Dynamic Changes in the Antioxidative Defense System in the Tea Plant Reveal the Photoprotection-Mediated Temporal Accumulation of Flavonoids under Full Sunlight Exposure.

To reveal the mechanisms underlying how light affects flavonoid metabolism and the potential role of flavonoids in protecting against photooxidative stress in tea leaves, tea plants adapted to low-light conditions were exposed to full sunlight over 48 h. There was an increase in the activities of catalase (CAT) and superoxide dismutase (SOD) as well as greater accumulation of reactive oxygen species, lutein, tocopherols, ascorbate and malondialdehyde, suggestive of a time-dependent response to photooxidative stress in tea leaves. Analysis of the time dependency of each element of the antioxidant system indicated that carotenoids and tocopherols exhibited the fastest response to light stress (within 3 h), followed by SOD, CAT and catechin, which peaked at 24 h. Meanwhile, flavonols, vitamin C and glutathione showed the slowest response. Subsequent identification of the main phytochemicals involved in protecting against oxidative stress using untargeted metabolomics revealed a fast and initial accumulation of nonesterified catechins that preceded the increase in flavonol glycosides and catechin esters. Gene expression analysis suggested that the light-induced accumulation of flavonoids was highly associated with the gene encoding flavonol synthase. Ultraviolet B (UV-B) irradiation further validated the time-dependent and collaborative effects of flavonoids in photoprotection in tea plants. Intriguingly, the dynamics of the metabolic response are highly distinct from those reported for Arabidopsis, suggesting that the response to light stress is not conserved across plants. This study additionally provides new insights into the functional role of flavonoids in preventing photooxidative stress and may contribute to further improving tea quality through the control of light intensity.

Epigenetic mapping of the Arabidopsis metabolome reveals mediators of the epigenotype-phenotype map.

Identifying the sources of natural variation underlying metabolic differences between plants will enable a better understanding of plant metabolism and provide insights into the regulatory networks that govern plant growth and morphology. So far, however, the contribution of epigenetic variation to metabolic diversity has been largely ignored. In the present study, we utilized a panel of Arabidopsis thaliana epigenetic recombinant inbred lines (epiRILs) to assess the impact of epigenetic variation on the metabolic composition. Thirty epigenetic QTL (QTLepi) were detected, which partly overlap with QTLepi linked to growth and morphology. In an effort to identify causal candidate genes in the QTLepi regions and their putative trans-targets, we performed in silico small RNA and qPCR analyses. Differentially expressed genes were further studied by phenotypic and metabolic analyses of knockout mutants. Three genes were detected that recapitulated the detected QTLepi effects, providing evidence for epigenetic regulation in cis and in trans These results indicate that epigenetic mechanisms impact metabolic diversity, possibly via small RNAs, and thus aid in further disentangling the complex epigenotype-phenotype map.

Azolla ferns testify: seed plants and ferns share a common ancestor for leucoanthocyanidin reductase enzymes.

Questions about in vivo substrates for proanthocyanidin (PA) biosynthesis and condensation have not been resolved and wide gaps in the understanding of transport and biogenesis in 'tannosomes' persist. Here we examined the evolution of PA biosynthesis in ferns not previously reported, asking what PAs are synthesised and how. Chemical and gene-expression analyses were combined to characterise PA biosynthesis, leveraging genome annotation from the floating fern Azolla filiculoides. In vitro assay and phylogenomics of PIP-dehydrogenases served to infer the evolution of leucoanthocyanidin reductase (LAR). Sporophyte-synthesised (epi)catechin polymers, averaging only seven subunits, accumulated to 5.3% in A. filiculoides, and 8% in A. pinnata biomass dry weight. Consistently, a LAR active in vitro was highly expressed in A. filiculoides. LAR, and paralogous fern WLAR-enzymes with differing substrate binding sites, represent an evolutionary innovation of the common ancestor of fern and seed plants. The specific ecological niche of Azolla ferns, a floating plant-microbe mat massively fixing CO2 and N2 , shaped their metabolism in which PA biosynthesis predominates and employs novel fern LAR enzymes. Characterisation of in vivo substrates of these LAR, will help to shed light on the recently assigned and surprising dual catalysis of LAR from seed plants.

