Natural variation meets synthetic biology: Promiscuous trichome-expressed acyltransferases from Nicotiana.

Acylsugars are defensive, trichome-synthesized sugar esters produced in plants across the Solanaceae (nightshade) family. Although assembled from simple metabolites and synthesized by a relatively short core biosynthetic pathway, tremendous within- and across-species acylsugar structural variation is documented across the family. To advance our understanding of the diversity and the synthesis of acylsugars within the Nicotiana genus, trichome extracts were profiled across the genus coupled with transcriptomics-guided enzyme discovery and in vivo and in vitro analysis. Differences in the types of sugar cores, numbers of acylations, and acyl chain structures contributed to over 300 unique annotated acylsugars throughout Nicotiana. Placement of acyl chain length into a phylogenetic context revealed that an unsaturated acyl chain type was detected in a few closely related species. A comparative transcriptomics approach identified trichome-enriched Nicotiana acuminata acylsugar biosynthetic candidate enzymes. More than 25 acylsugar variants could be produced in a single enzyme assay with four N. acuminata acylsugar acyltransferases (NacASAT1-4) together with structurally diverse acyl-CoAs and sucrose. Liquid chromatography coupled with mass spectrometry screening of in vitro products revealed the ability of these enzymes to make acylsugars not present in Nicotiana plant extracts. In vitro acylsugar production also provided insights into acyltransferase acyl donor promiscuity and acyl acceptor specificity as well as regiospecificity of some ASATs. This study suggests that promiscuous Nicotiana acyltransferases can be used as synthetic biology tools to produce novel and potentially useful metabolites.

Understanding the structure and composition of recalcitrant oligosaccharides in hydrolysate using high-throughput biotin-based glycome profiling and mass spectrometry.

Novel Immunological and Mass Spectrometry Methods for Comprehensive Analysis of Recalcitrant Oligosaccharides in AFEX Pretreated Corn Stover. Lignocellulosic biomass is a sustainable alternative to fossil fuel and is extensively used for developing bio-based technologies to produce products such as food, feed, fuel, and chemicals. The key to these technologies is to develop cost competitive processes to convert complex carbohydrates present in plant cell wall to simple sugars such as glucose, xylose, and arabinose. Since lignocellulosic biomass is highly recalcitrant, it must undergo a combination of thermochemical treatment such as Ammonia Fiber Expansion (AFEX), dilute acid (DA), Ionic Liquid (IL) and biological treatment such as enzyme hydrolysis and microbial fermentation to produce desired products. However, when using commercial fungal enzymes during hydrolysis, only 75-85% of the soluble sugars generated are monomeric sugars, while the remaining 15-25% are soluble recalcitrant oligosaccharides that cannot be easily utilized by microorganisms. Previously, we successfully separated and purified the soluble recalcitrant oligosaccharides using a combination of charcoal and celite-based separation followed by size exclusion chromatography and studies their inhibitory properties on enzymes. We discovered that the oligosaccharides with higher degree of polymerization (DP) containing methylated uronic acid substitutions were more recalcitrant towards commercial enzyme mixtures than lower DP and neutral oligosaccharides. Here, we report the use of several complementary techniques that include glycome profiling using plant biomass glycan specific monoclonal antibodies (mAbs) to characterize sugar linkages in plant cell walls and enzymatic hydrolysate, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) using structurally-informative diagnostic peaks offered by negative ion post-secondary decay spectra, gas chromatography followed by mass spectrometry (GC-MS) to characterize oligosaccharide sugar linkages with and without derivatization. Since oligosaccharides (DP 4-20) are small, it is challenging to mobilize these molecules for mAbs binding and characterization. To overcome this problem, we have applied a new biotin-coupling based oligosaccharide immobilization method that successfully tagged most of the low DP soluble oligosaccharides on to a micro-plate surface followed by specific linkage analysis using mAbs in a high-throughput system. This new approach will help develop more advanced versions of future high throughput glycome profiling methods that can be used to separate and characterize oligosaccharides present in biomarkers for diagnostic applications.

