Molecular Cloning, Expression and Characterisation of a Bacterial Myrosinase from Citrobacter Wye1.

Glucosinolates are plant natural products which on degradation by myrosinases give rise to the beneficial bioactive isothiocyanates. Recently, a myrosinase activity was detected in a Citrobacter strain isolated from soil. This enzyme was purified enabling its amino acid sequence and gene sequence (cmyr) to be determined. In order to study this myrosinase it was necessary to establish an expression system that would enable future work such as a structural determination of the protein to be carried out. The myrosinase gene was amplified, cloned and expressed in Escherichia coli with a 6XHis-tag. The heterologous expression of cmyr enabled relatively large amounts of myrosinase to be produced (3.4 mg cmyr/100 ml culture). Myrosinase activity was determined by mixing substrate and enzyme and determining glucose release. Optimum pH and temperature were determined to be pH 6.0 and 25 °C for the Ni-NTA purified protein. The kinetic parameters of the purified myrosinase were determined using sinigrin as a substrate. Km and Vmax were estimated as 0.18 mM and 0.033 mmol/min/mg respectively for sinigrin under optimum conditions and compared to other kinetic data for myrosinases. The substrate specificity of myrosinase was determined having the highest affinity for sinigrin followed by glucoiberin, progoitrin, glucoerucin, glucoraphanin and glucotropaeolin.

Chitin nanofibers trigger membrane bound defense signaling and induce elicitor activity in plants.

The present study demonstrated that chitin-based nanofibers (CNFs) trigger the chitinase genes (PGIP1 and CaChi2), while elevating salicylic acid that can protect plants against pathogens. Cross-talk between this genetic induction and salicylic-acid-mediated immune response was also observed, which may arm a plant against multiple pathovars. Crab and mushroom based CNFs were synthesized by electrospinning and ball milling techniques. Plants (mung bean, Vigna radiata) (pepper, Capsicum annuum) were pre-inoculated with CNFs and treated with the pathogens Scrolotium rolfsii for pepper and Macrophomina phaseolina for mung bean and shrimp-based CNFs were used as a control. Treated plants had elevated levels of chitinase genes in response to CNFs at inoculation concentrations <10 mg/mL both in soil and media, to protect them against the pathogenic fungal disease. After 24 h of exposure to the pathogens, qRT-PCR showed genes class II chitinase gene (CaChi2) and polygalacturonase inhibitor protein 1 (PGIP1) to be up-regulated in both root and shoot at 0.1 and 1 mg/mL of inoculation, respectively. The ball milled mushroom CNFs were sufficient to trigger the membrane based enzymes with less diameter (≥15 nm) to be most efficient versus others. In vitro analysis showed IC50 of ball milled mushroom CNFs to be most efficient in limiting the growth of fungal biomass. Further trigger-like effects were prominent in reducing pathogenic fungal spread in both species.

Reduced metabolites of nitroaromatics are distributed in the environment via the food chain.

Increased industrial processes have introduced emerging toxic pollutants into the environment. Phytoremediation is considered to be a very useful, economical and ecofriendly way of controlling these pollutants, however, certain pollutants can potentially travel through the food chain and accumulate at hazardous levels. Four isomers of dinitrotoluenes (DNT) were investigated and observed their potential toxicity towards A. thaliana. Two different aphid species (generalist and specialist) were allowed to feed on plants treated with DNTs and toxicity to aphids determined. Reduced metabolites of DNT (in both plant and aphids) were recovered and quantified through GC-MS analyses. 2,6-DNT was observed to be the toxic of the DNTs tested. Complete metabolism of DNTs to their reduced products was never achieved for higher concentrations. Regioselectivity was observed in the case of 2,4-DNT, with 4A2NT as the dominant isomer. Feeding aphids showed a similar toxicity pattern for DNT isomers as host plants. Metabolites were recovered from the body of aphids, demonstrating the potential transport of metabolites through the food chain. Plants show varied toxicity responses towards the DNT isomers. Aphids fed on A. thaliana plants treated with DNTs were shown to have ANTs present, which reflects the propagation of DNT metabolites through the food chain.

