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.

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.

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.

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.

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.