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.     

Metabolic engineering in Nicotiana benthamiana reveals key enzyme functions in Arabidopsis indole glucosinolate modification

Indole glucosinolates, derived from the amino acid Trp, are plant secondary metabolites that mediate numerous biological interactions between cruciferous plants and their natural enemies, such as herbivorous insects, pathogens, and other pests. While the genes and enzymes involved in the Arabidopsis thaliana core biosynthetic pathway, leading to indol-3-yl-methyl glucosinolate (I3M), have been identified and characterized, the genes and gene products responsible for modification reactions of the indole ring are largely unknown. Here, we combine the analysis of Arabidopsis mutant lines with a bioengineering approach to clarify which genes are involved in the remaining biosynthetic steps in indole glucosinolate modification. We engineered the indole glucosinolate biosynthesis pathway into Nicotiana benthamiana, showing that it is possible to produce indole glucosinolates in a noncruciferous plant. Building upon this setup, we demonstrate that all members of a small gene subfamily of cytochrome P450 monooxygenases, CYP81Fs, are capable of carrying out hydroxylation reactions of the glucosinolate indole ring, leading from I3M to 4-hydroxy-indol-3-yl-methyl and/or 1-hydroxy-indol-3-yl-methyl glucosinolate intermediates, and that these hydroxy intermediates are converted to 4-methoxy-indol-3-yl-methyl and 1-methoxy-indol-3-yl-methyl glucosinolates by either of two family 2 O-methyltransferases, termed indole glucosinolate methyltransferase 1 (IGMT1) and IGMT2.     

Comparative quantitative trait loci mapping of aliphatic, indolic and benzylic glucosinolate production in Arabidopsis thaliana leaves and seeds

Secondary metabolites are a diverse set of plant compounds believed to have numerous functions in plant-environment interactions. Despite this importance, little is known about the regulation of secondary metabolite accumulation. We are studying the regulation of glucosinolates, a large group of secondary metabolites, in Arabidopsis to investigate how secondary metabolism is controlled. We utilized Ler and Cvi, two ecotypes of Arabidopsis that have striking differences in both the types and amounts of glucosinolates that accumulate in the seeds and leaves. QTL analysis identified six loci determining total aliphatic glucosinolate accumulation, six loci controlling total indolic glucosinolate concentration, and three loci regulating benzylic glucosinolate levels. Our results show that two of the loci controlling total aliphatic glucosinolates map to biosynthetic loci that interact epistatically to regulate aliphatic glucosinolate accumulation. In addition to the six loci regulating total indolic glucosinolate concentration, mapping of QTL for the individual indolic glucosinolates identified five additional loci that were specific to subsets of the indolic glucosinolates. These data show that there are a large number of variable loci controlling glucosinolate accumulation in Arabidopsis thaliana.     

Benzoic acid glucosinolate esters and other glucosinolates from Arabidopsis thaliana

The spectacular recent progress in Arabidopsis thaliana molecular genetics furnishes outstanding tools for studying the formation and function of all metabolites in this cruciferous species. One of the major groups of secondary metabolites in A. thaliana is the glucosinolates. These hydrophilic, sulfur-rich glycosides appear to serve as defenses against some generalist herbivores and pathogens, and as feeding and oviposition stimulants to specialist herbivores. To help study their biosynthesis and role in plant-insect interactions, we wanted to determine the complete glucosinolate content of A. thaliana. In previous studies, 24 glucosinolates had been identified from ecotype Columbia. We reinvestigated Columbia as well as additional ecotypes and mutant lines, and identified 12 further glucosinolates, including five novel compounds. Structures were elucidated by MS and NMR spectroscopy of their desulfated derivatives, and by enzymatic cleavage of the attached ester moieties. Four of the novel glucosinolates are benzoate esters isolated from the seeds. In all but one of these compounds, esterification is on the glucose moiety rather than the side chain, a very unusual feature for glucosinolates. Among additional glucosinolates identified were the first non-chain elongated, methionine-derived glucosinolate from A. thaliana and the first compounds that appear to be derived from leucine.     

