Plant Myrosinase Research Articles

Broccoli Consumption Impacts the Human Gastrointestinal Microbiota

BackgroundThe human gastrointestinal (GI) microbiota is increasingly linked to health outcomes; however, our understanding of how specific foods alter the microbiota is limited. Cruciferous vegetables such as broccoli have been associated with cancer prevention as a result of their high levels of glucosinolates, especially glucoraphanin. The activation of glucoraphanin to its isothiocyanate form, sulforaphane, is dependent on the enzyme myrosinase. Myrosinase is naturally found in broccoli, but is degraded by cooking. It has been hypothesized that plasma sulforaphane observed in persons eating cooked broccoli may be related to glucoraphanin hydrolysis by microbial myrosinase. Myrosinase activity is greater in rodents who regularly consume cruciferous vegetables compared to those who do not, suggesting a priming effect on the microbiota. However, it remains to be discovered which members of the microbiota are responsible for this action in clinical populations.ObjectiveWe aimed to determine the impact of broccoli consumption on the GI microbiota of healthy adult men and women as a means to understand the role of microbes in glucosinolate hydrolysis.MethodsA controlled feeding, randomized, crossover study consisting of two 17‐day treatment periods separated by a 24‐day washout was conducted in healthy adults (n=18). Participants were fed at weight maintenance, and received a Brassica‐free control diet or the same diet with 200 g of cooked broccoli and 20 g of fresh daikon radish (as a source of plant myrosinase) per day. Fecal samples were collected at baseline and at the end of each treatment period for microbial analysis. Following DNA isolation, bacterial, archaeal, and fungal barcoded amplicon pools were generated using a Fluidigm Access Array system prior to high‐throughput sequencing on an Illumina MiSeq. Data were analyzed using QIIME 1.8.0 and 1.9.1 and SAS 9.4.ResultsBeta diversity analysis based on 97% operational taxonomic unit composition and abundances indicated that bacterial communities were impacted by treatment (p=0.03). Broccoli consumption resulted in a significant change in the proportion of Bacteroidetes relative to Firmicutes, whereby participants consuming broccoli increased this ratio by 37% from baseline to end, compared to a 5% reduction during the control period (p=0.02). Bacteroides increased by 6% following broccoli consumption and decreased by 2% in the control group, but the difference between these two groups was not statistically significant (p=0.15).ConclusionThese novel results reveal that broccoli consumption affects the diversity and composition of the GI microbiota of healthy adults. These data help fill the gap in knowledge related to the role of bacterial hydrolysis of phytonutrients. The increase in Bacteroides spp. is particularly relevant because Bacteroides thetaiotaomicron has been shown in vitro to utilize glucosinolates. Additional study is ongoing to determine if phenotypic responses are related to archaea and body mass index.Support or Funding InformationFunding provided by the National Cancer Institute and the USDA.

Arabidopsis Myrosinase Genes AtTGG4 and AtTGG5 Are Root-Tip Specific and Contribute to Auxin Biosynthesis and Root-Growth Regulation.

