Identification of Glucosinolates in Seeds of Three Brassicaceae Species Known to Hyperaccumulate Heavy Metals

Plants from the Brassicaceae family are known to contain secondary metabolites called glucosinolates. Our goal was to establish by LC/MS the glucosinolate profile of seeds of three Brassicaceae species known to hyperaccumulate heavy metals. We investigated Alyssum fallacinum auct. non Hausskn ., Iberis intermedia Guers ., and Noccaea caerulescens (J. Presl & C. Presl ) F. K. Mey . Our results indicate that A. fallacinum seeds contain glucoiberin and glucoibervirin, which had not been previously identified in this plant. Furthermore, we report for the first time the presence of glucoiberin, glucoibervirin, glucotropaeolin, and sinigrin in I. intermedia. We have detected for the first time glucoconringiin in N. caerulescens. In addition, glucosinalbin, 4‐hydroxyglucobrassicin, and glucomoringin were also detected.     

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

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

Leaf surface wax layers of Brassicaceae lack feeding stimulants for Phaedon cochleariae

Numerous reports have indicated that glucosinolates are important stimulants for specialist herbivores feeding on Brassicaceae, and that these metabolites might be present on the plant surface and thereby detectable by an alighting insect. We investigated the outermost layer of leaves of two species of Brassicaceae, Brassica napus L. var. ‘Martina’ and Nasturtium officinale R. Br., using two highly selective extraction methods. When the epicuticular wax layer was mechanically removed with gum arabic, no glucosinolates were detectable in the lower and upper leaf surfaces. Extracting the leaf surfaces with a threefold short rinse with chloroform/methanol/water (2 : 1 : 1 vol/vol/vol) led to varying results, depending on the light conditions under which plants had been kept in the period prior to extraction. In plants kept under light, glucosinolates were detectable in a first extraction in minor concentrations, with increasing amounts in a second and third extraction. In plants kept in darkness, glucosinolates were almost absent in the first extraction. We postulate that the polar glucosinolates are washed from the inner leaf tissue through open stomata to the outside during solvent extraction, but are not naturally present in the outermost wax layer. The response of the crucifer specialist Phaedon cochleariae (F.) (Coleoptera: Chrysomelidae) to leaf surfaces of the host plants B. napus and N. officinale and to a glucosinolate was tested. Adults preferred both the adaxial and abaxial leaf surfaces of host plants that had been treated with gum arabic in order to remove the epicuticular waxes over intact surfaces. Waxes may therefore prevent direct contact with the stimulants. Sinigrin (allyl glucosinolate) and/or surface extracts of N. officinale leaves applied on Pisum sativum leaf discs did not evoke feeding, but feeding did occur when total leaf extracts of B. napus or N. officinale were applied on this non‐host. We conclude that glucosinolates might only act as feeding stimulants for P. cochleariae in concert with compounds other than surface waxes.     

Uptake and turn-over of glucosinolates sequestered in the sawfly Athalia rosae

Larvae of the sawfly Athalia rosae sequester glucosinolates from their various host plants of the Brassicaceae into their hemolymph for defensive purposes. We found that the glucosinolate concentration in the insect varies in a fluctuating manner during larval development. Analyses of larvae which had been offered diets with different glucosinolate profiles showed that there is an equilibrium between a rapid uptake of glucosinolates into the hemolymph and a continuous turn-over. Injection of glucotropaeolin into the hemolymph and ingestion of the same amount resulted in similar levels of intact glucosinolates recovered from larvae after different periods of time. This indicates that hemolymph glucosinolates are the principal source for glucosinolate degradation. Feeding experiments with [14C]-labeled glucotropaeolin revealed that the majority of the ingested glucosinolate is excreted as one or more unidentified metabolite(s) within 14 h. We found no indication for the presence of an insect myrosinase, or sulfatase in A. rosae, which have been shown to be involved in glucosinolate metabolism in other specialists feeding on Brassicaceae. Furthermore, the metabolism of sinalbin in A. rosae seems to result in different products than its metabolism in the caterpillar Pieris rapae. Obviously, A. rosae has yet another way of coping with the glucosinolates.     

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.     

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.     

