Green synthesis of silver nanoparticles using Rhodiola imbricata and Withania somnifera root extract and their potential catalytic, antioxidant, cytotoxic and growth-promoting activities.

This study presents the development of a sustainable production process of environmentally benign silver nanoparticles (AgNPs) from aqueous root extract of Rhodiola imbricata (RI) and Withania somnifera (WS) for mitigating environmental pollution and investigating their potential applications in agriculture and biomedical industry. RIWS-AgNPs were characterized using several analytical techniques (UV-Vis, DLS, HR-TEM, SAED, EDX and FTIR). The antioxidant and anticancer activity of RIWS-AgNPs were estimated by DPPH and MTT assay, respectively. UV-Vis and DLS analysis indicated that equal ratio of RIWS-extract and silver nitrate (1:1) is optimum for green synthesis of well-dispersed AgNPs (λmax: 430 nm, polydispersity index: 0.179, zeta potential: - 17.9 ± 4.14). HR-TEM and SAED analysis confirmed the formation of spherical and crystalline RIWS-AgNPs (37-42 nm). FTIR analysis demonstrated that the phenolic compounds are probably involved in stabilization of RIWS-AgNPs. RIWS-AgNPs showed effective catalytic degradation of hazardous environmental pollutant (4-nitrophenol). RIWS-AgNPs treatment significantly increased the growth and photosynthetic pigments of Hordeum vulgare in a size- and dose-dependent manner (germination (77%), chlorophyll a (12.62 ± 0.07 µg/ml) and total carotenoids (7.05 ± 0.04 µg/ml)). The DPPH assay demonstrated that RIWS-AgNPs exert concentration-dependent potent antioxidant activity (IC50: 12.30 µg/ml, EC50: 0.104 mg/ml, ARP: 959.45). Moreover, RIWS-AgNPs also confer strong cytotoxic activity against HepG2 cancer cell line in dose-dependent manner (cell viability: 9.51 ± 1.55%). Overall, the present study for the first time demonstrated a green technology for the synthesis of stable RIWS-AgNPs and their potential applications in biomedical and agriculture industry as phytostimulatory, antioxidant and anticancer agent. Moreover, RIWS-AgNPs could potentially be used as a green alternative for environmental remediation.

Comparative transcriptomics uncovers differences in photoautotrophic versus photoheterotrophic modes of nutrition in relation to secondary metabolites biosynthesis in Swertia chirayita.

Swertia chirayita is a high-value medicinal herb exhibiting antidiabetic, hepatoprotective, anticancer, antiediematogenic and antipyretic properties. Scarcity of its plant material has necessitated in vitro production of therapeutic metabolites; however, their yields were low compared to field grown plants. Possible reasons for this could be differences in physiological and biochemical processes between plants grown in photoautotrophic versus photoheterotrophic modes of nutrition. Comparative transcriptomes of S. chirayita were generated to decipher the crucial molecular components associated with the secondary metabolites biosynthesis. Illumina HiSeq sequencing yielded 57,460 and 43,702 transcripts for green house grown (SCFG) and tissue cultured (SCTC) plants, respectively. Biological role analysis (GO and COG assignments) revealed major differences in SCFG and SCTC transcriptomes. KEGG orthology mapped 351 and 341 transcripts onto secondary metabolites biosynthesis pathways for SCFG and SCTC transcriptomes, respectively. Nineteen out of 30 genes from primary metabolism showed higher in silico expression (FPKM) in SCFG versus SCTC, possibly indicating their involvement in regulating the central carbon pool. In silico data were validated by RT-qPCR using a set of 16 genes, wherein 10 genes showed similar expression pattern across both the methods. Comparative transcriptomes identified differentially expressed transcription factors and ABC-type transporters putatively associated with secondary metabolism in S. chirayita. Additionally, functional classification was performed using NCBI Biosystems database. This study identified the molecular components implicated in differential modes of nutrition (photoautotrophic vs. photoheterotrophic) in relation to secondary metabolites production in S. chirayita.

