Chemical Diversity of Wild-Growing and Cultivated Common Valerian (Valeriana officinalis L. s.l.) Originating from Poland.

Common valerian is a medicinal plant. The underground organs of this species are used as a mild sedative and sleeping aid. Poland is one of the largest producers of this raw material in Europe, with local cultivar 'Lubelski' as a primary cultivated form. Although valerian is the subject of more or less deliberate selection carried out by farmers, it is still genetically unstable. The aim of this study was to determine the diversity of the 'Lubelski' cultivar originating from four regions of Poland (forms: L1-L4) in relation to wild-growing populations of the species. The plants were assessed in terms of the mass of underground organs and the content of valerenic acids and essential oils (EOs). The content of valerenic acids was determined using HPLC, whereas the content of EOs was determined using hydrodistillation. The composition of EOs was assessed using GC-MS GC-FID. The ploidy level of the analyzed objects was determined as well. Wild-growing populations (diploids) were characterized by lower masses of underground organs and lower contents of valerenic acid than cultivated forms (tetraploids). However, they produced higher contents of EOs. All the cultivated forms were strongly diversified with respect to the analyzed traits, including the mass of the roots (CV 49-75%), the content of valerenic acids (CV 18-55%), and the content of EOs (CV 28-57%). A total of 44 compounds were identified in the EOs. The dominant compound of both wild-growing populations and the 'Lubelski' forms were: α-fenchene, bornyl acetate, and valerenal. Among 'Lubelski' forms, the most interesting seems to be the L2 form, which was characterized by a relatively high yield and high content of valerenic acids and EOs. Thus, it appears to be a promising source of objects for further valerian cultivar improvement.

Genetic and Chemical Diversity of Commercial Japanese Valerian.

In order to investigate the relationship between the chemical composition of essential oils and haplotypes of the psbA-trnH intergenic spacer region of chloroplast DNA (psbA-trnH) in Valerianae Fauriei Radix (Japanese Valerian; JV), we analyzed the DNA sequence and GC-MS metabolome of JV from Japanese markets and of herbal specimens from related species. DNA analysis revealed that JV products from Japan consisted of three haplotypes, namely AH-1, -2 and -5 reported in our previous study. The GC-MS metabolome revealed five chemotypes (J1, J2, C, K and O), of which J1, J2 and C were detected in the JV products from Japan. Chemotypes J1 and J2, with kessyl glycol diacetate (KGD) as the main volatile component, were found in the products of Japanese origin whereas chemotype C, with 1-O-acetyl-2,10-bisaboladiene-1,6-diol (ABD), was found in the products of Chinese and Korean origin. The haplotypes were correlated with the chemotypes: haplotype AH-1 for chemotype J1, AH-2 for chemotype J2 and AH-5 for chemotype C, suggesting that the chemical diversity of JV is not attributed to the environmental factors rather to the genetic factors. Since KGD and ABD were reported to have sedative effects and nerve growth factor (NGF)-potentiating effects, respectively, understanding the chemotypes and selecting an appropriate one would be important for the application of JV. The psbA-trnH haplotypes could be useful DNA markers for the quality control and standardization of JV.

Biosynthesis of valerenic acid by engineered Saccharomyces cerevisiae.

OBJECTIVE: To produce valerenic acid (VA) in Saccharomyces cerevisiae by engineering a heterologous synthetic pathway. RESULT: Valerena-4,7(11)-diene synthase (VDS) derived from Valeriana officinalis (valerian) was expressed in S. cerevisiae to generate valerena-4,7(11)-diene as the precursor of VA. By overexpressing the key genes of the mevalonate pathway ERG8, ERG12 and ERG19, and integrating 4 copies of MBP (maltose-binding protein)-VDS-ERG20 gene expression caskets into the genome, the production of valerena-4,7(11)-diene was improved to 75 mg/L. On this basis, the cytochrome P450 monooxygenase LsGAO2 derived from Lactuca sativa was expressed to oxidize valerena-4,7(11)-diene to produce VA, and the most effective VA production strain was used for fermentation. The yield of VA reached 2.8 mg/L in the flask and 6.8 mg/L in a 5-L bioreactor fed glucose. CONCLUSIONS: An S. cerevisiae strain was constructed and optimized to produce VA, but the valerena-4,7(11)-diene oxidation by LsGAO2 is still the rate-limiting step for VA synthesis that needs to be further optimized in future studies.

