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Glandular trichomes (GTs) are epidermal structures that provide the first line of chemical defense against arthropod herbivores and other biotic threats. The most conspicuous structure on leaves of cultivated tomato (Solanum lycopersicum) is the type-VI GT (tVI-GT), which accumulates both flavonoids and volatile terpenoids. Although these classes of specialized metabolites are derived from distinct metabolic pathways, previous studies with a chalcone isomerase 1 (CHI1)-deficient mutant called anthocyanin free (af) showed that flavonoids are required for terpenoid accumulation in tVI-GTs. Here, we combined global transcriptomic and proteomic analyses of isolated trichomes as a starting point to show that the lack of CHI1 is associated with reduced levels of terpenoid biosynthetic transcripts and enzymes. The flavonoid deficiency in af trichomes also resulted in the upregulation of abiotic stress-responsive genes associated with DNA damage and repair. Several lines of biochemical and genetic evidence indicate that the terpenoid defect in af mutants is specific for the tVI-GT and is associated with the absence of bulk flavonoids rather than loss of CHI1 per se. A newly developed genome-scale model of metabolism in tomato tVI-GTs helped identify metabolic imbalances caused by the loss of flavonoid production. We provide evidence that flavonoid deficiency in this cell type leads to increased production of reactive oxygen species (ROS), which may impair terpenoid biosynthesis. Collectively, our findings support a role for flavonoids as ROS-scavenging antioxidants in GTs.

Monoterpene synthases catalyze the first committed step in the biosynthesis of monoterpenes and are in part responsible for the enormous structural diversity among this class of metabolites. Here, we explore the structure-function relationships underlying the formation of limonene enantiomers in limonene synthases that bind geranyl diphosphate as a common substrate. On the basis of analyses that consider both crystal structure data and amino acid sequence divergence, we identified candidate active site residues with potential roles in catalyzing reactions that involve accommodating reaction intermediates of opposite enantiomeric series. We demonstrate that spearmint (-)-limonene synthase [which generates99% (-)-limonene over (+)-limonene] can be converted into a mutant enzyme, by exchanging four residues (C321S, N345I, I453V, and M458V), which produces (+)-limonene with reversed enantiospecificity [80% (+)-limonene and 3% (-)-limonene; the remainder are mostly bicyclic monoterpenes]. This study provides the foundation for a more in-depth understanding of the formation of enantiomeric series of monoterpenes, which can have vastly different olfactory properties.

Three species of the genus Equisetum (E. arvense, E. hyemale, and E. telmateia) were selected for an analysis of chemical diversity in an ancient land plant lineage. Principal component analysis of metabolomics data obtained with above-ground shoot and below-ground rhizome extracts enabled a separation of all sample types, indicating species- and organ-specific patterns of metabolite accumulation. Follow-up efforts indicated that galactolipids, carotenoids, and flavonoid glycosides contributed positively to the separation of shoot samples, while stryrylpyrone glycosides and phenolic glycosides were the most prominent positive contributors to the separation of rhizome samples. Consistent with metabolite data, genes coding for enzymes of flavonoid and galactolipid biosynthesis were found to be expressed at elevated levels in shoot samples, whereas a putative styrylpyrone synthase gene was expressed preferentially in rhizomes. The current study builds a foundation for future endeavors to further interrogate the organ and tissue specificity of metabolism in the last living genus of a fern family that was prevalent in the forests of the late Paleozoic era.

Glandular trichomes are specialized anatomical structures that accumulate secretions with important biological roles in plant-environment interactions. These secretions also have commercial uses in the flavor, fragrance, and pharmaceutical industries. The capitate-stalked glandular trichomes of Cannabis sativa (cannabis), situated on the surfaces of the bracts of the female flowers, are the primary site for the biosynthesis and storage of resins rich in cannabinoids and terpenoids. In this study, we profiled nine commercial cannabis strains with purportedly different attributes, such as taste, color, smell, and genetic origin. Glandular trichomes were isolated from each of these strains, and cell type-specific transcriptome data sets were acquired. Cannabinoids and terpenoids were quantified in flower buds. Statistical analyses indicated that these data sets enable the high-resolution differentiation of strains by providing complementary information. Integrative analyses revealed a coexpression network of genes involved in the biosynthesis of both cannabinoids and terpenoids from imported precursors. Terpene synthase genes involved in the biosynthesis of the major monoterpenes and sesquiterpenes routinely assayed by cannabis testing laboratories were identified and functionally evaluated. In addition to cloning variants of previously characterized genes, specifically CsTPS14CT [(−)-limonene synthase] and CsTPS15CT (β-myrcene synthase), we functionally evaluated genes that encode enzymes with activities not previously described in cannabis, namely CsTPS18VF and CsTPS19BL (nerolidol/linalool synthases), CsTPS16CC (germacrene B synthase), and CsTPS20CT (hedycaryol synthase). This study lays the groundwork for developing a better understanding of the complex chemistry and biochemistry underlying resin accumulation across commercial cannabis strains.

The plant kingdom supports an extraordinary chemical diversity, with terpenoids representing a particularly diversified class of secondary (or specialized) metabolites. Volatile and semi-volatile terpenoids in the C10-C20 range are often formed in specialized cell types and secretory structures. In the angiosperm lineage, glandular trichomes play an important role in enabling the biosynthesis and storage (or in some cases secretion) of functionalized terpenoids. The 'decoration' of a terpenoid scaffold with functional groups changes its physical and chemical properties, and can therefore affect the perception of a specific metabolite by other organisms. Because of the ecological implications (e.g. plant-herbivore interactions) and commercial relevance (e.g. volatiles used in the flavor and fragrance industries), terpenoid functionalization has been researched extensively. Recent successes in the cloning and functional evaluation of genes as well as the structural and biochemical characterization of enzyme catalysts have laid the foundation for an improved understanding of how pathways toward functionalized monoterpenes may have evolved. In this review, we will focus on an up-to-date account of functionalization reactions present in glandular trichomes.