Fine mapping of a thrips resistance QTL in Capsicum and the role of diterpene glycosides in the underlying mechanism.

KEY MESSAGE: A major thrips resistance QTL in Capsicum was fine-mapped to a region of 0.4 Mbp, and a multidisciplinary approach has been used to study putative underlying mechanisms. Resistance to thrips is an important trait for pepper growers. These insects can cause extensive damage to fruits, flowers and leaves on field and greenhouse grown plants worldwide. Two independent studies in Capsicum identified diterpene glycosides as metabolites that are correlated with thrips resistance. In this study, we fine-mapped a previously defined thrips resistance QTL on chromosome 6, to a region of 0.4 Mbp harbouring 15 genes. Two of these 15 candidate genes showed differences in gene expression upon thrips induction, when comparing plants carrying the resistance allele in homozygous state to plants with the susceptibility allele in homozygous state for the QTL region. Three genes, including the two genes that showed difference in gene expression, contained a SNP that was predicted to lead to changes in protein structure. Therefore, these three genes, i.e. an acid phosphatase 1 (APS1), an organic cation/carnitine transporter 7 (OCT7) and an uncharacterized locus LOC107874801, are the most likely candidates for playing a role in thrips resistance and are a first step in elucidating the genetic basis of thrips resistance in Capsicum. In addition, we show that the diterpene glycoside profiles did not differ between plants with the resistance and susceptibility allele for the chromosome 6 QTL, suggesting that these compounds do not play a role in the resistance conferred by the genes located in the major thrips resistance QTL studied.

Stimulated biosynthesis of delphinidin-related anthocyanins in tea shoots reducing the quality of green tea in summer.

BACKGROUND: Greater proportions of purple tea buds and leaves usually appear in the summer, which seriously affects the color and taste quality of green tea products, yet the metabolism of purple tea shoots in summer remains unclear. Here, the metabolomic profiles and gene expression of related flavonoid metabolic pathways in the purple and normal green shoots of 'Longjing 43', and the quality of green tea made with these two phenotypes, were analyzed and compared. RESULTS: Differential metabolites identified using high-performance liquid chromatography-Orbitrap/mass spectrometry indicated that anthocyanin biosynthesis in purple leaves was enriched, with higher levels of anthocyanidins (delphinidin-hexose-coumaroyl showed the greatest increase), proanthocyanidins (oligomers of catechins) and kaempferol glycoside. Expression patterns of the genes ANR, ANS, FLS, LAR, C4H, PAL, CHI, CHS and DFR revealed that the metabolism of anthocyanin is positively regulated by high temperature and/or light levels in summer. Gas chromatography-mass spectrometry results showed that, in purple tea shoots, the metabolism of carbohydrates was enriched whereas that of amino acids was diminished, while their mannose, fructose, d-galactose, sorbose and d-glucose contents were more than double those found in green leaves. A sensory evaluation confirmed that a greater quantity of purple shoots had a greater negative impact on green tea quality because of a bitter taste and dark color (leaves and infusions were tested). CONCLUSIONS: These results highlight the need for and possibility of improving commercial tea quality via cultivation that controls the temperature or light of tea gardens during the summer. © 2019 Society of Chemical Industry.

Association between vitamin content, plant morphology and geographical origin in a worldwide collection of the orphan crop Gynandropsis gynandra (Cleomaceae).