Structurally Guided Reprogramming of Valerenadiene Synthase.

Valerena-1,10-diene synthase (VDS) catalyzes the conversion of the universal precursor farnesyl diphosphate into the unusual sesquiterpene valerena-1,10-diene (VLD), which possesses a unique isobutenyl substituent group. In planta, one of VLD's isobutenyl terminal methyl groups becomes oxidized to a carboxylic acid forming valerenic acid (VA), an allosteric modulator of the GABAA receptor. Because a structure-activity relationship study of VA for its modulatory activity is desired, we sought to manipulate the VDS enzyme for the biosynthesis of structurally diverse scaffolds that could ultimately lead to the generation of VA analogues. Using three-dimensional structural homology models, phylogenetic sequence comparisons to well-characterized sesquiterpene synthases, and a substrate-active site contact mapping approach, the contributions of specific amino acid residues within or near the VDS active site to possible catalytic cascades for VLD and other sesquiterpene products were assessed. An essential role of Tyr535 in a germacrenyl route to VLD was demonstrated, while its contribution to a family of other sesquiterpenes derived from a humulyl route was not. No role for Cys415 or Cys452 serving as a proton donor to reaction intermediates in VLD biosynthesis was observed. However, a gatekeeper role for Asn455 in directing farnesyl carbocations down all-trans catalytic cascades (humulyl and germacrenyl routes) versus a cisoid cascade (nerolidyl route) was demonstrated. Altogether, these results have mapped residues that establish a context for the catalytic cascades operating in VDS and future manipulations for generating more structurally constrained scaffolds.

Structural Mutations in the Organellar Genomes of Valeriana sambucifolia f. dageletiana (Nakai. ex Maekawa) Hara Show Dynamic Gene Transfer.

Valeriana sambucifolia f. dageletiana (Nakai. ex Maekawa) Hara is a broad-leaved valerian endemic to Ulleung Island, a noted hot spot of endemism in Korea. However, despite its widespread pharmacological use, this plant remains comparatively understudied. Plant cells generally contain two types of organellar genomes (the plastome and the mitogenome) that have undergone independent evolution, which accordingly can provide valuable information for elucidating the phylogenetic relationships and evolutionary histories of terrestrial plants. Moreover, the extensive mega-data available for plant genomes, particularly those of plastomes, can enable researchers to gain an in-depth understanding of the transfer of genes between different types of genomes. In this study, we analyzed two organellar genomes (the 155,179 bp plastome and the 1,187,459 bp mitogenome) of V. sambucifolia f. dageletiana and detected extensive changes throughout the plastome sequence, including rapid structural mutations associated with inverted repeat (IR) contraction and genetic variation. We also described features characterizing the first reported mitogenome sequence obtained for a plant in the order Dipsacales and confirmed frequent gene transfer in this mitogenome. We identified eight non-plastome-originated regions (NPRs) distributed within the plastome of this endemic plant, for six of which there were no corresponding sequences in the current nucleotide sequence databases. Indeed, one of these unidentified NPRs unexpectedly showed certain similarities to sequences from bony fish. Although this is ostensibly difficult to explain, we suggest that this surprising association may conceivably reflect the occurrence of gene transfer from a bony fish to the plastome of an ancestor of V. sambucifolia f. dageletiana mediated by either fungi or bacteria.

Identification of key genes associated with secondary metabolites biosynthesis by system network analysis in Valeriana officinalis.