Sterols serve essential functions as membrane constituents and hormones (brassinosteroids) in plants, while non-sterol triterpenoids have been implicated in defense responses. Surprisingly little is known about the sterol and triterpene profiles in different plant organs. To enhance our understanding of organ-specific sterol and triterpene accumulation, we quantified these metabolite classes in four different organs (root, stem, leaf, seed) ofArabidopsis thaliana(L.). Based on these data sets we developed kinetic mathematical models of sterol biosynthesis to capture flux distribution and pathway regulation in different organs. Simulations indicated that an increased flux through the sterol pathway would not only result in an increase of sterol end products but also a concomitant build-up of certain intermediates. These computational predictions turner out to be consistent with experimental data obtained with transgenic plants ectopically overexpressing 3-hydroxy-3-methylglutary-coenzyme A reductase (HMG1gene). The opportunities and limitations of incorporating mathematical modeling into the design of approaches to engineer sterol biosynthesis are discussed.

Taxol (paclitaxel) is a very widely used anticancer drug, but its commercial sources mainly consist of stripped bark or suspension cultures of members of the plant genus Taxus. Taxol accumulates as part of a complex mixture of chemical analogs, termed taxoids, which complicates its production in pure form, highlighting the need for metabolic engineering approaches for high-level Taxol production in cell cultures or microbial hosts. Here, we report on the characterization of acyl-activating enzymes (AAEs) that catalyze the formation of CoA esters of different organic acids relevant for the N-substitution of the 3-phenylisoserine side chain of taxoids. On the basis of similarities to AAE genes of known function from other organisms, we identified candidate genes in publicly available transcriptome data sets obtained with Taxus × media. We cloned 17 AAE genes, expressed them heterologously in Escherichia coli, purified the corresponding recombinant enzymes, and performed in vitro assays with 27 organic acids as potential substrates. We identified TmAAE1 and TmAAE5 as the most efficient enzymes for the activation of butyric acid (Taxol D side chain), TmAAE13 as the best candidate for generating a CoA ester of tiglic acid (Taxol B side chain), TmAAE3 and TmAAE13 as suitable for the activation of 4-methylbutyric acid (N-debenzoyl-N-(2-methylbutyryl)taxol side chain), TmAAE15 as a highly efficient candidate for hexanoic acid activation (Taxol C side chain), and TmAAE4 as suitable candidate for esterification of benzoic acid with CoA (Taxol side chain). This study lays important groundwork for metabolic engineering efforts aimed at improving Taxol production in cell cultures.

The pseudoalkaloid diterpene Taxol® (paclitaxel) emerged as a best-selling anti-cancer drug in the mid-1990s. The compound attracted considerable interest because of its unique mechanism to stabilize microtubules, thus reducing dynamicity and ultimately promoting mitotic arrest. Taxol was originally isolated from members of the genus Taxus. Over the last 50 years, close to 600 metabolites with taxane scaffolds were isolated from various Taxus species and their structures reported. The present review article provides an overview of the known chemical diversity of taxanes, with an emphasis on the functionalization of diterpene scaffolds. The implications of the occurrence of chemically diverse taxane metabolites for unraveling Taxol biosynthesis and enabling pathway engineering are discussed as well.

Adventitious root cultures were developed from Tripterygium regelii, and growth conditions were optimized for the abundant production of diterpenoids, which can be collected directly from the medium. An analysis of publicly available transcriptome data sets collected with T. regelii roots and root cultures indicated the presence of a large gene family (with 20 members) for terpene synthases (TPSs). Nine candidate diterpene synthase genes were selected for follow-up functional evaluation, of which two belonged to the TPS-c, three to the TPS-e/f, and four to the TPS-b subfamilies. These genes were characterized by heterologous expression in a modular metabolic engineering system in Escherichia coli Members of the TPS-c subfamily were characterized as copalyl diphosphate (diterpene) synthases, and those belonging to the TPS-e/f subfamily catalyzed the formation of precursors of kaurane diterpenoids. The TPS-b subfamily encompassed genes coding for enzymes involved in abietane diterpenoid biosynthesis and others with activities as monoterpene synthases. The structural characterization of diterpenoids accumulating in the medium of T. regelii adventitious root cultures, facilitated by searching the Spektraris online spectral database, enabled us to formulate a biosynthetic pathway for the biosynthesis of triptolide, a diterpenoid with pharmaceutical potential. Considering the significant enrichment of diterpenoids in the culture medium, fast-growing adventitious root cultures may hold promise as a sustainable resource for the large-scale production of triptolide.

Premise of research. The genus Citrus includes species that are among the most important tree fruit crops. Although fruit juice is the primary product, monoterpenoid essential oil obtained from the peel is an important value-added commodity. Peel monoterpenes are synthesized by subepidermal secretory cavities that consist of glandular cells surrounding an extracellular oil storage space. Several previous studies have focused on early secretory cavity development, ending with the initiation of secretion. In order to better understand the process of monoterpene formation, transport, and storage, it is important to obtain detailed information concerning plant oil glands during all phases of development.Methodology. TEM was performed on secretory cavities isolated from the exocarp of grapefruit (Citrus × paradisi Macfad. ‘Duncan’) preserved by microwave-assisted fixation or high-pressure freezing and freeze substitution. Immunocytochemistry was employed to localize (+)-limonene synthase. Tomography was used t...