MAIN CONCLUSION: The variability in nutrient content and morphology in Gynandropsis gynandra is associated with the geographic origin of the accessions and provides a basis for breeding for higher levels of vitamin C, carotenoids or tocopherols in higher-yielding cultivars. We examined the variation in carotenoids, tocopherols and ascorbic acid as well as morphological traits in a worldwide germplasm of 76 accessions of the orphan leafy vegetable Gynandropsis gynandra (Cleomaceae) using greenhouse experiments and high-performance liquid chromatography analysis. The levels of carotenoids and tocopherols accumulating in the leaves varied significantly across accessions and were linked with the geographical origin and morphological variation. The main carotenoids included lutein, ß-carotene, α-carotene and violaxanthin. A twofold to threefold variation was observed for these compounds. The main tocopherols detected were α-tocopherol and γ-tocopherol with a 20-fold variation. A ninefold variation in vitamin C concentration and independent of geographical origin was observed. Overall, the accessions were grouped into three clusters based on variation in nutrient content and morphology. West African accessions were short plants with small leaves and with high tocopherol contents and relatively low carotenoid contents, Asian accessions were short plants with broad leaves and with relatively low carotenoid and high tocopherol contents, while East-Southern African plants were tall with high contents of both carotenoids and chlorophylls and low tocopherol contents. Carotenoids were positively correlated with plant height as well as foliar and floral traits but negatively correlated with tocopherols. The absence of a significant correlation between vitamin C and other traits indicated that breeding for high carotenoids or tocopherols content may be coupled with improved leaf yield and vitamin C content. Our study provides baseline information on the natural variation available for traits of interest for breeding for enhanced crop yield and nutrient content in Gynandropsis gynandra.

Pyrethric acid of natural pyrethrin insecticide: complete pathway elucidation and reconstitution in Nicotiana benthamiana.

In the natural pesticides known as pyrethrins, which are esters produced in flowers of Tanacetum cinerariifolium (Asteraceae), the monoterpenoid acyl moiety is pyrethric acid or chrysanthemic acid. We show here that pyrethric acid is produced from chrysanthemol in six steps catalyzed by four enzymes, the first five steps occurring in the trichomes covering the ovaries and the last one occurring inside the ovary tissues. Three steps involve the successive oxidation of carbon 10 (C10) to a carboxylic group by TcCHH, a cytochrome P450 oxidoreductase. Two other steps involve the successive oxidation of the hydroxylated carbon 1 to give a carboxylic group by TcADH2 and TcALDH1, the same enzymes that catalyze these reactions in the formation of chrysanthemic acid. The ultimate result of the actions of these three enzymes is the formation of 10-carboxychrysanthemic acid in the trichomes. Finally, the carboxyl group at C10 is methylated by TcCCMT, a member of the SABATH methyltransferase family, to give pyrethric acid. This reaction occurs mostly in the ovaries. Expression in N. benthamiana plants of all four genes encoding aforementioned enzymes, together with TcCDS, a gene that encodes an enzyme that catalyzes the formation of chrysanthemol, led to the production of pyrethric acid.

The effect of isolation methods of tomato pollen on the results of metabolic profiling.

INTRODUCTION: Untargeted metabolomics is a powerful tool to detect hundreds of metabolites within a given tissue and to compare the metabolite composition of samples in a comprehensive manner. However, with regard to pollen research such comprehensive metabolomics approaches are yet not well developed. To enable isolation of pollen that is tightly enclosed within the anthers of the flower, such as immature pollen, the current pollen isolation protocols require the use of a watery solution. These protocols raise a number of concerns for their suitability in metabolomics analyses, in view of possible metabolic activities in the pollen and contamination with anther metabolites. OBJECTIVES: We assessed the effect of different sample preparation procedures currently used for pollen isolation for their suitability to perform metabolomics of tomato pollen. METHODS: Pollen were isolated using different methods and the metabolic profiles were analysed by liquid chromatography-mass spectrometry (LC-MS). RESULTS: Our results demonstrated that pollen isolation in a watery solution led to (i) rehydration of the pollen grains, inducing marked metabolic changes in flavonoids, phenylpropanoids and amino acids and thus resulting in a metabolite profile that did not reflect the one of mature dry pollen, (ii) hydrolysis of sucrose into glucose and fructose during subsequent metabolite extraction, unless the isolated and rehydrated pollen were lyophilized prior to extraction, and (iii) contamination with anther-specific metabolites, such as alkaloids, thus compromising the metabolic purity of the pollen fraction. CONCLUSION: We conclude that the current practices used to isolate pollen are suboptimal for metabolomics analyses and provide recommendations on how to improve the pollen isolation protocol, in order to obtain the most reliable metabolic profile from pollen tissue.

