Reducing progoitrin and enriching glucoraphanin in Braasica napus seeds through silencing of the GSL-ALK gene family

The hydrolytic products of glucosinolates in brassica crops are bioactive compounds. Some glucosinolate derivatives such as oxazolidine-2-thione from progoitrin in brassica oilseed meal are toxic and detrimental to animals, but some isothiocyanates such as sulforaphane are potent anti-carcinogens that have preventive effects on several human cancers. In most B. rapa, B. napus and B. juncea vegetables and oilseeds, there is no or only trace amount of glucoraphanin that is the precursor to sulforaphane. In this paper, RNA interference (RNAi) of the GSL-ALK gene family was used to down-regulate the expression of GSL-ALK genes in B. napus. The detrimental glucosinolate progoitrin was reduced by 65 %, and the beneficial glucosinolate glucoraphanin was increased to a relatively high concentration (42.6 μmol g(-1) seed) in seeds of B. napus transgenic plants through silencing of the GSL-ALK gene family. Therefore, there is potential application of the new germplasm with reduced detrimental glucosinolates and increased beneficial glucosinolates for producing improved brassica vegetables.     

The ‘mustard oil bomb’: not so easy to assemble?! Localization, expression and distribution of the components of the myrosinase enzyme system

Glucosinolates are plant secondary metabolites that are hydrolysed by the action of myrosinases into various products (isothiocyanates, thiocyanates, epithionitriles, nitriles, oxazolidines). Massive hydrolysis of glucosinolates occurs only upon tissue damage but there is also evidence indicating metabolism of glucosinolates in intact plant tissues. It was originally believed that the glucosinolate–myrosinase system in intact plants was stable due to a spatial separation of the components. This has been coined as the ‘mustard oil bomb’ theory. Proteins that form complexes with myrosinases have been described: myrosinase-binding proteins (MBPs) and myrosinase-associated proteins (MyAPs/ESM). The roles of these proteins and their biological relevance are not yet completely known. Other proteins of the myrosinase enzyme system are the epithiospecifier protein (ESP) and the thiocyanate-forming protein (TFP) that divert the glucosinolate hydrolysis from isothiocyanate production to nitrile/epithionitrile or thiocyanate production. Some glucosinolate hydrolysis products act as plant defence compounds against insects and pathogens or have beneficial health effects on humans. In this review, we survey and critically assess the available information concerning the localization, both at the tissular/cellular and subcellular level, of the different components of the myrosinase enzyme system. Data from the model plant Arabidopsis thaliana is compared to that from other glucosinolate-producing Brassicaceae in order to show common as well as divergent features of the ‘mustard oil bomb’ among these species.     

HPLC-based kinetics assay facilitates analysis of systems with multiple reaction products and thermal enzyme denaturation

Glucosinolates are plant secondary metabolites abundant in Brassica vegetables that are substrates for the enzyme myrosinase, a thioglucoside hydrolase. Enzyme-mediated hydrolysis of glucosinolates forms several organic products, including isothiocyanates (ITCs) that have been explored for their beneficial effects in humans. Myrosinase has been shown to be tolerant of non-natural glucosinolates, such as 2,2-diphenylethyl glucosinolate, and can facilitate their conversion to non-natural ITCs, some of which are leads for drug development. An HPLC-based method capable of analyzing this transformation for non-natural systems has been described. This current study describes (1) the Michaelis–Menten characterization of 2,2-diphenyethyl glucosinolate and (2) a parallel evaluation of this analogue and the natural analogue glucotropaeolin to evaluate effects of pH and temperature on rates of hydrolysis and product(s) formed. Methods described in this study provide the ability to simultaneously and independently analyze the kinetics of multiple reaction components. An unintended outcome of this work was the development of a modified Lambert W(x) which includes a parameter to account for the thermal denaturation of enzyme. The results of this study demonstrate that the action of Sinapis alba myrosinase on natural and non-natural glucosinolates is consistent under the explored range of experimental conditions and in relation to previous accounts.     