Development and testing of Spektraris-NMR an online spectral resource, is reported for the NMR-based structural identification of plant natural products (PNPs). Spektraris-NMRallows users to search with multiple spectra at once and returns a table with alist of hits arranged according to the goodness of fit between query data and database entries. For each hit, a link to a tabulated alignment of 1H-NMR and 13C-NMR spectroscopic peaks (query versus database entry) is provided. Furthermore, full spectroscopic records and experimental meta information about each database entry can be accessed online. To test the utility of Spektraris-NMR for PNP identification, the database was populated with NMR data (total of 466 spectra) for ∼250 taxanes, which are structurally complex diterpenoids (including the anticancer drug taxol) commonly found in the genus Taxus. NMR data generated used was then generated with metabolites purified from Taxus cell suspension cultures to search Spektraris-NMR, and were able to identify eight taxanes with high confidence. A ninth isolated metabolite could be assigned, based on spectral searches, to a taxane skeletal class, but no high confidence hit was produced. Using various spectroscopic methods, this metabolite was characterized as the taxane 2-deacetylbaccatin IV, a novel taxane. These results indicate that Spektraris-NMR is a valuable resource for rapid and reliable identification of known metabolites and has the potential to contribute to de-replication efforts in the search for novel PNPs.

For nearly 30 years, coenzyme M (CoM) was assumed to be present solely in methanogenic archaea. In the late 1990s, CoM was reported to play a role in bacterial propene metabolism, but no biosynthetic pathway for CoM has yet been identified in bacteria. Here, using bioinformatics and proteomic approaches in the metabolically versatile bacterium Xanthobacter autotrophicus Py2, we identified four putative CoM biosynthetic enzymes encoded by the xcbB1, C1, D1, and E1 genes. Only XcbB1 was homologous to a known CoM biosynthetic enzyme (ComA), indicating that CoM biosynthesis in bacteria involves enzymes different from those in archaea. We verified that the ComA homolog produces phosphosulfolactate from phosphoenolpyruvate (PEP), demonstrating that bacterial CoM biosynthesis is initiated similarly as the phosphoenolpyruvate-dependent methanogenic archaeal pathway. The bioinformatics analysis revealed that XcbC1 and D1 are members of the aspartase/fumarase superfamily (AFS) and that XcbE1 is a pyridoxal 5'-phosphate-containing enzyme with homology to d-cysteine desulfhydrases. Known AFS members catalyze β-elimination reactions of succinyl-containing substrates, yielding fumarate as the common unsaturated elimination product. Unexpectedly, we found that XcbC1 catalyzes β-elimination on phosphosulfolactate, yielding inorganic phosphate and a novel metabolite, sulfoacrylic acid. Phosphate-releasing β-elimination reactions are unprecedented among the AFS, indicating that XcbC1 is an unusual phosphatase. Direct demonstration of phosphosulfolactate synthase activity for XcbB1 and phosphate β-elimination activity for XcbC1 strengthened their hypothetical assignment to a CoM biosynthetic pathway and suggested functions also for XcbD1 and E1. Our results represent a critical first step toward elucidating the CoM pathway in bacteria.

Schizonepeta tenuifolia Briquet, commonly known as Japanese catnip, is used for the treatment of colds, headaches, fevers, and skin rashes in traditional Asian medicine (China, Japan and Korea). The volatile oil and its constituents have various demonstrated biological activities, but there is currently limited information regarding the site of biosynthesis. Light microscopy and scanning electron microscopy indicated the presence of three distinct glandular trichome types which, based on their morphological features, are referred to as peltate, capitate and digitiform glandular trichomes. Laser scanning microscopy and 3D reconstruction demonstrated that terpenoid-producing peltate glandular trichomes contain a disk of twelve secretory cells. The oil of peltate glandular trichomes, collected by laser microdissection or using custom-made micropipettes, was demonstrated to contain (−)-pulegone, (+)-menthone and (+)-limonene as major constituents. Digitiform and capitate glandular trichomes did not contain appreciable levels of terpenoid volatiles. The yield of distilled oil from spikes was significantly (44%) higher than that from leaves, while the composition of oils was very similar. Oils collected directly from leaf peltate glandular trichomes over the course of a growing season contained primarily (−)-pulegone (>80% at 32 days after germination) in young plants, while (+)-menthone began to accumulate later (>75% at 80 days after germination), at the expense of (−)-pulegone (the levels of (+)-limonene remained fairly stable at 3–5%). The current study establishes the morphological and chemical characteristics of glandular trichome types of S. tenuifolia, and also provides the basis for unraveling the biosynthesis of essential oil in this popular medicinal plant.

The commercially important essential oils of peppermint (Mentha × piperita) and its relatives in the mint family (Lamiaceae) are accumulated in specialized anatomical structures called glandular trichomes (GTs). A genome-scale stoichiometric model of secretory phase metabolism in peppermint GTs was constructed based on current biochemical and physiological knowledge. Fluxes through the network were predicted based on metabolomic and transcriptomic data. Using simulated reaction deletions, this model predicted that two processes, the regeneration of ATP and ferredoxin (in its reduced form), exert substantial control over flux toward monoterpenes. Follow-up biochemical assays with isolated GTs indicated that oxidative phosphorylation and ethanolic fermentation were active and that cooperation to provide ATP depended on the concentration of the carbon source. We also report that GTs with high flux toward monoterpenes express, at very high levels, genes coding for a unique pair of ferredoxin and ferredoxin-NADP+ reductase isoforms. This study provides, to our knowledge, the first evidence of how bioenergetic processes determine flux through monoterpene biosynthesis in GTs.

