Essentials in the acquisition, interpretation, and reporting of plant metabolite profiles.

Plant metabolite profiling reveals the diversity of secondary or specialized metabolites in the plant kingdom with its hundreds of thousands of species. Specialized plant metabolites constitute a vast class of chemicals posing significant challenges in analytical chemistry. In order to be of maximum scientific relevance, reports dealing with these compounds and their source species must be transparent, make use of standards and reference materials, and be based on correctly and traceably identified plant material. Essential aspects in qualitative plant metabolite profiling include: (i) critical review of previous literature and a reasoned sampling strategy; (ii) transparent plant sampling with wild material documented by vouchers in public herbaria and, optimally, seed banks; (iii) if possible, inclusion of generally available reference plant material; (iv) transparent, documented state-of-the art chemical analysis, ideally including chemical reference standards; (v) testing for artefacts during preparative extraction and isolation, using gentle analytical methods; (vi) careful chemical data interpretation, avoiding over- and misinterpretation and taking into account phytochemical complexity when assigning identification confidence levels, and (vii) taking all previous scientific knowledge into account in reporting the scientific data. From the current stage of the phytochemical literature, selected comments and suggestions are given. In the past, proposed revisions of botanical taxonomy were sometimes based on metabolite profiles, but this approach ("chemosystematics" or "chemotaxonomy") is outdated due to the advent of DNA sequence-based phylogenies. In contrast, systematic comparisons of plant metabolite profiles in a known phylogenetic framework remain relevant. This approach, known as chemophenetics, allows characterizing species and clades based on their array of specialized metabolites, aids in deducing the evolution of biosynthetic pathways and coevolution, and can serve in identifying new sources of rare and economically interesting natural products.

Phytoalexins of the crucifer Barbarea vulgaris: Structural profile and correlation with glucosinolate turnover.

Phytoalexins are antimicrobial plant metabolites elicited by microbial attack or abiotic stress. We investigated phytoalexin profiles after foliar abiotic elicitation in the crucifer Barbarea vulgaris and interactions with the glucosinolate-myrosinase system. The treatment for abiotic elicitation was a foliar spray with CuCl2 solution, a usual eliciting agent, and three independent experiments were carried out. Two genotypes of B. vulgaris (G-type and P-type) accumulated the same three major phytoalexins in rosette leaves after treatment: phenyl-containing nasturlexin D and indole-containing cyclonasturlexin and cyclobrassinin. Phytoalexin levels were investigated daily by UHPLC-QToF MS and tended to differ among plant types and individual phytoalexins. In roots, phytoalexins were low or not detected. In treated leaves, typical total phytoalexin levels were in the range 1-10 nmol/g fresh wt. during three days after treatment while typical total glucosinolate (GSL) levels were three orders of magnitude higher. Levels of some minor GSLs responded to the treatment: phenethylGSL (PE) and 4-substituted indole GSLs. Levels of PE, a suggested nasturlexin D precursor, were lower in treated plants than controls. Another suggested precursor GSL, 3-hydroxyPE, was not detected, suggesting PE hydrolysis to be a key biosynthetic step. Levels of 4-substituted indole GSLs differed markedly between treated and control plants in most experiments, but not in a consistent way. The dominant GSLs, glucobarbarins, are not believed to be phytoalexin precursors. We observed statistically significant linear correlations between total major phytoalexins and the glucobarbarin products barbarin and resedine, suggesting that GSL turnover for phytoalexin biosynthesis was unspecific. In contrast, we did not find correlations between total major phytoalexins and raphanusamic acid or total glucobarbarins and barbarin. In conclusion, two groups of phytoalexins were detected in B. vulgaris, apparently derived from the GSLs PE and indol-3-ylmethylGSL. Phytoalexin biosynthesis was accompanied by depletion of the precursor PE and by turnover of major non-precursor GSLs to resedine. This work paves the way for identifying and characterizing genes and enzymes in the biosyntheses of phytoalexins and resedine.

Ancient Biosyntheses in an Oil Crop: Glucosinolate Profiles in Limnanthes alba and Its Relatives (Limnanthaceae, Brassicales).

The glucosinolate (GSL) profiles of four Limnanthaceae species, including the oil crop Limnanthes alba (meadowfoam), were investigated by an ultrahigh-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-QToF-MS/MS) analysis of desulfoGSLs after desulfation of native GSLs, supplemented by NMR of desulfated 2-hydroxy-2-methylpropylGSL and 3-methoxybenzylGSL. Leaves, roots, and seeds were investigated, providing an overview of biosynthetic capabilities in the genera Floerkea and Limnanthes. Methoxyl groups on benzylGSLs were in meta but not para positions; two 3,5-disubstituted benzylGSLs are tentatively proposed. 2-Hydroxy-2-methylpropylGSL was accompanied by an isomer that was not a previously reported GSL. The combined GSL profile of the family included GSLs derived from valine, leucine, isoleucine, phenylalanine, and tyrosine, and possibly methionine and tryptophan. Substituted indole GSLs and GSLs derived from chain-elongated amino acids or alanine were searched for but not detected. Hypothetic glycosides of GSLs were detected at low levels. Based on biochemical interpretation, we suggest biosynthetic schemes and gene families (CYP79C, GSOH) relevant for tailoring GSL profiles in Limnanthes crops.

