Myrosinase isogenes in wasabi (Wasabia japonica Matsum) and their putative roles in glucosinolate metabolism.

BACKGROUND: Wasabi, a Brassicaceae member, is well-known for its unique pungent and hot flavor which is produced from glucosinolate (GSL) degradation. Myrosinase (MYR) is a principle enzyme catalyzing the primary conversion of GSLs to GSL hydrolysis products (GHPs) which is responsible for plant defense system and food quality. Due to the limited information in relation to MYRs present in wasabi (Wasabia japonica M.), this study aimed to identify the MYR isogenes in W. japonica and analyze their roles in relation to GSL metabolism. RESULTS: In results, WjMYRI-1 was abundantly expressed in all organs, whereas WjMYRI-2 showed only trace expression levels. WjMYRII was highly expressed in the aboveground tissues. Interestingly, WjMYRII expression was significantly upregulated by certain abiotic factors, such as methyl jasmonate (more than 40-fold in petioles and 15-fold in leaves) and salt (tenfold in leaves). Young leaves and roots contained 97.89 and 91.17 µmol‧g-1 of GSL, whereas less GSL was produced in mature leaves and petioles (38.36 and 44.79 µmol‧g-1, respectively). Similar pattern was observed in the accumulation of GHPs in various plant organs. Notably, despite the non-significant changes in GSL production, abiotic factors treated samples enhanced significantly GHP content. Pearson's correlation analysis revealed that WjMYRI-1 expression significantly correlated with GSL accumulation and GHP formation, suggesting the primary role of WjMYRI-1-encoding putative protein in GSL degradation. In contrast, WjMYRII expression level showed no correlation with GSL or GHP content, suggesting another physiological role of WjMYRII in stress-induced response. CONCLUSIONS: In conclusions, three potential isogenes (WjMYRI-1, WjMYRI-2, and WjMYRII) encoding for different MYR isoforms in W. japonica were identified. Our results provided new insights related to MYR and GSL metabolism which are important for the implications of wasabi in agriculture, food and pharmaceutical industry. Particularly, WjMYRI-1 may be primarily responsible for GSL degradation, whereas WjMYRII (clade II) may be involved in other regulatory pathways induced by abiotic factors.

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.

Glucosinolates, a natural chemical arsenal: More to tell than the myrosinase story.

Glucosinolates are a group of thioglucosides that belong to the class of plant nitrogen-containing natural products. So far, very little biological activity has been associated with intact glucosinolates. The hydrolysis of glucosinolates has, for long, attracted attention because of the potent biological activity of the hydrolysis products. From allelopathic to antiparasitic, antimicrobial and antineoplastic effects, the activity spectrum of the degradation products of typical glucosinolates has been the subject of much research. The present review seeks to address the various means of glucosinolate degradation (thermal, enzymatic, or chemical degradation) and the ensuing products. It also aims to draw a comparative profile of the various antimicrobial effects of these degradation products to provide a further understanding of the biological function of these important compounds.

Genome-Wide Identification and Expression Analysis of ESPs and NSPs Involved in Glucosinolate Hydrolysis and Insect Attack Defense in Chinese Cabbage (Brassica rapa subsp. pekinensis).

Glucosinolates are secondary plant metabolites that are part of the plant's defense system against pathogens and pests and are activated via enzymatic degradation by thioglucoside glucohydrolases (myrosinases). Epithiospecifier proteins (ESPs) and nitrile-specifier proteins (NSPs) divert the myrosinase-catalyzed hydrolysis of a given glucosinolate to form epithionitrile and nitrile rather than isothiocyanate. However, the associated gene families have not been explored in Chinese cabbage. We identified three ESP and fifteen NSP genes randomly distributed on six chromosomes in Chinese cabbage. Based on a phylogenetic tree, the ESP and NSP gene family members were divided into four clades and had similar gene structure and motif composition of Brassica rapa epithiospecifier proteins (BrESPs) and B. rapa nitrile-specifier proteins (BrNSPs) in the same clade. We identified seven tandem duplicated events and eight pairs of segmentally duplicated genes. Synteny analysis showed that Chinese cabbage and Arabidopsis thaliana are closely related. We detected the proportion of various glucosinolate hydrolysates in Chinese cabbage and verified the function of BrESPs and BrNSPs in glucosinolate hydrolysis. Furthermore, we used quantitative RT-PCR to analyze the expression of BrESPs and BrNSPs and demonstrated that these genes responded to insect attack. Our findings provide novel insights into BrESPs and BrNSPs that can help further promote the regulation of glucosinolate hydrolysates by ESP and NSP to resist insect attack in Chinese cabbage.