Glucosinolate and Desulfo-glucosinolate Metabolism by a Selection of Human Gut Bacteria.

Glucosinolate (GSL) hydrolysis is mediated by the enzyme myrosinase which together with specifier proteins can give rise to isothiocyanates (ITCs), thiocyanates, and nitriles (NITs) in cruciferous plants. However, little is known about the metabolism of GSLs by the human gut flora. The aim of the work was to investigate the metabolic fates of sinigrin (SNG), glucotropaeolin (GTP), gluconasturtiin (GNT), and their corresponding desulfo-GSLs (DS-GSLs). Three human gut bacterial strains, Enterococcus casseliflavus CP1, Lactobacillus agilis R16, and Escherichia coli VL8, were chosen for this study. GNT was metabolized to completion within 24 h to phenethyl ITC and phenethyl NIT (PNIT) by all bacteria, except for L. agilis R16 which produced only PNIT. At least 80 % of GTP and SNG were metabolized by all bacteria within 24 h to the corresponding ITCs and NITs. The pH of media over time gradually became acidic for both L. agilis R16 and E. coli VL8, while for E. casseliflavus CP1 the media became slightly alkaline with NIT and ITC production occurring between pH 3.0 and 7.5. ITC production peaked between 4 and 10 h in most cases and gradually declined while NIT production increased and remained relatively constant over time. The total percentage products accounted for 3-53 % of the initial GSL. NITs were produced from DS-GSLs suggesting an alternative metabolism via desulfation for the food based GSLs. The metal ion dependency for NIT production for GNT and its DS form was investigated where it was shown that Fe(2+) increased NIT production, while Mg(2+) stimulated the formation of ITC.

Discovery of a Bacterial Glycoside Hydrolase Family 3 (GH3) ß-Glucosidase with Myrosinase Activity from a Citrobacter Strain Isolated from Soil.

A Citrobacter strain (WYE1) was isolated from a UK soil by enrichment using the glucosinolate sinigrin as sole carbon source. The enzyme myrosinase was purified using a combination of ion exchange and gel filtration to give a pure protein of approximately 66 kDa. The N-terminal amino acid and internal peptide sequence of the purified protein were determined and used to identify the gene, which, based on InterPro sequence analysis, belongs to the family GH3, contains a signal peptide, and is a periplasmic protein with a predicted molecular mass of 71.8 kDa. A preliminary characterization was carried out using protein extracts from cell-free preparations. The apparent KM and Vmax were 0.46 mM and 4.91 mmol dm(-3) min(-1) mg(-1), respectively, with sinigrin as substrate. The optimum temperature and pH for enzyme activity were 25 °C and 6.0, respectively. The enzyme was marginally activated with ascorbate by a factor of 1.67.

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.

Identification of Proteins Possibly Involved in Glucosinolate Metabolism in L. agilis R16 and E. coli VL8.

This study was aimed to identify sinigrin-induced bacterial proteins potentially involved in the metabolism of glucosinolate in two glucosinolate-metabolising bacteria Lactobacillus agilis R16 and Escherichia coli VL8. Sinigrin (2 mM) was used to induce the proteins in both bacteria under anaerobic incubation for 8 h at 30 °C for L. agilis R16 and 37 °C for E. coli VL8 and the controls without sinigrin were performed. Allyl isothiocyanate and allyl nitrile as two degradation products of sinigrin were detected in sinigrin-induced cultures of L. agilis R16 (27% total products) and E. coli VL8 (38% total products) from a complete sinigrin degradation in 8 h for both bacteria. 2D gel electrophoresis was conducted to identify induced proteins with at least twofold increased abundance. Sinigrin-induced L. agilis R16 and the control produced 1561 and 1543 protein spots, respectively. For E. coli VL8, 1363 spots were detected in sinigrin-induced and 1354 spots in the control. A combination of distinct proteins and upregulated proteins of 32 and 35 spots in L. agilis R16 and E. coli VL8, respectively were detected upon sinigrin induction. Of these, 12 and 16 spots from each bacterium respectively were identified by LC-MS/MS. In both bacteria most of the identified proteins are involved in carbohydrate metabolism, oxidoreduction system and sugar transport while the minority belong to purine metabolism, hydrolysis, and proteolysis. This indicated that sinigrin induction led to the expressions of proteins with similar functions in both bacteria and these proteins may play a role in bacterial glucosinolate metabolism.