Development and application of a virus-induced gene silencing protocol for the study of gene function in narrow-leafed lupin.

BACKGROUND: Lupins are promising protein crops with an increasing amount of genomic and transcriptomic resources. The new resources facilitate the in silico identification of candidate genes controlling important agronomic traits. However, a major bottleneck for lupin research and crop improvement is the in planta characterization of gene function. Here, we present an efficient protocol for virus-induced gene silencing (VIGS) to down-regulate endogenous genes in narrow-leafed lupin (NLL) using the apple latent spherical virus (ALSV). RESULTS: We identified ALSV as an appropriate VIGS vector able to infect NLL without causing a discernible phenotype. We created improved ALSV vectors to allow for efficient cloning of gene fragments into the viral genome and for easier viral propagation via agroinfiltration of Nicotiana benthamiana. Using this system, we silenced the visual marker gene phytoene desaturase (PDS), which resulted in systemic, homogenous silencing as indicated by bleaching of newly produced tissues. Furthermore, by silencing lysine decarboxylase (LaLDC)-a gene likely to be involved in toxic alkaloid biosynthesis-we demonstrate the applicability of our VIGS method to silence a target gene alone or alongside PDS in a 'PDS co-silencing' approach. The co-silencing approach allows the visual identification of tissues where silencing is actively occurring, which eases tissue harvesting and downstream analysis, and is useful where the trait under study is not affected by PDS silencing. Silencing LaLDC resulted in a ~ 61% or ~ 67% decrease in transcript level, depending on whether LaLDC was silenced alone or alongside PDS. Overall, the silencing of LaLDC resulted in reduced alkaloid levels, providing direct evidence of its involvement in alkaloid biosynthesis in NLL. CONCLUSIONS: We provide a rapid and efficient VIGS method for validating gene function in NLL. This will accelerate the research and improvement of this underutilized crop.

Glucosinolate profiles and phylogeny in Barbarea compared to other tribe Cardamineae (Brassicaceae) and Reseda (Resedaceae), based on a library of ion trap HPLC-MS/MS data of reference desulfoglucosinolates.

A library of ion trap MS2 spectra and HPLC retention times reported here allowed distinction in plants of at least 70 known glucosinolates (GSLs) and some additional proposed GSLs. We determined GSL profiles of selected members of the tribe Cardamineae (Brassicaceae) as well as Reseda (Resedaceae) used as outgroup in evolutionary studies. We included several accessions of each species and a range of organs, and paid attention to minor peaks and GSLs not detected. In this way, we obtained GSL profiles of Barbarea australis, Barbarea grayi, Planodes virginica selected for its apparent intermediacy between Barbarea and the remaining tribe and family, and Rorippa sylvestris and Nasturtium officinale, for which the presence of acyl derivatives of GSLs was previously untested. We also screened Armoracia rusticana, with a remarkably diverse GSL profile, the emerging model species Cardamine hirsuta, for which we discovered a GSL polymorphism, and Reseda luteola and Reseda odorata. The potential for aliphatic GSL biosynthesis in Barbarea vulgaris was of interest, and we subjected P-type and G-type B. vulgaris to several induction regimes in an attempt to induce aliphatic GSL. However, aliphatic GSLs were not detected in any of the B. vulgaris types. We characterized the investigated chemotypes phylogenetically, based on nuclear rDNA internal transcribed spacer (ITS) sequences, in order to understand their relation to the species B. vulgaris in general, and found them to be representative of the species as it occurs in Europe, as far as documented in available ITS-sequence repositories. In short, we provide GSL profiles of a wide variety of tribe Cardamineae plants and conclude aliphatic GSLs to be absent or below our limit of detection in two major evolutionary lines of B. vulgaris. Concerning analytical chemistry, we conclude that availability of authentic reference compounds or reference materials is critical for reliable GSL analysis and characterize two publicly available reference materials: seeds of P. virginica and N. officinale.

Comparison of glucosinolate diversity in the crucifer tribe Cardamineae and the remaining order Brassicales highlights repetitive evolutionary loss and gain of biosynthetic steps.