Alkaloids of the Genus Datura: Review of a Rich Resource for Natural Product Discovery.

The genus Datura (Solanaceae) contains nine species of medicinal plants that have held both curative utility and cultural significance throughout history. This genus' particular bioactivity results from the enormous diversity of alkaloids it contains, making it a valuable study organism for many disciplines. Although Datura contains mostly tropane alkaloids (such as hyoscyamine and scopolamine), indole, beta-carboline, and pyrrolidine alkaloids have also been identified. The tools available to explore specialized metabolism in plants have undergone remarkable advances over the past couple of decades and provide renewed opportunities for discoveries of new compounds and the genetic basis for their biosynthesis. This review provides a comprehensive overview of studies on the alkaloids of Datura that focuses on three questions: How do we find and identify alkaloids? Where do alkaloids come from? What factors affect their presence and abundance? We also address pitfalls and relevant questions applicable to natural products and metabolomics researchers. With both careful perspectives and new advances in instrumentation, the pace of alkaloid discovery-from not just Datura-has the potential to accelerate dramatically in the near future.

Specialized Metabolism in a Nonmodel Nightshade: Trichome Acylinositol Biosynthesis.

Plants make many biologically active, specialized metabolites, which vary in structure, biosynthesis, and the processes they influence. An increasing number of these compounds are documented to protect plants from insects, pathogens, or herbivores or to mediate interactions with beneficial organisms, including pollinators and nitrogen-fixing microbes. Acylsugars, one class of protective compounds, are made in glandular trichomes of plants across the Solanaceae family. While most described acylsugars are acylsucroses, published examples also include acylsugars with hexose cores. The South American fruit crop naranjilla (lulo; Solanum quitoense) produces acylsugars containing a myoinositol core. We identified an enzyme that acetylates triacylinositols, a function homologous to the last step in the acylsucrose biosynthetic pathway of tomato (Solanum lycopersicum). Our analysis reveals parallels between S. lycopersicum acylsucrose and S. quitoense acylinositol biosynthesis, suggesting a common evolutionary origin.

Camptotheca acuminata 10-hydroxycamptothecin O-methyltransferase: an alkaloid biosynthetic enzyme co-opted from flavonoid metabolism.

The medicinal plant Camptotheca acuminata accumulates camptothecin, 10-hydroxycamptothecin, and 10-methoxycamptothecin as its major bioactive monoterpene indole alkaloids. Here, we describe identification and functional characterization of 10-hydroxycamptothecin O-methyltransferase (Ca10OMT), a member of the Diverse subclade of class II OMTs. Ca10OMT is highly active toward both its alkaloid substrate and a wide range of flavonoids in vitro and in this way contrasts with other alkaloid OMTs in the subclade that only utilize alkaloid substrates. Ca10OMT shows a strong preference for the A-ring 7-OH of flavonoids, which is structurally equivalent to the 10-OH of 10-hydroxycamptothecin. The substrates of other alkaloid OMTs in the subclade bear little similarity to flavonoids, but the 3-D positioning of the 7-OH, A- and C-rings of flavonoids is nearly identical to the 10-OH, A- and B-rings of 10-hydroxycamptothecin. This structural similarity likely explains the retention of flavonoid OMT activity by Ca10OMT and also why kaempferol and quercetin aglycones are potent inhibitors of its 10-hydroxycamptothecin activity. The catalytic promiscuity and strong inhibition of Ca10OMT by flavonoid aglycones in vitro prompted us to investigate the potential physiological roles of the enzyme in vivo. Based on its regioselectivity, kinetic parameters and absence of 7-OMT flavonoids in vivo, we conclude that the major and likely only substrate of Ca10OMTin vivo is 10-hydroxycamptothecin. This is likely accomplished by Ca10OMT being kept spatially separated at the tissue levels from potentially inhibitory flavonoid aglycones, and flavonoid aglycones being rapidly glycosylated to non-inhibitory flavonoid glycosides.

