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.

Hybridization and complex evolution of Barbarea vulgaris and related species (Brassicaceae).

Barbarea, winter-cress, is a genus of 29 species in Brassicaceae, the mustard family, which has emerged as a model for evolution of plant defence and specialised metabolites. Notably, some Barbarea species have evolved the ability to produce triterpenoid saponins as the only ones in Brassicaceae, some of which make plants resistant to important herbivores. Resistance has, however, been lost in a distinct group of plants within B. vulgaris ssp. arcuata, which is genetically strongly diverged from other B. vulgaris plants. This divergence is not reflected present in taxonomy. Thus, a phylogeny is needed to understand evolution and defence in Barbarea. Here, we analysed the nuclear ITS and the plastid matK, ndhF, rps16, and psbA-trnH DNA regions from seven out of 29 Barbarea species, 57 accessions of B. vulgaris, 10 accessions of other Barbarea species, and eight outgroup species, in addition to sequences available from GenBank. All Barbarea species formed a highly supported monophyletic group, separated from sister genera. Several clades seem to have radiated within the genus with no simple branching pattern, and discordant nuclear and plastid DNA phylogenies indicate reticulate evolution and chloroplast capture. One of the complex patterns may have resulted from chloroplast capture of a non-Nordic Barbarea species not included in the study. Two pairs of species were almost identical, B. australis and B. grayi, and B. orthoceras and B. stricta. Despite hybridization, chloroplast capture, and incongruence among the plastid and nuclear DNA data, the high level of intraspecific diversity, coupled with lineage specificity, lead us to recognize three groups of Barbarea vulgaris: G-type (glabrous) and P-type (pubescent) individuals of the current B. vulgaris ssp. arcuata as two distinct groups and the current B. vulgaris ssp. vulgaris as the third. Despite the high molecular diversity below species level, the evolutionary history of the saponin-based resistance remains unsettled due to unresolved basal branching.

Reciprocal mutations of two multifunctional ß-amyrin synthases from Barbarea vulgaris shift α/ß-amyrin ratios.

In the wild cruciferous wintercress (Barbarea vulgaris), ß-amyrin-derived saponins are involved in resistance against insect herbivores like the major agricultural pest diamondback moth (Plutella xylostella). Enzymes belonging to the 2,3-oxidosqualene cyclase family have been identified and characterized in B. vulgaris G-type and P-type plants that differ in their natural habitat, insect resistance and saponin content. Both G-type and P-type plants possess highly similar 2,3-oxidosqualene cyclase enzymes that mainly produce ß-amyrin (Barbarea vulgaris Lupeol synthase 5 G-Type; BvLUP5-G) or α-amyrin (Barbarea vulgaris Lupeol synthase 5 P-Type; BvLUP5-P), respectively. Despite the difference in product formation, the two BvLUP5 enzymes are 98% identical at the amino acid level. This provides a unique opportunity to investigate determinants of product formation, using the B. vulgaris 2,3-oxidosqualene cyclase enzymes as a model for studying amino acid residues that determine differences in product formation. In this study, we identified two amino acid residues at position 121 and 735 that are responsible for the dominant changes in generated product ratios of ß-amyrin and α-amyrin in both BvLUP5 enzymes. These amino acid residues have not previously been highlighted as directly involved in 2,3-oxidosqualene cyclase product specificity. Our results highlight the functional diversity and promiscuity of 2,3-oxidosqualene cyclase enzymes. These enzymes serve as important mediators of metabolic plasticity throughout plant evolution.

Cruciferous Weeds Do Not Act as Major Reservoirs of Inoculum for Black Rot Outbreaks in New York State.