We review glucosinolate (GSL) diversity and analyze phylogeny in the crucifer tribe Cardamineae as well as selected species from Brassicaceae (tribe Brassiceae) and Resedaceae. Some GSLs occur widely, while there is a scattered distribution of many less common GSLs, tentatively sorted into three classes: ancient, intermediate and more recently evolved. The number of conclusively identified GSLs in the tribe (53 GSLs) constitute 60% of all GSLs known with certainty from any plant (89 GSLs) and apparently unique GSLs in the tribe constitute 10 of those GSLs conclusively identified (19%). Intraspecific, qualitative GSL polymorphism is known from at least four species in the tribe. The most ancient GSL biosynthesis in Brassicales probably involved biosynthesis from Phe, Val, Leu, Ile and possibly Trp, and hydroxylation at the ß-position. From a broad comparison of families in Brassicales and tribes in Brassicaceae, we estimate that a common ancestor of the tribe Cardamineae and the family Brassicaceae exhibited GSL biosynthesis from Phe, Val, Ile, Leu, possibly Tyr, Trp and homoPhe (ancient GSLs), as well as homologs of Met and possibly homoIle (intermediate age GSLs). From the comparison of phylogeny and GSL diversity, we also suggest that hydroxylation and subsequent methylation of indole GSLs and usual modifications of Met-derived GSLs (formation of sulfinyls, sulfonyls and alkenyls) occur due to conserved biochemical mechanisms and was present in a common ancestor of the family. Apparent loss of homologs of Met as biosynthetic precursors was deduced in the entire genus Barbarea and was frequent in Cardamine (e.g. C. pratensis, C. diphylla, C. concatenata, possibly C. amara). The loss was often associated with appearance of significant levels of unique or rare GSLs as well as recapitulation of ancient types of GSLs. Biosynthetic traits interpreted as de novo evolution included hydroxylation at rare positions, acylation at the thioglucose and use of dihomoIle and possibly homoIle as biosynthetic precursors. Biochemical aspects of the deduced evolution are discussed and testable hypotheses proposed. Biosyntheses from Val, Leu, Ile, Phe, Trp, homoPhe and homologs of Met are increasingly well understood, while GSL biosynthesis from mono- and dihomoIle is poorly understood. Overall, interpretation of known diversity suggests that evolution of GSL biosynthesis often seems to recapitulate ancient biosynthesis. In contrast, unprecedented GSL biosynthetic innovation seems to be rare.

Engineering and optimization of the 2-phenylethylglucosinolate production in Nicotiana benthamiana by combining biosynthetic genes from Barbarea vulgaris and Arabidopsis thaliana.

2-Phenylethylglucosinolate (2PE) derived from homophenylalanine is present in plants of the Brassicales order as a defense compound. It is associated with multiple biological properties, including deterrent effects on pests and antimicrobial and health-promoting functions, due to its hydrolysis product 2-phenylethyl isothiocyanate, which confers 2PE as a potential application in agriculture and industry. In this study, we characterized the putative key genes for 2PE biosynthesis from Barbarea vulgaris W.T. Aiton and demonstrated the feasibility of engineering 2PE production in Nicotiana benthamiana Domin. We used different combinations of genes from B. vulgaris and Arabidopsis thaliana (L.) Heynh. to demonstrate that: (i) BvBCAT4 performed more efficiently than AtBCAT4 in biosynthesis of both homophenylalanine and dihomomethionine; (ii) MAM1 enzymes were critical for the chain-elongated profile, while CYP79F enzymes accepted both chain-elongated methionine and homophenylalanine; (iii) aliphatic but not aromatic core structure pathway catalyzed the 2PE biosynthesis; (iv) a chimeric pathway containing BvBCAT4, BvMAM1, AtIPMI and AtIPMDH1 resulted in a two-fold increase in 2PE production compared with the B. vulgaris-specific chain elongation pathway; and (v) profiles of chain-elongated products and glucosinolates partially mirrored the profiles in the gene donor plant, but were wider in N. benthamiana than in the native plants. Our study provides a strategy to produce the important homophenylalanine and 2PE in a heterologous host. Furthermore, chimeric engineering of the complex 2PE biosynthetic pathway enabled detailed understanding of catalytic properties of individual enzymes - a prerequisite for understanding biochemical evolution. The new-to-nature gene combinations have the potential for application in biotechnological and plant breeding.

Characterization of Arabidopsis CYP79C1 and CYP79C2 by Glucosinolate Pathway Engineering in Nicotiana benthamiana Shows Substrate Specificity Toward a Range of Aliphatic and Aromatic Amino Acids.