Glucosinolates in <Emphasis Type="Italic">Brassica</Emphasis> foods: bioavailability in food and significance for human health

Glucosinolates are sulphur compounds that are prevalent in Brassica genus. This includes crops cultivated as vegetables, spices and sources of oil. Since 1970s glucosinolates and their breakdown products, have been widely studied by their beneficial and prejudicial biological effects on human and animal nutrition. They have also been found to be partly responsible for the characteristic flavor of Brassica vegetables. In recent years, considerable attention has been paid to cancer prevention by means of natural products. The cancer-protective properties of Brassica intake are mediated through glucosinolates. Isothyocianate and indole products formed from glucosinolates may regulate cancer cell development by regulating target enzymes, controlling apoptosis and blocking the cell cycle. Nevertheless, variation in content of both glucosinolates and their bioactive hydrolysis products depends on both genetics and the environment, including crop management practices, harvest and storage, processing and meal preparation. Here, we review the significance of glucosinolates as source of bioactive isothiocyanates for human nutrition and health and the influence of environmental conditions and processing mechanisms on the content of glucosinolate concentration in Brassica vegetables. Currently, this area is only partially understood. Further research is needed to understand the mechanisms by which the environment and processing affect glucosinolates content of Brassica vegetables. This will allow us to know the genetic control of these variables, what will result in the development of high quality Brassica products with a health-promoting activity.     

Hydrolysis products of glucosinolates in Brassica napus tissues as inhibitors of seed germination

Enzymatic hydrolysis of glucosinolates, a class of compounds found in Brassica species, results in a number of products with potential to inhibit seed germination. To investigate the impact of both volatile and water-soluble allelochemicals, germination bioassays using Lactuca sativa seeds were conducted with root and combined leaf and stem tissues of Brassica napus. Tissues in which glucosinolates were hydrolyzed to remove volatile glucosinolate degradation products were compared with intact tissues and water controls. Only tissues containing glucosinolates produced volatiles that inhibited germination. Volatiles were trapped and identified using GC-MS. Volatiles produced in greater quanitity from intact tissues than from tissues without glucosinolates were almost exclusively glucosinolate hydrolysis products. Water-soluble components also inhibited germination. Chemical analysis of extracts confirmed the presence of glucosinolate hydrolysis products, but indicated the involvement of additional allelochemicals, especially in leaf and stem tissues. Results support the proposal that glucosinolate-containing plant tissues may contribute to reductions in synthetic pesticide use if weed seeds are targeted.Abbreviations ITC isothiocyanates - CN organic cyanides - OZT oxazolidinethione - iRoot intact root tissue - iL&S intact leaf and stem tissue - hRoot hydrolyzed root tissue - hL&S hydrolyzed leaf and stem tissue     

Leaching of isothiocyanates through intact soil following simulated biofumigation

Biofumigation can be used as an alternative to conventional soil fumigation to control soil-borne pests. With biofumigation, plant tissue with a natural content of glucosinolates (cruciferous plants) is damaged and incorporated into the topsoil. When the plant tissue is damaged, the glucosinolates come into contact with the endogenous enzyme myrosinase, which catalyse the hydrolysis of glucosinolates into various products depending on the reaction conditions. Isothiocyanates are among the potential products formed from these reactions. We investigated if the isothiocyanates from rape plant material were leached through the soil to drain depth when a heavy rainstorm followed the biofumigation. We applied isothiocyanates from rape plant material (1,480 μmol m⁻²) to four large (0.6 m diameter, 1.0 m long) intact soil monoliths from a loamy and a sandy soil and conducted a leaching experiment under semi-field conditions. The soil monoliths were irrigated with 70–90 mm (10 mm h⁻¹) and the concentrations of three isothiocyanates (3-butenyl, 4-pentenyl and 2-phenethyl) were monitored in the leachate. Between 0 and 14.8 mmol isothiocyanates were leached for each mol of isothiocyanates applied during application of 70–90 mm irrigation. The distribution coefficient estimated from leached concentrations was 0.04–1.19 for 3-butenyl, 0.04–1.15 for 4-pentenyl isothiocyanate and 0.037–0.97 for 2-phenethyl isothiocyanate. The concentration of total isothiocyanates in the leachate was in the same order of magnitude as the LD₅₀ of isothiocyanates for sensitive aquatic organisms.     

High-performance liquid chromatography-based method to evaluate kinetics of glucosinolate hydrolysis by Sinapis alba myrosinase

Isothiocyanates (ITCs) are one of several hydrolysis products of glucosinolates, plant secondary metabolites that are substrates for the thioglucohydrolase myrosinase. Recent pursuits toward the development of synthetic non-natural ITCs have consequently led to an exploration of generating these compounds from non-natural glucosinolate precursors. Evaluation of the myrosinase-dependent conversion of select non-natural glucosinolates to non-natural ITCs cannot be accomplished using established ultraviolet–visible (UV–Vis) spectroscopic methods. To overcome this limitation, an alternative high-performance liquid chromatography (HPLC)-based analytical approach was developed where initial reaction velocities were generated from nonlinear reaction progress curves. Validation of this HPLC method was accomplished through parallel evaluation of three glucosinolates with UV–Vis methodology. The results of this study demonstrate that kinetic data are consistent between both analytical methods and that the tested glucosinolates respond similarly to both Michaelis–Menten and specific activity analyses. Consequently, this work resulted in the complete kinetic characterization of three glucosinolates with Sinapis alba myrosinase, with results that were consistent with previous reports.     

