Glucosinolate-myrosinase System Research Articles

Bio-efficacy of mustard seed extracts against Bihar hairy caterpillar Spilosoma obliqua (Erebidae: Lepidoptera) & assessment of mustard allelo-chemicals in response to mustard aphid Lipaphis erysimi (Aphididae: Hemiptera) infestation

Mustard seed contains glucosinolate-myrosinase system of substrate-enzyme metabolism, accompanied with compartmentalized cells. Upon rupture, enzyme myrosinase leads to breakdown of glucosinolates and its metabolized products like allyl, benzyl, phenyl type of isothiocyantes and many other types like nitriles are released. The myrosinase activity is tested in three globally utilized different seed coat color mustards viz., brown mustard (Brassica juncea), white mustard (Sinapis alba) and black mustard (Brassica nigra) to demonstrate the release of glucosinolates which are known to deter insect pests. Aqueous seed extracts of these mustard types were subjected to anti-feedant activity analysis and oil extracted from these mustard seeds was subjected to insecticidal activity analysis against Spilosoma obliqua 4th instars on castor leaves. Meanwhile, the mustard leaves in open field were allowed for natural aphid infestation during early months from January to March. Thereafter, the leaf twigs of white, black and brown mustard, infested and uninfested by aphids, were collected and myrosinase activity of plants was analyzed with varying levels of important pest inflicted biochemicals in plant, viz., Total phenols, ortho-dihydric phenols and flavonols. Gas Chromatography–Mass Spectrometry analysis of mustard oil used in insecticidal activity was performed to elucidate the role of biochemicals involved in action. These compounds along with fatty acids and sterols compounds are known to have anti-feedant & insecticidal activities. Aqueous extract components were also subjected to total glucosinolate analysis, spectrophotometrically for knowing their concentration, with highest in brown (85.81 µmol/g) followed by black (59.32 µmol/g) and lowest in white mustard (47.63 µmol/g). The amount of glucosinolates, free fatty acids & sterol compounds present in different coat color mustard seed types are being correlated here with their potential bioactivities against biological plant pests. Bio-control of potentially harmful insect pests is possible by utilizing anti-feeding & insecticidal potential of mustard seed extracts.

Identification of nitric oxide regulated low abundant myrosinases from seeds and seedlings of Brassica juncea

Myrosinases constitute an important component of the glucosinolate-myrosinase system responsible for interaction of plants with microorganisms, insects, pest, and herbivores. It is a distinctive feature of Brassicales. Multiple isozymes of myrosinases are present in the vacuoles. Active myrosinases are also present in the apoplast and the nucleus however, the similarity or difference in the biochemical properties with the vacuolar myrosinases are not known. Here, we have attempted to isolate, characterize, and identify myrosinases from seeds, seedlings, apoplast, and nucleus to understand these forms. 2D-CN/SDS-PAGE coupled with western blotting and MS have shown low abundant myrosinases (65/70/72/75 kDa) in seeds and seedlings and apoplast & nucleus of seedlings to exist as dimers, oligomers, and as protein complex. Nuclear membrane associated form of myrosinase was also identified. The present study for the first time has shown enzymatically active myrosinase-alpha-mannosidase complex in seedlings. Both 65 and 70 kDa myrosinase in seedlings were S-nitrosated. Nitric oxide donor treatment (GSNO) led to 25% reduction in myrosinase activity which was reversed by DTT suggesting redox regulation of myrosinase. These S-nitrosated myrosinases might be a component of NO signalling in B. juncea.

Synthesis and Biological Activity of Sulfamate-Adamantane Derivatives as Glucosinolate Sulfatase Inhibitors.