Exposure Assessment of Acetamide in Milk, Beef, and Coffee Using Xanthydrol Derivatization and Gas Chromatography/Mass Spectrometry.

Acetamide has been classified as a possible human carcinogen, but uncertainties exist about its levels in foods. This report presents evidence that thermal decomposition of N-acetylated sugars and amino acids in heated gas chromatograph injectors contributes to artifactual acetamide in milk and beef. An alternative gas chromatography/mass spectrometry protocol based on derivatization of acetamide with 9-xanthydrol was optimized and shown to be free of artifactual acetamide formation. The protocol was validated using a surrogate analyte approach based on d3-acetamide and applied to analyze 23 pasteurized whole milk, 44 raw sirloin beef, and raw milk samples from 14 different cows, and yielded levels about 10-fold lower than those obtained by direct injection without derivatization. The xanthydrol derivatization procedure detected acetamide in every food sample tested at 390 ± 60 ppb in milk, 400 ± 80 ppb in beef, and 39 000 ± 9000 ppb in roasted coffee beans.

Acylsugar Acylhydrolases: Carboxylesterase-Catalyzed Hydrolysis of Acylsugars in Tomato Trichomes.

Glandular trichomes of cultivated tomato (Solanum lycopersicum) and many other species throughout the Solanaceae produce and secrete mixtures of sugar esters (acylsugars) on the plant aerial surfaces. In wild and cultivated tomato, these metabolites consist of a sugar backbone, typically glucose or sucrose, and two to five acyl chains esterified to various positions on the sugar core. The aliphatic acyl chains vary in length and branching and are transferred to the sugar by a series of reactions catalyzed by acylsugar acyltransferases. A phenotypic screen of a set of S. lycopersicum M82 × Solanum pennellii LA0716 introgression lines identified a dominant genetic locus on chromosome 5 from the wild relative that affected total acylsugar levels. Genetic mapping revealed that the reduction in acylsugar levels was consistent with the presence and increased expression of two S. pennellii genes (Sopen05g030120 and Sopen05g030130) encoding putative carboxylesterase enzymes of the α/ß-hydrolase superfamily. These two enzymes, named ACYLSUGAR ACYLHYDROLASE1 (ASH1) and ASH2, were shown to remove acyl chains from specific positions of certain types of acylsugars in vitro. A survey of related genes in M82 and LA0716 identified another trichome-expressed ASH gene on chromosome 9 (M82, Solyc09g075710; LA0716, Sopen09g030520) encoding a protein with similar activity. Characterization of the in vitro activities of the SpASH enzymes showed reduced activities with acylsugars produced by LA0716, presumably contributing to the high-level production of acylsugars in the presence of highly expressed SpASH genes.

Pyruvate Kinase Isoform Switching and Hepatic Metabolic Reprogramming by the Environmental Contaminant 2,3,7,8-Tetrachlorodibenzo-p-Dioxin.

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) elicits dose-dependent hepatotoxicity that includes fat accumulation, inflammation, and fibrosis that may progress to hepatocellular carcinoma. To further investigate these effects, RNA-Seq data were integrated with computationally identified putative dioxin response elements, and complementary targeted metabolomic and aryl hydrocarbon receptor (AhR) ChIP-Seq data from female C57BL/6 mice gavaged with TCDD every 4 days for 28 days. Data integration using CytoKEGG with manual curation identified dose-dependent alterations in central carbon and amino acid metabolism. More specifically, TCDD increased pyruvate kinase isoform M2 (PKM2) gene and protein expression. PKM2 has lower catalytic activity resulting in decreased glycolytic flux and the accumulation of upstream intermediates that were redirected to the pentose phosphate pathway and serine/folate biosynthesis, 2 important NADPH producing pathways stemming from glycolysis. In addition, the GAC:KGA glutaminase (GLS1) protein isoform ratio was increased, consistent with increases in glutaminolysis which serves an anaplerotic role for the TCA cycle and compensates for the reduced glycolytic flux. Collectively, gene expression, protein, and metabolite changes were indicative of increases in NADPH production in support of cytochrome P450 activity and ROS defenses. This AhR-mediated metabolic reprogramming is similar to the Warburg effect and represents a novel advantageous defense mechanism to increase anti-oxidant capacity in normal differentiated hepatocytes.