Type and concentration of redox reagents influencing nitrile formation upon myrosinase (Brassica carinata)-catalyzed hydrolysis of glucosibarin

The ratio of isothiocyanates (ITCs) to nitriles formed in the myrosinase-catalyzed hydrolysis of glucosinolates is a key factor determining the physiological effect of glucosinolate containing plants and materials. In this context, the mechanism by which nitrile formation occurs is not well understood. In the present paper we have studied the effect of three redox reagents – Fe²⁺, glutathione (GSH) and ascorbic acid – on the profile of products obtained upon the hydrolysis of a model glucosinolate (glucosibarin ((2R)-2-hydroxy-2-phenylethylglucosinolate)) catalyzed by Brassica carinata myrosinase. A Micellar Electrokinetic Capillary Chromatography method that allows following on-line the hydrolysis of the glucosinolate, the formation of the degradation products and the oxidation of GSH was used. Increasing the concentration of Fe²⁺ and GSH (from 0.25- to 2-fold molar excess with respect to the glucosinolate) increased the ratio of nitrile ((2R)-2-hydroxy-2-phenylethylcyanide) to oxazolidine-2-thione ((5S)-5-phenyloxazolidine-2-thione), whereas increasing the concentration of ascorbic acid decreased this ratio. Low concentrations of ascorbic acid favored nitrile formation. A mechanism for nitrile formation involving a disulfide bond in the myrosinase complex is proposed.     

Interactions between glucosinolate- and myrosinase-containing plants and the sawfly Athalia rosae

Several insects have specialised on using Brassicaceae as host plants. Therefore, they evolved metabolic pathways to cope with the defensive glucosinolate–myrosinase system of their diet. Larvae of the turnip sawfly, Athalia rosae L. (Hymenoptera: Tenthredinidae), incorporate various glucosinolates from their hosts into their haemolymph. The ability to sequester these metabolites makes A. rosae a useful model system to study mechanisms of glucosinolate metabolism in this species compared to other specialists, and to study effects of sawfly feeding on levels of glucosinolates and their hydrolysing enzymes in plants. The levels of plant metabolites might in turn directly affect the performance of the insect. On the one hand, costs for glucosinolate uptake and avoidance of myrosinase activity were postulated. On the other hand, sequestration of glucosinolates can be part of the insect’s defence against several predators. Here, the findings on glucosinolate metabolic pathways are compared between different herbivores and the sawfly. The impact of different glucosinolate levels and myrosinase activities on the performance of A. rosae is discussed. Furthermore, effects of feeding by A. rosae larvae on the chemical composition and enzyme activities of various Brassicaceae species are summarised. Induction patterns vary not only between different plant species and cultivars but also due to the inducing agent. Finally, the plant–herbivore interactions are discussed with regard to the sawflies’ defence abilities against different carnivore guilds.     

Differing mechanisms of simple nitrile formation on glucosinolate degradation in Lepidium sativum and Nasturtium officinale seeds

Glucosinolates are sulphur-containing glycosides found in brassicaceous plants that can be hydrolysed enzymatically by plant myrosinase or non-enzymatically to form primarily isothiocyanates and/or simple nitriles. From a human health perspective, isothiocyanates are quite important because they are major inducers of carcinogen-detoxifying enzymes. Two of the most potent inducers are benzyl isothiocyanate (BITC) present in garden cress (Lepidium sativum), and phenylethyl isothiocyanate (PEITC) present in watercress (Nasturtium officinale). Previous studies on these salad crops have indicated that significant amounts of simple nitriles are produced at the expense of the isothiocyanates. These studies also suggested that nitrile formation may occur by different pathways: (1) under the control of specifier protein in garden cress and (2) by an unspecified, non-enzymatic path in watercress. In an effort to understand more about the mechanisms involved in simple nitrile formation in these species, we analysed their seeds for specifier protein and myrosinase activities, endogenous iron content and glucosinolate degradation products after addition of different iron species, specific chelators and various heat treatments. We confirmed that simple nitrile formation was predominantly under specifier protein control (thiocyanate-forming protein) in garden cress seeds. Limited thermal degradation of the major glucosinolate, glucotropaeolin (benzyl glucosinolate), occurred when seed material was heated to >120 °C. In the watercress seeds, however, we show for the first time that gluconasturtiin (phenylethyl glucosinolate) undergoes a non-enzymatic, iron-dependent degradation to a simple nitrile. On heating the seeds to 120 °C or greater, thermal degradation of this heat-labile glucosinolate increased simple nitrile levels many fold.     