The metabolism of methylsulfinylalkyl- and methylthioalkyl-glucosinolates by a selection of human gut bacteria.

SCOPE: Certain myrosinase-positive human gut bacteria can metabolize glucosinolates (GSLs) to produce isothiocyanates (ITC) as chemopreventive agents. We investigated glucoerucin, glucoiberin, and glucoraphanin (present in broccoli) metabolism by human gut strains. METHODS AND RESULTS: All tested bacteria metabolized glucoerucin to completion within 16 h to erucin and erucin nitrile (NIT). Lactobacillus agilis R16 metabolized only 10% of glucoiberin and glucoraphanin with no detectable products. Enterococcus casseliflavus CP1, however, metabolized 40-50% of glucoiberin and glucoraphanin producing relatively low concentrations of iberin and sulforaphane. Interestingly, Escherichia coli VL8 metabolized 80-90% of glucoiberin and glucoraphanin and also bioconverted glucoraphanin and glucoiberin to glucoerucin and glucoiberverin, respectively, producing erucin, erucin NIT, iberverin, and iberverin NIT from the two GSLs. The putative reductase enzyme in the cell-free extracts of this bacterium required both Mg(2+) and NAD(P)H as cofactors for bioconversion. The cell-free extract of E. coli VL8 containing the reductase enzyme was able to reduce both the GSL glucoraphanin and its hydrolysis product sulforaphane to glucoerucin and erucin/erucin NIT, respectively. CONCLUSION: The composition and metabolic activity of the human gut bacteria can indirectly impact on the potential chemopreventive effects of GSL-derived metabolites.

A trio of viral proteins tunes aphid-plant interactions in Arabidopsis thaliana.

BACKGROUND: Virus-induced deterrence to aphid feeding is believed to promote plant virus transmission by encouraging migration of virus-bearing insects away from infected plants. We investigated the effects of infection by an aphid-transmitted virus, cucumber mosaic virus (CMV), on the interaction of Arabidopsis thaliana, one of the natural hosts for CMV, with Myzus persicae (common names: 'peach-potato aphid', 'green peach aphid'). METHODOLOGY/PRINCIPAL FINDINGS: Infection of Arabidopsis (ecotype Col-0) with CMV strain Fny (Fny-CMV) induced biosynthesis of the aphid feeding-deterrent 4-methoxy-indol-3-yl-methylglucosinolate (4MI3M). 4MI3M inhibited phloem ingestion by aphids and consequently discouraged aphid settling. The CMV 2b protein is a suppressor of antiviral RNA silencing, which has previously been implicated in altering plant-aphid interactions. Its presence in infected hosts enhances the accumulation of CMV and the other four viral proteins. Another viral gene product, the 2a protein (an RNA-dependent RNA polymerase), triggers defensive signaling, leading to increased 4MI3M accumulation. The 2b protein can inhibit ARGONAUTE1 (AGO1), a host factor that both positively-regulates 4MI3M biosynthesis and negatively-regulates accumulation of substance(s) toxic to aphids. However, the 1a replicase protein moderated 2b-mediated inhibition of AGO1, ensuring that aphids were deterred from feeding but not poisoned. The LS strain of CMV did not induce feeding deterrence in Arabidopsis ecotype Col-0. CONCLUSIONS/SIGNIFICANCE: Inhibition of AGO1 by the 2b protein could act as a booby trap since this will trigger antibiosis against aphids. However, for Fny-CMV the interplay of three viral proteins (1a, 2a and 2b) appears to balance the need of the virus to inhibit antiviral silencing, while inducing a mild resistance (antixenosis) that is thought to promote transmission. The strain-specific effects of CMV on Arabidopsis-aphid interactions, and differences between the effects of Fny-CMV on this plant and those seen previously in tobacco (inhibition of resistance to aphids) may have important epidemiological consequences.