A Feedback-Insensitive Isopropylmalate Synthase Affects Acylsugar Composition in Cultivated and Wild Tomato.

Acylsugars are insecticidal specialized metabolites produced in the glandular trichomes of plants in the Solanaceae family. In the tomato clade of the Solanum genus, acylsugars consist of aliphatic acids of different chain lengths esterified to sucrose, or less frequently to glucose. Through liquid chromatography-mass spectrometry screening of introgression lines, we previously identified a region of chromosome 8 in the Solanum pennellii LA0716 genome (IL8-1/8-1-1) that causes the cultivated tomato Solanum lycopersicum to shift from producing acylsucroses with abundant 3-methylbutanoic acid acyl chains derived from leucine metabolism to 2-methylpropanoic acid acyl chains derived from valine metabolism. We describe multiple lines of evidence implicating a trichome-expressed gene from this region as playing a role in this shift. S. lycopersicum M82 SlIPMS3 (Solyc08g014230) encodes a functional end product inhibition-insensitive version of the committing enzyme of leucine biosynthesis, isopropylmalate synthase, missing the carboxyl-terminal 160 amino acids. In contrast, the S. pennellii LA0716 IPMS3 allele found in IL8-1/8-1-1 encodes a nonfunctional truncated IPMS protein. M82 transformed with an SlIPMS3 RNA interference construct exhibited an acylsugar profile similar to that of IL8-1-1, whereas the expression of SlIPMS3 in IL8-1-1 partially restored the M82 acylsugar phenotype. These IPMS3 alleles are polymorphic in 14 S. pennellii accessions spread throughout the geographical range of occurrence for this species and are associated with acylsugars containing varying amounts of 2-methylpropanoic acid and 3-methylbutanoic acid acyl chains.

Functionally divergent alleles and duplicated Loci encoding an acyltransferase contribute to acylsugar metabolite diversity in Solanum trichomes.

Glandular trichomes from tomato (Solanum lycopersicum) and other species in the Solanaceae produce and secrete a mixture of O-acylsugars (aliphatic esters of sucrose and glucose) that contribute to insect defense. Despite their phylogenetic distribution and diversity, relatively little is known about how these specialized metabolites are synthesized. Mass spectrometric profiling of acylsugars in the S. lycopersicum x Solanum pennellii introgression lines identified a chromosome 11 locus containing a cluster of BAHD acyltransferases with one gene (named Sl-ASAT3) expressed in tip cells of type I trichomes where acylsugars are made. Sl-ASAT3 was shown to encode an acyl-CoA-dependent acyltransferase that catalyzes the transfer of short (four to five carbons) branched acyl chains to the furanose ring of di-acylsucrose acceptors to produce tri-acylsucroses, which can be further acetylated by Sl-ASAT4 (previously Sl-AT2). Among the wild tomatoes, diversity in furanose ring acyl chains on acylsucroses was most striking in Solanum habrochaites. S. habrochaites accessions from Ecuador and northern Peru produced acylsucroses with short (≤C5) or no acyl chains on the furanose ring. Accessions from central and southern Peru had the ability to add short or long (up to C12) acyl chains to the furanose ring. Multiple ASAT3-like sequences were found in most accessions, and their in vitro activities correlated with observed geographical diversity in acylsugar profiles.

Relative mass defect filtering of mass spectra: a path to discovery of plant specialized metabolites.