QTL mapping of insect resistance components of Solanum galapagense.

KEY MESSAGE: QTLs for insect resistance parameters, trichome type IV development, and more than 200 non-volatile metabolites, including 76 acyl sugars, all co-locate at the end of Chromosome 2 of Solanum galapagense. Host plant resistance is gaining importance as more and more insecticides are being banned due to environmental concerns. In tomato, resistance towards insects is found in wild relatives and has been attributed to the presence of glandular trichomes and their specific phytochemical composition. In this paper, we describe the results from a large-scale QTL mapping of data from whitefly resistance tests, trichome phenotyping and a comprehensive metabolomics analysis in a recombinant inbred line population derived from a cross between the cultivated Solanum lycopersicum and the wild relative S. galapagense, which is resistant to a range of pest insects. One major QTL (Wf-1) was found to govern the resistance against two different whitefly species. This QTL co-localizes with QTLs for the presence of trichomes type IV and V, as well as all 76 acyl sugars detected and about 150 other non-volatile phytochemicals, including methyl esters of the flavonols myricetin and quercetin. Based on these results, we hypothesize that Wf-1 is regulating the formation of glandular trichome type IV on the leaf epidermis, enabling the production and accumulation of bioactive metabolites in this type of trichomes.

Metabolomics of Thrips Resistance in Pepper (Capsicum spp.) Reveals Monomer and Dimer Acyclic Diterpene Glycosides as Potential Chemical Defenses.

The development of pesticide resistance in insects and recent bans on pesticides call for the identification of natural sources of resistance in crops. Here, we used natural variation in pepper (Capsicum spp.) resistance combined with an untargeted metabolomics approach to detect secondary metabolites related to thrips (Frankliniella occidentalis) resistance. Using leaf disc choice assays, we tested 11 Capsicum accessions of C. annuum and C. chinense in both vegetative and flowering stages for thrips resistance. Metabolites in the leaves of these 11 accessions were analyzed using LC-MS based untargeted metabolomics. The choice assays showed significant differences among the accessions in thrips feeding damage. The level of resistance depended on plant developmental stage. Metabolomics analyses showed differences in metabolomes among the Capsicum species and plant developmental stages. Moreover, metabolomic profiles of resistant and susceptible accessions differed. Monomer and dimer acyclic diterpene glycosides (capsianosides) were pinpointed as metabolites that were related to thrips resistance. Sucrose and malonylated flavone glycosides were related to susceptibility. To our knowledge, this is the first time that dimer capsianosides of pepper have been linked to insect resistance. Our results show the potential of untargeted metabolomics as a tool for discovering metabolites that are important in plant - insect interactions.

Metabolite variation in the lettuce gene pool: towards healthier crop varieties and food.

INTRODUCTION: Lettuce (Lactuca sativa L.) is generally not specifically acknowledged for its taste and nutritional value, while its cultivation suffers from limited resistance against several pests and diseases. Such key traits are known to be largely dependent on the ability of varieties to produce specific phytochemicals. OBJECTIVES: We aimed to identify promising genetic resources for the improvement of phytochemical composition of lettuce varieties. METHODS: Phytochemical variation was investigated using 150 Lactuca genebank accessions, comprising a core set of the lettuce gene pool, and resulting data were related to available phenotypic information. RESULTS: A hierarchical cluster analysis of the variation in relative abundance of 2026 phytochemicals, revealed by untargeted metabolic profiling, strongly resembled the known lettuce gene pool structure, indicating that the observed variation was to a large extent genetically determined. Many phytochemicals appeared species-specific, of which several are generally related to traits that are associated with plant health or nutritional value. For a large number of phytochemicals the relative abundance was either positively or negatively correlated with available phenotypic data on resistances against pests and diseases, indicating their potential role in plant resistance. Particularly the more primitive lettuces and the closely related wild relatives showed high levels of (poly)phenols and vitamin C, thus representing potential genetic resources for improving nutritional traits in modern crop types. CONCLUSION: Our large-scale analysis of phytochemical variation is unprecedented in lettuce and demonstrated the ample availability of suitable genetic resources for the development of improved lettuce varieties with higher nutritional quality and more sustainable production.