Glucosinolates (GLSs) are amino acid-derived defense compounds characteristic of the Brassicales order. Cytochromes P450s of the CYP79 family are the entry point into the biosynthetic pathway of the GLS core structure and catalyze the conversion of amino acids to oximes. In Arabidopsis thaliana, CYP79A2, CYP79B2, CYP79B3, CYP79F1, and CYP79F2 have been functionally characterized and are responsible for the biosynthesis of phenylalanine-, tryptophan-, and methionine-derived GLSs, respectively. However, the substrate(s) for CYP79C1 and CYP79C2 were unknown. Here, we investigated the function of CYP79C1 and CYP79C2 by transiently co-expressing the genes together with three sets of remaining genes required for GLS biosynthesis in Nicotiana benthamiana. Co-expression of CYP79C2 with either the aliphatic or aromatic core structure pathways resulted in the production of primarily leucine-derived 2-methylpropyl GLS and phenylalanine-derived benzyl GLS, along with minor amounts of GLSs from isoleucine, tryptophan, and tyrosine. Co-expression of CYP79C1 displayed minor amounts of GLSs from valine, leucine, isoleucine, and phenylalanine with the aliphatic core structure pathway, and similar GLS profile (except the GLS from valine) with the aromatic core structure pathway. Additionally, we co-expressed CYP79C1 and CYP79C2 with the chain elongation and aliphatic core structure pathways. With the chain elongation pathway, CYP79C2 still mainly produced 2-methylpropyl GLS derived from leucine, accompanied by GLSs derived from isoleucine and from chain-elongated mono- and dihomoleucine, but not from phenylalanine. However, co-expression of CYP79C1 only resulted in GLSs derived from chain-elongated amino acid substrates, dihomoleucine and dihomomethionine, when the chain elongation pathway was present. This shows that CYP79 activity depends on the specific pathways co-expressed and availability of amino acid precursors, and that description of GLS core structure pathways as "aliphatic" and "aromatic" pathways is not suitable, especially in an engineering context. This is the first characterization of members of the CYP79C family. Co-expression of CYP79 enzymes with engineered GLS pathways in N. benthamiana is a valuable tool for simultaneous testing of substrate specificity against multiple amino acids.

Glucosinolate structural diversity, identification, chemical synthesis and metabolism in plants.

The glucosinolates (GSLs) is a well-defined group of plant metabolites characterized by having an S-ß-d-glucopyrano unit anomerically connected to an O-sulfated (Z)-thiohydroximate function. After enzymatic hydrolysis, the sulfated aglucone can undergo rearrangement to an isothiocyanate, or form a nitrile or other products. The number of GSLs known from plants, satisfactorily characterized by modern spectroscopic methods (NMR and MS) by mid-2018, is 88. In addition, a group of partially characterized structures with highly variable evidence counts for approximately a further 49. This means that the total number of characterized GSLs from plants is somewhere between 88 and 137. The diversity of GSLs in plants is critically reviewed here, resulting in significant discrepancies with previous reviews. In general, the well-characterized GSLs show resemblance to C-skeletons of the amino acids Ala, Val, Leu, Trp, Ile, Phe/Tyr and Met, or to homologs of Ile, Phe/Tyr or Met. Insufficiently characterized, still hypothetic GSLs include straight-chain alkyl GSLs and chain-elongated GSLs derived from Leu. Additional reports (since 2011) of insufficiently characterized GSLs are reviewed. Usually the crucial missing information is correctly interpreted NMR, which is the most effective tool for GSL identification. Hence, modern use of NMR for GSL identification is also reviewed and exemplified. Apart from isolation, GSLs may be obtained by organic synthesis, allowing isotopically labeled GSLs and any kind of side chain. Enzymatic turnover of GSLs in plants depends on a considerable number of enzymes and other protein factors and furthermore depends on GSL structure. Identification of GSLs must be presented transparently and live up to standard requirements in natural product chemistry. Unfortunately, many recent reports fail in these respects, including reports based on chromatography hyphenated to MS. In particular, the possibility of isomers and isobaric structures is frequently ignored. Recent reports are re-evaluated and interpreted as evidence of the existence of "isoGSLs", i.e. non-GSL isomers of GSLs in plants. For GSL analysis, also with MS-detection, we stress the importance of using authentic standards.

A high-density genetic map and QTL mapping of leaf traits and glucosinolates in Barbarea vulgaris.