The rapid identification of novel plant metabolites and assignments of newly discovered substances to natural product classes present the main bottlenecks to defining plant specialized phenotypes. Although mass spectrometry provides powerful support for metabolite discovery by measuring molecular masses, ambiguities in elemental formulas often fail to reveal the biosynthetic origins of specialized metabolites detected using liquid chromatography-mass spectrometry. A promising approach for mining liquid chromatography-mass spectrometry metabolite profiling data for specific metabolite classes is achieved by calculating relative mass defects (RMDs) from molecular and fragment ions. This strategy enabled the rapid recognition of an extensive range of terpenoid metabolites in complex plant tissue extracts and is independent of retention time, abundance, and elemental formula. Using RMD filtering and tandem mass spectrometry data analysis, 24 novel elemental formulas corresponding to glycosylated sesquiterpenoid metabolites were identified in extracts of the wild tomato Solanum habrochaites LA1777 trichomes. Extensive isomerism was revealed by ultra-high-performance liquid chromatography, leading to evidence of more than 200 distinct sesquiterpenoid metabolites. RMD filtering led to the recognition of the presence of glycosides of two unusual sesquiterpenoid cores that bear limited similarity to known sesquiterpenes in the genus Solanum. In addition, RMD filtering is readily applied to existing metabolomics databases and correctly classified the annotated terpenoid metabolites in the public metabolome database for Catharanthus roseus.

Sesquiterpenoid glycosides from glandular trichomes of the wild tomato relative Solanum habrochaites.

Profiles of terpenoid glycoside metabolites in glandular trichomes of Solanum habrochaites LA1777 leaves were generated using ultrahigh performance liquid chromatography/time-of-flight mass spectrometry with multiplexing of non-selective collision-induced dissociation (CID). Profiling data suggested a diverse group of 52 sesquiterpenoid glycosides, and fragment ions observed in both non-selective CID mass spectra and true tandem mass spectrometry (MS/MS) product ion spectra documented variation in extent of glycosylation and the presence of malonate or acetate esters. Up to 10 isomers were detected for some metabolites. Malonate and acetate esters of three sesquiterpene diol glucosides and one unmodified diglucoside were purified using reversed phase semipreparative HPLC and analyzed and identified using 1D and 2D NMR and mass spectrometry. All four of the isolated products were glucosides of campheranane-2,12-diol.

Chemical imaging of trichome specialized metabolites using contact printing and laser desorption/ionization mass spectrometry.

Cell transfer by contact printing coupled with carbon-substrate-assisted laser desorption/ionization was used to directly profile and image secondary metabolites in trichomes on leaves of the wild tomato Solanum habrochaites. Major specialized metabolites, including acyl sugars, alkaloids, flavonoids, and terpenoid acids, were successfully detected in positive ion mode or negative ion mode, and in some cases in both modes. This simple solvent-free and matrix-free sample preparation for mass spectrometry imaging avoids tedious sample preparation steps, and high-spatial-resolution images were obtained. Metabolite profiles were generated for individual glandular trichomes from a single Solanum habrochaites leaf at a spatial resolution of around 50 µm. Relative quantitative data from imaging experiments were validated by independent liquid chromatography-mass spectrometry analysis of subsamples from fresh plant material. The spatially resolved metabolite profiles of individual glands provided new information about the complexity of biosynthesis of specialized metabolites at the cellular-resolution scale. In addition, this technique offers a scheme capable of high-throughput profiling of metabolites in trichomes and irregularly shaped tissues and spatially discontinuous cells of a given cell type.

Metabolic Profiles Distinguish Non-Dampness-Phlegm and Dampness-Phlegm Patterns among Korean Patients with Acute Cerebral Infarction.