Spatially Resolved Plant Metabolomics: Some Potentials and Limitations of Laser-Ablation Electrospray Ionization Mass Spectrometry Metabolite Imaging.

Laser-ablation electrospray ionization (LAESI)-mass spectrometry imaging has been applied to contrasting plant organs to assess its potential as a procedure for performing in vivo metabolomics in plants. In a proof-of-concept experiment, purple/white segmented Phalaenopsis spp. petals were first analyzed using standard liquid chromatography-mass spectrometry analyses of separate extracts made specifically from the purple and white regions. Discriminatory compounds were defined and putatively annotated. LAESI analyses were then performed on living tissues, and these metabolites were then relocalized within the LAESI-generated data sets of similar tissues. Maps were made to illustrate their locations across the petals. Results revealed that, as expected, anthocyanins always mapped to the purple regions. Certain other (nonvisible) polyphenols were observed to colocalize with the anthocyanins, whereas others were found specifically within the white tissues. In a contrasting example, control and Cladosporium fulvum-infected tomato (Solanum lycopersicum) leaves were subjected to the same procedures, and it could be observed that the alkaloid tomatine has clear heterogeneous distribution across the tomato leaf lamina. Furthermore, LAESI analyses revealed perturbations in alkaloid content following pathogen infection. These results show the clear potential of LAESI-based imaging approaches as a convenient and rapid way to perform metabolomics analyses on living tissues. However, a range of limitations and factors have also been identified that must be taken into consideration when interpreting LAESI-derived data. Such aspects deserve further evaluation before this approach can be applied in a routine manner.

Non-smoky glycosyltransferase1 prevents the release of smoky aroma from tomato fruit.

Phenylpropanoid volatiles are responsible for the key tomato fruit (Solanum lycopersicum) aroma attribute termed "smoky." Release of these volatiles from their glycosylated precursors, rather than their biosynthesis, is the major determinant of smoky aroma in cultivated tomato. using a combinatorial omics approach, we identified the non-smoky glycosyltransferase1 (NSGT1) gene. Expression of NSGT1 is induced during fruit ripening, and the encoded enzyme converts the cleavable diglycosides of the smoky-related phenylpropanoid volatiles into noncleavable triglycosides, thereby preventing their deglycosylation and release from tomato fruit upon tissue disruption. In an nsgt1/nsgt1 background, further glycosylation of phenylpropanoid volatile diglycosides does not occur, thereby enabling their cleavage and the release of corresponding volatiles. Using reverse genetics approaches, the NSGT1-mediated glycosylation was shown to be the molecular mechanism underlying the major quantitative trait locus for smoky aroma. Sensory trials with transgenic fruits, in which the inactive nsgt1 was complemented with the functional NSGT1, showed a significant and perceivable reduction in smoky aroma. NSGT1 may be used in a precision breeding strategy toward development of tomato fruits with distinct flavor phenotypes.

Rapid and sustained systemic circulation of conjugated gut microbial catabolites after single-dose black tea extract consumption.

Gut microbial catabolites of black tea polyphenols (BTPs) have been proposed to exert beneficial cardiovascular bioactivity. This hypothesis is difficult to verify because the conjugation patterns and pharmacokinetics of these catabolites are largely unknown. The objective of our study was to identify, quantify, and assess the pharmacokinetics of conjugated BTP metabolites in plasma of healthy humans by means of an a priori untargeted LC-MS-based metabolomics approach. In a randomized, open, placebo-controlled, crossover study, 12 healthy men consumed a single bolus of black tea extract (BTE) or a placebo. The relative and, in several cases, absolute concentrations of a wide range of metabolites were determined using U(H)PLC-LTQ-Orbitrap-FTMS. Following BTE consumption, a kinetic response in plasma was observed for 59 BTP metabolites, 11 of these in a quantitative manner. Conjugated and unconjugated catechins appeared in plasma without delay, at 2-4 h, followed by a range of microbial catabolites. Interindividual variation in response was greater for gut microbial catabolites than for directly absorbed BTPs. The rapid and sustained circulation of conjugated catabolites suggests that these compounds may be particularly relevant to proposed health benefits of BTE. Their presence and effects may depend on individual variation in catabolic capacity of the gut microbiota.