Genomics and relative expression analysis identifies key genes associated with high female to male flower ratio in Jatropha curcas L.

Jatropha curcas, has been projected as a major source of biodiesel due to high seed oil content (42 %). A major roadblock for commercialization of Jatropha-based biodiesel is low seed yield per inflorescence, which is affected by low female to male flower ratio (1:25-30). Molecular dissection of female flower development by analyzing genes involved in phase transitions and floral organ development is, therefore, crucial for increasing seed yield. Expression analysis of 42 genes implicated in floral organ development and sex determination was done at six floral developmental stages of a J. curcas genotype (IC561235) with inherently higher female to male flower ratio (1:8-10). Relative expression analysis of these genes was done on low ratio genotype. Genes TFL1, SUP, AP1, CRY2, CUC2, CKX1, TAA1 and PIN1 were associated with reproductive phase transition. Further, genes CUC2, TAA1, CKX1 and PIN1 were associated with female flowering while SUP and CRY2 in female flower transition. Relative expression of these genes with respect to low female flower ratio genotype showed up to ~7 folds increase in transcript abundance of SUP, TAA1, CRY2 and CKX1 genes in intermediate buds but not a significant increase (~1.25 folds) in female flowers, thereby suggesting that these genes possibly play a significant role in increased transition towards female flowering by promoting abortion of male flower primordia. The outcome of study has implications in feedstock improvement of J. curcas through functional validation and eventual utilization of key genes associated with female flowering.

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.

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.

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.

A proposed biosynthetic pathway of picrosides linked through the detection of biochemical intermediates in the endangered medicinal herb Picrorhiza kurroa.

INTRODUCTION: Picrorhiza kurroa Royle ex Benth is an important medicinal herb used in the preparation of several herbal drug formulations due to the presence of picroside-I (P-I) and picroside-II (P-II) along with other iridoid-glucosides derivatives. OBJECTIVE: The endangered status of P. kurroa coupled with lack of information on biosynthesis of P-I and P-II necessitate deciphering the biosynthetic pathway for picrosides. METHODS: LC with electrospray ionisation (ESI) and quadrupole time of flight combined with MS/MS was used to detect intermediates and assemble the picrosides biosynthetic pathway in P. kurroa. RESULTS: The presence of catalpol and aucubin, the major backbone structures of picrosides, along with intermediate metabolites boschnaloside, bartsioside and mussaenosidic acid, was confirmed in ESI negative mode with pseudomolecular ion peaks, that is, m/z 361, m/z 343, m/z 345, m/z 329 and m/z 375 ions and their fragmentation patterns. CONCLUSION: The picrosides biosynthetic pathway is expected to provide a reliable platform towards understanding the molecular components (genes/enzymes) of P-I and P-II biosynthesis in P. kurroa for their eventual utilisation in various applications.

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.

Optimizing nutrient media conditions for continuous production of shoot biomass enriched in major medicinal constituents, amarogentin and mangiferin of endangered medicinal herb, Swertia chirayita.