Valeriana officinalis is a medicinal plant, a source of bioactive chemical compounds and secondary metabolites which are applied in pharmaceutical industries. The advent of ethnomedicine has provided alternatives for disease treatment and has increased demands for natural products and bioactive compounds. A set of preliminary steps to answers for such demands can include integrative omics for systems metabolic engineering, as an approach that contributes to the understanding of cellular metabolic status. There is a growing trend of this approach for genetically engineering metabolic pathways in plant systems, by which natural and synthetic compounds can be produced. As in the case of most medicinal plants, there are no sufficient information about molecular mechanisms involved in the regulation of metabolic pathways in V. officinalis. In this research, systems biology was performed on the RNA-seq transcriptome and metabolome data to find key genes that contribute to the synthesis of major secondary metabolites in V. officinalis. The R Package Weighted Gene Co-Expression Network Analysis (WGCNA) was employed to analyze the data. Based on the results, some major modules and hub genes were identified to be associated with the valuable secondary metabolites. In addition, some TF-encoding genes, including AP2/ERF-ERF, WRKY and NAC TF families, as well as some regulatory factors including protein kinases and transporters were identified. The results showed that several novel hub genes, such as PCMP-H24, RPS24B, ANX1 and PXL1, may play crucial roles in metabolic pathways. The current findings provide an overall insight into the metabolic pathways of V. officinalis and can expand the potential for engineering genome-scale pathways and systems metabolic engineering to increase the production of bioactive compounds by plants.

Identification of Valeriana fauriei and other Eurasian medicinal valerian by DNA polymorphisms in psbA-trnH intergenic spacer sequences in chloroplast DNA.

In order to differentiate among Valeriana fauriei Briq. and other Eurasian medicinal valerian (V. dioica L., V. hardwickii Wall., V. jatamansi Jones, and V. officinalis L.), we attempted to establish DNA markers. DNA sequences for the psbA-trnH intergenic spacer region of chloroplast DNA (psbA-trnH) and 18S ribosomal RNA, internal transcribed spacer 1 (ITS1), 5.8S ribosomal RNA, internal transcribed spacer 2 (ITS2), and 28S ribosomal RNA of nuclear DNA in V. fauriei and other Eurasian medicinal valerian were compared. Using partial sequences of psbA-trnH (nucleotide positions 1-75 from the 5' end of the intergenic spacer region), V. fauriei and other Eurasian medicinal valerian could be correctly identified to the species level. In addition, the partial sequences of psbA-trnH in V. fauriei contained five different haplotypes, and it was possible to distinguish the origins of valerian from Japan and Eurasia (China and Korea). On the other hand, individuals had heterogeneous sequences of ITS1 and ITS2, making it impossible to use direct sequencing and DNA markers of ITS1 and ITS2 to distinguish species and origins of V. fauriei and other Eurasian medicinal valerian.

De novo synthesis of the sedative valerenic acid in Saccharomyces cerevisiae.

Valeriana officinalis (Valerian) root extracts have been used by European and Asian cultures for millennia for their anxiolytic and sedative properties. However, the efficacy of these extracts suffers from variable yields and composition, making these extracts a prime candidate for microbial production. Recently, valerenic acid, a C15 sesquiterpenoid, was identified as the active compound that modulates the GABAA channel. Although the first committed step, valerena-4,7(11)-diene synthase, has been identified and described, the complete valerenic acid biosynthetic pathway remains to be elucidated. Sequence homology and tissue-specific expression profiles of V. officinalis putative P450s led to the discovery of a V. officinalis valerena-4,7(11)-diene oxidase, VoCYP71DJ1, which required coexpression with a V. officinalis alcohol dehydrogenase and aldehyde dehydrogenase to complete valerenic acid biosynthesis in yeast. Further, we demonstrated the stable integration of all pathway enzymes in yeast, resulting in the production of 140 mg/L of valerena-4,7(11)-diene and 4 mg/L of valerenic acid in milliliter plates. These findings showcase Saccharomyces cerevisiae's potential as an expression platform for facilitating multiply-oxidized medicinal terpenoid pathway discovery, possibly paving the way for scale up and FDA approval of valerenic acid and other active compounds from plant-derived herbal medicines.