Indole-3-acetonitrile production from indole glucosinolates deters oviposition by Pieris rapae

Like many crucifer-specialist herbivores, Pieris rapae uses the presence of glucosinolates as a signal for oviposition and larval feeding. Arabidopsis thaliana glucosinolate-related mutants provide a unique resource for studying the in vivo role of these compounds in affecting P. rapae oviposition. Low indole glucosinolate cyp79B2 cyp79B3 mutants received fewer eggs than wild type, confirming prior research showing that indole glucosinolates are an important oviposition cue. Transgenic plants overexpressing epithiospecifier protein, which shifts glucosinolate breakdown toward nitrile formation, are less attractive to ovipositing P. rapae females. Exogenous application of indol-3-ylmethylglucosinolate breakdown products to cyp79B2 cyp79B3 mutants showed that oviposition was increased by indole-3-carbinol and decreased by indole-3-acetonitrile (IAN). P. rapae larvae tolerate a cruciferous diet by using a gut enzyme to redirect glucosinolate breakdown toward less toxic nitriles, including IAN, rather than isothiocyanates. The presence of IAN in larval regurgitant contributes to reduced oviposition by adult females on larvae-infested plants. Therefore, production of nitriles via epithiospecifier protein in cruciferous plants, which makes the plants more sensitive to generalist herbivores, may be a counter-adaptive mechanism for reducing oviposition by P. rapae and perhaps other crucifer-specialist insects.     

Intraspecific variation in defense against a generalist lepidopteran herbivore in populations of Eruca sativa (Mill.)

Populations of Eruca sativa (Brassicaceae) from desert and Mediterranean (Med) habitats in Israel differ in their defense against larvae of the generalist Spodoptera littoralis but not the specialist Pieris brassicae. Larvae of the generalist insect feeding on plants of the Med population gained significantly less weight than those feeding on the desert plants, and exogenous application of methyl jasmonate (MJ) on leaves of the Med plants significantly reduced the level of damage created by the generalist larvae. However, MJ treatment significantly induced resistance in plants of the desert population, whereas the generalist larvae caused similar damage to MJ‐induced and noninduced plants. Analyses of glucosinolates and expression of genes in their synthesis pathway indicated that defense in plants of the Med population against the generalist insect is governed by the accumulation of glucosinolates. In plants of the desert population, trypsin proteinase inhibitor activity was highly induced in response to herbivory by S. littoralis. Analysis of genes in the defense‐regulating signaling pathways suggested that in response to herbivory, differences between populations in the induced levels of jasmonic acid, ethylene, and salicylic acid mediate the differential defenses against the insect. In addition, expression analysis of myrosinase‐associated protein NSP2 suggested that in plants of the desert population, glucosinolates breakdown products were primarily directed to nitrile production. We suggest that proteinase inhibitors provide an effective defense in the desert plants, in which glucosinolate production is directed to the less toxic nitriles. The ecological role of nitrile production in preventing infestation by specialists is discussed.     

The Arabidopsis Epithiospecifier Protein Promotes the Hydrolysis of Glucosinolates to Nitriles and Influences Trichoplusia ni Herbivory

Glucosinolates are anionic thioglucosides that have become one of the most frequently studied groups of defensive metabolites in plants. When tissue damage occurs, the thioglucoside linkage is hydrolyzed by enzymes known as myrosinases, resulting in the formation of a variety of products that are active against herbivores and pathogens. In an effort to learn more about the molecular genetic and biochemical regulation of glucosinolate hydrolysis product formation, we analyzed leaf samples of 122 Arabidopsis ecotypes. A distinct polymorphism was observed with all ecotypes producing primarily isothiocyanates or primarily nitriles. The ecotypes Columbia (Col) and Landsberg erecta (Ler) differed in their hydrolysis products; therefore, the Col x Ler recombinant inbred lines were used for mapping the genes controlling this polymorphism. The major quantitative trait locus (QTL) affecting nitrile versus isothiocyanate formation was found very close to a gene encoding a homolog of a Brassica napus epithiospecifier protein (ESP), which causes the formation of epithionitriles instead of isothiocyanates during glucosinolate hydrolysis in the seeds of certain Brassicaceae. The heterologously expressed Arabidopsis ESP was able to convert glucosinolates both to epithionitriles and to simple nitriles in the presence of myrosinase, and thus it was more versatile than previously described ESPs. The role of ESP in plant defense is uncertain, because the generalist herbivore Trichoplusia ni (the cabbage looper) was found to feed more readily on nitrile-producing than on isothiocyanate-producing Arabidopsis. However, isothiocyanates are frequently used as recognition cues by specialist herbivores, and so the formation of nitriles instead of isothiocyanates may allow Arabidopsis to be less apparent to specialists.     