Drought alters interactions between root and foliar herbivores.

Drought can alter plant quality and the strength of trophic interactions between herbivore groups, and is likely to increase in occurrence and severity under climate change. We hypothesized that changes in plant chemistry due to root herbivory and drought stress would affect the performance of a generalist and a specialist aphid species feeding on a Brassica plant. High drought stress increased the negative effect of root herbivory on the performance of both aphid species (30% decrease in fecundity and 15% reduction in intrinsic rate of increase). Aphid performance was greatest at moderate drought stress, though the two species differed in which treatment combination maximized performance. Nitrogen concentration was greatest in high and moderately drought-stressed plants without root herbivores and moderately drought-stressed plants under low root herbivore density, and correlated positively with aphid fecundity for both species. Glucosinolate concentrations increased 62% under combined drought stress and root herbivory, and were positively correlated with extended aphid development time. Root herbivory did not influence relative water content and foliar biomass under normal water regimes but they decreased 24 and 63%, respectively, under high drought stress. This study shows that drought can alter the strength of interactions between foliar and root herbivores, and that plant chemistry is key in mediating such interactions. The two aphid species responded in a broadly similar way to root herbivore and drought-stress treatments, which suggests that generalized predictions of the effects of abiotic factors on interactions between above- and below-ground species may be possible.

Isothiocyanate concentrations and interconversion of sulforaphane to erucin in human subjects after consumption of commercial frozen broccoli compared to fresh broccoli.

SCOPE: Sulforaphane (a potent anticarcinogenic isothiocyanate derived from glucoraphanin) is widely considered responsible for the protective effects of broccoli consumption. Broccoli is typically purchased fresh or frozen and cooked before consumption. We compared the bioavailability and metabolism of sulforaphane from portions of lightly cooked fresh or frozen broccoli, and investigated the bioconversion of sulforaphane to erucin. METHODS AND RESULTS: Eighteen healthy volunteers consumed broccoli soups produced from fresh or frozen broccoli florets that had been lightly cooked and sulforaphane thio-conjugates quantified in plasma and urine. Sulforaphane bioavailability was about tenfold higher for the soups made from fresh compared to frozen broccoli, and the reduction was shown to be due to destruction of myrosinase activity by the commercial blanching-freezing process. Sulforaphane appeared in plasma and urine in its free form and as several thio-conjugates forms. Erucin N-acetyl-cysteine conjugate was a significant urinary metabolite, and it was shown that human gut microflora can produce sulforaphane, erucin, and their nitriles from glucoraphanin. CONCLUSION: The short period of blanching used to produce commercial frozen broccoli destroys myrosinase and substantially reduces sulforaphane bioavailability. Sulforaphane was converted to erucin and excreted in urine, and it was shown that human colonic flora were capable of this conversion.

Plant nutrient supply determines competition between phytophagous insects.

Indirect competition is often mediated by plant responses to herbivore feeding damage and is common among phytophagous insect species. Plant-mediated responses may be altered by abiotic conditions such as nutrient supply, which can affect plant growth, morphology, and the concentration of primary and secondary metabolites. Nutrient supply can be manipulated by the type and amount of fertilizer applied to a plant. Brassica oleracea plants were grown in several types of fertilizer, including those commonly used in sustainable and conventional agricultural systems. The occurrence of indirect competition between two phytophagous species from different feeding guilds (a phloem-feeder and leaf-chewer) was assessed. The leaf-chewer reduced aphid populations on plants growing in most fertilizer treatments, but not on those in the ammonium nitrate fertilizer treatment, which caused the highest concentration of foliar nitrogen. The potential consequences of our findings are discussed for phytophagous species in conventional and sustainable agricultural systems.