Plant myrosinases (β-thioglucoside glucohydrolases) are classified into two subclasses, Myr I and Myr II. The biological function of Myr I has been characterized as a major biochemical defense against insect pests and pathogens in cruciferous plants. However, the biological function of Myr II remains obscure. We studied the function of two Myr II member genes AtTGG4 and AtTGG5 in Arabidopsis. RT-PCR showed that both genes were specifically expressed in roots. GUS-assay revealed that both genes were expressed in the root-tip but with difference: AtTGG4 was expressed in the elongation zone of the root-tip, while AtTGG5 was expressed in the whole root-tip. Moreover, myrosin cells that produce and store the Myr I myrosinases in aboveground organs were not observed in roots, and AtTGG4 and AtTGG5 were expressed in all cells of the specific region. A homozygous double mutant line tgg4tgg5 was obtained through cross-pollination between two T-DNA insertion lines, tgg4E8 and tgg5E12, by PCR-screening in the F2 and F3 generations. Analysis of myrosinase activity in roots of mutants revealed that AtTGG4 and AtTGG5 had additive effects and contributed 35% and 65% myrosinase activity in roots of the wild type Col-0, respectively, and myrosinase activity in tgg4tgg5 was severely repressed. When grown in Murashiege & Skoog (MS) medium or in soil with sufficient water, Col-0 had the shortest roots, and tgg4tgg5 had the longest roots, while tgg4E8 and tgg5E12 had intermediate root lengths. In contrast, when grown in soil with excessive water, Col-0 had the longest roots, and tgg4tgg5 had the shortest roots. These results suggested that AtTGG4 and AtTGG5 regulated root growth and had a role in flood tolerance. The auxin-indicator gene DR5::GUS was then introduced into tgg4tgg5 by cross-pollination. DR5::GUS expression patterns in seedlings of F1, F2, and F3 generations indicated that AtTGG4 and AtTGG5 contributed to auxin biosynthesis in roots. The proposed mechanism is that indolic glucosinolate is transported to the root-tip and converted to indole-3-acetonitrile (IAN) in the tryptophan-dependent pathways by AtTGG4 and AtTGG5, and IAN is finally converted to indole-3-acetic acid (IAA) by nitrilases in the root-tip. This mechanism guarantees the biosynthesis of IAA in correct cells of the root-tip and, thus, a correct auxin gradient is formed for healthy development of roots.

Cyanide detoxification in an insect herbivore: Molecular identification of β-cyanoalanine synthases from Pieris rapae

Cyanogenic compounds occur widely in the plant kingdom. Therefore, many herbivores are adapted to the presence of these compounds in their diet by either avoiding cyanide release or by efficient cyanide detoxification mechanisms. The mechanisms of adaptation are not fully understood. Larvae of Pieris rapae (Lepidoptera: Pieridae) are specialist herbivores on glucosinolate-containing plants. They are exposed to cyanide during metabolism of phenylacetonitrile, a product of benzylglucosinolate breakdown catalyzed by plant myrosinases and larval nitrile-specifier protein (NSP) in the gut. Cyanide is metabolized to β-cyanoalanine and thiocyanate in the larvae. Here, we demonstrate that larvae of P. rapae possess β-cyanoalanine activity in their gut. We have identified three gut-expressed cDNAs designated PrBSAS1-PrBSAS3 which encode proteins with similarity to β-substituted alanine synthases (BSAS). Characterization of recombinant PrBSAS1-PrBSAS3 shows that they possess β-cyanoalanine activity. In phylogenetic trees, PrBSAS1-PrBSAS3, the first characterized insect BSAS, group together with a characterized mite β-cyanoalanine synthase and bacterial enzymes indicating a similar evolutionary history.

Sulforaphane Bioavailability from Glucoraphanin-Rich Broccoli: Control by Active Endogenous Myrosinase.

Glucoraphanin from broccoli and its sprouts and seeds is a water soluble and relatively inert precursor of sulforaphane, the reactive isothiocyanate that potently inhibits neoplastic cellular processes and prevents a number of disease states. Sulforaphane is difficult to deliver in an enriched and stable form for purposes of direct human consumption. We have focused upon evaluating the bioavailability of sulforaphane, either by direct administration of glucoraphanin (a glucosinolate, or β-thioglucoside-N-hydroxysulfate), or by co-administering glucoraphanin and the enzyme myrosinase to catalyze its conversion to sulforaphane at economic, reproducible and sustainable yields. We show that following administration of glucoraphanin in a commercially prepared dietary supplement to a small number of human volunteers, the volunteers had equivalent output of sulforaphane metabolites in their urine to that which they produced when given an equimolar dose of glucoraphanin in a simple boiled and lyophilized extract of broccoli sprouts. Furthermore, when either broccoli sprouts or seeds are administered directly to subjects without prior extraction and consequent inactivation of endogenous myrosinase, regardless of the delivery matrix or dose, the sulforaphane in those preparations is 3- to 4-fold more bioavailable than sulforaphane from glucoraphanin delivered without active plant myrosinase. These data expand upon earlier reports of inter- and intra-individual variability, when glucoraphanin was delivered in either teas, juices, or gelatin capsules, and they confirm that a variety of delivery matrices may be equally suitable for glucoraphanin supplementation (e.g. fruit juices, water, or various types of capsules and tablets).