A thiocyanate-forming protein generates multiple products upon allylglucosinolate breakdown in Thlaspi arvense

Glucosinolates, amino acid-derived thioglycosides found in plants of the Brassicales order, are one of the best studied classes of plant secondary metabolites. Together with myrosinases and supplementary proteins known as specifier proteins, they form the glucosinolate–myrosinase system that upon tissue damage gives rise to a number of biologically active glucosinolate breakdown products such as isothiocyanates, epithionitriles and organic thiocyanates involved in plant defense. While isothiocyanates are products of the spontaneous rearrangement of the glucosinolate aglycones released by myrosinase, the formation of epithionitriles and organic thiocyanates depends on both myrosinases and specifier proteins. Hydrolysis product profiles of many glucosinolate-containing plant species indicate the presence of specifier proteins, but only few have been identified and characterized biochemically. Here, we report on cDNA cloning, heterologous expression and characterization of TaTFP, a thiocyanate-forming protein (TFP) from Thlaspi arvense L. (Brassicaceae), that is expressed in all plant organs and can be purified in active form after heterologous expression in Escherichia coli. As a special feature, this protein promotes the formation of allylthiocyanate as well as the corresponding epithionitrile upon myrosinase-catalyzed hydrolysis of allylglucosinolate, the major glucosinolate of T. arvense. All other glucosinolates tested are converted to their simple nitriles when hydrolyzed in the presence of TaTFP. Despite its ability to promote allylthiocyanate formation, TaTFP has a higher amino acid sequence similarity to known epithiospecifier proteins (ESPs) than to Lepidium sativum TFP. However, unlike Arabidopsis thaliana ESP, its activity in vitro is not strictly dependent on Fe²⁺ addition to the assay mixtures. The availability of TaTFP in purified form enables future studies to be aimed at elucidating the structural bases of specifier protein specificities and mechanisms. Furthermore, identification of TaTFP shows that product specificities of specifier proteins can not be predicted based on amino acid sequence similarity and raises interesting questions about specifier protein evolution.     

The enzymic and chemically induced decomposition of glucosinolates

While the myrosinase-glucosinolate system has been reviewed in recent years by a number of authors, little attention has been paid to the enzymic and non-enzymic degradation of glucosinolates. Non-enzymic degradation processes are particularly important in the processing of brassica vegetables with respect to both flavour and in the role of glucosinolates as precursors of anticancer compounds in the diet. This review highlights early empirical work on glucosinolate degradation along with more recent aspects related to current research on mechanism of glucosinolate degradation in plants, microbes and animals.     

ESP and ESM1 mediate indol-3-acetonitrile production from indol-3-ylmethyl glucosinolate in Arabidopsis

Glucosinolates are plant secondary metabolites that act as direct defenses against insect herbivores and various pathogens. Recent analysis has shown that methionine-derived glucosinolates are hydrolyzed/activated into either nitriles or isothiocyanates depending upon the plants genotype at multiple loci. While it has been hypothesized that tryptophan-derived glucosinolates can be a source of indole-acetonitriles, it has not been explicitly shown if the same proteins control nitrile production from tryptophan-derived glucosinolates as from methionine-derived glucosinolates. In this report, we formally test if the proteins involved in controlling aliphatic glucosinolate hydrolysis during tissue disruption can control production of nitriles during indolic glucosinolate hydrolysis. We show that myrosinase is not sufficient for indol-3-acetonitrile production from indol-3-ylmethyl glucosinolate and requires the presence of functional epithospecifier protein in planta and in vitro to produce significant levels of indol-3-acetonitrile. This reaction is also controlled by the Epithiospecifier modifier 1 gene. Thus, like formation of nitriles from aliphatic glucosinolates, indol-3-acetonitrile production following tissue disruption is controlled by multiple loci raising the potential for complex regulation and fine tuning of indol-3-acetonitrile production from indol-3-ylmethyl glucosinolate.     