Biosynthesis and accumulation of Amarogentin and Mangiferin from shoot culture of endangered herb Swertia chirayita helped in rescuing its natural population along with continuous production of quality rich herbal material. Although, presence of Amarogentin and Mangiferin had already been reported, but such studies did not elaborate the significant developmental stages at two varying temperature (15 ± 1 °C and 25 ± 1 °C) in shoot cultures of S. chirayita. Different developmental stages involved throughout from callus induction to complete regeneration of plant by using shoot cultures of S. chirayita, reveal different amounts of significant medicinal compounds having high pharmacological importance like bearing anti-diabetic and anti-cancerous properties. So in the present study, different developmental stages i.e. plant segment as leaf disc explants, initiation of callus formation, callus mass development, shoots primordial, manifold shoot formation and shoot elongation with complete growth were explored for accumulation of Amarogentin and Mangiferin. The Amarogentin content was 4.72 µg/mg at 15 ± 1 °C and 4.41 µg/mg at 25 ± 1 °C whereas Mangiferin content was 15.54 µg/mg at 15 ± 1 °C and 9.70 µg/mg at 25 ± 1 °C in leaf discs provided with the medium MS + 2,4D = 1 mg/L, 6BAP = 0.5 mg/L, TDZ = 0.5 mg/L, respectively. The accumulation of Amarogentin and Mangiferin started from callus cultures differentiating into shoots and reached to the detectable amount equivalent to actual leaf explants in fully grown shoots with content of 5.79 µg/mg at 15 ± 1 °C and 5.35 µg/mg at 25 ± 1 °C whereas 15.56 µg/mg at 15 ± 1 °C and 13.15 µg/mg at 25 ± 1 °C provided with the medium MS + IBA = 3 mg/L, KN = 1 mg/L, respectively. Maximum accumulation of bioactive compounds was observed in ≈3 months old in-vitro grown shoots at 15 ± 1˚ C wherein, the content of Amarogentin was ≈8.51 folds higher and Mangiferin was ≈4.09 folds higher than the ≈3 months old green house grown shoots. So, the in-vitro raised shoots of S. chirayita enriched with marker medicinal compounds would be utilized as ready to use raw material for pharmaceutical industries for herbal drug formulations and can be utilized to transfer under natural habitats for conserving its diminishing population.

Agro-Morphological Characterization and Nutritional Profiling of Traditional Himalayan Crop Landraces for Their Promotion Toward Mainstream Agriculture.

The northwest Indian Himalayas are often regarded as a biological hotspot for the presence of rich agro-biodiversity harboring locally adapted traditional crop landraces facing utter neglect owing to modern agricultural systems promoting high-yielding varieties. Addressing this challenge requires extricating the potential of such cultivars in terms of agro-morphological and nutritional attributes. In this study, 29 traditional crop landraces of maize (11), paddy (07), finger millet (03), buckwheat (05), and naked barley (03) were characterized and evaluated for target traits of interest. In maize, Chitkanu emerged as an early maturing landrace (107 days) with high concentrations of zinc (Zn), iron (Fe), and potassium (K), and Safed makki showed the highest 100-seed weight (28.20 g). Similarly, Bamkua dhan exhibited high concentrations of K and phosphorus (P), and Lamgudi dhan showed a high protein content (14.86 g/100 g) among paddy landraces. Ogla-I and Phapra-I showed high contents of protein (14.80 g/100 g) and flavonoids (20.50 mg/g) among buckwheat landraces, respectively, followed by Nei-I, which exhibited the highest protein content (15.66 g/100 g) among naked barley landraces. Most of the target traits varied significantly (p < 0.05) among evaluated samples, except those associated with finger millet landraces. The grouping pattern obtained by principal component analysis (PCA) and multidimensional scaling (MDS) was congruent with the geographical relationship among the crop landraces. This study led to the identification of elite crop landraces having useful variations that could be exploited in plant breeding programs and biofortification strategies for future crop improvement. Our endeavor would aid in conserving the depleting Himalayan agro-biodiversity and promoting versatile traditional crops toward mainstream agriculture vis-à-vis future nutritional security.

Performance Evaluation of Pavement Geomaterials Stabilized with Pond Ash and Brick Kiln Dust Using Advanced Cyclic Triaxial Testing.

An increase in the generation of industrial waste materials such as pond ash and brick kiln dust represents a serious threat to the earth's environment. The waste materials have novel characteristics and therefore their physical, chemical, mineralogical, morphological, mechanical, and toxicity characteristics were determined so that these materials can be incorporated as construction materials based on technical and environmental considerations. The purpose of this study is to utilize the wastes in the stabilization of clay geomaterial to outperform existing problems of inadequate strength and stiffness of the subgrade layer in flexible pavements. Mixtures of geomaterials, containing pond ash, brick kiln dust, and their combination were prepared and important engineering characteristics such as the plasticity, compaction, and strength of the mixtures were examined. The measurement of response to dynamic loading is a pre-condition for the accurate characterization of geomaterials used in pavement construction. Using advanced cyclic triaxial testing, this study evaluates the performance of pond ash and brick kiln dust in increasing the stiffness of the geomaterial under cyclic loading. To stimulate the worst field conditions, the stiffness and strength tests were performed under standard and four-day water-soaked conditions. The implementation of several stress-dependent models for the prediction of stiffness was examined. Pond ash and brick kiln dust were found to be effective in increasing the stiffness and strength of the geomaterial. The wastes were the most effectual when added in combination to the geomaterial. The characterization of the wastes was informative in understanding the governing mechanism prevalent in the waste stabilized mixtures. The toxicity characterization study revealed the non-toxic and non-hazardous nature of the waste materials, permitting their use in the construction of pavements. This study recommends the use of wastes in the subgrade of flexible pavements. Further research is needed for performance evaluation of the wastes on silt and sand geomaterials for their wider application.