In silico prediction and automatic LC-MS(n) annotation of green tea metabolites in urine.

The colonic breakdown and human biotransformation of small molecules present in food can give rise to a large variety of potentially bioactive metabolites in the human body. However, the absence of reference data for many of these components limits their identification in complex biological samples, such as plasma and urine. We present an in silico workflow for automatic chemical annotation of metabolite profiling data from liquid chromatography coupled with multistage accurate mass spectrometry (LC-MS(n)), which we used to systematically screen for the presence of tea-derived metabolites in human urine samples after green tea consumption. Reaction rules for intestinal degradation and human biotransformation were systematically applied to chemical structures of 75 green tea components, resulting in a virtual library of 27,245 potential metabolites. All matching precursor ions in the urine LC-MS(n) data sets, as well as the corresponding fragment ions, were automatically annotated by in silico generated (sub)structures. The results were evaluated based on 74 previously identified urinary metabolites and lead to the putative identification of 26 additional green tea-derived metabolites. A total of 77% of all annotated metabolites were not present in the Pubchem database, demonstrating the benefit of in silico metabolite prediction for the automatic annotation of yet unknown metabolites in LC-MS(n) data from nutritional metabolite profiling experiments.

System-wide hypersensitive response-associated transcriptome and metabolome reprogramming in tomato.

The hypersensitive response (HR) is considered to be the hallmark of the resistance response of plants to pathogens. To study HR-associated transcriptome and metabolome reprogramming in tomato (Solanum lycopersicum), we used plants that express both a resistance gene to Cladosporium fulvum and the matching avirulence gene of this pathogen. In these plants, massive reprogramming occurred, and we found that the HR and associated processes are highly energy demanding. Ubiquitin-dependent protein degradation, hydrolysis of sugars, and lipid catabolism are used as alternative sources of amino acids, energy, and carbon skeletons, respectively. We observed strong accumulation of secondary metabolites, such as hydroxycinnamic acid amides. Coregulated expression of WRKY transcription factors and genes known to be involved in the HR, in addition to a strong enrichment of the W-box WRKY-binding motif in the promoter sequences of the coregulated genes, point to WRKYs as the most prominent orchestrators of the HR. Our study has revealed several novel HR-related genes, and reverse genetics tools will allow us to understand the role of each individual component in the HR.

Identification of genes in the phenylalanine metabolic pathway by ectopic expression of a MYB transcription factor in tomato fruit.

Altering expression of transcription factors can be an effective means to coordinately modulate entire metabolic pathways in plants. It can also provide useful information concerning the identities of genes that constitute metabolic networks. Here, we used ectopic expression of a MYB transcription factor, Petunia hybrida ODORANT1, to alter Phe and phenylpropanoid metabolism in tomato (Solanum lycopersicum) fruits. Despite the importance of Phe and phenylpropanoids to plant and human health, the pathway for Phe synthesis has not been unambiguously determined. Microarray analysis of ripening fruits from transgenic and control plants permitted identification of a suite of coregulated genes involved in synthesis and further metabolism of Phe. The pattern of coregulated gene expression facilitated discovery of the tomato gene encoding prephenate aminotransferase, which converts prephenate to arogenate. The expression and biochemical data establish an arogenate pathway for Phe synthesis in tomato fruits. Metabolic profiling and ¹³C flux analysis of ripe fruits further revealed large increases in the levels of a specific subset of phenylpropanoid compounds. However, while increased levels of these human nutrition-related phenylpropanoids may be desirable, there were no increases in levels of Phe-derived flavor volatiles.

Differences in insect resistance between tomato species endemic to the Galapagos Islands.