Glucosinolates Are Mainly Absorbed Intact in Germfree and Human Microbiota-Associated Mice.

Chemoprotective or genotoxic effects of glucosinolates occurring in Brassica vegetables are attributed to their hydrolysis products formed upon tissue damage by plant myrosinase. Since Brassica vegetables, in which myrosinase has been heat-inactivated, still display bioactivity, glucosinolate activation has been attributed to intestinal bacteria. The aim of this study was to investigate whether this is true. Glucoraphanin (172 mg/kg body weight) and neoglucobrassicin (297 mg/kg body weight) were administered intragastrically to germ free and human microbiota associated (HMA) mice. Approximately 30% of the applied doses of glucoraphanin and neoglucobrassicin were excreted unchanged in the urine of both germ free and HMA mice. Isothiocyanates, sulforaphane, and erucin, formed from glucoraphanin, were mainly excreted as urinary N-acetyl-l-cysteine conjugates. N-Methoxyindole-3-carbinol formed from neoglucobrassicin was observed in small amounts in both germ free and HMA mice. Formation of DNA adducts from neoglucobrassicin was also independent from bacterial colonization of the mice. Hence, intestinal bacteria are involved in the bioactivation of glucosinolates in the gut, but their contribution to glucosinolate transformation in HMA mice is apparently very small.

Myrosinase in Brassicaceae: the most important issue for glucosinolate turnover and food quality

Glucosinolates and their degradation products, isothiocyanates, have been widely identified by their benefits to human nutrition. Plant myrosinase, is an enzyme found in Brassicaceae family with an essential role on the glucosinolates conversion to isothiocyanates. In this review, we highlight recent progress in myrosinase, with particular emphasis on the involvement on in vivo glucosinolates turnover and the regulation during plant development and in response to environmental changes. Also, the myrosinase as influenced by some intrinsic and extrinsic factors, during postharvest and food processing such as pH, temperature, and pressure is examined as a challenge for increasing isothiocyanates bioactivity.

Role of glycoside hydrolase genes in sinigrin degradation by E. coli O157:H7

This work examined Escherichia coli O157:H7 strain 02-0304 for putative genes responsible for sinigrin hydrolysis. Sinigrin is a glucosinolate present in Oriental mustard (Brassica juncea), and its hydrolysis is mediated in plants by the enzyme myrosinase. Sinigrin hydrolysis by plant or bacterial myrosinase yields allyl isothiocyanate (AITC) which is bactericidal. In silico analysis using public databases found sequence similarity between plant myrosinase and enzymes encoded by genes from β-glucosidase families in E. coli O157:H7. Specifically, 6-phospho-β-glucosidase encoded by the genes bglA and ascB (family 1), and chbF (family 4) present in E. coli O157:H7 showed the highest similarity. Polymerase chain reaction (PCR) confirmed the presence of bglA, ascB, and chbF in the clinical E. coli strain tested. Disruption of these genes in wild-type E. coli O157:H7 strain 02-0304 using lambda-red replacement created single and double mutants. The relative importance of each gene in the hydrolysis of sinigrin by E. coli O157:H7 was also assessed by comparing gene expression and sinigrin degradation rates among the E. coli O157:H7 wild-type strain and its mutants. The results suggested that the genes bglA and ascB play a substantial role in the degradation of sinigrin by E. coli O157:H7 strain 02-0304.

