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.     

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.     

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.     

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.     

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 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.     

植物激素与芥子油苷在生物合成上的相互作用

植物激素在植物的生长发育中起着关键性作用,芥子油苷是一类重要的次生代谢物质。植物激素与芥子油苷之间存在复杂的相互作用。生长素与吲哚类芥子油苷在生物合成上存在着相互作用。植物防卫信号分子与芥子油苷之间也存在相互作用,茉莉酸强烈诱导吲哚类芥子油苷生物合成相关基因CYP7982和CYP7983的表达,从而诱导吲哚-3-甲基芥子油苷和N-甲氧吲哚-3-甲基芥子油苷等吲哚类芥子油苷的生成,水杨酸和乙烯则能轻度诱导4-甲氧吲哚-3-甲基芥子油苷的生成。植物防卫信号转导途径相互作用以精细调节不同种类吲哚类芥子油苷的生成。     

Characterization of the Arabidopsis TU8 Glucosinolate Mutation,an Allele of TERMINAL FLOWER2

Glucosinolates are a group of defense-related secondary metabolites found in Arabidopsis and other cruciferous plants. Levels of leaf glucosinolates are regulated during plant development and increase in response to mechanical damage or insect feeding. The Arabidopsis TU8 mutant has a developmentally altered leaf glucosinolate profile: aliphatic glucosinolate levels drop off more rapidly, consistent with the early senescence of the mutant, and the levels of two indole glucosinolates are uniformly low. In TU8 seeds, four long-chain aliphatic glucosinolates have significantly increased levels, whereas the indolyl-3-methyl glucosinolate level is significantly reduced relative to wild type. Genetic mapping and DNA sequencing identified the TU8 mutation as tfl2-6, a new allele of TERMINAL FLOWER2 (TFL2), the only Arabidopsis homolog of animal HETEROCHROMATIN PROTEIN1 (HP1). TU8 (tfl2-6) has other previously identified tfl2 phenotypes, including an early transition to flowering, altered meristem structure, and stunted leaves. Analysis of two additional alleles, tfl2-1 and tfl2-2, showed glucosinolate profiles similar to those of line TU8 (tfl2-6).     

Cytochrome P450 CYP79B2 from Arabidopsis catalyzes the conversion of tryptophan to indole-3-acetaldoxime, a precursor of indole glucosinolates and indole-3-acetic acid

Glucosinolates are natural plant products known as flavor compounds, cancer-preventing agents, and biopesticides. We report cloning and characterization of the cytochrome P450 CYP79B2 from Arabidopsis. Heterologous expression of CYP79B2 in Escherichia coli shows that CYP79B2 catalyzes the conversion of tryptophan to indole-3-acetaldoxime. Recombinant CYP79B2 has a K(m) of 21 microm and a V(max) of 7.78 nmol/h/ml culture. Inhibitor studies show that CYP79B2 is different from a previously described enzyme activity that converts tryptophan to indole-3-acetaldoxime (Ludwig-Müller, J. , and Hilgenberg, W. (1990) Phytochemistry, 29, 1397-1400). CYP79B2 is wound-inducible and expressed in leaves, stem, flowers, and roots, with the highest expression in roots. Arabidopsis overexpressing CYP79B2 has increased levels of indole glucosinolates, which strongly indicates that CYP79B2 is involved in indole glucosinolate biosynthesis. Our data show that oxime production by CYP79s is not restricted to those amino acids that are precursors for cyanogenic glucosides. Our data are consistent with the hypothesis that indole glucosinolates have evolved from cyanogenesis. Indole-3-acetaldoxime is a precursor of the plant hormone indole-3-acetic acid, which suggests that CYP79B2 might function in biosynthesis of indole-3-acetic acid. Identification of CYP79B2 provides an important tool for modification of the indole glucosinolate content to improve nutritional value and pest resistance.     

Contribution of Glucosinolate Transport to Arabidopsis Defense Responses

Accumulation of glucosinolates, a class of defense-related secondary metabolites found almost exclusively in the Capparales, is induced in response to a variety of biological stresses. It is often assumed that elevated glucosinolate levels result from de novo biosynthesis, but glucosinolate transport from other parts of the plant to the site of herbivory or pathogen infection can also contribute to the defense response. Several studies with Arabidopsis and other crucifers have demonstrated that glucosinolates from vegetative tissue are transported to developing seeds. Here we discuss evidence that long-chain aliphatic glucosinolates are transported to the site of herbivory in response to Myzus persicae (green peach aphid) feeding on Arabidopsis.Key Words: glucosinolate, transport, graft, Arabidopsis, Myzus persicae, aphid     

Conservation and clade-specific diversification of pathogen-inducible tryptophan and indole glucosinolate metabolism in Arabidopsis thaliana relatives