TRADITIONAL KOREAN MEDICINE CLASSIFIES STROKE INTO FOUR SUBTYPE PATTERNS ACCORDING TO SYMPTOMATIC PATTERN IDENTIFICATION: Qi deficiency (QD), Yin deficiency (YD), Dampness-phlegm (DP), and Fire and Heat (FH). This study investigated the difference in metabolic profiles of plasma comparing subjects displaying non-DP and DP patterns. A total of 141 patients with cerebral infarction enrolled in this study were distributed as non-DP (N = 68) and DP (N = 73). Anthropometric parameters and symptom/sign index were measured. Metabolic profiling was performed using ultrahigh-performance liquid chromatography-mass spectrometry. The Ratio of subjects with slippery pulse was higher in DP pattern, but fine pulse was lower than that in non-DP pattern. As a result of metabolomics analysis, twenty-one metabolites displayed different levels between non-DP and DP patterns. Two were identified as lysophosphatidylcholines (LPCs), LPC(18:2), and LPC(20:3) having an unsaturated acyl chain and showed lower levels in DP pattern than in non-DP pattern (P = 0.015, 0.034, resp.). However, the saturated LPCs, LPC(18:0) and LPC(16:0), exhibited slight but statistically insignificant elevation in DP pattern. Our results demonstrated that plasma LPCs with polyunsaturated fatty acid groups were associated with DP pattern and suggest that variation of plasma lipid profiles may serve as potential biomarker for diagnosis of DP pattern.

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.

Striking natural diversity in glandular trichome acylsugar composition is shaped by variation at the Acyltransferase2 locus in the wild tomato Solanum habrochaites.

Acylsugars are polyesters of short- to medium-length acyl chains on sucrose or glucose backbones that are produced in secretory glandular trichomes of many solanaceous plants, including cultivated tomato (Solanum lycopersicum). Despite their roles in biotic stress adaptation and their wide taxonomic distribution, there is relatively little information about the diversity of these compounds and the genes responsible for their biosynthesis. In this study, acylsugar diversity was assessed for 80 accessions of the wild tomato species Solanum habrochaites from throughout the Andes Mountains. Trichome metabolites were analyzed by liquid chromatography-time of flight-mass spectrometry, revealing the presence of at least 34 structurally diverse acylsucroses and two acylglucoses. Distinct phenotypic classes were discovered that varied based on the presence of glucose or sucrose, the numbers and lengths of acyl chains, and the relative total amounts of acylsugars. The presence or absence of an acetyl chain on the acylsucrose hexose ring caused clustering of the accessions into two main groups. Analysis of the Acyltransferase2 gene (the apparent ortholog of Solyc01g105580) revealed differences in enzyme activity and gene expression correlated with polymorphism in S. habrochaites accessions that varied in acylsucrose acetylation. These results are consistent with the hypothesis that glandular trichome acylsugar acetylation is under selective pressure in some populations of S. habrochaites and that the gene mutates to inactivity in the absence of selection.

Medicinal plants: a public resource for metabolomics and hypothesis development.

Specialized compounds from photosynthetic organisms serve as rich resources for drug development. From aspirin to atropine, plant-derived natural products have had a profound impact on human health. Technological advances provide new opportunities to access these natural products in a metabolic context. Here, we describe a database and platform for storing, visualizing and statistically analyzing metabolomics data from fourteen medicinal plant species. The metabolomes and associated transcriptomes (RNAseq) for each plant species, gathered from up to twenty tissue/organ samples that have experienced varied growth conditions and developmental histories, were analyzed in parallel. Three case studies illustrate different ways that the data can be integrally used to generate testable hypotheses concerning the biochemistry, phylogeny and natural product diversity of medicinal plants. Deep metabolomics analysis of Camptotheca acuminata exemplifies how such data can be used to inform metabolic understanding of natural product chemical diversity and begin to formulate hypotheses about their biogenesis. Metabolomics data from Prunella vulgaris, a species that contains a wide range of antioxidant, antiviral, tumoricidal and anti-inflammatory constituents, provide a case study of obtaining biosystematic and developmental fingerprint information from metabolite accumulation data in a little studied species. Digitalis purpurea, well known as a source of cardiac glycosides, is used to illustrate how integrating metabolomics and transcriptomics data can lead to identification of candidate genes encoding biosynthetic enzymes in the cardiac glycoside pathway. Medicinal Plant Metabolomics Resource (MPM) [1] provides a framework for generating experimentally testable hypotheses about the metabolic networks that lead to the generation of specialized compounds, identifying genes that control their biosynthesis and establishing a basis for modeling metabolism in less studied species. The database is publicly available and can be used by researchers in medicine and plant biology.