Developing multifunctional crops by engineering Brassicaceae glucosinolate pathways.

Glucosinolates (GSLs), found mainly in species of the Brassicaceae family, are one of the most well-studied classes of secondary metabolites. Produced by the action of myrosinase on GSLs, GSL-derived hydrolysis products (GHPs) primarily defend against biotic stress in planta. They also significantly affect the quality of crop products, with a subset of GHPs contributing unique food flavors and multiple therapeutic benefits or causing disagreeable food odors and health risks. Here, we explore the potential of these bioactive functions, which could be exploited for future sustainable agriculture. We first summarize our accumulated understanding of GSL diversity and distribution across representative Brassicaceae species. We then systematically discuss and evaluate the potential of exploited and unutilized genes involved in GSL biosynthesis, transport, and hydrolysis as candidate GSL engineering targets. Benefiting from available information on GSL and GHP functions, we explore options for multifunctional Brassicaceae crop ideotypes to meet future demand for food diversification and sustainable crop production. An integrated roadmap is subsequently proposed to guide ideotype development, in which maximization of beneficial effects and minimization of detrimental effects of GHPs could be combined and associated with various end uses. Based on several use-case examples, we discuss advantages and limitations of available biotechnological approaches that may contribute to effective deployment and could provide novel insights for optimization of future GSL engineering.

Comprehensive Volatilome Signature of Various Brassicaceae Species.

To investigate in detail the volatilomes of various Brassicaceae species, landraces, and accessions, and to extract specific volatile markers, volatile aroma compounds were isolated from plant samples by headspace solid-phase microextraction and analyzed by gas chromatography/mass spectrometry (HS-SPME-GC/MS). The data obtained were subjected to uni- and multivariate statistical analysis. In general, two cabbage (Brassica oleracea L. var. capitata) landraces emitted the lowest amounts of volatiles generated in the lipoxygenase (LOX) pathway. Wild species Brassica incana Ten. and Brassica mollis Vis. were characterized by relatively high trans-2-hexenal/cis-3-hexen-1-ol ratio in relation to other investigated samples. A Savoy cabbage (Brassica oleracea L. var. sabauda) cultivar and three kale (Brassica oleracea L. var. acephala) accessions exhibited particular similarities in the composition of LOX volatiles, while the LOX volatilome fraction of B. incana and B. mollis partially coincided with that of another wild species, Diplotaxis tenuifolia L. Regarding volatiles formed in the glucosinolate (GSL) pathway, Savoy cabbage and wild species B. incana, B. mollis, and D. tenuifolia showed more intense emission of isothiocyanates than cabbage and kale. Diplotaxis tenuifolia showed a rather limited production of nitriles. The results of this study contribute to the general knowledge about volatile composition from various Brassicaceae species, which could be exploited for their better valorization. Future studies should focus on the influence of various environmental, cultivation, and post-harvest factors to obtain data with a higher level of applicability in practice.

Impact of an Agriphotovoltaic System on Metabolites and the Sensorial Quality of Cabbage (Brassica oleracea var. capitata) and Its High-Temperature-Extracted Juice.

To date, the impacts of agriphotovoltaic (APV) condition on the production yield of crop have been studied; however, the effect of APV production on the sensorial quality and consumer acceptability of the produce remains unexplored. Therefore, to address this knowledge gap, we cultivated "Winter Storm" cabbage under solar panels (20.16 kW) and in open field in 2020. The weight and diameter reduction rate of fresh cabbage grown under APV condition compared to open-field conditions were 9.7% and 1.2%, respectively. The levels of glucosinolates and their hydrolysis products were not significantly different in the fresh cabbage between the two conditions. The amount of volatile organic compounds, which may affect the perception of smell, were significantly higher in the cabbage juice prepared from the ones grown in open-field conditions than in the juice prepared from cabbages grown under APV conditions (n = 3, p < 0.01). However, untrained subjects could not distinguish the difference in the quality of the 2 sets of cabbage juices in the triangle test (n = 70, p = 0.724). Regardless of the distinguishing features of color, aroma, and taste, the subjects did not have any preference between the two different cabbage juices.