BACKGROUND: Barbarea vulgaris is a wild cruciferous plant and include two distinct types: the G- and P-types named after their glabrous and pubescent leaves, respectively. The types differ significantly in resistance to a range of insects and diseases as well as glucosinolates and other chemical defenses. A high-density linkage map was needed for further progress to be made in the molecular research of this plant. RESULTS: We performed restriction site-associated DNA sequencing (RAD-Seq) on an F2 population generated from G- and P-type B. vulgaris. A total of 1545 SNP markers were mapped and ordered in eight linkage groups, which represents the highest density linkage map to date for the crucifer tribe Cardamineae. A total of 722 previously published genome contigs (50.2 Mb, 30% of the total length) can be anchored to this high density genetic map, an improvement compared to a previously published map (431 anchored contigs, 38.7 Mb, 23% of the assembly genome). Most of these (572 contigs, 31.2 Mb) were newly anchored to the map, representing a significant improvement. On the basis of the present high-density genetic map, 37 QTL were detected for eleven traits, each QTL explaining 2.9-71.3% of the phenotype variation. QTL of glucosinolates, leaf size and color traits were in most cases overlapping, possibly implying a functional connection. CONCLUSIONS: This high-density linkage map and the QTL obtained in this study will be useful for further understanding of the genetic of the B. vulgaris and molecular basis of these traits, many of which are shared in the related crop watercress.

The Role of the Glucosinolate-Myrosinase System in Mediating Greater Resistance of Barbarea verna than B. vulgaris to Mamestra brassicae Larvae.

We investigated the influences of two structurally similar glucosinolates, phenethylglucosinolate (gluconasturtiin, NAS) and its (S)-2-hydroxyl derivative glucobarbarin (BAR), as well as their hydrolysis products on larvae of the generalist Mamestra brassicae (Lepidoptera: Noctuidae). Previous results suggested a higher defensive activity of BAR than NAS based on resistance toward M. brassicae larvae of natural plant genotypes of Barbarea vulgaris R. Br. (Brassicaceae) dominated by BAR. In the present study, the hypothesis of a higher defensive activity of BAR than NAS was tested by comparing two Barbarea species similarly dominated either by BAR or by NAS and by testing effects of isolated BAR and NAS on larval survival and feeding preferences. Larvae reared on leaf disks of B. verna (Mill.) Asch. had a lower survival than those reared on B. vulgaris P- and G-chemotypes. Leaves of B. verna were dominated by NAS, whereas B. vulgaris chemotypes were dominated by BAR or its epimer. In addition, B. verna leaves showed a threefold higher activity of the glucosinolate-activating myrosinase enzymes. The main product of NAS from breakdown by endogenous enzymes including myrosinases ("autolysis") in B. verna leaves was phenethyl isothiocyanate, while the main products of BAR in autolyzed B. vulgaris leaves were a cyclized isothiocyanate product, namely an oxazolidine-2-thione, and a downstream metabolite, an oxazolidin-2-one. The glucosinolates BAR and NAS were isolated and offered to larvae on disks of cabbage. Both glucosinolates exerted similar negative effects on larval survival but effects of NAS tended to be more detrimental. Low concentrations of BAR, but not of NAS, stimulated larval feeding, whereas high BAR concentrations acted deterrent. NAS only tended to be deterrent at the highest concentration, but the difference was not significant. Recoveries of NAS and BAR on cabbage leaf disks were similar, and when hydrolyzed by mechanical leaf damage, the same isothiocyanate-type products as in Barbarea plants were formed with further conversion of BAR to cyclic products, (R)-5-phenyloxazolidine-2-thione [(R)-barbarin] and (R)-5-phenyloxazolidin-2-one [(R)-resedine]. We conclude that a previously proposed generally higher defensive activity of BAR than NAS to M. brassicae larvae could not be confirmed. Indeed, the higher resistance of NAS-containing B. verna plants may be due to a combined effect of rather high concentrations of NAS and a relatively high myrosinase activity or other plant traits not investigated yet.

Glucosinolate turnover in Brassicales species to an oxazolidin-2-one, formed via the 2-thione and without formation of thioamide.

Glucosinolates are found in plants of the order Brassicales and hydrolyzed to different breakdown products, particularly after tissue damage. In Barbarea vulgaris R.Br. (Brassicaceae), the dominant glucosinolate in the investigated "G-type" is glucobarbarin, (S)-2-hydroxy-2-phenylethylglucosinolate. Formation of the nitrile from glucobarbarin was observed in vitro, while a previously suggested thioamide (synonym thionamide) was not confirmed. Resedine (5-phenyl-1,3-oxazolidin-2-one) was detected after glucobarbarin hydrolysis in crushed B. vulgaris leaves and siliques, but not in intact parts. The abundance increased for several hours after completion of hydrolysis. The corresponding 1,3-oxazolidine-2-thione (OAT), with the common name barbarin, was also formed, and appeared to be the precursor of resedine. Addition of each of two non-endogenous OATs, (S)-5-ethyl-5-methylOAT and (R)-5-vinylOAT (R-goitrin), to a leaf homogenate resulted in formation of the corresponding 1,3-oxazolidin-2-ones (OAOs), confirming the metabolic connection of OAT to OAO. Formation of OAOs was inhibited by prior brief heating of the homogenate, suggesting enzyme involvement. We suggest the conversion of OATs to OAOs to be catalyzed by an enzyme ("oxazolidinethionase") responsible for turnover of OAT formed in intact plants. Resedine had been reported as an alkaloid from another species - Reseda luteola L. (Resedaceae) - naturally containing the glucosinolate glucobarbarin. However, resedine was not detected in intact R. luteola plants, but formed after tissue damage. The formation of resedine in two families suggests a broad distribution of putative OATases in the Brassicales; potentially involved in glucosinolate turnover that needs myrosinase activity as the committed step. In agreement with the proposed function of OATase, several candidate genes for myrosinases in glucosinolate turnover in intact plants were discovered in the B. vulgaris genome. We also suggest that biotechnological conversion of OATs to OAOs might improve the nutritional value of Brassicales protein. HPLC-MS/MS methods for detection of these glucobarbarin products are described.