Exploring the utility of "next-generation" sequence data on inferring the phylogeny of the South American Valeriana (Valerianaceae).

This study aimed to investigate the phylogenetic utility of genotyping-by-sequencing (GBS) data in the southern South American subclade of Valerianaceae (Dipsacales). The variety of forms that has arisen in this clade, presumably over the past 5-10 million years, has all the signatures of an adaptive and rapid radiation. While the phylogeny of Valerianaceae has received a great deal of attention in the last decade, species relationships have been hard to resolve using traditional phylogenetic markers. Here, we collected high-throughput genomic sequence data from reduced-representation libraries obtained through GBS protocols. Putative orthologs were identified using within- and among-sample clustering using the computer software pyRAD. We recovered over 3000 loci for 14 species of southern South AmericanValeriana,with 140 loci present across all samples.We analyzed a set of phylogenetic trees generated from each locus using maximum likelihood methods, as well as multispecies coalescent (∗BEAST) methods. For comparative purposes, we also used a supermatrix approach to infer the phylogeny for these taxa. Across different methods and data sets, we recovered consistent relationships for the southern South American valerians that we sampled with varying degrees of support.

Metabolic engineering of Escherichia coli for production of valerenadiene.

Valeriana officinalis is a medicinal herb which produces a suite of compounds in its root tissue useful for treatment of anxiety and insomnia. The sesquiterpene components of the root extract, valerenic acid and valerena-1,10-diene, are thought to contribute to most of the observed anxiolytic of Valerian root preparations. However, valerenic acid and its biosynthetic intermediates are only produced in low quantities in the roots of V. officinalis. Thus, in this report, Escherichia coli was metabolically engineered to produce substantial quantities of valerena-1,10-diene in shake flask fermentations with decane overlay. Expression of the wildtype valerenadiene synthase gene (pZE-wvds) resulted in production of 12µg/mL in LB cultures using endogenous FPP metabolism. Expression of a codon-optimized version of the valerenadiene synthase gene (pZE-cvds) resulted in 3-fold higher titers of valerenadiene (32µg/mL). Co-expression of pZE-cvds with an engineered methyl erythritol phosphate (MEP) pathway improved valerenadiene titers 65-fold to 2.09mg/L valerenadiene. Optimization of the fermentation medium to include glycerol supplementation enhanced yields by another 5.5-fold (11.0mg/L valerenadiene). The highest production of valerenadiene resulted from engineering the codon-optimized valerenadiene synthase gene under strong Ptrc and PT7 promoters and via co-expression of an exogenous mevalonate (MVA) pathway. These efforts resulted in an E. coli production strain that produced 62.0mg/L valerenadiene (19.4mg/L/OD600 specific productivity). This E. coli production platform will serve as the foundation for the synthesis of novel valerenic acid analogues potentially useful for the treatment of anxiety disorders.

Expression of Terpenoid Biosynthetic Genes and Accumulation of Chemical Constituents in Valeriana fauriei.

Valeriana fauriei (V. fauriei), which emits a characteristic and unpleasant odor, is important in traditional medicine. In this study, the expression of terpenoid biosynthetic genes was investigated in different organs that were also screened for volatile compounds including valerenic acid and its derivatives. Specific expression patterns from different parts of V. fauriei were observed using quantitative real-time PCR (qRT-PCR). The highest transcript levels of biosynthetic genes involved in mevalonic acid (MVA) and methylerythritol phosphate (MEP) production were found in the stem. Although the amounts of volatile compounds were varied by organ, most of the volatile terpenoids were accumulated in the root. Gas chromatography mass spectrometry (GC-MS) analysis identified 128 volatile compounds, which represented 65.33% to 95.66% of total volatiles. Certain compounds were only found in specific organs. For example, isovalerenic acid and valerenic acid and its derivatives were restricted to the root. Organs with high transcript levels did not necessarily have high levels of the corresponding chemical constituents. According to these results, we hypothesize that translocation may occur between different organs in V. fauriei.