The glucosinolate-myrosinase system, exclusively found in the Brassicaceae family, is a main defense strategy against insect resistance. The efficient detoxification activity of glucosinolate sulfatases (GSSs) has successfully supported the feeding of Plutella xylostella on cruciferous plants. With the activity of GSSs hampered in P. xylostella, the toxic isothiocyanates produced from glucosinolates severely impair larval growth and adult reproduction. Therefore, inhibitors of GSSs have been suggested as an alternative approach to controlling P. xylostella. Herein, we synthesized eight adamantyl-possessing sulfamate derivatives as novel inhibitors of GSSs. Adam-20-S exhibited the most potent GSS inhibitory activity, with an IC50 value of 9.04 mg/L. The suppression of GSSs by Adam-20-S impaired glucosinolate metabolism to produce more toxic isothiocyanates in P. xylostella. Consequently, the growth and development of P. xylostella were significantly hindered when feeding on the host plant. Our study may help facilitate the development of a comprehensive pest management strategy that combines insect detoxification enzyme inhibitors with plant chemical defenses.

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.

Inhibitor of Glucosinolate Sulfatases as a Potential Friendly Insecticide to Control Plutella xylostella.

The glucosinolate-myrosinase system is a two-component defense system characteristic of cruciferous plants. To evade the glucosinolate-myrosinase system, the crucifer specialist insect, Plutella xylostella, promptly desulfates the glucosinolates into harmless compounds by glucosinolate sulfatases (GSSs) in the gut. In this study, we identified an effective inhibitor of GSSs by virtual screening, molecular docking analysis, and in vitro enzyme inhibition assay. The combined effect of the GSS inhibitor with the plant glucosinolate-myrosinase system was assessed by the bioassay of P. xylostella. We show that irosustat is a GSS inhibitor and the inhibition of GSSs impairs the ability of P. xylostella to detoxify the glucosinolate-myrosinase system, leading to the systematic accumulation of toxic isothiocyanates in larvae, thereby severely affecting feeding, growth, survival, and reproduction of P. xylostella. While fed on the Arabidopsis mutants deficient in myrosinase or glucosinolates, irosustat had no significant negative effect on P. xylostella. These findings reveal that the GSS inhibitor is a novel friendly insecticide to control P. xylostella utilizing the plant glucosinolate-myrosinase system and promote the development of insecticide-plant chemical defense combination strategies.

Effect of high pressure pretreatment on myrosinase-glucosinolate system, physicochemical and bacterial properties during fermentation of brine-pickled radishes

The myrosinase-glucosinolate system, physicochemical properties, and bacterial community were profiled during fermentation of high hydrostatic pressure (HHP) pretreated brine-pickled radishes; traditionally brine-pickled radishes were utilised as the control. Scanning electron microscopy (SEM) analysis revealed that 300 MPa pretreatment promoted brine infiltration in radish cells and damaged cellular microstructures, which activated the myrosinase-glucosinolate system. The conversion of glucosinolate (GLs) to isothiocyanates (ITC) increased and significantly enhanced the raphasatin and sulforaphene contents of pickled radish. However, 600 MPa pretreatment suppressed myrosinase activity. HHP pretreatment altered the natural radish bacterial communities by reducing the total bacterial and lactic acid bacteria counts. Lactobacillus spp. became the dominant bacterial genus within 15 d of fermentation. However, the destruction of cellular structures by HHP pretreatment also significantly decreased hardness and caused the dissolution of amino acids and TTA into brine. This caused reduced amino acid and TTA contents compared to the control group, as well as decreases in pH. HHP pretreatment suppressed the growth of spoilage bacteria (e.g. Pseudomonas, Staphylococcus, and Shewanella genera). This study provides new insight into the potential applications of HHP treatment in pickling, as it demonstrates that HHP can increase the ITC conversion rate of pickled radish, modify its physiochemical characteristics, and decrease microbial risk. Therefore, HHP is a promising preprocessing technique to be used for pickle manufacturing industry.