A tandem array of UDP-glycosyltransferases from the UGT73C subfamily glycosylate sapogenins, forming a spectrum of mono- and bisdesmosidic saponins.

KEY MESSAGE: This study identifies six UGT73Cs all able to glucosylate sapogenins at positions 3 and/or 28 which demonstrates that B. vulgaris has a much richer arsenal of UGTs involved in saponin biosynthesis than initially anticipated. The wild cruciferous plant Barbarea vulgaris is resistant to some insects due to accumulation of two monodesmosidic triterpenoid saponins, oleanolic acid 3-O-ß-cellobioside and hederagenin 3-O-ß-cellobioside. Insect resistance depends on the structure of the sapogenin aglycone and the glycosylation pattern. The B. vulgaris saponin profile is complex with at least 49 saponin-like metabolites, derived from eight sapogenins and including up to five monosaccharide units. Two B. vulgaris UDP-glycosyltransferases, UGT73C11 and UGT73C13, O-glucosylate sapogenins at positions 3 and 28, forming mainly 3-O-ß-D-glucosides. The aim of this study was to identify UGTs responsible for the diverse saponin oligoglycoside moieties observed in B. vulgaris. Twenty UGT genes from the insect resistant genotype were selected and heterologously expressed in Nicotiana benthamiana and/or Escherichia coli. The extracts were screened for their ability to glycosylate sapogenins (oleanolic acid, hederagenin), the hormone 24-epibrassinolide and sapogenin monoglucosides (hederagenin and oleanolic acid 3-O-ß-D-glucosides). Six UGTs from the UGT73C subfamily were able to glucosylate both sapogenins and both monoglucosides at positions 3 and/or 28. Some UGTs formed bisdesmosidic saponins efficiently. At least four UGT73C genes were localized in a tandem array with UGT73C11 and possibly UGT73C13. This organization most likely reflects duplication events followed by sub- and neofunctionalization. Indeed, signs of positive selection on several amino acid sites were identified and modelled to be localized on the UGT protein surface. This tandem array is proposed to initiate higher order bisdesmosidic glycosylation of B. vulgaris saponins, leading to the recently discovered saponin structural diversity, however, not directly to known cellobiosidic saponins.

Hydroxyl and Methoxyl Derivatives of Benzylglucosinolate in Lepidium densiflorum with Hydrolysis to Isothiocyanates and non-Isothiocyanate Products: Substitution Governs Product Type and Mass Spectral Fragmentation.

A system of benzylic glucosinolates was found and characterized in common pepperweed, Lepidium densiflorum Schrad. The major glucosinolate was the novel 4-hydroxy-3,5-dimethoxybenzylglucosinolate (3,5-dimethoxysinalbin), present at high levels in seeds, leaves, and roots. Medium-level glucosinolates were 3,4-dimethoxybenzylglucosinolate and 3,4,5-trimethoxybenzylglucosinolate. Minor glucosinolates included benzylglucosinolate, 3-hydroxy- and 3-methoxybenzylglucosinolate, 4-hydroxybenzylglucosinolate (sinalbin), the novel 4-hydroxy-3-methoxybenzylglucosinolate (3-methoxysinalbin), and indole-type glucosinolates. A biosynthetic connection is suggested. NMR, UV, and ion trap MS/MS spectral data are reported, showing contrasting MS fragmentation of p-hydroxyls and p-methoxyls. Additional investigations by GC-MS focused on glucosinolate hydrolysis products. Whereas glucosinolates generally yielded isothiocyanates, the dominating 3,5-dimethoxysinalbin with a free p-hydroxyl group produced the corresponding alcohol and syringaldehyde (4-hydroxy-3,5-dimethoxybenzaldehyde). After thermal deactivation of the endogenous myrosinase enzyme, massive accumulation of the corresponding nitrile was detected. This case study points out how non-isothiocyanate glucosinolate hydrolysis products are prevalent in nature and of interest in both plant-pathogen interactions and human health.