Transcriptome and metabolome analysis in shoot and root of Valeriana fauriei.

BACKGROUND: Valeriana fauriei is commonly used in the treatment of cardiovascular diseases in many countries. Several constituents with various pharmacological properties are present in the roots of Valeriana species. Although many researches on V. fauriei have been done since a long time, further studies in the discipline make a limit due to inadequate genomic information. Hence, Illumina HiSeq 2500 system was conducted to obtain the transcriptome data from shoot and root of V. fauriei. RESULTS: A total of 97,595 unigenes were noticed from 346,771,454 raw reads after preprocessing and assembly. Of these, 47,760 unigens were annotated with Uniprot BLAST hits and mapped to COG, GO and KEGG pathway. Also, 70,013 and 88,827 transcripts were expressed in root and shoot of V. fauriei, respectively. Among the secondary metabolite biosynthesis, terpenoid backbone and phenylpropanoid biosynthesis were large groups, where transcripts was involved. To characterize the molecular basis of terpenoid, carotenoid, and phenylpropanoid biosynthesis, the levels of transcription were determined by qRT-PCR. Also, secondary metabolites content were measured using GC/MS and HPLC analysis for that gene expression correlated with its accumulation respectively between shoot and root of V. fauriei. CONCLUSIONS: We have identified the transcriptome using Illumina HiSeq system in shoot and root of V. fauriei. Also, we have demonstrated gene expressions associated with secondary metabolism such as terpenoid, carotenoid, and phenylpropanoid.

Regulation of sesquiterpenoid metabolism in recombinant and elicited Valeriana officinalis hairy roots.

The medicinal properties of Valerian (Valeriana officinalis) root preparations are attributed to the anxiolytic sesquiterpenoid valerenic acid and its biosynthetic precursors valerenal and valerenadiene, as well as the anti-inflammatory sesquiterpenoid ß-caryophyllene. In order to study and engineer the biosynthesis of these pharmacologically active metabolites, a binary vector co-transformation system was developed for V. officinalis hairy roots. The relative expression levels and jasmonate-inducibility of a number of genes associated with sesquiterpenoid metabolism were profiled in roots: farnesyl pyrophosphate synthase (VoFPS), valerendiene synthase (VoVDS), germacrene C synthase (VoGCS), and a cytochrome P450 (CYP71D442) putatively associated with terpene metabolism based on sequence homology. Recombinant hairy root lines overexpressing VoFPS or VoVDS were generated and compared to control cultures. Overexpression of the VoFPS cDNA increased levels of the corresponding transcript 4- to 8-fold and sesquiterpene hydrocarbon accumulation by 1.5- to 4-fold. Overexpression of the VoVDS cDNA increased the corresponding transcript levels 5- to 9-fold and markedly increased yields of the oxygenated sesquiterpenoids valerenic acid and valerenal. Our findings suggest that the availability of cytoplasmic farnesyl diphosphate and valerenadiene are potential bottlenecks in Valeriana-specific sesquiterpenoid biosynthesis, which is also subject to regulation by methyl jasmonate elicitation.

Monoterpene biosynthesis potential of plant subcellular compartments.

Subcellular monoterpene biosynthesis capacity based on local geranyl diphosphate (GDP) availability or locally boosted GDP production was determined for plastids, cytosol and mitochondria. A geraniol synthase (GES) was targeted to plastids, cytosol, or mitochondria. Transient expression in Nicotiana benthamiana indicated local GDP availability for each compartment but resulted in different product levels. A GDP synthase from Picea abies (PaGDPS1) was shown to boost GDP production. PaGDPS1 was also targeted to plastids, cytosol or mitochondria and PaGDPS1 and GES were coexpressed in all possible combinations. Geraniol and geraniol-derived products were analyzed by GC-MS and LC-MS, respectively. GES product levels were highest for plastid-targeted GES, followed by mitochondrial- and then cytosolic-targeted GES. For each compartment local boosting of GDP biosynthesis increased GES product levels. GDP exchange between compartments is not equal: while no GDP is exchanged from the cytosol to the plastids, 100% of GDP in mitochondria can be exchanged to plastids, while only 7% of GDP from plastids is available for mitochondria. This suggests a direct exchange mechanism for GDP between plastids and mitochondria. Cytosolic PaGDPS1 competes with plastidial GES activity, suggesting an effective drain of isopentenyl diphosphate from the plastids to the cytosol.