Members of the genus Tripterygium are known to contain an astonishing diversity of specialized metabolites. The lack of authentic standards has been an impediment to the rapid identification of such metabolites in extracts. We employed an approach that involves the searching of multiple, complementary chromatographic and spectroscopic data sets against the Spektraris database to speed up the metabolite identification process. Mass spectrometry-based imaging indicated a differential localization of triterpenoids to the periderm and sesquiterpene alkaloids to the cortex layer of Tripterygium roots. We further provide evidence that triterpenoids are accumulated to high levels in cells that contain suberized cell walls, which might indicate a mechanism for storage. To our knowledge, our data provide first insights into the cell type specificity of metabolite accumulation in Tripterygium and set the stage for furthering our understanding of the biological implications of specialized metabolites in this genus.

Summary Glandular trichomes are anatomical structures specialized for the synthesis of secreted natural products. In this review we focus on the description of glands that accumulate terpenoid essential oils and oleoresins. We also provide an in-depth account of the current knowledge about the biosynthesis of terpenoids and secretion mechanisms in the highly specialized secretory cells of glandular trichomes, and highlight the implications for metabolic engineering efforts. Phylogenetic distribution of glandular trichomes

The paraveinal mesophyll (PVM) layer of soybean leaves, which contains cells with various unique ultrastructural properties, has been studied for decades, and several hypotheses regarding its functional role have been developed. Here, we describe a method for obtaining PVM cells using laser capture microdissection and pressure catapulting, subsequent isolation of RNA from these cells, and downstream microarray analysis. A cell type-specific transcriptome analysis was used to compare the gene expression patterns in PVM cells with those of a mesophyll cell type (palisade parenchyma) as a reference. Transcripts related to vegetative storage protein (Vsp) and certain vegetative lipoxygenase (Vlx) isoforms were significantly enriched in PVM cells, which is in accordance with prior work that demonstrated an accumulation of the corresponding proteins. Potential roles of Vsp and Vlx in phosphate mobilization and defense responses, respectively, are discussed. In addition, we found an enrichment of several transport-related genes in PVM cells. Building on our transcriptome data, we provide a fresh discussion of the hypothesis that the PVM plays a role in photoassimilate mobilization and translocation.

Epithelial cells (ECs) lining the secretory cavities of Citrus peel have been hypothesized to be responsible for the synthesis of essential oil, but direct evidence for such a role is currently sparse. We used laser-capture microdissection and pressure catapulting to isolate ECs and parenchyma cells (as controls not synthesizing oil) from the peel of young grapefruit (Citrus × paradisi ‘Duncan’), isolated RNA, and evaluated transcript patterns based on oligonucleotide microarrays. A Gene Ontology analysis of these data sets indicated an enrichment of genes involved in the biosynthesis of volatile terpenoids and nonvolatile phenylpropanoids in ECs (when compared with parenchyma cells), thus indicating a significant metabolic specialization in this cell type. The gene expression patterns in ECs were consistent with the accumulation of the major essential oil constituents (monoterpenes, prenylated coumarins, and polymethoxylated flavonoids). Morphometric analyses demonstrated that secretory cavities are formed early during fruit development, whereas the expansion of cavities, and thus oil accumulation, correlates with later stages of fruit expansion. Our studies have laid the methodological and experimental groundwork for a vastly improved knowledge of the as yet poorly understood processes controlling essential oil biosynthesis in Citrus peel.

Herein we report on an improved method for the microscale extraction of huperzine A (HupA), an acetylcholinesterase-inhibiting alkaloid, from as little as 3 mg of tissue homogenate from the clubmoss Huperzia squarrosa (G. Forst.) Trevis with 99.95% recovery. We also validated a novel UHPLC-QTOF-MS method for the high-throughput analysis of H. squarrosa extracts in only 6 min, which, in combination with the very low limit of detection (20 pg on column) and the wide linear range for quantification (20–10,000 pg on column), allow for a highly efficient screening of extracts containing varying amounts of HupA. Utilization of this methodology has the potential to conserve valuable plant resources. Copyright © 2012 John Wiley & Sons, Ltd.

The repeated removal of flower, fruit, or vegetative buds is a common treatment to simulate sink limitation. These experiments usually lead to the accumulation of specific proteins, which are degraded during later stages of seed development, and have thus been designated as vegetative storage proteins. We used oligonucleotide microarrays to assess global effects of sink removal on gene expression patterns in soybean leaves and found an induction of the transcript levels of hundreds of genes with putative roles in the responses to biotic and abiotic stresses. In addition, these data sets indicated potential changes in amino acid and phenylpropanoid metabolism. As a response to sink removal we detected an induced accumulation of γ-aminobutyric acid, while proteinogenic amino acid levels decreased. We also observed a shift in phenylpropanoid metabolism with an increase in isoflavone levels, concomitant with a decrease in flavones and flavonols. Taken together, we provide evidence that sink removal leads to an up-regulation of stress responses in distant leaves, which needs to be considered as an unintended consequence of this experimental treatment.

Numerous experiments have shown positive diversity effects on plant productivity, but little is known about related processes of carbon gain and allocation. We investigated these processes in a controlled environment (Montpellier European Ecotron) applying a continuous 13CO2 label for three weeks to 12 soil-vegetation monoliths originating from a grassland biodiversity experiment (Jena Experiment) and representing two diversity levels (4 and 16 sown species). Plant species richness did not affect community- and species-level 13C abundances neither in total biomass nor in non-structural carbohydrates (NSC). Community-level 13C excess tended to be higher in the 16-species than in the 4-species mixtures. Community-level 13C excess was positively related to canopy leaf nitrogen (N), i.e. leaf N per unit soil surface. At the species level shoot 13C abundances varied among plant functional groups and were larger in legumes and tall herbs than in grasses and small herbs and correlated positively with traits as leaf N concentrations, stomatal conductance and shoot height. The 13C abundances in NSC were larger in transport sugars (sucrose, raffinose-family oligosaccharides) than in free glucose, fructose and compounds of the storage pool (starch) suggesting that newly assimilated carbon is to a small portion allocated to storage. Our results emphasize that the functional composition of communities is key in explaining carbon assimilation in grasslands.