Influence of Cabbage (Brassica oleracea) Accession and Growing Conditions on Myrosinase Activity, Glucosinolates and Their Hydrolysis Products.

Glucosinolates are secondary plant metabolites present in Brassica vegetables. The endogenous enzyme myrosinase is responsible for the hydrolysis of glucosinolates, yielding a variety of compounds, including health-promoting isothiocyanates. The influence of cabbage accession and growing conditions on myrosinase activity, glucosinolates (GSL) and their hydrolysis products (GHPs) of 18 gene-bank cabbage accessions was studied. Growing conditions, cabbage morphotype and accession all significantly affected myrosinase activity and concentration of glucosinolates and their hydrolysis products. In general, cabbages grown in the field with lower growth temperatures had significantly higher myrosinase activity than glasshouse samples. Profile and concentration of glucosinolates and their hydrolysis products differed across the accessions studied. Aliphatic glucosinolates accounted for more than 60 % of total glucosinolates in most of the samples assessed. Nitriles and epithionitriles were the most abundant hydrolysis products formed. The results obtained showed that consumption of raw cabbages might reduce the amount of beneficial hydrolysis products available to the consumer, as more nitriles were produced from hydrolysis compared to beneficial isothiocyanates. However, red and white cabbages contained high concentrations of glucoraphanin and its isothiocyanate, sulforaphane. This implies that careful selection of accessions with ample concentrations of certain glucosinolates can improve the health benefits derived from raw cabbage consumption.

The Impact of Domestic Cooking Methods on Myrosinase Stability, Glucosinolates and Their Hydrolysis Products in Different Cabbage (Brassica oleracea) Accessions.

Glucosinolate hydrolysis products are responsible for the health-promoting properties of Brassica vegetables. The impact of domestic cooking on the myrosinase stability, glucosinolates and hydrolysis products in 18 cabbage accession was investigated. Cabbages were steamed, microwaved, and stir-fried before analysis. Cooking significantly affected myrosinase stability and glucosinolate concentrations within and between cabbage morphotypes. Myrosinase was most stable after stir-frying, with up to 65% residual activity. Steaming and microwaving resulted in over 90% loss of myrosinase activity in some accessions. Stir-frying resulted in the greatest decrease in glucosinolate concentration, resulting in up to 70% loss. Steamed cabbages retained the highest glucosinolates after cooking (up to 97%). The profile and abundance of glucosinolate hydrolysis products detected varied across all cooking methods studied. Cooking reduced the amounts of nitriles and epithionitriles formed compared to raw samples. Steaming led to a significant increase in the concentration of beneficial isothiocyanates present in the cabbage and a significantly lower level of nitriles compared to other samples. Microwaving led to a reduction in the concentrations of both nitriles and isothiocyanates when compared to other cooking methods and raw cabbage. The results obtained help provide information on the optimal cooking methods for cabbage, suggesting that steaming may be the best approach to maximising beneficial isothiocyanate production.

Glucosinolate catabolism during postharvest drying determines the ratio of bioactive macamides to deaminated benzenoids in Lepidium meyenii (maca) root flour.

Postharvest processing of maca (Lepidium meyenii Walp., Brassicaceae), a traditional high-altitude Andean root crop, involves slow field drying prior to milling into flour. The progressive tissue dehydration and release of hydrolytic enzymes and substrates from cellular compartments results in the slow accumulation of free monosaccharides, fatty acids and amino acids. A more complex, and faster, kinetic profile is that of glucosinolate breakdown. A number of reactive transient and stable accumulation products are generated during drying, some of which have noteworthy bioactive properties. Among these are macamides, inhibitors of endocannabinoid neurotransmitter degradation in mammalian nervous systems. They result from the condensation of benzyl amine, a glucosinolate hydrolysis product, with free fatty acids released from lipid hydrolysis. Recent research has focused on developing drying processes under controlled conditions that can modulate the biochemistry of glucosinolate hydrolysis to optimize the content of bioactive compounds in the root flour. Low temperature (35 °C) oven-drying of shredded maca roots under controlled air flow generates benzyl amine as primary accumulation product, accounting for up to 94% of hydrolyzed glucosinolate in the flour. Kinetic evidence suggests that both deaminated benzenoids and macamides are allocated from the benzylamine pool through amine oxidase activity or condensation with free fatty acids, accounting for the remaining hydrolyzed glucosinolate (<5%). These activities determine the allocation to either one of these pathways. Later stages of dehydration result in shifts in the molar ratios of deaminated benzenoids, the accumulation of benzoic acid esters and benzyl alcohol. We propose that these are the result of changes in the rates of the reductive and oxidative half-reactions of endogenous aldehyde dehydrogenases. It is the ratio of benzylamine deamination to amide formation that determines the eventual yields of macamides in relation to benzenoids and their esters in maca flour.