Dynamics of the glucosinolate–myrosinase system in tuber mustard (Brassica juncea var. tumida) during pickling and its relationship with bacterial communities and fermentation characteristics

Pickled tuber mustard is a traditional fermented pickle widely consumed in China, and it is characterized by the presence of glucosinolates (GSLs). To understand the biotransformation of GSLs in tuber mustard during pickling, the dynamics of the glucosinolate–myrosinase (GSL-MYR) system and its potential associations with bacterial communities and fermentation characteristics (i.e., salinity, titratable acidity [TAA], and pH) were investigated. In total, 18 GSLs were identified in fresh tuber mustard; 12 were aliphatic, 4 were indolic, and 2 were aromatic, with aliphatic sinigrin and aromatic gluconasturtiin being the dominating components. The pickling process resulted in complete degradation of GSLs, with isothiocyanates (ITCs) and nitriles being the main breakdown products. Total ITCs reached maximum concentrations on day 21–28, while total nitriles peaked at the end of pickling. Based on Spearman’s correlation analysis, our study showed that lactic acid bacteria (LAB) species might contribute to GSL transformation in pickled tuber mustard. Specifically, Weissella paramesenteroides, Pediococcus pentosaceus, and unclassified Lactococcus exhibited positive correlations with GSL contents (p < 0.01), suggesting that they might contribute to the increasing amounts of GSLs in the initial pickling, while the Lactobacillus-related populations that dominated in the later stages (i.e., Companilactobacillus alimentarius and Lactiplantibacillus Plantarum) were positively correlated with nitrile product concentrations. Moreover, redundancy analysis showed that pH and TAA had strong effect on myrosinase activity during tuber mustard pickling, which was dictated via the organic acids produced by microorganisms. This study provided a perspective for understanding the effect of fermentation on the transformation of tuber mustard GSLs.

The myrosinase-glucosinolate system to generate neoglycoproteins: A case study targeting mannose binding lectins

A convenient strategy for a ‘one-pot’ synthesis of neoglycoproteins (NGP) was developed using the myrosinase-glucosinolate couple, a natural enzyme-substrate system. This enzymatic reaction allowed us to generate an isothiocyanate in situ which then reacted with the lysine residues of bovine serum albumin protein (BSA) to produce multivalent neoglycoproteins. Using two models, glucomoringin which is a natural glucosinolate bearing a l-rhamnose unit, and an artificial glucosinolate specifically designed for mannose type lectins, an average of up to 17.8 and 28.7 carbohydrate residues could be respectively grafted onto the BSA protein. This process is comparable to commercial approaches using BSA-ManC without the disadvantage of handling harmful chemical reagents. Lectin binding screening (GLYcoPROFILE®) showed that among all NGPs synthesized, BSA-Man 16 gave similar and in some cases better affinities in comparison with commercial BSA-Manc towards various mannose-specific lectins.

The Cellular and Subcellular Organization of the Glucosinolate-Myrosinase System against Herbivores and Pathogens.

Glucosinolates are an important class of secondary metabolites in Brassicales plants with a critical role in chemical defense. Glucosinolates are chemically inactive but can be hydrolyzed by myrosinases to produce a range of chemically active compounds toxic to herbivores and pathogens, thereby constituting the glucosinolate–myrosinase defense system or the mustard oil bomb. During the evolution, Brassicales plants have developed not only complex biosynthetic pathways for production of a large number of glucosinolate structures but also different classes of myrosinases that differ in catalytic mechanisms and substrate specificity. Studies over the past several decades have made important progress in the understanding of the cellular and subcellular organization of the glucosinolate–myrosinase system for rapid and timely detonation of the mustard oil bomb upon tissue damage after herbivore feeding and pathogen infection. Progress has also been made in understanding the mechanisms that herbivores and pathogens have evolved to counter the mustard oil bomb. In this review, we summarize our current understanding of the function and organization of the glucosinolate–myrosinase system in Brassicales plants and discuss both the progresses and future challenges in addressing this complex defense system as an excellent model for analyzing plant chemical defense.

Glucosinolate Bioactivation by Apis mellifera Workers and Its Impact on Nosema ceranae Infection at the Colony Level.