Abscisic acid (ABA) has been implicated as a mediator in plant responses to various environmental stresses. To evaluate the transcriptional and metabolic events downstream of ABA perception, Arabidopsis thaliana seedlings were analyzed by transcript and metabolite profiling, and results were integrated, using the recently developed BioPathAt tool, in the context of the biochemical pathways affected by this treatment. Besides the up-regulation of pathways related to the biosynthesis of compatible solutes (raffinose family oligosaccharides and certain amino acids) as a response to ABA treatment, we also observed a down-regulation of numerous genes putatively localized to and possibly involved in the reorganization of cell walls, an association that had not been recognized previously. Metabolite profiling indicated that specific antioxidants, particularly alpha-tocopherol and L-ascorbic acid, were accumulated at higher levels in ABA-treated seedlings compared to appropriate controls. The transcription of genes involved in alpha-tocopherol biosynthesis were coordinately up-regulated and appeared to be integrated into a network of reactions controlling the levels of reactive oxygen species. Based upon the observed gene expression patterns, these redox control mechanisms might involve an ABA-mediated transition of mitochondrial respiration to the alternative, non-phosphorylating respiratory chain mode. The presented data herein provide indirect evidence for crosstalk between metabolic pathways and pathways regulating redox homeostasis as a response to ABA treatment, and allowed us to identify candidate genes for follow-up studies to dissect this interaction at the biochemical and molecular level. Our results also indicate an intricate relationship, at the transcriptional and possibly post-transcriptional levels, between ABA biosynthesis, the xanthophyll cycle, and ascorbic acid recycling.

SUMMARY A sensitive and robust mixed-mode high performance liquid chromatography-tandem mass spectrometry method was developed for the qualitative and quantitative determination of sugar phosphates, which are notoriously difficult to separate using reversed-phase materials. Sugar phosphates were separated on a Primesep SB column by gradient elution using aqueous ammonium formate and acetonitrile as mobile phases. Target analytes were identified by their precursor/product ions and retention times. Quantitative analysis was performed in negative ionization/multiple reaction monitoring mode with five different time segments. The method was validated by spiking authentic sugar phosphate standards into complex plant tissue extracts. Standard curves of neat authentic standards and spiked extracts were generated for concentrations in the low picomole to nanomole range, with correlation coefficients of R(2) > 0.991, and the degree of ion suppression in the presence of a plant matrix was calculated for each analyte. Analyte recoveries, which were determined by including known quantities of authentic standards in the sugar phosphate extraction protocol, ranged from 40.0% to 57.4%. The analytical reproducibility was assessed by determining the coefficient of variance based on repeated extractions/measurements (

Plant metabolomics has matured over the past 8 years. Plant biologists routinely use comprehensive analyses of plant metabolites to discover new responses to genetic or environmental perturbation, or to validate initial hypotheses on the function and in vivo action of gene products. The wealth of scientific findings has increasingly provoked interest to share and review raw or processed data from plant metabolomics reports. We here suggest a minimum of parameters to be reported in order to define details of experimental study designs in plant metabolomics studies.

Misexpression of the AtNPC1 - 1 and AtNPC1 - 2 genes leads to altered sphingolipid metabolism, growth impairment, and male reproductive defects in a hemizygous Arabidopsis thaliana (L.) double-mutant population. Abolishing the expression of both gene copies has lethal effects. Niemann-Pick disease type C1 is a lysosomal storage disorder caused by mutations in the NPC1 gene. At the cellular level, the disorder is characterized by the accumulation of storage lipids and lipid trafficking defects. The Arabidopsis thaliana genome contains two genes (At1g42470 and At4g38350) with weak homology to mammalian NPC1. The corresponding proteins have 11 predicted membrane-spanning regions and contain a putative sterol-sensing domain. The At1g42470 protein is localized to the plasma membrane, while At4g38350 protein has a dual localization in the plasma and tonoplast membranes. A phenotypic analysis of T-DNA insertion mutants indicated that At1g42470 and At4g38350 (designated AtNPC1-1 and AtNPC1-2, respectively) have partially redundant functions and are essential for plant reproductive viability and development. Homozygous plants impaired in the expression of both genes were not recoverable. Plants of a hemizygous AtNPC1-1/atnpc1-1/atnpc1-2/atnpc1-2 population were severely dwarfed and exhibited male gametophytic defects. These gene disruptions did not have an effect on sterol concentrations; however, hemizygous AtNPC1-1/atnpc1-1/atnpc1-2/atnpc1-2 mutants had increased fatty acid amounts. Among these, fatty acid α-hydroxytetracosanoic acid (h24:0) occurs in plant sphingolipids. Follow-up analyses confirmed the accumulation of significantly increased levels of sphingolipids (assayed as hydrolyzed sphingoid base component) in the hemizygous double-mutant population. Certain effects of NPC1 misexpression may be common across divergent lineages of eukaryotes (sphingolipid accumulation), while other defects (sterol accumulation) may occur only in certain groups of eukaryotic organisms.