Arbuscular mycorrhizal fungi might alleviate aluminium toxicity in banana plants

Under defined laboratory conditions it was shown that two glucosinolate-containing plant species, Tropaeolum majus and Carica papaya , were colonized by arbuscular mycorrhizal (AM) fungi, whereas it was not possible to detect AM fungal structures in other glucosinolate-containing plants (including several Brassicaceae). Benzylglucosinolate was present in all of the T. majus cultivars and in C. papaya it was the major glucosinolate. 2-Phenylethylglucosinolate was found in most of the non-host plants tested. Its absence in the AM host plants indicates a possible role for the isothiocyanate produced from its myrosinase-catalysed hydrolysis as a general AM inhibitory factor in non-host plants. The results suggest that some of the indole glucosinolates might also be involved in preventing AM formation in some of the species. In all plants tested, both AM hosts and non-hosts, the glucosinolate pattern was altered after inoculation with one of three different AM fungi ( Glomus mosseae , Glomus intraradices and Gigaspora rosea ), indicating signals between AM fungi and plants even before root colonization. The glucosinolate induction was not specifically dependent on the AM fungus. A time-course study in T. majus showed that glucosinolate induction was present during all stages of mycorrhizal colonization.     

Geographic and evolutionary diversification of glucosinolates among near relatives of Arabidopsis thaliana (Brassicaceae)

Glucosinolates are biologically active secondary metabolites that display both intra- and interspecific variation in the order Brassicales. Glucosinolate profiles have not been interpreted within a phylogenic framework and little is known regarding the processes that influence the evolution of glucosinolate diversity at a macroevolutionary scale. We have analyzed leaf glucosinolate profiles from members of the Brassicaceae that have diverged from Arabidopsis thaliana within the last 15 million years and interpreted our findings relative to the phylogeny of this group. We identified several interspecific polymorphisms in glucosinolate composition. A majority of these polymorphisms are lineage-specific secondary losses of glucosinolate characters, but a gain-of-character polymorphism was also detected. The genetic basis of most observed polymorphisms appears to be regulatory. In the case of A. lyrata, geographic distribution is also shown to contribute to glucosinolate metabolic diversity. Further, we observed evidence of gene-flow between sympatric species, parallel evolution, and the existence of genetic constraints on the evolution of glucosinolates within the Brassicaceae.     

An efficient protocol for genetic transformation of watercress (<Emphasis Type="Italic">Nasturtium officinale</Emphasis>) using <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>

Watercress (Nasturtium officinale) is a member of the Brassicaceae family and a rich source of glucosinolate, which has been shown to possess anticancer properties. To extract these compounds from N. officinale for study, a method was developed in which Agrobacterium rhizogenes was used to transfer DNA segments into plant genomes in order to produce hairy root cultures, which are a reliable source of plant compounds. The A. rhizogenes strain R1000 had the highest infection frequency and induces the most hairy roots per explant. Polymerase chain reaction and cytohistochemical staining methods were used to validate transgenic hairy roots from N. officinale. Glucosinolate from watercress hairy roots was separated and analyzed using high-performance liquid chromatography coupled to electrospray ionization mass spectrometry. Indolic glucosinolates, including glucobrassicin (0.01–0.02 μmol/g of DW) and 4-methoxyglucobrassicin (0.06–0.18 μmol/g of DW), as well as aromatic glucosinolate (gluconasturtiin) (0.06–0.21 μmol/g of DW), were identified virtually identical or more in transformed than wild type roots of N. officinale. Hairy root culture of watercress is a valuable approach for future efforts in the metabolic engineering of glucosinolate biofortification in plants, particularly, because indolic glucosinolates are the precursors of a potent cancer chemopreventive agent (indole-3-carbinol).     

High glucosinolate broccoli: a delivery system for sulforaphane

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

Suppressive Effect of Yamato-mana (Brassica rapa L. Oleifera Group) Constituent 3-Butenyl Glucosinolate (Gluconapin) on Postprandial Hypertriglyceridemia in Mice

We examined the bioactivity of Yamato-mana (Brassica rapa L. Oleifera Group) constituent glucosinolates and found that 3-butenyl glucosinolate (gluconapin) decreased the plasma triglyceride gain induced by corn oil administration to mice. However, phenethyl glucosinolate (gluconasturtiin) had little effect. 2-Propenyl glucosinolate (sinigrin) also reduced the plasma triglyceride level, which suggests that alkenyl glucosinolates might be promising agents to prevent postprandial hypertriglyceridemia.     