Enhanced Production of Phenolic Compounds in Compact Callus Aggregate Suspension Cultures of Rhodiola imbricata Edgew.

Rhodiola imbricata is a rare medicinal plant of the trans-Himalayan region of Ladakh. It is used for the treatment of numerous health ailments. Compact callus aggregate (CCA) suspension cultures of Rhodiola imbricata were established to counter extinction threats and for production of therapeutically valuable phenolic compounds to meet their increasing industrial demands. The present study also investigated the effect of jasmonic acid (JA) on production of phenolic compounds and bioactivities in CCA suspension cultures. CCA suspension cultures established in an optimized Murashige and Skoog medium supplemented with 30 g/l sucrose, 3 mg/l NAA, and 3 mg/l BAP showed maximum biomass accumulation (8.43 g/l DW) and highest salidroside production (3.37 mg/g DW). Upon 100 µM JA treatment, salidroside production (5.25 mg/g DW), total phenolic content (14.69 mg CHA/g DW), total flavonoid content (4.95 mg RE/g DW), and ascorbic acid content (17.93 mg/g DW) were significantly increased in cultures. In addition, DPPH-scavenging activity (56.32%) and total antioxidant capacity (60.45 mg QE/g DW) were significantly enhanced upon JA treatment, and this was positively correlated with increased accumulation of phenolic compounds. JA-elicited cultures exhibited highest antimicrobial activity against Escherichia coli. This is the first report describing the enhanced production of phenolic compounds and bioactivities from JA-elicited CCA suspension cultures of Rhodiola imbricata.

Influence of light quality on growth, secondary metabolites production and antioxidant activity in callus culture of Rhodiola imbricata Edgew.

Rhodiola imbricata is a rare medicinal herb well-known for its adaptogenic and antioxidant properties due to the presence of a diverse array of secondary metabolites, including phenylethanoids and phenylpropanoids. These secondary metabolites are generating considerable interest due to their potential applications in pharmaceutical and nutraceutical industries. The present study investigated the influence of light quality on growth, production of industrially important secondary metabolites and antioxidant activity in callus cultures of Rhodiola imbricata. Callus cultures of Rhodiola imbricata were established under different light conditions: 100% red, 100% blue, 100% green, RGB (40% red: 40% green: 20% blue) and 100% white (control). The results showed that the callus cultures grown under red light accumulated maximum amount of biomass (7.43 g/l) on day 21 of culture, as compared to other light conditions. Maximum specific growth rate (0.126 days-1) and doubling time (132.66 h) was observed in callus cultures grown under red light. Reverse phase-high performance liquid chromatographic (RP-HPLC) analysis revealed that the callus cultures exposed to blue light accumulated maximum amount of Salidroside (3.12 mg/g DW) on day 21 of culture, as compared to other light conditions. UV-Vis spectrophotometric analysis showed that the callus cultures exposed to blue light accumulated maximum amount of total phenolics (11.84 mg CHA/g DW) and total flavonoids (5.53 mg RE/g DW), as compared to other light conditions. Additionally, callus cultures grown under blue light displayed enhanced DPPH free radical scavenging activity (53.50%). Callus cultures grown under different light conditions showed no significant difference in ascorbic acid content (11.05-13.90 mg/g DW) and total antioxidant capacity (27.37-30.17 mg QE/g DW). The correlation analysis showed a positive correlation between total phenolic content and DPPH free radical scavenging activity in callus cultures (r = 0.85). Taken together, these results demonstrate the remarkable potential of light quality on biomass accumulation and production of industrially important secondary metabolites in callus cultures of Rhodiola imbricata. This study will open new avenues and perspectives towards abiotic elicitation strategies for sustainable growth and enhanced production of bioactive compounds in in-vitro cultures of Rhodiola imbricata.