BACKGROUND: The Galapagos Islands constitute a highly diverse ecosystem and a unique source of variation in the form of endemic species. There are two endemic tomato species, Solanum galapagense and S. cheesmaniae and two introduced tomato species, S. pimpinellifolium and S. lycopersicum. Morphologically the two endemic tomato species of the Galapagos Islands are clearly distinct, but molecular marker analysis showed no clear separation. Tomatoes on the Galapagos are affected by both native and exotic herbivores. Bemisia tabaci is an important introduced insect species that feeds on a wide range of plants. In this article, we address the question whether the differentiation between S. galapagense and S. cheesmaniae may be related to differences in susceptibility towards phloem-feeders and used B. tabaci as a model to evaluate this. RESULTS: We have characterized 12 accessions of S. galapagense, 22 of S. cheesmaniae, and one of S. lycopersicum as reference for whitefly resistance using no-choice experiments. Whitefly resistance was found in S. galapagense only and was associated with the presence of relatively high levels of acyl sugars and the presence of glandular trichomes of type I and IV. Genetic fingerprinting using 3316 SNP markers did not show a clear differentiation between the two endemic species. Acyl sugar accumulation as well as the climatic and geographical conditions at the collection sites of the accessions did not follow the morphological species boundaries. CONCLUSION: Our results suggest that S. galapagense and S. cheesmaniae might be morphotypes rather than two species and that their co-existence is likely the result of selective pressure.

Automatic chemical structure annotation of an LC-MS(n) based metabolic profile from green tea.

Liquid chromatography coupled with multistage accurate mass spectrometry (LC-MS(n)) can generate comprehensive spectral information of metabolites in crude extracts. To support structural characterization of the many metabolites present in such complex samples, we present a novel method ( http://www.emetabolomics.org/magma ) to automatically process and annotate the LC-MS(n) data sets on the basis of candidate molecules from chemical databases, such as PubChem or the Human Metabolite Database. Multistage MS(n) spectral data is automatically annotated with hierarchical trees of in silico generated substructures of candidate molecules to explain the observed fragment ions and alternative candidates are ranked on the basis of the calculated matching score. We tested this method on an untargeted LC-MS(n) (n ≤ 3) data set of a green tea extract, generated on an LC-LTQ/Orbitrap hybrid MS system. For the 623 spectral trees obtained in a single LC-MS(n) run, a total of 116,240 candidate molecules with monoisotopic masses matching within 5 ppm mass accuracy were retrieved from the PubChem database, ranging from 4 to 1327 candidates per molecular ion. The matching scores were used to rank the candidate molecules for each LC-MS(n) component. The median and third quartile fractional ranks for 85 previously identified tea compounds were 3.5 and 7.5, respectively. The substructure annotations and rankings provided detailed structural information of the detected components, beyond annotation with elemental formula only. Twenty-four additional components were putatively identified by expert interpretation of the automatically annotated data set, illustrating the potential to support systematic and untargeted metabolite identification.

Detoxification of α-tomatine by Cladosporium fulvum is required for full virulence on tomato.

· α-Tomatine is an antifungal glycoalkaloid that provides basal defense to tomato (Solanum lycopersicum). However, tomato pathogens overcome this basal defense barrier by the secretion of tomatinases that degrade α-tomatine into the less fungitoxic compounds ß-tomatine and tomatidine. Although pathogenic on tomato, it has been reported that the biotrophic fungus Cladosporium fulvum is unable to detoxify α-tomatine. · Here, we present a functional analysis of the glycosyl hydrolase (GH10), CfTom1, which is orthologous to fungal tomatinases. · We show that C. fulvum hydrolyzes α-tomatine into tomatidine in vitro and during the infection of tomato, which is fully attributed to the activity of CfTom1, as shown by the heterologous expression of this enzyme in tomato. Accordingly, ∆cftom1 mutants of C. fulvum are more sensitive to α-tomatine and are less virulent than the wild-type fungus on tomato. · Although α-tomatine is thought to be localized in the vacuole, we show that it is also present in the apoplast, where it is hydrolyzed by CfTom1 on infection. The accumulation of tomatidine during infection appears to be toxic to tomato cells and does not suppress defense responses, as suggested previously. Altogether, our results show that CfTom1 is responsible for the detoxification of α-tomatine by C. fulvum, and is required for full virulence of this fungus on tomato.