Evidence for nucleolus organizer regions as the units of regulation in nucleolar dominance in Arabidopsis thaliana interecotype hybrids

Nucleolar dominance describes the silencing of one parent's ribosomal RNA (rRNA) genes in a genetic hybrid. In Arabidopsis thaliana, rRNA genes are clustered in two nucleolus organizer regions, NOR2 and NOR4. In F(8) recombinant inbreds (RI) of the A. thaliana ecotypes Ler and Cvi, lines that display strong nucleolar dominance inherited a specific combination of NORs, Cvi NOR4 and Ler NOR2. These lines express almost all rRNA from Cvi NOR4. The reciprocal NOR genotype, Ler NOR4/Cvi NOR2, allowed for expression of rRNA genes from both NORs. Collectively, these data reveal that neither Cvi rRNA genes nor NOR4 are always dominant. Furthermore, strong nucleolar dominance does not occur in every RI line inheriting Cvi NOR4 and Ler NOR2, indicating stochastic effects or the involvement of other genes segregating in the RI mapping population. A partial explanation is provided by an unlinked locus, identified by QTL analysis, that displays an epistatic interaction with the NORs and affects the relative expression of NOR4 vs. NOR2. Collectively, the data indicate that nucleolar dominance is a complex trait in which NORs, rather than individual rRNA genes, are the likely units of regulation.     

Comparison of feeding behaviour of two Brassica pests Brevicoryne brassicae and Myzus persicae on wild and cultivated brassica species

Feeding behaviour of the specialist Brassicae aphid, Brevicoryne brassicae (L) (cabbage aphid) and the generalist, Myzus persicae, (Sulzer) (peach potato aphid) was monitored electronically on the susceptible cauliflower, Brassica oleracea var. botrytis cv Newton Seale, and a range of 17 Brassica species, B. carinata, B. juncea, B. nigra, B. macrocarpa, and B. villosa var. drepanensis and cultivated brassica varieties, B. oleracea, B. campestris and B. napus. Aphids, monitored for 10 h on the underside of leaves, performed recognisable feeding behaviour on all brassica species. The main differences in feeding behaviour, between M. persicae and B. brassicae, on the susceptible cauliflower Newton Seale, were fewer probes, shorter times to initially reach the phloem but longer times to establish sustained phloem ingestion and the longer times spent, by M. persicae, in xylem ingestion.Feeding behaviour on the range of brassica species tested indicated that generalist and specialist aphids are influenced differently by the host plant. A longer time spent in xylem ingestion was again the major difference in the feeding behaviour of the two aphids. In addition, rejection of passive phloem ingestion, by M. persicae, was not related so closely to increased time spent in non probing activities, as for B. brassicae. This observation indicates that M. persicae does not generally accept or reject brassica species due to the presence of phagostimulants, such as glucosinolates at the leaf surface or along the stylet pathway, unless the concentration is very high. Differences in feeding strategies employed by generalist and specialist aphids on the same plants are discussed.     