Role of camalexin, indole glucosinolates, and side chain modification of glucosinolate-derived isothiocyanates in defense of Arabidopsis against Sclerotinia sclerotiorum

Plant secondary metabolites are known to facilitate interactions with a variety of beneficial and detrimental organisms, yet the contribution of specific metabolites to interactions with fungal pathogens is poorly understood. Here we show that, with respect to aliphatic glucosinolate‐derived isothiocyanates, toxicity against the pathogenic ascomycete Sclerotinia sclerotiorum depends on side chain structure. Genes associated with the formation of the secondary metabolites camalexin and glucosinolate were induced in Arabidopsis thaliana leaves challenged with the necrotrophic pathogen S. sclerotiorum. Unlike S. sclerotiorum, the closely related ascomycete Botrytis cinerea was not identified to induce genes associated with aliphatic glucosinolate biosynthesis in pathogen‐challenged leaves. Mutant plant lines deficient in camalexin, indole, or aliphatic glucosinolate biosynthesis were hypersusceptible to S. sclerotiorum, among them the myb28 mutant, which has a regulatory defect resulting in decreased production of long‐chained aliphatic glucosinolates. The antimicrobial activity of aliphatic glucosinolate‐derived isothiocyanates was dependent on side chain elongation and modification, with 8‐methylsulfinyloctyl isothiocyanate being most toxic to S. sclerotiorum. This information is important for microbial associations with cruciferous host plants and for metabolic engineering of pathogen defenses in cruciferous plants that produce short‐chained aliphatic glucosinolates.     

Glucosinolates – biochemistry, genetics and biological activity

This paper provides a brief overview of the biochemistry, genetics andbiological activity of glucosinolates and their degradation products.These compounds are found in vegetative and reproductive tissues of16 plant families, but are most well known as the major secondarymetabolites in the Brassicaceae. Following tissue disruption, theyare hydrolysed to a variety of products of which isothiocyanates(`mustard oils') are the most prominent. The majority of geneticstudies have concentrated on reducing the levels of these compoundsin the seeds of oilseed Brassica crops due to antinutritionalfactors associated with 2-hydroxy-3-butenyl glucosinolate. However,current interest is concerned with the anticarcinogenic activity ofisothiocyanates derived from cruciferous vegetables and salad crops.     

High glucosinolate broccoli: a delivery system for sulforaphane

The development of hybrid broccoli genotypes with enhanced levels of 4-methylsulphinylbutyl glucosinolate, the precursor of anticarcinogenic isothiocyanate sulforaphane (SF), by introgressing genomic segments from the wild ancestor Brassica villosa is described. We demonstrate that to obtain enhanced levels of either 3-methylsulphinylpropyl or 4-methylsulphinylbutyl glucosinolate it is necessary to have B. villosa alleles in either a homozygous or heterozygous state at a single quantitative trait locus (QTL) on O2. The ratio of these two glucosinolates, and thus whether iberin or SF is generated upon hydrolysis, is determined by the presence or absence of B. villosa alleles at this QTL, but also at an additional QTL2 on O5. We further demonstrate that following mild cooking high glucosinolate broccoli lines generate about three fold higher levels of SF than conventional varieties. Commercial freezing processes and storage of high glucosinolate broccoli maintains the high level of glucosinolates compared to standard cultivars, although the blanching process denatures the endogenous myrosinase activity.     

Glucosinolates in oilseed rape: secondary metabolites that influence interactions with herbivores and their natural enemies

Glucosinolates are sulphur‐containing secondary metabolites characteristic of Brassicaceous plants. Glucosinolate breakdown products, which include isothiocyanates, are released following tissue damage when hydrolytic enzymes act on them. The isothiocyanates have toxic effects on generalist herbivores when they attempt to feed on oilseed rape, Brassica napus, and also function as repellents. However, specialist herbivores such as Brevicoryne brassicae aphids, flea beetles, Psylliodes chrysocephala and the Lepidopteran pest, Pieris rapae, are adapted to the presence of glucosinolates and thrive on plants containing them. They may do this by avoiding tissue damage to prevent the formation of isothiocyanates or by metabolising or tolerating glucosinolates. For many specialist herbivores, the isothiocyanates function as attractants and glucosinolates can even be sequestered for defence against predatory insects. Thus, these herbivores have evolved resistance to host‐plant secondary metabolites and this type of evolutionary history may have given some insects an enhanced ability to adapt to xenobiotics. In an agricultural context, this may make pests better able to evolve resistance to artificially applied pesticides. The effect of increased glucosinolate content in making oilseed rape cultivars more susceptible to specialist pests was highlighted in a seminal article in the Annals of Applied Biology in 1995. This review of the literature considers developments in this area since then.