Influence of Temperature, Glucose, and Iron on Sinigrin Degradation by Salmonella and Listeria monocytogenes

Factors, including pH, temperature, glucose concentration, and iron compounds, affect the activity of plant myrosinase and, consequently, endogenous glucosinolate degradation. These factors also may affect glucosinolate degradation by bacterial myrosinase. Therefore, this study examined the effect of temperature (4 to 21°C), glucose (0.05 to 1.0%), and iron (10 mM ferrous or ferric) on sinigrin degradation by Salmonella or Listeria monocytogenes cocktails in Mueller-Hinton broth and the effect of sinigrin degradation on bacterial viability. The degradation of sinigrin by both pathogens increased with higher temperatures (21 > 10 > 4°C). Salmonella and L. monocytogenes cocktails hydrolyzed 59.1 and 53.2% of sinigrin, respectively, at 21°C up to 21 days. Both iron compounds significantly enhanced sinigrin degradation by the pathogens. On day 7, sinigrin was not detected when the Salmonella cocktail was cultured with ferrous iron or when the L. monocytogenes cocktail was cultured in Mueller-Hinton broth containing ferric iron. In contrast, ferric and ferrous iron inhibited the activity of 0.002 U/ml myrosinase from white mustard by 63 and 35%, respectively, on day 1. Salmonella and L. monocytogenes cocktails were able to degrade >80% of sinigrin at 0.05 and 0.1% glucose; however, 0.25 to 1.0% glucose significantly reduced sinigrin degradation. Although both pathogens significantly degraded sinigrin, the allyl isothiocyanate (AITC) recoverable was ≤6.2 ppm, which is not inhibitory to Salmonella or L. monocytogenes. It is probable that the gradual hydrolysis of sinigrin to form AITC either did not produce an inhibitory level of AITC or the AITC formed was unstable in the aqueous medium and rapidly decomposed to new compounds that were less bactericidal against the pathogens.

Dietary broccoli mildly improves neuroinflammation in aged mice but does not reduce lipopolysaccharide-induced sickness behavior

Aging is associated with oxidative stress and heightened inflammatory response to infection. Dietary interventions to reduce these changes are therefore desirable. Broccoli contains glucoraphanin, which is converted to sulforaphane (SFN) by plant myrosinase during cooking preparation or digestion. Sulforaphane increases antioxidant enzymes including NAD(P)H quinone oxidoreductase and heme oxygenase I and inhibits inflammatory cytokines. We hypothesized that dietary broccoli would support an antioxidant response in brain and periphery of aged mice and inhibit lipopolysaccharide (LPS)–induced inflammation and sickness. Young adult and aged mice were fed control or 10% broccoli diet for 28 days before an intraperitoneal LPS injection. Social interactions were assessed 2, 4, 8, and 24 hours after LPS, and mRNA was quantified in liver and brain at 24 hours. Dietary broccoli did not ameliorate LPS-induced decrease in social interactions in young or aged mice. Interleukin-1β (IL-1β) expression was unaffected by broccoli consumption but was induced by LPS in brain and liver of adult and aged mice. In addition, IL-1β was elevated in brain of aged mice without LPS. Broccoli consumption decreased age-elevated cytochrome b-245 β, an oxidative stress marker, and reduced glial activation markers in aged mice. Collectively, these data suggest that 10% broccoli diet provides a modest reduction in age-related oxidative stress and glial reactivity, but is insufficient to inhibit LPS-induced inflammation. Thus, it is likely that SFN would need to be provided in supplement form to control the inflammatory response to LPS.