Varying responses of insect herbivores to altered plant chemistry under organic and conventional treatments.

The hypothesis that plants supplied with organic fertilizers are better defended against insect herbivores than those supplied with synthetic fertilizers was tested over two field seasons. Organic and synthetic fertilizer treatments at two nitrogen concentrations were supplied to Brassica plants, and their effects on the abundance of herbivore species and plant chemistry were assessed. The organic treatments also differed in fertilizer type: a green manure was used for the low-nitrogen treatment, while the high-nitrogen treatment contained green and animal manures. Two aphid species showed different responses to fertilizers: the Brassica specialist Brevicoryne brassicae was more abundant on organically fertilized plants, while the generalist Myzus persicae had higher populations on synthetically fertilized plants. The diamondback moth Plutella xylostella (a crucifer specialist) was more abundant on synthetically fertilized plants and preferred to oviposit on these plants. Glucosinolate concentrations were up to three times greater on plants grown in the organic treatments, while foliar nitrogen was maximized on plants under the higher of the synthetic fertilizer treatments. The varying response of herbivore species to these strong differences in plant chemistry demonstrates that hypotheses on defence in organically grown crops have over-simplified the response of phytophagous insects.

Modifying the alkylglucosinolate profile in Arabidopsis thaliana alters the tritrophic interaction with the herbivore Brevicoryne brassicae and parasitoid Diaeretiella rapae.

Arabidopsis thaliana was used as an experimental model plant to investigate a tritrophic interaction between the plant, a specialist aphid herbivore, Brevicoryne brassicae, and its natural enemy, the parasitoid Diaeretiella rapae. The A. thaliana ecotype Col-5 was transformed with a functional 2-oxoglutarate dependent dioxygenase (BniGSL-ALK) that converts 3-methylsulfinylpropylglucosinolate and 4-methylsulfinylbutylglucosinolate to 2-propenylglucosinolate and 3-butenylglucosinolate, respectively. This transformation results in a change in the glucosinolate hydrolysis profile where 3-butenylisothiocyanate, 2-propenylisothiocyanate and 5-vinyloxazolidine-2-thione are produced in contrast to the wild-type plant where 4-methylsulfinylbutylisothiocyanate is the main product. Performance of B. brassicae was affected negatively by transforming Col-5 with BniGSL-ALK in terms of mean relative growth rates. In a series of behavioral bioassays, naïve D. rapae females were able to discriminate between B. brassicae infested and uninfested Col-5 plants transformed with BniGSL-ALK, with parasitoids showing a preference for B. brassicae infested plants. By contrast, naïve D. rapae females were unable to discriminate between aphid infested and uninfested Col-5 plants. Subsequent air entrainments of B. brassicae infested Col-5 plants transformed with BniGSL-ALK further confirmed the presence of 3-butenylisothiocyanate in the headspace. By contrast, no glucosinolate hydrolysis products were recorded from similarly infested Col-5 plants.

Comparative innate responses of the aphid parasitoid Diaeretiella rapae to alkenyl glucosinolate derived isothiocyanates, nitriles, and epithionitriles.