The microsporidian fungus Nosema ceranae represents one of the primary bee infection threats worldwide and the antibiotic fumagillin is the only registered product for nosemosis disease control, while few alternatives are, at present, available. Natural bioactive compounds deriving from the glucosinolate–myrosinase system (GSL–MYR) in Brassicaceae plants, mainly isothiocyanates (ITCs), are known for their antimicrobial activity against numerous pathogens and for their health-protective effects in humans. This work explored the use of Brassica nigra and Eruca sativa defatted seed meal (DSM) GSL-containing diets against natural Nosema infection in Apis mellifera colonies. DSM patties from each plant species were obtained by adding DSMs to sugar candy at the concentration of 4% (w/w). The feeding was administered in May to mildly N. ceranae-infected honey bee colonies for four weeks at the dose of 250 g/week. In the treated groups, no significant effects on colony development and bee mortality were observed compared to the negative controls. The N. ceranae abundance showed a slight but significant decrease. Furthermore, the GSL metabolism in bees was investigated, and MYR hydrolytic activity was qualitatively searched in isolated bee midgut and hindgut. Interestingly, MYR activity was detected both in the bees fed DSMs and in the control group where the bees did not receive DSMs. In parallel, ITCs were found in gut tissues from the bees treated with DSMs, corroborating the presence of a MYR-like enzyme capable of hydrolyzing ingested GSLs. On the other hand, GSLs and other GSL hydrolysis products other than ITCs, such as nitriles, were found in honey produced by the treated bees, potentially increasing the health value of the final product for human consumption. The results are indicative of a specific effect on the N. ceranae infection in managed honey bee colonies depending on the GSL activation within the target organ.

Branched-chain amino acid synthesis and glucosinolate-myrosinase system during takuan-zuke processing of radish root.

The traditional Japanese fermented pickle, takuan-zuke, is produced via dehydration and salt-aging of the Japanese winter radish root (Raphanus sativus L.). It has been reported that γ-aminobutyric acid (GABA; antihypertensive factor) accumulates during this production. Herein, the results of the comprehensive study (metabolites, enzyme activity, and gene expression levels were investigated) on the mechanism of branched-chain amino acid (BCAA) synthesis and glucosinolate (GLS)-myrosinase system of daikon dehydration have been reported. BCAAs, GLS, and their precursors were temporarily upregulated, and the genes linked to BCAA, GLS, and isothiocyanate synthesis (BCAT4, MAM3, IPMDH1, RMB1, RMB2, and GRS1) were upregulated during daikon dehydration. BCAAs and GLS accumulated in daikon during dehydration owing to the upregulation of genes, encoding these synthases as a stress response. These results suggest that the biological response of daikon adds characteristic flavor and health functionality to takuan-zuke and helps optimize the processing parameters to produce pickles with improved health benefits. PRACTICAL APPLICATIONS: Takuan-zuke is a popular pickle in Japan, which is produced from a Japanese winter radish root (Raphanus sativus L.), commonly referred to as daikon in Japan. Takuan-zuke is produced by dehydrating daikon either by the process of salt-pressing (shio-oshi) or sun-drying (hoshi). The processes influence the variations in the nutritional value of the final product. The results reveal that both the daikon dehydration processes can be followed to generate increased amounts of health-promoting components (e.g., branched-chain amino acid and glucosinolate) in takuan-zuke.

Testing Eruca sativa defatted seed meal as a potential bioherbicide on selected weeds and crops

Exploiting typical allelopathic molecules released from the glucosinolate-myrosinase system could represent a sustainable strategy for weed management in the near future. The present study investigates the potential bioherbicidal efficacy of Eruca sativa cv. Nemat defatted seed meals (EDSM) in two independent experiments: the first one explored air release of volatile compounds (biofumigation), and the second one focused on the effectiveness of soluble compounds (soaking). Each experiment was carried out using two weed species – Silybum marianum (L.) Gaertn. (milk thistle) and Malva sylvestris L. (mallow) – and two crops frequently cultivated in the Mediterranean basin – Cynara cardunculus L. (cardoon) and Eruca sativa cv. Nemat (rocket) – as targets. The results suggested a new high-value application of EDSM with both methods on both weeds. Mallow was mainly injured by soaking, while milk thistle was mainly affected by biofumigation. An auto-toxic effect of EDSM was also noted on rocket seeds.

