with two temperature variants (15 °C and 25 °C). Afterward, callus cell lines were screened based on growth biomass and metabolites content accumulation. The results revealed, maximum callus growth index along with antioxidant potential (IC50-40.88 µg/mL) and total phenol content (41.35 µg/mg) were observed under dark 25 °C. However, under light 15 °C, highest accumulation of picroside II (0.58 µg/mg), cinnamic acid (0.15 µg/mg), p-hydroxy acetophenone (0.30 µg/mg), total flavonoids (77.30 µg/mg), nitrogen (7.06%), carbohydrates (18.03%), and protein (44.12%) were detected. Major reported metabolite in callus was picroside I (1.63 µg/mg) under dark 15 °C. For the first time, picroside III content (range 0.15-0.56 µg/mg) was also detected and quantified in leaf-derived calli. Expression profiling of picroside biosynthetic pathway genes showed a positive correlation with the observed metabolites. Furthermore, an optimized protocol of metabolites enriched callus biomass could be used as potential strategy for sustainable production of picrosides at commercial scale.

Comparative transcriptome analysis in different tissues of a medicinal herb, Picrorhiza kurroa pinpoints transcription factors regulating picrosides biosynthesis.

Transcriptional regulation of picrosides biosynthesis, the iridoid glycosides of an endangered medicinal herb, Picrorhiza kurroa, is completely unknown. P. kurroa plants obtained from natural habitat accumulate higher picrosides than in-vitro cultured plants, which necessitates identification of transcription factors (TFs) regulating their differential biosynthesis. The current study investigates complete spectrum of different TF classes in P. kurroa transcriptomes and discerns their association with picrosides biosynthesis. Transcriptomes of differential picroside-I content shoots and picroside-II content roots were mined for seven classes of TFs implicated in secondary metabolism regulation in plants. Key TFs were identified through in silico transcript abundance and qPCR analysis was performed to confirm transcript levels of TFs under study in differential content tissues and genotypes. Promoter regions of key picrosides biosynthetic pathway genes were explored to hypothesize which TFs can possibly regulate target genes. A total of 131, 137, 107, 82 and 101 transcripts encoding different TFs families were identified in PKS-25, PKS-15, PKSS, PKR-25 and PKSR transcriptomes, respectively. ERF-18, bHLH-104, NAC-25, 32, 94 and SUF-4 showed elevated expression in roots (up to 37 folds) and shoots (up to 195 folds) of plants obtained from natural habitat, indicating their role as activators of picrosides biosynthesis whereas, elevated expression of WRKY-17, 40, 71 and MYB-4 in low picrosides content conditions suggested their down-regulatory role. In silico analysis of key picrosides biosynthetic pathway gene promoter regions revealed binding domains for ERF-18, NAC-25, WRKY-40 and MYB-4. Identification of candidate TFs contributing towards picrosides biosynthesis is a pre-requisite for designing appropriate metabolic engineering strategies aimed at enhancing picrosides content in vitro and in vivo.

Exogenous feeding of immediate precursors reveals synergistic effect on picroside-I biosynthesis in shoot cultures of Picrorhiza kurroa Royle ex Benth.

In the current study, we asked how the supply of immediate biosynthetic precursors i.e. cinnamic acid (CA) and catalpol (CAT) influences the synthesis of picroside-I (P-I) in shoot cultures of P. kurroa. Our results revealed that only CA and CA+CAT stimulated P-I production with 1.6-fold and 4.2-fold, respectively at 2.5 mg/100 mL concentration treatment. Interestingly, feeding CA+CAT not only directed flux towards p-Coumaric acid (p-CA) production but also appeared to trigger the metabolic flux through both shikimate/phenylpropanoid and iridoid pathways by utilizing more of CA and CAT for P-I biosynthesis. However, a deficiency in the supply of either the iridoid or the phenylpropanoid precursor limits flux through the respective pathways as reflected by feedback inhibition effect on PAL and decreased transcripts expressions of rate limiting enzymes (DAHPS, CM, PAL, GS and G10H). It also appears that addition of CA alone directed flux towards both p-CA and P-I production. Based on precursor feeding and metabolic fluxes, a current hypothesis is that precursors from both the iridoid and shikimate/phenylpropanoid pathways are a flux limitation for P-I production in shoot cultures of P. kurroa plants. This work thus sets a stage for future endeavour to elevate production of P-I in cultured plant cells.