Contribution of endogenous plant myrosinase to the antimicrobial activity of deodorized mustard against Escherichia coli O157:H7 in fermented dry sausage

This work investigated the antimicrobial activity of residual endogenous plant myrosinase in Oriental and yellow mustard powders and a deoiled meal (which contained more glucosinolate than unextracted mustard powder of each type of mustard), against Escherichia coli O15:H7 during dry-fermented sausage ripening. When small amounts of “hot” mustard powder or meal containing endogenous plant myrosinase were added to fully-deodorized powders and a meal of the same type, pathogen reduction rates were enhanced. The higher glucosinolate level in the deoiled mustard meal enabled the use of 50% less mustard in dry sausage to achieve the mandatory ≥5logCFU/g reduction of E. coli O157:H7. The myrosinase-like activity present in E. coli O157:H7 contributed to glucosinolate hydrolysis in sausages with fully-deodorized, deoiled mustard meal, although the period necessary for a 5log pathogen reduction was 14d longer. Yellow mustard derivatives were more potently antimicrobial than Oriental mustard.

Comparison of bioactive phytochemical content and release of isothiocyanates in selected brassica sprouts

The consumption of brassica sprouts as raw vegetables provides a fair amount of glucosinolates (GLs) and active plant myrosinase, which enables the breakdown of GLs into health-promoting isothiocyanates (ITCs). This study reports the determination of the main constituents related to human health found in edible sprouts of two Brassica oleracea varieties, broccoli and Tuscan black kale, and two Raphanus sativus varieties, Daikon and Sango. Radish sprouts exhibited the highest ability to produce ITCs, with Daikon showing the greatest level of conversion of GLs into bioactive ITCs (96.5%), followed by Sango (90.0%). Tuscan black kale gave a value of 68.5%, whereas broccoli displayed the lowest with 18.7%. ITCs were not the exclusive GL breakdown products in the two B. oleracea varieties, since nitriles were also produced, thus accounting for the lower conversion observed. Measuring the release of plant ITCs is a valuable tool in predicting the potential level of exposure to these bioactive compounds after the consumption of raw brassica sprouts.

Biotransformation of glucosinolates from a bacterial perspective.

Abstract Epidemiological studies have shown an association between the consumption of cruciferous vegetables and a reduced risk of certain types of cancers, in particular, pancreatic, bladder and colorectal. This is thought to be the result of the conversion of glucosinolates (GSLs) present in the vegetables into bioactive isothiocyanates (ITCs) that in turn stimulate a host response involving detoxification pathways. Conversion of GSLs is catalysed by the enzyme myrosinase, co-produced by the plant but stored in separate tissue compartments and brought together when the tissue is damaged. Myrosinase activity can be lost during storage of vegetables and is often inactivated by cooking. In the absence of active plant myrosinase the host's gut bacteria are capable of carrying out a myrosinase-like activity on GSLs in the lower gut. Several micro-organisms are known to be capable of metabolizing GSLs leading to the production of ITCs and nitriles, and this review examines the bacterial biotransformation of GSLs and a role for the microbiota in their biotransformation.

Interaction of glucosinolate content of Arabidopsis thaliana mutant lines and feeding and oviposition by generalist and specialist lepidopterans

The diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae), is an insect specialized on glucosinolate-containing Brassicaceae that uses glucosinolates in host-plant recognition. We used wild-type and mutants of Arabidopsis thaliana (L.) Heynh. (Brassicaceae) to investigate the interaction between plant glucosinolate and myrosinase content and herbivory by larvae of the generalist Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) and the specialist P. xylostella. We also measured glucosinolate changes as a result of herbivory by these larvae to investigate whether herbivory and glucosinolate induction had an effect on oviposition preference by P. xylostella. Feeding by H. armigera and P. xylostella larvae was 2.1 and 2.5 times less, respectively, on apk1 apk2 plants (with almost no aliphatic glucosinolates) than on wild-type plants. However, there were no differences in feeding by H. armigera and P. xylostella larvae on wild-type, gsm1 (different concentrations of aliphatic glucosinolates compared to wild-type plants), and tgg1 tgg2 plants (lacking major myrosinases). Glucosinolate induction (up to twofold) as a result of herbivory occurred in some cases, depending on both the plant line and the herbivore. For H. armigera, induction, when observed, was noted mostly for indolic glucosinolates, while for P. xylostella, induction was observed in both aliphatic and indolic glucosinolates, but not in all plant lines. For H. armigera, glucosinolate induction, when observed, resulted in an increase of glucosinolate content, while for P. xylostella, induction resulted in both a decrease and an increase in glucosinolate content. Two-choice tests with wild-type and mutant plants were conducted with larvae and ovipositing moths. There were no significant differences in preference of larvae and ovipositing moths between wild-type and gsm1 mutants and between wild-type and tgg1 tgg2 mutants. However, both larvae and ovipositing moths preferred wild-type over apk1 apk2 mutants. Two-choice oviposition tests were also conducted with P. xylostella moths comparing undamaged plants to plants being attacked by larvae of either P. xylostella or H. armigera. Oviposition preference by P. xylostella was unaffected as a result of larval plant damage, even in the cases where herbivory resulted in glucosinolate induction.

A concise report on pharmacological and bioanalytical aspect of sinigrin

Sinigrin (Allylglucosinolate) is precursors of the anticancer compounds allyl isothiocyanate which is found in Brassica juncea, Phyllanthus emblica, Armoracia lapthifolia, Wasabia japonica and some other plants. Except its uses as a medicinal compound it aslo have nutraceutical value. It protects plant from fungi, nematodes, plant pathogens and herbivores. Allyl isothiocyanate is produced from sinigrin through plant myrosinase or microbial myrosinase in gut microflora. Sinigrin have various type of pharmacological activity such as anticancer, antimicrobial and it suppresses NO production in the macrophages. This review will provide a brief idea about isolation techniques, analytical aspect and pharmacological activity of sinigrin. The data were collected from the available literature source in regards with sinigrin and it can be helpful to the researchers to get some idea about this unique phytoconstituents.

Influence of NaCl and NaNO3 on sinigrin hydrolysis by foodborne bacteria.

The glucosinolate sinigrin (SNG) is converted by endogenous plant myrosinase or by bacterial myrosinase-like activity to form the potent antimicrobial allyl isothiocyanate. In order to use SNG as a natural antimicrobial precursor in food, it became important to better understand the ability of bacteria to synthesize the enzyme(s) and understand factors influencing this synthesis at a constant SNG concentration. Eight spoilage, pathogenic, or starter culture bacteria were grown separately in medium containing individual or combined salts with SNG. SNG degradation by the bacteria and the formation of its major degradation product, allyl isothiocyanate, were followed for 12 days at 30 or 35°C. The bacterial strains varied in their ability to metabolize SNG, and this was enhanced by NaCl and/or NaNO(3). SNG hydrolysis took place after 4 days, and the greatest amount occurred by day 12. At 12 days, Escherichia coli O157:H7 showed the greatest capacity to hydrolyze SNG (45.3% degradation), followed by Staphylococcus carnosus (44.57%), while Pseudomonas fluorescens was not active against SNG. The ability of tested strains to metabolize SNG, in decreasing order, was as follows: Escherichia coli O157:H7 > Staphylococcus carnosus > Staphylococcus aureus > Pediococcus pentosaceus > Salmonella Typhimurium > Listeria monocytogenes > Enterococcus faecalis > Pseudomonas fluorescens.

A micromolar O-sulfated thiohydroximate inhibitor bound to plant myrosinase.

The 1.6 A resolution structure of the micromolar competitive inhibitor S-(N,N-dimethylaminoethyl) phenylacetothiohydroximate-O-sulfate bound to Sinapis alba myrosinase, a plant thioglucosidase, is reported. Myrosinase and its substrates, the glucosinolates, are part of the plant's defence system. The sulfate group and the phenyl group of the inhibitor bind to the aglycon-binding site of the enzyme, whereas the N,N-dimethyl group binds to the glucose-binding site and explains the large improvement in binding affinity compared with previous compounds. The structure suggests ways to increase the potency and specificity of the compound by improving the interactions with the hydrophobic pocket of the aglycon-binding site.