Myzus persicae (green peach aphid) salivary components induce defence responses in Arabidopsis thaliana

Myzus persicae (green peach aphid) feeding on Arabidopsis thaliana induces a defence response, quantified as reduced aphid progeny production, in infested leaves but not in other parts of the plant. Similarly, infiltration of aphid saliva into Arabidopsis leaves causes only a local increase in aphid resistance. Further characterization of the defence-eliciting salivary components indicates that Arabidopsis recognizes a proteinaceous elicitor with a size between 3 and 10 kD. Genetic analysis using well-characterized Arabidopsis mutants shows that saliva-induced resistance against M. persicae is independent of the known defence signalling pathways involving salicylic acid, jasmonate and ethylene. Among 78 Arabidopsis genes that were induced by aphid saliva infiltration, 52 had been identified previously as aphid-induced, but few are responsive to the well-known plant defence signalling molecules salicylic acid and jasmonate. Quantitative PCR analyses confirm expression of saliva-induced genes. In particular, expression of a set of O -methyltransferases, which may be involved in the synthesis of aphid-repellent glucosinolates, was significantly up-regulated by both M. persicae feeding and treatment with aphid saliva. However, this did not correlate with increased production of 4-methoxyindol-3-ylmethylglucosinolate, suggesting that aphid salivary components trigger an Arabidopsis defence response that is independent of this aphid-deterrent glucosinolate.     

Myzus persicae (green peach aphid) feeding on Arabidopsis induces the formation of a deterrent indole glucosinolate

Cruciferous plants produce a wide variety of glucosinolates as a protection against herbivores and pathogens. However, very little is known about the importance of individual glucosinolates in plant defense and the regulation of their production in response to herbivory. When Myzus persicae (green peach aphid) feeds on Arabidopsis aliphatic glucosinolates pass through the aphid gut intact, but indole glucosinolates are mostly degraded. Although aphid feeding causes an overall decrease in Arabidopsis glucosinolate content, the production of 4-methoxyindol-3-ylmethylglucosinolate is induced. This altered glucosinolate profile is not a systemic plant response, but is limited to the area in which aphids are feeding. Aphid feeding on detached leaves causes a similar change in the glucosinolate profile, demonstrating that glucosinolate transport is not required for the observed changes. Salicylate-mediated signaling has been implicated in other plant responses to aphid feeding. However, analysis of eds5, pad4, npr1 and NahG transgenic Arabidopsis, which are compromised in this pathway, demonstrated that aphid-induced changes in the indole glucosinolate profile were unaffected. The addition of purified indol-3-ylmethylglucosinolate to the petioles of cyp79B2 cyp79B3 mutant leaves, which do not produce indole glucosinolates, showed that this glucosinolate serves as a precursor for the aphid-induced synthesis of 4-methoxyindol-3-ylmethylglucosinolate. In artificial diets, 4-methoxyindol-3-ylmethylglucosinolate is a significantly greater aphid deterrent in the absence of myrosinase than its metabolic precursor indol-3-ylmethylglucosinolate. Together, these results demonstrate that, in response to aphid feeding, Arabidopsis plants convert one indole glucosinolate to another that provides a greater defensive benefit.     

Modulation of CYP79 genes and glucosinolate profiles in Arabidopsis by defense signaling pathways

Glucosinolates are natural plant products that function in the defense toward herbivores and pathogens. Plant defense is regulated by multiple signal transduction pathways in which salicylic acid (SA), jasmonic acid, and ethylene function as signaling molecules. Glucosinolate content was analyzed in Arabidopsis wild-type plants in response to single or combinatorial treatments with methyljasmonate (MeJA), 2,6-dichloro-isonicotinic acid, ethylene, and 2,4-dichloro-phenoxyacetic acid, or by wounding. In addition, several signal transduction mutants and the SA-depleted transgenic NahG line were analyzed. In parallel, expression of glucosinolate biosynthetic genes of the CYP79 gene family and the UDPG:thiohydroximate glucosyltransferase was monitored. After MeJA treatment, the amount of indole glucosinolates increased 3- to 4-fold, and the corresponding Trp-metabolizing genes CYP79B2 and CYP79B3 were both highly induced. Specifically, the indole glucosinolate N-methoxy-indol-3-ylmethylglucosinolate accumulated 10-fold in response to MeJA treatment, whereas 4-methoxy-indol-3-ylmethylglucosinolate accumulated 1.5-fold in response to 2,6-dichloro-isonicotinic acid. In general, few changes were seen for the levels of aliphatic glucosinolates, although increases in the levels of 8-methylthiooctyl glucosinolate and 8-methylsulfinyloctyl glucosinolate were observed, particularly after MeJA treatments. The findings were supported by the composition of glucosinolates in the coronatine-insensitive mutant coi1, the ctr1 mutant displaying constitutive triple response, and the SA-overproducing mpk4 and cpr1 mutants. The present data indicate that different indole glucosinolate methoxylating enzymes are induced by the jasmonate and the SA signal transduction pathways, whereas the aliphatic glucosinolates appear to be primarily genetically and not environmentally controlled. Thus, different defense pathways activate subsets of biosynthetic enzymes, leading to the accumulation of specific glucosinolates.     