Variation in the glucosinolate content of vegetative tissues of Chinese lines of Brassica napus L

The glucosinolate content of leaves, stems and roots of a range of Chinese oilseed rape (Brassica napus L.) breeding lines was analysed. Total content and spectrum of individual glucosinolates varied widely, and there was no correlation between seed and vegetative tissue glucosinolate content. Lines with low seed glucosinolates (00) did not necessarily have low glucosinolate content in vegetative tissues; nor did high seed glucosinolate lines always have high vegetative tissue content. There was no correlation between the glucosinolate content of leaf, stem, and root in any given line. It appears that glucosinolate synthesis and accumulation is under tissue-specific control, and the mutation which blocks accumulation of glucosinolates in seeds does not influence other tissues. The responses of these lines to elicitors was also examined. Methyl jasmonate and salicylic acid treatments produced increases in leaf indolyl and aromatic glucosinolates respectively. However, the extent of such increases differed widely between the lines, and there were other, less consistent, effects on other classes of glucosinolate. There seems to be greater variation in glucosinolate accumulation in rape than has previously been reported, and the lines described here have considerable potential for evaluating the effects of manipulating glucosinolate profiles on pest and disease interactions.     

Prey‐mediated effects of glucosinolates on aphid predators

1. Plant resistance against herbivores can act directly (e.g. by producing toxins) and indirectly (e.g. by attracting natural enemies of herbivores). If plant secondary metabolites that cause direct resistance against herbivores, such as glucosinolates, negatively influence natural enemies, this may result in a conflict between direct and indirect plant resistance. 2. Our objectives were (i) to test herbivore‐mediated effects of glucosinolates on the performance of two generalist predators, the marmalade hoverfly (Episyrphus balteatus) and the common green lacewing (Chrysoperla carnea) and (ii) to test whether intraspecific plant variation affects predator performance. 3. Predators were fed either Brevicoryne brassicae, a glucosinolate‐sequestering specialist aphid that contains aphid‐specific myrosinases, or Myzus persicae, a non‐sequestering generalist aphid that excretes glucosinolates in the honeydew, reared on four different white cabbage cultivars. Predator performance and glucosinolate concentrations and profiles in B. brassicae and host‐plant phloem were measured, a novel approach as previous studies often measured glucosinolate concentrations only in total leaf material. 4. Interestingly, the specialist aphid B. brassicae selectively sequestered glucosinolates from its host plant. The performance of predators fed this aphid species was lower than when fed M. persicae. When fed B. brassicae reared on different cultivars, differences in predator performance matched differences in glucosinolate profiles among the aphids. 5. We show that not only the prey species, but also the plant cultivar can have an effect on the performance of predators. Our results suggest that in the tritrophic system tested, there might be a conflict between direct and indirect plant resistance.     

<Emphasis Type="Italic">MAM</Emphasis> gene silencing leads to the induction of C3 and reduction of C4 and C5 side-chain aliphatic glucosinolates in <Emphasis Type="Italic">Brassica napus</Emphasis>

Methylthioalkylmalate (MAM) synthases and their associated genes that have been extensively investigated in Arabidopsis control the side-chain elongation of methionine during the synthesis of aliphatic glucosinolates. A Brassica homolog of the Arabidopsis MAM genes was used in this study to analyze the role of MAM genes in B. napus through RNA interference (RNAi). The silencing of the MAM gene family in B. napus canola and B. napus rapeseed resulted in the reduction of aliphatic glucosinolates and total glucosinolate content. The results indicated that RNAi has potential for reducing glucosinolate content and improving meal quality in B. napus canola and rapeseed cultivars. Interestingly, MAM gene silencing in B. napus significantly induced the production of 2-propenyl glucosinolate, a 3-carbon side-chain glucosinolate commonly found in B. juncea mustard. Most transgenic plants displayed induction of 2-propenyl glucosinolate; however, the absolute content of this glucosinolate in transgenic B. napus canola was relatively low (less than 1.00 μmol g⁻¹ seed). In the high glucosinolate content progenies derived from the crosses of B. napus rapeseed and transgenic B. napus canola, MAM gene silencing strongly induced the production of 2-propenyl glucosinolate to high levels (up to 4.45 μmol g⁻¹ seed).     

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.     

Biochemical genetics of glucosinolate modification in Arabidopsis and Brassica

Fine mapping of the glucosinolate biosynthesis gene OHP, which regulates the conversion of 3-methylsulphinylpropyl to 3-hydroxypropyl glucosinolate, in an Arabidopsis thaliana Columbia × Landsberg erecta RI line population positioned the gene within 54 kb of DNA on chromosome IV. Sequence data identified a family of genes encoding 2-oxoglutarate-dependent dioxygenases in this region. A probe based on these genes co-segregated with ALK in Brassica oleracea,a gene regulating the synthesis of alkenyl glucosinolates. The reactions catalysed by the OHP and ALK enzymes utilise similar substrates and may have a common mechanism. Thus, these dioxygenases are prime candidates for controlling the side chain modification of glucosinolates. Received: 12 May 2000 / Accepted: 29 May 2000