The genome sequence of Barbarea vulgaris facilitates the study of ecological biochemistry.

The genus Barbarea has emerged as a model for evolution and ecology of plant defense compounds, due to its unusual glucosinolate profile and production of saponins, unique to the Brassicaceae. One species, B. vulgaris, includes two 'types', G-type and P-type that differ in trichome density, and their glucosinolate and saponin profiles. A key difference is the stereochemistry of hydroxylation of their common phenethylglucosinolate backbone, leading to epimeric glucobarbarins. Here we report a draft genome sequence of the G-type, and re-sequencing of the P-type for comparison. This enables us to identify candidate genes underlying glucosinolate diversity, trichome density, and study the genetics of biochemical variation for glucosinolate and saponins. B. vulgaris is resistant to the diamondback moth, and may be exploited for "dead-end" trap cropping where glucosinolates stimulate oviposition and saponins deter larvae to the extent that they die. The B. vulgaris genome will promote the study of mechanisms in ecological biochemistry to benefit crop resistance breeding.

Methyl Transfer in Glucosinolate Biosynthesis Mediated by Indole Glucosinolate O-Methyltransferase 5.

Indole glucosinolates (IGs) are plant secondary metabolites that are derived from the amino acid tryptophan. The product of Arabidopsis (Arabidopsis thaliana) IG core biosynthesis, indol-3-ylmethyl glucosinolate (I3M), can be modified by hydroxylation and subsequent methoxylation of the indole ring in position 1 (1-IG modification) or 4 (4-IG modification). Products of the 4-IG modification pathway mediate plant-enemy interactions and are particularly important for Arabidopsis innate immunity. While CYP81Fs encoding cytochrome P450 monooxygenases and IGMTs encoding indole glucosinolate O-methyltransferases have been identified as key genes for IG modification, our knowledge about the IG modification pathways is not complete. In particular, it is unknown which enzyme is responsible for methyl transfer in the 1-IG modification pathway and whether this pathway plays a role in defense, similar to 4-IG modification. Here, we analyze two Arabidopsis transfer DNA insertion lines with targeted metabolomics. We show that biosynthesis of 1-methoxyindol-3-ylmethyl glucosinolate (1MOI3M) from I3M involves the predicted unstable intermediate 1-hydroxyindol-3-ylmethyl glucosinolate (1OHI3M) and that IGMT5, a gene with moderate similarity to previously characterized IGMTs, encodes the methyltransferase that is responsible for the conversion of 1OHI3M to 1MOI3M. Disruption of IGMT5 function increases resistance against the root-knot nematode Meloidogyne javanica and suggests a potential role for the 1-IG modification pathway in Arabidopsis belowground defense.

Glucosinolate diversity within a phylogenetic framework of the tribe Cardamineae (Brassicaceae) unraveled with HPLC-MS/MS and NMR-based analytical distinction of 70 desulfoglucosinolates.

As a basis for future investigations of evolutionary trajectories and biosynthetic mechanisms underlying variations in glucosinolate structures, we screened members of the crucifer tribe Cardamineae by HPLC-MS/MS, isolated and identified glucosinolates by NMR, searched the literature for previous data for the tribe, and collected HPLC-MS/MS data for nearly all glucosinolates known from the tribe as well as some related structures (70 in total). This is a considerable proportion of the approximately 142 currently documented natural glucosinolates. Calibration with authentic references allowed distinction (or elucidation) of isomers in many cases, such as distinction of ß-hydroxyls, methylthios, methylsulfinyls and methylsulfonyls. A mechanism for fragmentation of secondary ß-hydroxyls in MS was elucidated, and two novel glucosinolates were discovered: 2-hydroxy-3-methylpentylglucosinolate in roots of Cardamine pratensis and 2-hydroxy-8-(methylsulfinyl)octylglucosinolate in seeds of Rorippa amphibia. A large number of glucosinolates (ca. 54 with high structural certainty and a further 28 or more suggested from tandem MS), representing a wide structural variation, is documented from the tribe. This included glucosinolates apparently derived from Met, Phe, Trp, Val/Leu, Ile and higher homologues. Normal side chain elongation and side chain decoration by oxidation or methylation was observed, as well as rare abnormal side chain decoration (hydroxylation of aliphatics at the δ rather than ß-position). Some species had diverse profiles, e.g. R. amphibia and C. pratensis (19 and 16 individual glucosinolates, respectively), comparable to total diversity in literature reports of Armoracia rusticana (17?), Barbarea vulgaris (20-24), and Rorippa indica (>20?). The ancestor or the tribe would appear to have used Trp, Met, and homoPhe as glucosinolate precursor amino acids, and to exhibit oxidation of thio to sulfinyl, formation of alkenyls, ß-hydroxylation of aliphatic chains and hydroxylation and methylation of indole glucosinolates. Two hotspots of apparent biochemical innovation and loss were identified: C. pratensis and the genus Barbarea. Diversity in other species mainly included structures also known from other crucifers. In addition to a role of gene duplication, two contrasting genetic/biochemical mechanisms for evolution of such combined diversity and redundancy are discussed: (i) involvement of widespread genes with expression varying during evolution, and (ii) mutational changes in substrate specificities of CYP79F and GS-OH enzymes.