The isoprenoid biosynthetic pathway provides intermediates for the synthesis of a multitude of natural products which serve numerous biochemical functions in plants: sterols (isoprenoids with a C30 backbone) are essential components of membranes; carotenoids (C40) and chlorophylls (which contain a C20 isoprenoid side-chain) act as photosynthetic pigments; plastoquinone, phylloquinone and ubiquinone (all of which contain long isoprenoid side-chains) participate in electron transport chains; gibberellins (C20), brassinosteroids (C30) and abscisic acid (C15) are phytohormones derived from isoprenoid intermediates; prenylation of proteins (with C15 or C20 isoprenoid moieties) may mediate subcellular targeting and regulation of activity; and several monoterpenes (C10), sesquiterpenes (C15) and diterpenes (C20) have been demonstrated to be involved in plant defense. Here we present a comprehensive analysis of genes coding for enzymes involved in the metabolism of isoprenoid-derived compounds in Arabidopsis thaliana. By combining homology and sequence motif searches with knowledge regarding the phylogenetic distribution of pathways of isoprenoid metabolism across species, candidate genes for these pathways in A. thaliana were obtained. A detailed analysis of the vicinity of chromosome loci for genes of isoprenoid metabolism in A. thaliana provided evidence for the clustering of genes involved in common pathways. Multiple sequence alignments were used to estimate the number of genes in gene families and sequence relationship trees were utilized to classify their individual members. The integration of all these datasets allows the generation of a knowledge-based metabolic map of isoprenoid metabolic pathways in A. thaliana and provides a substantial improvement of the currently available gene annotation.

Two independent pathways operate in plants for the synthesis of isopentenyl diphosphate and dimethylallyl diphosphate, the central intermediates in the biosynthesis of all isoprenoids. The mevalonate pathway is present in the cytosol, whereas the recently discovered mevalonate-independent pathway is localized to plastids. We have used isolated peppermint (Mentha piperita) oil gland secretory cells as an experimental model system to study the effects of the herbicides fosmidomycin, phosphonothrixin, methyl viologen, benzyl viologen, clomazone, 2-(dimethylamino)ethyl diphosphate, alendronate, and pamidronate on the pools of metabolites related to monoterpene biosynthesis via the mevalonate-independent pathway. A newly developed isolation protocol for polar metabolites together with an improved separation and detection method based on liquid chromatography-mass spectrometry have allowed assessment of the enzyme targets for a number of these herbicides.

Many plant natural products are synthesized in specialized cells and tissues. To learn more about metabolism in these cells, they have to be studied in isolation. Here, we describe a protocol for the isolation of epithelial cells that surround secretory cavities in Citrus fruit peel. Cells isolated using laser microdissection are suitable for RNA isolation and downstream transcriptome analyses.

The integration of mathematical modeling with analytical experimentation in an iterative fashion is a powerful approach to advance our understanding of the architecture and regulation of metabolic networks. Ultimately, such knowledge is highly valuable to support efforts aimed at modulating flux through target pathways by molecular breeding and/or metabolic engineering. In this article we describe a kinetic mathematical model of peppermint essential oil biosynthesis, a pathway that has been studied extensively for more than two decades. Modeling assumptions and approximations are described in detail. We provide step-by-step instructions on how to run simulations of dynamic changes in pathway metabolites concentrations.

The participation of the mevalonic acid (MVA) and 1-deoxy- d -xylulose 5-phosphate/2-C-methyl- d -erythritol-4-phosphate (DOXP/MEP) pathways in sesquiterpene biosynthesis of grape berries was investigated. There is an increasing interest in this class of terpenoids, since the oxygenated sesquiterpene rotundone was identified as the peppery aroma impact compound in Australian Shiraz wines. To investigate precursor supply pathway utilization, in vivo feeding experiments were performed with the deuterium labeled, pathway specific, precursors [5,5-2H2]-1-deoxy- d -xylulose and [5,5-2H2]-mevalonic acid lactone. Head Space-Solid Phase Micro Extraction-Gas Chromatography-Mass Spectrometry (HS-SPME-GC–MS) analysis of the generated volatile metabolites demonstrated that de novo sesquiterpene biosynthesis is mainly located in the grape berry exocarp (skin), with no detectable activity in the mesocarp (flesh) of the Lemberger variety. Interestingly, precursors from both the (primarily) cytosolic MVA and plastidial DOXP/MEP pathways were incorporated into grape sesquiterpenes in the varieties Lemberger, Gewurztraminer and Syrah. Our labeling data provide evidence for a homogenous, cytosolic pool of precursors for sesquiterpene biosynthesis, indicating that a transport of precursors occurs mostly from plastids to the cytosol. The labeling patterns of the sesquiterpene germacrene D were in agreement with a cyclization mechanism analogous to that of a previously cloned enantioselective (R)-germacrene D synthase from Solidago canadensis. This observation was subsequently confirmed by enantioselective GC–MS analysis demonstrating the exclusive presence of (R)-germacrene D, and not the (S)-enantiomer, in grape berries.

We report the development and testing of an accurate mass – time (AMT) tag approach for the LC/MS-based identification of plant natural products (PNPs) in complex extracts. An AMT tag library was developed for approximately 500 PNPs with diverse chemical structures, detected in electrospray and atmospheric pressure chemical ionization modes (both positive and negative polarities). In addition, to enable peak annotations with high confidence, MS/MS spectra were acquired with three different fragmentation energies. The LC/MS and MS/MS data sets were integrated into online spectral search tools and repositories (Spektraris and MassBank), thus allowing users to interrogate their own data sets for the potential presence of PNPs. The utility of the AMT tag library approach is demonstrated by the detection and annotation of active principles in 27 different medicinal plant species with diverse chemical constituents.

Treatment of Pinus sylvestris L. cell-suspension cultures with an elicitor preparation from the pine needle pathogen Lophodermium seditiosum, resulted in a severalhundredto thousandfold accumulation of the stilbenes pinosylvin and pinosylvin 3-O-methyl ether in methanolic cell extracts. There was a simultaneous induction of the biosynthetic enzymes phenylalanine ammonia-lyase (E.C. 4.3.1.5.) and stilbene synthase (pinosylvin-forming, E.C. 2.3.1.146). For the first time, an incorporation of stilbenes into the cell wall fraction as well as stilbene excretion into the extracellular space was demonstrated in addition to intracellular accumulation.