Kinetics of glucosinolate hydrolysis by myrosinase in Brassicaceae tissues: A high-performance liquid chromatography approach

Glucosinolates are a group of secondary metabolites occurring in all the vegetables belonging to the Brassicaceae family. Upon tissue damage, glucosinolates are hydrolyzed by myrosinase to a series of degradation products, including isothiocyanates, which are important for their health-promoting effects in humans. The glucosinolate-myrosinase system has been characterized in several Brassica species, of which white mustard (Sinapis alba) has been studied the most. In this study, a new HPLC-UV assay to evaluate the activities and kinetics of myrosinases in aqueous extracts, which closely represent the physiological conditions of plant tissues, was developed. This method was tested on myrosinases extracted from broccoli and cauliflower inflorescences, employing sinigrin and glucoraphanin as substrates. The results showed a strong inhibition of both enzymes at high substrate concentrations. The main issues related to kinetic analysis on the glucosinolate-myrosinase system were also elucidated.

Brassica juncea leaf cuticle proteome analysis shows myrosinase protein, antifreeze activity, and post-translationally modified secretory proteins

Plant cuticle, the site of perception of stress signals, is an extracellular hydrophobic barrier that covers the epidermis of the above-ground parts. This lipidic layer has been explored for its cutin and wax composition. However, reports on the cuticle proteins are scanty. Therefore, leaf cuticle proteins of Brassica juncea isolated using organic solvents (chloroform-methanol, 2:1(v/v)) were analyzed using gel based and quantitative shotgun proteomics. Out of 615 proteins identified, 27% (169) had signal peptides supporting extracellular localization. Bioinformatics tool, QuickGO predicted the involvement of these proteins in catabolism (21%), peptidase activity (13%), oxidoreductase (12%), defense response (9%), fatty acid binding (9%), nutrient reservoir activity (8%), chitin binding (7%) and lipid transport (2%). Myrosinase-catalyzed glucosinolate hydrolysis releases bioactive compounds, which contribute to plant defense. This system is termed as “mustard oil bomb”. Myrosinase and its associating protein, GDSL esterase/lipase ESM1 (involved in cuticle structuring and defense) were detected in the cuticle. GDSL-esterase/lipase ESM1 and β-glucanase (an antifreeze protein) showed in vitro activity. Analysis of cuticle extract by nanoliter osmometer-phase contrast microscopy detected antifreeze activity due to non-protein component. Post-translational modification analysis using PTM viewer predicted N-glycosylation (66%), N-terminal proteolysis (40%), and phosphorylation (32%) to be the dominant modification in the classical secretory proteins. N-glycosylation of myrosinase and GDSL esterase/lipase, ESM1 was confirmed by Con A affinoblotting. This study not only identified leaf cuticle proteins, but also laid the foundation for exploring the extracellular glucosinolate-myrosinase system, PTM crosstalk, and antifreeze activity as stress adaptive strategies in B. juncea.

The Imaging of Guard Cells of thioglucosidase (tgg) Mutants of Arabidopsis Further Links Plant Chemical Defence Systems with Physical Defence Barriers.