? A hallmark of the innate immune system of plants is the biosynthesis of low-molecular-weight compounds referred to as secondary metabolites. Tryptophan-derived branch pathways contribute to the capacity for chemical defense against microbes in Arabidopsis thaliana. ? Here, we investigated phylogenetic patterns of this metabolic pathway in relatives of A. thaliana following inoculation with filamentous fungal pathogens that employ contrasting infection strategies. ? The study revealed unexpected phylogenetic conservation of the pathogen-induced indole glucosinolate (IG) metabolic pathway, including a metabolic shift of IG biosynthesis to 4-methoxyindol-3-ylmethylglucosinolate and IG metabolization. By contrast, indole-3-carboxylic acid and camalexin biosyntheses are clade-specific innovations within this metabolic framework. A Capsella rubella accession was found to be devoid of any IG metabolites and to lack orthologs of two A. thaliana genes needed for 4-methoxyindol-3-ylmethylglucosinolate biosynthesis or hydrolysis. However, C. rubella was found to retain the capacity to deposit callose after treatment with the bacterial flagellin-derived epitope flg22 and pre-invasive resistance against a nonadapted powdery mildew fungus. ? We conclude that pathogen-inducible IG metabolism in the Brassicaceae is evolutionarily ancient, while other tryptophan-derived branch pathways represent relatively recent manifestations of a plant-pathogen arms race. Moreover, at least one Brassicaceae lineage appears to have evolved IG-independent defense signaling and/or output pathway(s).     

Genetic control of natural variation in Arabidopsis glucosinolate accumulation

Glucosinolates are biologically active secondary metabolites of the Brassicaceae and related plant families that influence plant/insect interactions. Specific glucosinolates can act as feeding deterrents or stimulants, depending upon the insect species. Hence, natural selection might favor the presence of diverse glucosinolate profiles within a given species. We determined quantitative and qualitative variation in glucosinolates in the leaves and seeds of 39 Arabidopsis ecotypes. We identified 34 different glucosinolates, of which the majority are chain-elongated compounds derived from methionine. Polymorphism at only five loci was sufficient to generate 14 qualitatitvely different leaf glucosinolate profiles. Thus, there appears to be a modular genetic system regulating glucosinolate profiles in Arabidopsis. This system allows the rapid generation of new glucosinolate combinations in response to changing herbivory or other selective pressures. In addition to the qualitative variation in glucosinolate profiles, we found a nearly 20-fold difference in the quantity of total aliphatic glucosinolates and were able to identify a single locus that controls nearly three-quarters of this variation.     

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.     

Indole Glucosinolates and Camalexin do not Influence the Development of the Clubroot Disease in Arabidopsis thaliana

Root galls of Brassicaceae caused by Plasmodiophora brassicae are dependent on increased auxin and cytokinin formation. In this study we investigated whether indole glucosinolates are involved in indole‐3‐acetic acid (IAA) biosynthesis in root galls, by using a genetic approach. The cytochrome P450 enzymes, CYP79B2 and CYP79B3, convert tryptophan to indole‐3‐acetaldoxime (IAOx), which is a precursor for indole glucosinolates and the phytoalexin camalexin in Arabidopsis thaliana. Root galls of the Arabidopsis ecotypes Wassilewskija (WS) and Columbia (Col) accumulated camalexin, WS at levels up to 320 μg/g dry weight. By contrast, camalexin was absent in root galls of cyp79b2/b3 double mutants. Infection rate and disease index as a measure of club development in mutant and wild‐type plants of the two ecotypes were investigated and no differences were found in gall formation. This demonstrates that camalexin is an ineffective inhibitor of P. brassicae and indole glucosinolates are not the source of elevated levels of IAA in galls, because free IAA levels in mutant galls were comparable with those in wild type.     

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.     

拟南芥莲座叶芥子油苷含量对水分胁迫的响应

芥子油苷(glucosinolates)是十字花科植物中一类含氮、含硫的次生代谢产物,与其水解产物在植物防御功能中有重要意义且与环境因子关系密切.通过控制供水的方式对营养生长时期的拟南芥幼苗进行水分胁迫,观察了土壤自然干旱对营养生长时期拟南芥莲座叶芥子油苷含量及组成的影响.结果表明,土壤自然干旱处理下,拟南芥莲座叶的芥子油苷总量从处理3 d起低于对照,且随着处理天数的增加与对照组的差异逐渐增大,脂肪族芥子油苷的响应均比较明显,与芥子油苷总量的变化趋势基本一致,而吲哚族芥子油苷对水分胁迫则不敏感.脂肪族中的4-甲基亚磺酰丁基芥子油苷(4-methylsulphinylbutyl GS,4MSOB)占脂肪族芥子油苷的比例最大,它的含量变化成为影响莲座叶中芥子油苷组合模式的主导因素.