Cruciferous weeds have been shown to harbor diverse Xanthomonas campestris pathovars, including the agronomically damaging black rot of cabbage pathogen, X. campestris pv. campestris. However, the importance of weeds as inoculum sources for X. campestris pv. campestris outbreaks in New York remains unknown. To determine if cruciferous weeds act as primary reservoirs for X. campestris pv. campestris, fields that were rotating between cabbage or had severe black rot outbreaks were chosen for evaluation. Over a consecutive 3-year period, 148 cruciferous and noncruciferous weed samples were collected at 34 unique sites located across five New York counties. Of the 148 weed samples analyzed, 48 X. campestris isolates were identified, with a subset characterized using multilocus sequence analysis. All X. campestris isolates originated from weeds belonging to the Brassicaceae family, with predominant weed hosts being shepherd's purse (Capsella bursa-pastoris), wild mustard (Sinapis arvensis), yellow rocket (Barbarea vulgaris), and pennycress (Thlaspi arvense). Identifying pathogenic X. campestris weed isolates was rare, with only eight isolates causing brown necrotic leaf spots or typical V-shaped lesions on cabbage. There was no evidence of cabbage-infecting weed isolates persisting in an infected field by overwintering in weed hosts; however, similar cabbage and weed X. campestris haplotypes were identified in the same field during an active black rot outbreak. X. campestris weed isolates are genetically diverse both within and between fields, but our findings indicate that X. campestris weed isolates do not appear to act as primary sources of inoculum for B. oleracea fields in New York.

Sex-specific spatial use of the winter foraging areas by Magellanic penguins and assessment of potential conflicts with fisheries during winter dispersal.

Magellanic penguins (Spheniscus magellanicus) disperse widely during winter and are a major consumer of marine resources over the Patagonian Shelf. Magellanic penguins were equipped with geolocators at Martillo Island in late February- early March 2017 and recaptured at the beginning of the next breeding season to recover the devices and to collect blood samples for stable carbon (δ13C) and nitrogen (δ15N) isotope analysis. We evaluated their whole winter dispersal and their trophic niche by sex during the last month of the winter dispersal. Also, we evaluated their spatial overlap with bottom trawl and shrimp fisheries using data from satellite fisheries monitoring. Penguins dispersed northwards up to 42°S and showed latitudinal spatial segregation between sexes during May to August (females were located further north than males). In contrast, during the last month of the winter dispersal females were located more southerly and showed lower trophic position than males. Also, females did not dive as deep as males during winter. We found high overlap between both fisheries and penguin's spatial use in regions with documented interaction. However, no sex-specific statistical differences with fisheries overlap were found. Our results highlight the importance of understanding the spatial domains of each sex and assessment of their potential conflicts with bottom trawl fishery and shrimp fishery during the winter period.

Evaluation of biological activities of Barbarea integrifolia and isolation of a new glucosinolate derivated compound.

The aim of the present study is to determine the potent biological activities and carry out isolation studies on Barbarea integrifolia. The antioxidant capacity of the species was evaluated by total phenolic content, FRAP, CUPRAC, and DPPH radical scavenging activity. Anticancer activity studies were performed by MTT assay in MDA-MB-231, MCF-7, Hep3B, PC-3, A549, HCT116, L-929 cell lines. It was observed that the remaining aqueous fraction has higher total phenolic content while higher activity in the CUPRAC and FRAP assays was displayed for the methanolic extract and chloroform fraction. The extracts showed anticancer activity as compared with vincristine. It was observed that chloroform fraction has the highest anticancer activity on MCF-7 cell line, while ethyl acetate fraction has the highest anticancer activity on Hep-3B and A549 cell lines. Methanolic extract has the highest anticancer activity on HCT116 and MDA-MB-23 cell lines. The isolation studies have been performed using several chromatographic methods. The chemical structures of compounds have been identified by means of 1H NMR, 13C NMR, 2D-NMR, and MS. Five major compounds, one steroid (ß-Sitosterol), one phenolic acid (Rosmarinic acid), one flavonol heteroside (kaempferol 7-O-α-l-rhamnoside-3-O-ß-d-(2-O-ß- d -glucosyl)-ß-d-glucoside), and two glucosinolates (Gluconasturtiin, Gluconasturtiin choline salt) have been isolated.

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.

Effect of Lactobacillus acidophilus Fermented Broths Enriched with Eruca sativa Seed Extracts on Intestinal Barrier and Inflammation in a Co-Culture System of an Enterohemorrhagic Escherichia coli and Human Intestinal Cells.