Human, Animal and Plant Health Benefits of Glucosinolates and Strategies for Enhanced Bioactivity: A Systematic Review.

Glucosinolates (GSs) are common anionic plant secondary metabolites in the order Brassicales. Together with glucosinolate hydrolysis products (GSHPs), they have recently gained much attention due to their biological activities and mechanisms of action. We review herein the health benefits of GSs/GSHPs, approaches to improve the plant contents, their bioavailability and bioactivity. In this review, only literature published between 2010 and March 2020 was retrieved from various scientific databases. Findings indicate that these compounds (natural, pure, synthetic, and derivatives) play an important role in human/animal health (disease therapy and prevention), plant health (defense chemicals, biofumigants/biocides), and food industries (preservatives). Overall, much interest is focused on in vitro studies as anti-cancer and antimicrobial agents. GS/GSHP levels improvement in plants utilizes mostly biotic/abiotic stresses and short periods of phytohormone application. Their availability and bioactivity are directly proportional to their contents at the source, which is affected by methods of food preparation, processing, and extraction. This review concludes that, to a greater extent, there is a need to explore and improve GS-rich sources, which should be emphasized to obtain natural bioactive compounds/active ingredients that can be included among synthetic and commercial products for use in maintaining and promoting health. Furthermore, the development of advanced research on compounds pharmacokinetics, their molecular mode of action, genetics based on biosynthesis, their uses in promoting the health of living organisms is highlighted.

Methyl Jasmonate Treatment of Broccoli Enhanced Glucosinolate Concentration, Which Was Retained after Boiling, Steaming, or Microwaving.

Exogenous methyl jasmonate (MeJA) treatment was known to increase the levels of neoglucobrassicin and their bioactive hydrolysis products in broccoli (Brassica oleracea var. italica), but the fate of MeJA-induced glucosinolates (GSLs) after various cooking methods was unknown. This study measured the changes in GSLs and their hydrolysis compounds in broccoli treated with MeJA and the interaction between MeJA and cooking treatments. All cooked MeJA-treated broccoli contained significantly more GSLs than untreated broccoli (p < 0.05). After 5 min of cooking (boil, steam, microwave), MeJA-treated broccoli still contained 1.6- to 2.3-fold higher GSL content than untreated broccoli. Neoglucobrassicin hydrolysis products were also significantly greater in steamed and microwaved MeJA-treated broccoli. The results show that exogenous MeJA treatment increases neoglucobrassicin and its hydrolysis compounds in broccoli even after cooking. Once the positive and negative effects of these compounds are better understood, the results of this experiment can be a valuable tool to help food scientists, nutrition scientists, and dieticians determine how to incorporate raw or cooked broccoli and Brassica vegetables in the diet.

Corrigendum: Identification and Characterization of Three Epithiospecifier Protein Isoforms in Brassica oleracea.

[This corrects the article on p. 1552 in vol. 10, PMID: 31921230.].

Identification and Characterization of Three Epithiospecifier Protein Isoforms in Brassica oleracea.