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.

Transcriptome-wide mining suggests conglomerate of genes associated with tuberous root growth and development in Aconitum heterophyllum Wall.

Tuberous roots of Aconitum heterophyllum constitute storage organ for secondary metabolites, however, molecular components contributing to their formation are not known. The transcriptomes of A. heterophyllum were analyzed to identify possible genes associated with tuberous root development by taking clues from genes implicated in other plant species. Out of 18 genes, eight genes encoding GDP-mannose pyrophosphorylase (GMPase), SHAGGY, Expansin, RING-box protein 1 (RBX1), SRF receptor kinase (SRF), ß-amylase, ADP-glucose pyrophosphorylase (AGPase) and Auxin responsive factor 2 (ARF2) showed higher transcript abundance in roots (13-171 folds) compared to shoots. Comparative expression analysis of those genes between tuberous root developmental stages showed 11-97 folds increase in transcripts in fully developed roots compared to young rootlets, thereby implying their association in biosynthesis, accumulation and storage of primary metabolites towards root biomass. Cluster analysis revealed a positive correlation with the gene expression data for different stages of tuberous root formation in A. heterophyllum. The outcome of this study can be useful in genetic improvement of A. heterophyllum for root biomass yield.

Detection of intermediates through high-resolution mass spectrometry for constructing biosynthetic pathways for major chemical constituents in a medicinally important herb, Swertia chirayita.

Swertia chirayita is an endangered medicinal herb widely used as an antidiabetic. It contains two major classes of metabolites, secoiridoids and xanthones, i.e. swertiamarin, mangiferin, amarogentin and amaroswerin. The biosynthetic pathways for these chemical constituents are not completely deciphered due to gaps and redundancy of routes proposed such as for mangiferin. The missing intermediates in pathways were detected through LC-ESI-QToF-HRMS/MS, including the detection of new secoiridoids, amaronitidin and gentiopicroside. The study also reports that the biosynthesis of amaronitidin occurs through the coupling of gentiopicroside and biphenyl acid derivatives such as amarogentin and amaroswerin. This study reports for the first time complete biosynthetic pathways for gentiopicroside, mangiferin, amarogentin, amaroswerin and amaronitidin in S. chirayita with the detection of intermediate metabolites iriflophenone, maclurin, deoxyloganic acid, loganic acid and 1,3,6,7-tetrahydroxy-9H-xanthen-9-one.

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.

Contents of therapeutic metabolites in Swertia chirayita correlate with the expression profiles of multiple genes in corresponding biosynthesis pathways.

Swertia chirayita, an endangered medicinal herb, contains three major secondary metabolites swertiamarin, amarogentin and mangiferin, exhibiting valuable therapeutic traits. No information exists as of today on the biosynthesis of these metabolites in S. chirayita. The current study reports the expression profiling of swertiamarin, amarogentin and mangiferin biosynthesis pathway genes and their correlation with the respective metabolites content in different tissues of S. chirayita. Root tissues of greenhouse grown plants contained the maximum amount of secoiridoids (swertiamarin, 2.8% of fr. wt and amarogentin, 0.1% of fr. wt), whereas maximum accumulation of mangiferin (1.0% of fr. wt) was observed in floral organs. Differential gene expression analysis and their subsequent principal component analysis unveiled ten genes (encoding HMGR, PMK, MVK, ISPD, ISPE, GES, G10H, 8HGO, IS and 7DLGT) of the secoiridoids biosynthesis pathway and five genes (encoding EPSPS, PAL, ADT, CM and CS) of mangiferin biosynthesis with elevated transcript amounts in relation to corresponding metabolite contents. Three genes of the secoiridoids biosynthesis pathway (encoding PMK, ISPD and IS) showed elevated levels (∼57-104 fold increase in roots), and EPSPS of mangiferin biosynthesis showed an about 117 fold increase in transcripts in leaf tissues of the greenhouse grown plants. The study does provide leads on potential candidate genes correlating with the metabolites biosynthesis in S. chirayita as an initiative towards its genetic improvement.