Lactic acid bacteria (LAB) "fermentates" confer a beneficial effect on intestinal function. However, the ability of new fermentations to improve LAB broth activity in preventing pathogen-induced intestinal inflammation and barrier dysfunction has not yet been studied. The objective of this study was to determine if broths of LAB fermented with Eruca sativa or Barbarea verna seed extracts prevent gut barrier dysfunction and interleukin-8 (CXCL8) release in vitro in human intestinal Caco-2 cells infected with enterohemorrhagic Escherichia coli (EHEC) O157:H7. LAB broths were assayed for their effects on EHEC growth and on Caco-2 viability; thereafter, their biological properties were analysed in a co-culture system consisting of EHEC and Caco-2 cells. Caco-2 cells infected with EHEC significantly increased CXCL8 release, and decreased Trans-Epithelial Electrical Resistance (TEER), a barrier-integrity marker. Notably, when Caco-2 cells were treated with LAB broth enriched with E. sativa seed extract and thereafter infected, both CXCL8 expression and epithelial dysfunction reduced compared to in untreated cells. These results underline the beneficial effect of broths from LAB fermented with E. sativa seed extracts in gut barrier and inflammation after EHEC infection and reveal that these LAB broths can be used as functional bioactive compounds to regulate intestinal function.

In Vitro Bioaccessibility and Bioavailability of Iron from Mature and Microgreen Fenugreek, Rocket and Broccoli.

Iron deficiency is a global epidemic affecting a third of the world's population. Current efforts are focused on investigating sustainable ways to improve the bioavailability of iron in plant-based diets. Incorporating microgreens into the diet of at-risk groups in populations could be a useful tool in the management and prevention of iron deficiency. This study analysed and compared the mineral content and bioavailability of iron from microgreen and mature vegetables. The mineral content of rocket, broccoli and fenugreek microgreens and their mature counterparts was determined using microwave digestion and ICP-OES. Iron solubility and bioavailability from the vegetables were determined by a simulated gastrointestinal in vitro digestion and subsequent measurement of ferritin in Caco-2 cells as a surrogate marker of iron uptake. Iron contents of mature fenugreek and rocket were significantly higher than those of the microgreens. Mature fenugreek and broccoli showed significantly (p < 0.001) higher bioaccessibility and low-molecular-weight iron than found in the microgreens. Moreover, iron uptake by Caco-2 cells was significantly higher only from fenugreek microgreens than the mature vegetable. While all vegetables except broccoli enhanced FeSO4 uptake, the response to ferric ammonium citrate (FAC) was inhibitory apart from the mature rocket. Ascorbic acid significantly enhanced iron uptake from mature fenugreek and rocket. Microgreen fenugreek may be bred for a higher content of enhancers of iron availability as a strategy to improve iron nutrition in the populace.

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 cytochrome P450 CYP72A552 is key to production of hederagenin-based saponins that mediate plant defense against herbivores.

Plants continuously evolve new defense compounds. One class of such compounds is triterpenoid saponins. A few species in the Barbarea genus produce saponins as the only ones in the large crucifer family. However, the molecular mechanism behind saponin biosynthesis and their role in plant defense remains unclear. We used pathway reconstitution in planta, enzymatic production of saponins in vitro, insect feeding assays, and bioinformatics to identify a missing gene involved in saponin biosynthesis and saponin-based herbivore defense. A tandem repeat of eight CYP72A cytochromes P450 colocalise with a quantitative trait locus (QTL) for saponin accumulation and flea beetle resistance in Barbarea vulgaris. We found that CYP72A552 oxidises oleanolic acid at position C-23 to hederagenin. In vitro-produced hederagenin monoglucosides reduced larval feeding by up to 90% and caused 75% larval mortality of the major crucifer pest diamondback moth and the tobacco hornworm. Sequence analysis indicated that CYP72A552 evolved through gene duplication and has been under strong selection pressure. In conclusion, CYP72A552 has evolved to catalyse the formation of hederagenin-based saponins that mediate plant defense against herbivores. Our study highlights the evolution of chemical novelties by gene duplication and selection for enzyme innovations, and the importance of chemical modification in plant defense evolution.

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.

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.

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.

Screening for Triterpenoid Saponins in Plants Using Hyphenated Analytical Platforms.