The paraveinal mesophyll (PVM) of soybean is a distinctive uniseriate layer of branched cells situated between the spongy and palisade chlorenchyma of leaves that contains an abundance of putative vegetative storage proteins, Vspα and Vspβ, in its vacuoles. Soybean vegetative lipoxygenases (five isozymes designated as Vlx(A–E)) have been reported to co-localise with Vsp in PVM vacuoles; however, conflicting results regarding the tissue-level and subcellular localisations of specific Vlx isozymes have been reported. We employed immuno-cytochemistry with affinity-purified, isozyme-specific antibodies to reinvestigate the subcellular locations of soybean Vlx isozymes during a sink limitation experiment. VlxB and VlxC were localised to the cytoplasm and nucleoplasm of PVM cells, whereas VlxD was present in the cytoplasm and nucleoplasm of mesophyll chlorenchyma (MC) cells. Label was not associated with storage vacuoles or any evident protein bodies, so our results cast doubt on the hypothesis that Vlx isozymes function as vegetative storage proteins.

The integration of mathematical modeling and experimental testing is emerging as a powerful approach for improving our understanding of the regulation of metabolic pathways. In this study, we report on the development of a kinetic mathematical model that accurately simulates the developmental patterns of monoterpenoid essential oil accumulation in peppermint ( Mentha × piperita ). This model was then used to evaluate the biochemical processes underlying experimentally determined changes in the monoterpene pathway under low ambient-light intensities, which led to an accumulation of the branchpoint intermediate (+)-pulegone and the side product (+)-menthofuran. Our simulations indicated that the environmentally regulated changes in monoterpene profiles could only be explained when, in addition to effects on biosynthetic enzyme activities, as yet unidentified inhibitory effects of (+)-menthofuran on the branchpoint enzyme pulegone reductase (PR) were assumed. Subsequent in vitro analyses with recombinant protein confirmed that (+)-menthofuran acts as a weak competitive inhibitor of PR ( K i = 300 μM). To evaluate whether the intracellular concentration of (+)-menthofuran was high enough for PR inhibition in vivo , we isolated essential oil-synthesizing secretory cells from peppermint leaves and subjected them to steam distillations. When peppermint plants were grown under low-light conditions, (+)-menthofuran was selectively retained in secretory cells and accumulated to very high levels (up to 20 mM), whereas under regular growth conditions, (+)-menthofuran levels remained very low (

To support their sessile and autotrophic lifestyle higher plants have evolved elaborate networks of metabolic pathways. Dynamic changes in these metabolic networks are among the developmental forces underlying the functional differentiation of organs, tissues and specialized cell types. They are also important in the various interactions of a plant with its environment. Further complexity is added by the extensive compartmentation of the various interconnected metabolic pathways in plants. Thus, although being used widely for assessing the control of metabolic flux in microbes, mathematical modeling approaches that require steady-state approximations are of limited utility for understanding complex plant metabolic networks. However, considerable progress has been made when manageable metabolic subsystems were studied. In this article, we will explain in general terms and using simple examples the concepts underlying stoichiometric modeling (metabolic flux analysis and metabolic pathway analysis) and kinetic approaches to modeling (including metabolic control analysis as a special case). Selected studies demonstrating the prospects of these approaches, or combinations of them, for understanding the control of flux through particular plant pathways are discussed. We argue that iterative cycles of (dry) mathematical modeling and (wet) laboratory testing will become increasingly important for simulating the distribution of flux in plant metabolic networks and deriving rational experimental designs for metabolic engineering efforts.

In plants, two spatially separated pathways provide the precursors for isoprenoid biosynthesis. We generated transgenic Arabidopsis (Arabidopsis thaliana) lines with modulated levels of expression of each individual gene involved in the cytosolic/peroxisomal mevalonate and plastidial methylerythritol phosphate pathways. By assessing the correlation of transgene expression levels with isoprenoid marker metabolites (gene-to-metabolite correlation), we determined the relative importance of transcriptional control at each individual step of isoprenoid precursor biosynthesis. The accumulation patterns of metabolic intermediates (metabolite-to-gene correlation) were then used to infer flux bottlenecks in the sterol pathway. The extent of metabolic cross talk, the exchange of isoprenoid intermediates between compartmentalized pathways, was assessed by a combination of gene-to-metabolite and metabolite-to-metabolite correlation analyses. This strategy allowed the selection of genes to be modulated by metabolic engineering, and we demonstrate that the overexpression of predictable combinations of genes can be used to significantly enhance flux toward specific end products of the sterol pathway. Transgenic plants accumulating increased amounts of sterols are characterized by significantly elevated biomass, which can be a desirable trait in crop and biofuel plants.

Recent developments in genomic and post-genomic technologies have led to the amassment of data describing genome sequences, transcript, protein and metabolite abundances, protein modifications, and protein-protein and protein-DNA interactions. Such technologies have vastly expanded the inventory of detectable molecular species and can be used to describe their interdependence, but they have yet to fulfill their promise in enhancing our knowledge of how flux through metabolic pathways is regulated. A convergence of traditional reductionistic and novel holistic experimental approaches could aid in elucidating flux control.