Sinigrin suppresses nitric oxide production in rats administered intraperitoneally with lipopolysaccharide

Sinigrin, a glucosinolate contained in cruciferous vegetables, is converted to allyl isothiocyanate by plant myrosinase or microbial myrosinase in gut microflora. The intake of sinigrin significantly decreases the urinary nitrate + nitrite level (a nitric oxide [NO] production marker) in rats administered intraperitoneally with lipopolysaccharide (LPS). This result demonstrates that sinigrin suppresses NO production and therefore likely has an antioxidative effect in vivo.

Characterization of a root-specific β-thioglucoside glucohydrolase gene in Carica papaya and its recombinant protein expressed in Pichia pastoris

Plant β-thioglucoside glucohydrolases (TGGs or myrosinases) are a young class of enzymes in the glycosyl hydrolase family 1 and have a narrow distribution. TGG genes have mainly been cloned from crucifers, while TGGs in other species have received little attention. The TGG gene CpTGG2 and its recombinant protein from papaya were characterized in this paper . This is the first plant TGG gene without unusual intron splicing borders, as present in all other available TGG genes. Phylogenetic analysis indicated that plant myrosinases are divided into two major lineages. CpTGG2 is located in the lineage constituted by AtTGG4–6 from Arabidopsis thaliana, while the rest of myrosinases (including MA, MB and MC subfamilies) are grouped into another lineage. RT-PCR analysis indicated that CpTGG2 was specifically expressed in the root. The recombinant CpTGG2 expressed in yeast had a subunit mass of 70 kDa, and had low basal TGG activity without addition of ascorbate. Low concentrations of ascorbate stimulated CpTGG2 activity, while high concentrations were inhibitory. CpTGG2 was active in broad pH and temperature ranges, similar to AtTGG4 and AtTGG5. The apparent K m and V max were 2.24 mM and 24.3 μmol min −1 mg −1 when sinigrin was the substrate. The calculated k cat/ K m value was 1.3 × 10 4 S −1 M −1 . Our results reshaped and expanded the myrosinase family structure and provided clues to the evolution of myrosinase genes.

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.

Identification of indole glucosinolate breakdown products with antifeedant effects on Myzus persicae (green peach aphid)

The cleavage of glucosinolates by myrosinase to produce toxic breakdown products is a characteristic insect defense of cruciferous plants. Although green peach aphids (Myzus persicae) are able to avoid most contact with myrosinase when feeding from the phloem of Arabidopsis thaliana, indole glucosinolates are nevertheless degraded during passage through the insects. A defensive role for indole glucosinolates is suggested by the observation that atr1D mutant plants, which overproduce indole glucosinolates, are more resistant to M. persicae, whereas cyp79B2 cyp79B3 double mutants, which lack indole glucosinolates, succumb to M. persicae more rapidly. Indole glucosinolate breakdown products, including conjugates formed with ascorbate, glutathione and amino acids, are elevated in the honeydew of M. persicae feeding from atr1D mutant plants, but are absent when the aphids are feeding on cyp79B2 cyp79B3 double mutants. M. persicae feeding from wild-type plants and myrosinase-deficient tgg1 tgg2 double mutants excrete a similar profile of indole glucosinolate-derived metabolites, indicating that the breakdown is independent of these foliar myrosinases. Artificial diet experiments show that the reaction of indole-3-carbinol, a breakdown product of indol-3-ylmethylglucosinolate, with ascorbate, glutathione and cysteine produces diindolylmethylcysteines and other conjugates that have antifeedant effects on M. persicae. Therefore, the post-ingestive breakdown of indole glucosinolates provides a defense against herbivores such as aphids that can avoid glucosinolate activation by plant myrosinases.