Recently the number of studies investigating triterpenoid saponins has drastically increased due to their diverse and potentially attractive biological activities. Currently the literature contains chemical structures of few hundreds of triterpenoid saponins of plant and animal origin. Triterpenoid saponins consist of a triterpene aglycone with one or more sugar moieties attached to it. However, due to similar physico-chemical properties, isolation and identification of a large diversity of triterpenoid saponins remain challenging. This study demonstrates a methodology to screen saponins using hyphenated analytical platforms, GC-MS, LC-MS/MS, and LC-SPE-NMR/MS, in the example of two different phenotypes of the model plant Barbarea vulgaris (winter cress), glabrous (G) and pubescent (P) type that are known to differ by their insect resistance. The proposed methodology allows for detailed comparison of saponin profiles from intact plant extracts as well as saponin aglycone profiles from hydrolysed samples. Continuously measured 1D proton NMR data during LC separation along with mass spectrometry data revealed significant differences, including contents of saponins, types of aglycones and numbers of sugar moieties attached to the aglycone. A total of 49 peaks were tentatively identified as saponins from both plants; they are derived from eight types of aglycones and with 2-5 sugar moieties. Identification of two previously known insect-deterrent saponins, hederagenin cellobioside and oleanolic acid cellobioside, demonstrated the applicability of the methodology for relatively rapid screening of bioactive compounds.

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.

The effect of injury on whole-plant senescence: an experiment with two root-sprouting Barbarea species.

BACKGROUND AND AIMS: Senescence is the process of losing fitness when growing old, and is shaped by the trade-off between maintenance and reproduction that makes reproduction more unsure and maintenance more costly with age. In repeatedly reproducing plants, reductions in growth and fertility are signs of senescence. Disturbance, however, provides an opportunity to reset the ageing clock and consequently potentially ameliorate senescence. METHODS: To test the effects of disturbance on traits closely related to fitness and thus to senescence, a long-term garden experiment was established with two short-lived perennial congeners,Barbarea vulgaris and Barbarea stricta, that differ in their ability to resprout after injury. In the experiment, five damage treatments were applied to plants in four different phenophases. KEY RESULTS: It was found that damage to the plant body significantly prolonged life span in B. vulgaris but decreased whole-life seed production in both species. High concentration of seed production in one growing season characterized short life spans. Both more severe damage and a more advanced phenological phase at the time of damage caused reproduction to be spread over more than one growing season and equalized per-season seed production. In terms of seed quality, average weight of a single seed decreased and seed germination rate increased with age regardless of damage. CONCLUSIONS: Although disturbance is able to reset the ageing clock of plants, it is so harmful to plant fitness that resprouting serves, at best, only to alleviate slightly the signs of senescence. Thus, in terms of whole-life seed production, injured plants were not more successful than uninjured ones in the two studied species. Indeed, in these species, injury only slightly postponed or decelerated senescence and did not cause effective rejuvenation.

Optimal foraging behavior and the thermal neutral zone of Peromyscus leucopus during winter: A test using natural and controlled ambient temperatures.

Endotherms foraging at temperatures outside of their thermal neutral zone (TNZ) pay an increased energetic cost. We asked if thermally-induced changes in foraging costs influence quitting harvest rate (QHR) of mice. We predicted that mice foraging during the winter would have a higher QHR in more costly colder conditions. We conducted our study with wild caught Peromyscus leucopus in an enclosure located in West Terre Haute, Indiana. We assayed changes in QHR using the forager's giving up density (GUD), which is the amount of uneaten seeds reaming in a tray after foraging activity. Each night from January 12th to March 13th, we assigned 4 trays as "cold trays" (at ambient temperature), and 4 trays as "hot trays" (trays with a ceramic heat element that increased the temperatures of feeding trays ca. 10-15°C). GUDs (and therfore QHRs) increased as a function of decreasing ambient temperature. Furthermore there was an interaction between tray temperature and ambient temperature; namely, on cool nights mice had lower GUDs in the "hot trays", but on warm nights mice had lower GUDs in the "cold trays". The TNZ for P. leucopus actively foraging during winter may be closer to the environmental average temperature than typically measured in the laboratory. Overall, these results support the idea that QHR is related to an animal's foraging in thermally challenged conditions. We present a unique way of measuring an animal's TNZ in the field using behavioral indicators.