Discerning picroside-I biosynthesis via molecular dissection of in vitro shoot regeneration in Picrorhiza kurroa.

KEY MESSAGE: Expression analysis of primary and secondary metabolic pathways genes vis-à-vis shoot regeneration revealed developmental regulation of picroside-I biosynthesis in Picrorhiza kurroa. Picroside-I (P-I) is an important iridoid glycoside used in several herbal formulations for treatment of various disorders. P-I is synthesized in shoots of Picrorhiza kurroa and Picrorhiza scrophulariiflora. Current study reports on understanding P-I biosynthesis in different morphogenetic stages, viz. plant segment (PS), callus initiation (CI), callus mass (CM), shoot primordia (SP), multiple shoots (MS) and fully developed (FD) stages of P. kurroa. Expression analysis of genes involved in primary and secondary metabolism revealed that genes encoding HMGR, PMK, DXPS, ISPE, GS, G10H, DAHPS and PAL enzymes of MVA, MEP, iridoid and shikimate/phenylpropanoid pathways showed significant modulation of expression in SP, MS and FD stages in congruence with P-I content compared to CM stage. While HK, PK, ICDH, MDH and G6PDH showed high expression in MS and FD stages of P. kurroa, RBA, HisK and CytO showed high expression with progress in regeneration of shoots. Quantitative expression analysis of secondary metabolism genes at two temperatures revealed that 7 genes HMGR, PMK, DXPS, GS, G10H, DAHPS and PAL showed high transcript abundance (32-87-folds) in FD stage derived from leaf and root segments at 15 °C compared to 25 °C in P. kurroa. Further screening of these genes at species level showed high expression pattern in P. kurroa (6-19-folds) vis-à-vis P. scrophulariiflora that was in corroboration with P-I content. Therefore, current study revealed developmental regulation of P-I biosynthesis in P. kurroa which would be useful in designing a suitable genetic intervention study by targeting these genes for enhancing P-I production.

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.

Mining NGS transcriptomes for miRNAs and dissecting their role in regulating growth, development, and secondary metabolites production in different organs of a medicinal herb, Picrorhiza kurroa.

MAIN CONCLUSION: This study is the first endeavor on mining of miRNAs and analyzing their involvement in development and secondary metabolism of an endangered medicinal herb Picrorhiza kurroa (P. kurroa ). miRNAs are ubiquitous non-coding RNA species that target complementary sequences of mRNA and result in either translational repression or target degradation in eukaryotes. The role of miRNAs has not been investigated in P. kurroa which is a medicinal herb of industrial value due to the presence of secondary metabolites, picroside-I and picroside-II. Computational identification of miRNAs was done in 6 transcriptomes of P. kurroa generated from root, shoot, and stolon organs varying for growth, development, and culture conditions. All available plant miRNA entries were retrieved from miRBase and used as backend datasets to computationally identify conserved miRNAs in transcriptome data sets. Total 18 conserved miRNAs were detected in P. kurroa followed by target prediction and functional annotation which suggested their possible role in controlling various biological processes. Validation of miRNA and expression analysis by qRT-PCR and 5' RACE revealed that miRNA-4995 has a regulatory role in terpenoid biosynthesis ultimately affecting the production of picroside-I. miR-5532 and miR-5368 had negligible expression in field-grown samples as compared to in vitro-cultured samples suggesting their role in regulating P. kurroa growth in culture conditions. The study has thus identified novel functions for existing miRNAs which can be further validated for their potential regulatory role.

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.

Differential proteomics of Picrorhiza kurrooa Royle ex Benth. in response to dark stress.