Polymethylated myricetin in trichomes of the wild tomato species Solanum habrochaites and characterization of trichome-specific 3'/5'- and 7/4'-myricetin O-methyltransferases.

Flavonoids are a class of metabolites found in many plant species. They have been reported to serve several physiological roles, such as in defense against herbivores and pathogens and in protection against harmful ultraviolet radiation. They also serve as precursors of pigment compounds found in flowers, leaves, and seeds. Highly methylated, nonglycosylated derivatives of the flavonoid myricetin flavonoid, have been previously reported from a variety of plants, but O-methyltransferases responsible for their synthesis have not yet been identified. Here, we show that secreting glandular trichomes (designated types 1 and 4) and storage glandular trichomes (type 6) on the leaf surface of wild tomato (Solanum habrochaites accession LA1777) plants contain 3,7,3'-trimethyl myricetin, 3,7,3',5'-tetramethyl myricetin, and 3,7,3',4',5'-pentamethyl myricetin, with gland types 1 and 4 containing severalfold more of these compounds than type 6 glands and with the tetramethylated compound predominating in all three gland types. We have also identified transcripts of two genes expressed in the glandular trichomes and showed that they encode enzymes capable of methylating myricetin at the 3' and 5' and the 7 and 4' positions, respectively. Both genes are preferentially expressed in secreting glandular trichome types 1 and 4 and to a lesser degree in storage trichome type 6, and the levels of the proteins they encode are correspondingly higher in types 1 and 4 glands compared with type 6 glands.

Comparative functional genomic analysis of Solanum glandular trichome types.

Glandular trichomes play important roles in protecting plants from biotic attack by producing defensive compounds. We investigated the metabolic profiles and transcriptomes to characterize the differences between different glandular trichome types in several domesticated and wild Solanum species: Solanum lycopersicum (glandular trichome types 1, 6, and 7), Solanum habrochaites (types 1, 4, and 6), Solanum pennellii (types 4 and 6), Solanum arcanum (type 6), and Solanum pimpinellifolium (type 6). Substantial chemical differences in and between Solanum species and glandular trichome types are likely determined by the regulation of metabolism at several levels. Comparison of S. habrochaites type 1 and 4 glandular trichomes revealed few differences in chemical content or transcript abundance, leading to the conclusion that these two glandular trichome types are the same and differ perhaps only in stalk length. The observation that all of the other species examined here contain either type 1 or 4 trichomes (not both) supports the conclusion that these two trichome types are the same. Most differences in metabolites between type 1 and 4 glands on the one hand and type 6 glands on the other hand are quantitative but not qualitative. Several glandular trichome types express genes associated with photosynthesis and carbon fixation, indicating that some carbon destined for specialized metabolism is likely fixed within the trichome secretory cells. Finally, Solanum type 7 glandular trichomes do not appear to be involved in the biosynthesis and storage of specialized metabolites and thus likely serve another unknown function, perhaps as the site of the synthesis of protease inhibitors.

Atmospheric pressure femtosecond laser imaging mass spectrometry.

A novel atmospheric pressure imaging mass spectrometry approach that offers improved lateral resolution (10 microm) using near-infrared femtosecond laser pulses for nonresonant desorption and ionization of sample constituents without the need of a laser-absorbing matrix is demonstrated. As a proof of concept the method was used to image a two-chemical pattern in paper. To demonstrate the ability of the approach to analyze biological tissue, a monolayer of onion epidermis was imaged allowing the chemical visualization of individual cells using mass spectrometry at ambient conditions for the first time. As the spatial resolution is currently limited by the limit of detection of the setup (approximately 500 fmol limit of detection for citric acid), improvements in sensitivity will increase the achievable spatial resolution.