Glucosinolates present in Brassicaceae play a major role in herbivory defense. Upon tissue disruption, glucosinolates come into contact with myrosinase, which initiates their breakdown to biologically active compounds. Among these, the formation of epithionitriles is triggered by the presence of epithiospecifier protein (ESP) and a terminal double bond in the glucosinolate side chain. One ESP gene is characterized in the model plant Arabidopsis thaliana (AtESP; At1g54040.2). However, Brassica species underwent genome triplication since their divergence from the Arabidopsis lineage. This indicates the presence of multiple ESP isoforms in Brassica crops that are currently poorly characterized. We identified three B. oleracea ESPs, specifically BoESP1 (LOC106296341), BoESP2 (LOC106306810), and BoESP3 (LOC106325105) based on in silico genome analysis. Transcript and protein abundance were assessed in shoots and roots of four B. oleracea vegetables, namely broccoli, kohlrabi, white, and red cabbage, because these genotypes showed a differential pattern for the formation of glucosinolate hydrolysis products as well for their ESP activity. BoESP1 and BoESP2 were expressed mainly in shoots, while BoESP3 was abundant in roots. Biochemical characterization of heterologous expressed BoESP isoforms revealed different substrate specificities towards seven glucosinolates: all isoforms showed epithiospecifier activity on alkenyl glucosinolates, but not on non-alkenyl glucosinolates. The pH-value differently affected BoESP activity: while BoESP1 and BoESP2 activities were optimal at pH 6-7, BoESP3 activity remained relatively stable from pH 4 to 7. In order test their potential for the in vivo modification of glucosinolate breakdown, the three isoforms were expressed in A. thaliana Hi-0, which lacks AtESP expression, and analyzed for the effect on their respective hydrolysis products. The BoESPs altered the hydrolysis of allyl glucosinolate in the A. thaliana transformants to release 1-cyano-2,3-epithiopropane and reduced formation of the corresponding 3-butenenitrile and allyl isothiocyanate. Plants expressing BoESP2 showed the highest percentage of released epithionitriles. Given these results, we propose a model for isoform-specific roles of B. oleracea ESPs in glucosinolate breakdown.

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.

Dynamic Modeling of Indole Glucosinolate Hydrolysis and Its Impact on Auxin Signaling.

Plants release chemicals to deter attackers. Arabidopsis thaliana relies on multiple defense compounds, including indol-3-ylmethyl glucosinolate (I3G), which upon hydrolysis initiated by myrosinase enzymes releases a multitude of bioactive compounds, among others, indole-3-acetonitrile and indole-3-acetoisothiocyanate. The highly unstable isothiocyanate rapidly reacts with other molecules. One of the products, indole-3-carbinol, was reported to inhibit auxin signaling through binding to the TIR1 auxin receptor. On the contrary, the nitrile product of I3G hydrolysis can be converted by nitrilase enzymes to form the primary auxin molecule, indole-3-acetic acid, which activates TIR1. This suggests that auxin signaling is subject to both antagonistic and protagonistic effects of I3G hydrolysis upon attack. We hypothesize that I3G hydrolysis and auxin signaling form an incoherent feedforward loop and we build a mathematical model to examine the regulatory network dynamics. We use molecular docking to investigate the possible antagonistic properties of different I3G hydrolysis products by competitive binding to the TIR1 receptor. Our simulations reveal an uncoupling of auxin concentration and signaling, and we determine that enzyme activity and antagonist binding affinity are key parameters for this uncoupling. The molecular docking predicts that several I3G hydrolysis products strongly antagonize auxin signaling. By comparing a tissue disrupting attack - e.g., by chewing insects or necrotrophic pathogens that causes rapid release of I3G hydrolysis products - to sustained cell-autonomous I3G hydrolysis, e.g., upon infection by biotrophic pathogens, we find that each scenario gives rise to distinct auxin signaling dynamics. This suggests that plants have different defense versus growth strategies depending on the nature of the attack.

Differences in the enzymatic hydrolysis of glucosinolates increase the defense metabolite diversity in 19 Arabidopsis thaliana accessions.

Plants of the order Brassicales produce glucosinolates (GS), a group of secondary metabolites that are part of an elaborate defense system. But it is not the GS itself rather its enzymatic hydrolysis products that cause the bioactive effects protecting the plants against pests and pathogens. Thus the enzymatic hydrolysis and a variety of additional influential factors determine the structural outcome of the GS degradation process. To evaluate the possible diversity of defense metabolites a range of 19 Arabidopsis thaliana accessions were selected showing divergence in their geographical origin, in their phenotype, and in their GS profile. These particular accessions accumulate several alkenyl GS, hydroxyalkyl GS, methylthioalkyl GS, and methylsulfinylalkyl GS in their rosette leaves whereas the indole GS contents are relatively invariant, as analyzed by UHPLC-DAD. After tissue disruption the enzymatic formation of GS hydrolysis products was examined and breakdown products were identified and quantified by GC-MS. Great differences in the amount and structure of volatile enzymatic degradation products could be observed in the different accessions, with strong variation in formation of epithionitriles, nitriles, and isothiocyanates. The occurrence of specific GS hydrolysis products was put in relation to relative gene expression profiles of myrosinases and specifier proteins as measured by RT-qPCR, and in relation to relative protein abundance of epithiospecifier protein. Dependent on the different GS profiles and reliant on degradation protein abundance and composition the ecotypes strongly varied in their ability to form isothiocyanates, nitriles and epithionitriles, thus increasing the plants' equipment of defense metabolites.