Picrorhiza (Picrorhiza kurrooa Royle ex Benth.) an important medicinal herb of western Himalayan region has been used to treat various diseases and disorders. Over-harvesting and lack of cultivation has led to its entry in Red Data Book as an endangered species. Further, its very restrictive habitat and lesser biomass production are major limitations for bringing it under commercial cultivation. All these issues necessitate deeper insights into mechanisms governing its growth and interaction with the environmental cues. Light may be one of the important factors to be studied for its role in regulating growth and adaptation of Picrorhiza as in natural habitat it prefers shady niches. Keeping this in view, proteome of Picrorhiza kept under light vis-à-vis under dark was analysed and compared. Leaf as well as root proteome of Picrorhiza was studied. Denaturing two dimensional gel electrophoresis and mass spectrometry techniques were used to detect and identify differentially expressed proteins, respectively. Twenty two proteins from leaf and 25 proteins from root showed differential expression levels under dark and light conditions. Among the differentially expressed proteins, majority were those involved in metabolism, protein synthesis, and stress and defense response. Other differentially expressed proteins were those involved in photosynthetic process, photorespiration and few proteins were with unknown function indicating that many different processes work together to establish a new cellular homeostasis in response to dark and light conditions. Proteins found to be differentially expressed under light vis-à-vis dark conditions suggested a range of biochemical pathways and processes being associated with response of plant to dark conditions. The identified proteins may be utilized for developing strategies for improving the biomass production/performance of Picrorhiza under varied light/dark habitats.

Translation initiation factor 5A in Picrorhiza is up-regulated during leaf senescence and in response to abscisic acid.

Translation initiation, the first step of protein synthesis process is the principal regulatory step controlling translation and involves a pool of translation initiation factors. In plants, from recent studies it is becoming evident that these translation initiation factors impact various aspects of plant growth and development in addition to their role in protein synthesis. Eukaryotic translation initiation factor eIF5A is one such factor which functions in start site selection for the eIF2-GTP-tRNAi ternary complex within the ribosomal-bound preinitiation complex and also stabilizes the binding of GDP to eIF2. In the present study we have cloned and analysed a gene (eIF5a) encoding eIF5A from Picrorhiza (Picrorhiza kurrooa Royle ex Benth.) a medicinal plant of the western Himalayan region. The full length eIF5a cDNA consisted of 838 bp with an open reading frame of 480 bp, 88 bp 5' untranslated region and 270 bp 3' untranslated region. The deduced eIF5A protein contained 159 amino acids with a molecular weight of 17.359 kDa and an isoelectric point of 5.59. Secondary structure analysis revealed eIF5A having 24.53% α-helices, 8.81% ß-turns, 23.27% extended strands and 43.40% random coils. pk-eIF5a transcript was found to be expressing during the active growth phase as well as during leaf senescence stage, however, highest expression was observed during leaf senescence stage. Further, its expression was up-regulated in response to exogenous application of abscisic acid. Both high intensity as well as low intensity light decreased the expression of pk-eIF5a. The findings suggest eIF5a to be an important candidate to develop genetic engineering based strategies for delaying leaf senescence.

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.

The GATA and SORLIP motifs in the 3-hydroxy-3-methylglutaryl-CoA reductase promoter of Picrorhiza kurrooa for the control of light-mediated expression.