The glucosinolate-myrosinase system is a well-known plant chemical defence system. Two functional myrosinase-encoding genes, THIOGLUCOSIDASE 1 (TGG1) and THIOGLUCOSIDASE 2 (TGG2), express in aerial tissues of Arabidopsis. TGG1 expresses in guard cells (GCs) and is also a highly abundant protein in GCs. Recently, by studying wild type (WT), tgg single, and double mutants, we showed a novel association between the glucosinolate-myrosinase system defence system, and a physical barrier, the cuticle. In the current study, using imaging techniques, we further analysed stomata and ultrastructure of GCs of WT, tgg1, tgg2 single, and tgg1 tgg2 double mutants. The tgg mutants showed distinctive features of GCs. The GCs of tgg1 and tgg1 tgg2 mutants showed vacuoles that had less electron-dense granular material. Both tgg single mutants had bigger stomata complexes. The WT and tgg mutants also showed variations for cell wall, chloroplasts, and starch grains of GCs. Abscisic acid (ABA)-treated stomata showed that the stomatal aperture was reduced in tgg1 single and tgg1 tgg2 double mutants. The data provides a basis to perform comprehensive further studies to find physiological and molecular mechanisms associated with ultrastructure differences in tgg mutants. We speculate that the absence of myrosinase alters the endogenous chemical composition, hence affecting the physical structure of plants and the plants’ physical defence barriers.

Effect of temperature and insect herbivory on the regulation of glucosinolate-myrosinase system in Lepidium latifolium.

The glucosinolate-myrosinase (GLS-MYR) system is an important component of plant-insect interactions. However, there is no report on its performance in field conditions where the plants are subjected to both abiotic and biotic pressures simultaneously. We investigated the GLS-MYR system in a Himalayan ecotype of Lepidium latifolium that is recognized for its adaptive potential in field conditions. In order to understand the independent contribution of temperature and Pieris brassicae herbivory on the components of the GLS-MYR system, different conditions were simulated in the growth chamber. During field conditions, the final GLS hydrolysis products were found to be regulated by the metabolic GLS levels, the temperature conditions, and the density of insect interactions. These factors influence the expression of the hydrolyzing and specifier proteins, which further affects the GLS hydrolysis products. Our results suggest that the production of hydrolysis products is differentially affected under field conditions. While allyl isothiocyanate is significantly (P ≤ 0.05) affected by temperature but not insect density, 1-cyano-2,3-epithiopropane is not affected by either. The study shows that the outcome of the GLS-MYR system in a plant is a consequence of the combinatorial effect of ecophysiological factors and the insect interactions that eventually decide the performance of a plant in an environment.

Glucosinolate Biosynthesis and the Glucosinolate–Myrosinase System in Plant Defense

Insect pests represent a major global challenge to important agricultural crops. Insecticides are often applied to combat such pests, but their use has caused additional challenges such as environmental contamination and human health issues. Over millions of years, plants have evolved natural defense mechanisms to overcome insect pests and pathogens. One such mechanism is the production of natural repellents or specialized metabolites like glucosinolates. There are three types of glucosinolates produced in the order Brassicales: aliphatic, indole, and benzenic glucosinolates. Upon insect herbivory, a “mustard oil bomb” consisting of glucosinolates and their hydrolyzing enzymes (myrosinases) is triggered to release toxic degradation products that act as insect deterrents. This review aims to provide a comprehensive summary of glucosinolate biosynthesis, the “mustard oil bomb”, and how these metabolites function in plant defense against pathogens and insects. Understanding these defense mechanisms will not only allow us to harness the benefits of this group of natural metabolites for enhancing pest control in Brassicales crops but also to transfer the “mustard oil bomb” to non-glucosinolate producing crops to boost their defense and thereby reduce the use of chemical pesticides.