Cruciferous plants (Brassicaceae) are characterized by the accumulation of a group of secondary metabolites known as glucosinolates that, following attack by pathogens or herbivores, may be hydrolyzed to one of a number of products including isothiocyanates and nitriles. Despite the range of hydrolysis products that may be produced, the toxicity of glucosinolates to pathogens and herbivores may be explained largely by the production of isothiocyanates. Isothiocyanates are also known to provide an indirect defense by acting as host finding cues for parasitoids of insect herbivores that attack crucifers. It has been speculated that nitriles may provide a similar indirect defense. Here, we investigate the olfactory perception and orientation behavior of the aphid parasitoid Diaeretiella rapae, to a range of alkenylglucosinolate hydrolysis products, including isothiocyanates, nitriles, and epithionitriles. Electroantennogram responses indicated peripheral odor perception in D. rapae females to all 3-butenylglucosinolate hydrolysis products tested. By contrast, of the 2-propenylglucosinolate hydrolysis products tested, only the isothiocyanate elicited significant responses. Despite showing peripheral olfactory detection of a range of 3-butenylglucosinolate hydrolysis products, naïve females oriented only to the isothiocyanate. Similarly, parasitoids oriented to 3-isothiocyanatoprop-1-ene, but not to the corresponding nitrile or epithionitrile. However, by rearing D. rapae either on Brassica nigra, characterized by the accumulation of 2-propenylglucosinolate, or Brassica rapa var rapifera, characterized by the accumulation of 3-butenylglucosinolate, altered the innate response of parasitoids to 3-isothiocyanatoprop-1-ene and 4-isothiocyanatobut-1-ene. These results are discussed in relation to the defensive roles of glucosinolate hydrolysis products and the influence of the host plant on aphid parasitoid behavior.

Towards global understanding of plant defence against aphids--timing and dynamics of early Arabidopsis defence responses to cabbage aphid (Brevicoryne brassicae) attack.

Insect feeding on plants causes a complex series of coordinated defence responses. Little is known, however, about the time-dependent aspect of induced changes. Here we present a time series-based investigation of Arabidopsis thaliana Ler subjected to attack by a specialist pest of Brassicaceae species, Brevicoryne brassicae. Transcriptome and metabolome changes were studied at 6, 12, 24 and 48 h after infestation to monitor the progress of early induced responses. The use of full-genome oligonucleotide microarrays revealed the initiation of extensive gene expression changes already during the first 6 h of infestation. Data indicated the involvement of reactive oxygen species (ROS) and calcium in early signalling, and salicylic acid (SA) and jasmonic acid (JA) in the regulation of defence responses. Transcripts related to senescence, biosynthesis of anti-insect proteins, indolyl glucosinolates (GS) and camalexin, as well as several uncharacterized to date WRKY transcription factors, were induced. Follow-up studies of defence-involved secondary metabolites revealed depositions of callose at the insects' feeding sites, a decrease in the total level of aliphatic GS, particularly 3-hydroxypropyl glucosinolate, and accumulation of 4-methoxyindol-3-ylmethyl glucosinolate 48 h after the attack. The novel role of camalexin, induced as a part of defence against aphids, was verified in fitness experiments. Fecundity of B. brassicae was reduced on camalexin-accumulating wild-type (WT) plants as compared with camalexin-deficient pad3-1 mutants. Based on experimental data, a model of plant-aphid interactions at the early phase of infestation was proposed.

Accumulation of glucosinolates by the cabbage aphid Brevicoryne brassicae as a defense against two coccinellid species.

Brassica nigra plants, characterized by high levels of sinigrin, and artificial aphid diets to which sinigrin was selectively added were used to rear the crucifer specialist, Brevicoryne brassicae. Aphids were provided as a food source to two species of polyphagous ladybird, Adalia bipunctata and Coccinella septempunctata. First instar A. bipunctata were unable to survive when fed with B. brassicae reared on B. nigra or diets containing 0.2% sinigrin, but when fed with aphids reared on diets containing 0% sinigrin, survival rates were high. By contrast, first instar C. septempunctata were able to survive when fed with aphids reared on B. nigra or artificial diets containing up to 1% sinigrin. However, the presence of sinigrin in the aphid diet decreased larval growth and increased the time necessary for larvae to reach second instar for this species of ladybird. These results indicate that the presence of sinigrin in the diet of B. brassicae makes this aphid unsuitable as a food source for A. bipunctata but not for C. septempunctata, although for this ladybird species, there appear to be costs associated with feeding on aphids that contain this secondary metabolite.