Aromatic Glucosinolate Biosynthesis Pathway in Barbarea vulgaris and its Response to Plutella xylostella Infestation.

The inducibility of the glucosinolate resistance mechanism is an energy-saving strategy for plants, but whether induction would still be triggered by glucosinolate-tolerant Plutella xylostella (diamondback moth, DBM) after a plant had evolved a new resistance mechanism (e.g., saponins in Barbara vulgaris) was unknown. In B. vulgaris, aromatic glucosinolates derived from homo-phenylalanine are the dominant glucosinolates, but their biosynthesis pathway was unclear. In this study, we used G-type (pest-resistant) and P-type (pest-susceptible) B. vulgaris to compare glucosinolate levels and the expression profiles of their biosynthesis genes before and after infestation by DBM larvae. Two different stereoisomers of hydroxylated aromatic glucosinolates are dominant in G- and P-type B. vulgaris, respectively, and are induced by DBM. The transcripts of genes in the glucosinolate biosynthesis pathway and their corresponding transcription factors were identified from an Illumina dataset of G- and P-type B. vulgaris. Many genes involved or potentially involved in glucosinolate biosynthesis were induced in both plant types. The expression patterns of six DBM induced genes were validated by quantitative PCR (qPCR), while six long-fragment genes were validated by molecular cloning. The core structure biosynthetic genes showed high sequence similarities between the two genotypes. In contrast, the sequence identity of two apparent side chain modification genes, the SHO gene in the G-type and the RHO in P-type plants, showed only 77.50% identity in coding DNA sequences and 65.48% identity in deduced amino acid sequences. The homology to GS-OH in Arabidopsis, DBM induction of the transcript and a series of qPCR and glucosinolate analyses of G-type, P-type and F1 plants indicated that these genes control the production of S and R isomers of 2-hydroxy-2-phenylethyl glucosinolate. These glucosinolates were significantly induced by P. xylostella larvae in both the susceptiple P-type and the resistant G-type, even though saponins are the main DBM-resistance causing metabolites in G-type plants. Indol-3-ylmethylglucosinolate was induced in the G-type only. These data will aid our understanding of the biosynthesis and induction of aromatic glucosinolates at the molecular level and also increase our knowledge of the complex mechanisms underpinning defense induction in plants.

Diversified glucosinolate metabolism: biosynthesis of hydrogen cyanide and of the hydroxynitrile glucoside alliarinoside in relation to sinigrin metabolism in Alliaria petiolata.

Alliaria petiolata (garlic mustard, Brassicaceae) contains the glucosinolate sinigrin as well as alliarinoside, a γ-hydroxynitrile glucoside structurally related to cyanogenic glucosides. Sinigrin may defend this plant against a broad range of enemies, while alliarinoside confers resistance to specialized (glucosinolate-adapted) herbivores. Hydroxynitrile glucosides and glucosinolates are two classes of specialized metabolites, which generally do not occur in the same plant species. Administration of [UL-(14)C]-methionine to excised leaves of A. petiolata showed that both alliarinoside and sinigrin were biosynthesized from methionine. The biosynthesis of alliarinoside was shown not to bifurcate from sinigrin biosynthesis at the oxime level in contrast to the general scheme for hydroxynitrile glucoside biosynthesis. Instead, the aglucon of alliarinoside was formed from metabolism of sinigrin in experiments with crude extracts, suggesting a possible biosynthetic pathway in intact cells. Hence, the alliarinoside pathway may represent a route to hydroxynitrile glucoside biosynthesis resulting from convergent evolution. Metabolite profiling by LC-MS showed no evidence of the presence of cyanogenic glucosides in A. petiolata. However, we detected hydrogen cyanide (HCN) release from sinigrin and added thiocyanate ion and benzyl thiocyanate in A. petiolata indicating an enzymatic pathway from glucosinolates via allyl thiocyanate and indole glucosinolate derived thiocyanate ion to HCN. Alliarinoside biosynthesis and HCN release from glucosinolate-derived metabolites expand the range of glucosinolate-related defenses and can be viewed as a third line of defense, with glucosinolates and thiocyanate forming protein being the first and second lines, respectively.