Genetic and biochemical diversity among Valeriana jatamansi populations from Himachal Pradesh.

Valeriana jatamansi Jones is an important medicinal plant that grows wild in Himachal Pradesh, India. Molecular and biochemical diversity among 13 natural populations from Himachal Pradesh was assessed using RAPD and GC-MS to know the extent of existing variation. A total of seven genetically diverse groups have been identified based on RAPD analysis which corroborated well with the analysis based on chemical constituents. The essential oil yield ranged from 0.6% to 1.66% (v/w). A negative correlation between patchouli alcohol and viridiflorol, the two major valued constituents, limits the scope of their simultaneous improvement. However, other few populations like Chamba-II and Kandi-I were found promising for viridiflorol and patchouli alcohol, respectively. The analysis of chemical constitution of oil of the populations from a specific region revealed predominance of specific constituents indicating possibility of their collection/selection for specific end uses like phytomedicines. The prevalence of genetically diverse groups along with sufficient chemical diversity in a defined region clearly indicates the role of ecology in the maintenance of evolution of this species. Sufficient molecular and biochemical diversity detected among natural populations of this species will form basis for the future improvement.

Molecular cloning and characterization of drimenol synthase from valerian plant (Valeriana officinalis).

Drimenol, a sesquiterpene alcohol, and its derivatives display diverse bio-activities in nature. However, a drimenol synthase gene has yet to be identified. We identified a new sesquiterpene synthase cDNA (VoTPS3) in valerian plant (Valeriana officinalis). Purification and NMR analyses of the VoTPS3-produced terpene, and characterization of the VoTPS3 enzyme confirmed that VoTPS3 synthesizes (-)-drimenol. In feeding assays, possible reaction intermediates, farnesol and drimenyl diphosphate, could not be converted to drimenol, suggesting that the intermediate remains tightly bound to VoTPS3 during catalysis. A mechanistic consideration of (-)-drimenol synthesis suggests that drimenol synthase is likely to use a protonation-initiated cyclization, which is rare for sesquiterpene synthases. VoTPS3 can be used to produce (-)-drimenol, from which useful drimane-type terpenes can be synthesized.

Evaluation of tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) hairy roots for the production of geraniol, the first committed step in terpenoid indole alkaloid pathway.

The terpenoid indole alkaloids are one of the major classes of plant-derived natural products and are well known for their many applications in the pharmaceutical, fragrance and cosmetics industries. Hairy root cultures are useful for the production of plant secondary metabolites because of their genetic and biochemical stability and their rapid growth in hormone-free media. Tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) hairy roots, which do not produce geraniol naturally, were engineered to express a plastid-targeted geraniol synthase gene originally isolated from Valeriana officinalis L. (VoGES). A SPME-GC-MS screening tool was developed for the rapid evaluation of production clones. The GC-MS analysis revealed that the free geraniol content in 20 hairy root clones expressing VoGES was an average of 13.7 µg/g dry weight (DW) and a maximum of 31.3 µg/g DW. More detailed metabolic analysis revealed that geraniol derivatives were present in six major glycoside forms, namely the hexose and/or pentose conjugates of geraniol and hydroxygeraniol, resulting in total geraniol levels of up to 204.3 µg/g DW following deglycosylation. A benchtop-scale process was developed in a 20-L wave-mixed bioreactor eventually yielding hundreds of grams of biomass and milligram quantities of geraniol per cultivation bag.

Characterization of two geraniol synthases from Valeriana officinalis and Lippia dulcis: similar activity but difference in subcellular localization.