Myrosinase-dependent and -independent formation and control of isothiocyanate products of glucosinolate hydrolysis.

Brassicales contain a myrosinase enzyme that hydrolyzes glucosinolates to form toxic isothiocyanates (ITC), as a defense against bacteria, fungi, insects and herbivores including man. Low levels of ITC trigger a host defense system in mammals that protects them against chronic diseases. Because humans typically cook their brassica vegetables, destroying myrosinase, there is a great interest in determining how human microbiota can hydrolyze glucosinolates and release them, to provide the health benefits of ITC. ITC are highly reactive electrophiles, binding reversibly to thiols, but accumulating and causing damage when free thiols are not available. We found that addition of excess thiols released protein-thiol-bound ITC, but that the microbiome supports only poor hydrolysis unless exposed to dietary glucosinolates for a period of days. These findings explain why 3-5 servings a week of brassica vegetables may provide health effects, even if they are cooked.

The crystal structure of the thiocyanate-forming protein from Thlaspi arvense, a kelch protein involved in glucosinolate breakdown.

Kelch repeat-containing proteins are involved in diverse cellular processes, but only a small subset of plant kelch proteins has been functionally characterized. Thiocyanate-forming protein (TFP) from field-penny cress, Thlaspi arvense (Brassicaceae), is a representative of specifier proteins, a group of kelch proteins involved in plant specialized metabolism. As components of the glucosinolate-myrosinase system of the Brassicaceae, specifier proteins determine the profile of bioactive products formed when plant tissue is disrupted and glucosinolates are hydrolyzed by myrosinases. Here, we describe the crystal structure of TaTFP at a resolution of 1.4 Å. TaTFP crystallized as homodimer. Each monomer forms a six-blade ß-propeller with a wide "top" and a narrower "bottom" opening with distinct strand-connecting loops protruding far beyond the lower propeller surface. Molecular modeling and mutational analysis identified residues for glucosinolate aglucone and Fe(2+) cofactor binding within these loops. As the first experimentally determined structure of a plant kelch protein, the crystal structure of TaTFP not only enables more detailed mechanistic studies on glucosinolate breakdown product formation, but also provides a new basis for research on the diverse roles and mechanisms of other kelch proteins in plants.

The selective cytotoxicity of the alkenyl glucosinolate hydrolysis products and their presence in Brassica vegetables.

Cruciferous vegetable consumption correlates with reduced risk of cancer. This chemopreventative activity may involve glucosinolates and their hydrolysis products. Glucosinolate-derived isothiocyanates have been studied for their toxicity and chemopreventative properties, but other hydrolysis products (epithionitriles and nitriles) have not been thoroughly examined. We report that these hydrolysis products differ in their cytotoxicity to human cells, with toxicity most strongly associated with isothiocyanates rather than epithionitriles and nitriles. We explored mechanisms of this differential cytotoxicity by examining the role of oxidative metabolism, oxidative stress, mitochondrial permeability, reduced glutathione levels, cell cycle arrest and apoptosis. 2-Propenylisothiocyanate and 3-butenylisothiocyanate both inhibited cytochome P450 1A (CYP1A) enzyme activity in CYP expressing MCL-5 cells at high cytotoxic doses. Incubation of MCL-5 cells with non-cytotoxic doses of 2-propenylisothiocyanate for 24h resulted in a dose-dependent inhibition of ethoxyresorufin O-deethylase, yet failed to affect CYP1A1 mRNA expression indicating interference with enzyme activity rather than inhibition of transcription. Increased reactive oxygen species (ROS) production was observed only for 2-propenylisothiocyanate treatment. 2-Propenylisothiocyanate treatment lowered reduced glutathione levels whereas no changes were noted with 3,4-epithiobutylnitrile. Cell cycle analysis showed that 2-propenylisothiocyanate induced a G2/M block whereas other hydrolysis products showed only marginal effects. We found that 2-propenylisothiocyanate and 3-butenylisothiocyanate induced cell death predominantly via necrosis whereas, 3,4-epithiobutylnitrile promoted both necrosis and apoptosis. Thus the activity of glucosinolate hydrolysis products includes cytotoxicity that is compound-class specific and may contribute to their putative chemoprotection properties.