Expression analysis of biosynthetic pathway genes vis-à-vis podophyllotoxin content in Podophyllum hexandrum Royle.

Podophyllum hexandrum Royle is known for its vast medicinal properties, particularly anticancer. It contains higher amount of podophyllotoxin (4.3 %), compared to Podophyllum peltatum (0.025 %) and other plant species; as a result, it has been used worldwide in the preparation of various drugs including anticancer, antimalarial, antiviral, antioxidant, antifungal, and so on. Currently, Etoposide (VP-16-213), Vumon® (Teniposide; VM-26), Etopophos®, Pod-Ben- 25, Condofil, Verrusol, and Warticon are available in the market. Due to highly complex synthesis and low cell culture yields of podophyllotoxin (0.3 %), the supply of raw material cannot be met due to increasing industrial demands. The knowledge on podophyllotoxin biosynthetic pathway vis-à-vis expression status of genes is fragmentary. Quantitative expression analysis of 21 pathway genes has revealed 9 genes, namely SD, PD, PCH, CM, CMT, CAD, CCR, C4H, and ADH, that showed increase in transcript abundance up to 1.4 to 23.05 folds, respectively, vis-à-vis podophyllotoxin content in roots (1.37 %) and rhizomes (3.05 %) of P. hexandrum. In silico analysis of putative cis-regulatory elements in promoter regions of overexpressed genes showed the presence of common Skn-1 motif and MBS elements in CMT, CAD, CCR, C4H, and ADH genes, thereby, suggesting their common regulation. The outcome of the study has resulted in the identification of suitable candidate genes which might be contributing to podophyllotoxin biosynthesis that can act as potential targets for any genetic intervention strategies aimed at its enhanced production.

Multiple genes of mevalonate and non-mevalonate pathways contribute to high aconites content in an endangered medicinal herb, Aconitum heterophyllum Wall.

Aconitum heterophyllum Wall, popularly known as Atis or Patis, is an important medicinal herb of North-Western and Eastern Himalayas. No information exists on molecular aspects of aconites biosynthesis, including atisine- the major chemical constituent of A. heterophyllum. Atisine content ranged from 0.14% to 0.37% and total alkaloids (aconites) from 0.20% to 2.49% among 14 accessions of A. heterophyllum. Two accessions contained the highest atisine content with 0.30% and 0.37% as well as the highest alkaloids content with 2.22% and 2.49%, respectively. No atisine was detected in leaves and shoots of A. heterophyllum, thereby, suggesting that the biosynthesis and accumulation of aconite alkaloids occur mainly in roots. Quantitative expression analysis of 15 genes of MVA/MEP pathways in roots versus shoots, differing for atisine content (0-2.2 folds) showed 11-100 folds increase in transcript amounts of 4 genes of MVA pathway; HMGS, HMGR, PMK, IPPI, and 4 genes of MEP pathway; DXPS, ISPD, HDS, GDPS, respectively. The overall expression of 8 genes decreased to 5-12 folds after comparative expression analysis between roots of high (0.37%) versus low (0.14%) atisine content accessions, but their relative transcript amounts remained higher in high content accessions, thereby implying their role in atisine biosynthesis and accumulation. PCA analysis revealed a positive correlation between MVA/MEP pathways genes and alkaloids content. The current study provides first report wherein partial sequences of 15 genes of MVA/MEP pathways have been cloned and studied for their possible role in aconites biosynthesis. The outcome of study has potential applications in the genetic improvement of A. heterophyllum.