Metabolic engineering of p-hydroxybenzylglucosinolate in Arabidopsis by expression of the cyanogenic CYP79A1 from Sorghum bicolor

Glucosinolates are natural products in cruciferous plants, including Arabidopsis thaliana. CYP79A1 is the cytochrome P450 catalysing the conversion of tyrosine to p-hydroxyphenylacetaldoxime in the biosynthesis of the cyanogenic glucoside dhurrin in sorghum. Both glucosinolates and cyanogenic glucosides have oximes as intermediates. Expression of CYP79A1 in A. thaliana results in the production of high levels of the tyrosine-derived glucosinolate p-hydroxybenzylglucosinolate, which is not a natural constituent of A. thaliana. This provides further evidence that the enzymes have low substrate specificity with respect to the side chain. The ability of the cyanogenic CYP79A1 to integrate itself into the glucosinolate pathway has important implications for an evolutionary relationship between cyanogenic glucosides and glucosinolates, and for the possibility of genetic engineering of novel glucosinolates.     

Indole glucosinolate breakdown and its biological effects

Most species in the Brassicaceae produce one or more indole glucosinolates. In addition to the parent indol-3-ylmethylglucosinolate (IMG), other commonly encountered indole glucosinolates are 1-methoxyIMG, 4-hydroxyIMG, and 4-methoxyIMG. Upon tissue disruption, enzymatic hydrolysis of IMG produces an unstable aglucone, which reacts rapidly to form indole-3-acetonitrile and indol-3-ylmethyl isothiocyanate. The isothiocyanate, in turn, can react with water, ascorbate, glutathione, amino acids, and other plant metabolites to produce a variety of physiologically active indole compounds. Myrosinase-initiated breakdown of the substituted indole glucosinolates proceeds in a similar manner to that of IMG. Induction of indole glucosinolate production in response to biotic stress, experiments with mutant plants, and artificial diet assays suggest a significant role for indole glucosinolates in plant defense. However, some crucifer-feeding specialist herbivores recognize indole glucosinolates and their breakdown products as oviposition and/or feeding stimulants. In mammalian diets, IMG can have both beneficial and deleterious effects. Most IMG breakdown products induce the synthesis of phase 1 detoxifying enzymes, which may in some cases prevent carcinogenesis, but in other cases promote carcinogenesis. Recent advances in indole glucosinolate research have been fueled by their occurrence in the well-studied model plant Arabidopsis thaliana. Knowledge gained from genetic and biochemical experiments with A. thaliana can be applied to gain new insight into the ecological and nutritional properties of indole glucosinolates in other plant species.     

Wound-induced increases in the glucosinolate content of oilseed rape and their effect on subsequent herbivory by a crucifer specialist

Damage to the oilseed rape plant (Brassica napus L.) by the cabbage stem flea beetle, Psylliodes chrysocephala L. (Coleoptera: Chrysomelidae) induces systemic changes to the glucosinolate profile, most noticeably an increase in the concentration of indole glucosinolates. When jasmonic acid was applied to the cotyledons of the plant, a similar effect was observed. Feeding tests with artificial substrates compared a glucosinolate fraction from jasmonic acid-treated plants with a similar fraction from untreated plants. In these tests, alterations to the glucosinolate profile increased the feeding of a crucifer-specialist feeder (P. chrysocephala). However, in whole plant tests, P. chrysocephala did not feed more on the jasmonic acid treated plants than on the controls. This implies that other aspects of the damage response are being induced by the jasmonic acid treatment and having a negative effect on subsequent herbivory.