Two geraniol synthases (GES), from Valeriana officinalis (VoGES) and Lippia dulcis (LdGES), were isolated and were shown to have geraniol biosynthetic activity with Km values of 32 µM and 51 µM for GPP, respectively, upon expression in Escherichia coli. The in planta enzymatic activity and sub-cellular localization of VoGES and LdGES were characterized in stable transformed tobacco and using transient expression in Nicotiana benthamiana. Transgenic tobacco expressing VoGES or LdGES accumulate geraniol, oxidized geraniol compounds like geranial, geranic acid and hexose conjugates of these compounds to similar levels. Geraniol emission of leaves was lower than that of flowers, which could be related to higher levels of competing geraniol-conjugating activities in leaves. GFP-fusions of the two GES proteins show that VoGES resides (as expected) predominantly in the plastids, while LdGES import into to the plastid is clearly impaired compared to that of VoGES, resulting in both cytosolic and plastidic localization. Geraniol production by VoGES and LdGES in N. benthamiana was nonetheless very similar. Expression of a truncated version of VoGES or LdGES (cytosolic targeting) resulted in the accumulation of 30% less geraniol glycosides than with the plastid targeted VoGES and LdGES, suggesting that the substrate geranyl diphosphate is readily available, both in the plastids as well as in the cytosol. The potential role of GES in the engineering of the TIA pathway in heterologous hosts is discussed.

Functional identification of valerena-1,10-diene synthase, a terpene synthase catalyzing a unique chemical cascade in the biosynthesis of biologically active sesquiterpenes in Valeriana officinalis.

Valerian is an herbal preparation from the roots of Valeriana officinalis used as an anxiolytic and sedative and in the treatment of insomnia. The biological activities of valerian are attributed to valerenic acid and its putative biosynthetic precursor valerenadiene, sesquiterpenes, found in V. officinalis roots. These sesquiterpenes retain an isobutenyl side chain whose origin has been long recognized as enigmatic because a chemical rationalization for their biosynthesis has not been obvious. Using recently developed metabolomic and transcriptomic resources, we identified seven V. officinalis terpene synthase genes (VoTPSs), two that were functionally characterized as monoterpene synthases and three that preferred farnesyl diphosphate, the substrate for sesquiterpene synthases. The reaction products for two of the sesquiterpene synthases exhibiting root-specific expression were characterized by a combination of GC-MS and NMR in comparison to the terpenes accumulating in planta. VoTPS7 encodes for a synthase that biosynthesizes predominately germacrene C, whereas VoTPS1 catalyzes the conversion of farnesyl diphosphate to valerena-1,10-diene. Using a yeast expression system, specific labeled [(13)C]acetate, and NMR, we investigated the catalytic mechanism for VoTPS1 and provide evidence for the involvement of a caryophyllenyl carbocation, a cyclobutyl intermediate, in the biosynthesis of valerena-1,10-diene. We suggest a similar mechanism for the biosynthesis of several other biologically related isobutenyl-containing sesquiterpenes.

Impact of geographic range on genetic and chemical diversity of Indian valerian (Valeriana jatamansi) from northwestern Himalaya.

An effort was made to determine the impact of geographic range on genetic richness and chemical constituents of Valeriana jatamansi Jones, an herb indigenous to the northwestern Himalaya. The genetic structure of 16 accessions from two major divisions of Uttarakhand state (Kumaon and Garhwal) was analyzed by ISSR markers. Overall genetic diversity among the populations was 45 %, with a cumulative range of 35-92 % similarity for most of the high-altitude plants and a comparatively narrow range, 50-88 %, for the population below the altitude of 1,800 m. Likewise, a remarkable predictability was evident from the chemical constituents on an individual basis. In principal component analysis, most of the accessions fall into two major groups and are classified as chemotypes based on the percentage of similar chemical constituents; these are mostly correlated to altitude. Geographic distance seems to influence the genetic and chemical variability, indicating the genetic inbreeding within the population.