The cabbage aphid: a walking mustard oil bomb.

The cabbage aphid, Brevicoryne brassicae, has developed a chemical defence system that exploits and mimics that of its host plants, involving sequestration of the major plant secondary metabolites (glucosinolates). Like its host plants, the aphid produces a myrosinase (beta-thioglucoside glucohydrolase) to catalyse the hydrolysis of glucosinolates, yielding biologically active products. Here, we demonstrate that aphid myrosinase expression in head/thoracic muscle starts during embryonic development and protein levels continue to accumulate after the nymphs are born. However, aphids are entirely dependent on the host plant for the glucosinolate substrate, which they store in the haemolymph. Uptake of a glucosinolate (sinigrin) was investigated when aphids fed on plants or an in vitro system and followed a different developmental pattern in winged and wingless aphid morphs. In nymphs of the wingless aphid morph, glucosinolate level continued to increase throughout the development to the adult stage, but the quantity in nymphs of the winged form peaked before eclosion (at day 7) and subsequently declined. Winged aphids excreted significantly higher amounts of glucosinolate in the honeydew when compared with wingless aphids, suggesting regulated transport across the gut. The higher level of sinigrin in wingless aphids had a significant negative impact on survival of a ladybird predator. Larvae of Adalia bipunctata were unable to survive when fed adult wingless aphids from a 1% sinigrin diet, but survived successfully when fed aphids from a glucosinolate-free diet (wingless or winged), or winged aphids from 1% sinigrin. The apparent lack of an effective chemical defence system in adult winged aphids possibly reflects their energetic investment in flight as an alternative predator avoidance mechanism.

Transcriptional responses of Arabidopsis thaliana ecotypes with different glucosinolate profiles after attack by polyphagous Myzus persicae and oligophagous Brevicoryne brassicae.

Plants are equipped with a range of defence mechanisms against herbivorous insects. In cruciferous species, jasmonic acid, salicylic acid, and ethylene along with glucosinolates and their hydrolysis products play important roles in plant protection and plant-insect communication. In turn, a number of herbivores have adapted to plants that contain glucosinolates. As a result of adaptation to their host plants, specialized insects may elicit different plant-inducible responses than generalists. Oligonucleotide microarrays and qRT-PCR analysis were used to characterize transcriptional profiles of Arabidopsis thaliana plants in response to infestation with a generalist aphid, Myzus persicae, or a cruciferous plant specialist, Brevicoryne brassicae. To find possible differences and similarities in molecular responses between plants differing in predominant glucosinolate hydrolysis products, three ecotypes of A. thaliana were chosen: Wassilewskija (Ws), Cape Verde Islands (Cvi), and Landsberg erecta (Ler), which, respectively, produce mainly isothiocyanates, epithionitriles, and nitriles. In all three ecotypes, general stress-responsive genes, genes belonging to octadecanoid and indole glucosinolate synthesis pathways were induced upon both generalist and specialist attack. By contrast, transcription of myrosinases, enzymes hydrolysing glucosinolates, was suppressed. The induction of the jasmonic acid synthesis pathway was strongest in Cvi, while the up-regulation of the indole glucosinolate synthesis pathway was highest in Ler, suggesting a slightly different defence strategy in these two ecotypes. Specialist and generalist infestations caused statistically significant differential regulation of 60 genes in Ws and 21 in Cvi. Among these were jasmonic acid and tryptophan synthesis pathway enzymes, and pathogenesis related protein (PR1). Insect no-choice experiments revealed lowered fitness of B. brassicae on Ler and Cvi in comparison to Ws, but no ecotype-dependent change in fecundity of M. persicae. Targeted studies employing constructs of GUS reporter gene under the control of promoters from CYP79B2 and CYP79B3 genes showed insect-specific induction of the indole glucosinolates synthesis pathway.