Novel glucosinolate metabolism in larvae of the leaf beetle Phaedon cochleariae

Plants of the Brassicales are defended by a binary system, in which glucosinolates are degraded by myrosinases, forming toxic breakdown products such as isothiocyanates and nitriles. Various detoxification pathways and avoidance strategies have been found that allow different herbivorous insect taxa to deal with the glucosinolate-myrosinase system of their host plants. Here, we investigated how larvae of the leaf beetle species Phaedon cochleariae (Coleoptera: Chrysomelidae), a feeding specialist on Brassicaceae, cope with this binary defence. We performed feeding experiments using leaves of watercress (Nasturtium officinale, containing 2-phenylethyl glucosinolate as major glucosinolate and myrosinases) and pea (Pisum sativum, lacking glucosinolates and myrosinases), to which benzenic glucosinolates (benzyl- or 4-hydroxybenzyl glucosinolate) were applied. Performing comparative metabolomics using UHPLC-QTOF-MS/MS, N-(phenylacetyl) aspartic acid, N-(benzoyl) aspartic acid and N-(4-hydroxybenzoyl) aspartic acid were identified as major metabolites of 2-phenylethyl-, benzyl- and 4-hydroxybenzyl glucosinolate, respectively, in larvae and faeces. This suggests that larvae of P. cochleariae metabolise isothiocyanates or nitriles to aspartic acid conjugates of aromatic acids derived from the ingested benzenic glucosinolates. Myrosinase measurements revealed activity only in second-instar larvae that were fed with watercress, but not in freshly moulted and starved second-instar larvae fed with pea leaves. Our results indicate that the predicted pathway can occur independently of the presence of plant myrosinases, because the same major glucosinolate-breakdown metabolites were found in the larvae feeding on treated watercress and pea leaves. A conjugation of glucosinolate-derived compounds with aspartic acid is a novel metabolic pathway that has not been described for other herbivores.

The phytopathogenic fungus Sclerotinia sclerotiorum detoxifies plant glucosinolate hydrolysis products via an isothiocyanate hydrolase

Brassicales plants produce glucosinolates and myrosinases that generate toxic isothiocyanates conferring broad resistance against pathogens and herbivorous insects. Nevertheless, some cosmopolitan fungal pathogens, such as the necrotrophic white mold Sclerotinia sclerotiorum, are able to infect many plant hosts including glucosinolate producers. Here, we show that S. sclerotiorum infection activates the glucosinolate-myrosinase system, and isothiocyanates contribute to resistance against this fungus. S. sclerotiorum metabolizes isothiocyanates via two independent pathways: conjugation to glutathione and, more effectively, hydrolysis to amines. The latter pathway features an isothiocyanate hydrolase that is homologous to a previously characterized bacterial enzyme, and converts isothiocyanate into products that are not toxic to the fungus. The isothiocyanate hydrolase promotes fungal growth in the presence of the toxins, and contributes to the virulence of S. sclerotiorum on glucosinolate-producing plants.

Inhibition of light-induced stomatal opening by allyl isothiocyanate does not require guard cell cytosolic Ca2+ signaling.

The glucosinolate–myrosinase system is a well-known defense system that has been shown to induce stomatal closure in Brassicales. Isothiocyanates are highly reactive hydrolysates of glucosinolates, and an isothiocyanate, allyl isothiocyanate (AITC), induces stomatal closure accompanied by elevation of free cytosolic Ca2+ concentration ([Ca2+]cyt) in Arabidopsis. It remains unknown whether AITC inhibits light-induced stomatal opening. This study investigated the role of Ca2+ in AITC-induced stomatal closure and inhibition of light-induced stomatal opening. AITC induced stomatal closure and inhibited light-induced stomatal opening in a dose-dependent manner. A Ca2+ channel inhibitor, La3+, a Ca2+chelator, EGTA, and an inhibitor of Ca2+ release from internal stores, nicotinamide, inhibited AITC-induced [Ca2+]cyt elevation and stomatal closure, but did not affect inhibition of light-induced stomatal opening. AITC activated non-selective Ca2+-permeable cation channels and inhibited inward-rectifying K+ (K+in) channels in a Ca2+-independent manner. AITC also inhibited stomatal opening induced by fusicoccin, a plasma membrane H+-ATPase activator, but had no significant effect on fusicoccin-induced phosphorylation of the penultimate threonine of H+-ATPase. Taken together, these results suggest that AITC induces Ca2+ influx and Ca2+ release to elevate [Ca2+]cyt, which is essential for AITC-induced stomatal closure but not for inhibition of K+in channels and light-induced stomatal opening.