Light upregulates the expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) in Picrorhiza kurrooa, an endangered medicinal herb. Upstream sequences of HMGR of P. kurrooa (PropkHMGR) were analyzed in relation to its role in light-mediated regulation of gene expression. GATA motif in PropkHMGR exhibited stronger DNA-protein interaction with the nuclear extract of dark-exposed plants in contrast to SORLIP that exhibited stronger binding with the nuclear extract of light-exposed plants. Analysis of PropkHMGR (PropkHMGR-D1, -1,059/-1) and its deletion fragments PropkHMGR-D2 (-825/-1), PropkHMGR-D3 (-651/-1), PropkHMGR-D4 (-452/-1), and PropkHMGR-D5 (-101/-1) in Arabidopsis thaliana showed PropkHMGR to regulate gene expression [ß-glucuronidase (GUS) was used as a reporter gene] at all the developmental stages but only in actively dividing tissues, excluding anthers. Whereas, PropkHMGR-D2 regulated GUS expression in relatively older seedlings but the expression was observed only in shoot apical meristem, root tips, and anthers. PropkHMGR-mediated gene expression was higher in dark as compared to that in the light in Arabidopsis across four temperatures studied. As opposed to the results in P. kurrooa, GATA motifs exhibited DNA-protein interaction with nuclear extract of light-exposed plants of Arabidopsis. SORLIP motifs in Arabidopsis also exhibited DNA-protein interaction with nuclear extract of light-exposed plants as in P. kurrooa. Data showed that (1) PropkHMGR regulated light-mediated gene expression and (2) GATA motif exhibited an inverse relationship between strength of DNA-protein interaction and the gene expression whereas the relationship was species specific for SORLIP.

Components of antioxidant system of Picrorhiza kurrooa exhibit different spatio-temporal behavior.

Antioxidant system is one of the important factors in regulating plant growth, development and adaptation. Thus, in order to have better insights into molecular mechanisms of growth and adaptation of a plant it is prerequisite to have known the status of various components of the antioxidant system of the plant. Here we studied the status of enzymatic and non-enzymatic components of the antioxidant system of picrorhiza (Picrorhiza kurrooa). Picrorhiza is an important medicinal herb of western Himalayan region and has been listed in the Red Data Book as an endangered species. Spatio-temporal analysis of ascorbic acid and glutathione in leaf, root and rhizome during different stages of development revealed differential status of these antioxidant molecules. Of the three tissues, ascorbic acid was found to be highest in leaves and lowest in roots. Interestingly, just opposite to that, glutathione was highest in roots and lowest in leaves. Using degenerate primers based approach followed by rapid amplification of complementary DNA (cDNA) ends method, full length cDNAs of three important genes namely Picrorhiza kurrooa ascorbate peroxidase (pkapx), Picrorhiza kurrooa monodehydroascorbate reductase (pkmdhar) and Picrorhiza kurrooa glutathione reductase (pkgr) of antioxidant system were cloned from picrorhiza. Complementary DNAs of pkapx, pkmdhar and pkgr contained 1,049, 2,016 and 1,664 bp, respectively. Expression analysis showed differential spatio-temporal expression of these genes. Expressions of all the three genes were found higher in roots as compared to rhizome and leaves. Temporal expression analysis of pkapx, pkmdhar and pkgr revealed differential transcript levels. Expression of pkapx exhibited negative correlation with the light intensity. Just opposite to the pkapx, expression pattern of pkgr revealed its positive correlation with light intensity. Expression pattern of pkmdhar revealed its light independent expression behavior. The findings may be useful to assess the role of cloned genes in picrorhiza growth, adaptation and can further be utilized for transgenic development for desired trait(s).

Plant regeneration, genetic fidelity, and active ingredient content of encapsulated hairy roots of Picrorhiza kurrooa Royle ex Benth.

Among five hairy root lines of Picrorhiza kurrooa that were established through Agrobacterium rhizogenes, one (H7) was selected for encapsulation due to high accumulation of picrotin and picrotoxinin (8.3 and 47.6 µg/g DW, respectively). Re-grown encapsulated roots induced adventitious shoots with 73 % frequency on MS medium supplemented with 0.1 µM 6-benzylaminopurine, following 6 months of storage at 25 °C. Regenerated plantlets had 85 % survival after 2 months. Regenerants were of similar morphotype having increased leaf number and branched root system as compared to non-transformed plants. The transformed nature of the plants was confirmed through PCR and Southern blot analysis. Genetic fidelity analysis of transformed plants using RAPD and ISSR showed 5.2 and 3.6 % polymorphism, respectively. Phytochemical analysis also showed that picrotin and picrotoxinin content were similar in hairy root line and its regenerants.

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.