Post-genomic era research is focusing on studies to attribute functions to genes and their encoded proteins, and to describe the regulatory networks controlling metabolic, protein synthesis and signal transduction pathways. To facilitate the analysis of experiments using post-genomic technologies, new concepts for linking the vast amount of raw data to a biological context have to be developed. Visual representations of pathways help biologists to understand the complex relationships between components of metabolic networks, and provide an invaluable resource for the integration of transcriptomics, proteomics and metabolomics data sets. Besides providing an overview of currently available bioinformatic tools for plant scientists, we introduce BioPathAt, a newly developed visual interface that allows the knowledge-based analysis of genome-scale data by integrating biochemical pathway maps (BioPathAtMAPS module) with a manually scrutinized gene-function database (BioPathAtDB) for the model plant Arabidopsis thaliana. In addition, we discuss approaches for generating a biochemical pathway knowledge database for A. thaliana that includes, in addition to accurate annotation, condensed experimental information regarding in vitro and in vivo gene/protein function.

In higher plants, two independent pathways are responsible for the biosynthesis of isopentenyl diphosphate and dimethylallyl diphosphate, the central five-carbon precursors of all isoprenoids. The cytosolic pathway, which involves mevalonate (MVA) as a key intermediate, provides the precursor molecules for sterols, ubiquinone, and certain sesquiterpenes, whereas the plastidial MVA-independent pathway is involved in the formation of precursors for the biosynthesis of isoprene, monoterpenes, diterpenes, carotenoids, abscisic acid, and the side chains of chlorophylls, tocopherols, and plastoquinone. Recent experiments provided indirect evidence for the presence of an export system for isoprenoid intermediates from the plastids to the cytosol in Arabidopsis thaliana. Here we report that isolated chloroplasts (from spinach, kale, and Indian mustard), envelope membrane vesicles, and proteoliposomes prepared from the solubilized proteins of envelope membranes (from spinach) are capable of the efficient transport of isopentenyl diphosphate and geranyl diphosphate. Lower rates of transport were observed with the substrates farnesyl diphosphate and dimethylallyl diphosphate, whereas geranylgeranyl diphosphate and mevalonate were not transported with appreciable efficiency. Our data suggest that plastid membranes possess a unidirectional proton symport system for the export of specific isoprenoid intermediates involved in the metabolic cross talk between cytosolic and plastidial pathways of isoprenoid biosynthesis.

In plants, the formation of isopentenyl diphosphate and dimethylallyl diphosphate, the central intermediates in the biosynthesis of isoprenoids, is compartmentalized: the mevalonate (MVA) pathway, which is localized to the cytosol, is responsible for the synthesis of sterols, certain sesquiterpenes, and the side chain of ubiquinone; in contrast, the recently discovered MVA-independent pathway, which operates in plastids, is involved in providing the precursors for monoterpenes, certain sesquiterpenes, diterpenes, carotenoids, and the side chains of chlorophylls and plastoquinone. Specific inhibitors of the MVA pathway (lovastatin) and the MVA-independent pathway (fosmidomycin) were used to perturb biosynthetic flux in Arabidopsis thaliana seedlings. The interaction between both pathways was studied at the transcriptional level by using GeneChip (Affymetrix) microarrays and at the metabolite level by assaying chlorophylls, carotenoids, and sterols. Treatment of seedlings with lovastatin resulted in a transient decrease in sterol levels and a transient increase in carotenoid as well as chlorophyll levels. After the initial drop, sterol amounts in lovastatin-treated seedlings recovered to levels above controls. As a response to fosmidomycin treatment, a transient increase in sterol levels was observed, whereas chlorophyll and carotenoid amounts decreased dramatically when compared with controls. At 96 h after fosmidomycin addition, the levels of all metabolites assayed (sterols, chlorophylls, and carotenoids) were substantially lower than in controls. Interestingly, these inhibitor-mediated changes were not reflected in altered gene expression levels of the genes involved in sterol, chlorophyll, and carotenoid metabolism. The lack of correlation between gene expression patterns and the accumulation of isoprenoid metabolites indicates that posttranscriptional processes may play an important role in regulating flux through isoprenoid metabolic pathways.

A systematic proteomic analysis of rice ( Oryza sativa ) leaf, root, and seed tissue using two independent technologies, two-dimensional gel electrophoresis followed by tandem mass spectrometry and multidimensional protein identification technology, allowed the detection and identification of 2,528 unique proteins, which represents the most comprehensive proteome exploration to date. A comparative display of the expression patterns indicated that enzymes involved in central metabolic pathways are present in all tissues, whereas metabolic specialization is reflected in the occurrence of a tissue-specific enzyme complement. For example, tissue-specific and subcellular compartment-specific isoforms of ADP-glucose pyrophosphorylase were detected, thus providing proteomic confirmation of the presence of distinct regulatory mechanisms involved in the biosynthesis and breakdown of separate starch pools in different tissues. In addition, several previously characterized allergenic proteins were identified in the seed sample, indicating the potential of proteomic approaches to survey food samples with regard to the occurrence of allergens.

Publisher Summary The peppermint oil gland secretory cell (complimentary) cDNA library has proven to provide a highly enriched source of candidate genes involved in essential oil biosynthesis. A functional genomics approach has successfully been employed to clone genes involved in the mevalonate-independent pathway of isoprenoid biosynthesis and in the peppermint-specific steps producing (-)-menthol and (-)-menthone. The optimization of (liquid chromatography mass spectrography ) LC-MS technology to profile phosphorylated carbohydrates and prenyl diphosphates has helped to establish that the reaction catalyzed by 1-deoxy-D-xylulose 5- phosphate reductoisomerase (DXR) is a slow step of the mevalonate-independent pathway in peppermint. Transgenic plants with either increased or decreased steady-state DXR transcript accumulation or enzyme activity have confirmed the correlation of DXR expression rate and essential oil yield, thus providing evidence for the utility of profiling biosynthetic intermediates to predict flux control through metabolic pathways.